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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.