Download DNA Sequencing as a Method for Larval Identification in Odonates

DNA Sequencing as a Method for Larval
Identification in Odonates
Adeline Harris
121 North St Apt 3
Farmington, ME 04938
[email protected]
Christopher Stevens
147 Main St Apt 1
Farmington, ME 04938
[email protected]
Faculty Sponsors
Dr. J.A. Doty
[email protected]
Dr. R.G. Butler
[email protected]
1 Introduction
Damselflies (Odonata: Zygoptera) are a diverse group of organisms ranging from the subarctic to
the tropical that play an important role in lentic and lotic ecosystems (Artiss et al 2001). They are
considered valuable biological indicators as they lay eggs in specific plant structures and sometimes on
specific plant species (Butler & deMaynadier 2008). Due to this fact, they are at an increased risk from
near-shore development and disturbance which may damage near-shore vegetation required for
reproduction (Butler & deMaynadier 2008). Unlike the dragonflies (Anisoptera), the majority of the
Zygoptera are weak flyers. Therefore any habitat disturbance may have large effect on their fitness. As a
result, quantifying their diversity is an important aspect of their conservation. Unfortunately, abundance is
often estimated by simply surveying adult populations (Fleck et al 2006; Rach et al 2007), which may
underestimate the actual diversity within a given body of water (Fleck et al 2006).
Monitoring the biodiversity of small, ecologically significant areasfor conservation depends on
reliable identification of species. Larval odonate identification in particular presents a major challenge to
scientists (Rach et al 2007). Identification of adults, especially males, is often quite easy and allows some
understanding of their diversity. Larval identification, however, is very difficult and sometimes
impossible for some species (Westfall & May 2006). Taxonomy and identification of zygopterans has
previously been largely based on morphological characteristics (Fleck et al 2006). This may present an
issue when determining phylogenies and identities of closely related species (Pilgrim et al 2002). This is
especially true when looking at larval specimens. Methods such as DNA barcoding allow for consistent
and reliable results, which complement traditional morphological identification (Rach et al 2007).
Assessment using DNA as a taxonomic tool will allow a more accurate evaluation of species identity and
diversity (Pilgrim et al 2002).
2 In addition to identification purposes, DNA sequencing is important in phylogenetic research.
The 16S rRNA and rDNA regions of the mitochondrial genome have often been used to identify odonate
species relationships (Misof et al 2000; Hasegawa & Kasuya 2006; Pilgrim & Dohlen 2008). In more
recent studies, researchers have used multiple genomic sequences that allow them to examine different
levels and patterns of variation for phylogenetic research (Chippindale et al 1999).
Phylogenies can also be used to trace patterns of biodiversity in species of damselflies over time
by looking at sequence divergences in DNA (Brown et al 2000). Local diversity is greatly influenced by
speciation and extinction in addition to ecological factors, and studies have shown that climatic patterns
have had large impacts on species radiation and diversification (Brown et al 2000; Turgeon et al 2005).
The only way to reliably determine historical radiations is through the use of fossil evidence or DNA.
However fossil evidence is often limited in terms of temporal scope and geographical scale, as climatic
variability plays a large role in the fossilization process (Turgeon et al 2005). DNA sequencing allows a
more robust phylogeny to be created that illustrates speciation events and helps to explain local and global
species diversity. The phylogenetic relationship between the two species concerned in this study places
them only one to three nodes apart on various parsimonious tree diagrams based upon both morphological
and genomic data (Brown et al 2000). In particular, the morphological tree places them closer together
than any of the genetic trees, suggesting that morphological similarity does not necessarily denote genetic
similarity (Brown et al 2000). The goal of this project is to amplify a sequence of DNA that will allow
reliable species determination for two congeneric damselflies (Enallagma ebrium and E. hageni), females
of which are morphologically indistinguishable as larvae.
3 Research Methods
Specimens were collected in Maine from June to August, 2007. These were previously identified
and preserved in ethanol [(70%) various sources]. Male individuals of each species will be selected for
DNA isolation and sequencing. For each individual, the legs will be removed and crushed using liquid
nitrogen. DNA will be extracted using a standard DNA extraction kit. Two primer pairs will be used to
amplify 28S rRNA on the mitochondrial genome. An automated sequencer will be used to sequence the
amplified fragments. In addition to sequencing done on the premises a sample will be sent to a
professional lab for sequencing to assure the correct sequence is isolated. Once a useable sequence has
been obtained, the procedure will be repeated with female specimens of E. ebrium and E. hageni, which
cannot be differentiated between morphologically.
