Download Parallel Session Abstracts

Fifth International
Barcode of Life Conference
第五届国际生命条形码大会
www.dnabarcodes2013.org
Abstracts Volume
27-31 October 2013
Kunming China 中国昆明
Contents
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Contents
Contents .................................................................................................................................................................... 1
Part I. Plenary Session Speakers and Abstracts ................................................................................................... 3
Session 1: iBOL Update ................................................................................................................................... 4
Session 2: Implications for Biodiversity I ...................................................................................................... 7
Session 3: Implications for Biodiversity II................................................................................................... 10
Session 4: Implications for Biodiversity III ................................................................................................. 14
Session 5: Ecological Implications ................................................................................................................ 16
Session 6: Ecosystem Implications ............................................................................................................... 19
Session 7: Societal Implications .................................................................................................................... 22
Session 8: Final Reflections ........................................................................................................................... 25
Part II. Parallel Session Abstracts: Oral presentation ....................................................................................... 28
Parallel Session 1: Plants I ............................................................................................................................ 29
Parallel Session 1: Fishes I ............................................................................................................................ 36
Parallel Session 1: Insects I ........................................................................................................................... 43
Parallel Session 1: Education ........................................................................................................................ 50
Parallel Session 1: Next Generation Sequencing......................................................................................... 56
Parallel Session 2: Pollinators....................................................................................................................... 61
Parallel Session 2: Plants II .......................................................................................................................... 66
Parallel Session 2: Data Analysis Methods .................................................................................................. 71
Parallel Session 2: Environmental Monitoring ........................................................................................... 77
Parallel Session 2: Amphibians and Reptiles............................................................................................... 82
Parallel Session 3: Informatics ..................................................................................................................... 87
Parallel Session 3: Medicinal Plants I .......................................................................................................... 94
Parallel Session 3: Marine Barcoding ........................................................................................................ 101
Parallel Session 3: Pests, Parasites, Etc. .................................................................................................... 108
Parallel Session 3: Insects II ....................................................................................................................... 115
Parallel Session 4: Other Invertebrates ..................................................................................................... 122
Parallel Session 4: Fishes II ........................................................................................................................ 127
Parallel Session 4: Plants III ....................................................................................................................... 132
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Contents
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Parallel Session 4: Environmental Barcoding ........................................................................................... 137
Parallel Session 4: Insects III ...................................................................................................................... 142
Parallel Session 5: Plant Methods .............................................................................................................. 146
Parallel Session 5: Fungi and Algae ........................................................................................................... 153
Parallel Session 5: International Collaboration ........................................................................................ 160
Parallel Session 5: Medicinal Plants II ...................................................................................................... 168
Part III. Posters and others................................................................................................................................. 171
Amphibians and Reptiles ............................................................................................................................ 172
Education...................................................................................................................................................... 175
Environmental Barcoding ........................................................................................................................... 179
Environmental Monitoring ......................................................................................................................... 182
Fishes............................................................................................................................................................. 187
Fungi and Algae ........................................................................................................................................... 198
Informatics ................................................................................................................................................... 207
Insects ........................................................................................................................................................... 209
Marine Barcoding ........................................................................................................................................ 223
Medicinal Plants .......................................................................................................................................... 232
Next Generation Sequencing....................................................................................................................... 241
Other Invertebrates ..................................................................................................................................... 242
Plants............................................................................................................................................................. 244
Pollinators..................................................................................................................................................... 256
Vertebrates ................................................................................................................................................... 257
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Plenary Session Speakers & Abstracts
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Part I. Plenary Session Speakers
and Abstracts
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Plenary Session Speakers & Abstracts
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Session 1: iBOL Update
De-Zhu Li (李德铢)
Prof. De-Zhu Li received his PhD from the Chinese Academy of
Sciences (CAS)’ Kunming Institute of Botany (KIB) in 1990. He
worked at the Royal Botanic Garden Edinburgh from 1993 to 1994
as a Ferguson Fellow and at the Cambridge University Botanic
Garden as Taxonomist from 1994 to 1996. He is now Director and
Professor of KIB/CAS, as well as Vice President of the Botanical
Society of China, and the Representative of China for the
International Barcode of Life project (iBOL). His research interests
focus on plant systematics, biogeography, and germplasm
conservation. He has authored some 260 papers, including 170 in
ISI-index journals, such as PNAS, Trends in Plant Science,
Molecular Ecology, New Phytologist, Molecular Phylogenetics and Evolution, and Taxon. He has
authored or contributed to nine monographs and books, including the Lonicera account of the
European Garden Flora and the bamboo account of the Flora of China, and is the editor of the
Chinese translation of Plant Systematics, A Phylogenetic Approach (3rd Edition).
Integrating DNA Barcoding and Genomics for Biodiversity Inventory and
Monitoring
De-Zhu Li
Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming, Yunnan 650201, China
E-mail: [email protected]
Half a century after the discovery of the double helix of DNA, and two years after completion of the
draft human genome, DNA barcoding was proposed to speed the documentation of biodiversity.
Coordinated efforts have been made to build a standardized barcode reference library for major
phyla of animals, as well as algae, fungi, plants and protists. DNA barcoding has already amplified
the inventory and understanding of global biodiversity since 2003. Meanwhile, next-generation
sequencing made it possible to accelerate the sequencing of barcodes, organelles and genomes. The
use of organelle-scale barcodes (plastid or mitochondrial genomes) is becoming feasible, while
meta-barcoding is addressing in-depth ecological and environmental questions. Recently, iFlora was
proposed to incorporated DNA barcodes, diagnostic morphological characters, digital images and
geo-referenced collections for inventory and monitoring of plant diversity. It seems that deep
integration of barcoding and genomics into biodiversity research and conservation will be the key
challenge for the barcode of life research community over the next decade in the face of global
change.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Plenary Session Speakers & Abstracts
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Session 1: iBOL Update
Gerhard Haszprunar
Gerhard Haszprunar is a Full Professor of Systematic Zoology at
the University of Munich (LMU) and heads as Director General all
natural history museums of Bavaria, Germany. Focusing on
Mollusca he is most interested in all aspects of integrative
phylogeny and taxonomy. He sees barcoding activities as a major
tool for taxonomic outreach to applicant and the broad public and to
focus taxonomic research on exiting taxa.
Barcoding Fauna Bavarica: Facts, Surprises, Visions
Gerhard Haszprunar
SNSB - Zoologische Staatssammlung München, Münchhausenstr. 21, 81247 München, Germany
E-mail: [email protected]
In 2009 the BFB-project has started to provide a barcode-catalogue of the whole Bavarian fauna
with about 35.000 species in the framework of iBOL. Currently about 12.000 species have been
barcoded showing high agreement with traditional taxonomy concerning insect groups, but also
revealing substantial gaps in knowledge concerning e.g. the molluscs. The currently proposed
second phase of the project will focus on remaining “difficult” groups, will widen to include also
lichens and fungi, will add applied aspects such as parasites, Red List or CITES taxa, and will
strengthen outreach activities.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Plenary Session Speakers & Abstracts
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Session 1: iBOL Update
Paul Hebert
Dr. Paul Hebert is the Scientific Director of iBOL and Director of the
Biodiversity Institute of Ontario. A native of Kingston, Ontario, Dr.
Hebert completed his undergraduate work at Queen’s University, his
PhD in genetics at Cambridge University, and postdoctoral
fellowships at the University of Sydney and at the Natural History
Museum in London. He took up a faculty position at the University
of Windsor in 1976 where he remained until repositioning to the
University of Guelph in 1990 where he holds a Canada Research
Chair in Molecular Biodiversity. Over his career, Dr. Hebert has
served as Director of the Great Lakes Institute at Windsor, as Chair
of the Department of Zoology at Guelph, and as Chairman of the
Huntsman Marine Science Centre in St. Andrews. He has published
more than 375 papers, has received varied national and international
scientific awards, and is a Fellow of the Royal Society of Canada. His 2003 paper proposing DNA
barcodes as a method to identify all species is regarded as the beginning of the global barcode
enterprise.
DNA Barcoding - Then, Now, Next
Paul Hebert
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
E-mail: [email protected]
Over the past decade, DNA barcoding has transitioned from academic battleground to major research
enterprise. For the first few years, reviewers were unkind, critics were vocal, and funding was scarce.
The situation has shifted; our community now oversees the largest research program ever undertaken in
biodiversity science. DNA barcodes have already been obtained from 2.5 million specimens, and it is
very likely that the International Barcode of Life Project will achieve its primary goal of delivering
barcode coverage for 500K species by late 2015. While this progress represents collective effort, some
nations and some places have gained more intensive analysis than others. These settings provide a first
indication of the ways in which DNA barcoding will improve our understanding of planetary
biodiversity. As we look to the future, it is clear that biodiversity science needs to transition from a
name-based discipline to one founded on an indexing system, a shift that occurred much earlier in other
scientific fields. Because of its role as a taxonomic registry, the Barcode Index Number (BIN) system
will support this transition, enabling rapid analysis of species diversity patterns. By coupling BINs with
the ability of next generation sequencers to process massive numbers of specimens, we can expect a
species count for the planet within a decade or so. By mapping species distributions with unprecedented
resolution, DNA barcoding will aid conservation action at a critical time, while enabling new
investigations in evolution, ecology, and genomics. While full realization of these impacts will require
augmented funding, new research facilities, and further diversification of the network of collaborators,
the next decade promises scientific discovery on a grand scale.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Plenary Session Speakers & Abstracts
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Session 2: Implications for Biodiversity I
Pete Hollingsworth
Pete Hollingsworth is Director of Science at the Royal Botanic
Garden Edinburgh, and is a visiting professor at the University of
Edinburgh and an honorary professor of the Kunming Institute of
Botany. His background is in plant population genetics and his
research interests include understanding plant diversification,
integration of genetic information into conservation programmes,
use of DNA data in taxonomy, and reintroduction biology.
Plant barcoding: current status, future directions
Pete Hollingsworth
Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh, EH3 5LR, UK
E-mail: [email protected]
Plants are dominant structural components of terrestrial ecosystems and underpin ecosystem
functions and provide extensive goods and services that are directly or indirectly used by humanity.
However, despite centuries of work to characterise plant diversity, there is still remarkable
uncertainty in estimates of global species numbers, and there remains incomplete floristic and
monographic coverage of taxa and regions. During this talk, I will give an overview of the progress
and potential of DNA barcoding to contribute to these challenges. I will firstly outline the scope and
scale of current plant barcoding activities. I will secondly, cover some of the major challenges for
plant barcoding. Finally, I will explore future opportunities for greater integration of DNA sequence
data into the plant taxonomy/biodiversity characterisation work flow.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Plenary Session Speakers & Abstracts
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Session 2: Implications for Biodiversity I
Michelle Van der Bank
Michelle van der Bank is an Associated Professor at the University
of Johannesburg (South Africa) and Head of the African Centre for
DNA Barcoding (ACDB). The ACDB mission is to fill the
knowledge gap and strengthen research frameworks for international,
regional and inter-institutional co-operation in Africa in the field of
DNA technology for biodiversity science. Her research group uses
molecular phylogenetics, comparative analyses and intensive
fieldwork to address questions relevant to conservation and our
understanding of biodiversity in Africa.
TreeBOL Africa: from barcodes to ecological applications
Michelle Van der Bank
Department of Botany and Plant Biotechnology, University of Johannesburg, Auckland Park 2006
South Africa
E-mail: [email protected]
We undertook a seven-year data collection, sampling over 50% of the estimated 2,486 woody shrub
and tree species in southern Africa, spanning 115 families and 661 genera of Gymnosperms and
Angiosperms. We then generated DNA sequences for all plants collected based on the core plant
barcodes (rbcLa and matK). Using this DNA database, we reconstructed the phylogenetic
relationships of the region’s woody flora using Bayesian inference and 28 independent fossil
calibrations. This phylogeny was used to address a wide range of questions across disciplines
including ecology, conservation biology, taxonomy, extinction risk, biogeography, and invasion
biology. In general, the results underline the potential significance of molecular phylogenetic
method in characterizing biodiversity, especially under the unprecedented rate of species loss.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Plenary Session Speakers & Abstracts
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Session 2: Implications for Biodiversity I
Cecilia Kopuchian
Cecilia Kopuchian is a researcher of the National Research Council
of Argentina (CONICET), working in the Ornithology Division of
the Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”
in Buenos Aires.Her research is focused in diverse aspects of avian
evolutionary biology, including communication, morphology,
speciation, and diversification patterns of Neotropical birds. She has
been involved in the project to barcode the birds of southern South
America since 2005, an activity that has been integrated with his
interests in evolutionary biology. She is currently also part of
projects aimed at barcoding other animal groups, with special
emphasis on lepidopterans.
Studying avian diversification in the southern Neotropics with DNA barcodes
Dario Lijtmae1, Cecilia Kopuchian2
División Ornitología, Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Buenos
Aires, Argentina
National Research Council of Argentina (CONICET), Buenos Aires, Argentina
E-mail: [email protected]
Even though southern South America comprises a diverse array of environments, including areas
with some of the highest rates of avian diversification, evolutionary patterns in this region have
barely been studied. We used DNA barcodes from 3,425 specimens belonging to 730 species of
Argentinean and Bolivian birds to analyze the patterns of diversification in this region and assess
the role of various diversifying factors. Comparison with birds of the Nearctic, the Palearctic and
lower latitudes of the Neotropics evidenced that diversification patterns in southern South America
are complex, consistent with the influence of glacial cycles as well as other factors more typical of
the Neotropical realm. Comparisons among different areas of southern South America showed that
Patagonian and Andean species are younger and less variable than more tropical or widely
distributed species, a pattern consistent with a more prominent role of glacial cycles in these areas.
Finally, seven percent of the species showed high intraspecific variation, usually including deeply
divergent lineages. The study of these cases with various algorithms permitted an initial assessment
of the presence of evolutionary independent lineages. Further analyses will indicate whether these
taxa include overlooked species, allowing a better understanding of avian diversity in this region.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Plenary Session Speakers & Abstracts
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Session 3: Implications for Biodiversity II
Bob Murphy
Prof. Robert Murphy received his Ph.D. in 1982 from the University of
California, Los Angeles (UCLA). After postdoctoral positions at
UCLA in the genetics of freshwater fishes and the use of flow
cytometry to diagnose forms of human cancers, he accepted
employment at the Royal Ontario Museum, Toronto Canada, where he
is the Sr. Curator of Herpetology. Among various appointments, he is a
professor in the Department of Ecology and Evolutionary Biology at
the University of Toronto and a Visiting Senior International Professor
in the Chinese Academy of Science stationed in the Laboratory of
Molecular Evolution & Genome Diversity, Kunming Institute of
Zoology. He serves as the co-chair of Cold Code, and the amphibian
and nonavian reptile committees of Genome 10K. His interests and
publications span from classical taxonomy to molecular evolution and genomics.
Cold Code: What’s in a Term?
Bob Murphy1, 2
1
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese
Academy of Sciences, Kunming, Yunnan 650223, China
2
Department of Natural History, Royal Ontario Museum, Toronto, Ontario, Canada
E-mail: [email protected]
DNA barcoding initiatives have been developed for many groups of organisms, including some
clusters that fall outside of taxonomic affiliations. Until recently, all vertebrate groups were
represented in these initiatives except for amphibians and nonavian reptiles. Funded by China, an
initiative was developed for these taxa. As a marketing strategy, the moniker ‘Cold Code’ was
chosen for the cold-blooded barcoding of species that Linnaeus considered to be ‘foul and
loathsome creatures”. The development of Cold Code was initially announced in 2012 and the
dedicated website www.coldcode.org became active in 2013. The project has been received
enthusiastically and the development of the website and Cold Code continues. Part of Cold Code
involves the free barcoding of the first 10 specimens of a species and the international community is
increasingly taking advantage of this opportunity. Cold Code not only interacts actively with iBOL
and CBOL, it has applications to law enforcement initiatives.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Plenary Session Speakers & Abstracts
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Session 3: Implications for Biodiversity II
Alfried Vogler
Dr. Alfried Vogler is Professor of Molecular Systematics at Imperial
College London and the Natural History Museum. He obtained a
PhD in Bacterial Genetics in 1988, before moving into molecular
taxonomy for his postdoc at the American Museum of Natural
History. He has been active in DNA barcoding since the legendary
‘Banbury I’ meeting in 2003 and was a co-organizer of the 1st
International Barcode of Life conference held in London, UK. His
main interests are DNA approaches to understand the evolution and
patterns of diversity of insects.
New approaches to assess patterns of biodiversity with DNA barcoding
Alfried Vogler1, 2
1
Department of Entomology, Natural History Museum, London SW7 5BD, United Kingdom
2
Division of Biology, Imperial College London, Ascot SL5 7PY, United Kingdom
E-mail: [email protected]
With the rapidly growing barcode database, more ambitious programs can be launched for sampling
and to investigate patterns of biodiversity. This talk will propagate a ‘site-based’ approach to DNA
barcoding, i.e. to conduct DNA-based detailed inventories of taxonomically or ecologically chosen
target groups at specifically chosen localities. This approach simplifies the logistics and
standardization of sampling and permits the application of spatially defined models of taxonomic
richness and turnover. We demonstrate the results from such studies on large communities of insects
across selected sampling sites, which revealed striking regularities in the spatial distribution of
species and genotypes. These results should stimulate the synthesis of geographic data from the
barcode database. In addition, faster methods are needed to assess the species diversity in entire
communities, e.g. as those obtained by standardized trapping of tropical insects. We propose the use
of whole-mitochondrial genomes as a system for biodiversity assessment, in an approach termed
mitochondrial metagenomics. Mitogenomes retain many advantages of the COI marker but are
more easily accessible to next-generation sequencing technology and are stronger marker for
phylogenetics. The growing library of mitogenomes can be linked to the existing taxonomy via
standard COI barcodes. Several pilot studies will be presented which show that the mitogenome
approach simplifies the characterisation of taxonomically complex samples. The procedure has
great potential for global monitoring of biodiversity.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Plenary Session Speakers & Abstracts
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Session 3: Implications for Biodiversity II
Michael Balke
Dr. Michael Balke received his MSc and PhD from Free
University Berlin and completed his post-doctoral work at the
Natural History Museum in London, the National University of
Singapore and the Zoological State Collection Munich
(SNSB-ZSM). At present, he is a curator of Coleoptera at ZSM.
He has also be involved in short stays at the USNM (NMNH) in
Washington DC, the Natural History Museum in Vienna, and the
South Australian Museum in Adelaide. His field trips have been
mainly to tropical countries around the world, with a focus on
New Guinea and France. Michael started as a beetle taxonomist
30+ years ago, but has since developed keen interest in
evolutionary biology, ecology and of course, molecular
systematics, molecular biodiversity assessment; he was one of
the coordinators of Barcoding Fauna Bavarica.
Taxonomy. Faster, Better
Michael Balke
SNSB - Zoologische Staatssammlung München, Münchhausenstr. 21, 81247 München, Germany
E-mail: [email protected]
The term “turbo-taxonomy” was recently coined for an approach combining cox1 sequences,
concise morphological descriptions by an expert taxonomist, and high-resolution digital imaging to
streamline the formal description of larger numbers of new species. We further developed this
approach and demonstrate the functionality of a species-description pipeline by naming 101 new
species of hyper diverse New Guinea Trigonopterus weevils in the open-access journal ZooKeys.
We conclude that fast track taxonomy will not only increase speed, but also sustainability of global
species inventories. It will be of great practical value to all the other disciplines that depend on a
usable taxonomy and will change our perception of global biodiversity. While this approach is
certainly not suitable for all taxa alike, it is the tool that will help to tackle many hyper diverse
groups and pave the road for more sustainable comparative studies. Finally, we will present a
proposed large-scale pilot study in Indonesia, and argue that fast track taxonomy and the BIN
system will have to work hand in hand to provide objective, sustainable data in such a large
framework where it is hopeless to assume that traditional tools will be able to inform stakeholders
about the contents of a given area.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Plenary Session Speakers & Abstracts
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Session 4: Implications for Biodiversity III
Jan Pawlowski
Dr. Jan Pawlowski is a Professor of Biology at the Department of
Genetics and Evolution, University of Geneva. He is leading the
research group on molecular evolution and ecology of microbial
eukaryotes (protists). His scientific interests include evolutionary
history of protists, origin of their diversification and their symbiotic
interactions. His group is involved in several projects dealing with
phylogenomics and metagenetic analysis of marine eukaryotes
diversity, using next generation sequencing (NGS) technologies. He
is particularly interested in genetic diversity of bottom-dwelling
organisms in the deep-sea and Polar Regions, focusing on
foraminifera, which constitute one of the major groups of marine
meiobenthos, commonly used as bio indicators of marine pollution.
His laboratory hosts the largest collection of DNA isolated from
foraminifera and deep-sea sediment samples. Large part of his activities is devoted to DNA
barcoding: he coordinates the Protist Working Group of the International Barcode of Life (iBOL),
and is leading the Swiss Barcode of Life Network. Currently, he is working on NGS-based
assessment of foraminiferal richness and its applications for bio monitoring and survey of marine
environment. http://genev.unige.ch/en/users/Jan-Pawlowski/unit
Barcoding protists: the unseen eukaryotic majority
Jan Pawlowski
Department of Genetics and Evolution, University of Geneva, CH-1205 Geneva, Switzerland
Email: [email protected]
Protists or micro eukaryotes comprise multiple lineages of mainly single-cell organisms, grouped
together with animals, plants and fungi into a few super-groups of eukaryotes. Free-living, parasitic
and symbiotic protists play a key ecological role in various types of environment. Yet, because of
their commonly small size and a lack of distinctive morphological features, they often go unseen
and remain the most inconspicuous fraction of eukaryotes. About 75,000 species belonging to
different groups of protists have been formally described. The NGS environmental surveys of
eukaryotic diversity, however, suggest that there are more than a million of genetically divergent
protistan taxa. Identification of these taxa and their placement in a universal morpho-genetic
framework of protist taxonomy is the main task of Protist Working Group (ProWG), created in 2011
by a group of experts in protist taxonomy and ecology. Based on multiple studies exploring protist
genetic diversity, the ProWG recommended the two-step barcoding approach, which comprises a
preliminary identification using a universal 18S rDNA barcode followed by a species-level
assignment using a group-specific barcode. Defining the specific barcodes and documenting the
morphology of thousands uncultivable species are the main challenges for the future of protist
barcoding.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Plenary Session Speakers & Abstracts
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Session 4: Implications for Biodiversity III
Gary Saunders
Dr. Gary Saunders is a Professor and Research Chair at the
University of New Brunswick, Canada. His research focuses on
aspects of marine algal systematics and biogeography. He has
collected extensively, which has greatly facilitated seaweed barcode
surveys from remote regions, and is recognized as a leader in the
field of protistan barcoding.
Global DNA barcode surveys reveal would-be endemics as widely distributed
species highlighting the importance of long-distance dispersal in seaweed
biogeography and speciation
Gary Saunders
Department of Biology, University of New Brunswick, Fredrickton, Canada
E-mail: [email protected]
Traditionally scientists have established species inventories for an area by examining the
morphology of individuals. Although appropriate for many organisms, this approach commonly
fails for seaweeds. Molecular-assisted alpha taxonomy has generated a novel appreciation of
seaweed diversity with many previously overlooked species being uncovered; taxonomic
conundrums corrected and introduced species reported. Using this approach we surveyed the marine
algal flora of Churchill during 2006-2007 and uncovered ~12 ‘cryptic endemic species’. Explaining
endemic species in this recently deglaciated region spawned notions of undiscovered refugia.
Similar surveys in Haida Gwaii from 2009-2012, a known glacial refugium, also turned up ~30
‘cryptic endemics’, but unexpectedly provided novel records for some of the Churchill ‘endemics’ –
the latter apparently Pacific to Churchill in their distribution and not Arctic endemics at all. In a
similar dethroning, barcode data generated for Californian collections from 2010 uncovered
virtually all of the Haida Gwaii ‘endemics’, these species apparently having a disjunct distribution
from California to northern British Columbia (not encountered during sampling from 2004 to 2011
in southern British Columbia with ~4000 barcode records). Hypotheses will be presented to explain
and explore these disjunct distributional patterns and the advantages of global over local barcode
projects will be stressed.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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Plenary Session Speakers & Abstracts
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Session 4: Implications for Biodiversity III
Zhu-Liang Yang (杨祝良)
After received his Ph. D. and a prize for the best dissertation from the
Department of Biology of the Universität Tübingen, Germany, Prof. Dr.
Yang has been working as a mycologist at the Key Laboratory of
Biodiversity and Biogeography, Kunming Institute of Botany, Chinese
Academy of Sciences for 15 years. He has over 20 years of experience in
the collecting, studying and identification of mushrooms (Fungi) in China,
and has published two monographs and over 80 scientific papers in
international scientific journals, such as Fungal Genetics and Biology,
Fungal Diversity, Mycologia, Mycological Research, and Molecular
Ecology. He received a rewarded fund for his scientific studies from the
National Science Fund for Distinguished Young Scholars of the National
Natural Science Foundation of China in 2005. He has been acting as the
editor-in-Chief of Fungal Diversity since 2013.
DNA barcoding of economically important mushrooms from China
Zhu-Liang Yang
Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy
of Sciences, Kunming, Yunnan 650201, China
E-mail: [email protected]
Wild mushrooms are important natural resources both ecologically and economically. As saprophytes or
ectomycorrhizal partners, they play vital roles in ecosystems. As gourmet food or traditional medicine,
humans have collected mushrooms since ancient times. However, dramatic poisonings or even deaths from
consuming poisonous mushrooms are frequently reported in China and in many other countries. It is true that
separating edible mushrooms from the poisonous ones is often difficult even for professional taxonomists, let
alone for non-experts. In order to establish a standard DNA barcode for edible and poisonous mushrooms in
China, several candidate markers were tested, including the nuclear large subunit ribosomal RNA gene
(nLSU), the internal transcribed spacer (ITS) region, the translation elongation factor 1-alpha (tef1-a), the
largest and second largest subunits of RNA polymerase II (rpb1 and rpb2), the mitochondrial cytochrome c
oxidase 1 (COI), and the mitochondrial large subunit of ribosomal RNA (mtLSU). We used eukaryotic
general primers of these genes and tested over 1000 samples of about 300 common species of wild edible
and poisonous mushrooms in China. Our data showed that the sequences of nLSU and tef1-α in combination
with either ITS or rpb2 sequences can be effective at identifying these mushroom species. In contrast,
although COI, mtLSU and rpb1 have higher sequencing success rate than the four other markers, the
inter-specific sequence variations of these three genes are usually low. Considering the PCR amplification
and sequencing success rates, the patterns of intra- and inter-specific sequence variations, and the correct
species identification rates, our data suggest that ITS can serve as the primary barcode in most cases, with
nLSU, tef1-a and rpb2 as supplementary barcodes. Our analyses of the multi-gene sequences have also
unveiled a large number of new taxa of amanitas, boletes (porcinii), morels, and several other groups of
mushrooms. Since the approach that we have employed is an integrated one which combined DNA
sequences, detailed morphological characterizations of a large number of new taxa with voucher specimens
and high-resolution digital images provided by expert taxonomists, sequences generated in this study could
serve as a reference database for future practical applications and scientific studies related to edible and
poisonous mushrooms in China.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
15
Plenary Session Speakers & Abstracts
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Session 5: Ecological Implications
Graham Stone
Dr. Graham Stone is Professor of Ecology at the University of
Edinburgh. His research group studies many aspects of natural
biological communities – particularly pollination networks and
plant-herbivore-parasitoid interactions. His overall goal is to
understand how natural communities assemble through time and
space. He uses a wide range of molecular techniques to understand
community composition, population history, and patterns in the
evolution of phenotypic traits.
Using DNA barcode data for interacting species to reconstruct the history of
community assembly
Graham Stone
Institute of Evolutionary Biology, University of Edinburgh, Edinburgh EH9 3JT, United Kingdom
E-mail: [email protected]
DNA barcode data are now available for many species in many biogeographic regions. In addition
to their use in taxon identification, barcode data for multiple species can be analysed to reconstruct
patterns of community assembly and regional changes in population size. We illustrate the potential
of this approach by using barcode data to reconstruct assembly across the Western Palearctic of an
insect community comprising 31 species of oak-feeding insect herbivores and their chalcid
parasitoids (Stone et al. 2012 Current Biology 22, 1-6). We show that this community assembled
through a series a westwards range expansion pulses through the Pleistocene in each of the
herbivore and parasitoid guilds. Herbivores escaped some of their parasitoids for many millennia,
but the community reassembled after parasitoid pursuit. Such multispecies analysis may be possible
with existing data, although this may be limited by frequent restriction of barcode sampling to few
individuals. Multispecies analyses are best achieved using geographically wide and deep sampling
of barcodes in suites of interacting species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
16
Plenary Session Speakers & Abstracts
—————————————————————————————————————————————
Session 5: Ecological Implications
Beth Clare
Beth Clare completed her Ph.D. at the University of Guelph in
Canada on bat systematics and molecular evolution. She then held a
post-doctoral fellowship at the University of Bristol in England
developing protocols for measuring food web dynamics with
molecular tools. In January of 2013 Beth took a Lectureship in
Ecological Genomics in the School of Biological and Chemical
Sciences at Queen Mary University of London. Her main research
area is the large-scale analysis of food webs using DNA barcoding
and next generation sequencing. She has worked on food web
analyses in Costa Rica, Jamaica, England, Eastern Europe, Canada,
Belize, Swaziland, Mauritius and The Philippines with insectivores,
seed dispersers and pollinators. Most recently she has been working with large insectivorous
ensembles, the impact of invasive shrews on skinks in Mauritius and the reconstruction of ancient
ecosystems. She is particularly interested in mechanisms of species' coexistence, the degree of
flexibility in trophic interactions and the role of both factors in promoting ecosystem stability.
Detecting trophic interactions with barcodes: from ancient ecosystems to
modern food webs
Beth Clare
School of Biological and Chemical Sciences, Queen Mary University of London, London, United
Kingdom
E-mail: [email protected]
DNA barcoding has changed the way we measure biodiversity. However, counting species is a
simple task compared to quantifying their interactions. These connections form the structure of
ecological communities and understanding how they change through time and space is fundamental
to understanding ecosystem function and response to disturbance. High throughput sequencing, in
combination with DNA barcoding, has changed our view of ecological networks by providing a
rapid mechanism to identify species-level relationships. These ecological genomic approaches are
particularly powerful when applied to hyper-diverse tropical communities where thousands of
species may interact through predation, parasitism, herbivory, pollination and seed dispersal. We
have used these methods to analyze competitive food webs and mutualistic interactions among
thousands of contemporary tropical and temperate species and in ancient extinct ecosystems. Our
analysis has revealed previously unrecognized adaptations, trophic niche switching and functional
redundancies. These cases are challenging our view of “generalists” and “specialists” and raising
questions about trophic niche specialization and resource competition. In this presentation I will
provide an overview of how ecological genomics can be applied to document species’ interactions
from a diversity of life including small mammals, spiders, reptiles and plants.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
17
Plenary Session Speakers & Abstracts
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Session 5: Ecological Implications
Tomas Roslin
Dr. Tomas Roslin works as a Senior Lecturer at the University of
Helsinki, Finland. His lab studies the effects of habitat modification
and climate change on biotic interactions at all levels: between
individuals, populations and communities. In particular, he has
striven to understand how anthropogenic impacts on trophic
interactions will ultimately affect ecosystem functioning. The work
of his group spans systems from the High Arctic through agricultural
landscapes and fragmented forests of the temperate zone to the high
biodiversity of the tropics. As a specific approach to conducting
large-scale experiments in ecology, the team has focused on public
involvement through citizen science.
Barcoding an Arctic food web
Tomas Roslin
Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland
E-mail: [email protected]
If we want to understand the abundance and distribution of species, then we need to understand how
they interact with each other. Food webs offer excellent tool for this – but if in constructing them,
we mix species with each other or fail to resolve the true links between them, then the resultant
impression of web structure will be biased. DNA barcodes now provide promising tools for
accurately describing the architecture of natural webs. In this talk, I will describe our efforts to use
them in dissecting the structure of one of the simplest food webs on the globe, that of the
Zackenberg Valley in Northeast Greenland. With a total terrestrial fauna and flora of only around
500 and 163 species, respectively, this community allows the comprehensive barcoding of all its
members. I will show how the application of barcode-based techniques to even a subset of the web
fundamentally changes our view of how interactions are structured.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
18
Plenary Session Speakers & Abstracts
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Session 6: Ecosystem Implications
Mehrdad Hajibabaei
Dr. Mehrdad Hajibabaei is an expert in biodiversity genomics and
bioinformatics. He has conducted DNA barcoding research since 2003
including pioneering work on developing high-throughput technologies for
DNA barcoding and recovery of DNA barcodes from degraded samples. Dr.
Hajibabaei is currently an Assistant Professor at the Biodiversity Institute
of Ontario & Department of Integrative Biology of the University of
Guelph, Canada, specializing on development and application of
next-generation genomics technologies for rapid and accurate analysis of
biological diversity from genes to ecosystems. He has worked on all major
groups of organisms and has published numerous high profile articles in
first-class scientific journals. Dr. Hajibabaei has served in advisory and
review panels for various international organizations and funding agencies
and has collaborated with regulatory agencies and biotech industry. He
currently leads Biomonitoring 2.0 (www.biomonitoring2.org), a large-scale
applied genomics project--involving 7 research groups and 2 federal agencies--which employs
next-generation sequencing technologies as well as sophisticated bioinformatics tools for comprehensive
assessment of biological diversity in environmental samples with direct linkage to environmental monitoring
of Alberta Oil Sands.
Ecosystem Assessment and Biomonitoring through Next-Generation Sequencing
Analysis of DNA Barcodes
Mehrdad Hajibabaei
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
E-mail: [email protected]
All human activities, from agriculture to urbanization to natural resource and energy extraction, can impact the
environment and the natural ecosystems contained therein. Understanding and monitoring these impacts allows
wise decision-making with regards to management of our environmental footprint, and balance between our
societal needs and sustainability plans. Changes in the physical and chemical characteristics of an environment
can be measured to provide the basis for environmental assessment. However, ecological and biological
consequences of environmental change can only be achieved through direct observation of living organisms in
their natural setting. Given the key role of biodiversity as a measure of the ability of ecosystems to sustain
anthropogenic activities, measuring and reporting on biodiversity remains a costly and inconsistent activity,
limited by a reliance on outmoded and impractical techniques. Biodiversity genomics is focused on the use of
genomics information for understanding biological diversity from genes to organisms to species to whole
ecosystems. For example, DNA barcodes can provide the basis for species identification in all domains of life.
Biomonitoring 2.0 utilizes massively parallelized next-generation sequencing (NGS) technologies to extract
sequence information directly from environmental samples such as soil, water and sediments. Taxonomic
information is generated through comparative analysis of sequence data against available reference databases.
Additionally, phylogenetic analysis of sequences can provide genetic diversity measures even in the absence of
species-level reference sequences. This approach has been applied for large-scale ecosystem monitoring in various
settings. The next phase of Biomonitoring 2.0 involves utilizing targeted transcriptomics and meta-transcriptomics
to assess functional consequences of environmental change.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
19
Plenary Session Speakers & Abstracts
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Session 6: Ecosystem Implications
Douglas Yu
Douglas Yu is a Reader in Conservation Biology at the University of
East Anglia and a Principal Investigator at the Kunming Institute of
Zoology. His lab works on using high-throughput sequencing and
DNA taxonomy to solve a range of environmental management
problems, including restoration ecology, surveillance monitoring,
and endangered species detection. He also runs research projects in
the evolution of symbiosis and bushmeat hunting.
Reliable, verifiable,
metabarcoding
and
efficient
metabarcoding
of
biodiversity
via
Douglas Yu1, 2
1
Ecology, Environment, and Conservation Center (ECEC), Kunming Institute of Zoology, Chinese
Academy of Sciences, Kunming, Yunnan 650223, China
2
School of Biological Sciences, University of East Anglia, Norwich, Norfolk NR4 7TJ, United
Kingdom
E-mail: [email protected]
To manage and conserve biodiversity, one must know what is being lost, where, and why, as well as
which remedies are likely to be most effective. Metabarcoding technology can characterize the
species compositions of mass samples of eukaryotes or of environmental DNA. I report on how we
have tested the reliability of metabarcoding data by comparing against three high-quality standard
datasets collected in Malaysia (tropical), China (subtropical), and the UK (temperate), that
comprised 55,813 arthropod and bird specimens identified to species level with the expenditure of
2,505 person-hours of taxonomic expertise. The metabarcode and standard datasets exhibit
statistically correlated alpha- and beta-diversities, and the two datasets produce similar policy
conclusions for two conservation applications: restoration ecology and systematic conservation
planning. Compared with standard biodiversity datasets, metabarcoded samples are taxonomically
more comprehensive, many times quicker to produce, less reliant on taxonomic expertise, and
auditable by third parties, which is essential for dispute resolution. I also report on the application of
metabarcoding to other problems in ecology, including ecosystem services provisioning, validation
of remote sensing imagery, and endangered species detection.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
20
Plenary Session Speakers & Abstracts
—————————————————————————————————————————————
Session 6: Ecosystem Implications
Baoli Zhu (朱宝利)
Dr. Baoli Zhu is a Professor in the University of Chinese Academy
of Sciences, Principal Investigator in the Institute of Microbiology,
Head of the Beijing City Key Laboratory of Microbial Drug
Resistance and Resistome. Dr. Baoli Zhu obtained his PhD in
Biochemistry in Beijing Agricultural University in 1992. In 1995, he
became a postdoc in The Babraham Institute at Cambridge, UK,
working on cattle genome mapping. He started to work on Bacterial
Artificial Chromosome libraries for a variety of animal genomes in
BACPAC Resources Center at Children’s Hospital Oakland in 1999.
He returned to Beijing in 2006 and begins to work on Microbial
Genomics at The Institute of Microbiology, Chinese Academy of
Sciences and has been Head of Joint Microbial Genomic Center
during 2006-2008. His research focus is on (1) comparative
genomics on microbes and the evolution of bacterial virulence; (2)
metagenomic studies of human microbiota related to human health; (3) drug resistance genes in
bacterial genomes; (4) development of technology for clinical test of pathogens based on bacterial
genomic studies.
Microbial DNA Barcodes in Metagenomic Studies
Xi Yang1, Yongfei Hu1, Chunguang Luan1, Na Lv1, Jing Li1, Junjie Qin2, Jun Wang2, Baoli Zhu1
1
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology,
Chinese Academy of Sciences, Chaoyang District, Beijing 100101, China
2
BGI-Shenzhen, Yantian District, Shenzhen 518083, China
E-mail: [email protected]
DNA Barcode has been widely used in species identification in fungi, plant and animal kingdoms, but
encounters difficulties in applying in microbial world specially when dealing with the metagenomes that
have been defined as the sum of genomes of microbes in a given ecosystem, where the bacteria is the
major habitant. The number of bacterial species is enormous and it has been estimated to be more than
one million and only a portion of it (few thousands) is cultured and well characterized, and the majority
of it is uncultured and not identified. Besides, the bacterial genomes are highly diversified that has been
the hurdle for DNA barcode applications. The 16S RNA gene has been used for bacteria classification in
the past and present, but has the disadvantages of inability to identify bacteria beyond genus level. It has
limited its use as a DNA barcode and in metagenomic analysis.
There are few existing genotyping methods that can identify bacteria at species level or beyond but
are labor intensive. Due to their laborious nature, these methods cannot be used for metagenomic
analysis. Recently, the whole metagenomic sequencing data has been used for bacterial classification,
and few single genes such as cpn60 have also been proposed as a DNA barcode to be used in bacterial
identification at species level. In this study, we tested different methods for metagenomic analysis
using the 4.4 million human gut metagenomic gene catalog, and found that the cpn60 genes could be
used as a DNA barcode gene for metagenomic analysis.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
21
Plenary Session Speakers & Abstracts
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Session 7: Societal Implications
David E. Schindel
Dr. David E. Schindel was trained as an invertebrate paleontologist
and geologist (B.S. in Geology, University of Michigan 1973; Ph.D.
in Geological Sciences, Harvard University, 1979). He was a member
of Yale University’s Department of Geology & Geophysics and
Curator of Invertebrate Fossils in the Yale Peabody Museum from
1978 to 1986. From 1986-2004 he directed a variety of funding
programs in the U.S. National Science Foundation (systematic
biology research, natural history museum collection improvement,
elementary school science education, interdisciplinary research
centers). From 1998 to 2004, Dr. Schindel served as the National
Science Foundation’s European representative, based in the US
Embassy in Paris. He joined the Smithsonian Institution in 2004 as
CBOL's founding director and he is the Principal Investigator of the
Global Impact Award from Google Giving that supports the Barcode of Wildlife Project.
Regulatory and Forensic Uses of BARCODE Records and the Barcode of Wildlife Project
David E. Schindel
Consortium for the Barcode of Life, Smithsonian Institution, Washington, DC 20013, United States
E-mail: [email protected]
‘What are the obstacles to large-scale adoption of barcoding?’ was the topic of discussion session at
the Fourth International Barcode of Life Conference (Adelaide, December 2011). The lack of a
reliable reference database was the unanimous reason, not the lack of a handheld barcorder.
Potential users from government agencies and private companies reported that even though the
BARCODE data standard improved documentation it did not guarantee that barcodes would
provide reliable taxonomic identifications. BARCODE records in GenBank can't be used with
confidence because species identifications are often missing or have no clear level of reliability. An
unknown portion of the BARCODE records may have been identified solely through other
BARCODE records, not independent evidence. The Consortium for the Barcode of Life (CBOL)
responded to this input in two ways. First, we are developing revisions to the BARCODE data
standard to provide metadata on taxonomic reliability. Second, CBOL found funding from Google’s
Global Impact Award program for the Barcode of Wildlife Project (BWP) on endangered species, in
which barcode data must meet high legal standards. Several levels of BARCODE records will be
created in BWP: Platinum, Gold and Silver. BWP is working with six developing partner countries
to develop a reference database of 50,000 records from 10,000 species – 2,000 species that are
protected by national laws and 8,000 of their close relatives and look-alikes. These two activities
will demonstrate how the barcode community can produce more fully documented
‘platinum-quality’ barcode data that will attract the interest and support of regulatory agencies.
CBOL hopes to engage taxonomists, research groups, biorepositories and barcoding labs as
participants in BWP if they are willing and able to meet these higher standards.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
22
Plenary Session Speakers & Abstracts
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Session 7: Societal Implications
Linda Santschi
Dr. Linda Santschi is the Scientific Co-Director of Coastal Marine
Biolabs, a research-based educational organization located in
Ventura Harbor, California. Over the last 5 years, her laboratory has
pioneered the use of DNA barcoding as a new tool to innovate life
science education and place students at the intersection of science,
nature, and technology. She is presently the Co-Investigator on a
national project that enables high school and undergraduate students
to generate and share reference barcode data with the iBOL
community.
Education and the Barcode of Life: New opportunities to engage students in
deep explorations of nature, science and technology
Linda Santschi
Coastal Marine Biolabs, Integrative Biosciences Program, Ventura, CA, United States
E-mail: [email protected]
Education has gained significant prominence among the social aspects of the iBOL project.
Nevertheless, efforts to exploit the full potential of DNA barcoding to enhance life science
instruction have been met by a number of barriers. The absence of a customized and centralized
informatics platform where the educational community can seamlessly intersect with the iBOL
research community has represented the most significant stumbling block to student engagement in
DNA barcoding. This challenge has been compounded by fragmentary information on the concepts
and methods of DNA barcoding, pervasive misconceptions regarding barcode data standards and
their implications for student participation in iBOL, and a dearth of curriculum materials for use in
school/university classroom settings. In an effort to scale our own educational programming and
support emerging educational initiatives centered on DNA barcoding, we launched the Education
and Barcode of Life (eBOL) Community Web Portal (www.EducationandBarcoding.org).
Developed in collaboration with science professionals, the eBOL Community Web Portal integrates
powerful new resources for educators to overcome these barriers and engage their students in
transformative educational experiences. The portal also establishes a framework to facilitate the
formation of a professional community committed to advancing DNA barcoding as a new tool to
innovate life science education.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
23
Plenary Session Speakers & Abstracts
—————————————————————————————————————————————
Session 7: Societal Implications
Tania Bubela
Dr. Tania Bubela is an Associate Professor in the School of Public
Health, University of Alberta. Her research program in knowledge
management and governance of translational life sciences brings
together her legal training (JD University of Alberta), her PhD in
conservation biology and expertise in genetics and molecular
biology. Her research program focuses on large collaborative science
networks in genomics, gene therapy, and stem cell biology,
addressing barriers to the effective translation of new technologies.
These are varied and include legal and ethical issues, effective
communication of risks and benefits among stakeholder groups,
commercialization and regulation. Her research is funded by
Genome Canada, Canadian Institutes of Health Research, the Stem
Cell Network, and Alberta Innovates Health Solutions. She has over
70 publications in journals such as Nature, Nature Biotechnology, PLoS Biology, Science
Translational Medicine, and Cell Stem Cell.
International collaborative barcoding research: moving the debate forward on
access and benefit sharing of genetic resources to promote biodiversity
Tania Bubela
School of Public Health, University of Alberta, Edmonton, Alberta, Canada
E-mail: [email protected]
The Convention on Biological Diversity and associated Nagoya Protocol focus international
attention on equitable and mutually beneficial research relationships that utilize genetic resources. A
2013 multi-stakeholder workshop in Mexico City discussed best practices in negotiating Access and
Benefit Sharing (ABS) agreements for the barcoding community. DNA barcoding provides an
example of non-commercial research with a goal of conserving biodiversity, but a survey of the
international barcoding community indicated that concerns about improper use of data and samples
were one factor impeding sharing.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
24
Plenary Session Speakers & Abstracts
—————————————————————————————————————————————
Session 8: Final Reflections
Da-Wei Huang (黄大卫)
Dr. Da-Wei Huang is a professor of Institute of Zoology, Chinese
Academy of Sciences. His recent major interests lie in
biosystematics and evolution. He recently focuses on the model
system of fig plants and their associated pollinating and parasitic
wasps. For biosystematics, he is especially interested in sexual
dimorphism and male polymorphism, which challenge the
morphological species delimitation. His research addresses the
problems concerning DNA barcoding, such as heteroplasmy,
NUMTs, endosymbionts. For evolution, he devotes himself to both
gene and genome level. He is driven craze by cross-domain events,
such as horizontal gene transfer. In short, He likes mobile elements
in various biological levels.
He received his PhD in the Institute of Zoology, Chinese
Academy of Sciences in 1989. To date, he has published 209 papers, 2 monographs and 2 books.
His publications were mainly in the field of taxonomy of Chalcidoidea before 2003 and some in
ecology of fig wasps from 2004 to 2009. His recent publications were involved in evolution of fig
wasps including gene evolution, Wolbachia, DNA barcoding and comparative genomics.
Final Reflection: China Focus and beyond Conference
Da-Wei Huang
Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy
of Sciences, Chaoyang District, Beijing 100101, China
E-mail: [email protected]
This talk will focus on the development of DNA barcoding in China. I will summarize the Chinese
barcoding experiences, the major part reflected in the conference and the rest beyond it. I will
discuss some scientific or administrative problems regarding to barcoding researches in China.
Furthermore, some suggestions will be raised for a promising future.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
25
Plenary Session Speakers & Abstracts
—————————————————————————————————————————————
Session 8: Final Reflections
John Kress
Dr. W. John Kress received his education at Harvard University (B.
A., 1975) and Duke University (Ph. D. 1981) where he studied
tropical biology, ethnobotany, evolution, and plant systematics He is
a Fellow of the American Association for the Advancement of
Science and has been the Executive Director of the Association for
Tropical Biology and Conservation since 1997. Dr. Kress has served
as an Adjunct Professor of Biology at the Xishuangbanna Tropical
Botanical Garden, Chinese Academy of Sciences, and received the
Caiyun International Cooperation Award from Yunnan Province in
2006. In addition to his research position, Dr. Kress currently holds
the appointment of Director of the Consortium for Understanding
and Sustaining a Biodiverse Planet at the Smithsonian. As Director
of the Biodiversity and Sustainability Consortium, Dr. Kress brings
together scientists from numerous fields to address broad research projects, which explore some of
the largest questions in biology with significant social impact. Through the Grand Challenges
Consortia, interactions among scholars in different but complementary fields have greatly increased
at the Smithsonian resulting in the promotion and facilitation of new avenues of scientific endeavors.
His most current books are DNA Barcodes: Methods and Protocols (with David Erickson;
Humana-Springer-Verlag) and The Ornaments of Life: Coevolution and Conservation in the Tropics
(with Ted Fleming; Univ. Chicago Press).
DNA Barcodes for Ecology, Evolution, and Conservation
John Kress
Department of Botany, Smithsonian Institution, Washington, DC 20013, United States
E-mail: [email protected]
The use of DNA barcodes is entering a new phase of application as more and more investigations
employ these genetic markers to problems in the ecology and evolution of natural systems. The
suite of DNA barcode markers now applied to specific taxonomic groups of organisms are proving
invaluable for understanding species boundaries, community ecology, functional trait evolution,
trophic interactions, and the conservation of biodiversity. The application of Next Generation
Sequencing technology will greatly expand the versatility of DNA barcodes across the Tree of Life,
across habitats, and across geographies as new methodologies are explored and developed. All of
these applications of DNA barcodes will greatly enhance our understanding of how nature functions,
especially in the face of radical climate change and the massive alteration of habitats.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
26
Plenary Session Speakers & Abstracts
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Session 8: Final Reflections
Richard Lane
As Director of Science at the Natural History Museum in London
(2003-2011), Dr. Richard Lane set the strategic direction of the
scientific activities of the NHM and contributed to establishing
international partnerships in biodiversity, e.g. SciColl. He was a
founding member of the Consortium for the Barcode of Life and
host of the First International Conference on Barcoding in London.
His personal research has focused on insect vectors of disease and
has been consultant to a number of governments for the World
Health Organization. As Director of International Programmes at the
Wellcome Trust he was responsible for major research programmes
and partnerships in biomedical, clinical and social sciences. He has
been closely associated with the ethics of international research.
Since retiring from the NHM in 2011, he contributes to the scientific
advisory boards of several organisations in the UK and internationally and consults to governments
and institutions outside the UK.
DNA Barcoding - where from and where to?
Richard Lane
Independent Consultant
E-mail: [email protected]
DNA barcoding of life is a bold and ambitious project to enable identification of any eukaryotic
organism from anywhere by anyone. Initially, it faced many challenges, some known others
unknown
• scientific – is a short standardised sequence diagnostic at species level?
• technical – can a database and associated bioinformatics tools be built? Can DNA be extracted and
sequenced in the field?
• sociological – strong resistance by some of the scientific community and contrasting expectations
of the user community, restrictions on movement of DNA; fostering global participation.
