Telenomus podisi: one species, or more? THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Kelsey Rae Bowers Graduate Program in Evolution, Ecology and Organismal Biology The Ohio State University 2015 Master's Examination Committee: Dr. Norman F. Johnson, advisor Dr. Marymegan Daly Dr. John V. Freudenstein Copyright by Kelsey Rae Bowers 2015 Abstract Parasitoid wasps are a collection of hymenopterans whose larvae feed on the body of a host, eventually killing it (Godfray 1994). The parasitoid wasp Telenomus podisi Ashmead (Hymenoptera: Platygastridae, Telenominae) parasitizes the eggs of both phytophagous and predaceous pentatomids, including a number of important agricultural pests (e.g., Temerak and Whitcomb 1984, Schaefer and Panizzi 2000). Comparisons of strains from Maryland and Brazil has begun to raise questions as to whether T. podisi is a single species (Borges et al. 2003). This study considers populations of T. podisi throughout the species range to determine if there is evidence to support the hypothesis of T. podisi as more than one species. Cytochrome oxidase I is sequenced for 50 specimens of T. podisi from Saskatchewan, Ohio, Tennessee, Arkansas, Nebraska, Panama, Brazil and the Dominican Republic, along with one specimen each of Telenomus sechellensis Kieffer, Telenomus grenadensis Ashmead, and Trissolcus basalis Wollaston. A number of species delimitation methods are considered, including DNA barcoding with pairwise distances, the General Mixed Yule Coalescent Model (GMYC) and Poisson Tree Process Model (PTP) (Fujisawa and Barraclough 2013, Zhang et al. 2013). DNA barcoding attempts to detect the transition between interspecific and intraspecific variation by a gap in the distribution of genetic distances, and this gap is clear in the histogram of p- distances (Hebert et al., 2004). Mean p-distances between specimens from Ohio and Brazil fall within the rightmost curve in the distribution, suggesting that these populations ii may be separate species. A maximum likelihood tree and the results from GMYC and PTP suggest that the situation may be even more complicated. Both support the hypothesis of T. podisi as more than one species; PTP identifies 16 different species and GMYC 14. While a clade consisting of the majority of specimens from the continental US is identified as one species in both methods (support=0.905 in bPTP), so is a clade of two Ohio specimens (0.927), a clade consisting of two Ohio specimens and one from Texas (0.859) and a clade of 2-4 specimens from Texas, delimited differently depending on the method (0.247). Other potential species identified corresponded with geographic region, including a clade from Panama (0.944), a clade formed by two individuals from Brazil (0.801), and one by the two specimens from the Dominican Republic (0.898). A number of amino acid changes unique to these clades are identified, including a number of changes that distinguish the specimens from Ohio and Texas that are not included in the continental US clade. Results from pairwise distance data, GMYC, PTP and analysis of fixed sites all suggest that Telenomus podisi may include more than one species. Increased sampling, introduction of multiple loci, and further study of morphological differences within the group is necessary. iii Dedication To my husband, my family, and my friends: thank you for believing in me. iv Acknowledgments Foremost I would like to thank my advisor Dr. Norman Johnson for his guidance and expertise. I am grateful to have been able to learn from you. Thanks also to Dr. Alejandro Valerio, whose humor, friendship and advice was indispensable for me as a new graduate student, and my committee members Dr. Meg Daly and Dr. John Freudenstein. You listened and gave me advice during the most difficult points of my graduate career, you helped me to graduate. Thank you. I would also like to thank Corey Ross, who has never stopped helping me since I called the Evolution, Ecology and Organismal Biology office unable to find my way to teaching orientation on my first day. Without you I would have been lost. And finally, thank you Nick Skomrock; you know exactly how much you’ve done for me as a friend and fellow graduate student. v Vita May 2008 .......................................................Sidney High School May 2008 .......................................................A.S. Edison Community College June 2011 ......................................................B.S. Biological Science, Wright State University 2011 to 2012 .................................................University Fellowship, The Ohio State University 2012 to 2013 ..................................................Graduate Teaching Associate, Center for Life Sciences Education, The Ohio State University 2013 to 2014 ..................................................Metro Fellowship, Metro Early College High School 2014 to present ..............................................Graduate Teaching Associate, Center for Life Sciences Education, The Ohio State University Fields of Study Major Field: Evolution, Ecology and Organizmal Biology vi Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita ..................................................................................................................................... vi List of Tables ..................................................................................................................... ix List of Figures ..................................................................................................................... x Introduction ......................................................................................................................... 1 Parasitoid Wasps and Telenomus podisi ......................................................................... 1 DNA Barcoding............................................................................................................... 6 Species Concepts ............................................................................................................. 8 Biological Control ......................................................................................................... 12 Methods............................................................................................................................. 15 Results ............................................................................................................................... 19 Pairwise Distances......................................................................................................... 19 Population Genetics Methods........................................................................................ 21 Fixed Sites and Haplotype Network.............................................................................. 23 vii Discussion ......................................................................................................................... 25 DNA Barcoding and Pairwise Distances ...................................................................... 25 Maximum Likelihood Tree ........................................................................................... 31 GMYC and PTP Analysis ............................................................................................. 32 Fixed Sites and Haplotype Network Analysis............................................................... 34 Future Directions ........................................................................................................... 36 Conclusion ........................................................................................................................ 38 References ......................................................................................................................... 40 Appendix: Results and Data.............................................................................................. 45 viii List of Tables Table 1. Mean pairwise distances between Telenomus podisi from [ 1] Texas, [2] Ohio, [3] Arkansas, [4] Nebraska, [5] Brazil, [6] Tennessee, [8] Panama, [9] Dominican Republic, and individuals of [10] Trissolcus basalis, [11] Telenomus sechellensis, and [12] Telenomus grenadensis. Calculated in MEGA version 5 (Tamura et al. 2011). ....... 20 Table 2. Mean pairwise distances within groups of Telenomus podisi from different regions, Trissolcus basalis, Telenomus sechellensis and Telenomus grenadensis. Calculated in MEGA version 5 (Tamura et al. 2011). Groups with only one specimen are marked not calculated (n/c). .............................................................................................. 20 Table 3. Data for all specimens included in the analysis. Accension numbers are pending inclusion to GenBank. ....................................................................................................... 50 Table 4. Number of mutations between individuals in the minimum spanning network (Bandelt et al. 1999) constructed in PopART 1.7 (http://popart.otago.ac.nz) .................. 54 ix List