Scholarship Funds and Project Outcomes
This is a joint proposal for two separate Wilson Scholarships in which each individual is in
charge of a separate aspect of the project. Adeline Harris will be in charge of DNA extraction, isolation,
and sequencing regarding E. ebrium and Christopher Stevens will be in charge of DNA extraction,
isolation, and sequencing regarding E. hageni as all aspects of the project will be extremely time
consuming. Results for both species will be compiled and used to identify indistinguishable female
specimens and a joint presentation will be given on symposium day detailing the results and implications
of the study. In addition, if results are significant, an attempt will be made to publish them in a peer
reviewed journal such as the Journal of Insect Conservation or Odonatologica. The additional funds for
research materials will be required for both the scholarships as our needs for extraction and polymerase
chain reaction (PCR) kits and other chemicals will likely reach $600 (see supplies).
4 Supplies Needed
DNA isolation kit - ~$340
Primers - ~$22/primer
Agarose gel - ~$70
Ethidium Bromide - ~$21/g
PCR purification kit - $96
Micropipet loading tips for sequencer gel plate - ~ $50
Total
~$600
5 References
Artiss, T., T.R. Shultz, D.A. Polhemus, and C. Simon. Molecular phylogenetic analysis of the dragonfly
genera Libellula, Ladona, and Plathemis (Odonata: Libullulidae) based on mitochondrial
cutochrome oxidase I and 16S rRNA sequence data. 2001. Molecular Phylogenetics and
Evolution, 18:3 pp348-361.
Brown, J.M., M.A. McPeek, and M.L. May. A phylogenetic perspective on habitat shifts and diversity in
the North American Enallagama damselflies. 2000. Systematic Biology, 49:4 pp679-712.
Butler, R.G., P.G. deMaynadier. The significance of littoral and shoreline habitat integrity to the
conservation of lacustrine damselflies (Odonata). 2008. Journal of Insect Conservation, 12:
pp23-36.
Chippindale, P.T., V.K. Dave, D.H. Whitmore, and J.V. Robinson. Phylogenetic relationships of North
American damselflies of the genus Ischnura (Odonata: Zygoptera: Coenagrionidae) based on
sequences of three mitochondrial genes. 1999. Molecular Phylogenetics and Evolution, 11:1
pp110-121.
Fleck, G., M. Brenk, and B. Misof. DNA taxonomy and the identification of immature insect stages: the
true larva of Tauriphila argo (Hagen 1869) (Odonata: Anisoptera: Libellulidae)
Hasegawa, E., and E. Kasuya. 2006. Phylogenetic analysis of the insect order Odonata using 28S and 16S
rDNA sequences: a comparison between data sets with different evolutionary rates.
Entomological Science, 9:pp55-66.
Misof, B., C.L. Anderson, and H. Hadrys. A phylogeny of the damselfly genus Calopteryx (Odonata)
using mitochondrial 16S rDNA markers. 2000. Molecular Phylogenetics and Evolution, 15:1
pp5-14.
Pilgrim, E.M., S.A. Roush, and D.E. Krane. Combinging DNA sequences and morphology in systematics:
testing the validity of the dragonfly species Cordulegaster bilineata. 2002. Heredity, 89:
pp184-190.
Pilgrim, E.M., C.D. Von Dohlen. Phylogeny of the Sympetrinae (Odonata: Libellulidae): further evidence
of the homoplasious nature of wing venation. 2008. Systematic Entomology, 33:pp159-174.
Rach, J., R. DeSalle, I.N. Sarkar, B. Schierwater, and H. Hadrys. Character-based DNA barcoding allows
discrimination of genera, species, and populations in Odinata. 2008. The Royal Society,
275:pp237-247.
Turgeon, J., R. Stoks, R.A. Thum, J.M. Brown, and M.A. McPeek. Simultaneous quaternary radiations of
three damselfly clades across the Holarctic. 2005. The American Naturalist, 165:4:ppE76-107.
Westfall, M.J., and M.L. May. 2006. Damselflies of North America. Scientific Publishers. Gainesville,
Florida.
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