Having made extraordinary progress, re-examining the initial proposition of barcoding is timely;
‘why do we need to identify an organism, what do we really want to know?’
To meet the needs of science and society at large, we need to know a lot about a few species and a
little about a lot of species. For pests, disease vectors, invasive organisms and ecological indicators
we need speed and precision within a regulatory framework. To measure large scale changes, focus
is on detecting differences in many taxa, whatever their names, over time and space. Refining
barcoding’s basic proposition in the light of the ‘proofs of principle’ now established will help its
tactical and strategic development.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
27
Parallel Session Abstracts: Oral presentation
—————————————————————————————————————————————
Part II. Parallel Session Abstracts:
Oral presentation
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
28
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Plants I
Barcode UK: A complete national resource for people, plants and the planet.
Natasha de Vere1,2, Tim Rich3, Col Ford1, Matt Hegarty2, Helena Davies1, Joe Moughan1, Adelaide
Griffith1, Laura Jones1, Aoife Sweeney1, Eleanor Brittain1, Hannah Garbett4, Jenny Hawkins5, Les
Baillie5, Andrew Lucas6, Dan Forman6, Pete Hollingsworth7, Michelle Hollingsworth7, Laura
Forrest7, Heather McHaffie7.
1
National Botanic Garden of Wales, Llanarthne, Carmarthenshire, SA32 8HG, UK
2
Aberystwyth University, Institute of Biological, Environmental and Rural Sciences, UK
3
National Museum Wales, UK
4
University of South Wales, Faculty of Advanced Technology, UK
5
Cardiff University, School of Pharmacy and Pharmaceutical Sciences, UK
6
Swansea University, SERT, UK
7
Royal Botanic Garden Edinburgh, UK
E-mail: [email protected]
We have DNA barcoded over 90% of the native flowering plants and conifers of the United
Kingdom, using the core DNA barcode markers rbcL and matK. This represents the most complete
DNA barcode coverage of any country in the world. We have also investigated the utility of using
the nuclear marker ITS2 for DNA barcoding temperate floras, assessing universality, sequence
quality and discrimination for the most species rich families of UK plants. We investigate the
potential problem of multiple copies by analysing a subset of species with 3 different primer
combinations to look for consistency of product. We are developing a wide range of applications
that make full use of our DNA barcode research platform. We use both 454 and Illumina next
generation sequencing approaches to tackle diverse questions ranging from phylogenetic
community ecology to investigating the pollination service provided by hoverflies. We are also
using DNA barcoding to investigate the medicinal properties of honey. Honey samples collected
from throughout the UK are tested for their ability to kill the hospital-acquired infection MRSA.
The honey is then DNA barcoded using 454 and Illumina sequencing in order to investigate whether
the floral composition of the honey affects its antimicrobial properties.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
29
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Plants I
DNA barcoding the tree Flora of subtropical Dinghushan nature reserve at south
China
Juan Liu1, 2, Xue-jun Ge1, Steven Newmaster3
1
Key Laboratory of Plant Resource Conservation and Sustainable Utilization, South China
Botanical Garden, the Chinese Academy of Sciences, Guangzhou China 510650
2
University of the Chinese Academy of Sciences, Beijing 100049
3
Biodiversity Institute of Ontario (BIO), University of Guelph, Ontario, Canada, N1G 2W1
E-mail: [email protected] and [email protected]
DNA barcoding has been proven to be effective to identify species across many plant groups
especially for the local flora. But few studies on floristic barcoding including species-rich genera
sampling on a diverse taxonomic scale was conducted. We built the DNA barcoding database for a
subtropical forest in Dinghushan National Nature Reserve using four regions, rbcL, matK,
trnH-psbA and ITS and evaluated their performance for species assignment. The sample consisted
of 549 tree species (980 individual) from 84 family 267 genera. 429 species from 80 families and
226 genera was of 2 representatives. Our results showed that rbcL+matK received a high
identification for genus or family recognition (80.17%, 95.24%), but performed poorly at the
species level (58.80%). The species discrimination of rbcL+matK for monotypic genera was much
higher than for multi-species genera (86.67% vs. 28.0%). For the polytypic genera with more than
four species, ITS showed higher identification level than trnH-psbA (46% vs. 32.67%). Our work
on woody species of DNNR demonstrated that proposed core plant barcode, rbcL+matK, was less
successful in diverse tropical region due to poor performance for species from polytypic genera.
ITS (ITS2) provided more powerful ability to discriminate closely related species than trnH-psbA.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
30
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Plants I
DNA barcoding a complex prairie flora using the rbcL-matK gene regions
Kevin S. Burgess, Robert B. Futrell, John A. Barone
Columbus State University, Columbus, GA 31907, United States
E-mail: [email protected]
Used in conjunction, the rbcL+matK gene regions have yielded relatively high species
discrimination at a relatively low cost in previous studies. Here, we examined the effectiveness of
these barcodes to distinguish species in a complex prairie flora that includes a high proportion of
polytypic genera. 475 samples representing 204 species from 108 genera in 31 families were
collected from the Black Belt prairie region of Alabama and Mississippi. Following high-throughput
sequencing at the Canadian Centre for DNA Barcoding, contiguous sequences were downloaded
from BOLD Systems for all-to-all BLASTn analyses. Although sequencing success was relatively
high for rbcL (91%) and matK (68.2%), complete coverage for both regions was marginal (62.9%).
Species resolution was relatively low for rbcL (44.0%) and matK (43.1%) but increased for the
combined barcode (51.0%). Monotypic genera were 2.8 times more likely to be successfully
distinguished than polytypic taxa (mean species resolution was 88.9% for monotypic taxa and 35.7%
for polytypic taxa). Furthermore, there was a significant, negative correlation between the number of
species per genera and percent species resolution. The results suggest that for floras with a high
number of closely related species, additional gene regions will be required to supplement the
rbcL+matK barcode.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
31
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Plants I
RbcL gene barcoding and phylogenetic analysis of species of Phoebe/Machilus
and Ficus in Xishuangbanna rainforest of China
Yu Song1, Xue Liang2, Xueliang Zhang2, Mengjie Qiu3, Bin Liu2, 4
1
Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, China; 2Center
of Systematic Genomics, Xinjiang Institute of Ecology and Geography, Chinese Academy of
Sciences, Urumqi, China; 3Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing,
China; 4Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China. E-mail:
[email protected].
Tropical rainforests are the hotspots of biodiversity and remain the most complicated forest
ecosystem. In China, the tropical rainforests are mainly located in Yunnan and Hainan Provinces,
whereas Xichuangbanna rainforest in Yunnan is unique on its own stance. In this study, both rbcL
and matK were applied as standard plant DNA barcodes and were successfully used for over 50% of
the collected samples of Phoebe/Machilus (Lauraceae) and Ficus (Moraceae) in this region and for
the first time phylogenetic markers were assigned to these species. In order to increase the coverage
we extended the markers to the full-length of these genes, it also give more room for designing of
more suitable primers and for increasing the efficiency of PCR amplification. With this
advancement, nearly all samples were successfully amplified and characterized for the full gene
sequence of rbcL. With this improved gene barcode we were able to identify both Phoebe/Machilus
samples and Ficus samples in several distinctive groups. Further full-length gene sequencing
analysis will reveal detailed phylogenetic relationship among these species. We demonstrated that
standard plant DNA barcodes can be used to the identification of trees in tropical rainforest and
full-length gene sequences of these markers have further increasing interpreting power and more
convenient to use as an added benefit. Wider application of DNA barcodes should facilitate the
species study and conservation and utility of rich biodiversity and natural resources of our
ecosystems.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
32
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Plants I
DNA barcoding and species identification across tropical and temperate forest
plots
Nancai Pei1, Bufeng Chen1, Xiangcheng Mi2, Keping Ma2, Xue-Jun Ge3, Wan-Hui Ye3, Zhanqing
Hao4, Dingliang Xing4, David L. Erickson5, Nathan G. Swenson6, Jinglong Zhang7, Shawn Lum8, W.
John Kress5
1
Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou 510520, PR
China; 2State Key Laboratory of Vegetation and Environmental Change, Institute of Botany,
Chinese Academy of Sciences, Beijing 100093, PR China; 3Key Laboratory of Plant Resource
Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of
Sciences, Guangzhou 510650, PR China; 4Institute of Applied Ecology, Chinese Academy of
Sciences, Shenyang 110016, PR China; 5Department of Botany, MRC-166, National Museum of
Natural History, Smithsonian Institution, P.O. Box 37012, Washington, DC 20013-7012, USA;
6
Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824, USA;
7
Flora Conservation Department, Kadoorie Farm and Botanic Garden, Lam Kam Road, Tai Po, N.T.,
Hongkong; 8National Institute of Education of Nanyang Technological University (Singapore).
E-mail: [email protected]
DNA barcoding is beginning to fulfill its promise to enhance research on taxonomy and biodiversity
for both animals and plants. The species diversity decreases with the increase of the latitude
gradient, then we hypothesize that the forest plot located in higher latitude will be barcoded easier
and possessed with greater rate of correct identification. We compared the results of rbcL, matK and
psbA-trnH from three temperate forests (SERC in USA, 104 species; SCBI in USA, 54 species; and
CBS in China, 61 species), two subtropical forest plots (DHS in China, 181 species; GTS in China,
150 species), and four tropical forest plots (BT in Singapore, 278 species; NRS in French Guiana,
195 species; BCI in Panama, 276 species; and LFDP in Puerto Rico, 85 species), to evaluate the
universality and species correct discrimination of the proposed DNA barcodes across these nine
forest plots. We determine the uniqueness of a barcode sequence using TaxonDNA method. At the
local scale (i.e., the individual of the nine or eight forest plots), the rates of correct identifications of
rbcL varied from 61% in the DHS plot to 90% in the Luquillo plot, matK from 77% in the DHS plot
to 92% in the BT plot, and psbA-trnH from 86% in the DHS plot to 98% in the Luquillo plot. At the
regional scale (i.e., the individual of the three climate regions), the rates of correct identifications of
rbcL varied from 68% in the subtropics to 79% in the temperate zone, matK from 77% in the
subtropics to 89% in the tropics, and psbA-trnH from 90% in the subtropics to 96% in the tropics.
At the global scale (i.e., the nine or eight forest plots combined), the rates of correct identifications
of rbcL, matK and psbA-trnH were greater than 72%, 85% and 94 %, respectively. We suggest that
a mixture of slower and faster evolving barcode regions favors discriminating plant species in the
given geographic location and facilitates reconstructing robust community phylogenies in highly
diverse forests across tropics and temperate zone.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
33
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Plants I
Study on DNA barcoding of Smilacaceae and reveal the limits between species in
the family
Zhechen Qi, Zhongda Shao, Chengxin Fu
Laboratory of Systematic & Evolutionary Botany and Biodiversity, Institute of Plant Sciences,
Zhejiang University, China, Hangzhou 310058, China
E-mail: [email protected] and [email protected]
Smilacaceae consisting of two genera, Smilax and Heterosmilax, are cosmopolitan distributed and
taxonomically complex monocot family with about 200 species. Based on DNA barcoding theory
and method, combined with over 20 years morphological and taxonomical research data, we studied
DNA barcodes of Smilacaceae focused on species in China using molecular systematic techniques.
We discussed the identification ability of standard DNA barcodes in the Smilacaceae, and evaluated
the function and significance of DNA barcoding for revealing species boundaries. The main results
are as follows: ITS + rbcL are effective DNA barcodes combination in Smilacaceae. In this study,
two cpDNA barcodes rbcL, matK and nrDNA barcode ITS were selected as barcode markers. Then
we sample 227 individuals representing 75 species to assess the effectiveness and universality of
these barcode markers in Smilacaceae. The results of barcoding gap test showed that no barcode
gap in rbcL, matK, and ITS. Wilcoxon signed rank tests were used to evaluate the intraspecific and
interspecific variations. The result showed that ITS>>rbcL>>matK for interspecific and
ITS>>rbcL=matK for intraspecific. All the results of NJ, UPMGA, ML and MP analysis showed
that single ITS has the highest recognition rate and are able to discriminate 59.7% of species, while
rbcL and matK are 42.7%, 36%, respectively. The recognition rate of the combination of
rbcL+matK+ITS and rbcL+ITS can discriminate 69.4% of species, which were higher than each
single fragment. According to the cost and efficiency, rbcL + ITS will be an ideal combination
fragments to identify the species of Smilacaceae. Incomplete interspecific differentiation may be a
negative cause of identification in DNA barcoding. About 30% of the species cannot be identified in
our study. On one hand, some of them are closely related species complex group, which are not
genetically differentiated; On the other hand, misidentification and taxonomical issue of some
species would also affect the identification effect of the DNA barcodes. Finally, the systematic tree
reconstructed by these barcodes can be used as an effective identification reference for unidentified
Smilax species.
This research was supported by a grant of Knowledge Innovation of Chinese Academy of Sciences (Grant No.
KSCX2-EWZ-1), and a key project of the National Natural Science Foundation of China (Grant No. 30830011).
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
34
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Plants I
DNA barcoding of Rhododendron species in a biodiversity hotspot
Lian-Ming Gao1, Li-Jun Yan1, De-Zhu Li1, 2
1
Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese
Academy of Sciences
2
Plant Germplasm and Genomics Centre, Germplasm Bank of Wild Species, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, China
E-mail: [email protected]
Rhododendron is the biggest genus in Ericaceae with about 1000 species. Three global biodiversity
hotspots located in the Sino-Himalayas, where is one of the diversification and distribution center of
Rhododendron including 317 species (53.8% of total species in China), of which 210 are endemic
(35.6%). Total of 365 individuals of 136 species representing all the subgenera and sections of
Rhododendron were sampled from Sino-Himalayas for DNA barcoding to address the species
discriminatory effectiveness using the four candidate barcodes (i.e. rbcL, matK, trnH-psbA and ITS)
in the biodiversity hotspot. All the four DNA barcodes showed high levels of PCR and sequencing
success. Four methods (PWG, Distance, Blast and tree-building) were performed for species
identification analysis. Of which the ‘Blast’ method exhibits the highest species discrimination
ability based on both the single barcode and combinations of 2-4 barcode. The methods of PWG
and distance provide the similar performance on species identification. Among the four DNA
barcodes, trnH-psbA showed the highest species discrimination rate with 24%, followed by matK
(19.8%) and ITS (15.3%), while rbcL with lowest rate (10.3%) on PWG method. Combinations of
three DNA barcodes can significant improve the discriminatory power than those of two-barcode
combinations. The combination of matK, trnH-psbA and ITS provides the highest discriminatory
ability with 44.2% species resolution, same as the combination of all the four DNA barcodes.
Overall, DNA barcoding showed the low species discriminatory effectiveness for the rapid radiation
diversification genus Rhododendron in the biodiversity hotspot.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
35
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Fishes I
Barcoding fishes from the Argentine Antarctic Peninsula and adjacents Islands
E. Mabragaña1, S. M. Delpiani1, J. J. Rosso1, M. González-Castro1, R. Hanner2, J. M. Díaz de
Astarloa1
1
Laboratorio BIMOPE, Instituto de Investigaciones Marinas y Costeras, Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Mar del Plata
(Argentina)
2
Biodiversity Institute of Ontario (Canada)
E-mail: [email protected]
The Antarctic fish fauna has a remarkably high level of endemism (c.a 95% of around 320 of the
species recorded are endemic). Although several taxonomic studies have been carried out on
Antarctic fishes, the number of species is probably underestimated as some taxa and regions have
not been deeply explored. In addition, recent molecular studies showed incomplete species
discrimination within some representatives of Notothenidae, Artedidraconidae and Liparidae. As
part of a global project conducted by CONICET on the systematics and biology of Antarctic
organisms, DNA barcodes of fish species around Antarctic Peninsula and adjacent Islands were
obtained and concordance with traditional morphological identification was explored. A total of 228
specimens, belonging to 28 different putative species from 7 families, were obtained from research
cruises conducted by the R/V Puerto Deseado (Argentina) during summers 2011/12. Barcode
sequences were subjected to distance based and spectral clustering (BIN) analyses, which revealed
that nearly all species exhibit unique barcodes or clusters of closely related haplotypes. Interspecific
divergence (D) was relatively high except in some species within Artedidraconidae, Liparidae,
Channichthidae and Zoarcidae (D<3%). On the other hand, 26 different BINs were recognized, with
only a few species of the genera Pogonophryne (D=1.8%) and Paraliparis (D= 0.6%) lacking
barcode resolution. Our results highlight the utility of the BIN system for Antarctic fishes and the
concordance between morphological and DNA based identification approaches.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
36
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Fishes I
Progress in the DNA barcoding of fishes in southern Africa
Monica Mwale, Tuuli Mäkinen, Paul Skelton, Ernst Swartz
South African Institute for Aquatic Biodiversity, Grahamstown, South Africa
E-mail: [email protected]
The DNA barcoding of fishes at the South Africa Institute for Aquatic Biodiversity (SAIAB) has
concentrated on setting up a reference library of regional species from the Western Indian Ocean
(WIO) and freshwater species mainly from southern Africa. These projects were supported through
the CCDB (University of Guelph) pre 2009 and then later through the IDRC/IBOL research
program. These projects have generated over 2200 marine and 1500 freshwater fish records. This
funding support and effort has facilitated national interest in the DNA barcoding of fishes. This
includes projects on the barcoding of invasive fish species and other less studied taxonomic groups
through the South African National Biodiversity Institute (SANBI) and the National Research
Foundation (NRF). Additional funding support for field collections from other regional
programmes (WIOMSA and ACEP) has enabled sampling from the WIO (Maldives, Mozambique,
Comoros and Kenya) and major river systems (Okavango, Zambezi and Kwanza Rivers). New
projects have also been initiated on other aquatic organisms including amphibians and aquatic
insects. The purpose is to provide data that can be used to address research gaps in aquatic
biodiversity with a focus on taxonomic revisions, resource use (assessment of stocks and
conservation status), and ecosystem description.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
37
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Fishes I
Validation of DNA barcodes and proteome fingerprints for the identification of
fish species from the Northern European shelf
Thomas Knebelsberger1, Filipe Costa2, Silke Laakmann1, Monica Landi2, Ralf Thie3
1
Senckenberg Research Institute, German Center for Marine Biodiversity Research (DZMB),
Frankfurt, Germany
2
Department of Biology, University of Minho, Braga, Portugal
3
Biocenter Grindel und Zoological Museum, University of Hamburg, Hamburg, Germany
E-mail: [email protected]
The accurate identification of species is a pivotal component in many fields of biological research
and conservation efforts. The fish fauna of the North Sea, a marginal sea of the Atlantic Ocean
located between Great Britain, Scandinavia, Germany, the Netherlands, and Belgium, is rather
well-known since it has been monitored for decades by a series of European-wide fish surveys.
While the morphological identification of adult fish specimens is easy for most of the species, the
identification of larvae and eggs is still a challenging issue. In this context, the application of
molecular methods for species identification is essential. In fish, several DNA based identification
methods have been developed over the last decades, whereas DNA barcoding constitutes one of the
most promising techniques by using partial cytochrome c oxidase subunit I (COI) sequences for
standardized species identification. In our research we tested the barcoding approach for about 50%
of the fish fauna of the North Sea. Furthermore we examined the potential of proteome fingerprints
using the technology of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) for discrimination of species. This technology may constitute an alternative or
supplementary approach for a rapid, cost-effective species identification.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
38
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Fishes I
DNA Barcoding helps to identify early life stages of the Anguilliform fishes of the
Mesoamerican Reef System
Vásquez-Yeomans L1., Morales S1., Victor B2., and Valdez-Moreno M1.
1
El Colegio de la Frontera Sur, Av. Centenario Km 5.5, Chetumal Quintana Roo, México, 77014
2
Ocean Science Foundation, 405 Glenwood, Irvine, CA 9604 and Guy Harvey Research Institute,
Nova Southeastern University, 8000 North Ocean Drive, Dania Beach, FL 33004, USA.
E-mail: [email protected]
The eel-like fishes of the Order Anguilliformes (in the superorder Elopomorpha) comprise an
extremely diverse group, with over 800 species presently known. The group is characterized by the
distinctive flat larval stage called the leptocephalus. Larval identification is difficult, both because
of the large number of species and the similarity of eggs and early leptocephali and the changing
morphology at metamorphposis. We present the results of a DNA barcoding study of these larvae
and eggs collected from coastal samples and oceanographic cruises along the Mesoamerican Reef
(NOAA Gordon-Gunter 2006-2007). In total, 151 specimens were sequenced after we identified the
larvae morphologically to the lowest taxonomic level possible. Approximately 85% of eggs and
larval sequences matched with a >97% similarity to reference sequences in the BOLD ID engine.
Six families (Congridae, Chlopsidae, Moringuidae, Muraenidae, Nettastomatidae and Ophichthidae)
were identified, including 27 genera and 44 species. New information about early stages of several
species of Anguilliform is presented with precise data about spawning times and locations in the
Mesoamerican Reef System.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
39
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Fishes I
DNA barcoding of family Serranidae in Malaysian waters
Rajiv Ravi, Ahmad Faisal Ghazali, Darlina Md Naim, Zary Shariman Yahaya, Siti Azizah Mohd
Nor
School of Biological Sciences, Universiti Sains Malaysia, 11800 USM Pulau Pinang, Malaysia.
E-mail: [email protected]
DNA barcoding is an accurate method in identification of fish and fish products to assist in
managing fisheries for long term sustainability and conservation. In this study, a 621 bp region of
COI was sequenced for 43 individuals in a preliminary survey. Eight species were recorded in this
study namely; Epinephelus areolatus, E. akaara, E. poecilonotus, E. quoyanus, E. ongus,
Plectropomus leopardus, Cephalopholis farmosa, and C. cyanostigma. This is a small fraction of the
54 putative Malaysian species of family Serranidae documented in FishBase and 68 in a Malaysian
study based on morphological characteristics. Kimura two parameter (K2P) mean distances within
species, genus and family were 1.805%, 16.203%, 19.614%, respectively. Mean GC composition of
codon position 3 was 37.47%. All described species formed monophyletic clusters in the
Neighbor-joining phylogenetic tree. Genus Epinephelus formed a monophyletic clade and was more
closely related to Cephalopholis. This study has established a DNA barcode database of the marine
fish family Serranidae in Malaysia. However, work is in progress in identifying all other putative
fish species as recorded in previous studies based traditional taxonomic keys.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
40
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Fishes I
DNA barcoding of the Lake Tanganyika cichlid fauna of the rocky shores
Floris Breman1, Van Steenberge Maarten1,2, Kurt Jordaens1, Jos Snoeks1,2
1
Royal Museum for Central Africa, JEMU, Leuvensesteenweg 13, B-3080 Tervuren, Belgium
2
Catholic University of Leuven, Laboratory of Animal Diversity and Systematics, Charles
Deberiotstraat 32, B-3000 Leuven, Belgium
E-mail: [email protected]
The Lake Tanganyika (LT) cichlid fauna has been the subject of intense evolutionary studies. Yet,
DNA barcoding as an identification tool has not been evaluated. We have obtained 399 DNA
barcodes of 95 species of LT cichlids from 11 tribi, and the usefulness of DNA barcoding as an
identification tool for this group was evaluated. Morphological species assignment was done using
morphological identification keys but treated in three different ways: viz. 1) specimens were
identified to the species level, 2) specimens were identified to the species level but only described,
or well-delimited, species were considered (to account for ill-defined/delimited species in the older
literature), and 3) specimens were grouped in morphological species complexes for recently
evolved species. We used an ad hoc DNA based distance threshold (based on each separate library
rather than a fixed value) for each of the libraries and then determined DNA barcode identification
success. Our results show that DNA barcoding performs poorly in LT-cichlids, when compared to
other fish groups. Even when problematic species were lumped in complexes the ID success
remained low. Potential causes for this low identification success include an incomplete knowledge
of the taxonomy, hybridization and incomplete lineage sorting.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
41
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Fishes I
Hyphessobrycon (Characifomes: Characidae): DNA barcoding analysis and
phylogenetic relationships
Claudio De Oliveira, Tatiane Casagrande Mariguela, Fausto Foresti
Universidade Estadual Paulista, São Paulo, Brazil
E-mail: [email protected]
The family Characidae is the most speciose in the Neotropical area. Taxonomic studies shown that
the genus Hyphessobrycon has 127 species widely distributed and in many cases, very difficult to
identify using traditional tools. Additionally, several studies suggest that this genus in polyphyletic
although a phylogeny with a significant number species of the group was not done yet. In the
present study sequences of the COI gene of 549 specimens of Hyphessobrycon were analyzed to
check the ability of these sequences to flag different species. Fifty-two Hyphessobrycon species
were also sequenced for five more genes (CytB, 16sRNA, Rag1, Rag2, Myh6) for the study of the
relationships among this genus and other Characidae species. COI analysis showed that several
species were split in more than one cluster which resulted in an intra-specific genetic distance
ranging from 0 to 44.9% and an inter-specific genetic distance ranging from 0 to 43.1%. Among all
sequences obtained 125 Barcoding Identification Numbers (BINs) were found and several species
with more than one cluster were identified as H. eques, H. herbertaxelrodi, H. luetkenii and H.
vilmae. Considering that only about 50 nominal species were identified in our taxonomic studies we
have that 60% of the putative species represent species of difficult identification or new species.
Phylogenetic analysis among 50 species of Hyphessobrycon and about 100 species of different
Characidae genera showed that all species of Hyphessobrycon belong to the currently named Clade
C but the genus is polyphyletic, with species divided in nine groups, all them mixed with other
Characidae species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
42
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Insects I
Use of DNA barcodes to solve aphid taxonomic problems: a case study in
Greenideinae (Hemiptera: Aphididae)
Qinghua Liu1,2, Liyun Jiang1, Gexia Qiao1,2
1
Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy
of Sciences, Chaoyang District, Beijing 100101, China
2
University of Chinese Academy of Sciences, Beijing 100049, China.
E-mail: [email protected], [email protected]
Species of the aphid subfamily Greenideinae are distributed mainly throughout Southeast Asia and
include some important agricultural and horticultural pests. Accurate species circumscription and
identification in this group is very difficult because similar morphological traits are shared among
congeneric species. DNA barcoding is a useful tool for species identification in a diverse range of
taxa. In the present study, to test the efficiency of DNA barcoding in Greenideinae, we analyzed 214
samples covering 42 species belonging to 9 genera using two mitochondrial gene fragments (COI
barcode fragment and Cytb gene fregment). The results show that DNA barcoding is a useful
species identification method in this subfamily. Both genes can correctly identify most species using
NJ tree analyses and distance-based analyses. Based on the molecular and morphological evidence,
the validity of two species, Mollitrichosiphum rhusae Ghosh and Schoutedenia emblica (Patel &
Kulkarni), were questioned. Further analysis of COI barcode fragments show that Greenidea psidii
van der Goot, an invasive species in Hawaii, is possibly from China. This is a preliminary DNA
barcoding study in Greenideinae, and comprehensive sampling is needed to test and confirm the
usefulness of DNA barcoding in this group.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
43
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Insects I
Expanding the frontiers of the All-Leps initiative: Barcoding the Butterflies of
Argentina
Cecilia Kopuchian 1,2, Ezequiel O. Nuñez Bustos1, Pablo D. Lavinia 1, Darío A. Lijtmaer 1, Natalia C.
García 1, Pablo L. Tubaro1
1
Museo Argentino de Ciencias Naturales ¨Bernandino Rivadavia¨ - MACN-CONICET, Argentina
2
Centro de Ecología Aplicada del Litoral- CECOAL-CONICET, Argentina
E-mail: [email protected]
Although almost half of the existing DNA barcode sequences correspond to lepidopterans, the
species from southern South America are poorly represented. Since December 2010, the Argentine
Museum of Natural Science (MACN) is participating in the global initiative that is generating the
barcodes of the worldwide Lepidoptera species. In this context, we have generated more than 1900
COI sequences from 400 species of Butterflies, representing more than the 30% of the diurnal
lepidopterans from Argentina. The mean intraspecific genetic variability was 0.5%, while the mean
divergence between congeneric species was close to 6%, suggesting that DNA barcodes are very
efficient for the identification of the Argentinean butterflies species. We detected several cases with
high intraspecific genetic variability in COI, some of which present genetic structure. These species
were flagged as interesting cases to be studied more deeply. Moreover, as a consequence of the
intense collecting field work that we have been carrying on, we have registered species that
nowadays are rare or even not previously cited for Argentina. Apart from the genetic database that
is being assembled, this greatly contributes to the generation of a very valuable collection for the
study of the Lepidoptera of southern South America.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
44
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Insects I
Barcoding old type specimens: Obstacles, solutions, and current results
Sean Prosser 1, Jeremy DeWaard 1, Scott Miller, Paul Hebert
1
Biodiversity Institute of Ontario, University of Guelph, Guelph, ON, Canada
2
Smithsonian Institute, Washington, DC, United States
E-mail: [email protected]
The recovery of barcode data from type specimens is critical for the resolution of cryptic species
complexes, as well to ensure the appropriate use of names for contemporary specimens. In cases of
rare or extirpated species, type specimens may be the only source of genetic material, thus making
them invaluable for barcode library construction. A large-scale DNA barcoding project was initiated
in late 2012 with the goal of developing an inexpensive and effective means of recovering barcode
data from type specimens of insects ranging in age from 50-150 years. Analysis has focused on
1700 specimens, most belonging to two families of Lepidoptera, Geometridae and Xyloryctidae.
The refinement of protocols harnessing ancient DNA techniques combined with a
medium-throughput platform has allowed for the recovery of 100-200 bp sections of the COI
barcode region from most individuals. Although the sequences are short, they are, in most cases,
sufficient to enable their unambiguous connection to one representative of a particular species
complex. Aside from discussing these results, the prospects for protocol improvements will be
considered.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
45
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Insects I
DNA barcodes for species identification of the malaria mosquitoes (Anopheles
dirus complex)
Uraiwan Arunyawat1, Umarin Boonkue1, Theeraphap Chareonviriyaphap2
1
Department of Genetics, Faculty of Science, Kasetsart University, Ladyaow Chatuchak Bangkok
10900, Thailand
2
Department of Entomology, Faculty of Agriculture, Kasetsart University, Ladyaow Chatuchak
Bangkok 10900, Thailand
E-mail: [email protected]
Anopheles dirus complex is widely distributed as important malaria vectors in Southeast Asia
region. Morphological similarity among the Dirus sibling species is often difficult to identify
mosquitoes within the complex species, which usually lead to species misidentification. In this
study, allele-specific PCR was developed to identify the cryptic species within Anopheles dirus
complex in Thailand. The allele-specific primers were designed based on the nucleotide
polymorphisms in mitochondrial cytochrome oxidase subunit I sequences. The results showed that
these primers clearly amplified different size of PCR products for species within the Dirus group.
The designed DNA barcodes were validated with field-collected Anopheles mosquitoes throughout
Thailand, suggesting that the allele-specific DNA method could be effectively identified member of
the Dirus species complex.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
46
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Insects I
Barcoding beetles: preliminary results from 1832 species of North European
Coleoptera
Mikko Pentinsaari, Marko Mutanen
Department of Biology, University of Oulu, 90570 Oulu, Finland
E-mail: [email protected]
With about 400 000 described species, beetles (Coleoptera) are the largest known group of animals.
Considering this fact, surprisingly few barcode studies, especially taxonomically comprehensive
ones, have been carried out on them. The North European beetle fauna is perhaps the most
thoroughly studied in the world by means of traditional taxonomy. Therefore, it provides an
excellent opportunity to test the utility of DNA barcodes in species-level identification and as a tool
for preliminary species delimitation in beetles. So far, I have sampled 6232 North European
specimens representing more than 2000 species, out of which 5234 specimens (1832 species) have
been successfully barcoded. Based on preliminary analyses, DNA barcodes perform spectacularly
well in identifying beetles to species level. Between-species divergences in Coleoptera appear to be
much higher than in a similar, comprehensive sample of Finnish Lepidoptera. Within Carabidae, for
example, the mean genetic distance between species is about 15 %, while the mean distance within
species is only 0.4 %. Despite a long history of intensive taxonomic research, several possible cases
of previously undiscovered species have been found in the data. The cases where barcode-based
identification fails all represent closely related species pairs or groups.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
47
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Insects I
Species-level para- and polyphyly in DNA barcode gene trees in European
Lepidoptera
Marko Mutanen1, Peter Huemer2, Axel Hausmann3, Andreas H. Segerer3, H. Charles J. Godfray4,
Carlos Lopez-Vaamonde5, Vlad Dinca6, Roger Vila7, Camiel Doorenweerd8, Leif Aarvik9,
Rodolphe Rougerie5, Erik J. van Nieukerken8, Konstantin A. Efetov10, Gerhard Tarmann11,
Thibaud Decaens12, Arild Johnsen9, Rutger Vos13, Reza Zahiri14, Sujeevan Ratnasingham14, Ole
Karsholt15, Paul D.N. Hebert14
1
University of Oulu, Oulu, Finland
Tiroler Landesmuseen-Betriebsgesellschaft, 6020 Innsbruck, Austria
3
Bavarian Natural History Collections, Zoological Collection of the State of Bavaria, 81247 Munich,
Germany
4
University of Oxford, Oxford OX1 2JD, United Kingdom
5
INRA Orléans - Unité de Recherche en Zoologie Forestière, 45075 Ardon, France
6
Department of Zoology, Stockholm University, SE-106 91 Stockholm, Sweden
7
Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), 08003 Barcelona, Spain
8
Naturalis Biodiversity Centre, Department of Terrestrial Zoology, 2333 CR Leiden, Netherlands
9
Natural History Museum, University of Oslo, 0313 Oslo, Norway
10
Crimean State Medical University, Crimea, Ukraine
11
Tiroler Landesmuseen, Ferdinandeum Naturwissenschaftliche Abteilung, 6020 Innsbruck, Austria
12
Université de Rouen, 76821 Mont-Saint-Aignan, France
13
Naturalis Biodiversity Center, 2333 CR Leiden, Netherlands
14
Biodiversity Institute of Ontario, University of Guelph, Guelph, ON N1G 2W1, Canada
15
Natural History Museum of Denmark, DK-1350 Copenhagen, Denmark
E-mail: [email protected]
2
Gene trees are representations of gene genealogies. Although gene genealogy may differ from that
of a species, both are usually correlated and sets of gene trees are frequently used to infer
relationships among taxa. Gene trees of the DNA barcode region (a fragment of the COI gene) are
widely used nowadays in alpha-taxonomy since they bear valuable information regarding species
relationships and because they powerfully discriminate species. This is complicated, however, by
the fact that species do not always form monophyletic units in gene trees. In a recent literature
survey, about 26% of arthropod species were documented to show species-level paraphyly or
polyphyly in mitochondrial gene trees. Here, we report the frequency of non-monophyly in DNA
barcode gene trees in a wide variety of European lepidopteran species and based on an extensive
sampling (over 40K sequenced individuals). We observed many potential cases of historical
introgression between species, as well as other cases of presumably young species where lineage
sorting was incomplete. However, a significant proportion of these cases may represent artifacts
caused by misidentifications or inaccurate taxonomy such as oversplitting or lumping.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
48
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Insects I
Existence of Species Complex Largely Reduced Barcoding Success for Invasive
Species of Tephritidae: a case study on Bactrocera spp
Fan Jiang1,*, Qian Jin2,*, Aibing Zhang2*, Zhihong Li1*
1
Department of Entomology, College of Agronomy and Biotechnology, China Agricultural
University, Yuanmingyuan West Road 2, Haidian District, 100193 Beijing, China
2
College of Life Sciences, Capital Normal University, Beijing 100048, China
*These authors contributed equally to this work
E-mail: [email protected], [email protected]; [email protected]
The Tephritidae, fruit flies, is one of the most economically important pests with many species
complexes. However, the effectiveness of DNA barcoding for identifying fruit flies remains largely
unknown although DNA barcoding has gradually been verified as an effective tool to identify
species in wide range of taxonomic groups. In this study, we evaluated the standard cytochrome c
oxidase I (COI) gene for diagnosis of fruit flies using 1426 sequences for 73 species of Bactrocera
collected from the Barcode of Life Database (BOLD) and Plant Quarantine and Invasion Biology
Lab of China Agricultural University (CAUPQL). Tree-based (Neighbor-joining (NJ)) and
distance-based methods, such as, Best Match (BM), Best Close Match (BCM) and Minimum
Distance (MD) methods were used to evaluate barcoding success rates. Our results indicate that the
existence of species complex largely reduced barcoding success for Tephritidae, e.g. relatively low
success rates based on BM (74.4%), BCM (74.4%) and MD (75.6%) were obtained when sequences
from species complex were included in the analysis, whereas significantly large success rates were
achieved with species complex removed from the dataset – BM (98.08%), BCM (97.4%) and MD
(98.6%).
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
49
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Education
DNA Barcoding in Schools - The School Malaise Trap Program
Dirk Steinke
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
E-mail: [email protected]
Early 2013 the Biodiversity Institute of Ontario teamed up with grade 6 and grade 12 students from
schools across Southern Ontario to explore the insect diversity in their schoolyards through DNA
Barcoding. The program involved 60 schools in 42 cities, 77 classrooms, and some 2000 students
that were introduced to the life of a biologist and DNA Barcoding.
A total of 81 traps on average collected 1,180 specimens for the collecting period. Out of 95,500
specimens 22,646 were selected to be barcoded. Our final dataset was made up of 19,501
barcodes representing 1,392 species collected in a two week period in early spring.
Aside from highlighting some of the scientific results this presentation will introduce the
educational program and how it relates to the provincial curriculum.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
50
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Education
The public face of barcoding: How iBOL and DNA barcoding are represented in
newspapers from around the world
Janis Geary, Emma Camicioli, Tania Bubela
School of Public Health, University of Alberta, Edmonton Alberta, Canada
E-mail: [email protected]
Barcoding has the potential to aid in conserving biodiversity, monitoring trade, and protecting
consumers, but these benefits rely on sharing genetic resources such as data and materials. Policy
decisions related to barcoding can be influenced by media representations of barcoding, as the
media shapes and reflects public discourse. Our research examines media representations of iBOL
and DNA barcoding in newspapers to determine how barcoding technology and implications of
sharing genetic resources are portrayed to the public. We searched for relevant English language
newspaper articles from Canadian Newstand, Factiva and Lexis Nexis (2003-2012). Our search
returned 1266 articles from 21 countries, including Canada (361), USA (291), UK (199), India (95),
and China (93). The most common theme represented the scientific advancement of barcoding
(842), although many articles mentioned bio-conservation (465) or food mislabelling (355). Many
articles (905) mentioned barcoding without any explanation of barcoding methods. Only 6 articles
mentioned issues related to sharing genetic resources (either biopiracy or bioprospecting), and 29
referred to an international treaty related to sharing genetic resources. Media representations of
iBOL and DNA barcoding emphasize the potential applications of barcoding, but fail to adequately
describe barcoding methods or implications of sharing genetic resources.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
51
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Education
The ECOTROP field-school: integrating hands-on DNA barcoding into an
education program for the census of invertebrate biodiversity in the Lopé
National Park, Gabon
Thibaud Decaëns1, Stephan Ntie2, Olivia Scholtz3, David Porco4, Rodolphe Rougerie5, Emelie
Arlette Apinda-Legnouo6, Myriane Andeime Eyene7, Philippe Le Gall8, Carlos Lopez Vaamonde9,
Marlucia B. Martins10, Nicolas Moulin11, Maurice Tindo12, Michel Veuille13, David Sebag14, the
ECO-TROP team
1
University Rouen, France
2
University Masuku, Gabon
3
WCS, Gabon
4
University Rouen, France
5
INRA-Orléans, France
6
IRET-CENAREST, Gabon
7
IRAF-CENAREST, Gabon
8
IRD, Cameroun
9
INRA-Orléans, France
10
MPEG-Belém, Brazil
11
NMEntomologie
12
Univ Douala, Cameroun
13
EPHE, France
14
IRD/Univ Rouen, Cameroun
E-mail: [email protected]
In biodiversity hotspots such as tropical forests of Central Africa, invertebrates represent the bulk of
global diversity. They are however very poorly known, and their abundance and diversity has
remained so far an unbeatable challenge impeding their use as indicators for ecological and
conservation studies. When taxonomic knowledge is scarce and partial, the use of DNA barcoding
is a pragmatic and efficient way to delineate species and document local or regional richness and
species assemblages. Here, we present a summary of the knowledge gained after implementing
DNA barcoding in local surveys of invertebrate biodiversity in Gabonese forests and savannas.
Sampling took place over three consecutive years within the framework of ECOTROP, a
field-school in tropical ecology and paleoecology. Students attending ECOTROP gained training in
fieldwork and study design, and became involved in the front-end processing of samples for DNA
barcoding. In total, more than 3500 Lepidoptera, 500 soil arthropods, 1000 earthworms and 100
mantids were sampled for DNA barcoding. The results from preliminary analyses are presented,
revealing an outstanding level of diversity within these groups (e.g. 44, 272 and 1330 MOTUs of
earthworms, soil arthropods and moths, respectively – most of which likely undescribed) and
interesting patterns of species assemblages.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
52
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Education
DNA barcoding market survey: a high school citizen science project
Amanda Naaum, Robert Hanner
Department of Integrative Biology, Centre for Biodiversity Genomics, University of Guelph,
Canada
E-mail: [email protected]
As a method of species identification, DNA barcoding has many applications and is finding its way
into classrooms in Canada given its intersection with fundamental concepts in the high school
curriculum (e.g. biodiversity, taxonomy and evolution) and their intersection with modern
biotechnology. The goal of this project was to provide tools for high school teachers to introduce
DNA barcoding to their students. In partnership with Let’s Talk Science – a Canadian non-profit
organization dedicated to science literacy and outreach - six modules were created covering
concepts related to DNA barcoding. They were specifically tailored to complement the existing
curriculum in molecular genetics and included instructions for how to conduct a seafood market
survey using DNA barcoding. Seafood samples from Ontario and Manitoba were collected and
identified using DNA barcoding and BOLD. Twenty-four percent market substitution was detected,
including evidence of suspected adulteration with economic, health and conservation impacts.
Educators reported increased student engagement as a result of the real-world impact of the project.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
53
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Education
Capacity building in modern taxonomy as an instrument for knowledge transfer
in IPBES
Torbjørn Ekrem1, Elisabeth Stur1, Tuuli Mäkinen2, Roger Bills2, Michelle Hamer3, Carlos de la
Rosa4 and Endre Willassen5
1
NTNU University Museum, Department of Natural History, Trondheim, Norway
2
South African Institute of Aquatic Biology, Grahamstown, South Africa
3
South African National Biodiversity Institute, Pretoria, South Africa
4
La Selva Biological Station, Organization for Tropical Studies, Heredia, Costa Rica
5
University Museum of Bergen, Natural History Collections, Bergen, Norway
E-mail: [email protected]
Biodiversity science, management and conservation rely on our ability to recognise species and
other taxonomic entities in nature. Basic taxonomic knowledge of organismal life is also crucial for
sustainable development of natural resources and ecosystem services, key areas for the United
Nations’ Intergovernmental Platform on Biodiversity and Ecosystem Services (IPBES).
Nevertheless, we know far less about the true biodiversity than we need to properly understand the
dynamics of terrestrial, aquatic and marine ecosystems. This project aims to increase our capacity to
discover and document species diversity by applying DNA barcoding in knowledge transfer
activities among partners in South Africa, West Africa, Costa Rica and Norway. The project will
also highlight DNA barcoding as a tool in biodiversity management to facilitate broader use of the
DNA barcode library. With funding from the Norwegian Environment Agency and JRS Biodiversity
Foundation, three hands-on workshops were held in South Africa, Norway and Costa Rica in 2013.
The invited participants received both a theoretical introduction and practical experience with DNA
barcoding and the BOLD database. Although capacity building and knowledge transfer is the main
goal of this initiative, the project also has as goal to produce > 5000 DNA barcodes from the
participating countries in Africa and Central America.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
54
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Education
Education and skills development in barcoding and taxonomic practice
David Castle
School of Social and Political Science, University of Edinburgh, Edinburgh EH1 1LZ, United
Kingdom
Email: [email protected]
Taxonomy may be an old and venerable science whose contributions to biodiversity knowledge are
uncontestable, yet support for museum and university-based taxonomy has been withering at
precisely the time when awareness of threats to global biodiversity has grown. Traditional
taxonomic practices can be conserved, but options for expanding, modernizing, and making
taxonomy more relevant to practical issues are available. Nevertheless, many taxonomists reject
methods like barcoding because they are regarded as threats, not supports, to taxonomic practice.
Arising from the dispute between traditional taxonomy and barcoding are identifiable education
objectives. These include awareness of the potential and limitations of high-throughput,
semi-automated method of assigning unique identifiers to taxa. Furthermore the aims, methods and
objectives of barcoding need explication to address concerns about the methods and technology, the
use of barcoding in biodiversity science, and the expectations of how barcoding will expand
contributors and users of taxonomy.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
55
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Next Generation Sequencing
Environmental monitoring in genomics era
Xin Zhou
China National GeneBank, BGI-Shenzhen, Shenzhen, Guangdong, China
E-mail: [email protected]
Nearly all big leaps in applied science have been initiated by technology innovation. Environmental
monitoring aims to rapidly access and monitor changes in habitat quality using environmental
factors, such as biological community composition. Similarly, this important domain has benefited
from novel developments in molecular biology and genomics. While classic DNA barcoding has
opened up a new path to understand community based on its molecular features, metabarcoding
empowered by next-generation-sequencing technologies provides a more applicable approach to
rapidly characterizing biodiversity in a high-throughput manor. Recent developments in
metabarcoding have also borrowed much experience from genomics. For instance, we now are able
to take advantage of very short NGS reads and recover full-length DNA barcodes from tissue
mixtures. Additionally, enabled by deep sequencing capacity of the NGS platforms, direct
sequencing of the entire arthropod community without amplification of any specific DNA markers
has become possible. The improvements in sequencing and informatics also provide an opportunity
to expand the current single-marker metabarcoding to utilize full mitochondrial information. This
presentation will introduce some of these new trends in the field of metabarcoding that will
revolutionize the field of environmental monitoring.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
56
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Next Generation Sequencing
The verification and application of metabarcoding
Yinqiu Ji1, Louise Ashton2, Yong Tang3, Akihiro Nakamura2,4, Roger Kitching2, Xiaoyang Wang1,
Martin Lott5, Brent C. Emerson6, Douglas W. Yu1,7
1
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese
Academy of Sciences, Kunming, Yunnan 650223 China
2
Environmental Futures Centre and Griffith School of Environment, Griffith University, Nathan,
Queensland 4111 Australia
3
Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Meng La, Yunnan
666303 China
4
Queensland Museum, South Brisbane, Queensland 4101 Australia; [email protected]
5
School of Computing Sciences, University of East Anglia, Norwich, Norfolk NR47TJ, United
Kingdom
6
Island Ecology and Evolution Research Group, IPNA-CSIC, C/Astrofísico Francisco Sánchez 3,
38206 La Laguna, Tenerife, Canary Islands, Spain
7
Department of Biology, University of Florida, Gainesville, Florida 32611 USA
E-mail: [email protected]
Metabarcoding is a rapid method of biodiversity assessment that combines two technologies: DNA
barcoding and high-throughput DNA sequencing. Here we validate metabarcoding by testing it
against the traditional ecological dataset from the rainforest of south-western China. The
metabarcoding and traditional datasets show statistically correlated alpha- and beta-diversities, and
give us very similar results. Because of its high efficiency and reliability, metabarcoding could be
used for more projects which need to do biodiversity monitoring.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
57
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Next Generation Sequencing
Establishing an high definition biodiversity map in Amazonian rainforest
Eric Coissac1, Aurélie Bonin1, Ludovic Gielly1, Amaia Iribar-Pelozuelo2, Melanie Roy2, Heidy
Schimann3, Lucie Zinger2, Jérome Chave2, Pierre Taberlet1
1
Laboratoire d’Ecologie Alpine, CNRS UMR 5553, Université Joseph Fourier, BP 53, 38041
Grenoble Cedex 09, France.
2
Evolution & Diversité Biologique, CNRS UMR 5174, Université Paul Sabatier, 118 route de
Narbone, 31062 Toulouse Cedex 09, France.
3
Ecologie des Forêts Tropicales, INRA UMR, Campus agronomique, BP 709, 97387 Kourou,
French GuianaE-mail: [email protected]
Rainforests are emblematic biodiversity hotspots. But assessing the diversity and spatial distribution
of all organisms living in such environments remains highly challenging with traditional approaches.
DNA metabarcoding allows detecting a broad range of taxa by using an environmental barcode
targeting all eukaryotes (18S rDNA) and a combination of specific markers for plants (trn-L-P6
loop), fungi (ITS1), and some animals like termites (12S rDNA). We are applying this technique to
establish a high definition biodiversity map of a 12-hectare permanent plot at the Nouragues
Research Station (French Guiana, CNRS), where soil samples were collected every 10 m. Soil DNA
was extracted from each sample, and each marker cited above was amplified by PCR and sequenced
using Illumina technology. A preliminary high-resolution map of the local soil biodiversity was
obtained from a 1-hectare subplot (sampled every 5 m). This map revealed that taxa distributions
were generally congruent amongst the different markers used and with field observations. At such
reduced spatial scales, we also observed a high micro-heterogeneity of taxa richness for all the
groups surveyed, as well as the covariation of the taxa turnover between plants and some clades of
fungi.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
58
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Next Generation Sequencing
Using next-generation sequencing technology to accelerate DNA barcoding
Shadi Shokralla1, Joel Gibson1, Hamid Nikbakht1, Daniel H. Janzen2, Winnie Hallwachs2 and
Mehrdad Hajibabaei1
2
Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50
Stone Road East, Guelph, ON, Canada
2
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, United States
E-mail: [email protected]
DNA barcoding is an efficient method to aid in detecting undescribed and cryptic species and to
address complex ecological interactions. The conventional means of generating DNA barcodes is
through Sanger sequencing of DNA barcode loci in individual specimens. Here we demonstrate the
potential application of next generation sequencing (NGS) platforms for parallel acquisition of
DNA barcode sequences from large batches (e.g. hundreds) of specimens simultaneously.
Specimens are tagged in PCR stage using barcoded primers to facilitate separating their associated
DNA barcode sequences after NGS analysis. We perform a side-by-side comparison of the Sanger
sequencing versus NGS in generating DNA barcodes for bulk specimens of insects. Protocol
simplicity coupled with higher throughput and lower cost per sequence makes this approach
potentially useful in large high throughput barcoding projects using a single or multiple barcoding
markers.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
59
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 1: Next Generation Sequencing
Metabarcoding characterises the biodiversity response to environmental
management in UK plantation forest and farmland ecosystems
Catharine Bruce1, Nadia Barsoum2, Chenxue Yang3, Yinqui Ji3, Douglas Yu1,3
1
University of East Anglia, Norwich, UK
2
Forest Research, UK Forestry Commission
3
Kunming Institute of Zoology, Kunming, China
E-mail: [email protected]
Even in well-known ecosystems, the taxonomic impediment has made it impractical to monitor
biodiversity. Therefore, policy decisions are based on a narrow range of indicator groups, sampled
infrequently, which are not necessarily representative of the true state of nature.
