Download Conclusion - National University of Singapore

Orthoptera in Singapore: diversity, new species
and the predatory katydids
Tan Ming Kai (A0087366R)
Supervisors: Prof. Rudolf Meier and Dr. Hwang Wei Song
Department of Biological Sciences, Faculty of Science, National University of Singapore
Introduction
•
•
•
•
•
•
Objectives
Orthoptera include grasshoppers, crickets and katydids
Diverse taxa: ~ 27000 species worldwide 1
Master of deception and known for “singing” abilities
Knowledge gaps on Orthopteran diversity in Singapore still to be filled
More new species still awaiting discovering
Diet of orthopterans poorly known with traditional diet analysis laborious,
incomprehensive and low resolution
Part 1: Diversity of Orthoptera in SIngapore
2. Species discovery: Describing three new species of scaly crickets and discovering cryptic pygmy
grasshoppers using molecular and ecological data
3. Molecular ecology: Pioneering diet assessment of predatory katydids Hexacentrus unicolor via
metabarcoding
Scaly cricket Micrornebius from Singapore
Part 2: Species discovery: cryptic
grasshoppers and scaly crickets
Material and Methods
• First checklist for Singapore compiled:
1. 1,498 specimens deposited, mainly from
I. Historical collection by D. H. Murphy (1954-2005)
II. Active field collection (2010-2015)
2. 240 research articles on taxonomy of Orthoptera and global
database Orthoptera Species File
• ~240 high resolution images taken using Visionary Digital System
and uploaded online (http://lkcnhm.nus.edu.sg/APS)
• Synonym lists, identifying features and biological notes added
Results
•
•
•
•
1. Diversity of Orthoptera in Singapore: Determining orthopteran diversity of Singapore and
compiling images and taxonomic information for public access
Material and Methods
• Hennigian Species Concept applied for species
hypothesis
• Morphology, DNA and ecology used for testing
species boundaries
I. Morphometric analyses using PCA
II. Male genitalia dissection
III. Phylogenetic analysis of COI and ITS2 datasets
IV. Microhabitat analysis using PCA
• Taxonomy reviewed and published in Zootaxa 2
• Number of Micrornebius recorded from Singapore
increased from 2 to 5
• 3 species new to science described
Micronebius eclipsus
Cryptic pygmy grasshopper from Nee Soon
245 species
50 Caelifera (grasshoppers)
195 Ensifera (crickets, katydids and allies)
All major lineages of Orthoptera found in Singapore
•
•
No distinct morphological differences
Morphometric analysis: no clear groupings
Micronebius distinctus
Micronebius mandai
Scale bar: 10 mm
• 2 distinct lineages based on COI data • ITS2 results more complicated – require
• 12.7% pairwise divergence between
further analysis
the lineages
• NGS revealed multiple copies in ITS2
RAx-ML Tree
MP Tree
• Using paralogs for species
delimitation fundamentally wrong
• Paralogs in ITS2 reflected similar
clustering as homologs in COI
Orthopterans species page in LKCNHM’s
Animals & Plants Web
• Microhabitat analysis:
• Species 1 found in
forested habitat
• Species 2 found in
grassy habitat
N = 41
Possible explanations:
concerted evolution 3
• Conclusion: 2 cryptic species based on COI and ecological data
Part 3: Molecular ecology: diet analysis
via metabarcoding
Material and Methods
• 30 Hexacentrus unicolor dissected to remove mid- and
hindguts  gut lining removed to obtain gut content
 DNA extraction of gut content
• Amplify COI (313bp) using tagged primers
• 3-8 PCR replicates per specimen to reduce PCR bias
and amplify cryptic prey multiplexed and
SureCleaned
• COI sequenced using Illumina MiSeq
• Sequences blasted against GenBank database for prey
identification
Preliminary results
• Metabarcoding
revealed newly
documented prey
items from gut
content:
• Grasshopper
Xenocatantops
humilis (98%)
• Flesh fly Ravinia
pusiola (89%)
• Metabarcoding
findings
support field
observation:
Grasshopper
Oxya sp. (100%)
• Unknown prey
identified via
metabarcoding:
Moth Hypena
spp. (Erebidae)
(94%)
• Potential for prey identification at higher resolution
• Field observation + metabarcoding gut content = comprehensive diet
breadth
Conclusion
• Most comprehensive understanding of the
diversity of Orthoptera in Singapore available
• 4 new species described: 3 scaly crickets and 1
pygmy grasshopper
• Diet breadth of Hexacentrus unicolor potentially
more comprehensively documented than before
Acknowledgements
I am thankful of Yeo Huiqing for field assistance; Darren Yeo, Amrita Srivathsan, Wong Wing
Hing, Zheng Yujie and Sujatha Sunil for help with bioinformatics; Ang Yuchen for help with
APS; Chong Kwek Yan for help with statistics; Sigfrid Ingrisch for the review of manuscript 2
and Sergey Storozhenko for help on Tetrigidae. Research permit: NP/RP10-073.
References
1 Eades, D. C., Otte, D., Cigliano, M. M., & Braun, H. (2015). Orthoptera Species File. Version 5.0/5.0.
2 Tan, M. K. (2014). New species of small scaly crickets of genus Micrornebius (Orthoptera:
Mogoplistidae; Mogoplistinae) from Singapore. Zootaxa.
3 Ruiz‐Estévez, M. et al. (2015). Non‐random expression of ribosomal DNA units in a grasshopper
showing high intragenomic variation for the ITS2 region. Insect molecular biology.