We use CO1 metabarcoding to gather representative biodiversity data for flying arthropods in UK
plantation forest and farmland ecosystems. We demonstrate that this produces useful information
for evaluating management options and allows the identification of species that are associated with
particular environmental variables. Key findings from the forest study include: (1) there is a very
fast turnover of arthropod communities over a seven week period, which has implications for
sampling design; (2) within a varied matrix of plantation forest stands, monocultures can make an
important contribution to regional biodiversity; and (3) in our study system, regional biodiversity
can be maximised by maximising the variation in ratio of conifer/deciduous trees, density of
undergrowth, and tree species diversity. In the agricultural study, we show that different
agri-environment management systems are characterised by different communities of flying
arthropods and soil fauna.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
60
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 2: Pollinators
Barcoding reveals that all Apidae bee species in Fiji, Vanuatu and Samoa
represent anthropogenic dispersals
Scott V. C. Groom1, Hien T. Ngo2, Sandra M. Rehan3, Posa Skelton4, Mark I. Stevens5, Michael P.
Schwarz1
1
School of Biological Sciences, Flinders University, GPO Box 2100, SA 5001, Adelaide, Australia
2
Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada
3
Department of Biology, University of Pennsylvania, 433 S. University Avenue, Philadelphia, PA
19104, U.S.A.
4
Secretariat for the Pacific Regional Environmental Programme.
5
South Australian Museum, GPO Box 234, SA 5000, and School of Earth and Environmental
Sciences, University of Adelaide, SA 5005, Adelaide, Australia
E-mail: [email protected]
Museum records going back the early 1900s indicate the presence of multiple genera of the
long-tongued bee gamily Apidae in the south west Pacific and at least some of these were thought to
be indigenous to the region. Here we use barcode sequences to show that all such species are
likely to have been introduced by humans. Source regions include Australia, S.E. Asia and Central
America. Extensive collections also show that one species, Braunsapis puangensis, has been
introduced to Fiji sometime in the last 40 years, and has become highly abundant in Viti Levu.
The multiple accidental introductions of these Apidae species is probably due to their nesting
substrates, which include dead plant stalks such as straw, and wood, which could have been moved
across ocean barriers by maritime trade. The very high abundance of Braunsapis in Fiji might be
ecologically benign, but there is also a possibility that it could disrupt endemic plant-pollinator
relationships. There is an urgent need to explore this possibility.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
61
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 2: Pollinators
The campaign to barcode the bees of the world
Laurence Packer
Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada
E-mail: [email protected]
The campaign to barcode the bees of the world was inaugurated at York University in May 2008.
Progress has been rapid with clusters representing approximately 25% of the world's bee species
having at least one good barcode sequence and most genera having at least one species sampled.
This includes some of the most rarely collected genera as well as several undescribed ones.
However, a considerable amount of additional taxonomic work is required for most taxa from
most parts of the world. In this talk I will outline progress so far, present some illustrative
examples and discuss the issues that arise with such an ambitious project.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
62
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 2: Pollinators
Asian bee DNA barcode database, ABeeBOL, and a DNA barcode study of two
closely related species of Asian Andrena (Chlorandrena)
Osamu Tadauchi, Ryuki Murao
Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, JAPAN
E-mail: [email protected]
DNA barcode data of bees have been rapidly accumulated after BEEBOL, a committee promoting
DNA barcoding of bees was organized by Prof. Packer of York University, Canada in 2008. We
started to construct a DNA barcode database of Asian bees, ABeeBOL. Each record consists of 19
items in the format of the Darwin Core of the Global Biodiversity Information Facility (GBIF),
including scientific name, country, collection locality, collection date, collector, etc. of the specimen
as well as its barcode data. We will upload our data to BOLD System as well as continue to
accumulate Asian bee DNA barcode data. One of our studies related to DNA barcoding is also
presented. Two closely related species, Andrena (Chlorandrena) knuthi Alfken and A. (C.)
okinawana Matsumura et Uchida, occurring in Japan, Korea and China were analyzed. The
differences between the two species are a few and especially difficult in female. In the Tsushima Is.
(Japan) situated between Japan and Korean Peninsula the two species are found. We examined their
habitat in the island in detail and analyzed DNA barcodes of material collected from the Tsushima
Island, Korea and China. The result showed the two species are good species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
63
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Pollinators
Coalescent analyses indicate parallel responses of bees to past climate change in
three isolated archipelagos of the south western Pacific
Scott Groom1, Mark Stevens2, Michael Schwarz1
1
School of Biological Sciences, Flinders University, GPO Box 2100, SA 5001, Adelaide, Australia
2
South Australian Museum, GPO Box 234, SA 5000, and School of Earth and Environmental
Sciences, University of Adelaide, SA 5005, Adelaide, Australia
E-mail: [email protected]
The impacts of glacial cycles on the spatial distribution and population size of populations have
been shown in numerous taxa, both terrestrial and marine, across various regions. Despite
representing an engine room of global climates, the impact of climate fluctuations on the
development of biota in the Pacific Ocean is poorly understood. Here we use DNA barcodes to
examine how population sizes of the key bee fauna in the neighbouring south west Pacific
archipelagos of Fiji, Vanuatu and Samoa have fluctuated over the Quaternary. We show that all
three faunas suffered massive population declines, roughly corresponding in time to the last glacial
maximum, followed by rapid expansion in the subsequent inter-glacial period. This demonstrates
that some key ecosystem elements in tropical island habitats can be strongly affected in parallel
ways, despite their historical isolation. As key pollinators in these island systems, such changes may
have been critical in the development of the biodiverse Pacific island flora. Our results indicate that
future climate change scenarios may have alarming consequences for Pacific island plant
communities and for agriculture involving pollinator-dependent crops.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
64
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Pollinators
Aridification in Australia and the genesis of a xeric bee fauna: the need for a
barcoding approach to resolve cryptic species
Rebecca Dew, Michael Schwarz
School of Biological Sciences, Flinders University, GPO Box 2100, SA 5001, Adelaide, Australia
E-mail: [email protected]
Environmental factors are thought to have been major drivers in the evolution of eusociality, a
complex social behaviour involving distinct queen and worker castes. Most eusocial origins
occurred deep in evolutionary history, without extant transitional species to determine the process
behind this social shift. The allodapine bees, however, demonstrate a range of social behaviours,
with only one highly eusocial species, Exoneurella tridentata. In fact the three other described
Exoneurella species all display only weak social traits. Two species, E. lawsoni and E. eremophila
show some shared parental care, a potential precursor to eusociality. These two species and the
eusocial E. tridentata live in arid environments, suggesting that pressures of xeric ecosystems may
be driving eusocial evolution in this group. Recently a number of undescribed Exoneurella species
were identified from both arid and non-arid environments. Similarly revision of populations of
species has suggested the presence of cryptic species. These new species provide the opportunity to
explore social evolution in this genus and examine if aridification may have driven eusociality. We
will use barcodes to determine the presence of separate species and examine genetic variability
within populations of different social behaviours. Here I present our initial data from cryptic
Exoneurella species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
65
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Plants II
Genetic Variability in Eggplant Solanum L. and Related Species in Southern
Nigeria as Revealed by Nuclear and Chloroplast Genes
Mutiu Sifiu1, Oluwatoyin Ogundipe2, Bola Oboh2
1
National Center for Genetic Research and Biotechnology, Abuja-FCT, Nigeria
2
Department of Botany,University of Lagos, Lagos, Nigeria
E-mail: [email protected]
The common name “Eggplant” is given to vegetable Solanum L. which has been used as
nomenclature to refer to several Solanum species important for human diet and health. Despite the
importance of this genus, its taxonomy and phylogenetic relationships among these taxa are
currently unclear due to its large size and tropical center of diversity. This study assessed the genetic
diversity in forty nine eggplant and related Solanum species’ genetic resources in Southern Nigeria
using one nuclear (ITS) and two chloroplast genes. DNA sequence data from the three regions (ITS,
trnl C- trnl D and trnl E – trnl F) on analysis gave a high level of genetic variability (polymorphism)
among the samples studied. The highest bootstrap value of 100 was observed between S.
macrocarpon L. and S. torvum Sw., and between S. macranthum A.Rich. and S. indicum L.,
closely followed by a value of 99 between S. aethiopicum L. and S. dasyphyllum Schum. & Thonn.
All these are indications of a close relationship between these species and a possibility of a common
ancestor is strongly proposed. S. torvum separated out from S. melongena, and even where they
grouped together they have a very low bootstrap value of 3, which is an indication of distant
relatedness. The high level of intra and inter specific variations displayed within eggplant
accessions and between its relatives as reported in this study could be effectively used in genetic
improvement of cultivated eggplant varieties as well as in-situ and ex-situ conservation.
KEYWORDS: Eggplant, Variability, ITS, Chloroplast, Polymorphism.
Acknowledgement: CRC 2005/03 (UNILAG)
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
66
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Plants II
Progress of iFlora in China: building a rapid identification system for the
national protected key wild plants
Wen-Bin Yu1, Hong Wang1, Lian-Ming Gao1, Jun-Bo Yang2, Yu-Hua Wang3, Ting-Shuang Yi2, Jie
Cai2, Chun-Xia Zeng2, En-De Liu1, Xiang-Yun Yang2, De-Zhu Li1,2
1
Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese
Academy of Sciences
2
Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming, Yunnan 650201, China
3
Science and Technology Information Center, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming, Yunnan 650201, China
4
Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese
Academy of Sciences, Kunming, Yunnan 650201, China
E-mails: [email protected], [email protected]
The first batch of the Catalogue of the National Protected Key Wild Plants (NPKWP) was
promulgated by the Ministry of Forestry and the Ministry of Agriculture in September 1999 and
authorized by the State Council of China. This Catalogue includes 305 species with representatives
from 92 families and 194 genera, which were placed in two protection categories. The NPKWP is
the key reference employed by Chinese administrative departments to protect and manage
endangered and/or economic wild plants. Because DNA barcoding makes it possible to identify
vegetative plants and small tissue fragments, it allows administrative staff to identify NPKWP
without specialists. As an important part of the Next-Generation Flora (iFlora) project led by the
Kunming Institute of Botany, Chinese Academy of Sciences, we built a rapid identification system
for the NPKWP. Firstly, we collected all of the plant species on the NPKWP and their closely
related species and then sequenced the core DNA barcoding loci. This work led to the development
of a system based on DNA barcodes, as well as on morphological descriptions, photos, and
geographic information. For species identification, a user just inputs a query barcode sequence into
the platform. And it rapidly provides the most closely related species (NPKWP or not). The user can
then use morphological features, photos, and geographic information in the system to obtain a final
identification.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
67
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Plants II
PCR primers for ITS regions of plants with improved universality and
specificity
Tao Cheng1,2,* Li Lei3,* Chao Xu,1,2 Changhao Li1,2 Yaping Zhang4, Shiliang Zhou1
1
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese
Academy of Sciences, Beijing 100093, China
2
University of Chinese Academy of Sciences, Beijing 100049, China
3
Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506-5502, USA
4
College of Landscape Architecture, Sichuan Agricultural University, Chengdu 611130, China
*These authors contributed equally to this work.
E-mail: [email protected], [email protected]
The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA is one of the most
commonly used genes in plant molecular studies, especially in phylogeny and DNA barcoding, and
has been recommended as a core plant DNA barcode. Contrary to its popularity, the universality and
specificity of PCR primers are not satisfactory, which have brought many troubles to the users. By
thoroughly surveying and analyzing the sequences of ITS and its adjacent genes deposited in public
databases, we designed both universal and plant-specific PCR primers for amplifying ITS1, ITS2
and the whole ITS. In silico analysis of the primer pairs based on the public sequence databases
indicated that 1) the newly designed universal primer pairs are suitable for over 95% of plants; 2)
the plant-specific primer pairs are suitable for over 90% of plants without any amplification of fungi.
We used a total of 209 species from 96 angiosperm families, 11 gymnosperm families, 30 fern
families, 16 moss families and 6 fungi to test the performances of our primers. The in vitro PCR
gave very similar results to those from the in silico analyses. We are expecting that the new
universal ITS primers will find their wide applications in biodiversity assessments, and the new
plant-specific ITS primers will prevent DNA fragments of animals or fungi from amplifications in
DNA barcoding, molecular systematics and molecular ecology.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
68
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Plants II
Identification of Arbutus pavarii based on DNA barcoding techniques: can it be
differentiated from closely related Canary Island and Mediterranean species?
Ahmed Gawhari1, Stephen Jury1, Alastair Culham1
1
School of Biological Sciences, University of Reading, Reading, United Kingdom
E-mail: [email protected]
Arbutus pavarii, endemic to Libya, has always been treated taxonomically as a distinct species, but
has been confused with Arbutus unedo. Worldwide there are 20 species of Arbutus distributed
around the world, of which four species occur in the Canary Islands and Mediterranean region from
North Africa to the Middle East. Here the use of DNA barcoding is explored to establish whether A.
pavarii is distinct in DNA sequence from it is Old-World congeners in the Canary Islands and
Mediterranean. All Arbutus specimens were sampled from the herbarium of the University of
Reading (RNG). DNA fragments of the regions psbA-trnH, matK and internal transcribed spacer
ITS were explored to establish identity. TaxonDNA has been used to determine proportion of
intra-and interspecific divergences and to determine the proportion of species that could be
identified, and the presence, or absence, of a barcoding gap for the species is discussed.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
69
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Plants II
Cryptic Tamarix species identification using DNA Barcoding
Xuewei Jiang1,2, Steven G Newmaster2, Shanmughanandhan Dhivya2,3 and, Xinan Pang1,
Ramalingam Sathishkumar3, Subramanyam Ragupathy2
1
College of Life Science, Xinjiang Production & Construction Corps Key Laboratory of Protection
and Utilization of Biological Resources in Tarim Basin, Tarim University, Alar 843300, Xinjiang,
People’s Republic of China
2
Center for Biodiversity Genomics, University of Guelph, Guelph, Ontario, Canada
3
Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India
E-mail: [email protected]
The genus Tamarix is ecologically and economically significant in Tarim basin, China. The Tamarix
species complex is relatively difficult to distinguish due to phenotypic similarities, extensive
interspecific hybridization and introgression. Our research seeks to investigate the genomic
diversity of Tamarix complex, addressing a key uncertainty that will provide a framework for a
DNA barcoding method that could be used for fast, sensitive, and accurate identification of Tamarix
complex. In order to segregate the native wild species of Tamarix, 18 population representing 6
native species were collected from Tarim basin. DNA barcoding was performed using ITS2 and
rbcL barcode regions. An effective ITS2 DNA barcode was able to make 100% accurate
identification of all Tamarix species. We found considerable interspecific variation among 18
population representing six species of Tamarix using nuclear region ITS2. The species resolution
was much higher when the tiered approach (ITS + rbcL) was performed. The development of a
DNA barcoding system for Tamarix complex will facilitate Tamarix diversity assessment and will
have broad ecological and economical applications.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
70
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Data Analysis Methods
Species-specific identification from incomplete sampling: applying DNA
barcodes to distinguish species-rich genera
Wei Zhang
Marine College, Shandong University at Weihai; Weihai 264209, China
E-mail: [email protected]
Comprehensive sampling is crucial to DNA barcoding, but it is rarely performed because materials
are usually unavailable. In practice, only a few rather than all species of a genus are required to be
identified. Thus identification of a given species using a limited sample is of great importance in
current application of DNA barcodes. Here, we selected ten species-rich genera to explore whether
DNA barcodes can provide reliable specific-species discrimination in the context of incomplete
sampling. Based on their previous phylogenetic framework, we firstly sampled representative
species from each primary evolutionary clade of the genus and then added species and individuals
in the clade that nested within the unknown species–until it was formed a monophyletic group. The
tree-building and modified barcode gap methods were employed to assess species resolution. The
results showed that specific species can be successfully identified. We conclude that a
specific-species could be identified from a phylogenetic framework using incomplete
sampling–through this, DNA barcoding will greatly benefit the current fields of its application.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
71
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Data Analysis Methods
VIP Barcoding: composition vector-based
identification based on DNA barcoding
software
for
rapid
species
Long Fan1, Jerome H.L. Hui1, Zu Guo Yu2,3, Ka Hou Chu1
1
School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
2
School of Mathematics and Computational Science, Xiangtan University, Hunan 411105, China
3
School of Mathematical Sciences, Queensland University of Technology, Brisbane Q4001,
Australia
E-mail: [email protected], [email protected]
DNA barcoding has emerged to be an integral part of modern taxonomy. However, software for
analyzing large and multi-locus barcoding datasets is scarce. BLAST is currently the fastest tool
capable to handle large databases (e.g. >5,000 sequences), but its accuracy is a concern and has
been criticized for its local optimization. The other more accurate software require sequence
alignment or complex calculations which are time-consuming in dealing with large datasets during
data preprocessing or search stage. In this context, we present VIP Barcoding, a user-friendly
software for large-scale and multi-locus DNA barcoding. It adopts a hybrid, two-stage algorithm.
First, an alignment-free composition vector method is utilized to reduce searching space by
screening a reference database. The alignment-based K2P distance nearest neighbor method is then
employed to analyze the smaller dataset generated in the first stage. In comparison to other software,
VIP Barcoding has (i) higher accuracy than Blastn and several alignment-free methods, and (ii)
higher scalability than alignment-based distance methods and character-based methods. These
results suggest that this platform is able to deal with both large-scale and multi-locus barcoding data
with accuracy, and can contribute to the DNA barcoding for modern taxonomy. VIP Barcoding is
free and available at http://msl.sls.cuhk.edu.hk/vipbarcoding/.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
72
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Data Analysis Methods
Benchmarking the barcode index number (BIN) system: an evaluation of the
refined single linkage (RESL) algorithm
Sujeevan Ratnasingham, Paul Hebert
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
E-mail: [email protected]
Because many animal species are undescribed, and because the identification of known species is
often difficult, interim taxonomic nomenclature has often been used in biodiversity analysis. By
assigning individuals to presumptive species, called operational taxonomic units (OTUs), these
systems speed investigations into the patterning of biodiversity and enable studies that would
otherwise be impossible. Although OTUs have conventionally been separated through their
morphological divergence, DNA-based delineations are not only feasible, but have important
advantages. OTU designation can be automated, data can be readily archived, and results can be
easily compared among investigations. The popularity of this approach has lead to the development
of multiple methods for algorithmic OTU generation. A common challenge faced by all available
methods is the computational complexity of analyzing the growing volume of data resulting form
falling sequencing costs. Some approaches trade accuracy for computational speed while others
maintain a high accuracy but are limited to small data sets. We present a novel method of OTU
generation, based on COI barcodes, called RESL (Refined Single Linkage), that addresses this
challenge by excelling at both accuracy and speed. RESL exploits the network structure in
distance matrices to cluster sequences. We evaluate the performance of our solution by comparing
groups of specimens identified to a species through prior taxonomic work and those inferred from
the analysis of COI sequence variation using RESL.
We subsequently compare the performance
of RESL against four established (ABGD, CROP, GMYC, jMOTU) algorithms. Our benchmark
shows RESL to be highly competitive in accuracy but excels in speed, completing analysis 500
times faster than the second fastest algorithm.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
73
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Data Analysis Methods
A comparison of four DNA barcode-based species delineation methods – a story
by gelechioid moths
Mari Kekkonen1, Lauri Kaila 2, Marko Mutanen 3, Paul Hebert4
1
Finnish Museum of Natural History, University of Helsinki
2
Finnish Museum of Natural History
3
University of Oulu
4
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
E-mail: [email protected]
The great number of undescribed species strongly calls for efficient means to enhance taxonomic
workflows. DNA barcode-based species delineation is a promising option, providing putative
species (operational taxonomic units, OTUs) to be used in further studies such as phylogenetic
analyses and taxonomic revisions with additional data. The benefits of DNA-based delimitation are
convincing as it offers increased speed, repeatability, and taxonomic accuracy by aiding the
recognition of most cryptic species from the beginning. In this study, we compare four methods,
Barcode Index Number (BIN), parsimony networks, Automatic Barcode Gap Discovery (ABGD),
and General Mixed Yule-coalescent (GMYC), using two data sets of fairly well-known gelechioid
moths. The two groups are different, because most Finnish gelechiine species are genetically
distinct, while Australian elachistine species show much lower divergences making them a
challenging group. This study aims (i) to test the congruence of OTU counts between the methods
and previous taxonomic works and (ii) to compare the content of OTUs and described species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
74
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 2: Data Analysis Methods
Supervised DNA barcode species classification:
results
analysis, comparison and
Emanuel Weitschek1,2, Giulia Fiscon2,3, Giovanni Felici2
1
Department of Computer Science and Automation, University Roma Tre, Italy
Institute of Systems Analysis and Computer Science, National Research Council, Italy
3
Department of Computer, Control, and Management Engineering, Sapienza University, Italy
E-mail: [email protected]
2
Background
Species classification with DNA Barcode sequences has proven effective on different organisms.
Indeed, specific gene regions have been shown as Barcode in the main life kingdoms, i.e. the gene
cytochrome c oxidase subunit I (COI) in animals, regions rbcL, matK in plants and ITS in fungi.
The DNA Barcode sequence classification problem assigns an unknown specimen to a known
species by analyzing its Barcode. This task has to be supported with reliable methods and
algorithms.
Methods
In this work the efficacy of supervised machine learning methods to classify species with DNA
Barcode sequences is shown. The Weka machine learning software, which includes a collection of
supervised classification methods, is adopted to address the task of DNA Barcode analysis.
Classifiers families (trees, rules, lazy learners, Bayesian and functions) are tested on public
available synthetic and real data sets belonging to the animals, fungi and plants kingdoms. In
particular, the function based method support vector machines (SMO), the rule based RIPPER
(JRIP), the classification trees based C4.5, and the Bayesian based method Naïve Bayes are
considered. Additionally, the classification results are compared with traditional and well
established DNA Barcode classification methods, as phylogenetic trees (NJ, PAR), similarity based
(BLAST), and character based (DNA-BAR, BLOG).
Results and discussion
An integrated software that converts the DNA Barcode FASTA sequences to the Weka format was
developed, adapting different input formats to permit the execution of the experiments. Analysis
results show that Support Vector Machines (SVM) and Naïve Bayes methods outperform on
average the other considered classifiers (synthetic and real data sets), although they do not provide a
clear and compact human interpretable classification model. Ruled based methods, as BLOG and
RIPPER have inferior classification performances, but the user is provided with the diagnostics
positions and nucleotide assignments. On simulated data the supervised machine learning methods
obtain superior classification performances with respect to the traditional DNA Barcode
classification methods (fig. 1). On real data the classification performances are at a comparable
level to the other methods (fig. 2).
Conclusion
The classification analysis shows that supervised machine learning methods are promising
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
75
Parallel Session Abstracts – Monday, 28 October
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candidates for handling with success the DNA Barcoding species classification problem, obtaining
excellent classification performances. Finally, the DNA Barcoding community is provided with a
powerful tool to perform species classification.
Fig. 1 Classification performances (accuracy) for the synthetic data sets
Fig. 2 Classification performances (accuracy) for the real data sets
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
76
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Environmental Monitoring
Barcoding trophic interactions for deep-sea ecosystem
Hsuan-Wien Chen1, 2, Sz-Chuen Wang2, 3, Hsing-Juh Lin2, 4, Kwang-Tsao Shao2
1
Department of Biological Resources, National Chiayi University, Chiayi, Taiwan, China
2
Biodiversity Research Center, Academia Sinica, Taipei, Taiwan, China
3
Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, China
4
Department of Life Science, National Chung-Hsing University, Taichung, Taiwan, China
E-mail: [email protected]
Deep sea is indeed the last frontier on our planet Earth. In addition to its wealthy but unrevealed
biodiversity, the structure and function of this unique ecosystem are even less recognized. One
important feature to distinguish deep-sea ecosystem is to illustrate its trophic structure, namely, to
portray who is eating whom in the deep ocean. To uncover those hidden interactions, demersal and
benthic communities were sampled in deep waters off Taiwan. The stomach contents of two
dominant deepwater fish, Coelorinchus kishinouyei and C. leptorhinus, were used to test if DNA
barcoding provides better resolution for identifying prey items than the conventional morphological
approach does. As a result, 197 prey items that isolated from 13 fish stomachs were used to generate
131 sequences, representing 64 unique haplotype of the CO1 fragment. Sequence blast against those
in BOLD and Genbank resulted in 17 matches at species level (similarity>96%, include 7 matched
with the predator fish itself). Among those matches, more than 50% cases, the barcoding approach
improved prey item’s taxonomic resolutions obtained from morphologies along; while in the other
cases, samples were either misidentified or contaminated with predator’s DNA. The tree-based
comparisons were also applied and lead to additional prey item identifications. With the limited
match rate, more or less than 30%, our study showed that DNA barcoding did provide effectiveness
and higher taxonomic resolution on recognizing potential trophic interactions in deep-sea
environment; however, the lack of barcode data for marine benthos encumbered its promising wide
applications in further food web/ecosystem studies.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
77
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Environmental Monitoring
Quantifying Terrestrial Biodiversity: The Canadian National Park Malaise Trap
Program
Jeremy deWaard, Stephanie deWaard, Jayme Sones, and Paul Hebert
Biodiversity Institute of Ontario, University of Guelph, Guelph, ON, Canada
E-mail: [email protected]
Water quality assessments routinely employ information on the species composition of invertebrate
communities as an index of biological integrity. By contrast, assessments of terrestrial environments
lack a widely accepted protocol to derive a biotic index, and instead generally rely on surveys of a
few indicator taxa (birds, vascular plants). This approach disregards an important reality – the vast
majority of species in terrestrial ecosystems are arthropods, whose responses to disturbance are
unmeasured. We are addressing this gap through a time- and cost-efficient approach for terrestrial
environmental assessments based on the DNA barcode analysis of specimens collected by Malaise
traps. While samples will eventually be analyzed with next-generation sequencers, Sanger-based
analysis is currently critical to maintain the link between each barcode record and its source
specimen to create a barcode reference library based on carefully identified specimens. This
approach was tested in the summer of 2012 with the deployment of Malaise traps in 14 of Canada’s
National Parks. Specimens from these traps were processed without pre-sorting in a 384 well-based
analytical pipeline that utilized the Barcode Index Number (BIN) system to further enhance the
efficiency of workflows such as specimen photography to species lacking such coverage. The
barcode analysis of nearly 300K specimens revealed over 20K BINs, roughly one third of all insect
species known from Canada. We discuss how a terrestrial biotic index might be calculated using the
results obtained from this approach.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
78
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Environmental Monitoring
Tracking Ghosts: using environmental DNA barcodes to detect invasive and
endangered species
Chris Wilson1, Jennifer Bronnenhuber1, Margaret Boothroyd2, Natasha Serrao3, Kristyne Wozney1,
Caleigh Smith1
1
Aquatic Research and Development Section, Ontario Ministry of Natural Resources, Trent
University, Peterborough, Ontario K9J 7P8
2
Biology Program, Trent University, Peterborough, Ontario K9J 7P8
3
Environmental and Resource Studies Program and Department of Biology, Trent University,
Peterborough, Ontario K9J 7P8
E-mail: [email protected]
Effective management of both endangered and invasive species requires sensitive detection of
species occurences, often at low abundances. In aquatic environments, detection of rare species
such as endangered taxa or early-stage invasive species can be further confounded by site
accessibility, sampling gear, and capture efficiency, with direct observation being difficult at best.
Discriminating between detection failure (null) versus true absence (zero) can be problematic, but
has significant consequences for species and habitat management. A novel application of DNA
barcoding has been to develop species-specific detection in aquatic habitats from environmental
DNA (eDNA). Experimental eDNA trials in a controlled environment to determine species
occurrence confirmed the taxonomic specificity and spatial, temporal, and quantitative sensitivity of
eDNA detection. eDNA surveillance is currently being used in Ontario to track the presence or
absence of aquatic endangered fish species in natural habitats, as well as to survey Ontario waters of
Lake Erie for the potential occurrence of Asian carps (bighead, silver, and grass carp). The
combined results indicate that environmental DNA is a reliable method for species detection in
freshwater systems, and can be used as an effective sampling technique for detecting both invasive
and endangered freshwater species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
79
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Environmental Monitoring
DNA barcoding a new natural park in China
Wenhui Song, Shenzhou Yang, Xu Su, Shanlin Liu, Xin Zhou
China National Genebank, BGI-Shenzhen, Shenzhen, China, 518083
E-mail: [email protected]
Most of China’s biodiversity is protected in natural parks. Although many new parks are being
established, a large proportion of the native biodiversity remains unknown, both morphologically
and molecularly. Recent studies suggest DNA barcoding can expedite and complement
morphology-based approaches in biodiversity surveys, providing a rapid path to characterize largely
unknown fauna, such as that in China. We integrate a DNA barcoding approach in routine survey
and management in the newly established Old Creek Nature Reserve, Sichuan Province, China.
Survey for insect diversity is part of the top priority for this new reserve. Intensive sampling using
light traps, kick nets, Malaise traps and hand-picking has been deployed with a focus on
environmental indicator groups, e.g., lepidotperans, mayflies, stoneflies, and caddisflies. More than
6,000 insect specimens have been collected covering the majority of flying season and individually
barcoded. All specimens are morphologically identified to the finest level, while representatives of
all morpho-species are sequenced. The integration of DNA barcoding into routine biodiversity
survey has greatly accelerated the progress of registering insect fauna for the new (and largely
unknown) natural park. Sanger barcodes built for the target region also serve as a useful reference
for applying metabarcoding in future biodiversity monitoring programs.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
80
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Environmental Monitoring
DNA barcoding of urban environments: A look at the invertebrate diversity of
eThekwini (South Africa)
Sandi Willows-Munro
University of KwaZulu-Natal, Durban, South Africa
E-mail: [email protected]
Rates of urbanization in Africa are the highest in the world. By 2025, more than half of the African
population will be urban. Habitat loss and fragmentation associated with urbanization is recognized
as a major component of biodiversity loss. Urban areas have lower biodiversity when compared to
natural areas and few DNA barcode studies have focused exclusively on biodiversity within urban
environments. The eThekwini region is a metropolitan municipality covering a land area of 2297
km2 including the coastal city of Durban and surrounding towns. Although eThekwini is located
within the biodiversity rich Maputaland-Pondoland-Albany region, the area is heavily urbanized.
To integrate and conserve areas of high biodiversity within the city, a network of open spaces has
been established linking nature reserves and undeveloped pieces of privately and
municipality-managed land. This network of natural habitats within the city act as reservoirs for
local diversity. Invertebrate taxa were collected from several of these open spaces within the city of
Durban and DNA barcoded (mitochondrial COI). This talk will include some preliminary analyses
of the DNA barcode species inventory for eThekwini, highlighting the unexpectedly high species
richness recovered for the region and will also describe the human capacity developed during the
project.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
81
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Amphibians and Reptiles
Cytonuclear discordance in Vietnamese bent-toed geckos (Cyrtodactylus
irregularis species complex) with concerns on combining mitochondrial and
nuclear DNA sequence data
Sang Ngoc Nguyen1,2,3, Wei-Wei Zhou1, Thanh-Ngan Thi Le4, Anh-Dao Thi Tran4,5, Jie-Qiong Jin1,
Ba Dinh Vo6, Luan Thanh Nguyen6, Tao Thien Nguyen7, Truong Quang Nguyen8, Dat Duc Hoang3,
Nikolai L. Orlov9, Jing Che1, Robert W. Murphy1,10, Ya-Ping Zhang1,11
1
State Key Laboratory of Genetic Resources and Evolution, and Yunnan Laboratory of Molecular
Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences,
Kunming, China
2
Graduate University of Chinese Academy of Sciences, Beijing, China
3
Institute of Tropical Biology, the Vietnamese Academy of Science and Technology (VAST), Ho
Chi Minh, Vietnam
4
Vietnam National University, University of Science, Ho Chi Minh, Vietnam
5
Zoologisches Forschungs museum Alexander Koenig, Bonn, Germany
6
Hue University, College of Science, Hue, Vietnam
7
Vietnam National Museum of Nature, VAST, Hanoi, Vietnam
8
Institute of Ecology and Biological Resources, VAST, Hanoi, Vietnam
9
Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia
10
Centre for Biodiversity and Conservation Biology, Royal Ontario Museum, Toronto, Canada
11
Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming,
China
E-mail: [email protected]
Examples of cytonuclear discordance (CD) are increasing. This raises concerns about combining
data from the two genomes to reconstruct phyletic history. Further, we sought an efficient means to
detect CD and explored the efficacy of DNA barcoding based on the gene cytochrome c oxidase
subunit I (COI) and two nuclear DNA (nDNA) loci: RPL35 and RAG1. Our analyses used 164
samples of Vietnamese bent-toed geckos of the Cyrtodactylus irregularis species complex (Reptilia:
Gekkonidae) collected from 24 localities. DNA barcoding inferred the presence of eight described
species and identified at least 13 unknown lineages, among which seven matrilines were deeply
divergent; K2P genetic distances between sites averaged 16.6±4.6%. Isolation by distance explained
most of the cryptic diversity. The matrilineal genealogy and nDNA phylogeny were largely
concordant in resolving major lineages, but not their relationships. Analyses of both genomes
suggested the presence of at least six undescribed cryptic species. Geckos at Ta Kou Mountain, had
a complex history involving the unification of two divergent lineages. This important history would
not have been detected using analyses of nDNA markers alone because of gene flow. Further, the
extent of CD suggested that combining the two genomes for a single analysis was undesirable.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
82
Parallel Session Abstracts – Monday, 28 October
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Parallel Session 2: Amphibians and Reptiles
Cryptic diversity in ex situ tropical amphibian conservation program revealed
by DNA barcoding
Andrew J. Crawford1,2,3, Catalina Cruz3, Edgardo Griffith4, Heidi Ross4, Roberto Ibáñez1,2, Karen R.
Lips1,5, Amy C. Driskell6, Eldredge Bermingham1 And Paul Crump7
1
Tropical Research Institute, Apartado 0843–03092, Panama, Republic of Panama
2
Círculo Herpetológico de Panamá, Apartado 0824–00122, Panama, Republic of Panama
3
Departamento de Ciencias Biológicas, Universidad de los Andes, A.A. 4976, Bogotá, Colombia
4
El Valle Amphibian Conservation Center, El Valle de Anton, Coclé, Republic of Panama
5
Department of Biology, University of Maryland, College Park, MD 20742, USA
6
Laboratories of Analytical Biology, Smithsonian Institution, Suitland, MD 20746, USA
7
Department of Conservation and Science, Houston Zoo, Houston, TX 77030, USA
E-mail: [email protected]
Amphibians constitute a diverse yet incompletely characterized clade of vertebrates, in which new
species are still being described at a high rate. Amphibians are also increasingly endangered, due in
part to disease-driven threats of extinctions. As an emergency response, conservationists have begun
ex situ assurance colonies for priority species. The abundance of cryptic amphibian diversity,
however, could hinder ex situ conservation. In this study we used DNA barcoding to survey
mitochondrial DNA (mtDNA) variation in captive populations of ten species of Neotropical
amphibians maintained in an ex situ assurance program at El Valle Amphibian Conservation Center
(EVACC) in the Republic of Panama. We combined mtDNA sequences with genetic data from
presumably conspecific wild populations from Panama, and applied genetic distance-based and
character-based analyses to identify cryptic lineages. We found that three of ten species harbored
substantial cryptic genetic diversity within EVACC, and an additional three species harbored cryptic
diversity among wild populations, but not in captivity. Ex situ conservation efforts focused on
amphibians are therefore vulnerable to an incomplete taxonomy leading to misidentification among
cryptic species. DNA barcoding may therefore provide a simple, standardized protocol to identify
cryptic diversity readily applicable to any amphibian community.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
83
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 2: Amphibians and Reptiles
Exploring and understanding the diversity of Chinese Amphibians by DNA
Barcoding
Jing Che1, Hong-man Chen1, Jie-qiong Jin1, Yun-yu Wang2, Zhi-yong Yuan1, Nikolay A. Poyarkov
Jr.3, Jin-zhong Fu4, Ding-qi Rao1, Jia-tang Li5, Jian-ping Jiang5, Yu-chi Zheng5, Xiao-mao Zeng5,
Wen-zhi Wang2, Li-jun Wang6, Hai-peng Zhao7, Hai-tao Zhao8, Hai-tao Shi6, Robert M. Murphy2,9,
Ya-ping Zhang1,10
1
State Key Laboratory of Genetic Resources and Evolution, and Yunnan Laboratory of Molecular
Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences,
Kunming 650223, China
2
Southern China DNA Barcoding Center, and Animalia Resources Bank Kunming Institute of
Zoology, Chinese Academy of Sciences, Kunming 650223, China
3
Department of Vertebrate Zoology, Moscow MV Lomonosov State University, Moscow 119991,
Russia
4
Department of Integrative Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada;
5
Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China;
6
Department of Biology, Hainan Normal University, Haikou 571158, China
7
School of Life Science, Henan University, Kaifeng 475001, China
8
Department of Environment and Life Science, Bijie College, Guizhou 551700, China
9
Centre for Biodiversity and Conservation Biology, Royal Ontario Museum, 100 Queen’s Park,
Toronto M5S 2C6, Canada
10
Laboratory for Conservation and Utilization of Bioresources, Yunnan University, Kunming
650091, China
E-mail: [email protected]
The Kunming Institute of Zoology, Chinese Academy of Sciences, has been leading the initiative to
DNA barcode the amphibians of China as part of the Cold Code project. Our field investigations
have covered the major areas of China and most species were sampled from across their
distributions. To date, more than 333 of 408 Chinese species of amphibians, which exceeds 80% of
the total amphibian fauna, were sequenced for COI. The dataset included more than 6100 sequences.
More than 250 species (over 61%) were sampled from type localities and this initiative continues
where possible. Analyses identified lineages and discovered many unconfirmed candidate species.
The amphibian diversity of China appeared to be underestimated. We identified regions and taxa
requiring integrated surveys and the analyses resulted in the prioritizing of taxa and regions for
conservation efforts. The integrative amphibian inventory provided insights into and served to guide
future taxonomic initiatives. Moreover, the impressive dataset facilitated a more complete
understand the evolutionary history of the amphibian fauna. These and future discoveries will allow
assessments of spatial patterns of amphibian richness and endemism in China.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
84
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 2: Amphibians and Reptiles
Progress towards DNA barcoding amphibians from neotropics
Mariana Lyra, Célio Haddad
Universidade Estadual Paulista-UNESP, Campus de Rio Claro, Departamento de Zoologia, Av 24A,
N 1515, Bela Vista CEP 13506-900, Rio Claro, SP, Brazil
E-mail: [email protected]
Amphibian diversity is currently facing serious population declines and/or extinctions worldwide.
Paradoxically, several new species are continuously being described, suggesting that in many cases
species are being lost before they are described or before we know their existence. The use DNA
Barcoding may help to accelerate taxonomic researches by providing an initial perspective on
species genetic diversity. The greatest diversity of amphibians on the planet is found in Brazil (~940
spp) and in the last few years we have DNA barcoding Brazilian species. So far we have analyzed
COI sequences from more than 3900 individuals, comprising more than 580 species of frogs, toads,
caecilians and salamander (60% of described species from Brazil). We have found more than 1000
DNA barcodes clusters (BINs) within our data, suggesting that amphibian diversity in neotropics is
highly underestimated. The results show that the use of DNA barcodes may help to make a better
estimative of amphibian diversity and to formulate a more precise evaluation of global amphibian
decline. We hope that these data can contribute effectively for conservation and management of
Brazilian species. Financial support: FAPESP, CNPq and Chinese Academy of Sciences
(COLDCODE initiative).
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
85
Parallel Session Abstracts – Monday, 28 October
—————————————————————————————————————————————
Parallel Session 2: Amphibians and Reptiles
DNA barcoding of Comoran squamate reptiles
Oliver Hawlitschek1, Zoltán T. Nagy2, Johannes Berger1, Frank Glaw1
1
Zoologische Staatssammlung München, Münchhausenstr. 21, 81247 Munich, Germany
2
Royal Belgian Institute of Natural Sciences, JEMU, Rue Vautier 29, B-1000 Brussels, Belgium
E-mail: [email protected]
Recently, DNA barcoding became increasingly common as a method for species identification in
biodiversity inventories. Here we present a DNA barcoding study of squamates of the Comoros, a
poorly studied group of oceanic islands close to and mostly colonized from Madagascar. Our
dataset includes 27 of the 29 currently recognized squamate species, including 17 of the 18 endemic
species. Some species considered endemic to the Comoros according to current taxonomy were
found to cluster with non-Comoran lineages, probably due to poorly resolved taxonomy. All other
species for which more than one barcode was obtained corresponded to distinct clusters useful for
species identification by barcoding. In most species, even island populations could be distinguished.
Two cryptic species were identified using the DNA barcoding approach. The obtained barcoding
topology, a Bayesian tree based on COI sequences of 5 genera, was compared with available
multigene topologies, and in 3 cases, major incongruences between the two topologies became
evident. We conclude that in the case of the squamates of the Comoros Islands, DNA barcoding has
proven an efficient way of detecting isolated populations and promising starting points for
subsequent research.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
86
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 3: Informatics
The Barcode of Traditional Chinese Medicine Data System
Linchun Shi, Jingyuan Song, Hui Yao, Shilin Chen
Institute of Medicinal Plant Development, Chinese Academy of Medicinal Sciences, Peking Union
Medical College, Hai Dian District, Beijing 100094, China
E-mail: [email protected]
The Barcode of Traditional Chinese Medicine Data System is an informatics workbench aiding the
collection, analysis and identification of traditional Chinese medicine materials. This data system is
freely available to any researcher with interests in DNA barcoding of traditional Chinese medicine
materials. By now, it includes17,529 ITS2 sequences belonging to 9,884 species, 2,088 psbA-trnH
sequences from 1408 species and 1,543 CO1 sequences of 245 species. All of these sequences have
been carefully verified and covered most of the species listed in the Chinese Pharmacopoeia and
their adulterants. Here, we provide a brief introduction to the key elements of this data system and
discuss their functional capabilities.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
87
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 3: Informatics
Information system for Chinese DNA barcode data
Li Liu, Di Liu, Juncai Ma
Institute of microbiology, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, China
E-mail: [email protected]
DNA barcoding is a novel concept for taxonomic identification using short, specific genetic markers
and has been applied to study a large number of eukaryotes. The huge amount of data output
generated by DNA barcoding requires well-organized information systems. For this purpose, we
developed the global mirror system of DNA barcode data (BOLDMirror, http://www.boldmirror.net)
and information system for Chinese DNA barcode data (http://data.barcodeoflife.cn). The
information system has data submission, data representation and statistics modules, and also
provides some programs to analysis barcode data. The information system can run on the LAMP
(Linux + Apache + MySQL + PHP) environment. Unitil now, there are more 10 thousands samples
and over 23 thousands sequences in the information system.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
88
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 3: Informatics
BOLD Mobile: taking the first steps towards mobile barcoding
Sujeevan Ratnasingham1, Sarah McMullin2, Tim McConnell2, Dean Chan1, Paola Pierossi1,
Mohammad El-Bastami1, Michael Morawski1, David Loop2, Boris Kravchenko2, Aayushya
Agarwal2, William Zhang2, Sharath Sundar2, Emma Cullen2, James Buchanan2
1
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
2
SAP Waterloo Emerging Technologies Lab, SAP, Waterloo, Ontario, Canada
A mobile barcoding device has been a core component of the vision of DNA barcoding from the
outset. The development of such a tool would completely break down barriers to taxonomic
knowledge and empower researchers, educators, and citizen-scientists in utilizing DNA sequencing
technology to explore biodiversity and accurately answer questions about species identity and
distributions.
Multiple biotech firms actively pursue the miniaturization of sequencing
instruments but such efforts do not address the information technology (IT) demands of mobile
barcoding. To address this gap, we have performed database analysis and user-experience research
to identify IT needs and workflow requirements for mobile barcoding. Through this process, we
have developed a proof-of-concept mobile application, called BOLD Mobile, through which users
in the field can collect specimens, enter metadata, photograph the organism and its environment,
and have the data directly uploaded to BOLD using standard mobile devices. Additionally, users
can search the BOLD database for and access information on barcode coverage to inform their
sampling activities in the field. The application is being developed through a collaboration
between SAP and BOLD with the goal increasing contributions to iBOL and increasing utilization
of the barcode reference library.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
89
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 3: Informatics
European BOLD mirror
Vincent Robert1, Jan van Tol2, Gerard Verkleij1, Duong Vu1, Nathalie van de Wiele1, Carlo
Brouwer1, Benjamin Stielow1, Pedro Crous1
CBS-KNAW, Fungal Biodiversity Center, Uppsalalaan 8, 3584 CT Utrecht, Netherlands
Naturalis Biodiversity Center , Darwinweg 2, 2333 CR Leiden, Netherlands
E-mail: [email protected]
At the beginning of the iBOL project, it was agreed that Canada BOLD based data should be
mirrored in at least two additional websites in China and in Europe. While the Chinese was quickly
started, the European mirror has been delayed until now because we wanted to offer different
functionalities to the end-users. Thanks to the NCB project, which is a large and ambitious project
subsidized by the Dutch government, we have been able to implement a configurable system that
aims at providing collection and molecular (DNA barcodes) information to the general public on
collections available from NCB Naturalis (Plants and Animals mainly) and CBS-KNAW (Fungi).
Generated data are fully and freely available for third parties via databases accessible through one
or several websites. The system could be used and extended to third parties databases in the future.
Since we are using the BioloMICS software, one can design, update and modify the structure of the
databases on the fly to adapt to the needs of the curators and the end-users. Polyphasic or
multi-locus identifications are possible and constitute a major advantage over other systems when
identifying closely related organisms requiring more than one locus. The new system will be
unveiled during the Barcoding meeting in Kunming and some of its original functionalities will be
demonstrated.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
90
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 3: Informatics
Multi locus clustering opimization
Douglas Chesters
Institute of Zoology, Chinease Academy of Sciences, Chaoyang District, Beijing 100101, China
E-mail: [email protected]
Grouping DNA sequences at the species level requires specification of parameters for clustering.
The most optimal parameters are usually derived from data in which species memberships are
already known, and then these are applied to new data. We demonstrate the complexities of this
process where multiple loci are used, and introduce a heuristic method which allows the user to
search for a set of parameters which return expected species units as combined over different loci.
Species clustering is performed concurrently with this heuristic parameter optimization, to give
delineations with reduced reliance on specific parameters.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
91
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Informatics
Development of bioinformatic analysis tool set for DNA barcoding analysis
Chang Liu, Linchun Shi, Xincun Wang, Liang Huang, Xiaoqian Liu
Institute of Medicinal Plant Development China, Chinese Academy of Medical Sciences, Beijing
100094, China
E-mail: [email protected]
During the last few years, our groups have published several papers on the selection of DNA
barcoding markers, the development of web servers that support DNA barcoding analyses, the
usage of QR code as the representation of DNA barcodes and etc. In this presentation, we descried
the integration of these validated tools into a tool set that consists two categories of modules. The
first category of tools includes modules “Retrieve”, “AutoAnnotate” and “Identify”. The first
modules allow the retrieval of particular sets of DNA barcode sequences. The “AutoAnnotate”
module accepts an input sequence from the users and annotates the sequence automatically. The
“Identify” module performs species identification using BLAST or BLAST+P distance methods.
The second category is for advanced data analysis, such as “GetStatistics”, “MannualAnnotate”,
“CompareMarkers” and “CheckPerformance”. The “GetStatistics” module calculates various interand intra distances for the input sequences. The “MannualAnnotate” module allows users to provide
their own Hidden Markov Models (HMM) to annotate the sequences. The “CompareMarkers”
module supports the comparison of various markers for a particular taxon group. The
“CheckPerformance” module allows users to check the species identification efficiencies of various
markers. We are eager to collaborate with various researchers in this rapid developing field.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
92
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Informatics
Assessing errors in dna barcode sequence records through the use of frequency
matrices
Taryn Athey1, Robert Hanner2, Paul McNicholas1
1
Department of Mathematics and Statistics, University of Guelph, Guelph, ON, Canada
2
Centre for Biodiversity Genomics & Department of Integrative Biology, University of Guelph, ON,
Canada
E-mail: [email protected]
Errors in DNA sequence databases derive from varied sources (e.g., contamination,
misidentification, non-specific amplification, and sequence assembly errors) and left undetected,
they threaten to compromise subsequent analyses. Because DNA barcode data is used to infer
species identity, error detection is a crucial step in making sure the reference data is “fit-for-use” in
socio-economically relevant (e.g., regulatory) applications. However, it is important to not flag
biological variants as errors, to obtain a true measure of evolutionary diversity. We employed a
frequency matrix approach (coded as an R package) to assess the occurrence of each nucleotide in
each position of the DNA barcode sequence among four test data sets (birds, fishes, true bugs, and
noctuid moths). Nucleotides that occur with a frequency of less than 0.1% are named very low
frequency variants (VLFs). Our results demonstrate that VLFs typically occur at the beginning or
end of a sequence. Those sequences that contain VLFs can be flagged for further investigation as
potential sources of error in sequence databases. Features of the code, including ways of assessing
whether VLFs are due to biological variants or sequencing error, will be discussed.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
93
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Medicinal Plants I
DNA barcoding system for Chinese medicinal materials
Shilin Chen1,2, Jingyuan Song1, Hui Yao1, Xiaohui Pang1, Jianping Han1
1Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union
Medical College, Beijing 100193, China
2Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing
100700, China
E-mail: [email protected]
Traditional Chinese medicine (TCM) has played an important role in the health care system in
China for thousands of years, which have been widely used in clinical applications. The accurate
identification of Chinese medicinal materials is essential for safe application of TCM. DNA
barcoding technology uses a short and standardized gene region to identify species, which can
achieve a rapid and accurate identification for medicinal materials. Our research group has been
involved in DNA barcoding of Chinese medicinal materials for six years and has obtained
substantial achievements. Firstly, we compared different DNA regions using a wide range of taxa
and proposed that the ITS2 and psbA-trnH regions can be used separately as the standard and
complementary barcodes for identification of medicinal plants. The effectiveness of ITS2 and
psbA-trnH for species identification has been validated by our many case studies. Secondly, we
collaborated with other research groups to propose that ITS/ITS2 should be incorporated into the
core barcode for seed plants, which has been widely recognized. Thirdly, we discussed the impact
of “paralogous copies problem” on the identification ability of ITS2. Fourthly, we suggested that the
ITS2 region can be used as a complementary barcode for COI to identify animal species. Fifthly, we
constructed database and web server for barcoding Chinese herbs based on a two-locus combination
of ITS2 + psbA-trnH (http://its2-plantidit.dnsalias.org and http://psba-trnh-plantidit.dnsalias.org).
Sixthly, we applied ITS2 barcode to identify many medicinal materials, including crude drug, drug
powder and herbal pieces. The ITS2 barcode can successfully authenticate medicinal materials from
cortex, whole plant, root, vine and flower. Seventhly, we have published a book called DNA
Barcoding Chinese Medicinal Materials, which included many studies on identification of 208
herbs and their 1000 adulterants. Based on the above research, we have established the systems for
barcoding medicinal plant materials based on a two-locus combination of ITS2+psbA-trnH and for
authenticating medicinal animal materials based on COI+ITS2. Moreover, we wrote the guiding
principle of DNA barcoding technique, which has been added to the supplement of Chinese
Pharmacopoeia. In conclusion, DNA barcoding is an efficient tool for authentication of medicinal
materials, and our studies will contribute for the quality control and safe pharmaceutical use of
TCM.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
94
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Medicinal Plants
On the methods and principles of identification of herbal medicinal materials
using DNA barcodes
Qing-Jun Yuan1, Wen-Jing Zhang1, Dan Jiang1, Shu-Jiau Chiou2, Lu-Qi Huang1
1
State Key Laboratory of Dao-di Herbs, National Resource Center of Chinese Materia Medica,
China Academy of Chinese Medical Sciences, Beijing 100700, China
2
Biomedical Technology and Device Research Laboratories, Industrial Technology Research
Institute, Hsinchu, 30011, Taiwan, China
E-mail: [email protected], [email protected], [email protected]
DNA barcoding has been increasingly used in identification of herbal medicinal materials and
accuracy of it is relevant to the safety of human life and economic interests. However, widespread
use and accuracy of it have been affected because of some issues remained: mainly focusing on
original plant but not materia medica, incompletely sampling due to just restricted medicinal species,
lacking concept of population, and having no full realization for genetic mixture induced by
cultivation. In view of the issues, we proposed a set of methods and principles for identification of
herbal medicinal materials using DNA barcodes: 1) Identification between authenticity and
adulteration by DNA barcodes direct-PCR methods, which was demonstrated by an example
authenticating between Agkistrodon acutus (Qishe) and its adulteration; 2) Identification between
official herb and substitute by DNA barcodes phylogeny-based methods, which was elucidated by a
case discriminating medicinal species and materia medica in Angelica; 3) Identification among
multiple species origin of one official herbs by DNA barcodes population-genetics-based methods,
which was illuminated by a study differentiating among multiple species origin of official Golden
Thread (Huanglian). The methods and principles are based on how closely identified species related,
where main evolution mechanism affecting genetic delimitation of species, such as incomplete
lineage sorting, hybridization, introgression, etc. were considered fully. Meanwhile, the studying
method identifying materia medica using DNA barcodes was also explored. Such information will
not only yield a protocol reliably identifying herbal medicinal materials using DNA barcodes, but
also facilitate practical application of molecular identification of materia medica.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
95
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Medicinal Plants
DNA barcoding medicinal plants from pakistan
Melanie Schori, AlexaRae Kitko, Zabta K. Shinwari, Allan M. Showalter
Ohio University, Athens, OH 45701, United States
E-mail: [email protected]
In Pakistan, herbal products are a primary source of medicines, yet quality control for raw plant
material sold to individuals or companies is very limited. Large-scale cultivation of medicinal plants
is not prevalent, so the bulk of raw material is imported from other countries or collected from the
wild, with no provenance. Roots, bark, twigs, leaves, flowers, and seeds are sold under common
names in local languages, so the potential for misidentifications and mixed collections is high.
Misidentifications or adulteration of authenticated materials can lead to reduced effectiveness of
herbal products or accidental poisonings. Barcoding provides a way to confirm the identification of
raw plant material and establish a level of quality assurance. We have generated barcodes from 43
medicinal plant species, representing 23 different families, using the rbcL, matK, and psbA-trnH
spacer regions. Six market samples have been documented as adulterated or entirely misidentified,
including Matricaria recutita var. chamomilla and Hyssopus officinalis. Comparisons of medicinal
plant barcodes with those of sister species have indicated that the psbA-trnH spacer is most useful
for distinguishing species, followed by matK and rbcL.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
96
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Medicinal Plants
Improved PCR and barcoding data analysis
Melanie Schori, Wei Lin, Allan Showalter
Ohio University, Athens, OH 45701, United States
E-mail: [email protected]
DNA barcoding of medicinal plants is an effective way to identify adulterated or contaminated
market materials, but it can be quite challenging, both in generating barcodes and analyzing the data
to determine discrimination power. Successful PCR of barcoding regions is often inhibited by
desirable secondary metabolites in the medicinal plants. However, modifications of extraction
methods, primer sequences, and the use of an engineered polymerase can usually overcome PCR
problems. Sequence data were obtained for medicinal plants from 24 different families at rates of
96% for rbcL and 79% for matK. Data were then compared to sister species sequences in GenBank
to assess which gene region was most informative for the medicinal species of interest.
Unfortunately, many sequences in GenBank contain errors. An algorithm was developed to “clean”
alignments and identify problematic sequences, facilitating their removal or correction. A second
algorithm can then determine how many positions can be used to perfectly discriminate between
pairs of species, thus highlighting which gene region is most useful for identifying species within a
particular genus.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
97
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Medicinal Plants
Authentication of Chinese medicinal materials using DNA barcodes
Pang-Chui Shaw, Ka-Lok Wong, Li-Li Jiang, Yat-Tung Lo, Sally Yuk-Lau Wong and Paul Pui-Hay
But
State Key Laboratory of Phytochemistry and Plant Resources in West China (CUHK), Institute of
Chinese Medicine and School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.
T., Hong Kong, China
E-mail: [email protected]
Traditional Chinese medicine (TCM) has been used for treatment for centuries in China and Asian
countries. The efficacy of TCM is critically dependent on the use of the correct materials.
Unfortunately, substitutes and adulterants are introduced intentionally or accidentally from time to
time. Chinese medicinal materials are usually dried or processed; therefore, morphological
identification may not be accurate. DNA-based identification system represents a promising
approach to resolve this impediment. In this presentation, examples of our group successfully using
DNA barcodes in TCM identification are introduced. These include distinguishing Hedyotis
diffusa from other 13 closely related Hedyotis species by unique ITS sequences and the
identification of Acontium and Aristolochia species through chloroplast trnH-psbA. Besides, the
study of Cordyceps gunnii, an adulterant of a valuable herb Cordyceps, showed the samples
identified as C. gunnii in China using organleptic criteria is likely a misidentification, which
indicates that DNA barcoding can help to clarify the sample origin. With the gathered DNA
sequences, we have generated the first medicinal materials DNA barcode database. Our group has
also developed protocols for quick authentication by isothermal DNA amplification and for
analyzing DNA in highly processed medicinal materials.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
98
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Medicinal Plants
DNA Barcoding of Neopicrorhiza scrophulariiflora (Pennell.) Hong. using four
standard DNA barcodes
Gaurav Gyanwali 1,2, Sangita Shrestha1, Rajani Malla 2, Tribikram Bhattaraii2, Jyoti Maharjan1
1
Molecular Biotechnology Unit, Nepal Academy of Science and Technology, Patan 44700, Nepa
2
Central Department of Biotechnology, Tribhuvan University, Kirtipur 44618, Nepal
E-mail: [email protected]
More than 1950 Medicinal and Aromatic Plants (MAPs) have been reported to be found in various
landscapes of Nepal ranging from Terai (below 100 asl), Mid Hills to Himalayan regions (>4000
asl). These MAPs, however, suffer rapid decline due to urbanization, overexploitation for trade,
unscientific collection, land degradation and more recently climate change. The proper management,
utilization and conservation of these plants are direly imperative. Neopicrorhiza scrophulariiflora
(Kutki) is a high value vulnerable (VU) medicinal plant of Nepal according to IUCN (1996) and
CAMP (2002). DNA barcoding and molecular phylogeny of this species was assessed using four
standard barcode sequences (ITS, matK, rbcL and trnH-psbA). A total of eight accessions from
three different locations were analyzed. Sequence analysis was done using Codon code Aligner v.
4.2.1 and phylogenetic analysis was performed using MEGA v. 5.2.2. The amplification efficiency
of matK and rbcL was found to be 100% while that of nrITS and trnH-psbA was found to be 87.5%.
Sequence analysis of the accessions showed no variable sites within the samples with nrITS, matK
and rbcL sequences while four variable sites were observed in case of trnH-psbA sequence. A
maximum parsimony tree length generated using trnH-psbA sequence is 143 with consistency Index
(CI) 1.000, retention index (RI) 1.000 and Composite index of 1.000 for all sites.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
99
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Medicinal Plants
Identification of medicinal species and materia medica within Angelica
(Umbelliferae) using DNA barcodes based on thoroughly sampled phylogeny
Qing-Jun Yuan1†, Bin Zhang1,3†, Wen-Jing Zhang1, Tsai-Yun Lin2, Nian-He Wang4, Shu-Jiau
Chiou5*, Lu-Qi Huang1*
1
National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical
Sciences, Beijing 100700, China
2
Department of Life Science and Institute of Bioinformatics and Structural Biology, National Tsing
Hua University, Hsinchu 30013, Taiwan
3
Department of Pharmacy, Affiliated Hospital of National Research Center for Rehabilitation
Technical Aids, Beijing 100176, China
4
Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China
5
Biomedical Technology and Device Research Laboratories, Industrial Technology Research
Institute, Hsinchu, 30011, Taiwan, China
†
Contributed equally
E-mail: [email protected], [email protected], [email protected]
Accuracy of DNA barcoding depends on thoroughly sampled phylogeny. However, incomplete
sampling is noticeable for DNA barcoding in identifying medicinal plants due to just restricted
medicinal species. In this study, we evaluated the utility of four candidate plant DNA barcoding
regions (rbcL, matK, trnH-psbA and ITS) in Angelica based on comprehensive species sampling,
including medicinal and non-medicinal species. Ninety-four accessions of leaves were analysed,
representing 23 species or varieties and covering most of the distribution range of Angelica in Chian.
The discriminating power of the four markers at the species level was 22% (rbcL), 43% (matK),
35% (trnH-psbA) and 78% (ITS). For ITS, the interspecific distance was more than 50 times the
intraspecific distance (0.00008%), which exhibited the biggest barcoding ‘‘gap’’ between intra- and
interspecific variation in the four markers. Thus, ITS is the best choice for DNA identification of
Angelica species. Further conduction of ITS to the decoction pieces of A. sinensis and A. biserrata
revealed that the decoction pieces of them in the market were commonly adulterated with those of
Leristicum officinale. The result testing the impact of processing procedures on DNA barcoding
materia medica indicates that higher than 60°C temperature baking from crude drugs to decoction
pieces has an immediate effect on barcoding decoction pieces of A. dahurica. ITS2 could be
considered as complementary barcode for difficultly amplified materia medica because of easier
PCR-amplification and sequence. The approaches reported here will provide new insights into
identification of medicinal plants and their materia medica using DNA barcodes.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
100
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Marine Barcoding
Three in one: DNA barcoding reveals an overlooked species complex in Chinese
Sternaspis (Polychaeta)
Hong Zhou1, 2, Paul Hebert 2, Er Hua 1, Xiaoshou Liu 1, Xinyu Sun 1, Zhinan Zhang1
1
Ocean University of China, 5 Yushan Road, Qingdao 266003, China
2
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
E-mail: [email protected]
The polychaete genus Sternaspis includes 13 species, most known from a few localities. However, S.
scutata, a species described from the Mediterranean Sea, has long been viewed as possessing a
nearly cosmopolitan distribution. Prior studies have, for example, indicated that it is the sole
Sternaspis in Chinese waters where it comprises an important component of the benthic fauna.
However, barcode analysis of specimens from the Bohai Sea revealed three sympatric clades with
pairwise K2P distances ranging from 18.7- 22.6%. Morphological study indicated that one lineage
was S. costata, a species previously known from the northwest Pacific. The other two lineages
morphologically resemble S. scutata, but they are deeply divergent from each other (22.6%), and
from specimens collected in Alaska (22.6-23.8%) and Chile (20.2-21.3%). Moreover, 16S rDNA
sequence from S. scutata collected near its type locality is deeply divergent from those of both
Chinese lineages. Based on this analysis, it is apparent that specimens assigned to this
‘cosmopolitan’ species actually represent at least five taxa, and that S. scutata is unlikely to occur in
Chinese waters.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
101
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Marine Barcoding
Known and unknown in the Arctic: a marine biodiversity portrait from
Churchill (Manitoba, Canada)
Adriana Radulovici, Paul Hebert
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
E-mail: [email protected]
DNA barcoding has been used in marine biodiversity studies during the last decade as a tool to
accelerate the inventory of life. A growing database of DNA sequences is currently being used to
answer various research questions despite a lack of taxonomic assignment (i.e., Linnaean species
names) for all specimens. Here we present the progress in barcoding the marine fauna from a
Canadian subarctic site (Churchill, Manitoba), data accumulated as part of an ongoing project to
barcode the Churchill biota. At present, the marine database includes ~2,000 DNA barcodes
representing 10 phyla (Arthropoda, Annelida, Brachiopoda, Chordata, Cnidaria, Echinodermata,
Mollusca, Nematoda, Nemertea, Priapulida). The level of taxonomic validation (i.e., specimens
identified to genus/species level by experts or by using GenBank/BOLD ID engine) varied between
33% (Nematoda) and 100% (Chordata, Echinodermata, Mollusca). Consequently, we assessed
diversity as richness of genetic clusters identified through different algorithms: BIN, ABGD,
jMOTU, GMYC. Similarity analyses between marine ecoregions of Canada (adding Alaska) were
conducted by using BINs as proxies for species, revealing affinities between Arctic and Atlantic
fauna. Although the inventory of life follows traditional standards and the Linnaean nomenclature,
BINs can be used for various analyses, while awaiting formal species validation.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
102
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Marine Barcoding
A DNA barcode-based appraisal of Gammarus (Crustacea: Amphipoda) from
the Mediterranean Sea indicates noteworthy hidden diversity
Maria Sara Ferreira1, Murat Sezgin2, Valentina Iannilli3, Gordon S. Karaman4, Filipe O. Costa1
1
Centre of Molecular and Environmental Biology (CBMA), Department of Biology / University of
Minho, Campus de Gualtar 4710-057 Braga, Portugal
2
Sinop University Fisheries Faculty, Marine Biology and Ecology Department, Sinop, Turkey
3
Unità Tecnica AGRI-ECO, ENEA C.R. Casaccia Via Anguillarese, 301 00123 Roma, Italy
4
Montenegrin Academy of Sciences and Arts, Riste Stijovica 5 Str. 2000 Podgorica Crna Gora,
Montenegro
E-mail: [email protected]
We investigated DNA barcode sequence variation among 4 dominant species (G. aequicauda, G.
crinicornis, G. insensibilis, and G. locusta) of the so-called “locusta-complex” of the Mediterranean
Sea. Specimens were collected from multiple locations in the Black Sea, Mediterranean Sea and
European Atlantic coasts. DNA extraction, amplification and bi-directional sequencing of the DNA
barcode region of cytochrome oxidase I gene (COI-5P) followed published protocols. Based on the
genetic divergences and the clustering pattern in a neighbor-joining tree, we identified 4 and 5
clearly separated lineages within each of the G. insensibilis and G. aequicauda phylogroups,
respectively. In both cases completely sorted Mediterranean Sea and Black Sea lineages were
apparent, diverging (Kimura-2-parameter) in average 14% in the case of G. aequicauda, and 15%
among G insensibilis sub-lineages. Each of these two G. insensibilis lineages split further, notably
with one of the splits sorting Atlantic and Mediterranean lineages (average divergence 5%). Three
of the lineages identified in G. aequicauda are singletons and await additional data to allow further
inferences. According to the available information, established species of marine members of
Gammarus display usually less than 2% divergence in COI-5P barcodes. Our findings therefore
indicate the presence of evolutionarily significant hidden diversity among Mediterranean
Gammarus.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
103
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Marine Barcoding
DNA barcodes of the common fouling barnacle model, Amphibalanus amphitrite
reveals three distinct clades distributed in the world
Hsi-Nien Chen1, Ling Ming Tsang2, Benny K.K. Chan1, 3
1Institute of Ecology and Evolutionary Biology, National Taiwan University, Taiwan, China
2Institute of Marine Biology, National Taiwan Ocean University, Taiwan, China
3Biodiversity Research Center, Academia Sinica, Taiwan, China
E-mail: [email protected]
Amphibalanus amphitrite (= Balanus amphitrite) is a well-known model barnacle species in
biofouling studies. A. amphitrite is reported distributing in tropical and warm temperate waters
worldwide, probably because their larvae are being transported through human-mediated dispersal
including ballast water and long line shipping routes. However, the morphology of A. amphitrite is
highly variable and has been identified into five subspecies worldwide. Therefore, it is critical to
study whether A. amphitrite has a genetically global homogenous population or contained a cryptic
species complex due to variable appearance. In the present study, DNA barcode analysis revealed A.
amphitrite has three distinct phylogenetic clades (no morphological differences) in the world. There
are two worldwide clades distributed in both temperate and tropical region while the
Australia-Malay clade is only distributed in Australia to Malaysian region. Such genetic divergence
pattern is believed from mixing of original genetically differentiated native A. amphitrite
populations through human mediated dispersal.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
104
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Marine Barcoding
Barcoding Ocean realms: The North Sea
Michael Raupach1, Jan Beermann2, Thomas Knebelsberger1, Alexander Kieneke1, Silke Laakmann1,
Hermann Neumann2, Inga Mohrbeck1, Thorben Hofmann1, Pedro Martinez Arbizu1
1
Senckenberg am Meer, 26382 Wilhelmshaven, Germany
2
Alfred Wegener Institu, D-27498 Helgoland, Germany
E-mail: [email protected]
During the last years, the effectiveness of DNA barcoding for animal species identification has been
proven in many studies, analyzing both vertebrate and invertebrate taxa. In terms of marine
organisms, most barcoding studies focus on economically relevant species, for example fish, as well
as on the documentation of hotspots of species diversity, e.g. tropical coral reefs or almost
unexplored deep sea regions. In contrast to this, species diversity of “well-known” habitats is nearly
neglected.
As part of our running project we started to build up a comprehensive DNA barcode library of for
the metazoan taxa of the North Sea, an ecologically as well as economically highly important
marine ecosystem of North Western Europe. Beside typical taxa of marine barcoding studies, e.g.
fish or decapod crustaceans, our on-growing library also includes groups that are often ignored, e.g.
echinoderms, cnidarians, gastrotrichs, and pycnogonids. Summarizing our results, our given data
clearly support the use of DNA barcodes to identify species inhabiting the North Sea. Furthermore,
our sequence data give also evidence for the influence of Pleistocene glaciations on the genetic
structure of some selected taxa.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
105
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Marine Barcoding
High genetic variability in Korean Thais populations (Muricidae: Mollusca)
revealed by DNA barcoding analysis: molecular evidence for long-distance
dispersal events
Sang-Hwa Lee, Junhee Lee, Joong-Ki Park
Program in Cell Biology and Genetics and Department of Parasitology, College of Medicine,
Chungbuk National University, Cheongju 361-763, Republic of Korea
E-mail: [email protected]
Thais Röing, 1798 is among the most frequently found gastropod genera worldwide on intertidal
rocky shores including those of Japan, China, Taiwan and Korea. This group contains some
environmentally important model species for assessing the marine pollution by organotin
compounds, but its taxonomy is much complicated because of morphological variations in their
shell characters. To assess relationships and genetic diversity of Korean Thais species, we
performed DNA barcoding analysis based on mtDNA COI sequences. Phylogenetic trees from
different analytic methods showed that T. bronni and T. luteostoma are very closely related than
either of these to T. clavigera. Sequence comparisons revealed that there are high degree of
within-species divergence and genotypic diversity for each of the three Korean species: Some
mtDNA genotypes with different geographic origins are widely distributed across the
geographically distant populations (sharing the same genotypes between upstream Chinese
populations and downstream Korean populations). This finding, together with the high level of
within-species genetic divergence, suggests that there is a continuous influx of Thais recruits by
long-distance dispersal from different geographic origins. For better understanding of the potential
mechanisms operating on the genetic structure of downstream populations, characterization of
genetic structure for potential source populations based on extensive sampling is further required.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
106
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Marine Barcoding
Dna barcode in the study of pontellid copepod diversity in the gulf of California.
Juan Ramón Beltrán Castro, Sergio Hernández-Trujillo
Instituto Politécnico Nacional. CICIMAR, Av.IPN s/n, Col. Playa Sta. Rita, 23096 La Paz, B.C.S.
México E-mail: [email protected]
Although sequences have been obtained for several copepod species worldwide, in Mexico is
starting to associate morphology to genetics using CO1. In order to relate the Linnaean taxonomy
with genetic sequence of pontellid copepods from the Gulf of California, we show preliminary
results. So far, have been identified morphologically 4 genera and 10 species, of which Calanopia
elliptica, Calanopia minor, Pontellopsis armata and Pontellopsis brevis are new records for the study
area. We have obtained genetic sequences of Calanopia elliptica, Pontellopsis armata, Labidocera
acuta, Labidocera acutifrons, Pontellina plumata and Labidocera johnsoni. Preliminary analysis
shows that the two-parameter model of Kimura, or K2P, to species had maximum divergence of
1.39%, a low of 0% and averaged 0.14%, while the genus level was the maximum divergence
24.02%, with a low of 21.28% and 22.44% average. Bootstrap values on nodes that group the
species are high (95.7% to 100%) which indicates consistency. The greatest differences were
observed in Pontellina plumata and lowest in Labidocera johnsoni and Calanopia elliptica, where
the last two species had 0%, confirming the identity of individuals of these species. So far, the
usefulness of this molecular tool can be linked successfully with morphology of pontellid copepods.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
107
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Pests, Parasites, Etc.
Assessing DNA barcode coverage for the first global species list of human
ectoparasites, endoparasites and vectors of disease
Danielle Ondrejicka, Robert Hanner
Biodiversity Institute of Ontario, University of Guelph, Guelph, ON, Canada
E-mail: [email protected]
An urgent gap in taxonomic knowledge pertains to medically important pathogens and zoonotic
disease vectors. Despite an early call to barcode these taxa, they remain under represented and
efforts to characterize them are hampered by the lack of an available checklist. This study compiled
the first comprehensive species list of human endoparasites, ectoparasites and vectors of disease,
totaling 1332 entries from across 14 phyla, 30 classes, 55 orders, 156 families, 380 genera and 1305
named species. To measure progress, this list was compared against the barcode sequence library.
Fewer than 40% of the entries on the checklist were represented on BOLD. Of them, 22.2% had
between one and ten barcodes and 17.4% had greater than 10 barcodes. Notably, more than half of
the available sequences derive from GenBank and lack barcode-relevant meta data. This level of
barcode coverage is significantly less than other taxonomic groups of socio-economic importance
and is unacceptable considering that over one billion people are infected with neglected tropical
diseases. Our checklist and the analysis of gaps in barcode coverage demonstrates the need for a
coordinated campaign to barcode these dangerous species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
108
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Pests, Parasites, Etc.
A Holarctic barcoding survey of parasites (Digenea: Diplostomidae) in the eyes
of freshwater fish
Sean Locken1, David Marcogliese1
1
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
2
Environment Canada, St. Lawrence Centre, Montreal, Quebec, Canada
E-mail: [email protected]
Metacercariae of digenetic trematodes in the cosmopolitan genus Diplostomum are common and
pathogenic in the eyes of freshwater fish. Diversity and taxonomy within Diplostomum are poorly
known, as are host-parasite relationships. In this study, the diversity and distribution of
Diplostomum was characterized with barcodes from 1700 specimens collected across North
America and Eurasia and, in a subset of specimens, ribosomal spacer (ITS) sequences. The spatial
scale of sampling had significant but small effects on CO1 divergence between specimens.
Maximum CO1 divergence within species was associated with geographic extent of species and the
number of specimens sequenced, with effects levelling off after <1000 km and ≈200 specimens.
Neither mean genetic divergence within species nor minimum distance to heterospecific neighbours
was associated with sampling effort or spatial extent of species. The most widely distributed species,
D. spathaceum, occurred in snails and fish 8000 km apart in Europe and China. The clarity of the
barcode gap in CO1 divergence contrasts markedly with a continuum of distances seen in ITS.
Diplostomum species infecting an immunologically inert site, the lens of the eye, were less
numerous and host specific than species inhabiting immunologically active tissues, suggesting
immune pressure may limit host range and cause speciation.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
109
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Pests, Parasites, Etc.
Barcode of helminths in Mexico
Virginia Leon-Regagnon, Maria Guadalupe Velarde-Aguilar
Instituto de Biologia, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico
E-mail: [email protected]
Barcoding of parasites in Mexico has been focused mainly in helminth parasites of wild vertebrates.
The taxonomic groups that have been targeted more intensively are nematodes, trematodes and
acanthocephalans. Barcodes have been used to investigate different aspects of their biology, as the
linkage of different larval stages, or the differentiation of species that are morphologically very
similar, or their phylogenetic relationships. The availability of effective primer sets to amplify the
COI region in these groups has limited the number of studies using barcodes, and the design of new
primer sets that more effectively amplify this region in a wider diversity of helminth taxa will
contribute to the increase of the reference barcode library of helminths worldwide.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
110
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Pests, Parasites, Etc.
DNA Barcoding Applications for Pest Identification and Control
Amanda Naaum1, Andrew Frewin1, Robert Foottit 2, Eric Maw2, Robert Hanner1
1
Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada
2
Invertebrate Biodiversity, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada
E-mail: [email protected]
The accurate identification of pest species is an essential component of integrative pest management.
Due to their size and cryptic life-cycles, many pests can be difficult to identify to the level of
species which may hinder their management. Additionally, the identifications of immature life
stages is often challenging or in the case of eggs, impossible by traditional methods. Fortunately,
DNA barcode sequences are accumulating for many pests of economic significance (see:
www.plantpestbarcoding.org) and they can be used to extend species-level identification
capabilities to eggs, larvae and even fragmentary remains that are not amenable to traditional
identification methods. However, DNA barcoding primarily relies on Sanger-based DNA
sequencing, which currently requires that a specimen be submitted to a properly equipped lab for
sequencing. By exploiting patterns of species-specific differences among available barcode
sequences, portable PCR-based molecular diagnostic assays can be developed for specific target
taxa while the use of non-destructive DNA extraction protocols allow the specimen to be retained
for further study, if necessary. These principles will be exemplified through the discussion of recent
case studies involving varied agricultural pests.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
111
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Pests, Parasites, Etc.
Quarantine screening for bacteria and fungi from wheat siftage using NGS
Chengran Zhou, Shanlin Liu, Xin Zhou
BGI-Shenzhen, Shenzhen, Guangdong 518083, China
E-mail: [email protected]
Bacteria and fungi contain important quarantine organisms that may cause significant damage to
local crops. The classic screening protocol requires isolation and culturing of individual microbes
followed by morphological or molecular identification. This procedure is time consuming and many
of the quarantine microbes, however, cannot be cultured in laboratory. The
next-generation-sequencing technology allows for analyzing microbial community composition. We
extracted DNA from wheat siftage obtained from the Shenzhen custom and amplified the 16S
ribosomal V3 gene for the bacteria and the ITS1 gene for the fungi. PCR products were sequenced
using Illumina Hiseq 2000, producing 1.79Gb and 1.4Gb data for the bacteria and fungi,
respectively. As a result, more than 2000 and 3000 species were identified for the two microbial
groups, respectively, including a series of quarantine sensitive species. Our work reveals
surprisingly high diversity of microbes in the crop products and provides a new pipeline for routine
quarantine examination for the custom.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
112
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Pests, Parasites, Etc.
Identification of forensically important Diptera from Belgium and France using
the Barcode of Life Data Systems (BOLD) and GenBank
Gontran Sonet1, Kurt Jordaens2,3, Yves Braet4, Luc Bourguignon4, Eréna Dupont4, Thierry
Backeljau1,3, Marc De Meyer2, Stijn Desmyter4
1
Royal Belgian Institute of Natural Sciences, JEMU, Vautierstraat 29, 1000 Brussels, Belgium
2
Royal Museum for Central Africa, JEMU, Leuvensesteenweg 13, 3080 Tervuren, Belgium
3
University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
4
National Institute of Criminalistics and Criminology, Vilvoordsesteenweg 100, 1120 Brussels,
Belgium
E-mail: [email protected]
Post-mortem interval can be estimated using fly larvae that feed on corpses. The identification of
these flies is crucial in forensic casework and can be facilitated by DNA barcoding. We sequenced
COI DNA fragments from 85 adult flies representing 16 species and three families of forensic
interest (Calliphoridae, Muscidae and Fanniidae). All haplotypes were used as queries to search for
most similar sequences in GenBank and BOLD and to evaluate the accuracy of the identifications
provided by these libraries and their search options. All databases allowed a correct identification of
the queries but a few forensically important fly species were not represented in the libraries and
some of the identifications were ambiguous. Including the early-released sequences of BOLD in the
searched database allows 1) to increase the number of specimens that can be identified and 2) to
detect and correct barcodes that are erroneously annotated (e.g. from misidentified specimens).
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
113
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Pests, Parasites, Etc.
DNA barcoding of confiscated Pangolin scales in Hong Kong: implications for
wildlife conservation
Huarong Zhang, Peter Feng Yang, Gary Ades, Gunter Fischer
Kadoorie Farm & Botanic Garden, Hong Kong SAR, China
E-mail: [email protected]
All eight species of the Pangolin are threatened by illegal trade of their meat and medicinal use of
their scales. International trade in the four species of Asian Pangolin (Manis spp.) is not permitted
under CITES (the Convention on International Trade in Endangered Species of Wild Fauna and
Flora), while international trade in the four African species is possible with permitting under CITES.
Pangolin scales are frequently seized by customs in Hong Kong and passed to the Agriculture,
Fisheries and Conservation Department (AFCD). Information such as species and origins of the
seized scales is important for law enforcement and biological conservation. Unfortunately,
morphological characters are not sufficient to identify the confiscated scales to species. This study
using scales passed to Kadoorie Farm & Botanic Garden by the AFCD, evaluates the discrimination
capability of the mitochondrial gene, cytochrome c oxidase I (COI) as DNA barcode to identify the
confiscated Pangolin scales to species using identified tissue of Manis pentadactyla and Manis
javanica as a reference. High quality sequences were recovered from most of the Pangolin scale
samples, showing significant genetic variation revealed in a Bayesian Phylogenetic analysis. Manis
pentadactyla and Manis javanica were clearly separated in two clades, whereby most of the
confiscated samples were identified as Manis javanica. This study highlights the utility of DNA
barcoding for species identification to better monitor the wildlife trade and to enhance law
enforcement. This work will also contribute toward the barcode sequence database for Pangolin
species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
114
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Insects II
Selection and assessment of DNA barcodes for aphids (Hemiptera: Aphididae)
Rui Chen1,2, Liyun Jiang1, Lin Liu1,2, Qinghua Liu1,2, Juan Wen1,2, Ruiling Zhang1,2, Xingyi Li1,2,
Yuan Wang1,2, Fumin Lei1, Gexia Qiao1
1
Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy
of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China;
([email protected])
2
University of Chinese Academy of Sciences, No. 19, Yuquan Road, Shijingshan District, Beijing
100049, China.
E-mail: [email protected]
To evaluate the utility of COI and other barcode genes in aphids, an experimental research based on
taxonomy was conducted. Over 80 species of Lachninae (Hemiptera: Aphididae) were chosen and
the results obtained using COI, COII, and Cytb were compared. COI and COII demonstrated a
greater PCR amplification efficiency than Cytb. Species identification using COII showed a higher
frequency of success and yielded lower intra- and higher inter-specific genetic divergence.
Therefore, a COII-based identification system seems to be more effective in identifying lachnine
species. The primary endosymbiont of aphids Buchnera has a higher evolutionary rate and
inter-specific divergence than its co-diverging hosts, making it a potential tool for resolving the
ambiguities in aphid taxonomy. We compared the effectiveness of two different DNA regions,
Buchnera gnd and mitochondrial COI, for discrimination of over 100 aphid species. The mean
inter-specific divergence of gnd was significantly higher than the mean intra-specific variation, with
nearly no overlapping distributions between the intra- and inter-specific samples. The results show
that gnd can effectively identify the species in Aphididae. Gnd can be used as a potentially effective
barcode for aphid species identification. Besides, we also recommend a two-locus combination of
gnd+COI as the aphid barcode.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
115
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Insects II
Species boundaries in the economically important Mexican grasshopper genus
Sphenarium (Orthoptera: Pyrgomorphidae)
Alejandro Zaldívar-Riverón, Carlos Pedraza-Lara, Eduardo Nuple-Juárez
Instituto de Biologia, National Autonomous University of Mexico, 04510 Mexico, Mexico
E-mail: [email protected]
The grasshopper genus Sphenarium (Pyrgomorphidae) is a small group of orthopterans currently
composed of six species and eight recognised forms, with all except one being endemic to Mexico.
Members of Sphenarium are of considerable economic interest because they represent pests in
agriculture, and because they are employed for human consumption since prehispanic times.
However, despite its considerable economic relevance, the taxonomy of this genus has never been
revised. Here we show the preliminary results of a study that investigates the species boundaries in
Sphenarium based on morphological and mitochondrial (barcoding locus) and nuclear (28S) DNA
sequence data. Examination of about 200 specimens from all recognised forms has revealed that
their morphology and genetic divergence for the barcoding locus are not congruent with their
current taxonomy, but instead are more concordant with specific geographic areas. In particular, our
data indicate that some isolated populations originally assigned to the widely distributed S. p.
purpurascens actually are both genetically and morphologically well differentiated. Also, we have
discovered a species diversification spot for the genus in the state of Guerrero. This ongoing work
remarks the utility of applying integrative taxonomic approaches for the rapid study of poorly
known invertebrate taxa.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
116
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Insects II
DNA barcoding of four Tortricidae Species (Lepidoptera: Tortricoidea:
Tortricidae) from China
Xiaoye Li1, Lu Qian 2, Yulin An2, Maohua Chen1
1
College of Plant Protection, Key Laboratory of Crop Pest Integrated Pest Management on the
Loess Plateau of Ministry of Agriculture, Key Laboratory of Plant Protection Resources and Pest
Management of Ministry of Education, Northwest A&F University, Yangling, Shaanxi 712100,
China
2
Animal Plant and Food Inspection Center, Jiangsu Entry-Exit Inspection and Quarantine Bureau,
Nanjing, Jiangsu 210001, China
E-mail: [email protected], [email protected]
Four Tortricidae species: Cydia pomonella, Grapholita (Cydia) molesta, Grapholita (Cydia)
funebrana, Grapholita (Cydia) dimorpha, are all the serious pest insects in fruit orchards in China.
As identification of small tortricid larvae by morphological characters and damage symptoms
is diffcult, even for experienced taxonomists, an easy and accurate identification method that allows
differentiation of the four species, particularly at early larval instars is needed. DNA Barcoding
analysis based on can successfully identify the four tortricid internal fruit feeders of major
economic importance in China and beyond. In the current study, we amplified and sequenced the
mitochondrial cytochrome oxidase subunit I (COI) and the second internal transcribed spacer (ITS2)
of the nuclear ribosomal RNA (rDNA) as barcodes of the specimens which were collected from 17
different localities in China. Preliminary results from DNA barcoding of these four species showed
high interspecific sequence variation, and COI played a better role than ITS2 in identifying
congeneric species. Our research suggests that DNA barcoding is a useful method not only for
taxonomical purposes, but also for ecological purposes. The consequences of DNA barcoding for
ecological studies of moth communities is discussed in the article as well.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
117
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Insects II
Molecular evolutionary rates correspond with major transitions in insect
evolution
T. Fatima Mitterboeck, Sarah J. Adamowicz
Department of Integrative Biology & Biodiversity Institute of Ontario, University of Guelph,
Guelph, ON, Canada
E-mail: [email protected]
Growing public databases of DNA sequence data present new opportunities for broad-scale
investigation of patterns of molecular evolution and macroevolution across life. We present data on
molecular evolutionary rates and biological shifts in insects, such as flight loss and
terrestrial-aquatic habitat transitions. Our analysis of numerous independent instances of flight loss
in various insect orders shows that flightless lineages have faster molecular evolutionary rates than
related flight-capable lineages. Among available nuclear and mitochondrial protein-coding genes,
this pattern was apparent in the mitochondrial genes, with a significant pattern for the cytochrome c
oxidase subunit I (COI) gene. This pattern may be explained by relaxed selective constraints on
metabolism in flightless lineages, possibly in combination with an influence of reduced effective
population size. We present emerging results for terrestrial-aquatic habitat transitions analysed from
various insect orders, including Lepidoptera, Coleoptera, Hemiptera, Trichoptera, Mecoptera, and
Diptera.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
118
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Insects II
DNA barcoding reveals that the reverse latitudinal gradient of Gracillariidae
leaf-miners is an artifact of tropical under-sampling
Carlos Lopez-Vaamonde1, David C. Lees2, Akito Kawahara3, Rodolphe Rougerie4, Issei Ohshima5,
Atsushi Kawakita6, Jurate De Prins7
1
INRA-Orléans, UR633, Unité de Zoologie, Orléans, France,
2
Department of Zoology, Cambridge University, CB2 3EJ, UK
3
Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611 USA
4
INRA, UR633 Zoologie Forestière, F-45075 Orléans, France
5
Department of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan
6
Center for Ecological Research, Kyoto University, Kyoto, Japan
7
Entomology Section, Royal Museum for Central Africa, Leuvensesteenweg 13, B-3080 Tervuren,
Belgium
E-mail: [email protected]
Higher taxa often show increasing species richness towards tropical low latitudes, a pattern known
as the latitudinal biodiversity gradient (LBG). A rare reverse LBG (with greater richness towards
temperate high latitudes) is exhibited by Gracillariidae moths, in which most described species,
occur in northern temperate areas. We carried out the first assessment of gracillariid species
diversity in two Neotropical regions to test whether the relatively low tropical species diversity of
this family is genuine or caused by insufficient sampling and a strong taxonomic impediment. Field
surveys in six French Guianan and one Ecuadorian site produced 516 gracillariid specimens that
were DNA barcoded to facilitate identification and to match larvae inside leaf-mines with adults.
Species
delineation
from
sequence
data
was
approximated
using
Automatic-Barcode-Gap-Discovery and Refined-Single-Linkage-Analysis through the Barcode
Index Number system, and the proportion of described/undescribed species was estimated after
comparison with types. Strikingly, at least 85% of the species collected as adults were found to be
undescribed. The results from both our molecular and morphological analyses indicate that the
current reverse LBG seen in this group is an artifact of insufficient sampling and a strong taxonomic
deficit in the Neotropics.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
119
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 3: Insects II
The Geometrid Moths of Europe: What BINs can tell us about mega-diverse
faunas
Axel Hausmann1, Marko Mutanen2, Paul Hebert3
1
Bavarian State Collection of Zoology Munich, Munich, Germany
2
University of Oulu, Oulu, Finland
3
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
E-mail: [email protected]
The iBOL project has now gathered more than 70,000 DNA barcode records for geometrid moths
from sites around the world. More than 10,000 of these barcodes derive from European geometrids,
with an additional 3000 barcodes from conspecific individuals in adjacent countries. Altogether
these records provide coverage for 892 of the species recognized by traditional taxonomy (91% of
the European fauna of 985 geometrid species). With additional sequencing of old specimens in
collections, coverage should rise to 972 (98.7%) species by late 2013.
In this study we compare the species recognized by traditional taxonomy with the DNA barcode
clusters delineated by the “Barcode Index Number (BIN)” system. The overall count is quite
similar - BOLD assigns the 13,000 sequence records from 892 European species of geometrids to
1100 BINs. There is usually a very close match (often 100%) with traditional species when results
are examined at a country level. However, the BIN count is about 23% higher than the species count
when viewed at a continental scale because regional lineages are placed in different BINs. We
conclude that DNA barcoding is a powerful method for biodiversity assessment, and that the BIN
system corresponds closely with the species (and often ‘subspecies’) boundaries recognized through
traditional taxonomy. The results of this study encourage the use of DNA barcoding and BIN
assignments for biodiversity investigations, particularly in geographic regions and animal groups
that have received little taxonomic attention.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
120
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session: Insects II
Barcoding the moths of Argentina: unveiling this complex, highly diverse and
mostly ignored group of Lepidoptera.
Pablo D. Lavinia1, Ezequiel O. Nuñez Bustos 1, Cecilia Kopuchian 1,2, Darío A. Lijtmaer 1, Pablo L.
Tubaro1
1
Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” (MACN–CONICET), Argentina.
2
Centro de Ecología Aplicada del Litoral (CECOAL–CONICET), Argentina
E-mail: [email protected]
Lepidopterans constitute the second most diverse group of insects and represent almost half of the
sequences of the entire barcode library. However, until 2010 lepidopterans had not been barcoded in
Argentina. In addition, even though moths represent over 80% of the described species of this order,
there is only a rough estimate of 37,000 species in the Neotropics and no official count in Argentina.
In this context, we started to barcode the Lepidoptera of Argentina in 2010 and to assess the
diversity of moths in 2012, including the generally ignored Microlepidopterans. The analysis of
almost 2,500 COI sequences from around 800 species of moths showed that the interspecific
divergence was 14 to 24-fold higher than the intraspecific divergence, which is consistent with the
barcode gap reported for other regions. In particular, we found various cases of deep intraspecific
divergence (>2%) and species pairs with low sequence distance (<2%), although these cases did not
compromise COI-based identifications. We discuss if there is an association between geographic
structure and these divergences. Finally, our dataset includes 868 barcode clusters, 209 of which are
new to BOLD. We thus believe that this study will reveal part of the hidden diversity within this
group.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
121
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 4: Other Invertebrates
Dragonfly barcoding and the evolution and conservation of freshwater
biodiversity
Klaas-Douwe B. Dijkstra, Frank R. Stokvis
Naturalis Biodiversity Centre, PO Box 9517, 2300 RA Leiden, Netherlands
E-mail: [email protected]
Earth’s most concentrated and threatened biodiversity occurs in freshwater: covering only <1% of
Earth’s surface, it harbors 10% of known animal species, of which >60% are insects. Odonata
(dragonflies and damselflies) is the fourth-largest radiation of aquatic insects and, due to their
popularity and well-resolved taxonomy, the only order for which a global status assessment is
feasible. Africa’s unparalleled diversity of aquatic habitats is among the most untouched in the
world, but the growth rates of its economy and population are also unrivalled, and the environment
will change faster here than anywhere else. Detailed knowledge of the status, distribution and
ecology of African Odonata is being developed for freshwater management and samples of 81% of
the 730 species are available. As part of the All Odonata Barcode Initiative, COI has been
sequenced for nearly 6000 of the over 10,000 specimens available, of which 38% are African, plus
extensive material from tropical Asia and New Guinea. The data have great application in taxonomy
and conservation, but also in evolutionary research. The unprecedented dataset allows the first
quantification of diversification in such a dynamic environment and can ultimately predict impacts
of changing climate or land-use, map ecological sensitivity, and other conservation applications.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
122
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 4: Other Invertebrates
Biodiversity of springtails (Collembola) is underestimated even in simple
Antarctic ecosystems
Ian Hogg, Kristi Bennett, Gemma Collins
Department of Biological Sciences, University of Waikato, Hamilton, New Zealand
E-mail: [email protected]
We used mitochondrial DNA (COI) sequences to examine the levels of genetic variability within
and among populations of five endemic springtail (Arthropoda: Collembola) species along a
latitudinal gradient in the Ross Sea region of Antarctica. This was the first re-evaluation of several
areas, including the central and southern Transantarctic Mountains (>84oS), in almost 50 years. We
tested the hypothesis that genetic divergences would occur among populations within the
fragmented Antarctic landscape. COI sequences showed high levels of divergence at both small
(<15km) and large (>300km) spatial scales for three of the five species. For example,
Gomphiocephalus hodgsoni, a widespread and common species showed 7.6% sequence divergence
on opposite sides of the Mackay Glacier (77oS) and >8% with sites near the Beardmore Glacier
(84oS). High levels of sequence divergences were also found for the more range-restricted species
Neocryptopygus nivicolus (77oS) and Antarctophorus subpolaris (85oS). We conclude that
glaciation in Antarctica has promoted and maintained the levels of diversity observed among
populations of springtails and that isolation has occurred even on relatively small spatial scales.
Levels of divergence are likely to reflect the presence of previously unknown or cryptic species and
conservation efforts should be directed towards protecting and preserving the biotic integrity of
fragmented landscapes in Antarctica.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
123
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session: Other Invertebrates
Antarctic nematodes, rotifers and tardigrades: assessing morphological and
molecular diversity at a continental scale
Alejandro Velasco-Castrillón1, Mark I. Stevens1,2
1
Australian Centre for Evolutionary Biology and Biodiversity, School of Earth and Environmental
Science, The University of Adelaide, North Terrace, Adelaide, South Australia, SA 5005, Australia
2
South Australian Museum, GPO Box 234, Adelaide SA 5000, Australia
E-mail: [email protected]
Antarctic terrestrial life has been described as some of the simplest on Earth, and is restricted to soil
meiofaunal communities, composed mainly of rotifers, tardigrades and nematodes. Numerous
studies have hypothesised that the lack of diversity is due to the extreme environmental conditions
and thought to be driven mainly by abiotic factors. Morphologically, it has been difficult to make
accurate large scale assessments of the patterns and processes of Antarctic Meiofaunal biodiversity.
Recent studies have combined habitat information with molecular data to examine relationships
among populations, and to delineate species boundaries and dispersal patterns further. However,
species diversity of these ecologically-important animals is still limited because original taxonomic
work has not been revised in current years despite descriptions of new species and re-description of
known species. The limiting factor for meiofauna is that species diagnosis is difficult in many cases
due to the conservative morphology of these animals. Here we present preliminary findings of
morphological and molecular studies of meiofaunal groups that have been barcoded for the COI
mitochondrial gene across Antarctica. Our data suggests that a molecular strategy is preponderant to
discern among cryptic species and to delineate species boundaries (widespread vs endemic) for
meiofaunal groups. Our data shows hidden diversity for Antarctic meiofauna represented by over
100 mitochondrial lineages and 240 unique haplotypes, revealing potential new undescribed species
based on the COI gene. Our results indicate extreme ranges in diversity, distribution range and
habitat requirements for the targeted groups; with bdelloid rotifers as the most diverse and
widespread group across a wider range of habitats, followed by tardigrades and nematodes.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
124
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 4: Other Invertebrates
What the barcodes can tell us about freshwater zooplankton
Elías-Gutiérrez Manuel1, Miracle Rosa2, Vicente Eduardo2, Silva Briano Marcelo3
1
El Colegio de la Frontera Sur, Chetumal Unit, Chetumal, Mexico
2
Universitat de Valencia, 46010 Valencia, Spain
3
Universidad Autónoma de Aguascalientes, Aguascalientes, Mexico
E-mail: [email protected]
The DNA barcoding of main groups of freshwater zooplankton (herein Cladocera, Copepoda,
Ostracoda and Rotifera) has not been an easy task. New primers have been developed, and many
species still are a challenge. In spite of this problem, so far 772 Barcode Index Numbers (BIN),
belonging to species from these groups can be found in the BOLD database. Most of them come
from five countries (Mexico, Canada, Spain, United States and Bolivia). An analysis of these
species indicates that only few are widely distributed. All other have restricted distributions even in
North America, the most studied area.The only confirmed widespread species in North America is
Moina macrocopa. Many taxa seem to be restricted to particular environments as semidesert,
highlands, cold temperate, or tropical regions. Intercontinental distributions could be due to recent
introductions as the case of Daphnia laevis, Acanthocyclops americanus and Brachionus sp.
Almenara. Among confirmed introduced species in America are Daphnia lumholtzi and Daphnia
magna (used in cultures in Mexico and Canada). Part of this complicated puzzle has been slowly
disentangled confirming the presence of cryptic species as the complexes represented by
Mastigodiaptomus albuquerquensis, Lebereis davidi, Scapholeberis armata and Leptodiaptomus
novamexicanus.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
125
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 4: Other Invertebrates
The first eukaryotic organism described with a fully sequenced transcriptome,
DNA barcode and micro computed tomography
Lyubomir Penev1,2, Pavel Stoev2,3, Ana Komerički4, Nesrine Akkari5, Shanlin Li6, Xin Zhou6, Scott
Edmunds6,7, Chris Hunter6,7, Alexander M. Weigand4,8, David Porco9, Marzio Zapparoli10, Teodor
Georgiev2, Daniel Mietchen2, 11, Dave Roberts12, Vincent Smith12
1
Institute for Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia,
Bulgaria
2
Pensoft Publishers, Sofia, Bulgaria
3
National Museum of Natural History Museum, Sofia, Bulgaria
4
Croatian Biospeleological Society, Zagreb, Croatia
5
Natural History Museum of Denmark, Copenhagen, Denmark
6
BGI-Shenzhen, Shenzhen, China
7
GigaScience, BGI HK Ltd., Tai Po, Hong Kong, China
8
Goethe-University, Institute for Ecology, Evolution and Diversity, Frankfurt am Main, Germany
9
Université de Rouen - Laboratoire ECODIV, Mont Saint Aignan Cedex, France
10
Universita degli Studi della Tuscia, Dipartimento di Protezione delle Piante, Viterbo, Italy
11
Museum für Naturkunde – Leibniz-Institut für Evolutions- und Biodiversitätsforschung,
Invalidenstraße 43, 10115 Berlin, Germany
12
The Natural History Museum, Cromwell Road, London, United Kingdom
E-mail: [email protected]
We demonstrate how new species descriptions can be enhanced by applying molecular methods,
novel computing and imaging technologies. A cave-dwelling centipede of the taxon Eupolybothrus
(Chilopoda: Lithobiidae), found in a remote karst region in Knin, Croatia, is the first eukaryotic
species that in addition to its traditional morphological characteristics, is described with its fully
sequenced transcriptome, DNA barcode (i.e., mitochondrial Cytochrome C Oxidase Subunit I gene;
COI), and detailed anatomical X-ray by microtomography (micro-CT) scan. This pilot project
illustrates a workflow of producing, storing, publishing and disseminating large data sets associated
with the description of a new taxon. It was developed in collaboration between several research
institutions and driven by BGI-Shenzhen, GigaScience and Pensoft Publishers. The study will be
published in the novel Biodiversity Data Journal, launched within the EU-funded project ViBRANT
(www.vbrant.eu) as the first journal to provide an integrated workflow for article authoring,
peer-review and the open access publication of data and text within a single online collaborative
platform. The large-scale data handling, management and storage was provided by the GigaScience
GigaDB database (http://dx.doi.org/10.5524/100063), with transcriptomic and annotation data made
publically available to the most stringent metadata standards in GigaDB and the relevant datatype
specific repositories.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
126
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 4: Fishes II
Calibrating snakehead diversity with DNA barcodes: expanding taxonomic
coverage to enable identification of potential and established invasive species
Natasha Serrao1,2,, Dirk Steinke 1, Robert Hanner1,2
1
Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph ON, N1G
2W1, Canada
2
Department of Integrative Biology, University of Guelph, 50 Stone Road East, Guelph ON, N1G
2W1, CanadaE-mail: [email protected]
Detecting and documenting the occurrence of invasive species outside their native range requires
tools to support their identification. DNA barcoding provides a potent method for identifying
invasive species, as it allows for species identification at all life stages, including fragmentary
remains. Snakehead fishes are a diverse group of opportunistic predators endemic to Asia and
Africa that pose significant threats as aquatic invasive species: at least three snakehead species
(Channa argus, C. maculata, and C. marulius) are thought to have entered North America through
the aquarium and live-food fish markets, and have established populations that pose threats to
indigenous freshwater biodiversity. The objectives of this study were to assemble a library of DNA
barcode sequences derived from expert identified reference specimens in order to determine the
identity of the invasive species found in North America using DNA barcodes. Barcodes were
obtained from 121 tissue samples representing 25 species and combined with public records from
GenBank for a total of 36 putative species. Multiple divergent clusters were observed within C.
gachua, C. marulius, C. punctata and C. striata suggesting the potential presence of cryptic species
diversity within these lineages. Our findings demonstrate that DNA barcoding is a valuable tool for
snakehead identification and provides a useful framework for inferring invasion pathways analysis.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
127
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 4: Fishes II
DNA barcoding of elasmobranchs and teleosts from India
Wazir S. Lakra, A. Pavan-Kumar, Gireesh-Babu P., Divya Viswambharan, Dhaval Bamaniya,
A.K. Jaiswar, Aparna Chaudhari, Gopal Krishna, S.K. Chakraborty
Central Institute of Fisheries Education, Deemed University (ICAR), Versova, Mumbai-400061,
India
E-mail: [email protected]
India is bestowed with rich biodiversity and recognized as one of the mega biodiversity hot spot.
However, limited number of taxonomists in the country hampers the progress in research on
biodiversity characterization and documentation. Further, the traditional morphological tools for
species identification are constrained by phenotypic plasticity, life stage specific identification cues
and the occurrence of cryptic species. DNA barcoding has emerged as a global standard method for
the identification of biological species in recent years. The Central Institute of Fisheries Education
(CIFE), Mumbai being the apex organization in the field of fisheries, is actively involved in
documenting aquatic biodiversity through DNA barcodes. The authors have generated over 250
DNA barcodes for 75 species including elasmobranchs representing 4 orders and 9 families and
teleosts representing 6 orders and 17 families. The mean intraspecific and inter-specific distances
values for teleosts are 0.446 and 12.24%, respectively. In the case of elasmobranchs, the average
interspecific and intraspecific distance values are found to be 8.6% and 0.3% for sharks and 12.4%
and 0.63% for rays using K2P method. The barcodes generated in the present study clearly
discriminated all specimens up to species level with high congeneric distance values than the
conspecific distance values. Phylogentic relationships among selected groups were studied by multi
gene approach. The Neighbour – joining tree revealed distinct clusters shared by the species of
same genera with significant bootstrap values (95-100%). Our study strongly validates the efficacy
of DNA barcoding in characterizing aquatic biodiversity for sustainable management and
utilization.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
128
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 4: Fishes II
A Review of the freshwater fish diversity in the mediterranean biodiversity
hotspot using dna barcoding
Matthias Geiger, Fabian Herder, Jörg Freyhof
Sektion Ichthyologie, Zoologisches Forschungsmuseum Museum Alexander Koenig, Leibniz
Institute for Animal Biodiversity (ZFMK), Bonn, Germany
E-mail: [email protected]
Biodiversity Hotspots are areas holding exceptionally rich biotas, serving the need for prioritizing
conservation efforts. The Mediterranean Biodiversity Hotspot is one of the most important
temperate biodiversity areas, with a remarkable diversity of freshwater fishes. Actually, 397 out of
526 species are recognized as endemic. In order to assess their diversity, and to construct a
molecular identification system supporting conservation, we constructed a DNA barcode library for
498 Mediterranean freshwater fish species (98% extant species coverage) based on 3171 individuals.
The congruence of clusters with morphological species ranged from 45 – 85% of species and was
highly dependent on the method used to delineate clusters. Genetic discontinuities suggest the
existence of up to 64 candidate species, potentially increasing fish diversity by <12%. Shared
haplotypes among species highlight the limits of a barcoding approach, although mismatches tended
to occur in recent (Pleistocene- or Holocene-evolved) lineages. We found reduced reliability of
DNA barcoding to identify species unambiguously in a highly biogeographically structured area,
but much greater accuracy when applied at the catchment scale. This scale effect has important
implications for barcoding assessments, and our results suggest that fairly simple identification
pipelines can be applied in local freshwater fish monitoring. Inventorying and management of
large-scale diversity requires higher analytical and conceptual efforts. The barcoding of 98% of
freshwater fish species of the Mediterranean Biodiversity Hotspot represents a success story for
biodiversity research in the geopolitically complex region. The database will enable the recognition
of conservation units within the Hotspot and will strongly support the ongoing exploration of the
global freshwater fish diversity.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
129
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session: Fishes II
Building a reference library for Malaysian marine fishes
Siti Azizah Mohd Nor1,2, Noor Adelyna Mohammed Akib1,2, Masazurah A. Rahim1,2, Rajiv Ravi 1
1
School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia
2
Centre of Marine and Coastal Studies, USM, Penang, Malaysia
E-mail: [email protected]
More than 2000 fish specimens were obtained from local markets or fish landing sites throughout
the four major surrounding seas of Malaysia namely Strait of Malacca, South China Sea, Sulu Sea
and Suluwesi Sea. In addition bottom trawling has also been initiated to collect fishes from their
natural habitats. A neighbour-joining analysis revealed that the data from this approach paralleled
the morphological identification in more than 95% of the species surveyed. Most species exhibited
low intraspecific genetic distances (0.31%), about 43-fold lower than the distance among species
within a genus. However, higher intraspecific divergences ranging from 2.2% to 7.6% were
observed in detailed study of several families suggesting overlooked diversity. On the other hand,
several species-pair exhibited barcode divergences of <1% warranting further investigation of their
taxonomic status and underlying causal factors. This library will provide a basis for further studies
of this group from all life stages, seafood forensics and estimations about species composition and
richness in this biodiversity hotspot region ecosystem.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
130
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 4: Fishes II
DNA barcoding of fishes collected from Red Sea coastal waters of Tabuk, Saudi
Arabia
Subrata Trivedi1, Raafat Affan1, Abdulrahman Hirab A Alessa1, Bishal Dhar2, Pradosh Mahadani2 ,
Sankar K Ghosh2
1
Department of Biology, Faculty of Science, University of Tabuk, Ministry of Higher Education,
Kingdom of Saudi Arabia.
2
Department of Biotechnology, Assam University, Silchar-788011, Assam, India
E-mail: [email protected], [email protected]
Fishes are important animal protein sources for human consumption. The proper identification of
fish species is not only of interest for taxonomy and systematics, but also of prime importance in
management of fisheries and authentication of food products. DNA barcoding is an efficient way of
species identification. DNA barcoding promises fast, accurate species identifications by focusing
analysis on a short standardized segment of the genome. In this study, the standard ~ 650 bp region
near the 5′ end of the mitochondrial cytochrome oxidase subunit1 gene (COI) was sequenced in
marine fish specimens collected from Red Sea coastal waters of Tabuk region in Saudi Arabia. After
sequencing the ORF was checked, followed by submission of the sequences to global GenBank
(GenBank accession numbers KF434771 - KF434776). Bioinformatics analysis was carried out to
elucidate the phylogenetic relationships among the different fish species. Since these fish species
are also available in the market, this data can be useful in authentication of the fish and fish
products.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
131
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 4: Plants III
The multigene tiered approach for barcoding plants: a cost-effectiveness analysis
for choosing secondary tier regions.
Subramanyam Ragupathy, Steven Newmaster
Centre for Biodiversity Genomics, Biodiversity Institute of Ontario (BIO), University of Guelph,
Guelph, Ontario, Canada
E-mail: [email protected]
The multigene tiered approach is based on the use of a common, easily amplified, and aligned
region (rbcL) that can act as a scaffold on which to place secondary tier data from a highly variable
noncoding region. Here we present a cost-effectiveness analysis for choosing secondary tier regions
based on barcoding data from 4,500 species of plants spanning artic, temperate and tropical floras
collected within barcoding facilities in Canada, China and India. The aim of our project is to
provide 1) a common framework for making decisions on choosing regions, and 2) a basis for
comparing secondary tier regions based on expected cost of barcoding each region within the
context of the expected effective gain in species resolution. We expect that this research will evolve
with further analysis from participating labs who share the vision of building a multigene tiered
model for barcoding plants in which existing data is utilized in providing the most efficient regions
for successive tiers. This would provide routine barcoding labs with the ability to choose the most
cost effective regions for specific plant taxa already in the model while allowing researchers to
evaluate other regions for new taxa that can be added to the model.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
132
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 4: Plants III
Chloroplast capture and genetic diversification in Osmorhiza (Apiaceae)
Ting-shuang Yi1, Gui-hua Jin1, Jun Wen2
1
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, China
2
National Museum of Natural History, MRC 166, Smithsonian Institution, Washington, DC 20013,
United States
E-mail: [email protected]
Osmorhiza Raf. (Apiaceae) contains eleven species disjunctly distributed in temperate Asia, western
North America, eastern North America, and South to Central America. Maximum parsimony and
Bayesian inference were employed to analyze sequences of three nuclear loci and nine plastid loci
from all eleven recognized Osmorhiza species. The nuclear ITS and ETS and the plastid data well
resolved the infrageneric relationships, yet the two phylogenies were largely incongruent.
Comparisons of nuclear and plastid phylogenies revealed eight cases of introgression of chloroplast
genome: from O. berteroi to O. depauperata (Wen 7350 and Wen 7400), O. glabrata (Wen 7464 and
Teillier et al. 2419), and O. brachypoda (Wen 7114 and Wen 7122); from O. depauperata to O.
bipatriata (Lowry & Warnock 3182); from a possible extinct lineage to O. berteroi (Wen 7397).
Chloroplast capture commonly occurs at different taxa and taxonomic level, spurious phylogenetic
relationships could be obtained if only plastid data was applied. Agreeing with the conclusions of a
few previous studies, we strongly suggested the combined application of both cpDNA and nuclear
markers in barcoding to reduce the spurious results.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
133
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 4: Plants III
Genetic diversity and DNA barcode identification of thirteen endemic Nepenthes
species of the Philippines based on rbcL, matK and 18s rDNA sequences
Rachel Acil1, Ma. Genaleen Diaz2, Victor Amoroso3, Rita Laude2, Merlyn Mendioro2, Antonio
Laurena2
1
PBGB Division, International Rice Research Institute, Los Baños, Laguna, Philippines/Graduate
School, University of the Philippines Los Baños, Laguna, Philippines
2
Genetics and Molecular Biology Division, Institute of Biological Sciences, University of the
Philippines Los Baños, Laguna, Philippines
3Central Mindanao University, Bukidnon, Philippines
E-mail: [email protected]
Using three different modified CTAB extraction methods, the DNA from silica gel-dried young
leaves of thirteen endemic Nepenthes species was successfully isolated. Molecular analysis was
done using rbcL, matK, and a nuclear gene, 18s rDNA, which were amplified and sequenced. Using
BLASTN, the samples were all correctly identified up to genus level based on available rbcL, matK
and 18s rDNA gene sequences. The intra- and intergroup genetic distance between sequences in
each gene were computed using Kimura-2-Parameter method. The 18s rDNA gene sequences had
the highest intergroup genetic divergence of 0.03844, followed by matK (0.01729) while rbcL had
the lowest (0.00181). For intragroup genetic distance, N. alata has the highest for rbcL gene, N.
merrilliana for matK, and N. bellii for 18s rDNA gene. Mean nucleotide diversity was also
computed using Jukes-Cantor model, 18s rDNA has the highest mean diversity within, in entire, and
inter- population. The phylogenetic tree for each gene sequences was constructed using maximum
parsimony with 700 and 1000 bootstrap replications for rbcL and matK respectively. For 18s rDNA,
maximum likelihood method with 1000 bootstrap replicates was used. The tree based on the matK,
and concatenated rbcL and matK sequences formed distinctive clades of the three individuals of
each Nepenthes species. Based on haplotype analysis, rbcL and matK gene can discriminate one
and five species respectively.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
134
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 4: Plants III
DNA barcoding of Swertia chirayita (Roxb. Ex Fleming) H. Karst populations of
Nepal using four standard markers
Surendra Neupane1,2, J. Sijapati 1 , T. Bhattarai 1, J. K. Chhipi Shrestha1, Sangita Shrestha1
1
Molecular Biotechnology Research Unit, Nepal Academy of Science and Technology, Lalitpur,
Nepal
2
Central Department of Biotechnology, Tribhuvan University, Kathmandu, Nepal
E-mail: [email protected]
Swertia chirayita (Roxb. Ex Fleming) H. Karst is considered vulnerable medicinal plant indigenous
to the temperate Himalayan landscapes. Around 30 species of Swertia are reported to be found in
Nepal with nine species in national and international trade. Authentication of S. chirayita species is
crucial from conservation, utilization and human health safety aspects. In this study we sampled 18
accessions (three accessions for each of six populations) to investigate the utility of the four
potential barcoding loci i.e., rbcL, matK, trnH-psbA, and internal transcribed spacer (ITS). All four
loci were easy to amplify and sequence. In the four tested DNA regions, rbcL had the most variable
sites (19) followed by trnH-psbA (12), matK (5) and ITS had the least (3). Since the intraspecific
genetic variability of ITS is low, it is preferred for barcoding in Swertia chirayita. Because of high
interspecific variability and high “Best Match” value (95.83%), rbcL marker was proposed for
Swertia species discrimination. Along with this, phylogenetics among the accessions of different
populations was also studied. Neighbour-joining and Maximum Parsimony trees were generated for
different loci using K2-P parameter distance. The consistency index (CI) values of trees based on
ITS, rbcL, trnH-psbA, matK were found to be 0.991, 0.965, 0.964, 0.900 respectively.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
135
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 4: Plants III
DNA barcodes of Korean Apiaceae (Umbelliferae)
Kyeonghee Kim1, Sang-Hun Oh2, Chong-Wook Park1
1
School of Biological Sciences, Seoul National University, Seoul 151-747, Korea
2
Department of Biology, Daejeon University, Daejeon 300-716, Korea
E-mail: [email protected], [email protected]
Apiaceae (Umbelliferae) is a large, cosmopolitan family of flowering plants with about 3600
species that includes many economically important species for food, spice and medicine. In Korea,
67 species in 33 genera are distributed. While the Apiaceae are easily recognized by sheathing
petioles, umbellate inflorescence, and fruits of schizocarps, identification of a species is often
problematic when the materials lack certain set of characters, such as fruits, crucial for species
identification. Five chloroplast DNA regions (matK, rbcL, atpF-atpH, psbK-psbI and psbA-trnH)
were investigated for DNA barcodes for accurate and rapid identification of the Korean Apiaceae.
Eighty-four accessions of 67 species were examined and performance of species discrimination was
analyzed based on neighbor-joining trees. Resolving power of species using a single region was
relatively low, ranging from 0.47 (rbcL) to 0.81 (psbA-trnH), and increased when multiple regions
were used. The combination of three regions (matK+rbcL+atpF-atpH) and four regions
(matK+rbcL+atpF-atpH+psbK-psbI), both resulted in resolving power of 0.93. When all five
regions were included, resolving power was 0.96, indicating that all the species examined were
discriminated except for two species. These include Pimpinella brachycarpa and P. hallaisanensis,
and further study is necessary on the taxonomic identity of the two species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
136
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 4: Environmental Barcoding
PCR free approach for environmental DNA barcoding using a capture system
coupled with next-generation sequencing technology
Shadi Shokralla, Mehrdad Hajibabaei
Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph,
Guelph, ON, Canada N1G 2W1
E-mail: [email protected]
Environmental DNA analysis through the use of specific gene markers such as DNA barcodes has
been a key application of next-generation sequencing technologies in ecological and environmental
research. However, the real-world application of these technologies, especially in routine
monitoring programs, demands optimized protocols and workflows capable of producing unbiased
and reproducible results. An important consideration involves the bias associated with the initial
PCR amplification. Primer sequence, number of amplification cycles and the used annealing
temperature can potentially bias the sequencing results qualitatively and quantitatively. Recently, a
number of target enrichment technologies have been introduced to facilitate capturing target
genomic regions prior to NGS analysis mostly for medical genomics applications. Here we present
a custom target enrichment system designed for capturing CO1-barcode diversity in terrestrial
arthropods (e.g. Malaise traps) and benthic macro-invertebrates (e.g. kicknets). We tested this
system for both types of samples in parallel to amplicon-based NGS analysis on MiSeq and
Roche-454 platforms.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
137
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 4: Environmental Barcoding
Rapid construction of a comprehensive mitogenome library – Mitochondrion
10K project
Min Tang1, Meihua Tan1, Yiyuan Li2, Shenzhou Yang1, songwenhui1, Xu Su1, Shanlin Liu1, Xin
Zhou1
1
China National Genebank, BGI-Shenzhen, Shenzhen, China, 518083
2
Department of Biological Sciences, University of Notre Dame, IN 46556
E-mail: [email protected]
Classic DNA barcoding and metabarcoding approaches have been relying on one or two standard
genes to differentiate species. A recent trend in metabarcoding is to directly sequence the mixed
genomic-DNA without having to rely on PCR amplification, which typically introduces artifacts
due to biased primer efficiencies. One obvious challenge to this new approach is that the majority of
NGS sequences cannot be utilized for species identification due to the lack of reference for
non-COI genes. Here we develop a new NGS approach to rapidly build a reference library for
whole mitogenomes in a cost-efficient fashion. In this pilot experiment, we sequence a mixture of
50 animal species without individual tagging or PCR. With only 1 failure, most protein-coding MT
genes were successfully recovered for each of the mixed species. Our new pipeline delivers
high-quality assemblies even from highly degraded DNA. And closely related species (e.g.,
co-genors) can be successfully assembled separately. Based on the success of this pilot test, we
propose a new initiative – MT10K – with an aim to sequence whole mitogenomes for 10,000
animal species. Such dataset will significantly improve our knowledge on mitogenomes across
animals and expand the application of metabarcoding to utilize full set of MT DNA.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
138
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 4: Environmental Barcoding
Characterizing the composition
metabarcoding approach
of
the
rumen
microbiome:
a
DNA
Aurelie Bonin1, Frédéric Boyer1, Eric Coissac1, Ilma Tapio2, Seppo Ahvenjärvi2, Tomasz Stefański2,
Johanna Vilkki2, Kevin Shingfield3, Pierre Taberlet1
1
Laboratoire d'Ecologie Alpine, France
2
MTT Agrifood Research, Finland
E-mail: [email protected]
The rumen microbiome plays a central role in the ability of ruminants to digest plant material,
which has major implications for nutrient supply to the animal and for the production of
fermentation end-products such as methane. Therefore, we need a more comprehensive
understanding of the factors influencing rumen taxonomic composition, including host genome and
diet. Here, we present a DNA metabarcoding approach to characterize the different taxa of bacteria,
archaea, fungi and protozoa present in the rumen. DNA metabarcoding consists in (1) amplifying
short DNA fragments (“barcodes”) from a complex mixture of organisms using primers specifically
designed for a particular taxonomic group of interest; (2) sequencing these barcodes using
next-generation sequencing technologies; and (3) assigning the obtained sequences to the
corresponding taxa based on a reference sequence database.
In this study, we designed the most efficient primer pairs to amplify each rumen target taxon. For
this purpose, we drew upon publicly available sequence data, while taking into consideration
current next-generation sequencing constraints. Then we illustrated the usefulness of DNA
metabarcoding in an experiment investigating the influence of the host species on the rumen
microbiome ecosystem, where rumen contents have been transferred from cows to reindeers and
sampled periodically afterwards.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
139
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 4: Environmental Barcoding
Using soil fauna to measure aboveground biodiversity
Chenxue Yang
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese
Academy of Sciences China, Kunming, Yunnan 650223, China
E-mail: [email protected]
Metabarcoding of bulk arthropod samples shows great promise as a rapid, taxonomically
comprehensive, and reliable method for measuring biodiversity. However, sampling is still a
bottleneck. For instance, Malaise trap samples typically require a week, exposing traps to theft and
disturbance. We ask if soil fauna, which can be collected in a few minutes per sample, can
differentiate habitats in the same ways as aboveground arthropods. We processed two datasets. In
Mengsong, Yunnan, China, we compare Malaise-trap, leaf-litter, and soil samples from 28 sites
covering a gradient of closed-canopy forest, open-canopy forest, and tea plantation. The
Malaise-trap samples were metabarcoded for COI, and the soil and leaf-litter samples were
metabarcoded for 18S. The soil samples were first filtered in water to concentrate soil fauna. We
find that the three sample types produce NMDS ordinations that are significantly pairwise
correlated (Procrustes tests). Also, all three sample types weakly and non-significantly differentiate
the three habitat types, which is consistent with considerable cross-habitat dispersal. In Vu Quang
and Bach Ma, Vietnam, we compare canopy-fogging and leaf-litter samples from Acacia plantations
and natural rainforest. The canopy-fogging samples were metabarcoded for COI, and the leaf-litter
samples were metabarcoded for 18S. The leaf-litter samples were first processed through Winkler
funnels to concentrate the fauna. We find that the two sample types produce NMDS ordinations that
are strongly correlated. Also, both datasets strongly differentiate Acacia and natural forest
communities in Bach Ma but not in Vu Quang. Thus, even though leaf-litter and aboveground
samples are highly taxonomically divergent, they contain similar ecological information, and this
could accelerate sampling for large-scale biodiversity surveys.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
140
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 4: Environmental Barcoding
Genetic identification of zooplankton for aquatic environmental monitoring
Steve Woods1, Jonathan Banks2, Gemma Collins1, Nathan Watson1, Clare Beet2, Ian Duggan1, Ian
Hogg1
1
University of Waikato, New Zealand
2
Cawthron Institute, New Zealand
E-mail: [email protected]
Zooplankton communities are commonly used to assess water quality. However, morphological
assessment of zooplankton for environmental assessments requires a high level of taxonomic
expertise and is time consuming. Here, our aim was to use DNA barcoding to expedite the routine
monitoring of zooplankton communities. We tested and compared COI and 28S in order to find a
suitably short and easily amplified fragment that would reliably identify species using current next
generation sequencing platforms. Individual zooplankton species have been sequenced and added to
the Barcode of Life Datasystems (BOLD) database for reference. To date, reference libraries have
been developed for New Zealand freshwater rotifers, copepods and cladocerans. Both gene regions
produced unique sequences for each species. However, more samples were successfully amplified
and sequenced using the 28S gene compared with the COI gene. We now aim to characterise
aquatic communities using DNA from environmental samples with next-generation sequencing
approaches such as the Illumina platform. This approach can then be used to rapidly assess
zooplankton diversity in aquatic environments as well as to monitor water quality.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
141
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 4: Insects III
Australian Sphingidae – DNA barcodes challenge current species boundaries
and distributions
Rodolphe Rougerie1, Ian J. Kitching2, Jean Haxaire3, Scott E. Miller4, Axel Hausmann 5, Paul D. N.
Hebert6
1
INRA, Department Forest, Grassland and Freshwater Ecology, UR633, Forest Zoology Unit,
Orléans, France
2
Department of Life Sciences, Natural History Museum, London, UK
3
Honorary Attaché, Muséum National d’Histoire Naturelle de Paris, Le Roc, Laplume, France
4
National Museum of Natural History, Smithsonian Institution, Washington, USA
5
Bavarian State Collection of Zoology, Munich, Section Lepidoptera, Munich, Germany
6
University of Guelph, Biodiversity Institute of Ontario, Guelph, Ontario, Canada
This work examines the extent of taxonomic and biogeographical uncertainty in a well-studied group
of Australian Lepidoptera, the hawkmoths (Sphingidae). We analyzed the diversity of these moths
through the comparative analysis of their DNA barcodes, supplemented by morphological
re-examinations and sequence information from a nuclear marker in selected cases. The results from
the analysis of Australian sphingids were then placed in a broader context by including conspecifics
and closely related taxa from outside Australia to test taxonomic boundaries.
Our results led to the discovery of six new species, four of which have now been described, and
revealed one additional invalid case of synonymy. Overall, our study establishes the occurrence of 75
species of Australian hawkmoths although two require description. The analysis of records from
outside Australia challenge the validity of current taxonomic boundaries in several species, including
Agrius convolvuli, a common species which has gained adoption as a model system. Our work has
revealed a higher level of endemism than previously recognized. Most (91%) Australian sphingids
are endemic to the continent (47%) or to Australia, the Pacific Islands and the Indo-Papuan region
east of Wallace’s Line (44%). Only seven species (9%) have ranges that extend beyond this major
biogeographical barrier toward SE Asia and other regions of the Old World.
As apparent from our results, taxonomic errors produced significant misconceptions concerning the
distribution patterns in a group of insects that was considered well known taxonomically. Because
DNA barcoding represents a straightforward way to identify taxonomic discrepancies, it is a powerful
approach to alleviate the Linnean shortfall, improving the accuracy of primary diversity data in
biogeography and conservation studies.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
142
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 4: Insects III
DNA barcodes and bins reveal nearly 4500 undescribed species of Australian
Lepidoptera
Paul Hebert1, Jeremy deWaard1, Mari Kekkonen2, Rodolphe Rougerie3, John La Salle4, Sujeevan
Ratnasingham1
1
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
2
Finnish Museum of Natural History, 00100 Helsinki, Finland
3
University of Rouen, 76821 Mont-Saint-Aignan, France
4
Atlas of Living Australia, CSIRO Ecosystem Sciences, Canberra, ACT 2601, Australia
Although natural history museums are massive repositories of identified specimens, they typically
play only an indirect role in supporting the assignment of taxonomic names to new collections.
DNA barcoding provides the opportunity to mobilize taxonomic knowledge from museums in a
format that enables specimen identification. The present study demonstrates the efficacy of this
approach by analyzing 45,000 specimens of Lepidoptera from the Australian National Insect
Collection. This work has created a DNA barcode reference library for nearly 6500 known species,
and 4500 taxa that are undescribed. The latter taxa gained delineation through the Barcode Index
Number (BIN) system, a registry for sequence clusters that correspond closely to species. The
efficacy of BINs in discriminating species of Australian Lepidoptera was validated by the analysis
of 12 assemblages with strong taxonomy. BIN counts were subsequently used to estimate the
number of undescribed species within 74 families, highlighting those in particular need of
taxonomic attention. Analysis of collection dates for the undescribed taxa indicate that they have
resided in the ANIC for an average of 31 years, making clear the disconnection between species
discovery and registration.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
143
Parallel Session Abstracts – Wednesday, 30 October
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Parallel Session 4: Insects III
Butterflies (DNA barcodes) as biodiversity indicators
John-James Wilson, Sing Kong-Wah, Khairunnisa Syaripuddin, Karen-Chia Huey-Min
University of Malaya, Kuala Lumpur, Malaysia
E-mail: [email protected]
The use of butterfly DNA barcodes as indicators of biodiversity will be evaluated against 4 key
criteria:
1. Easy to survey? A well-known ecology should allow for the design of effective sampling
protocols that can be standardized and deployed in a cost- and time-efficient manner.
2. Taxonomically tractable? The species must be easy to identify even by non-specialists,
facilitating comparisons between surveys conducted at different times, different locations and by
different researchers.
3. Found everywhere, but species are specialized? Butterflies must be present at all sites with stable
population sizes, but exhibit different species composition at different sites.
4. Patterns of diversity reflected in other groups? Butterflies should act as a biodiversity “umbrella”;
meaning conservation of butterfly species would benefit numerous co-occurring species from other
groups.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
144
Parallel Session Abstracts – Wednesday, 30 October
—————————————————————————————————————————————
Parallel Session 4: Insects III
DNA barcode enabled ecological research in Papua New Guinea
Scott E. Miller and many co-authors
Smithsonian Institution, National Museum of Natural History, Washington, DC 20560, Unted States
E-mail: [email protected]
We present examples of the multiple lines of research supported by DNA barcoding of the
Lepidoptera (butterflies and moths) of Papua New Guinea. Barcoding has become an integral part
of a large-scale study of the ecology and biogeography of lowland rainforests in Papua New Guinea.
The foundation of which has been characterizing the insects, particularly Lepidoptera, and woody
plants. As well as being a great tool for pin-pointing diversity in taxonomically difficult groups, the
barcode data has been instrumental in: identifying caterpillars, detecting host-parasitoid interactions,
and identifying host plants from caterpillar stomach contents. We have demonstrated that barcodes
add great value to a taxonomic dataset making it useful to a broad range of research enterprises.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
145
Parallel Session Abstracts – Thursday, 31 October
—————————————————————————————————————————————
Parallel Session 5: Plant Methods
Application of next-generation genome sequencing to DNA barcoding of
mid-west Australian Acacia species
Anna Williams1,2, Paul Nevill2,1, Laura M. Boykin3, Ian Small3, Joe Miller4, Siegfried Krauss2,1
1
School of Plant Biology, The University of Western Australia, Crawley, WA 6009, Australia
2
Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, West Perth, WA 6005,
Australia
3
Australian Research Council Centre of Excellence in Plant Energy Biology, The University of
Western Australia, Crawley, WA 6009, Australia
4
Centre for Australian National Biodiversity Research, CSIRO Plant Industry, The Australian
National Herbarium, GPO Box 1600, Canberra, ACT 2601, Australia
E-mail: [email protected]
Australia is home to over 1,000 species of Acacia and over 100 of these occur in the mid-west
region of Western Australia. The mid-west is currently the focus of intensive and increasing mining
activity and yet many Acacia species are highly endemic to the region making their conservation a
priority. The morphological similarity seen among many of these species makes identification a
challenge and the genus would therefore benefit greatly from DNA barcoding. The poor resolution
of the standard barcodes in some genera and the decreasing costs and increasing power of next
generation sequencing technologies has prompted suggestions that traditional barcoding needs to
embrace this new technology. In this study we conducted low-depth, high throughput sequencing of
total DNA preparations using an Illumina HiSeq. A single lane of Illumina HiSeq sequencing
enabled us to obtain almost complete chloroplast genome and nuclear ITS sequences from 96
mid-west Acacia specimens (both fresh and dried) suitable for barcoding and phylogenetic analysis.
Preliminary results suggest that low-depth sequencing of total DNA has great cost-effective
potential for assisting the rapid and accurate assessment of biodiversity.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
146
Parallel Session Abstracts – Thursday, 31 October
—————————————————————————————————————————————
Parallel Session 5: Plant Methods
ycf1, the most promising chloroplast DNA barcode for land plants
Wenpan Dong, Shiliang Zhou
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese
Academy of Sciences, Beijing 100093, China
E-mail: [email protected]
Highly variable DNA barcodes are the prerequisite for its successful application in species
discriminations. Unfortunately, although several regions of chloroplast genomes have been
proposed as candidate barcodes for plants, none of them can pass most tests at species level. They
all suffer low resolutions at species level. Are there no highly variable regions in the chloroplast
genomes? It has been shown that there are highly variable regions in chloroplast genomes [Dong W.
et al. 2012. PLoS ONE 7(4):e35071. doi:10.1371/journal.pone.0035071]. Two regions located in
ycf1 (called ycf1a and ycf1b) were found to be too variable to design universal primers at that time
when very few ycf1 sequences were available. The questions now are (1) if universal primers could
be designed, and (2) how well the ycf1a or ycf1b would perform. To answer the two questions, we
generated 749 sequences representing four groups of plants, i.e. mosses, ferns, gymnosperms and
angiosperms. These sequences were combined and aligned together with those downloaded from
GenBank. We found that the franking sequences of ycf1a in ferns and ycf1b in mosses,
gymnosperms and angiosperms are quite conservative for designing universal primers for the
groups. We thus designed universal primers for each groups of plants. The primers were verified
working very well. PCR successes using were 91.43%, 72.73% and 98.17% for mosses,
gymnosperm and angiosperm, respectively, and 74.36% for ferns. We conducted two tests to
evaluate the performances of ycf1 in species discriminations using matK and rbcLb (a newly
proposed region of rbcL) as controls. First, we sampled nearly all woody plants cultivated in our
botanical garden and sequenced ycf1b region. The species discriminatory powers are 72.79% for
ycf1b, 57.56% for matK, and 58.02% for rbcLb. The performance of ycf1b is no less a marker than
rbcLb+matK (71.73%). Second, we tested their performances in Pinus, Calycanthaceae, Iris,
Paeonia sect. Mutan, Prunus sect. Armeniaca, Quercus and Panax which are species rich or well
sampled. Correspondingly to the order of taxa listed above, the species discriminatory powers are
30.53%, 74.07%, 62.22%, 38.46%, 52.38%, 5.26%, and 69.57% for matK; 28.78%, 44.44%,
57.78%, 8.00%, 0.00%, 21.05%, and 82.61% for rbcLb; and 64.49%, 66.67%, 71.11%, 88.46%,
100.00%, 36.84%, and 86.96% for ycf1b. Ycf1b was only slightly outperformed by matK in
Calycanthaceae. We concluded that ycf1 is the most promising chloroplast DNA barcode for plants.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
147
Parallel Session Abstracts – Thursday, 31 October
—————————————————————————————————————————————
Parallel Session 5: Plant Methods
Plant DNA Barcoding: From Gene to Genome
Xiwen Li1, Yang Yang1, Robert J Henry 2, Maurizio Rossetto3, Shilin Chen4, Yitao Wang1
1
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical
Sciences, University of Macau, Macau, 999078, China
2
Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Brisbane
QLD 4072, Australia
3
National Herbarium of NSW, The Royal Botanic Gardens and Domain Trust, Mrs Macquaries
Road, Sydney NSW 2000, Australia
4
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing
100700, China
E-mail: [email protected], [email protected], [email protected]
DNA barcoding has been widely recognized and applied in animals but the lack of a universal and
effective barcode has created barriers to broad adoption in plant identification. Because of the
inherent limitations of the lack of adequate variations of traditional DNA barcodes, recent barcoding
studies have placed high hope on the use of whole chloroplast genome sequences enabled by
improving DNA sequencing technologies. While this can already deliver a reliable barcode for
accurate plant identification it is not yet resource effective and does not yet offer the advantages of
speed of analysis offered by traditional barcodes to simple laboratory facilities. In this paper, we
review the development of traditional candidate barcodes and discuss the feasibility of using the
chloroplast genome as a super-barcode. We here advocate a new point of view of DNA barcoding
that combines for selected groups of plants the best use of traditional barcodes and super-barcodes
for efficient plant identification. Specific barcodes might enhance our ability to distinguish closely
related plants at the species and population levels.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
148
Parallel Session Abstracts – Thursday, 31 October
—————————————————————————————————————————————
Parallel Session: Plant Methods
Discriminating plants using the DNA barcode rbcLb: an appraisal based on a
large dataset
Wenpan Dong, Tao Cheng, Changhao Li, Shiliang Zhou
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese
Academy of Sciences, Beijing 100093, China
E-mail: [email protected]
The ideal DNA barcode for plants remains to be discovered. The candidate barcode rbcL has met
considerable skepticism and suspicion since it was proposed. In fact, the variability within this gene
has never been fully explored across all plant groups from algae to flowering plants, and its
performance as a barcode has not been adequately tested. By analyzing all rbcL sequences currently
available from GenBank, we attempted to determine how well a region of rbcL performs as a
barcode in species discrimination. We found that the region rbcLb was more variable than the
frequently used region rbcLa. Both universal and plant group-specific primers were designed to
amplify rbcLb. The performance of both rbcLa and rbcLb was tested in several ways. Using BLAST,
both regions successfully identified all families and nearly all genera. Successful
species-identification rates varied significantly among plant groups, ranging from 24.58% to
85.50% for rbcLa and from 36.67% to 90.89% for rbcLb. In species-rich families, successful
species discrimination ranged from 5.19% to 96.33% for rbcLa and from 22.09% to 98.43% for
rbcLb. In species-rich genera, successful species discrimination ranged from 0 to 88.73% for rbcLa
and from 2.04% to 100% for rbcLb. Both regions performed better for lower plants than for higher
plants, but rbcLb performed significantly better than rbcLa overall, especially for angiosperms.
Considering its applicability across plants, easy and unambiguous alignment, high primer
universality, high sequence quality and high species-discrimination power for lower plants, we
suggest rbcLb as a plant barcode.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
149
Parallel Session Abstracts – Thursday, 31 October
—————————————————————————————————————————————
Parallel Session 5: Plant Methods
New approach to dna barcoding fresh plant and herbarium specimens with
semiconductor sequencing: A phyloplastome model with hops (Humulus,
Cannabaceae)
Jeffrey R. Boutain1, David K. Visi2, Michael S. Allen3, Jianchu Xu4, Ernest Small5, Kim E.
Hummer6, Will C. McClatchey1,7,8
1
Botany Department, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA
2
Department of Forensic and Investigative Genetics, University of North Texas Health Science
Center, Fort Worth, Texas 76107, USA
3
Department of Forensic and Investigative Genetics, University of North Texas Health Science
Center, 3500 Camp Bowie, Fort Worth, Texas 76107, USA
4
Kunming Institute of Botany, Chinese Academy of Sciences, and World Agroforestry Centre,
China and East Asia, Kunming, Yunnan 650201, China;
5
Agriculture and Agri-Food Canada, Science and Technology Branch, Ottawa, Ontario K1A 0C6,
Canada
6
USDA ARS National Clonal Germplasm Repository Corvallis, Corvallis, Oregon 97333, USA
7
Botanical Research Institute of Texas, Fort Worth, Texas 76107, USA
8
Environmental Studies Program, Texas Christian University, Fort Worth, Texas 76107, USA
E-mail: [email protected]
Semiconductor sequencers generate whole genomes quickly and inexpensively; therefore, an
optimal barcode for plant species is the plastome. A plastome phylogeny of three hops (Humulus L.),
including taxonomic varieties in the Humulus lupulus L. species complex, is presented. DNA was
extracted from both fresh and herbarium material. Sequencing was completed on the Ion Torrent
Personal Genome Machine (PGM), bioinformatics were performed with CLC bio Genomics
Workbench (GW), and phylogenetic inference was conducted with BEAST2. An 8-plexed
sequencing run using a 318 chip for 200 base pair reads resulted in 4,528,200 sequence reads with a
mean read length of 154 bp, representing 697 Mb of data. Reads from each sample were assembled
into draft plastomes with the reference Cannabis sativa L. Consensus sequences for each sample
were aligned with MAFFT7 and then loaded to BEAST2 with the Reversible-jump Based
substitution estimated for the Yule Model tree at 100 million generations. High posterior
probabilities from BEAST2 support a polyphyletic tree with basal East Asian taxa and monophyly
of the genus. Compared to single or several chloroplast regions used to barcode plants, a
phyloplastome model supports the cohesiveness of the H. lupulus species complex.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
150
Parallel Session Abstracts – Thursday, 31 October
—————————————————————————————————————————————
Parallel Session 5: Plant Methods
Potential use of low-copy nuclear genes in DNA barcoding: a comparison with
plastid genes in two Hawaiian plant radiations
Yohan Pillon1, Jennifer Johansen1, Tomoko Sakishima1, Srikar Chamala2,3, W. Brad Barbazuk2,3,
Eric H. Roalson4, Elizabeth A. Stacy1
1
Department of Biology, University of Hawaii at Hilo, Hilo, HI, 96720, USA
2
Department of Biology, University of Florida, Gainesville, FL 32611, USA
3
Genetics Institute, University of Florida, Gainesville, FL 32610, USA
4
School of Biological Sciences, Washington State University, Pullman, WA 99164-4236
E-mail: [email protected]
DNA barcoding of land plants has relied traditionally on a small number of markers from the plastid
genome. In contrast, low-copy nuclear genes have received little attention as DNA barcodes
because of the absence of universal primers for PCR amplification. From pooled-species 454
transcriptome data we identified two variable intron-less nuclear loci for each of two species-rich
genera of the Hawaiian flora: Clermontia (Campanulaceae) and Cyrtandra (Gesneriaceae) and
compared their utility as DNA barcodes with that of plastid genes. We found that nuclear genes
showed an overall greater variability, but also displayed a high level of heterozygosity, intraspecific
variation, and retention of ancient alleles. Thus, nuclear genes displayed fewer species-diagnostic
haplotypes compared to plastid genes and no interspecies gaps. The apparently greater coalescence
times of nuclear genes are likely to limit their utility as barcodes, as only a small proportion of their
alleles were fixed and unique to individual species. In both groups, species-diagnostic markers from
either genome were scarce on the youngest island; a minimum age of ca. two million years may be
needed for a species flock to be barcoded. For young plant groups, nuclear genes may not be a
superior alternative to slowly evolving plastid genes.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
151
Parallel Session Abstracts – Thursday, 31 October
—————————————————————————————————————————————
Parallel Session 5: Plant Methods
Sample sequence analysis for the discovery of genetic relatedness, genome
composition and genome evolution in flowering plants
Jeff Bennetzen1,2, Srini Chaluvadi2, Jeremy DeBarry2, Matt Estep2, Justin Vaughn2, Tushar Tushar3,
Latha Rangan3
1
Kunming Institute of Botany, Kunming, Yunnan 650201, China
2
Department of Genetics, University of Georgia, Athens, GA 30602, United States
3
Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India
E-mail: [email protected]
We have found that a very small sample of genomic DNA sequence, such as a few million reads
from an Illumina run, can uncover a great deal of information about basic genome structure in
flowering plants. If the samples are chosen with a systematic intent, then these results can also
indicate how genomes are related and both the tempo and nature of genomic change. Specialized
software and annotation protocols have been developed for this purpose and applied to members of
the Poaceae, Orobanchaceae and Zingiberaceae lineages.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
152
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: Fungi and Algae
DNA Barcoding of Marine Red Macroalgae in Korea: Advances and prospects
Myung Sook Kim, Mi Yeon Yang, Hyung Woo Lee and Young Ho Koh
Department of Biology, Jeju National University, Jeju 690-756, Korea
E-mail: [email protected]
Rhodophyta is a monophyletic group currently divided into six classes with approximately 6000
species. Taxonomic identification of red algae is, however, notoriously difficult due to their
relatively simple morphologies, phenotypic plasticity, and complex life-histories. A more recent
trend is the use of DNA barcoding as an identification tool and the COI gene is commonly used for
red algal DNA barcoding. In the Korean algal inventory, marine red algal flora consists of 600 taxa
at species and infraspecific level. We are screening the Rhodophyta diversity, in particular the order
Halymeniales, Rhodymeniales, Plocamiales, and Gracilariales, to test the effectiveness of DNA
barcoding. To accomplish this main goal, COI barcode region is being sequenced with other
molecular marker, UPA as supplements. So far over 3000 specimens have been collected, and 500
sequences were generated with different molecular markers. Our study demonstrate that DNA
barcoding can provide an efficient method for species-level identifications and contributes
powerfully to taxonomic and biodiversity research.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
153
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: Fungi and Algae
Using DNA barcodes to detect indoor fungal composition
Helena Korpelainen, Tea Huotari and Maria Pietiläinen
Department of Agricultural Sciences, University of Helsinki, Finland
E-mail: [email protected]
Indoor human environments contain a variety of microbes, some of which are detrimental to human
health. Problems develop in damp buildings, where various materials become wet for extended
periods of time. Such dampness provides the moisture that supports the growth of bacteria and fungi
(i.e., mould). Mould in buildings is positively associated with several allergic and respiratory effects,
and certain moulds are toxigenic, meaning that they can produce mycotoxins. Recent media
attention has increased public awareness and concern over exposure to moulds at homes, and in
workplaces, schools etc. Indoor fungi are traditionally determined by culture-dependent methods,
which inevitably underestimate diversity and bias results towards fungi that grow well on generic
fungal growth media. To increase precision in analyses on indoor fungi and to provide useful data
for end-users, we developed a project, where we collected fungal samples from buildings with a
known or suspected mould problem (day care centers) and from control buildings, and conducted
DNA barcoding (ITS) using NGS methods. Problem buildings will be examined both before and
after renovation, and also seasonal variation in fungal diversity will be considered. In this
presentation, we will introduce our methodological approach and preliminary results of fungal
diversity indoors.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
154
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: Fungi and Algae
Does fungal community control wood decomposition?
Chunyan Yang1, Weijie Liu2, Xiaoyang Wang1, Chenxue Yang1, Douglas Allen Schaefer2, Douglas
Wailie Yu1
1
Kunming Institute of Zoology,Chinese Academy of Sciences, Kunming, Yunnan 650223,China
2
Xishuangbanna Tropical Botanical Garden,Chinese Academy of Sciences, Menglun, Mengla,
Yunnan 666303, China
E-mail: [email protected]
Decomposition of wood debris (WD) results in a large return of CO2 to the atmosphere,
contributing an amount of CO2 similar to that of global fossil-fuel combustion. Thus, it is important
to better understand factors controlling WD decomposition. Previous studies have shown that half
or less of the variation in WD decay rates can be attributed to environmental factors such as
temperature and moisture. As wood decomposition depends on interactions among fungal
decomposers of WD, these interactions might transcend those environmental factors. Simplified
fungal communities constructed in WD suggest that species richness is negatively related to
decomposition. Our ongoing WD decomposition research in the Ailao Mountains of southwestern
China supports earlier studies showing that environmental factors explain up to half of the
variability. However, the >300 WD pieces we have measured show eight-fold variation at the
individual-piece scale, even though they are decomposing in the same forest environment. From
454 pyrosequencing, we have found a strong (R2 > 0.75) negative relationship between the
Shannon diversity of fungal OTUs and WD decay over two years. In a separate study of >90 WD
pieces at Ailao, we found a one-month 50% reduction in decay rate during the warm/wet season in
September, when environmental factors do not limit WD decay. All these observations are
consistent with strong negative interactions within the WD fungal community, but broader tests are
needed. We plan to explore relationships between WD fungal communities and decay rates at Ailao
and other sites. Decay rates alter the persistence of WD as habitat for many organisms in forests, as
well as its CO2 return to the atmosphere. If WD fungal communities exert the strongest controls on
those factors, our research will be the first to demonstrate it.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
155
Parallel Session Abstracts – Thursday, 31 October
—————————————————————————————————————————————
Parallel Session 5: Fungi and Algae
The ISHAM ITS database for Human/Animal Pathogenic Fungi - pros and cons
of the ITS region as universal fungal DNA barcoding marker
Laszlo Iriny1, Michael Arabatzis2, Stéphane Ranque3, Dirk Stuppe4, Laurence Delhaes5, Ian Arthur6,
Dea Garcia-Hermoso7, Carolina Firacative1, Gary Pan1, William King1, Maria Macinante1, Marie
Desnos-Ollivier7, Mauro de Medeiros Muniz8, Manoel Marques Evangelista de Oliveira8,
Anne-Cécile Normand3, Carole Cassagne3, Charles Mary3, Renaud Piarroux3, Daniel
Estrada-Barcenas9, Benjamine Stielow10, Vincent Robert10, Célia Pais11, Francoise Drommer7,
Conchita Toriello12, Maria Lucia Taylor9, Célia Maria de Ameida Soares13, Rosely M.
Zancope-Oliveira7, Arnaldo Colombo14, Aristea Velegraki2, Sybren de Hoog10, Josep Cano15,
Wieland Meyer1; Email: [email protected].
1
Molecular Mycology Research Laboratory, CIDM, Sydney Medical School-Westmead Hospital,
The University of Sydney, Westmead Millennium Institute, Sydney, NSW, Australia
2
Mycology Laboratory, Medical School, National and Kapodistrian University of Athens, Greece
3
Aix Marseille Université, IP-TPT UMR MD3, and APHM, CHU Timone-Adultes, Parasitologie et
Mycologie, Marseille, France
4
BCC/IHEM Biomedical Fungi and Yeast Collection, Brussels, Belgium
5
BDEEP-EA4547, CIIL, Institut Pasteur de Lille, Université de Lille 2, Lille, France
6
Mycology Laboratory, Department of Microbiology and Infectious Diseases, PathWest Laboratory
Medicine WA, QEII Medical Centre, Nedlands, Western Australia, Australia
7
National Reference Center for Invasive Mycoses and Antifungal Molecular Mycology Unit,
Institute Pasteur, Paris, France
8
Laboratório de Micologia/Setor de Imunodiagnóstico Instituto de Pesquisa Clínica Evandro
Chagas (IPEC) Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
9
Laboratorio de Inmunologia de Hongos, Departamento de Microbiología-Parasitología, Facultad
de Medicina, Universidad Nacional Autonóma de México, México
10
CBS-KNAW, Utrecht, The Netherlands
11
Universidade Federal de Goiás, Instituto de Ciências Biológicas, Departamento de Bioquímica e
Biologia Molecular, Golas, Brazil
12
Laboratorio de Micología Básica, Depto. de Microbiología y Parasitología, Facultad de Medicina,
Universidad Nacional Autónoma de México, México D.F., México
13
Centre of Molecular and Environmental Biology (CBMA), Biology Department, School of
Sciences, University of Minho, Braga, Portugal
14
Special Mycology Laboratory, Department of Medicine, Federal University of São Paulo, São
Paulo, Brazil; 15Universidad Rovira I Virgili, Reus, Spain
E-mail: [email protected]
Correct and fast identification of the causative agents of human mycoses is of great importance for
the adequate choice of early and efficient anti-fungal treatments. However, traditional
morphological and biochemical based idenitification of human pathogenic fungi is time-consuming,
laborious and needs specially trained personal. To overcome those limits the Internal Transcribed
Spacer (ITS) regions of the ribosomal DNA gene cluster have been used for molecular identification
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
156
Parallel Session Abstracts – Thursday, 31 October
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of human pathogenic fungi long before it had been selected as the official fungal DNA barcode in
2012. Besides the large number of ITS sequences that have been deposited in GenBank the
identification of human pathogenic fungi is often ambiguous and the obtained species names are not
always reliable, because of the vast number of incorrect sequences deposited, the limited taxonomic
range covered and the limited strain diversity represented. To surmount this problem, a number of
quality control ITS databases have been created to ensure the proper identification of fungi. As the
result of a global collaboration between 13 medical mycology reference laboratories within the
ISHAM working group for “DNA barcoding of human/animal pathogenic fungi” a quality control
database has been established, that currently contains ITS sequences of approximately 2000 strains
representing
380
human
pathogenic
species,
and
is
accessible
via
the
http://www.mycologylab.org/BioloMICSID.aspx. These ITS sequences will also be part of the new
GenBank reference sequence database. In general the ITS1/2 region has a high probability to
successfully identify an agent of human mycosis to the species level, however, the intra-species
variation did range from 0-8.5% depending on the species under investigation, raising the question:
“Is the ITS region the most appropriate locus for fungal barcoding?” Based on these findings
alternative genetic loci are currently under investigation to either be used as secondary DNA
barcode in species where the resolution of the ITS is not sufficient or even totally replace it with a
more powerful DNA barcoding marker.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
157
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: Fungi and Algae
One fungus, which genes?
Benjamin Stielow, Michel de Vries, Pedro Crous, Marizeth Groenewald, Gerard Verkley, Vincent
Robert
CBS-KNAW, Fungal Biodiversity Center, Uppsalalaan 8, 3584 CT Utrecht, Netherlands
E-mail: [email protected]
During the course of the EMbaRC project (European Consortium of Microbial Resource Centers),
work package JRA2.2.1, alternative gene targets from available fully sequenced genomes were
identified and their suitability to be used as supplementary fungal barcodes tested. A total of 54
gene sections were identified via different methods, but only 15 were recognized as putative novel
candidate regions. Based on the data retrieved from the in silico analyses, more than 80 primers (in
more than 100 combinations) were manually designed based on the most universally conserved
sites. These primers, corresponding to nine distinct regions, were first tested and the PCR and
sequencing conditions optimized, on DNA extracts from genome sequenced strains and or species
available in the CBS collection. With this study we aim to provide precise quantitative estimates, as
to which gene section(s), coupled with the most efficient universal primer pair(s) provides optimal
species resolution among the investigated orders of the fungal kingdom, to eventually be proposed
as additional barcodes to supplement the universal fungal barcode ITS.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
158
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: Fungi and Algae
Discovery of new species using DNA barcode and cohesion-based approaches in
lichen-forming fungi
Thorsten Lumbsch, Steven Leavitt, Sittiporn Parnmen
The Field Museum, Chicago, IL 60605, United States
E-mail: [email protected]
A growing body of evidence suggests a substantial number of unrecognized species are likely
hidden within traditional phenotype-based species in lichen-forming fungi. Molecular genetics has
revolutionized our ability to assess traditional species concepts and provides additional tools for
robust species delimitation. In this presentation we discuss the use of cohesion-based methods to
better understand delimitation of species using examples from different genera of lichen-forming
fungi, including the genera Cladia, Montanelia, and Rhizoplaca (all Lecanorales) and how DNA
barcode methods are used to confirm and rapidly identify the distinct lineages. The internal
transcribed spacer (ITS) region has been chosen as barcode marker for fungi. We tested the
potential of ITS to identify distinct lineages in the Cladia aggregata complex, a group of lichenized
fungi exhibiting remarkable morphological and chemical diversity. Our recent studies using
multilocus DNA sequence data and coalescent-based species delimitation methods supported a 12
species delimitation scenario. We evaluated the ratio of the intra- and interspecific genetic distances
of ITS in these 12 putative species. All 12 putative species showed a lower ratio of intraspecific
variation than interspecific variation, supporting the hypothesis that these represent distinct lineages.
In Rhizoplaca melanophthalma sensu lato we identified six distinct lienages using support from the
coalescent-based species delimitation method implemented in the program Bayesian Phylogenetics
and Phylogeography (BPP) as the diagnostic feature distinguishing the species. also assessed
intraspecific genetic distances within the six R. melanophthalma sensu lato species. While
intraspecific genetic distances within the five new species were less than or equal to the lowest
interspecific pairwise comparison values, an overlap in genetic distances within the R.
melanophthalma sensu stricto clade suggests the potential for additional undiscovered species. In
the genus Montanelia we also foudn that the current phenotype-based species concept is not
consistent with results of phylogenetic studies based on multi-locus data set and we demonstrate
how DNA barcoding can rapidly identify the species hidden under the current wide concept of taxa
in the genus.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
159
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: International Collaboration
Barcoding, biosecurity and protection of endangered species in South Africa
Olivier Maurin1, Michelle Hamer2, Guy Preston3, Michelle van der Bank1
1
The African Centre for DNA Barcoding, University of Johannesburg, Johannesburg, South Africa
2
The South African National Biodiversity Institute, Pretoria, South Africa
3
Department of Environmental Affairs, Cape Town, South Africa
E-mail: [email protected]
South Africa has an extremely rich biodiversity with ca. 20 456 species occurring in the region. Of
these, some 2 577 taxa are threatened with regional and global extinction. These threats are mainly
through agriculture, use of resources for medicinal purposes, expanding population, and national
and international trade of endangered species. The spread of invasive species are also of growing
concern due to their negative impacts on biodiversity and huge economic burden. In light of the
challenges opposing accurate identification, alternative solutions need to be explored within the
country. Thus the department of Environmental Affairs, the South African National Biodiversity
Institute, and the African Centre for DNA Barcoding initiated two large-scale projects to use DNA
barcoding to identify biological material entering the country at O.R. Tambo International Airport
and as a forensics tool to fight wildlife crime. These projects are funded and supported through
IDRC/iBOL, department of Environmental Affairs South Africa, University of Johannesburg, and
CBOL through the Google Impact Awards. This presentation highlights challenges facing the
country, various programmes initiated in response to these, preliminary results, as well as human
capacity development, which are devoted to boosting skills development under young South
Africans.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
160
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: International Collaboration
The role of German institutions in assembling DNA barcode data from countries
in the southern hemisphere
Axel Hausmann1, Gerhard Haszprunar1, Lars Hendrich1, Stefan Schmidt1, Thomas von Rintelen2,
Christoph Haeuser2, Paul D.N. Hebert3
1
Bavarian Natural History Collections, Zoological Collection of the State of Bavaria, 81247 Munich,
Germany
2
Museum für Naturkunde, 10115 Berlin, Germany
3
Biodiversity Institute of Ontario Canada, University of Guelph, Guelph, Ontario, Canada
E-mail: [email protected]
German research institutions support the inventory and assessment of biodiversity in several
tropical countries through DNA barcoding. We present examples of ongoing projects in South
America, Africa, and the Indo-Pacific region undertaken by German institutions in close
collaboration with local counterparts in which barcodes for more than 20K species have been
assembled. These initiatives were enabled by the involvement of taxonomists from German
natural history museums, with funding from German and Canadian research agencies, and technical
support from sequencing facilities in Canada. In the near future, major projects are planned in Peru,
several African countries, and Indonesia
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
161
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: International Collaboration
Organization of the DNA Barcoding project in a developing country: the case of
Argentina
Pablo Luis Tubaro
Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” (MACN – CONICET), Argentina
E-mail: [email protected]
The participation of Argentina as a regional node of the iBOL project is supported by the National
Research Council (CONICET). The key elements of the project include: a) five medium scale
barcode laboratories that transmit both lysis and PCR plates to the Biodiversity Institute of Ontario
(BIO) for sequencing; b) the annual training workshops hosted at the MACN that cover all stages of
the barcoding process and are attended by 80-90 researchers and Ph.D. students each year (about
10% of them coming from other countries in the region); c) a research vessel that has been deployed
to collect marine specimens, particularly aiding Fish-BOL; and d) the iBOL Argentina fund that
supports about a hundred small collecting projects to produce new materials (tissues + vouchers).
As a result of this system, Argentina is making major contributions to barcode coverage on birds,
fish, bees and Lepidoptera and is also generating barcodes of other groups of animals, plants and
fungi. In addition, the IDRC project has supported highly qualified personnel to help process newly
collected specimens and has also enabled activities in Bolivia. Twelve Argentinian students have
also received advanced training at BIO. Finally, Argentina is actively participating in the global
Malaise program.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
162
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: International Collaboration
Building the biodiversity macroscope in Bolivia: a collaborative network
between two South American neighboring counties in the International Barcode
of Life project
Cecilia Kopuchian
Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” (MACN–CONICET), Argentina.
E-mail: [email protected]
The Neotropics are among the most biodiverse regions of the world and are therefore particularly
important for the goals of the International Barcode of Life project. In this context, in 2008 MACN
in Argentina started a collaboration with the Colección Boliviana de Fauna in Bolivia to barcode the
biodiversity of Southern South America. This collaborative network was supported by Canadian
(BIO, IDRC) and U.S. (Richard Lounsbery Foundation) funds. Bolivia is a developing country with
a small scientific community. Its territory presents a rich and not fully known biodiversity,
distributed in several ecoregions in a wide altitudinal gradient going from 90 to 6500 masl. Since it
became a regional node in 2009, Argentina has been working with Bolivian Institutions from La Paz,
Cochabamba and Santa Cruz to obtain genetic barcodes of vertebrate and invertebrate species
which are contained in more than 10 BOLD projects. Also, more than 20 Bolivian researchers and
students were trained in Bolivia and Argentina in barcode techniques and in field work. Moreover,
collection campaigns and existing Museum collections were economically and logistically
supported. Nowadays, the future of this fruitful collaboration mainly relies on the resolution of
permit restrictions that are being implemented by the new Bolivian authorities.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
163
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: International Collaboration
Building global DNA barcoding capacity: the importance of international
development agencies
Alex Borisenko
Biodiversity Institute of Ontario Canada, University of Guelph, Guelph, Ontario, Canada
E-mail: [email protected]
One of iBOL’s long-term goals is to build DNA barcoding capacity in its participating nations: the
ability to perform all stages of the analytical chain and to facilitate a broad gamut of applications. It
builds on the existing strengths of each member and takes advantage of technological innovations
within the entire barcoding community. These efforts can be undermined by the scarcity of funding
for biodiversity research, especially in developing nations. International development agencies can
help overcome this challenge if they direct funds to support general operations at the lead
institutions of national or regional iBOL nodes.
From 2010-2013, a $2.2M grant from the Canadian International Development Research Centre
(IDRC) supported barcoding activities in two regional and five national iBOL node countries. This
support helped catalyze government funding commitments for barcoding research within two major
recipients, Argentina and South Africa. Although international development agencies are unlikely to
provide sustained funding for barcoding activities, they can give the national and regional iBOL
nodes the time and momentum necessary to secure government support and to garner interest from
prospective regulatory and business partners. The IDRC success story provides a good example of
global biodiversity research leadership to aid agencies in other developed nations.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
164
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: International Collaboration
Current progress of Mexico in the iBol Project, the MEXBOL network
Elías-Gutiérrez Manuel1 & León-Regagnon Virginia 2
1
El Colegio de la Frontera Sur, Chetumal Unit, Chetumal, Mexico
2
Estación de Biología Chamela, Instituto de Biología, National Autonomous University of Mexico,
04510 Mexico, Mexico
E-mail: [email protected]
Mexico, with a surface of 2000000 Km2 and 10000 km of littoral zone, occupies the fourth place in
biodiversity of the world. Since 2005 several Mexican researchers got involved in the activities of
DNA barcoding: they organized the III International Barcode of Life Conference in Mexico City
and by 2010, the Mexican Barcode of Life Network started activities, currently including 113
formal members from 20 academic institutions. This network has been supported by the National
Council of Science and Technology and the National Commision for the Knowledge and Use of the
Biodiversity, in collaboration with the Biodiversity Institute of Ontario. In the begining, a National
Laboratory for DNA Barcoding (with three nodes) was developed, together with an organizational
strategy to support all interested researchers. As a result, this country is nowadays among the top
five countries in the generation of barcodes for Acanthocephala, Annelida, several arthropod groups
(as Crustacea), Cnidaria, Nematoda, Platyhelminthes, Rotifera, Pinophyta, and Fungi. Two special
numbers in specialized journals have been developed, and more than 70 papers on the topic have
been published, including some practical applications. We can conclude that DNA barcoding has
been an important impulse to the biodiversity research in this country.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
165
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: International Collaboration
iBOLColombia advances and perspectives
Mailyn Gonzalez1, Andrea Paz1, Santiago Madriñan2, Silvia Restrepo2
1
Instituto de investigación de recursos biológicos Alexander von Humboldt, Bogotá, Colombia
2
Universidad de los Andes, Bogotá, Colombia
E-mail: [email protected]
Colombia´s outstanding biodiversity is largely undiscovered and severely threatened. Standardized
and reliable tools of species identification such as DNA barcodes are urgently needed in an effort to
describe and conserve the vast biodiversity of Colombia. In 2008 we started a first attempt to
integrate institutional efforts to conform the iBOLColombia network.
Major difficulties
encountered in the development of the barcoding initiative were legal permits and funding among
others. In 2011, seven universities and three environmental national institutions where participating
in the iBOLColombia network. In a recent effort to build the DNA barcode library of Colombian
biodiversity we have generated the first 1000 DNA barcodes of endangered flora and fauna species.
Among the generated barcodes we have species of birds listed in the CITES appendix, species of
plants representative of emblematic ecosystems such as Paramo and Amazonia and fungi species
linked to pathogen infections or symbioses in plants. Recent modifications in the Colombian
legislation promise to encourage genetic studies and the generation of DNA barcodes sequences.
The network will thus play a major role in structuring this new explosion of information
maximizing availability for the global scientific community.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
166
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: Medicinal Plants II
DNA barcoding Chinese medicinal Bupleurum
Zhi Chao, Jing Liao, Li Liu, Zhenbiao Liang
School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong
510515, China
E-mail: [email protected]
We tested four markers (ITS2, psbA-trnH, matK, rbcL) in order to evaluate the candidate DNA
barcodes for distinguishing Bupleuri Radix (Chaihu) from its adulterants. 51 individual plant
samples of Bupleurum representing 19 species were collected from different areas in China. All
the candidate barcode regions were attempted to be amplified and sequenced, and then were
assessed by the success rate of PCR amplification and sequencing, the differential intra- and
inter-specific divergences, the DNA barcoding gap and the ability to discriminate species. The
results showed that ITS2 produced the better results among the four loci. The identification
efficiency of ITS2 was 73.68%, after combined with psbA-trnH the efficiency improved to 83.33%.
In addition, we further evaluated the efficiency of the ITS2 to discriminate among species of
Bupleurum using a large database from GENBANK, which included 223 samples from 74 species,
ITS2 successfully discriminated 64.13% of the samples at the species level. In conclusion, the
ITS2 could be taken as a potentially useful barcode for Bupleurum species, and psbA-trnH as
supplementary locus.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
167
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: Medicinal Plants II
Purity test on crude drug using genetic information: The future of crude drug
identification in the Japanese Pharmacopoeia.
Haruka Asahina1, Junichiro Shinozaki2, Motoyoshi Satake3
1
Graduate school of Humanities and Sciences, Ochanomizu University, Bunkyo, Tokyo 112-0012,
Japan
2
Showa Pharmaceutical University, Machida, Tokyo 194-0042, Japan
3
Institute of Environmental Science for Human Life, Ochanomizu University, Bunkyo, Tokyo
112-0012, Japan
E-mail: [email protected]
The first step in the quality assurance of herbal medicine is the testing of the right plant sources.
Methods for differentiating the original plant species of crude drugs, such as by inner- and
outer-morphological methods, organoleptic tests, and chemical methods are described in the
Japanese Pharmacopoeia. However, due to recent changes of plant cultivating areas, further mixing
of species is expected. Therefore, more powerful methods for differentiation are required.
Among various differentiation methods, morphological and chemical methods have been used in
the differentiation of two groups of Atractylodes ( Group"Soujutsu": A. lancea & A. chinensis,
Group "Byakujutsu" : A. japonica & A. ovate). On the other hand, many studies finally concluded
that genetic tests using the ITS-1 region was the most reliable for separating these four species.
With consideration on this topic by the JP Commission, two practical purity tests for Group
"Byakujutsu" using genetic information were mentioned in the Reference Information of JP 16th
edition. We will discuss past and future differentiation methods for plant origin of crude drugs in the
JP, and the possibility of medicinal Dendrobium identification using DNA barcoding.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
168
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: Medicinal Plants II
Development of a standard reference material (SRM) DNS barcode library for
herbal product authentication
Sathishkumar Ramalingam, Dhivya Shanmughanandhan
Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University,
Coimbatore-641 046, Tamil Nadu, India
E-mail: [email protected]
Traditional medicinal practices in India involve the treatment of diseases with drugs derived from
plants. Due to various reasons, herbal products are adulterated and substituted with alternative plant
based ingredients. Our study, thus, aimed at securing quality products in the global marketplace and
protecting consumers from health risks associated with fake product substitution and contamination.
For this, we used DNA barcoding to test the authenticity of i) 30 herbal products representing 3
different manufactures and 30 different species and ii) 100 herbal species that were used to identify
the unknown herbal products and leaf samples, thus creating a standard reference material (SRM)
herbal barcode library. We recovered DNA barcodes from various herbal products (91%) and leaf
samples (100%), with 95% species resolution using a tiered approach (rbcL+ITS2). Most of the
products tested contained DNA barcodes from plant species not listed on the labels. Although we
were able to authenticate almost half of the products, one-third of these also contained contaminants
and or fillers not listed on the label. Some of the contaminants we found pose serious health risks to
consumers. Therefore the use of a SRM for testing herbal products available in the market place
could provide a relatively cheap method for herbal product authentication.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
169
Parallel Session Abstracts – Thursday, 31 October
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Parallel Session 5: Medicinal Plants II
DNA barcoding of high value medicinal and aromatic plants of Sagarmatha
National Park (SNP), Nepal
Jagat Krishna Chhipi Shrestha, Smita Shrestha, Jyoti Maharjan, Neesha Rana, Jaishree Sijapati,
Sangita Shrestha
Nepal Academy of Science and Technology (NAST), Khumaltar, Lalitpur, Nepal
E-mail: [email protected]
Nepal is rich in Medicinal and Aromatic Plants (MAPs) biodiversity. Over 1950 MAPs with their
associated indigenous Traditional Knowledge (TK) have already been reported to be found in
various low land Terai landscapes (below 100 masl) to high Himalayan landscapes (>4000 masl) of
Eastern, Central and Western Nepal. However, these valuable resources are rapidly depleting due to
rapid urbanization, overexploitation, land degradation and prevailing climate change. Hence, if
these resources are not judiciously managed, conserved and utilized, they will be lost forever. As
high altitude flora are more vulnerable to climate change effects, newly emerging DNA barcoding
techniques are best options for biomonitoring in spatial and temporal scales. As an initiation, NAST
has started a project of DNA barcoding of high value MAPs of SNP region. DNA Barcoding of
Podophyllum hexandrum, Pedicularis gracilis, Prunus avium, Plantago erosa, Bistorta vivipara,
Rhododenron spp., Salix sp., Oxytropis sp., Fagopyrum sp. and other species using potential
standard DNA barcodes for land plants (nuclear rDNA ITS and cpDNA matK, rbcL and trnH-psbA)
have been performed. The sequences were edited, aligned and analyzed using Sequencher v.4.1.4
and Codon Code Aligner v.4.2.2. Multiple species of Rhododendron were employed in a
phylogenetic study using Mega v.5.2.2. Possibility of using different DNA barcodes in species
identification and authentication of MAPs were explored.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
170
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Part III. Posters and others
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
171
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Amphibians and Reptiles
DNA barcoding of Ranidae frogs of Peninsular Malaysia
Zalina Awang1, Amirah Hurzaid1, Ibrahim Jaafar1 and Siti Azizah Mohd Nor2, 3
1
Biological Sciences Program, School of Distance Education, Universiti Sains Malaysia,
11800 Minden, Penang, Malaysia.
2
School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia.
3
Centre of Marine and Coastal Studies, Universiti Sains Malaysia, 11800 Minden, Penang,
Malaysia.
E-mail: [email protected]
We employed the mitochondrial 16S rRNA gene which has been established as the DNA barcoding
marker for frogs and related taxa, to investigate its utility in species identification and elucidating
phylogenetic relationships of ranid frogs from three genera (Amolops, Odorrana, Hylarana) found
in Peninsular Malaysia. In total, liver tissue samples of 108 representative samples were obtained
from eleven presumed species within the Ranidae group; Amolops larutensis, Odorrana hosii,
Hylarana labialis, H. banjarana, H. erythraea, H. luctuosa, H. laterimaculata, H. glandulosa, H.
picturata, H. nigrovittata and H. nicobariensis. A final fragment length of 490 bp was obtained after
alignment and editing of ambiguous sites revealing a total of 191 variable sites (172 parsimony
informative sites). The Neighbour Joining (NJ), Maximum Parsimony (MP), Maximum Likelihood
(ML) and Bayesian analyses produced similar topologies and strongly supported the monophyletic
assemblages of the three genera of ranid frogs. In general, molecular approach successfully
differentiated and classified all eleven ranid frogs into their own genus and species group with
minimal overlap.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
172
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Amphibians and Reptiles
Identification of Crocodile production through DNA barcode
Fengjuan Guo, Jianchun Yang, Shijia Hu, Yongshi Li
South China wildlife species Identification Center, Guangdong Entomological Institute, Guangzhou,
Guangdong 510260, China
E-mail: [email protected]
DNA barcode has been extensively used in species identification and taxonomical research.. In this
paper, a batch of smuggled crocodile products were identified using DNA barcode. Three types of
the crocodile products (dried crocodile meat, crocodile penis and dried crocodile skin) were selected
as the samples for species identification. Mitochondrial DNA was extracted and cytochrome
oxidase subunit I gene fragments of each sample was amplified using the universal primers.
Alignment analysis with the data of eighteen homologous sequences of five different crocodile
species in Genebank revealed that the three unknown samples had the highest sequences similarity,
which was 99.6%~100%, with the sequences of crocodylus siamensis. The genetic distance that was
computed by MEGA4.0 between thoses samples and crocodylus siamensis was 0.000~0.003, which
was the minimum. The phylogenetic tree were constructed by P-distance model and
Neighbor-joining method, and Varanus bengalensis was set as the outgroup. The result indicated
that all of the three samples come from crocodylus siamensis. Consequently, it convincingly
demonstrated that the samples were crocodylus siamensis products, which provides a powerful
evidence for the execution of the customs.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
173
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Data Analysis Methods
Automating the calculation of the ad hoc distance thresholds for DNA barcoding
identification using two R functions
Gontran Sonet1, Kurt Jordaens2,3, Zoltán T. Nagy1, Floris C. Breman2, Marc De Meyer2, Thierry
Backeljau2,3, Massimiliano Virgilio2
1
Royal Belgian Institute of Natural Sciences, JEMU, Vautierstraat 29, 1000 Brussels, Belgium
Royal Museum for Central Africa, JEMU, Leuvensesteenweg 13, 3080 Tervuren, Belgium
3
University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
E-mail: [email protected]
2
The probability of erroneous DNA barcoding identification is higher when identification is based on
a large distance between the query (the barcode sequence of the specimen to identify) and its best
match in a reference barcode library. Yet, the number of these incorrect identifications can be
reduced by setting a distance threshold above which identification is considered unreliable. To this
end, it has been proposed to use an ad hoc distance threshold producing identifications with a
user-defined error probability (e.g. 5%). Here, we propose two R scripts to automate the calculation
of ad hoc distance thresholds for reference libraries of DNA barcodes. Both scripts, a user manual
and an example file are freely available upon request on the JEMU website
(http://jemu.myspecies.info).
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
174
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Education
Exploring invertebrate diversity in Gabonese ecosystems through DNA
barcoding
Thibaud Decaëns1, David Porco1, Myrianne Andeime Eyene2, Stephan Ntie3, Rodolphe Rougerie4,
Olivia Scholtz5, David Sebag6, Samuel W James7, the ECOTROP team
1
Univ of Rouen, France
2
IRAF-CENAREST, Libreville, Gabon
3
University Masuku, Gabon
4
INRA, Orléans, France
5
WCS, Gabon
6
University of Rouen/IRD, Cameroun
7
University of Iowa, USA
E-mail: [email protected]
In Gabon, natural ecosystems are renowned for their charismatic species of vertebrates and plants.
By contrast, invertebrates are poorly known and generally absent from conservation plans, despite
being several orders of magnitude more diverse and dominant in biomass. This shortfall is critical
and largely caused by our inability to sort and identify these organisms. DNA barcodes offer a way
of documenting basic descriptive metrics of invertebrate diversity, even in the absence of a
taxonomic framework. By integrating their use into the research and educational program
ECOTROP, scientific objectives are paired with the training of students whose participation and
increased awareness is expected to promote the consideration and description of these organisms.
To illustrate the potential of this approach, we present the results of a sampling campaign focused
on earthworms from three regions of Gabon: in Libreville and the National Parks of La Lopé and
Ivindo. So far, we generated DNA barcodes for 771 specimens representing 83 Molecular
Operational Taxonomic Units. Using these as surrogates for species diversity and assemblage
composition, we describe earthworm diversity at local and regional scales, with special attention to
impacts of land use and forest dynamics, and to the spread of invasive species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
175
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Education
The public face of barcoding: how iBOL and DNA barcoding are represented in
newspapers from around the world
Janis Geary, Emma Camicioli, Tania Bubela
School of Public Health, University of Alberta, Edmonton Alberta, Canada
E-mail: [email protected]
Barcoding has the potential to aid in conserving biodiversity, monitoring trade, and protecting
consumers, but these benefits rely on sharing genetic resources such as data and materials. Policy
decisions related to barcoding can be influenced by media representations of barcoding, as the
media shapes and reflects public discourse. Our research examines media representations of iBOL
and DNA barcoding in newspapers to determine how barcoding technology and implications of
sharing genetic resources are portrayed to the public. We searched for relevant English language
newspaper articles from Canadian Newstand, Factiva and Lexis Nexis (2003-2012). Our search
returned 1266 articles from 21 countries, including Canada (361), USA (291), UK (199), India (95),
and China (93). The most common theme represented the scientific advancement of barcoding
(842), although many articles mentioned bio-conservation (465) or food mislabelling (355). Many
articles (905) mentioned barcoding without any explanation of barcoding methods. Only 6 articles
mentioned issues related to sharing genetic resources (either biopiracy or bioprospecting), and 29
referred to an international treaty related to sharing genetic resources. Media representations of
iBOL and DNA barcoding emphasize the potential applications of barcoding, but fail to adequately
describe barcoding methods or implications of sharing genetic resources.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
176
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Education
DNA Barcodes: their application and use in a regulatory organisation
Diane Jones
Plant Health & Environment Laboratory, Ministry for Primary Industries, New Zealand
E-mail: [email protected]
DNA barcoding has become a standard molecular technique for species identification. The New
Zealand Government agency, Ministry for Primary Industries (MPI) is a regulatory organisation
tasked with protecting New Zealand from invasive organisms. Accurate and timely identification
of organisms arriving at New Zealand’s borders is essential. MPI has adopted the use of DNA
barcodes to identify organisms encountered at New Zealand’s borders particularly in instances
where morphological identification is difficult or impossible e.g. immature insect lifestages. The
benefits and challenges faced with using DNA barcodes in a regulatory organisation, the potential
solutions and the development of an ‘inhouse’ MPI Border barcode database will be presented.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
177
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Education
Promoting DNA barcoding in Belgium through the BeBoL network
Zoltán T. Nagy1, Gontran Sonet1, Kurt Jordaens2, Floris C. Breman2, Johan De Gruyter2, Marc De
Meyer2,3, Thierry Backeljau1,3, Belgian Network for DNA Barcoding of Life
1
Royal Belgian Institute of Natural Sciences, JEMU, Rue Vautier 29, B-1000 Brussels, Belgium
2
Royal Museum for Central Africa, JEMU, Leuvensesteenweg 13, B-3080 Tervuren, Belgium
3
coordinator of the BeBoL network
E-mail: [email protected]
The Belgian Network for DNA Barcoding of Life (BeBoL) has been established in 2011. Its main
task is to facilitate, centralize and coordinate DNA barcoding activities in Belgium, to assist in
exchanging experience and providing training opportunities in the field of DNA barcoding and
molecular systematics in the widest sense (bebol.myspecies.info). This network includes 21 partners
from Belgian universities and research institutes, as well as CBOL and ECBOL. Recently, three
major activities have been held. In September 2012, the BeBoL network involving the Joint
Experimental Molecular Unit (JEMU) of the Royal Belgian Institute of Natural Sciences and the
Royal Museum for Central Africa organized the Third European Conference for the Barcode of Life
(ECBOL3) in Brussels with almost 100 international participants from 19 countries. In 2013, a
symposium on metabarcoding of environmental samples and a workshop on ViBRANT tools for the
DNA taxonomist have been organized. Original papers of the network and participants of the
ECBOL3 meeting will be presented in a special issue of the journal ZooKeys. We aim to publish
this issue in autumn/winter 2013, if possible simultaneously to this 5th International Barcode of
Life Conference.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
178
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Environmental Barcoding
BanglaBOL- the DNA barcoding initiative in Bangladesh for molecular
taxonomy and biodiversity conservation
Badrul Bhuiya
Department of Zoology, University of Chittagong, Bangladesh
E-mail: [email protected]
DNA barcodes have been proved to be a vital tool not only for conservation but also for monitoring
species. BanglaBOL, the Bangladesh Barcode of Life, was initiated during a DNA barcoding
training workshop held in Chittagong in 2012 which organized around 30 interested
biotechnologists from around the country. DNA of agromyzid (Diptera) leaf miner insect pests of 42
different vegetable and cereal crops of Bangladesh have been extracted and sequenced in the newly
established Barcoding Lab at the University of Chittagong. A number of biological control parasitic
insect agents (Hymenoptera) were also recorded from these pests and sequenced. A year long
survey conducted in 42 districts of Bangladesh revealed that a good number of pest species are
common in the country. Scientists at the Chittagong Veterinary and Animal Sciences University are
working for DNA barcoding of the Reptilia of Bangladesh. Recently DNA of an amphibian was
extracted and awaiting sequencing and barcoding which was believed to be extinct from the
Oriental Region. The insect biodiversity of Bangladesh is being studied through barcoding of a
museum collection in the Zoology Department, University of Chittagong. The Department of
Zoology at the University of Dhaka has recently taken initiative to establish a molecular lab for
biotechnology research which is expected to conduct barcoding as well. For monitoring and
conservation strategy of the Bangladesh Biodiversity it is expected that barcoding will reveal
accurate species names of the so far known organisms in near future.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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179
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Environmental Barcoding
A genomic toolbox for soil biodiversity assessment
Thibaud Decaëns1, David Porco1, Sylvain Dolédec2, Lise Dupont3, Aurélie Husté1, Johan Oszwald4,
Jean-Pierre Rossi5, Rodolphe Rougerie6, David Sebag7, G. Blagoev8, C. Young9, Shadi Shokralla8,
Mehrdad Hajibabaei8
1
University of Rouen, France
2
University of Lyon 1, France
3
University Paris 6, France
4
University of Rennes, France
5
IRNA, Montpellier, France
6
INRA, Orléans, France
7
University of Rouen/IRD, Cameroun
8
University of Guelph, Canada
9
Carnegie Museum of Natural History, United States
E-mail: [email protected]
The biodiversity of soil animal communities is still poorly known, small body-sized and large
invertebrates of the macrofauna, suffering both from a strong taxonomic deficit. The coupling of
DNA barcoding and environmental sequencing is now providing innovative and promising tools to
avoid this impediment and fasten the description of soil biodiversity. In this poster we present
preliminary results from the GENOSOIL-Fr project, which aimed to develop innovative molecular
methods allowing biodiversity estimations for a broad taxonomic spectrum of soil organisms. In a
first step, we built comprehensive reference libraries of DNA barcodes (CO1) for a range of soil
invertebrates. The second step was the analysis of invertebrate and soil samples taken in contrasted
ecosystems to compare species richness estimations obtained using classical identification (based
on morphological characters) or genomic approaches (using the Molecular Operational Taxonomic
Units as surrogates to species). Molecular approaches included (1) systematic DNA barcoding of
soil invertebrates; (2) pyrosequencing (using Roche 454) of bulk samples composed of invertebrate
fragments; (3) pyrosequencing of soil environmental samples.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
180
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Environmental Barcoding
Barcoding microbe by restriction profiling of 23S rDNA
Parvathi.J.R, Mrunal Gokhale, Sunita Singh
1
Department of Biotechnology and Bioinformatics, Padmashree Dr. D. Y. Patil University, CBD
Belapur, Navi Mumbai, Maharashtra, India
E-mail: [email protected]
Micro-diverse clusters of 23S rDNA were perused to differentiate Escherichia coli from its colony
morphovars and establish the clonal nature by targeting regions of Single Nucleotide
Polymorphisms. An alternative en-route for bacterial detection by coupling broad-range PCR with
restriction analysis was ventured over routine molecular methods. Collating and evaluating the
intra-genic and inter-genic diversity of ribosomal rRNA repeats using Multiple Sequence Alignment
across the selected bacterial strains was followed by restriction profiling against 201 conventional
restriction enzymes. 2 broad range primer sets; 23SP1880 & 23S P2682 were designed; each
amplifying a separate region common to the referred strains but differing in restriction profile.
Amplified Ribosomal DNA restriction Analysis (ARDRA) of 23SP1880 with BfaI helped to
discriminate E. coli from members of Enterobacteriaceae family, whereas ARDRA of 23S P2682
with Hae III facilitated in establishing predominant clones. Analysis of restriction profiles of latter
against dendogram constructs of whole genome data of 57 E. coli strains confirmed that the
polymorphic patterns of the “core” genome can be validated at a pan-genomic level. The generic
nature, conservation and barcode “gap” of 23S rDNA makes it as an ideal choice for molecular
diagnostics in bacteria and can substitute 16S rDNA.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
181
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Environmental Monitoring
Developing molecular approaches for assessing stream health: DNA barcoding
of Ephemeroptera, Plecoptera, Trichoptera (EPT) taxa in New Zealand
Ian D. Hogg1, C. Beet1, K.R. Bennett1, G. Collins1, B.J. Smith2, J.C. Banks1,3, P.D.N. Hebert4
1
Department of Biological Sciences, University of Waikato, Hamilton, New Zealand
2
National Institute of Water and Atmospheric Research, Hamilton, New Zealand
3
Cawthron Institute, Nelson, New Zealand
4
Biodiversity Institute of Ontario, University of Guelph, Ontario, Canada
E-mail: [email protected]
We are using mitochondrial DNA (COI) gene sequences to examine species diversity for the New
Zealand mayflies (Ephemeroptera), stroneflies (Plecoptera) and caddisflies (Trichoptera), the ‘EPT’
taxa. All taxa are common inhabitants of stream ecosystems and are frequently used in assessments
of stream health such as the Macroinvertebrate Community Index (MCI). However, accurate
identification of taxa for these indices is often time consuming and can require considerable
taxonomic expertise. A molecular-based approach to the MCI or similar indices could speed up the
routine assessment and monitoring of stream invertebrates for water quality analyses. As a first step,
we have been working to build reference libraries for the EPT taxa. To date we have analysed over
600 individuals of caddisflies covering a putative 130 species. Several species have shown high
levels of “intraspecific” divergence, with genetically distinct populations. Sequence divergences in
these cases are as high as 9.92% (between the North and South Island populations of Pycnocentria
evecta). Efforts are currently underway to complete the roughly 250 species of New Zealand
caddisfly as well as the remaining species of mayfly (n= 40 species) and stonefly (n= 104 species).
We are currently testing next generation sequencing (NGS) platforms (e.g. Illumina, Ion Torrent)
based on these reference libraries to develop community-based indices of stream health. In the
interim, we conclude that the diversity of New Zealand caddisflies, and likely other aquatic taxa, is
underestimated and would benefit from re-evaluation using a combined morphological and
molecular approach.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
182
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Environmental Monitoring
Application of metagenomics in assessing the impact of desertification on the soil
microbial population and diversity in the front line states of Nigeria.
Christie O. Onyia1, Nwadiuto Esiobu2, Beatrice Ojiego1, Patience Jideofor1, B. Mwajim3, B. O.
Solomon1
1
National Biotechnology Development Agency, Abuja, Nigeria
2
Florida Atlantic University, Florida, United States
3
Biotechnology Centre, University of Maiduguri, Maiduguri, Nigeria
E-mail: [email protected]
Desertification has continued to cause innumerable social and economic consequences to the people
living in the Frontline States of Nigeria. The country loses thousands of square kilometers of its
land mass and unaccountable amount of genetic resources to the advancing desert. Variations in
climatic conditions, as a result of the current global phenomenon known as “Climate Change” have
been identified as the most important natural cause of desertification and drought in Nigeria. These
phenomena have led to poverty, social strife, resource-use conflicts and migration. Flora and fauna
have been badly depleted, while wildlife is highly endangered. Microorganisms have been
neglected as important component of the total biodiversity, hence the dearth of information on
microbial resources of the hot, arid zones. This project is designed to investigate the microbial
dynamics of the area and possible impact of desertification on their population and or diversity.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
183
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Environmental Monitoring
Changing perspectives on the diversity of bats (Chiroptera) at Ulu Gombak
since the establishment of the field studies centre by lord medway in 1965
Kong-Wah Sing, Khairunnisa Syaripuddin and John-James Wilson
Museum of Zoology, Institute of Biological Sciences, Faculty of Science, University of Malaya,
50603 Kuala Lumpur, Malaysia
E-mail: [email protected]
Ulu Gombak Forest Reserve is a selectively logged forest located at the Pahang-Selangor border. A
field studies centre was established at the western edge of the reserve by Medway in 1965. Ulu
Gombak had previously been reported as the single locality with the highest species richness of bats
in the Old World. In light of recent studies demonstrating extensive numbers of cryptic bat species,
diversity assessments at Ulu Gombak would benefit from reexamination. In this study we examine
changing perspectives on bat diversity at Ulu Gombak since the establishment of the Field Studies
Centre, and particularly, how assessments of species richness change with the incorporation of DNA
barcoding into bat surveys. One hundred and sixty records of bats at Ulu Gombak were extracted
from literature and from the Museum of Zoology, University of Malaya collection. Fifty-two
morphological species of bats had been recorded at Ulu Gombak between 1962 and 2012 which
was equivalent to one additional species record every two years throughout this period. During
surveys at Ulu Gombak in 2012/2013 DNA barcodes were obtained from 45 bats. The DNA
barcodes were assigned to seven species. Four of these were dark taxa, previously reported species
which lack formal description, in the genera Cynopterus and Hipposideros. Additionally, a deep
DNA barcode divergence (4.2%) from conspecifics from Indonesia strongly suggested the presence
of a cryptic species of Chironax which had not been reported previously. These five species were
added to the cumulative checklist for Ulu Gombak taking the total to 57 species of bats. The high
number of cryptic species uncovered supports the prediction that the number of bat species in Ulu
Gombak is significantly underestimated. The projected number of 89 bat species provides a
benchmark for future, more intensive, surveys using multiple trapping methods and covering a
larger area of the reserve, but critically, incorporating DNA barcoding for species recognition.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
184
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Environmental Monitoring
A new bio-nano-assembly for bio molecules capture
Biao Zhang, Genliang Han, Liping Ma, Yuzhe Song
Institute of Sensor Technology , Gansu Academy of Sciences, Lanzhou, Gansu, China
E-mail: [email protected]
A bio-nano-assembly (BNA) is prepared. The BNA has a magnetic core attached thereto specific
bio acceptors or bio probes. This structure can be used to capture specific target bio molecules or
bio-species from complex environment. The captured bio molecules can be sorted, extracted and
guided with the aid of magnetic cores by using magnetic fields. As such, extraction, purification,
and capture of target bio-molecules or species, and then the following sequencing or
characterizations can be significantly simplified.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
185
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Environmental Monitoring
Arthropods as the indicator taxon for China’s protected areas management
Kai Zhang1,2*, Siliang Lin3*, Douglas W. Yu1,4, Haisheng Jiang3, Hesheng Wang5, Haihao Fu6,
Chenxue Yang1,2, Xiaoyang Wang1,2, Yinqiu Ji1, Chunyan Yang1
1
Ecology, Conservation, and Environment Center (ECEC), State Key Laboratory of Genetic
Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming
650223, China
2
University of the Chinese Academy of Sciences, Beijing100049, China
3
Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, School
of Life Sciences, South China Normal University, Guangzhou 510631, China
4
School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich,
Norfolk NR 47 TJ, UK
5
Yinggeling Nature Reserve, Baisha 572800, China
6
Datian National Nature Reserve, Dongfang572600, China
* These authors contributed equally
E-mail: [email protected], [email protected]
Incorporating biodiversity into the measurement of the effectiveness of China’s protected areas (PAs)
is needed to avoid the “empty forests” or “green deserts”. Arthropods are potentially good indicator
because arthropods comprise >2/3 of the Earth’s total species diversity and are abundant in all
terrestrial PAs. Moreover, arthropods are easily collected by standard methods (e.g. malaise traps),
and rapid identification is now possible with metabarcoding. Previous studies also showed that
arthropod assemblage closely correlated with plant species composition in grasslands, indicating
that arthropod community is responsive to the plant community variation. Here we further test such
relationship in tropical forests. We established 29 50×50m plots in the rain forest of Yinggeling
Nature Reserve, and 12 3×3m plots in the scrub forest of Datian Nature Reserve, Hainan Island,
China. We surveyed all the woody trees with DBH > three centimeters in Yingeling, and all the
flowering plants in Datian. Arthropods were collected with malaise traps, and were further
identified by metabarcoding. We compared the tree/flowering plant and the arthropod assemblages,
and found significant correlations. In conclusion, arthropod communities respond to plant
community for a wide range of habitats. We recommend arthropods as indicator taxon to assess the
effectiveness of China’s PAs.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
186
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Fishes
DNA barcodes characterizing intertidal Tridentiger gobies of Zhoushan Islands
Cuiping Gu, Yongjiu Chen
Zhejiang Ocean University, Changzhi Island, Zhoushan 316022, P.R. China
E-mail: [email protected]
Tridentiger is one of the goby genera native to coastal marine environments of West Pacific. In the
sea of Zhoushan Archipelago, two species, T. barbatus (shokihaze goby) and T. trigonocephalus
(chameleon goby) are generally recognized. In this study, DNA sequences of mitochondrial
cytochrome C subunit I (COI) and nuclear ryanodine receptor 3 (Ryr3) genes were barcoded for a
total of 203 Tridentiger samples collected from six intertidal areas of Zhoushan Islands and two
reference sites. DNA barcodes based on both COI and Ryr3 phylogenies characterize three species,
T. barbatus, T. trigonocephalus, and T. bifasiatus. Split from T. trigonocephalus that is mostly found
in sandy and rocky shore, T. bifasiatus (shimofuri goby) is reported for the first time on the islands
and abundant in various habitats. The average distance of Kimura-2-parameter (K-2-P) based on
COI data is 0.005 within species, and 0.166 between congeners within Tridentiger; Ryr3 data
suggest that on average, K-2-P distance is 0.003 within species and 0.021 between congeners. The
indices of haplotype and nucleotide diversity on the genus level vary across islands and habitats.
Overall, the levels of COI in the archipelago are within 0.69-0.88 (h) and 0.021-0.087 (π), while
those of Ryr3 vary within 0.37-0.87 (h) and 0.003-0.011 (π). On average, the two northern remote
islands host significantly higher levels of genus diversity than others. In addition, the two islands
exhibited significant genetic distinctiveness. Perhaps, the disparities in local diversity benefit from
lower levels of industrious development on the islands.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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187
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Fishes
DNA Barcoding of the endemic fish species from the upper Yangtze River
Yan Huang, Zuogang Peng
School of Life Science, Southwest University, Beibei, Chongqing 400715, P. R. China
E-mail: [email protected], [email protected]
DNA barcoding has been reported as significant potential for species-level identification in many
taxa. In the present study, we have bi-directionally sequenced the standard 652 bp barcode region of
mitochondrial cytochrome c oxidase I (COI) gene from 244 specimens (23 species, 18 genera, 5
families and 3 orders) of the endemic fish species from the upper Yangtze River to evaluate whether
DNA barcodes can distinguish these species efficiently. Results showed that the average Kimura
2-parameter (K2P) distance within species, genera, families and orders were 0.79%, 4.95%, 12.41%
and 20.77%, respectively. Overall, 87% (with Botia superciliaris, B. reevesae, Sinibrama taeniatus
and Acipenser dabryanus, exceptionally) of these species each formed monophyletic clusters using
COI region on the neighbor-joining (NJ) tree with high bootstrap value. Within several taxa, we
suspected that recent speciation or introgression hybridization may have occurred leading to
barcoding ineffectively (e.g. B. superciliaris and B. reevesae), nonrandom and impure hybridization
in artificial breeding process may have extended intraspecies genetic differentiation (e.g. A.
dabryanus) or the morphological identification may have been erroneous (e.g. Coreius guichenoti).
The present study evidenced that endemic fish species from the upper Yangtze River can be
efficiently and accurately identified using DNA barcoding.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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188
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PFishes
Applying DNA barcoding on the identification and recruitment studies of
damselfish (Pomacentridae) larvae from Taiwan
Hui-Ling Ko, Ching-Yi Chen, Kwang-Tsao Shao
Biodiversity Research Center, Academia Sinica, Nankang, Taipei, Taiwan, China
E-mail: [email protected]
Although the identifications of larval fishes are much easier than fish eggs, many larval fishes still
could not be identified to the species level. This impedes the study of community ecology of larval
fish and their life history including spawning period and ground. The aim of this study is to use
DNA barcoding to identify damselfish larvae and acquire the information of their recruitment data.
During 2005 and 2012, we used the light trap to collect late-stage larval fishes at night and plankton
net to collect early-stage larval fishes in Kenting, northern coast of Taiwan and Dongsha Islands in
the South China Sea. A total of 650 damselfish larvae were identified by the morphological
characters. All specimens were sorted into 30 different types by these characters. Among these types,
the CO1 sequences showed that 26 types could be identified to the species level (8 genera in 26
species) and the rest four types were only recognized to the genus level. After identifying our
specimens to the species level, we can then compile diagnostic keys and illustration of larval fishes
which were verified by DNA barcode. Meanwhile, we can get better understanding of the early life
history of damselfish and their spawning season in the coastal waters of Taiwan. This information is
useful and important to the conservation and restoration of coral reef fishes.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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189
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Fishes
Species composition of marine fish eggs in the East China Sea by DNA
barcoding
Han-Yang Lin1, Kwang-Tsao Shao1, I-Shiung Chen2
1
Biodiversity Research Center, Academia Sinica, Taipei, Taiwan, China
2
Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, China
E-mail: [email protected]
A total of 8,933 fish eggs were towed by 1000μm ichthyoplankton net at 25 stations in the East
China Sea in July, 2009. All fish eggs were classified to 87 morpho-types. Among them, only 26
types could be identified to 10 taxa (belonging to 7 order, 9 families, 5 genera and 4 species) and
the other 61 types couldn’t. After DNA barcoding, 73 types could be identified to 45 taxa
(belonging to 10 order, 28 families, 35 genera and 32 species), but the remaining 14 types still
couldn’t be identified. Nine of the fifteen dominant species (account for over 2% of total) in this
study were economic species, in other words, the East China Sea is an important spawning ground
for many economic species. In addition, the assemblages of fish eggs fitted the pattern of surface
water mass properties (e.g. temperature, chlorophyll a and salinity). The study determines that
applying DNA barcoding can solve the problem of morphological method, and provide important
information about fish life story, spawning ground and spawning season for the use of fishery
management and conservation.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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190
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Fishes
Potential bomarker for gnetic ientification and pylogeny of Tor spp. bsed on
ATP6 & ATP 8 gnes
Mohamed Yunus Norfatimah1,2, S.H Nor Farah1, M.L. Siti Noor Hajjar3, L.K Teh4, M.Z Salleh4,
M.N Mat Isa5, Siti Azizah Mohd Nor 3
1
Faculty of Applied Sciences, Universiti Teknologi MARA Shah Alam, 40450, Malaysia
2
School of Biological Sciences, Universiti Sains Malaysia Penang 11800, Malaysia
3
Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA Shah Alam, 40450,
Malaysia
4
Pharmacogenomic Centre, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam,
42300, Malaysia
5
Malaysia Genome Institute, Kajang, 43000, Malaysia
E-mail: [email protected]
The main objective of this research was to analyze the new species-diagnostic biomarker (DNA
barcoding) for the endangered Malaysian Mahseer (kelah), Tor tambroides based on ATP6 & ATP8
genes to investigate the presence of undefined or misidentified species which may also be subject to
exploitation. Genomic DNA of a single individual sample was isolated from the caudal fin using a
tissue DNA extraction kit (Biotools, Madrid, Spain). The new biomarker with primer code ID
TT8642-F and TT8642-R has been developed from NGS data and registered at Barcode of Life
Database (boldsystem.org). 690bp ATP6 & ATP 8 genes were successfully amplified using these
registered primers. 21 samples collected which including Tor tambroides, Tor douronensis and
closely related species, Neolissochilus hexagonolepis were amplified and sequenced. All
haplotype sequences were aligned using Clustal W in Molecular Evolutionary Genetic Analysis
(MEGA) version 5. Sequence divergences were calculated using pairwise distance (p-distance)
model. Neighbour-Joining tree of p-distance were generated to give the pattern of divergences
between species. The phylogenetic diagram clearly discriminate Tor spp and Neolissochilus spp into
their major clade. All the sequences successfully barcoded and deposited in the BOLD systems.
Generally, the finding from this research will provide beneficial information and knowledge that
could be applied to DNA barcoding specifically for species identification and aquaculture industry.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
191
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Fishes
Cryobanking of wild animal and fish barcode project in Taiwan
Kwang-Tsao Shao, H.L. Ko, H.Y. Lin, C.H. Chang, P.L. Lin, Y.C. Lin
Biodiversity Research Center, Academia Sinica, Nankang, Taipei, Taiwan, China
E-mail: [email protected]
“Cryobanking of wild animal genetic materials and barcode from Taiwan” project was sponsored by
Forest Bureau, COA since 2005. In the past 8 years, a total number of 11,084 individuals and 3,205
species, including 4 species of lancelet, 1,387 species of fish, 101 amphibians and reptiles, 199
birds, 85 mammals and 1,419 insects and invertebrates have been deposited at various museums.
All specimen data can be browsed from the Taiwan cryobank and barcode database
(http://cryobank.sinica.edu.tw or http://bol.taibif.tw). Since 2012, we welcome exchange tissue
samples and no longer provide them freely.
A summary of what we have learned so far from our fish barcode studies in Taiwan in the past
few years include:
1. DNA barcode helped us to find out many new species from Taiwan including Synodontidae,
Pinguipedidae, Gobiidae and Cynoglossidae etc.
2. Only by using the DNA barcode can fish eggs and larvae be identified to the species level and
increase their accuracy of identification.
3. DNA barcode result can be used to compile a more reliable diagnostic keys or encyclopedia for
fish eggs and larvae.
4. Help government to identify possible smuggling samples quite frequently which is useful for
the management of ecological conservation.
5. Many ecological monitoring programs now include the item of DNA barcode of fish eggs and
larvae such as in long term ecological studies and in environmental impact assessment.
6. A new three-year NSC project was lunched in this August. The study goals include establish an
integrated fish egg and larvae barcode database from Taiwan and its surrounding waters, publish a
new book of Taiwan fish eggs and larvae using the species that have been verified by DNA barcode,
and get a whole picture of early life history of marine fishes in Taiwan, especially their spawning
season and grounds for future fishery resources management and restoration use.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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192
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Fishes
Molecular identification and phylogenetics of seahorse, Hippocampus kuda
(Bleeker 1852) using 16S rRNA and COI mitochondrial gene sequences.
Karan Veer Singh1, A Gopalakrishnan2, Wazir Singh Lakra3, Abhishek Malakar2
1
NBAGR, Karnal, India
2
NBFGR, Lucknow, India
3
CIFE, Mimbai, India
E-mail: [email protected]
There are 34 valid species of seahorses reported across the world. They are most commonly found
in shallow tropical and temperate waters all over the world. They prefer to live in sheltered areas
such as seagrass beds, coral reefs, or mangroves. The partial sequences of 16S rRNA and
Cytochrome oxidase subunit I (COI) portion of mitochondrial genes were analyzed for species
identification and phylogenetic relationship in seahorse Hippocampus kuda distributed along the
east and west coast of India. The present study on sequence analysis of H. kuda for 16S rRNA and
COI gene segment showed similarity with no apparent variation. A 592 bp amplified product
obtained for 16S rRNA gene segment and 655 bp of COI was in the expected size range. The
analysis of the 16S rRNA gene revealed only 5 unique haplotype with 4 variable, while COI had 8
unique haplotypes with 25 variable nucleotide sites. It seems that the 16S rRNA gene had a much
slower rate of evolution compared to COI and the selective pressure exerted on this gene kept its
sequence conserved.
A total of 655 base pairs of COI gene fragments were successfully sequenced from H. kuda.
The nucleotide sequence comparison showed 8 unique haplotypes with 25 variable, 13 parsimony
informative and 12 singleton sites. Among the 25 polymorphic sites, 18 were transitions and only
seven were transversion with an average transition / transversion ratio as 2.57%. Majority of the
changes occurred in the 3rd codon position. The mean number of nucleotide position was A =
25.7% T= 33.7%, G= 17.6% and C = 22.9% of the 8 haplotypes, four (H1, H2, H3, H4) were
specific to the east coast (Palk Bay and Gulf of Mannar), two to Kerala coast (H5, H6) and the
remaining two (H7, H8) to Goa. The COI haplotypes showed fixed differences between east and
west coast; and sharing of haplotypes between Palk Bay and Gulf of Mannar population along the
east coast. The COI amplified gene segment is highly conserved and did not show any sequence
variation in the H. kuda samples between the east and the west coast regions of India, which makes
COI a more suitable marker for the genetic identification of the species. A NJ tree separated the COI
haplotypes of populations into two main groups. Three major lineages were identified and clustered
in 2 groups. One group included the haplotypes of east coast (Palk Bay and Gulf of Mannar)
supported by high bootstrap values, whereas the second group included the lineage of west coast
(Kerala coast and Goa). It seems that clustering of haplotypes correspond to specific geographical
population. The construction of phylogenetic relationships employing the maximum-likelihood
method produced similar topologies that were obtained using different weighting schemes. The fact
that no haplotypes was shared between the populations, suggest an interruption of geneflow for an
efficient number of generations in Indian waters as observed by Lourie and Vincent in 2004. The
significant pair-wise comparison of ST and the AMOVA values between sample collection site and
the absence of common haplotypes between the populations indicated the occurrence of distinct
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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193
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population structure in H. kuda in Indian waters. This is in conformity with the study of Lourie and
Vincent in 2004.
The large differences amongst west coast and east coast populations of H. kuda observed in the
present study (supported by significant ST and AMOVA and no sharing of haplotypes) were
expected as Teske et al. in 2005 also reported striking differences in terms of genetic diversity
between the Tamil Nadu and Ratnagiri/Goa lineages of H. kuda using 380 bp sequence data of
mtDNA control region (CR). Our results also indicate low dispersal ability of seahorses as reported
in earlier studies. It further reflects genetic adaptation to specific climatic or environmental
conditions, prolonged isolation of populations, or possibly repeated extinction and recolonisation
events by small founding population. Teske et al. in 2005 attributed the strikingly different
oceanographic conditions between east and west coasts of India as a major factor for the large
differences in genetic diversity of H. kuda populations from Indian waters.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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194
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Fishes
An integrative taxonomical approach to describe the larvae of Eucinostomus
jonesii (Pisces Gerreidae)
Martha Valdez-Moreno1, Dalia Cázarez-Carrillo2, Lourdes Vásquez-Yeomans1
1
El Colegio de la Frontera Sur-Unidad Chetumal Av. Centenario Km 5.5 Col. Pacto Obrero,
Chetumal, Quintana Roo, Mexico
2
Comité de Sanidad e Inocuidad Acuícola y Pesquero del Estado de Q. Roo A.C., Mexico
E-mail: [email protected]
Through integrative taxonomy approach, we could resolve the taxonomical status of all ontogenetic
stages of Eucinostomus jonesii (an important commercial fish in Mexico). Most problems to be
solved are related with the larval phases. Because of this reason, there are few and poor descriptions
of these organisms in these stages. The aims of this study was to use the barcodes to identify the
larvae of E. jonesii matching the COI sequence of the identified adults of this species and develop a
morphological and osteological descriptions of the larvae. In total 66 adults and 205 larvae of
gerreids were collected from the northeast and southeast of Yucatan Peninsula. After barcoding, we
identified 11 adults and 33 larvae of E. jonesii. All of them gave high quality sequences with 652 pb.
The described larvae correspond to the postflexion stage (9.6 mm and 11.6 mm). For the
osteological description, 22 larvae were cleared and stained. The skull was in formation and
ossification process, the exoccipital and basioccipital bones were indistinguishable at this stage. The
jaws were more developed than the neurocranium region. Teeth present, the branchial arches are
one of the best developed regions. The caudal fin showed two different conditions.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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195
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Fishes
Defining an elegant seamless solution for the identification of fish species using
the COI sequencing as a molecular barcode
Kamini Varma, Steve Berosik, Achim Karger, Michael Wenz, Stephen Sharp, Athena
Panagiotakakos, Peter Christey and Caifu Chen
Lifetech Inc, South San Francisco, CA 994080, United States
E-mail: [email protected]
The Fish Barcode of Life Initiative (FISH-BOL) is creating a standardized library of all fish species,
allowing the end user to use species identification for all potential uses, including flagging
previously unrecognized species and perhaps most importantly, enabling identifications where
traditional methods are not applicable. To this end, molecular approaches utilize a DNA barcode
based on ~700bp of mitochondrial gene cytochrome oxidase I to identify and discriminate between
different fish samples. One application for using these molecular approaches is to ensure that
domestically processed and imported seafood is safe, wholesome and properly labeled as seafood is
one of the most highly traded commodities in the world. A variety of validated protocols that use
piecemeal solutions have been released by the FDA and CCDB to aid the end user in using the
COI gene sequencing to discriminate between different fish species. In order to streamline the end
to end process, we have designed a one-stop solution allowing the user to proceed from tissue
sampling to data analysis. We present validated data using different methods for DNA purification,
optimized PCR and sequencing conditions on the ABI 3500xl sequencing platform with an
integrated secondary data analysis.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
196
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Fishes
DNA barcoding Mugilidae of Zhejiang Coast
Zetan Xu, Yongjiu Chen
Zhejiang Ocean University, Changzhi Island, Zhoushan, Zhejiang 316022, China
E-mail: [email protected]
Mugilidae is a worldwide distribution species which have a lot of disputes of taxonomy and
nomenclature of this particular family based on morphological identification. In this study, 598 bp
region of mitochondrial cytochrome C subunit I (COI) gene were obtained from 163 Mugilidae
samples which collected from 13 localities in Zhejiang coast. Seven species were found through
DNA barcode from these sequences; Liza affinis (Günther, 1861), Liza haematocheila (Temminck
& Schlegel, 1845), Chelon subviridis (Valenciennes, 1836), Chelon macrolepis (Smith, 1846),
Moolgarda seheli (Forsskål, 1775), Moolgarda cunnesius (Valenciennes, 1836), Mugil cephalus
Linnaeus (1758), among which C.subviridis, C.macrolepis, M.seheli and M. cunnesius are
described for the first time in Zhejiang Coast, and L.affinis is the most common species which
distributed in every location.The average Kimura-2-parameter (K-2-P) distance between individuals
within species, between species within genera, between genera within family were 0.1%, 13.3%,
20.5%, respectively. In addition, the result of molecular phylogenetic relationship of all these
species based on COI gene by Bayesian inference and neighbor joining analyses showed that the
genus Liza has close affiliation with Chelon and grouped together form a monophyletic group, and
Mugil and Moolgarda were set into another two different clades. The phylogenetic relationships of
these four genera support the previous taxonomic classification.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
197
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Fungi and Algae
DNA barcoding of some filamentous Rhodomelaceae (Ceramiales, Rhodophyta)
from Korea
Young Ho Koh, Byeongseok Kim and Myung Sook Kim
Department of Biology, Jeju National University, Jeju, Korea
E-mail: [email protected]
DNA barcode is a useful tool for species identification, which is the relatively short length of the
region used in a 660-base fragment at the 5’ end of cox1 (COI-5P). In the Korean algal inventory,
filamentous Rhodomelaceae is composed of 11 genera (Ardissonula, Bostrychia, Dasyclonium,
Enelittosiphonia, Herposiphonia, Leveillea, Lophocladia, Neosiphonia, Polysiphonia, Pterosiphonia
and Symphyocladia) and 52 species. They are very difficult to identify using morphological
characteristics only because most species have microscopic thallus size, high morphological
variation and necessary to observe the reproductive structure. We are screening the diversity of
filamentous Rhodomelaceae using DNA barcoding and morphological observation. We currently
confirmed six genera of filamentous Rhodomelaceae by COI DNA barcoding and described their
morphology. This result is necessary to accumulate more samples and DNA barcoding data
continuously for understanding species delimitation of filamentous Rhodomelaceae.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
198
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Fungi and Algae
Species delimitation of Korean green macroalgae applying DNA barcoding
Hyung Woo Lee1, Chun Jung Kwon2, Myung Sook Kim1
1
Department of Biology, Jeju National University, Jeju 690-756, Korea
2
Department of Ecological Engineering, Pukyong National University, Busan, Korea
E-mail: [email protected]
DNA barcoding has proven to be a useful tool for resolving taxonomic problems and reaching a
new discovery of cryptic species. Green macroalgae is difficult to identify accurately due to a few
diagnostic characteristics and morphological variations. In the literature, 25 genera and 96 species
of green algae have been reported in Korea, but their species delimitation still remains unclear. To
identify species of green macroalgae accurately, we applied different DNA barcoding markers
according to the taxa; 18S rDNA for Cladophorales, rbcL exon 1 and tufA for Ulvales and
Bryopsidales. As a result, we confirmed 30 species in 7 genera, such as Bryopsis, Codium,Caulerpa,
Cladophora, Chaetomorpha, Ulva, and Umbraulva. 18S rDNA marker provided the information of
phylogenetic relationships as well as DNA barcoding data in the genera Cladophora and
Chaetomorpha. Both rbcL and tufA have sequenced with highly successful rates (more than 90 %)
and provided good resolution for identifying Korean Bryopsidales and Ulvales. This result is to
enhance our knowledge on the usefulness of green macroalgal DNA barcoding.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
199
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Fungi and Algae
Identification of commercial “Lingzhi” (Ganoderma) using DNA barcoding
based on ITS2 Loci
Baosheng Liao, Xiaochen Chen, Jingyuan Song, Shilin Chen, Jianping Han
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking
Union Medical College, Beijing 100094, China
E-mail: [email protected], [email protected]
1
Since recorded in Shengnong’s Compendium of Material Medica, “Lingzhi” has been medicinally
applied more than 2,000 years in China. Currently, “Chizhi” and “Zizhi” are the widely used species.
There are many “Lingzhi” products in the worldwide market. “Chizhi” had been named as
“Ganoderma lucidum” for decades in China. But recent studies have shown that the cultivated
G.lucidum in East Asia is a different species to the original G. lucidum from Europe. The present
study aims to identify the commercial G.lucidum and G. sinense from other related Ganoderma
species based on DNA barcoding using ITS2 loci. 74 samples including wild collection, crude drug,
mycelias and extract collected from various localities and markets. The ITS2 sequences were
amplified and sequenced successfully and aligned by CodonCode Aligner V3.71. The intra- and
inter-specific distance was estimated by MEGA 5.1 as well as phylogeny reconstruction using K2P
model. The result support that the cultivated G. lucidum in China is different from the original G.
lucidum in Europe. “Chizhi” and “Zizhi” clustered into two clades respectively separated with other
Ganodrma species. Thus, fruiting body or commodity of G. lucidum and G. sinense could be
distinguished from other Ganoderma species successfully by comparing the ITS2 region.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
200
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Fungi and Algae
A global genetic backbone for the fungi: The CBS-KNAW barcoding project
Benjamin Stielow, Michel de Vries, Pedro Crous, Vincent Robert, Duong Vu, Marizeth Groenewald,
Gerard Verkley
CBS-KNAW, Fungal Biodiversity Center, Uppsalalaan 8, 3584 CT Utrecht, Netherlands
E-mail: [email protected]
The CBS collection currently hosts more than 90.000 strains of filamentous fungi and yeasts. This
number is steadily increasing at the rate of close to 3000 new acquisitions per year. Identification,
delimitation and classification of all fungal strains are routinely subjected to DNA barcoding.
Considering the heritage of this world renowned collection to the scientific community, DNA
barcode data for 90.000 fungal strains will reflect a milestone in understanding fungal evolution and
in quality assurance for furnished strains. Thus, the primary aim of this project is to generate two
standardized DNA identifiers (ITS/partial 28S) for all strains in the fungal collection. Scientifically
and strategically, the generated ‘barcodes’ will tremendously improve our ability to provide
guidance to future projects and innovative research.
With support of lab robotics, a fully automated pre-PCR sequencing pipeline was set up which
currently produces up to 10.000 individual sequences per month (since early summer 2012). With
more than 200.000 Sanger reads already produced and 53.000 DNA extracts (more than 2/3 of the
public collection) a global genetic backbone for kingdom Fungi appears to be achievable in the near
future.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
201
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Fungi and Algae
DNA barcoding of soil fungi from manaslu conservation area (MCA), Nepal
Khanal Sunita1,2, Rajani Malla2, Sangita Shrestha1
1
Nepal Academy of Science and Technology (NAST), Khumaltar, Lalitpur, Nepal.
2
Central Department of Biotechnology (CDBt), Tribhuvan University, Kirtipur, Kathmandu, Nepal.
E-mail: [email protected]
Fungal floras in soils or associated with plant roots have been studied worldwide. In this study, total
of fifty-nine fungi were isolated from the soil of Manaslu Conservation Area (MCA) collected from
1700 to 4300 m (asl). They were identified with morphological characteristics. Of the isolated spp.
Penicillium and Aspergillus being dominant as well as industrially and medically important were
selected and characterized by using nuclear ribosomal ITS region and D1/D2 domains of large
subunit (28S) ribosomal DNA. The sequencing of these regions facilitated the identification of
fungi at species level. The sequences were identical to Penicillium ochrochloron, , Penicillium
aurantiogriseum strain DAOM 214787, Penicillium aurantiogriseum strain CBS 324.89, Penicillium
ruqulosum, Penicillium chrysogenum, Penicillium simplicissimum, Aspergillus parasticus,
Aspergillus protuberus, Aspergillus niger, and Aspergillus flavus. The sequences were edited,
aligned and analyzed using Codon Code Aligner v.4.2.2 and MEGA v.5.2.2. The inter- and intraspecies difference in nucleotides in D1/D2 regions was found between 8 to 299bp. The Maximum
Parsimony (MP) tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm with
most parsimonious tree length of 490 and parsimony-informative sites of 301. The consistency
index and the retention index value is 0.906122 and 0.936022 respectively. This data suggested that
the D1/D2 domain can be used for the identification of filamentous fungi and related species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
202
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Fungi and Algae
Development of real-time PCR approach for rapid detection of Antrodia
cinnamomea in foods
Hsiu-Wei Tsuei, Yuan-Hsin Chang, Zih-Ling Jia, Che-Yang Lin, Hsu-Yang Lin, Lih-Ching Chiueh,
Daniel Yang-Chih Shih
Food and Drug Administration, Ministry of Health and Welfare, Executive Yuan, Taiwan, China
E-mail: [email protected]
Antrodia cinnamomea is a unique medicinal fungus in Taiwan. Many functional foods claimed to
contain Antrodia cinnamomea-derived ingredients. In order to avoid fraudulent mislabeling, a
reliable real-time PCR assay was developed for differentiating Antrodia cinnamomea from other
fungus species. Specific primers and TaqMan probe based on barcode marker internal transcribed
spacers (ITS) of nuclear ribosomal DNA were designed for detection of Antrodia cinnamomea in
this study. The specificity of the method was evaluated by testing Antrodia cinnamomea, Antrodia
salmonea, Antrodia malicola, medicinal fungus and medicinal plants. The results showed only
Antrodia cinnamomea obtained specific reaction and no cross-reaction was observed with other
species used in this study. Sensitivity tests revealed this method was sensitive in detecting the low
levels of target DNA (120 fg/uL). Furthermore, this specific method was applied to 33 commercial
samples comprising capsules, tea bags and drinks, and experimental data indicated the method
could successfully identify the Antrodia cinnamomea-derived ingredients. In conclusion, the
real-time PCR detection method developed herein was a rapid, sensitive and applicable detection
tool for accurate identification of Antrodia cinnamomea ingredients in foods.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
203
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Fungi and Algae
Reassessing species diversity of the red algal order Gigartinales from Korea
using DNA barcoding
Mi Yeon Yang1, Jeong Hyung Lee2, Myung Sook Kim1
1
Department of Biology, Jeju National University, Jeju 690-756, Korea
2
Department of Management Information Systems, Dong-A University, Busan, Korea
E-mail: [email protected]
Gigartinales is one of the most diverse red algal order globally distributed and has an economical
importance as a primary source of carrageenans in the phycocolloid industry. In spite of difficulty of
the accurate identification, the morphological species concept in the red algae is commonly
employed. In the literature, 24 genera and 62 species of Gigartinales have been reported in Korea
based on morphological description only. Here we investigated the diversity of the Gigartinales
from Korea using DNA barcoding approach which is a powerful tool for the species discrimination.
We estimated the inter- and intra-specific divergence within the Gigartinales order by using the COI
barcoding marker. We have identified 11 families, 15 genera and 30 species until now with several
cryptic species. COI gene provided a high resolution to identify closely related species of the order
Gigartinales. These findings have significant implications for the successful design of biodiversity
surveys along the Korean coasts and molecular species characteristics should be complement
morphological identification for improving biodiversity assessments.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
204
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Fungi and Algae
Colletotrichum species from red-fleshed dragon fruit (Hylocereus polyrhizus) in
Malaysia
Latiffah Zakaria, Suzianti Iskandar Vijaya
School of Biological Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia
E-mail: [email protected]
Red-fleshed dragon fruit (H. polyrhizus) has been reported to be infected by fungal diseases that
affected the production and reduced the yield. Sixty isolates of Colletotrichum were successfully
isolated from the stem anthracnose lesions of red-fleshed dragon fruit (H. polyrhizus). Based on
sequencing of ITS regions and β-tubulin sequences, two Colletotrichum species were identified, C.
gloeosporioides and C. trunctaum. Pathogenicity test was successfully carried out confirming that
C. gloeosporioides and C. truncatum are the causal pathogens of anthracnose on red-flesh dragon
fruit stem, thus Koch’s postulates was fulfilled. This study is the first scientific report of C.
truncatum as one of the causal pathogens of anthracnose of red flesh dragon fruit in Malaysia.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
205
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Fungi and Algae
Molecular identification of black aspergilli from sandy beach
Teh Li Yee, Latiffah Zakaria
School of Biological Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia.
E-mail: [email protected]
Black aspergilli or Aspergillus section Nigri are predominantly found in soil where they either act as
decomposers or exist in dormant state, and are difficult to be identified based solely on their
morphological characters. The black aspergilli isolates were isolated from sandy beach of Batu
Ferringhi, Penang, Peninsula Malaysia, and identified using ITS regions and ß - tubulin sequences.
Based on BLAST search of ITS regions and ß - tubulin sequences, five Aspergillus species were
identified, namely A. niger, A. tubingensis, A. carbonarius, A. ibericus and A. aculeatus with 99% to
100% similarity. Phylogenetic tree of combined dataset of ITS regions and ß - tubulin showed that
the two closely related biseriate species; A. niger and A. tubingensis were grouped in two separate
clades. Two large-conidia producing species, A. carbonarius and A. ibericus were distinctively
separated into two sub-clades while the uniseriate species; A. aculeatus also formed separate clade.
The present study contribute to the knowledge on the diversity of black aspergilli in Malaysia and
shows that by using ITS regions and ß - tubulin sequences, the members of black aspergilli can be
successfully characterized and distinguished from other closely related species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
206
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Informatics
The Norwegian Barcode of Life Network (NorBOL)
Torbjørn Ekrem1, Inger G. Alsos2, Arild Johnsen3, Endre Willassen4, Elisabeth Stur1, Gunnhild
Marthinsen3, Andreas J. Kirchhefer2, Katrine Kongshavn4, Aina Mærk Aspaas1, Erik Boström1 and
Xiaolong Lin1
1
NTNU University Museum, Department of Natural History, NO-7491 Trondheim, Norway.
2
Tromsø University Museum, NO-90370 Tromsø, Norway
3
University of Oslo, Natural History Museum, P.O. Box 1172 Blindern, NO-0318 Oslo, Norway
4
University Museum of Bergen, Natural History Collections, P.O. Box 7800, NO-5020 Bergen,
Norway
E-mail: [email protected]
NorBOL (www.norbol.org) was formed in 2007 as a national network to 1) advance barcoding of
Norwegian and Arctic biodiversity, 2) raise funding, 3) curate barcode reference material, 4)
coordinate and initiate new barcoding projects, and 5) increase public awareness of DNA barcoding
and barcoding results in Norway. NorBOL is a regional node within iBOL, with a particular
responsibility for polar regions. NorBOL is coordinated by the NTNU Museum of Natural History
and Archaeology in Trondheim and connects 16 institutions, including all four major natural history
museums as well as all major research institutes in Norway. Despite strong support among research
institutions, substantial external funding was only first achieved in 2012 through a grant from the
Norwegian Biodiversity Information Centre (2012-2015). Since then, barcoding progress of the
Norwegian fauna, flora and fungi has increased and the Barcode of Life Data Systems database
currently holds more than 10000 DNA barcodes of more than 3400 species from Norway. The target
for NorBOL is set to 20,000 species over 6 years. NorBOL currently targets barcoding of museum
collections, vascular plants of the north, coastal marine invertebrates, and inventory projects
supported by the Norwegian Taxonomy Initiative.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
207
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Informatics
Assembling mitochondrial genomes from 1 000 insect transcriptomes
Meihua Tan, Shanlin Liu, Xin Zhou
BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China
E-mail: [email protected], [email protected]
Mitochondrial genome plays a pivotal role in many biological research fields, such as
phylogenomics, population genetics, as well as the newly emerged mito-metabarcoding, which
evaluates biodiversity via non-amplified genomic DNA. However, available resource of
mitochondrial genomes is limited, with only ~400 species from * insect orders due to the absence of
a cost-efficient method. The 1000 Insect Transcriptome Evolution (1KITE) project aims to unveil
the evolution of insects by sequencing transcriptomes of 1000 insect species across all major
lineages. Mitochondrial coding genes are expected to present in high copy numbers in
transcriptomes, thus providing a new route to collect MT genes for a wide range of insect taxa.
Approximately 500 insect transcriptomes have been screened for mitochondrial gene fragments so
far. On average, ca. 10 protein coding MT genes have been successfully assembled for each species.
Our pipeline opens up a completely new way to construct MT genomes across a wide taxonomic
range. This new resource for MT genes will significantly contribute to global effort on the
construction of MT genomic references, such as the MT10K project. The promise of this rapidly
growing database will then improve new technologies in biodiversity sciences, such as PCR-free
metabarcording.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
208
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Insects
DNA barcodes suggest that head lice, Pediculus humanus (Pediculidae:
Phthiraptera), are a complex of four cryptic species
Muhammad Ashfaq, Paul Hebert
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
E-mail: [email protected]
The head louse (Pediculus humanus capitis) and body louse (P. humanus humanus) are obligate
human ectoparasites which differ in ecology, but show no genetic divergence. However, prior
sequence analysis has indicated that P. humanus includes three clades (A, B, C). Clade A has a
global distribution, while clade B occurs in Europe, North America, Central America, and Australia.
The final clade (C) is thought to occur in both Africa and South Asia. Pairwise sequence
divergences at COI range from 4.9% - 9.5% among these three clades. NJ analysis of DNA
barcodes from 461 head lice from different localities in Pakistan coupled with 269 records from
BOLD revealed two clusters corresponding to lineages, A, B. However, cluster C was partitioned
into two lineages (C1, C2) with a minimum divergence of 2.3%, one from Africa (C1) and the other
from South Asia (C2). Most of the lice (94%) from Pakistan matched lineage C2, a lineage
previously known from only three individuals. Since few sequences are available from Africa, more
specimens are being analyzed from this continent as well as from the New World.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
209
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Insects
DNA barcodes: helping to elucidate the role of mosquito species in flavivirus
transmission in the southern area of Mar Chiquita Lake, central Argentina
Clara I. Berrón1, Viviana E. Ré1, Darío A. Lijtmaer2, Natalia G. Diez3, Marina Stein4, Andrés
Visintin5,6 , Pablo L. Tubaro2, Walter R. Almirón5,6 , Marta S. Contigiani1
1
Instituto de Virología “Dr. JM Vanella” (INVIV), Facultad de Ciencias Médicas, Universidad
Nacional de Córdoba, Córdoba, Argentina
2
Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” (MACN-CONICET), Buenos
Aires, Argentina
3
Facultad de Humanidades y Ciencias, Universidad Nacional del Litoral, Santa Fe, Argentina
4
Instituto de Medicina Regional, Universidad Nacional del Nordeste, Resistencia, Argentina
5
Centro de Investigaciones Entomológicas de Córdoba, Facultad de Ciencias Exactas, Físicas y
Naturales, Universidad Nacional de Córdoba, Córdoba, Argentina
6
Instituto de Investigaciones Biológicas y Tecnológicas. CONICET – Universidad Nacional de
Córdoba, Córdoba, Argentina
E-mail: [email protected], [email protected]
Saint Louis encephalitis virus and West Nile virus (Flaviviridae; Flavivirus) cause neurological
diseases in humans. Transmission cycles of both viruses involve birds and ornithophilic mosquitoes
of the genus Culex. In the southern area of Mar Chiquita Lake there is serological and molecular
evidence of circulation of both viruses. We studied the mosquito feeding patterns in order to
contribute to identify the vector species. The project was based on three components: 1) the
generation of the barcode library for the mosquito species of the study site, 2) the identification of
the engorged mosquitoes using these barcodes and 3) the identification of the mosquito blood meal
sources using the vertebrate barcodes available on BOLD (using primarily the “Birds of Argentina”
project). Mosquitoes were processed at the INVIV, MACN and BIO/CCDB. Engorged females were
successfully identified. Ochlerotatus, Psorophora and Mansonia genera fed exclusively on mammals
(7 host species), while mosquitoes belonging to Culex genus fed mainly on birds (22 host species).
This analysis provided important clues about the mosquito and bird species that might be implicated
in the transmission cycles of these viruses at the study site. DNA barcodes thus proved to be very
useful for flavivirus research in Argentina.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
210
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Insects
DNA barcoding and the effect of spatial distance on the genetic variation of
African Noctuoidea (Insecta; Lepidoptera)
Johan De Gruyter1, Kurt Jordaens1,2, Luc De Bruyn2,3, Zoltán T. Nagy1,4, Gontran Sonet1,4, Floris C.
Breman1
1
Royal Museum for Central Africa, JEMU, Leuvensesteenweg 13, B-3080 Tervuren, Belgium
2
University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
3
Species Diversity, Research Institute for Nature and Forest (INBO), Kliniekstraat 25, B-1070
Brussels, Belgium
4
Royal Belgian Institute of Natural Sciences, JEMU, Rue Vautier 29, B-1000 Brussels, Belgium
E-mail: [email protected]
This study aims at evaluating the use of DNA barcoding to identify African species of the
superfamily Noctuoidea (Insecta; Lepidoptera). Three objectives were tested: (1) Can the DNA
barcode fragment of the mitochondrial cytochrome c oxidase subunit I (COI) be used to distinguish
species in this group? (2) Has spatial distance an influence on the intraspecific sequence divergence?
and (3) How well do the Barcode of Life Database Systems (BOLD) and GenBank perform in
identifying African noctuoids? One hundred and twenty three specimens of 66 noctuoid species,
caught at four localities in Tanzania (Morogoro, Ruaha National Park, Udzungwa National Park,
Saadani National Park), were DNA barcoded. This dataset was supplemented with noctuoid barcode
sequences from BOLD so that the final dataset comprised 627 COI haplotypes of 115 species. One
third of the examined species could not be unambiguously identified using DNA barcodes,
indicating that DNA barcoding alone cannot be relied upon to correctly identify these species.
Despite most species having a widespread distribution, intraspecific sequence divergence only
increased with spatial distance in 11 species. Many pest species are well represented in the public
databases BOLD and GenBank, and provide reliable identifications. This is not true for
lesser-known noctuoid species. Also, several potentially incorrect identifications were discovered.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
211
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Insects
DNA barcoding and molecular differentiation of West European and North
American Phormia regina (Diptera; Calliphoridae)
Kurt Jordaens1,2, Gontran Sonet3, Yves Braet4, Luc Bourguignon4, Frankie Goovaerts2, Thierry
Backeljau2,3, Marc De Meyer1 & Stijn Desmyter4
1
Royal Museum for Central Africa, JEMU, Leuvensesteenweg 13, 3080 Tervuren, Belgium
2
University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
3
Royal Belgian Institute of Natural Sciences, JEMU, Vautierstraat 29, 1000 Brussels, Belgium
4
National Institute of Criminalistics and Criminology, Vilvoordsesteenweg 100, 1120 Brussels,
Belgium
E-mail: [email protected]
Phormia regina (the black fly) is a common Holarctic blowfly species which serves as a primary
indicator taxon to estimate forensic post mortem intervals. It is also a major research model in
physiological studies on insect development. Previous studies have shown a sequence divergence of
up to 4.3% in the mitochondrial COI gene between W European and N American P. regina
populations. We DNA barcoded 44 P. regina specimens from six N American and 17 W European
populations and confirmed this intercontinental 4% mean sequence divergence, while sequence
divergence within each continent was a ten-fold lower. Comparable mean mtDNA sequence
divergences were observed for cyt b (5.3 %) and for COII (3.7 %), but mean divergence was lower
for 16S (0.6%). In contrast, intercontinental divergence at nuclear DNA was very low (0.1% for
both 28S and ITS2). These results suggest a substantial amount of mtDNA differentiation between
populations from both continents. This might call for a taxonomic re-evaluation and should be taken
into account when using the "species" in forensic casework or experimental research.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
212
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Insects
DNA barcoding and genetic divergence of Japanese non-biting midges in
Chironomus (Insecta: Diptera: Chironomidae)
Nnatsuko Kondo1, Ryuhei Ueno1, Kako Ohbayashi1, Veronica Golygina2, Kenji Takamura1
1
National Institute for Environmental Studies (NIES), Tsukuba, Ibaraki Prefecture, Japan
2
Russian Academy of Sciences (RAS), Moscow, Russia
E-mail: [email protected]
Non-biting midges (Insecta: Diptera: Chironomidae) has a large diversity in their adaptation to
environmental conditions, and hence they are promising indicators for aquatic and ecotoxicological
monitoring. Although the potential of this approach can be limited by the difficulty in their
morphological identification, DNA barcoding offers a solution. We focused on genus Chironomus,
which is characterized by its worldwide distribution and abundance among Chironomidae. Seven
Japanese Chironomus species were subjected to DNA barcoding. The all seven species were clearly
distinguished by barcoding of 658 bp of cytochrome c oxidase subunit I (COI). We calculated
genetic divergences among these seven species and additional 43 Chironomus species found on the
International Nucleotide Sequence Database, with the Kimura 2-parameter (K2P) model on the
basis of their sequences. The calculated K2P genetic divergences within and between species were
2.0% and 15.0% on average, respectively. The magnitudes of these divergences were not different
from those for other Dipteran groups. We found no definitive threshold that may separates the
within- and between-species genetic divergences. Based on the results, we discuss the possible
reclassification of some Chironomus species inhabiting Japan.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
213
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Insects
A first step in DNA barcoding hybotid flies (Diptera, Empidoidea)
Zoltán T. Nagy1, Gontran Sonet1, Jonas Mortelmans2, Camille Vandewynkel3, Patrick Grootaert2
1
Royal Belgian Institute of Natural Sciences, JEMU, Rue Vautier 29, B-1000 Brussels, Belgium
2
Royal Belgian Institute of Natural Sciences, Entomology, Rue Vautier 29, B-1000 Brussels,
Belgium
3
Université de Limoges, Laboratoire des Sciences de l’eau et environnement, Faculté des Sciences
et Techniques, Avenue Albert Thomas, 23, F-87060 Limoges, France
E-mail: [email protected]
Hybotidae is a large family of dance flies including about 2000 described species worldwide. Yet,
the global diversity of hybotids remains scarcely assessed and very few DNA barcodes are available
for this family. Within the framework of a pilot study, we barcoded 339 specimens of Old World
hybotids belonging to 163 species and 22 genera. Samples came from the Palaearctic (mainly
Belgium, France, Portugal and Russian Caucasus), the Afrotropic (Democratic Republic of the
Congo) and the Oriental realms (Singapore and Thailand). Thereby, we optimized lab protocols for
barcoding hybotids. Although most taxa could be well distinguished using COI sequences, the study
also revealed 1) some undescribed taxa within a group of morphologically very similar or identical
specimens, 2) an absence of divergence in COI sequences between some morphologically distinct
species and 3) high ‘intraspecific’ divergences between samples separated by large spatial distances.
We conclude that in hybotids, DNA barcoding could be useful to identify species, provided that
taxon sampling and spatial scale of sampling is taken into account. Moreover, extra efforts are
needed to increase the representation of hybotids in barcoding databases.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
214
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Insects
Barcoding the Lepidoptera of Argentina: An example on how to build up a
barcode library in short term in a developing country.
Pablo D. Lavinia
Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” (MACN–CONICET), Argentina
E-mail: [email protected]
Despite the fact that the All-Leps campaign represents almost half of the barcode library, until 2010
there were no projects aiming to barcode the lepidopterans of Argentina. Seeking to fill this gap in
the global initiative, we have undertaken this task through a system based on three crucial aspects:
national and international funding, intense field work effort and advanced training of Argentinian
students at the Biodiversity Institute of Ontario (BIO). I will discuss this strategy, which has
allowed us to generate over 4,000 lepidopteran BOLD records in two and a half years, placing
Argentina among the top 20 countries in the campaign. Particularly, a sole four-month training
period at the BIO made possible the generation of COI sequences of 1,900 moth specimens, which
gave significant momentum to the barcoding of the Argentinian moths. This training has been
beneficial for the MACN, which now has another student qualified to work in the barcoding
laboratory and in data analysis. Finally, this project represents another gain for researchers working
on this group: the existence of a Lepidoptera tissue collection with associated vouchers that expands
the collection already present at the Entomology Division at MACN, whose specimens constitute
our next target to be barcoded.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
215
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Insects
The genetic differences of COI and TaqMan-MGB probe detection for different
geographic populations of Gypsy moth
Lu Qian1 Yulin An1, Mei Xu1, Junxian Song2
1
Jiangsu Entry-Exit Inspection & Quarantine Bureau of the P. R. C., Nanjing 210001, China
2
Liyang plant protection and quarantine station, Liyang 213300, China
E-mail: [email protected]
Gypsy moth which was divided into the European-and Asian sub-type is an important forest pest
and has important quarantine significance. In order to reveal the type of Europe and Asia-based
genetic differences, we measured 25 different geographic samples gypsy moth COI gene sequence
which from China, Russia, Mongolia, Japan and the United States. With the DNASTAR software
analysis, gypsy moth COI gene is 1531bp long. There are 14 stable different base sites between
Asian and American populations, where is only transformation between the bases, no transversion,
insertion or deletion sites. Only one single base makes the difference in the amino acid site at the
406bp base sequence. Constructed Upgm phylogenetic tree basing COI gene indicated that
European type (U.S. population) and Asian type were divided into two branches. Japan population
has a far distantly relationship more than other Asian populations species, formed a separate branch.
According to the single base difference of 406bp, we designed MGB probe and successfully
distinguish the type of Europe and Asia gypsy moth using allele typing method. The study provides
a scientific basis for genetic differentiation of gypsy moth and made a new method to distinct
different geographical populations of gypsy moth.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
216
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Insects
DNA barcoding identification for medical insects at frontier ports
Deyi Qiu, Qiaoyun Yue, Jia Hu, Jian Chen, Dexing Liu, Xiaoya Wei, Guoxiong Liu
Zhongshan Entry-Exit Inspection and Quarantine Bureau Technology Center, Zhongshan, China
E-mail: [email protected]
Due to carry many kinds of pathogen, medical insects are closely related to people’s health and are
the most important quarantined objects at the frontier ports. Along with the rapid development of
international trade and tourism, invasive medical insect species often are smuggled unintended in
the entry or exit goods. In order to stop the spreading of the invasive species and its carried
pathogen, rapid identification of the species is very necessary and important. In the daily inspection
and surveillance, morphology incomplete adult and non adult individuals such as egg, larvae and
pupae are often intercepted, the most popular method of species identification based on morphology
charactertics of the adults is not sufficient for this purpose, hatching the egg or pupae to adult is
quite common at the laboratory, but by doing this, the long identification period increases the
spreading chances for the medical insects and its carried pathogen, and will increase the threaten to
ecological and human health. DNA barcoding, a technique with great potential application for the
fast and accurate identification of medical insects can solve the problem. At present, we are
undertaking the National science and technology support program ‘DNA barcoding identification of
the medical vectors and its techniques demonstration’. Once there is sufficient data in the data base.
It could be widely used to identify species and should help to shorten the identification period.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
217
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Insects
DNA barcodes promote future use of Chironomidae (Diptera) in freshwater
biomonitoring
Elisabeth Stur, Torbjørn Ekrem, Kaare Aagaard
NTNU University Museum, Department of Natural History, NO-7491 Trondheim, Norway.
E-mail: [email protected]
The family Chironomidae is frequently the most species rich and abundant group of insects in
freshwater habitats. Different species often have distinctive habitat requirements, thus chironomids
are well-suited for freshwater biomonitoring. However, due to difficult taxonomy and unknown
larvae for many species, Chironomidae often are too resource demanding to be incorporated in
freshwater assessments despite their obvious advantages. The Atna River in Central Norway has
been a reference system for watersheds in Norway since 1985 and continuous monitoring activity
has made this pristine alpino-boreal river system one of the best documented in Norway. Here we
provide DNA barcodes of Chironomidae species from the upper part of the river to promote future
biomonitoring using a genetic identification system. 484 barcodes from 140 species representing
149 BINs, 43 genera and 5 subfamilies were recorded. Both cryptic species and species new to
science were detected with the aid of DNA barcodes. We estimate that we have recorded about 90%
of the chironomid diversity in the upper course of the river. The number of Chironomidae
species/BINs outnumbers the collective species number of other aquatic insect groups. Thus, future
monitoring of the macroinvertebrates in Atna River would gain much from including molecular
diversity of chironomids.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
218
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Insects
DNA barcodes of the Japanese Merodon hoverflies (Diptera, Syrphidae): high
genetic variation in the native species and low variation in the invasive congener
Mitsuaki Sutou, Motomi Ito
University of Tokyo, Bunkyo, Tokyo 113-8654, Japan
E-mail: [email protected]
The genus Merodon includes about 160 species of medium-sized hoverflies worldwide. Most are
distributed in the Mediterranean region, although several species occur in Central and East Asia.
Japan is home to a single native species of this genus: M. kawamurae. It is distributed in central and
western Japan. In addition, the European species M. equestris (the large narcissus fly) invaded
central and eastern Japan in the latter half of the 20th century.
This study compared the DNA barcodes of these two species using the universal primers LCO1490
and HCO2198. We sequenced 32 individuals of M. kawamurae collected in Hyogo (western Japan),
Saitama, and Hachioji (central Japan), and found that they possessed seven haplotypes. High
genetic divergence was also found between the populations of western and central Japan of this
species. Conversely, we sequenced 56 individuals of M. equestris collected in Yokohama, Tokyo,
Saitama, Hachioji (central Japan), and Sendai (eastern Japan), and found that 55 out of 56 had the
same haplotype. In the rural areas of central Japan surveyed in this study (Saitama and Hachioji),
the native and invasive species of Merodon occur sympatrically. We discuss potential competition
of these two species for larval and adult food resources.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
219
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Insects
A Comprehensive DNA Barcode Library for North American Crambidae
(Lepidoptera: Pyraloidea)
Zhaofu Yang1, 3, Jean-François Landry2, Yalin Zhang1, Paul D. N. Hebert3
1
Key laboratory of Plant Protection Resources and Pest Management, Ministry of Education;
Entomological Museum, Northwest A & F University, Yangling, China
2
Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Centre, C.E.F., Ottawa,
Ontario K1A 0C6, Canada
3
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
E-mail: [email protected]
The Crambidae is among the largest and most diverse insect families in Lepidoptera, with 14
subfamilies are recognized includes 1,018 genera and 9,666 described species that inhabit all
terrestrial habitat types (Regier et al., 2013; Nuss et al., 2003–2011). The larvae show the most
diverse among subfamilies, with habits including detritivory, coprophagy, predation and parasitism
(Regier et al., 2013). Over 800 species have been described in America north of Mexico, the
majority of which occur in the Atlantic and southeastern states, or southern Texas and Arizona.
An effort is underway to gather COI barcode library for all Lepidoptera species in North
America and records are now available for more than 5,000 species. Our work focuses on
assembling a comprehensive DNA barcode library for the crambids of Canada, Mexico, and the
United States. We gathered over 10,500 sequences representing 637 nominal and 124 provisional
species, accounting for four fifth of the entire regional fauna. Our results indicate that 87 per cent of
species forming distinct, cohesive clusters providing reliable identification. Undifferentiated or
overlapping clusters with higher intraspecific and lower interspecific divergence are indeed
indispensible to need thorough taxonomic scrutiny and further analysis, which is most likely to
reveal cryptic diversity, clarify unrecognized synonymy and to infer the relationships between
species in the Crambidae.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
220
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Insects
Morphology and DNA barcoding study reveal: Hebardina concinna (Blattodea:
Blattidae) has macropterous and brachypterous polymorphisms
Keliang Wu1, Qiaoyun Yue1,2, Jia Hu1, Deyi Qiu1, Dexing Liu , Xiaoya Wei1, Jian Chen1
1
Zhongshan Entry-Exit Inspection and Quarantine Bureau Technology Center, Zhongshan, China
Guangdong Entry-Exit Inspection and Quarantine Bureau Technology Center, Guangzhou, China
E-mail: [email protected]
2
Although the macropterous and brachypterous dimorphism wings of Hebardina concinna (Haan,
1842) were described by many authors since it was published, but its most important morphology
identification character-male genitalia was never described, because of the lackless of the male
genitalis descriptions, these two phenotypes are mistaken as two different species by many people.
A detailed morphological description of male genitalia structures is provided for the first time in
this paper. Male genitalia structures of both the macropterous and brachypterous specimens were
compared carefully, DNA barcoding were sequenced for these two phenotypes as well, and both the
morphology and molecular results show that the macropterous and brachypterous phenotypes are
the same species. Reasons for dimorphism wings of this species are briefly discussed. General
morphology characteristics of this species are redescribed and phylogeny trees of these related
species are built in this paper as well.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
221
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Insects
Use of DNA sequences for identification of possible biotypes of the fruit borer
Neoleucinodes elegantalis (Guenée) (Lepidoptera: Crambidae), an important
pest of Andean solanaceous fruits
Patricia Zapata1, OscarCastañeda1, Gerardo Gallego1, Ana Elizabeth Diaz2, WilsonVásquez3, Harold
Suárez-Baron1 y Joe Tohme1
1
Agrobiodiversity and Biotechnology Project, International Center for Tropical Agriculture (CIAT).
Cali, Colombia
2
Colombian Corporation for Agricultural Research (CORPOICA), La Selva. Rionegro, Colombia
3
INIAP-CORPOINIAP, Ecuador
E-mail: [email protected]
In Colombia, Venezuela, Ecuador, Brazil and Honduras, the tomato borer, Neoleucinodes
elegantalis, is the most important fruit-related plague of the Solanaceae family. A total of 292
adult individuals from Colombia, Ecuador y Honduras, were characterized using molecular
techniques by comparing the partial sequence of the gene Cytocrome c oxidase I (COI), 18S rDNA
gene and geographic information systems. The analysis of the COI showed good sensitivity,
achieving an initial differentiation of 44 possible haplotypes, with some association to specific life
zones, but without apparent relation to the host type. Divergence between groups was set at
0.1-3.6%, and the greatest were found in one specific group, which was distributed all along the
Western Cordillera, and was also apparently geographically isolated from the rest of the populations
of N. elegantalis. This group was located in an area with very high anthropogenic disturbance by
insecticides; this could be acting as a factor that increases genetic distances between the groups
through local extinctions i.e. distribution gaps. Currently, morphologic variability, reproductive
compatibility and response to pheromones are being studied to complement the molecular analyses.
Now, our efforts are focused on the evaluation of 18S gen to compare and complement the results
obtained from COI sequences.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
222
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Marine Barcoding
Barcoding Hydromedusae from Beibu Gulf, China: an efficient tool for local
biodiversity assessment
Jinru He1, Lianming Zheng1, 2, 3, Yuanshao Lin1, 2, Wenqing Cao1, 2, Dangni Zhang1
1
College of Ocean and Earth Sciences, Xiamen University, Xiamen 361005, China
2
Marine Biodiversity and Global Change Research Center (MBIGC), Xiamen University, Xiamen
361005, China
3
Fujian Provincial Key Laboratory for Coastal Ecology and Environmental Studies (CEES),
Xiamen University, Xiamen 361005, China
E-mail: [email protected]
There is few works about the marine biodiversity was conducted in Beibu Gulf, which is one of the
most important fishing zones in China. From July 2006 to November 2007, four cruises were
carried out to produce a assessment of marine zooplankton biodiversity in Beibu Gulf.
Hydromedusae are one of the most important group of zooplankton, however, identification of
hydromedusae species is challenging, due to the scarcity of distinct morphological characters and
phenotypic plasticity. In this study, mtCOI and 16S sequences were used to barcode 441 specimens
of 51common hydromedusae species in Beibu Gulf. The intra-specific genetic divergence ranged
0~3.3%, with the mean value as 0.4% for COI, and varied as 0%~1.6%, with the average as 0.3%
for 16S, respectively. The inter-specific genetic divergence ranged from 4.5% to 47.5%, with the
average of 20.4% for COI, and varied from 6.2% to 64.2%, with the mean value of 22.3% for 16S,
respectively. A distinct barcoding gap was detected for all species in both markers, indicating that
both COI and 16S could barcode hydromedusae in this area. The present work showed two markers
reliable for species identification in a relatively wide taxonomic range, and contributed to
biodiversity assessment in the Northern South China Sea.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
223
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Marine Barcoding
16S rRNA is a better choice than COI for DNA barcoding Campanulariidae
(Cnidaria: Hydrozoa: Leptothecata)
Jinru He1, Lianming Zheng1, 2, 3, Yuanshao Lin1, 2, Wenqing Cao1, 2, Dangni Zhang1
1
College of Ocean and Earth Sciences, Xiamen University, Xiamen 361005, China
2
Marine Biodiversity and Global Change Research Center (MBIGC), Xiamen University, Xiamen
361005, China
3
Fujian Provincial Key Laboratory for Coastal Ecology and Environmental Studies (CEES),
Xiamen University, Xiamen 361005, China
E-mail: [email protected]
Identification of Campanulariidae species is challenging, due to their complex life cycles and
phenotypic plasticity. DNA barcoding provides an efficient method for species identification,
however, the choice between mitochondrial COI and 16S as a standard barcode for hydrozoans is
subject to debate. Herein, we directly compared the barcode potential of COI and 16S in
Campanulariidae using 287 sequences from 51 species. Analysis of K2-P genetic distances
documented the mean intraspecific/interspecific variation for COI and 16S to be 0.021/0.147 and
0.012/0.135, respectively. An obvious “barcoding gap” was detected for all species in both markers
and all individuals of a species clustered together in both the COI and 16S trees. These results
suggested that the species within the studied taxa can be efficiently and accurately identified by
COI and 16S. Furthermore, our results detected that 16S was shown to be a better phylogenetic
marker for Campanulariidae at the subfamily level: Clytiinae, Campanulariinae and Obeliinae
formed a monophyletic clade respectively in the 16S tree; On the constrast, all three subfamilies
appeared polyphyletic in the COI tree. Considering the resolution and effectiveness for species
identification and phylogenetic analyses of Campanulariidae, 16S is a better choice for barcoding
Campanulariidae.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
224
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Marine Barcoding
Molecular identification of sea slugs from Korea based on mitochondrial COI
sequences
Daewui Jung1, Jongrak Lee2, Chang-Bae Kim1
1
Department of Life Science, Sangmyung University, Seoul 110-743, Korea
2
Marine Biodiversity Research Institute, INTHESEA KOREA Inc., Jeju 697–110, Korea
E-mail: [email protected], [email protected]
The Opisthobranchia, also known as sea slugs, is an infraclass of gastropods including
approximately 6,000 described species. They are highly interested by means of beautiful appearance
with diverse body color and species-specific defensive mechanism deterring predators. However its
identification is not easy task by its similar external morphology and various body color variation.
Molecular identification is considered as an alternative way. The aim of the study is to estimate the
validity of mitochondrial cytochrome c oxidase I (COI) gene sequences from 45 species of Korean
opisthobranch mollusks for an identification tool. Intraspecific and interspecific distances were
calculated based on K2P parameter with sequence data from related species obtained by BLAST
search. In addition, phylogenetic trees were reconstructed to know whether related species clustered
and identify diagnostic nucleotides. These results showed molecular identification by using COI
sequences is an effective identification tool for Korean opisthobranch mollusks even though more
species and specimens should be analyzed.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
225
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Marine Barcoding
Integrating morphology, DNA barcodes and proteome fingerprints for the
identification of marine metazoan species
Silke Laakmann, Thomas Knebelsberger
Senckenberg Research Institute, German Center for Marine Biodiversity Research (DZMB)
E-mail: [email protected]
Valid species identification represents a central issue in many fields of biological research. For more
than one decade the application of DNA barcoding demonstrates the successful connection of
morphological diagnostic characters with species-specific mitochondrial COI sequence divergence
for a broad range of metazoan taxa. However, for the analysis of high specimen numbers more rapid
and cost-effective species identification methods are required. In our studies we tested the potential
of proteome fingerprints as supplementary or alternative species-specific marker system. In this
context, we analysed proteome fingerprints of different metazoan taxa on the basis of
matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The
correct species identification of the analysed specimens was validated by the adjustment of
diagnostic morphological characters and the correlating COI sequences. We present first insights in
the species cluster of different pelagic and benthic metazoan taxa originating from the North Sea
and adjacent waters. Until now we were able to demonstrate valid species clusters for different taxa,
congeners, life history stages, sibling species, and for specimens from different sampling sites.
Since the application of MALDI-TOF MS requires less sample preparation steps and low
consumable costs compared to DNA barcoding lab procedures, we recommend proteomic
fingerprinting for rapid species identification.
Key word: proteome fingerprints valid species discrimination
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
226
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Marine Barcoding
DNA barcoding of marine molluscs in China
Qi Li, Hong Yu, Lingfeng Kong, Xiaodong Zheng, Ruihai Yu
College of Fisheries, Ocean University of China, Qingdao 266003, China
E-mail: [email protected]
To explore alternative mitochondrial barcode regions for mollusks, we compared the efficacy of 12
mitochondrial protein-coding genes (except ATP8) from 239 mitochondrial genomes of 138
molluscan species. Three barcoding methods (distance, monophyly and character-based methods)
were used in species identification. The distance-based method for all the 12 genes performed
poorly in terms of species identification with success rate from 75%-83%, especially for the
identification of closely related species. The character-based method provided higher resolution
(91.7%-100%) than the monophyly-based method (75-95.8%). The neighbour-joining phylogenetic
tree of the 12 genes could not distinguish the closely related species of mussels, while the
character-based method of NAD3, NAD4 and NAD5 genes could successfully recover these
problematic species. There were no significant differences in intra- or interspecific variability
among genes, with the high species-recovery rate (> 90%) as a whole. Besides the widely accepted
metazoan barcode region (COX1 gene), the other 11 mitochondrial genes are all suitable barcode
regions for mollusks. Moreover, we found that several genes including ATP6, NAD2, NAD4L and
NAD6 had experienced severe substitution saturation and would be inappropriate for phylogenetic
reconstruction.
Because of the conserved “universal” primer sites for PCR, we still selected the universal COX1
barcoding region to investigate how DNA barcode complement taxonomy and explore species
diversity in Mollusca, with high level of phenotypic variability. A total of 1108 specimens of 257
Mollusca species collected from the coast of China were included for analyses. As predicted,
distance and monophyly-based methods were very helpful in most species identification but the
character-based method showed some advantages especially in closely related taxonomic entities.
All specimens identified to species level based on morphological traits possessed more than three
diagnostic characters, indicating that COX1 barcoding is effective in Mollusca species delimitation
and could assist taxonomy. Furthermore, 14 putative hidden or cryptic species (5.45%) were
uncovered in traditional morphospecies, suggesting the potential cryptic diversity in Mollusca, and
also the value of DNA barcoding in disclosing hidden species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
227
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Marine Barcoding
DNA barcoding of marine invertebrates in Korea: Current progress and
prospects
Joong-Ki Park1, Won Kim2
1
Program in Cell Biology and Genetics and Department of Parasitology, College of Medicine,
Chungbuk National University, Cheongju 361-763, Republic of Korea
2
School of Biological Sciences, Seoul National University, Seoul 151-747, Republic of Korea
E-mail: [email protected]
According to the most recent survey of the Census of Marine Life, approximately 5,200 marine
invertebrate species are reported to exist in Korean seas. DNA barcoding strategies offer great
efficiency for identification of the hyper-diverse taxonomic groups of marine invertebrates that
normally require time-intensive processes by many taxonomic experts. The Korean Marine Barcode
of Life Initiative (‘KMBOL’) was launched in 2013, supported from the Ministry of Ocean and
Fisheries of Republic of Korea with the aims of: 1) exploring marine biodiversity; 2) providing a
new tool for rapid, accurate species identification of hyperdiverse marine taxonomic groups; and 3)
establishing a long-term national plan to collect, manage and analyze DNA barcode data for the
marine life in Korea. In this presentation, details of DNA barcode records and target genes, and
taxonomic information for each marine invertebrate group registered in the Barcode DB system are
discussed. Based on current progress in this research program, it is anticipated that DNA barcoding
for Korean marine living organisms will accelerate sharing of collaborative expertise and global
standards with international working groups, and this should bring about the next-generation
platform for exploring marine biodiversity and facilitating more effective conservation efforts in
marine ecosystems.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
228
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Marine Barcoding
Boring barcoding: Marine shipworms, pillbugs and gribbles from New Zealand
Courtney Rayes, Ian Hogg, Ian Duggan, James Beattie
Department of Biological Sciences, University of Waikato, Hamilton, New Zealand
E-mail: [email protected]
Known for the significant destruction they inflict on marine infrastructure, marine wood borers
present a major worldwide concern. Three families of marine wood borer from two phyla occur in
New Zealand; Teredinidae (Mollusca), and Sphaeromatidae and Limnoriidae (Arthropoda),
otherwise known as shipworms, pillbugs and gribbles, respectively. Of the known species in New
Zealand, a number are considered non-indigenous or are of uncertain origin. Although assumed to
have invaded New Zealand via wooden ships, introductions are historic, and it is possible that some
were present in New Zealand prior to human colonization. Field sampling for wood borers has been
undertaken at historically important ports, encompassing previously identified genetically divergent
regions of the North Island, New Zealand. Next steps involve the analysis of the COI gene locus to
determine genetic diversity and geographical structuring of wood borer taxa, in attempt to elucidate
their status as native or non-indigenous. Furthermore, DNA sequences will assist in the
identification of shipworm species, which offer little morphological distinction.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
229
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Marine Barcoding
Molecular approach to identify puffer fish from Malaysian waters
Danial Hariz Zainal-Abidin1, Masazurah A. Rahim1,2, Siti Azizah Mohd Nor1
1
School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia
2
Fisheries Research Institute, 11960 Batu Maung, Penang, Malaysia
E-mail: [email protected]
This study was conducted to generate the DNA barcodes of Malaysian puffer fishes based on the
amplification of a 637 bp mitochondrial cytochrome oxidase subunit I (COI) gene with a template
of total DNA from muscle tissues. Thirteen pufferfish species collected from Malaysian waters were
barcoded; Arothron hispidus, A. manilensis, A. reticularis, A. stellatus, Aluterus monoceros,
Cauthigaster rivulata, Diodon holocanthus, Lagocephalus lunaris, L. sceleratus, L. wheeleri,
Tetraodon nigroviridis, Takifugu oblongus, and Xenopterus naritus. A neighbor joining tree
confirmed the monophyly of each species. The data would permit accurate identification of
morphologically similar species with different levels of toxicity.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
230
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Marine Barcoding
High genetic diversity in geographical population of Apostichopus japonicus and
its implication for regional sea cucumber identification
Wei Zhang, Hong Zhao
Marine College, Shandong University at Weihai; Weihai 264209, China i
E-mail: [email protected]
Sea cucumber (Apostichopus japonicus), is an important aquaculture species in China, Japan and
Korea both as nutritive seafood and traditional medicine. The nutritional ingredient and commercial
value of this species are varied with their producing areas. However, it’s difficult to ascertain their
origin. Here the COI sequences were used to investigate the genetic variation of 17 sea cucumber
wild populations from China, Japan and Korea based on 330 samples. The results showed that the
haplotype diversity was 0.913 and nucleotide diversity was 2.39% in these sea cucumber
populations, indicating a high level of genetic diversity. In addition, the tree-building analysis,
together with the barcode gap method supported seven main lineages of sea cucumber, mainly
corresponding to their geographical distribution. These results reveal that sea cucumber
geographical race can be successfully identified using COI barcode. Our study firstly provided a
geographical population level case for the discriminatory power of DNA barcodes. We thus suggest
that intra-species level barcode can be explored and applied in some important economic species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
231
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Medicinal Plants
Sequencing chloroplast genome of Aconitum kusnezoffii (Ranunculaceae) by
using the third generation platform
Xiaochen Chen1, Baosheng Liao1, Lili Wang1 Jingyuan Song1, Jianping Han1, Shilin Chen1
1
Center for Computational Biology and Bioinformatics, Institute of Medicinal Plant Development,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR
China
2
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing
100700, PR China
E-mail: [email protected]
Chloroplasts possess their own genome and genetic system that play a leading role to the
photosynthesis process. Chloroplast genome (cp genome) was once suggested as the super-barcoder
to identify specific difficult organism in the 2nd DNA barcoding conference. With the development
of the sequencing technique, the cost has decreased much more, which makes the cp genome as the
super-barcoder possible. The sequencing of the first chloroplast genome Nicotiana tabacum
heralded a new age of chloroplast studies in photobiology, phylogenetic biology, evolutionary
biology, species identification and even chloroplast genetic engineering. Until now, 304 complete
cp genome sequences were deposited in GenBank (By 2013-7-14), most of which were sequenced
by the next generation sequencing (NGS). In the year of 2011, the third generation sequencing
platform based on the single-molecule real-time (SMRT) sequencing approach was released into
commercial market by Pacific Biosciences Company. With the aiming of experiencing the reported
advantages of the SMRT sequencing approach, long reads, high accuracy, shortest run time etc., we
have determined the cp genome of Aconitum kusnezoffii (Caowu), which has the hotspot of its
toxic components and the great value of phylogeny studies of the Ranunculaceae family. The
complete cp genome of Aconitum kusnezoffii is 156,749 bp in length with the common
quadripartite structure including a large single-copy region (LSC) of 87,630 bp and a small
single-copy region (SSC) of 16,941 bp divided by two inverted repeats (IRs) of 26,089 bp. It
contains 130 genes including 84 protein-coding genes, 34 tRNA genes and eight rRNA genes. Four
forward, five inverted and eight tandem repeats were identified in the cp genome. For SSR analysis,
the longest poly structure is a 20 T-repetition Located in the intergenic spacer (petA, psbJ). It could
be inferred that the SSR loci contribute to the A+T richness of the cp genome. The satisfactory
accuracy of the SMRT sequence is 99.336% on average validated by Sanger sequencing via six
selected genes from the cp genome. Our results prove that the third generation sequencing
technology is a powerful tool and can be used for sequencing of the cp genomes rapidly and
economically.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
232
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Medicinal Plants
Using ITS/ITS2 barcodes to identify Zanthoxmli Pericarpium
Dian-Yun Hou 1,2, Hui Yao 1, Jing-Yuan Song 1, Pei Yang 1, Hong Zhou 1, Shi-Lin Chen1,3
1
The National Engineering Laboratory for Breeding of Endangered Medicinal Materials,
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union
Medical College, Beijing 100193, China
2
Agricultural College, Henan University of Science and Technology, Luoyang 471003, China
3
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing
100700, China
E-mail: [email protected],
Zanthoxmli Pericarpium coming from the desiccative pericarp of Zanthoxylum schinifoliun and Z.
bungeanum is very rich in our country, and can be taken as a food and medicine. Because of the
high market demand of the Zanthoxmli Pericarpium, many of other related species have been
misused as Zanthoxmli Pericarpium. To ensure the quality and therapeutic effects of Zanthoxmli
Pericarpium, it is very important to identify Zanthoxmli Pericarpium and its closely related species.
In this study, the accuracy and stability of ITS/ITS2 barcode has been evaluated in identification of
Zanthoxmli Pericarpium. ITS regions were amplified and the ITS2 sequences obtained by using the
hidden Markov model (HMM) based on annotation method from the ITS sequences. The inter- and
intra- specific variation of the Zanthoxmli Pericarpium and its adulterants were analyzed.
Zanthoxmli Pericarpium has been identified through the Species Identification System for
Traditional Chinese Medicine and neighbor-joining (NJ) phylogenetic trees. The lengths of
ITS/ITS2 sequence of Zanthoxmli Pericarpium were 618-620bp and 224 bp respectively. The
Zanthoxmli Pericarpium and its adulterants can be easily differentiated according the the Species
Identification System for Traditional Chinese Medicine and the NJ trees methods. Therefore,
ITS/ITS2 regions as DNA barcodes can stably and accurately distinguish Zanthoxmli Pericarpium
and its adulterants.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
233
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Medicinal Plants
Identification of Crocus sativus and its adulterants by using DNA barcoding
technique
Weijuan Huang1, Chunlin Long1, 2
1
College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, China
2
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
E-mail: [email protected]
Crocus sativus, commonly known as saffron, is a traditional and expensive herb. The dried stigmas
of saffron have many different uses. In herbal medicine, it has an outstanding effect in treating
irregular and painful menstruation. However, due to the similar morphological characters and high
market demands, it has often been misused, adulterated or intentionally introduced as saffron in
medicinal market, pharmacy and online store. In this study, 13 samples called as saffron were
collected from 12 different provinces of China. Then we compared three candidate DNA barcoding
markers by using trnH-psbA, rbcL and ITS2 sequences from nuclear and chloroplast. Based on
sequence alignments and analysis, we found there three samples were not Crocus sativus, but two
Carthamus tinctorius (100% supporting rate) and one Chrysanthemum x morifolium (98%
supporting rate). Except for rbcL, trnH-psbA and ITS2 can distinguish Crocus sativus from other
species respectively but both of them have a low identification rate. Our results suggest that three
combined loci are superior barcodes for the identification of Crocus sativus from common
substitutes or adulterants.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
234
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Medicinal Plants
To construct a Taxonomy System For Medicinal Plants from Sellaginella with
digital morphological characteristics Based on DNA bacorde technique
Yimei Liu, Shilin Chen, Keli Chen
1
Hubei University of Chinese Medicine, Wuhan, Hubei 430065, China
2
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing
100700, China
E-mail: [email protected]
In spite of the world has had very detailed information about morphological characteristics of each
species ， but the work to establish a classification system based on digital morphological
characteristics has not yet to begin. Selaginella is a primitive and important taxa group in the ferns,
the medicinal plants from the group have important function and there are some difficulties in the
species identification and infrageneric classification.
In this paper, chloroplast matK, rbcL, plasmid trnH-psbA, and nuclear ITS, ITS2 gene were selected
as the candidate sequences, based on which a phylogenetic tree for the medicinal plants from
Sellaginella was created using bioinformatics methods. Then the genetic distance between species
and the position species in this tree branch system were used as objective scale, to construct a
taxonomy system with the same phylogenetic tree structure but based on morphological
characteristics. So according to the phylogenetic tree can be determined the status and importance
of each morphological characteristic in the structure, and their order occurring in the species
differentiation. And so the morphological characteristics are entrusted with the digitized classified
information, which can be used to resolve objectively some dispute in the morphological
classification, and explore a new method to find resource plant combining morphological and
genetic features .
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
235
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Medicinal Plants
Identification of medical plants of 24 Ardisia species from China using the matk
genetic marker
Yimei Liu, Shilin Chen, Keli Chen
1
Hubei University of Chinese Medicine, Wuhan, Hubei 430065, China
2
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing
100700, China
E-mail: [email protected]
Ardisia is a group of famous herbs in China, which has been used as medical plants for more than
900 years. However, the species from the genus are so analogous that it is difficult to discriminate
them just by morphological characteristics. DNA barcoding is a new technique that uses a shot and
standard fragments of DNA sequences to identify species quickly, accurately and objectively. In this
context, we tried to screen an applicable sequence to discriminate 24 species of Ardisia. Four
regions (psbA-trnH, ITS2, matK, rbcL), which were intensively recommended as candidate DNA
barcodes, were tested for their ability to identify 54 samples of 24 species from genus Ardisia. The
success rates of PCR amplification and sequencing, differential intra- and interspecific divergences,
DNA barcoding gap and identification efficiency were used to evaluate the discrimination ability of
these candidate sequences using BLAST1 and nearest Distance methods. It was found that the PCR
amplification efficiency were 100% (psbA-trnH), 100% (ITS2), 100% (rbcL) and 88.9% (matK)
respectively for collected 54 plant samples belonging to 24 species of Ardisia, and all the 4
sequences exhibited significant difference between intra- and interspecific divergences (P<0.05).
The identification efficiency using BLAST1 method were 70.4% (psbA-trnH), 51.9% (ITS2),
27.8% (rbcL), 98.1% (matK), and the efficiency using nearest Distance method were 44.4%
(psbA-trnH), 51.9% (ITS2), 27.8 (rbcL), 91.7% (matK). The results showed that matK produced the
highest rate of successful identification at the species level, that it can correctly discriminate 21
Chinese medicinal species from Ardisia. Therefore, it is proposed that the matK region is a
promising DNA barcode for the genus Ardisia.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
236
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Medicinal Plants
DNA barcoding the commercial Chinese caterpillar fungus
Li Xiang1,2, Jingyuan Song1, Tianyi Xin1, Yingjie Zhu1, Linchun Shi1, Xiaolan Xu1, Xiaohui Pang1,
Hui Yao1, Wenjia Li3, Shilin Chen1,2
1
National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of
Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing, China
2
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China
3
HEC Pharm Co., Ltd, Guangdong, China
E-mail: [email protected]
Chinese caterpillar fungus (Ophiocordyceps sinensis) has been widely used as tonic in Asian
medicine. Considering its curative effect and high cost, various counterfeit versions of O. sinensis
have been introduced and are commercially available. These counterfeits have morphological
characteristics that are difficult to distinguish based on morphology alone, thereby causing
confusion and threatening its safe use. In this study, internal transcribed spacer (ITS) sequences as a
DNA barcode was analyzed and assessed for rapid and accurate identification of 131 O. sinensis
samples and 12 common counterfeits and closely related species. Results showed that sufficient ITS
sequence differences, also known as “barcode gaps”, existed to distinguish between O.sinensis and
counterfeit species. ITS sequence correctly identified 100% of the samples at the species and genus
level using the Basic Local Alignment Search Tool 1 and the nearest distance method. Furthermore,
O. sinensis, counterfeits, and closely related species can be successfully identified using tree-based
methods including maximum parsimony, neighbor-joining, and maximum likelihood analysis.
These results indicated that DNA barcoding could be used as a fast and accurate identification
method to distinguish O. sinensis from counterfeits and closely related species to ensure its safe use.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
237
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Medicinal Plants
Market supervision of the “Plateau Ginseng” using ITS2 as DNA Barcode
Tianyi Xin1, Jing-Yuan Song1, Hui Yao1, Shi-Lin Chen1,2
1
The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of
Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union
Medical College, Beijing 100193, China;
2
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing
100700, China
E-mail: [email protected]
Rhodiolae crenulatae Radix et Rhizoma, known as "Plateau Ginseng" in the Orient, was originally a
traditional Tibetan medicine, which derives from the Rhodiola crenulata (Crassulaceae) according
to the Pharmacopoeia of the PR China (2010 edition). With the research progresses in recent years,
it has surprisingly become a major force as an environment adaptability medicine for the treatment
of acute high altitude disease. However, there are many kinds of Rhodiola species are sold as
Rhodiolae crenulatae Radix et Rhizoma in the market. According to the WHO, the adulteration of
herbal medicine is a threat to consumer safety. Our research aims to investigate the authentication of
Rhodiolae crenulatae Radix et Rhizoma and its adulterants using the internal transcribed spacer 2
(ITS2) as DNA barcode. In this study, 58 medicinal materials samples (eight adulterants) were
utilized to get the ideal barcode. Based on the ITS2 barcode, the neighbor-joining (NJ) tree,
BLAST1, and nearest distance methods were able to differentiate the Rhodiolae crenulatae Radix et
Rhizoma and its adulterants. Therefore, we present the ITS2 barcode is a powerful tool for the
market supervision of Rhodiolae crenulatae Radix et Rhizoma and its adulterants.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
238
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Medicinal Plants
Identification of Medicinal Plants in Genus Polygonatum using DNA Barcode
Pei Yang1, Hong Zhou1, Shan Xu1, Dianyun Hou1, Jingyuan Song1, Shilin Chen1,2, Hui Yao1*
1
The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of
Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing 100193, China
2
Institute of Chinese MateriaMedica, China Academy of Chinese Medical Sciences, Beijing 100700,
China
E-mail: [email protected], [email protected]
Plants of genus Polygonatum (Liliaceae) have been widely used in traditional medicine to cure
many diseases for a long history. However, there's still some controversy about the classification
and identification of the species in Polygonatum lead to the misuse of medicine materials. As a
recent aid to traditional taxonomic identification, DNA barcoding was chosen to assess the
contribution to the taxonomy under the genus Polygonatum. In this study, fifty-five individual plant
samples of Polygonatum were collected. Five proposed DNA barcoding sequences (ITS, ITS2,
matK, rbcL, psbA-trnH) were used to evaluate the discrimination ability to the species of
Polygonatum. The PCR amplification efficiency, extent of specific genetic divergence, DNA
barcoding gap were assessed. The results showed that ITS2 /ITS of Polygonatum failed in PCR
amplification while more than fifty sequences for matK, rbcL,psbA-trnH were obtained. The matK
successfully identified 75% of all samples compared with rbcL and psbA-trnH. Therefore, matK is
recommended as the best DNA barcoding candidate. It also showed that the combination of two or
three loci presented a higher success rate of species discrimination than one locus alone. Therefore,
we suggested that the first choice of single DNA barcode locus to authenticate plants in genus
Polygonatum was matK, and psbA-trnH could be a candidate for loci combination.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
239
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Medicinal Plants
Identification of Cortex Acanthopanacis and its adulterants using DNA
barcoding
Sha Zhao1,2, Xiaohui Pang2, Hui Yao2, Jingyuan Song2, Shilin Chen1,2,3
1
School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China;
2
The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of
Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing 100193, China;
3
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing
100700, China
E-mail: [email protected], [email protected]
The traditional Chinese medicine Cortex Acanthopanacis (Wujiapi) is from the dried root barks of
Acanthopanax gracilistylus, which is listed officially in Chinese Pharmacopoeia. At present, Cortex
Acanthopanacis is widely used for treating arthritis, rheumatism, lameness, paralysis and liver
diseases in clinical applications. However, some closely related species of Acanthopanax
gracilistylus were used as Cortex Acanthopanacis because of their similar morphological feature in
some places, which brought risks to the drug safety. Similarly, Cortex Periplocae was inadvertently
misused as Cortex Acanthopanacis, which may cause incidences of poisoning. In order to ensure the
quality and safety of Cortex Acanthopanacis in medication, we used the DNA barcodes ITS2 and
psbA-trnH to identify it and its adulterants in this study. In total, 50 samples belonging to 8 species
from Cortex Acanthopanacis and its adulterants were collected. The ITS2 and psbA-trnH regions
were amplified and sequenced. The genetic distances of two regions were calculated using MEGA
5.0. BLAST1, nearest distance and phylogenetic tree (NJ-tree) methods were used to evaluate the
identification efficiency of the two barcodes. Our conclusion reveals that the intra-specific genetic
distances of Cortex Acanthopanacis are lower than the inter-specific genetic distances between
Cortex Acanthopanacis and its adulterants. Both the ITS2 and psbA-trnH barcodes are appropriate
to identify Cortex Acanthopanacis and its adulterants.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
240
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Next Generation Sequencing
Monitoring forest biodiversity using DNA metabarcoding: high-throughput
species identification of saproxylic beetles
Rodolphe Rougerie1, Mehrdad Hajibabaei2, Christophe Bouget3, Carlos Lopez-Vaamonde1
1
NRA, Department Forest, Grassland and Freshwater Ecology, UR633, Forest Zoology Unit,
Orléans, France
2
University of Guelph, Guelph, ON, Canada
3
Institute for Engineering in Agriculture and Environment CEMAGREF, Domaine des Barres, F-45
290 Nogent-sur-Vernisson, France
E-mail: [email protected]
Saproxylic beetles – associated with dead wood or with other insects, fungi and microorganisms
that decompose it – play a major role in forest nutrient cycling. They are important ecosystem
service providers and are used as key bio-indicators of old-growth forests. In France alone, where
the present study took place, there are about 2500 species distributed within 71 families. This high
diversity represents a strong challenge for specimen sorting and identification.
We have developed a DNA metabarcoding approach to facilitate and enhance the monitoring of
saproxylic beetles as indicators in ecological studies. We use Illumina Mi-Seq to recover species
diversity from bulk samples collected in interception traps. Sampling was designed to account for a
variety of conditions and parameters possibly affecting the experiments. These include species
diversity, relative abundance and biomass, sampling medium and preservation methods.
DNA-based species identification derived from available records in public databases and from a
newly assembled DNA barcode library for European species identified by expert taxonomists
currently comprising 580 species (855 records in 47 families).
Preliminary results show the potential of DNA metabarcoding to accurately identify saproxylic
beetles from bulk samples collected with interception traps. Our approach opens new avenues of
research using these insects as forest indicators.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
241
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Other Invertebrates
DNA barcoding of great salt lake invertebrates
Son Nguyen, Jonathan B. Clark
Department of Zoology, Weber State University, Ogden, Utah, USA
E-mail: [email protected]
Great Salt Lake (GSL), in northern Utah, is one of the largest lakes in the United States, with a total
surface area of 4400 square kilometers. Arthropods constitute the most conspicuous and abundant
animals inhabiting the waters of GSL. These include two principle species of brine flies (abundant
in littoral areas); the corixid (Coleoptera) Trichocorixa verticalis (found in bays and along island
margins); and the pelagic brine shrimp, Artemia franciscana. The dynamic of interactions among
these invertebrates is poorly studied and yet is an important influence in the community structure of
this saline lake. T. verticalis is the main predator of brine shrimp and a shrimp density is
associated with an increase in protozoa and phytoplankton, which are a principle food source of
both brine fly larvae and adults. This study compares the utility of the internal transcribed spacer
region (ITS-1) size polymorphism and the mitochondrial cytochrome oxidase I gene in identifying
GSL invertebrates. Data on intraspecific and interspecific sequence variation for both loci are
discussed for selected species.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
242
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Other Invertebrates
DNA Barcoding and Geometric Morphometric Analyses confirm rediscovery
and revalidation of two cryptic Nearctic spiders of genus Araneus (Araneae:
Araneidae)
Gergin Blagoev1, Anna Šestáková2, Yuri M. Marusik3, Alex Borisenko1
1
Biodiversity Institute of Ontario, University of Guelph, Canada
Department of Zoology, Comenius University, Bratislava, Slovakia
3
Institute for Biological Problems of the North, Magadan, Russia
E-mail: [email protected]
2
Araneus is the largest genus in the order Araneae with over than 660 species. It was never
comprehensively studied and appears to have at least two thirds of the species misplaced.
A common orb-weaver, A. marmoreus Clerck, 1757, is a widely distributed and morphologically
variable Holarctic spider that was thought to be a single species with many synonyms (Platnick,
2013). DNA barcoding analysis showed that A. marmoreus splits into three deeply divergent but
compact haplogroups, corresponding to three non-overlapping geographic areas: Paleacrtic;
Western and Central USA; and Canada and Northern USA. This was corroborated by GMA of male
palpal sclerites, embolus lamella and median apophysis. Aside from the fine characters above, the
tree haplogroups are morphologically similar. Examination of the original descriptions and
illustrations revealed two available names for the Nearctic clusters of A. marmoreus: Epeira obesa
Hentz, 1847 and E. insularis Hentz, 1847, with matching diagnostic features. In addition, A.
insularis can be distinguished by abdominal coloration pattern. Combined molecular and
morphometric evidence supports rising A.obesus and A. insularis to species rank. This reappraisal
brings a new perspective to our understanding of orb-weaver speciation in the Nearctic and
reaffirms the utility of DNA barcoding in revealing cryptic alpha-diversity.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
243
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Plants
The potential of rbcL, trnH-psbA and matK regions as DNA barcode for ten
varities of Malaysian cassava plant
Ruzainah Ali, Asvini Sri Nagappan, Abdul Manan Dos Mohamad
Universiti Kuala Lumpur-MICET, Kuala Lumpur 50250, Malaysia
E-mail: [email protected]
DNA barcodes is a fast molecular identification method that can be used for plant species
identification with DNA extraction from minuscule tissue samples. The DNA barcode is a very
short standardized DNA sequence in a distinguished gene for example using rbcL, trnH-psbA, and
matK (Aron et al., 2008). In this research, DNA barcode method is used to identify a few varieties
of cassava (Manihot esculenta) in Malaysia.
The advantages of this identification are to ensure
the usage of the right, safe and high quality of raw material plant. Samples collected from
MARDI Serdang consisted of a few varieties - Sri Kanji 1, Medan, Black Twig, Perintis, Sri Kanji
2, and Sri Pontian . Each of these varieties has its own usage for examples in food industries and
starch production (Dixon et al., 2003: Sriroth et al., 2000, Tanujari, 2004). Three different regions
which are rbcL, matK, and trnH-psbA regions were investigated on their ability to differentiate each
variety or plant identification.
From the PCR amplification, rbcl region shows high success
results compared to trnH-psbA and matK. Genetic variance analysis using MEGA5 that illustrate
on conserved, variable, parsimony informative and singleton showed that rbcl provides high value
of conserved sites and trnH-psbA with high variable sites. This concludes that trnH-psbA barcode
more powerful to differentiate the cassava varieties compared to rbcL region. Successful DNA
sequences obtained shows that matK has lowest sequencing success despite its ability in high
species discrimination meanwhile rbcL barcode which has the ability to identify most of varieties
with high successful amplification. Further studies will be execute to identify others potential
barcode in identifying these varieties in more detail.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
244
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Plants
Plant DNA Barcoding is very effective to identify tropical rainforest trees in
plot-based inventories in Southwest China
Xiuqin Ci, Jie Li
Xishuangbanna Tropical Botanical Garden,Chinese Academy of Sciences, Menglun, Yunnan
666303, China
E-mail: [email protected], [email protected]
Large-scale plant diversity inventories are principal time-consuming using traditional methods and
are dependent on the availability of taxonomic expertise, which is a resource in decline. In addition,
identification of tropical plants in most cases remains a challenge even for experts. DNA barcoding,
as one of the most economic and reliable molecular approaches, showed its effectiveness in some
regions. In order to evaluate power and accuracy of DNA barcoding in tropical rainforest of
Southwest China, we chose four regions - rbcL, matK, trnH-psbA and ITS to identify trees in 20
hectares dynamics plot in Xishuangbanna. In this study, we collected 1019 individuals to evaluate
universality statistics of primers and sequence recovery. Our results showed that rbcL was easy to
be amplified and sequenced, while ITS had a low rate of sequencing success. In addition, we built a
database of 858 individuals including 372 species to evaluate species resolution in 20-ha plot. In the
four tested DNA regions, ITS had the most variable information, and rbcL had the least. ITS
achieved the highest rate of correct identification than other similar researches regardless of
algorithms (neighbor joining and BLAST). The core barcode rbcL + matK showed a lower species
resolution (62.85% for blast and 59.42% for NJ tree) and the combination of tree or four markers
will increase success rate of identification. Moreover, DNA barcoding showed good ability of
species assignment for unknown species in this plot. So we concluded that DNA barcoding is very
effective to identify tropical rainforest trees in plot-based inventories in Southwest China.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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245
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Plants
DNA Barcoding of Podophyllum hexandrum Royle of Nepal using standard DNA
Barcode sequences
Rajesh Lamichhane1,2, Tribikram Bhattarai 2, Rajani Malla 2, Sangita Shrestha1
1,2
Molecular Biotechnology Unit, Nepal Academy of Science and Technology (NAST), Khumaltar,
Lalitpur, Nepal
2
Central Department of Biotechnology (CDBt), Tribhuvan University, Kirtipur, Kathmandu, Nepal
E-mail: [email protected]
Nepal being a Himalayan country is hub to many medicinal and aromatic plants including
Podophyllum hexandrum Royle (Himalayan Mayapple). The indiscriminate collections of the plant
rhizomes from the wild for the valuable anticancer drug podophyllotoxin have rendered these plants
endangered. Although CITES (appendix II) and the Nepalese government has forbidden in the
trades and other related processes, the populations of Podophyllum hexandrum Royle continues to
decline. DNA barcoding has been using for the study of phylogenetics. We analyzed 12 accessions
from four different populations covering three developmental regions; eastern, central and western
using the four candidate DNA barcodes from both nuclear and plastid genomes [matK, rbcL,
trnH-psbA, and internal transcribed spacer (ITS)]. Altogether 37 high quality sequences were
generated, all equally efficient in species identification using NCBI genebank and BLAST. Of the
four tested regions, ITS has the highest number of variable sites (9/661) followed by matk (1/829),
whereas, rbcL has none. ITS2 region of ITS was found to consist more variable sites of ITS (4/243).
Besides, phylogenetic studies were done by MEGA V.5.2.2. At population level, ITS gave the
highest within and between group mean distance. Correct identifications according to "Best Match"
was highest for ITS (87.5%) followed by matK (76.0%) and rbcL (65.38%).
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
246
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Plants
The utility of dna barcodes in plant living collections management: case study of
Orchids in a Tropical Botanical Garden
Ji-hong Li, Pankaj Kumar, Stephan Gale, Huarong Zhang, Feng Yang, Gunter Fischer
Kadoorie Farm and Botanic Garden, Lam Kam Road, Tai Po, New Territories, Hong Kong, China
E-mail: [email protected]
Botanic gardens are repositories of global plant diversity and have a crucial role to play in the ex
situ conservation of locally threatened taxa. DNA barcoding has great potential to support
management decisions that shape the composition of these living collections. In particular,
barcoding can assist in (i) quantifying phylogenetic diversity; (ii) assessing the representability of a
collection relative to the local or regional flora; (iii) identifying gaps in taxa and lineages that are
important for conservation; (iv) gauging whether sufficient genetic diversity has been captured
within an ex situ collection of special conservation significance; (v) revealing cryptic taxa and cases
of misidentification; and (vi) highlighting biases (desirable or otherwise) in a collection’s
composition with respect to traits such as attractiveness for display, ease of cultivation, and
potential in breeding and cultivar development. As a case study of these aspects in the management
of a large, diverse and historic collection of tropical plants, we generated ITS and matK sequence
data for 453 orchid accessions held at Kadoorie Farm and Botanic Garden in Hong Kong, China.
Here we outline some of the ways in which we are utilising our findings to inform approaches in
taxonomy, ecology, conservation, horticulture and plant breeding.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
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247
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Plants
Identification of the medical plants candidates in Korean Aconitum L. species by
DNA barcode sequencing
Chae Eun Lim1, Chong-Wook Park2
1
National Institute of Biological Resources, Incheon 404-708, South Korea
2
Department of Biology, Daejeon University, Daejeon 300-716, Korea
E-mail: [email protected]
The Genus Aconitum has been well known as pharmaceutical or poisonous plants in the world.
Furthermore, they are also notorious for their taxonomical difficulties. Recently, DNA barcode
psbA-trnH intergenic spacer region was proposed to determine the genetic variation among Chinese
Aconitum species. In the present study, we have examined the psbA-trnH intergenic spacer region
to differentiate the medicine candidates in Korean Aconitum species. In the neighbor joining tree
obtained from psbA-trnH intergenic spacer region, A. jaluense complex is combined with A.
carmichaelii and A. kusnezoffii. These two Chinese Aconites were a well-known toxic and potent
herbal medicine. Additionally, we attempted to discover a new barcode sequence for identification
of Aconitum species in Korea. The neighbor joining tree obtained from combined sequences of the
two intergenic spacer regions revealed that it supports the intersubgeneric relationships in the Genus
Aconitum L. based on the morphological characters
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
248
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Plants
Testing DNA barcodes to identify species in the genus of Gentiana
Juan Liu1,2, Xue-Jun Ge1
1
Key Laboratory of Plant Resource Conservation and Sustainable Utilization, South China
Botanical Garden, the Chinese Academy of Sciences, Guangzhou 510650 China.
2
University of the Chinese Academy of Sciences, Beijing 100049
E-mail: [email protected] and [email protected]
One of the biggest challenges for DNA barcoding is to discriminate the closely related species.
Using supplementary markers has been suggested to increase the species discriminatory ability. The
non-coding gene trnH-psbA and the internal transcribed spacers from nuclear ribosomal DNA (ITS)
are more promising obvious choices. In this study, we assessed the performance of two
supplementary loci and two proposed barcodes to discriminate the species of Gentiana, the genus
with rapid radiation speciation. 73 individuals representing 27 Gentiana species were sampled and
22 individuals from 7 closely related genera and 8 species were included. The results showed that
ITS got much higher species resolution (57.89%) than trnH-psbA (33.33%), and higher than the
combination of rbcL and matK (40.74%). Among possible combinations with ITS, four regions
combination obtained the best performance (63.16%) but only marginal returns than ITS alone. The
combination of matK and ITS got the second higher success rate but equal to that of ITS. We
support ITS as supplementary loci for DNA barcoding and argue that this marker could be as a core
barcode for some taxa.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
249
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Plants
Rapid identification of species and iFlora: application of DNA barcoding in a
large temperate genus Pedicularis (Orobanchaceae)
Min-Lu Liu1,3, Wen-Bin Yu1, De-Zhu Li1,2, Hong Wang1
1Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese
Academy of Sciences, Kunming, Yunnan 650201, China
2 Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming, Yunnan 650201, China
3 University of Chinese Academy of Sciences, Beijing 100049, China
E-mail: [email protected], [email protected]
DNA barcoding involves species identification using a short and standardized DNA region. This
method has many advantages in comparison with the traditional morphology-based identification,
including higher effectivity, sensitivity, reliability and standardization. In this study, we chose the
whorled-/opposite-leaved species of Pedicularis as a case to test the utility of DNA barcoding for
rapid identification. Four candidate DNA barcoding loci (three chloroplast: rbcL, matK, trnH-psbA,
and one nuclear: ITS) were evaluated using 43 species and 163 accessions by tree-based and
distance methods. Our results show that species identification rates of tree-based and disatance
methods using the nuclear ITS are 81.4% and 89.57%, respectively. The rates surpass those of three
chloroplastic barcodes and all combination barcodes. In addition, the nuclear ITS can help to
identify some species those are difficult to identify using morphological traits. Practicality of DNA
barcoding in Pedicularis indicates that the next-generation intelligent Flora can fulfill the rapid and
accurate identification of species using DNA sequencing technologies, and it can provide key
information for traditional taxonomy and investigations of ecology, evolutionary biology,
population genetics, conservation genetics, etc.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
250
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Plants
DNA barcoding of aid plants In Nigeria
Oluwatoyin Ogundipe1, Temitope Adeyemi1, Akeem Kadiri1, Adebayo Ogunkanmi1, Paul Lyam2,
Sunday Aladele2
1
Department of Botany,University of Lagos, Lagos, Nigeria
2
National Center for Genetic Research and Biotechnology, Abuja-FCT, Nigeria
E-mail: [email protected]
Africa's biodiversity is one of the most extraordinary in the world, but also one of the most
threatened by human activities (population growth, over-exploitation, logging) and global change
(desertification, climatic warming). While biodiversity is disappearing alarmingly fast, there have
also been fantastic technological developments that can help reverse biodiversity loss – but the
problem is that these novel technologies are not easily accessible by countries rich in biodiversity
but constrained in resources. This research is a CARGS project awarded to the University of Lagos,
Nigeria in collaboration with the National Center for Genetic Research and Biotechnology
(NACGRAB). It focuses on the application of DNA barcoding technique to solve the problems of
species identification, desertification and inter-boundary migration of species, genetic erosion of
valuable resources and the problem of ever changing climatic condition with particular emphasis on
preservation of the plant samples as well as generation of DNA barcodes for identification purposes.
Extracted DNA samples were deposited at the DNA Bank of the University of Lagos, Nigeria and
voucher specimens were deposited at the University of Lagos Herbarium, Lagos, Nigeria. A
hundred and forty DNA barcodes were generated using the coding matK and rbcL genes. These
were deposited together with their voucher information in the Barcode of Life database. This
research is a great contribution to the taxonomy of arid plants especially in Nigeria.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
251
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Plants
Conservation of tree genetic resources of northeastern Lagos Nigeria
Oluwatoyin Ogundipe, George Nodza, Temitope Adeyemi
Department of Botany,University of Lagos, Lagos, Nigeria
E-mail: [email protected]
This study investigates the rate of concurrent depletion on the remnant flora growing in the
northeastern part of Lagos, southwestern Nigeria. Tree species growing in this area is not spared
from advancing civilization, which have resulted in inevitable loss of genetic resources. Hence,
molecular technique is adopted in an effort to conserve the genetic resources of the tree species.
Samples were collected at random and identified. A total of 66 tree species was recorded.
Genomic DNA was extracted from fresh leaves samples following modified CTAB DNA extraction
protocol. The DNA when viewed on 1% agarose revealed bands of high molecular weight. Also,
Spectrophotometric check on the genomic DNA showed a good quality with a ratio of 1.7 to 1.8.
The purified DNA was dissolved in buffer and store at -80°C in the established DNA Bank at the
University of Lagos. This can be used for further investigations including understanding genetic and
evolutionary relationships between taxa, functional analysis of genes, comparative genomics, DNA
barcoding and plant breeding.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
252
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Plants
Phylogenetic and genomic relationships among melon populations based on
RAPD
Oluwatoyin Ogundipe, Dolapo Oshingboye, Temitope Adeyemi
Department of Botany,University of Lagos, Lagos, Nigeria
E-mail: [email protected]
Melons belong to the family Cucurbitaceae; they are one of the most important cash crops globally.
Their demand in Nigeria is very high been used for several preparations, notably soup, since they
are thought to increase nutrient in the body. This study was conducted to determine the genetic
diversity of melon populations in south western Nigeria. Eighteen melon cultivars including;
Cucumis melo, Citrullus lanatus and Lagenaria siceraria species were assessed using both seed
morphology and molecular characters. These revealed that the samples have sharply pointed seeds
with elliptical/oval shape. Also analysis of the extracted genomic DNA using 1% agarose revealed
high molecular weight DNA bands of 1.7 – 2.0 absorbance ratios in 88.89% of the samples. Further
RAPD analysis of the DNA using three different primers revealed a wide genetic base with
monomorphic bands, which were used to generate UPGMA dendograms. The dendograms showed
that the examined melons are monophyletic with sample AD001 and AD002 showing distinct
morphometric evaluation with 18.00 and 16.94 similarity coefficient respectively and samples
AD003 and AD004 having similarity coefficient of 2.06. This study has helped in understanding the
extent of variation and relatedness among the 18 melon cultivars. This would enable breeders make
informed decisions on the selections of melon for improved breeding. This research would also
serve as a basis for further characterization of the melon populations in Nigeria.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
253
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Plants
RbcL gene barcoding and phylogenetic analysis of species of Phoebe/Machilus
and Ficus in Xishuangbanna rainforest of China
Yu Song1, Xue Liang2, Xueliang Zhang2, Mengjie Qiu3, Bin Liu2, 4
1
Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, China
2
Center of Systematic Genomics, Xinjiang Institute of Ecology and Geography, Chinese Academy
of Sciences, Urumqi, China
3
Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China
4
Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
E-mail: [email protected]
Tropical rainforests are the hotspots of biodiversity and remain the most complicated forest
ecosystem. In China, the tropical rainforests are mainly located in Yunnan and Hainan Provinces,
whereas Xichuangbanna rainforest in Yunnan is unique on its own stance. In this study, both rbcL
and matK were applied as standard plant DNA barcodes and were successfully used for over 50% of
the collected samples of Phoebe/Machilus, lauraceae and Ficus, moraceae in this region and for the
first time phylogenetic markers were assigned to these species. In order to increase the coverage we
extended the markers to the full-length of these genes, it also give more room for designing of more
suitable primers and for increasing the efficiency of PCR amplification. With this advancement,
nearly all samples were successfully amplified and characterized for the full gene sequence of rbcL.
With this improved gene barcode we were able to identify both Phoebe/Machilus samples and Ficus
samples in several distinctive groups. Further full-length gene sequencing analysis will reveal
detailed phylogenetic relationship among these species. We demonstrated that standard plant DNA
barcodes can be used to the identification of trees in tropical rainforest and full-length gene
sequences of these markers have further increasing interpreting power and more convenient to use
as an added benefit. Wider application of DNA barcodes should facilitate the species study and
conservation and utility of rich biodiversity and natural resources of our ecosystems.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
254
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Plants
DNA barcoding of Malus
Jiahui Sun, Shiliang Zhou
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese
Academy of Sciences, Beijing 100093, China
E-mail: [email protected]
Many species in Malus are of paramount economic importance. Apples (M. pumila) prevail over
other cultivated fruit trees throughout the world. (Malus spectabilis) and many other species in the
genus are beloved ornamentals. There are 30-55 species and very many cultivars in Malus.
Identification of Malus species is a big problem even for some taxonomic experts due to continuity
of variations of morphological characters and apomixes. The situation becomes even worse recently
when more and more inter-specific hybrids are being created. Reliable identification of Malus
species was often made an appeal to molecular markers. To find out reliable candidates of DNA
barcode for Malus species, we screened 10 chloroplast regions (matK, psbA-trnH, rbcL, rpl16,
rpl20-rps12, rps16, trnC-ycf6, trnL-F, trnS-G, and ycf1) and the nuclear Sbe1 using 40 samples of
36 species. We found that all these genes performed very badly. By contrast, Sbe1, rbcL and rps16
are the worst, and matK and rpl20-rps12 are the best. Our work suggested that the frequently used
genes are unsuitable to being DNA barcodes of Malus. The best candidate barcode of Malus
remains to be sought from either chloroplast or nuclear genomes.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
255
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Pollinators
Megachilidae in the south west Pacific: the impact of a recent bee fauna on
ecosystem development and stability
Scott Groom1, Sarah Hayes1, Hien Ngo2, Mark Stevens3, Michael Schwarz1
1
School of Biological Sciences, Flinders University, GPO Box 2100, SA 5001, Adelaide, Australia
2
Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada
3
South Australian Museum, GPO Box 234, SA 5000, Adelaide, Australia
E-mail: [email protected]
The megachilid bees of the south west Pacific islands represent a large proportion of all described
species for the region. The other most prevalent group, the halictine bees, arrived during the late
Pleistocene suggesting a critical pollinator only arrived in the Pacific very recently. Here we use
DNA barcodes to show the likely origin of the megachilid bees was actually more recent with many
potential introductions since human colonisation of the islands. This has broad implications for the
early development of terrestrial ecosystems in the Pacific, and how they have been impacted since
the arrival of a now abundant pollinator.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
256
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Vertebrates
DNA Barcoding: Challenge and opportunities for faunal diversity conservation
and management in Bangladesh.
Md. Sagir Ahmed, Gulshan Ara Latifa
Advanced fisheries and DNA Barcoding Lab, Department of Zoology, University of Dhaka, Dhaka
1000, Bangladesh
Email: [email protected]
E-mail: [email protected]
Bangladesh is a tropical country of large alluvial plan with rich faunal diversity. The country having
an area of 147,570 sq km, contains 266 inland fishes, 442 marine fishes, 34 amphibians, 133 inland
reptiles, 17 marine reptiles, 388 resident birds, 300 migratory birds, 110 inland mammals and 03
marine mammals. This is really an extraordinary situation that such a great diversity still exists in
an unusually overpopulated country. Unfortunately, there is no stock taking and regular monitoring
system exists for this invaluable resources. The review of the status of the vertebrate fauna revealed
that 54 inland fishes, 08 amphibians 58 inland reptiles, 41 resident birds and 40 inland mammals
have come under different categories of threat in Bangladesh. The country has already lost more
than 12 vertebrate fauna during the last century. The lack of proper database on our biodiversity is
one of the greatest impediments for utilization and safeguarding of our interests. DNA barcoding
is an efficient method for species-level identification. The country now needs to maintain the
genetic identity as well as integrity of species in their natural habitats. Hence documentation of
genetic variation and diversity is of vital significance to evolve conservation strategies with long
term impact.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
257
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Vertebrates
Using DNA barcoding approach to track poultry feed (meat and bone meal)
derived from swine sources in Bangladesh
A. Z. Siddiki, M. I. Uddin, K. M. R. Uddin, K. M. Kamaruddin
Poultry Research and Training Centre (PRTC), Chittagong Veterinary and Animal Sciences
University (CVASU), Khulshi-4225, Chittagong, Bangladesh.
E-mail: [email protected]
The poultry sector is an emerging sector in Bangladesh where feed ingredients are imported every
year. The predominantly Muslim country has restrictions in pork farming as Muslims does not eat
pork due to religious obligations. Bangladesh prohibits the importation of protein concentrate made
from bone meal, meat meal and other tissues derived from pork origin because of religious taboos
and contamination with some contagious diseases like BSE, Salmonella, etc. A porcine detection
methodology based on deoxyribonucleic acid (DNA) extraction and polymerase chain reaction
(PCR) amplification of a specific porcine fragment was used based on the detection of three
different porcine mitochondrial genes (DNA barcoding approach). A simple modified technique was
used to extract DNA from protein concentrate by using commercially available kit and a series of
incubation steps were applied before PCR analyses. The optimized method is in practice to identify
pork in protein concentrate submitted to our laboratory for quality control tests. Toll now, out of 100
samples 17 were found positive indicating contamination with porcine materials. The study
highlights the role of DNA barcoding in successfully identifying adulteration and necessitates
further optimization.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
258
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Vertebrates
DNA barcoding reveals market substitution in commercial yak jerky in China
Yun-Yu Wang, Kai He, Wenzhi Wang,Ya-Ping Zhang
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese
Academy of Sciences China, Kunming, Yunnan 650223, China
E-mail: [email protected]
Food safety is becoming a major concern especially in Chinese consumers’ opinion. In the trade of
livestock products, especially the market of meat jerky, the species authenticity is complicated
because of loss of morphological features after food industrial processing. Here, we used a DNA
barcoding approach for the species identification of 30 commercial yak jerkies sold in China market.
DNA barcoding using cytochrome C oxidase 1 (COI) and 16S ribosomal RNA (16S) genes reveals
species substitution in 21 samples (70%). Among those, 3 (10%) were processed from taxa
Soricidae (Shrew), which maybe potentially dangerous due to toxic or hurtful for consumers. The
results showed serious commercial frauds in the yak jerky market and highlights DNA barcoding as
an effective tool for food traceability.
Fifth International Barcode of Life Conference – 27-31 October 2013 – Kunming
Abstracts Volume
259