CHARACTERIZING BILLBUG (SPHENOPHORUS SPP.) SEASONAL BIOLOGY USING DNA BARCODES AND A SIMPLE MORPHOMETRIC ANALYSIS by Marian M. Rodriguez-Soto A Thesis Submitted to the Faculty of Purdue University In Partial Fulfillment of the Requirements for the degree of Master of Science Department of Entomology West Lafayette, Indiana May 2021 THE PURDUE UNIVERSITY GRADUATE SCHOOL STATEMENT OF COMMITTEE APPROVAL Dr. Douglas S. Richmond, Co-chair Department of Entomology Dr. Laramy S. Enders, Co-chair Department of Entomology Dr. Linda J. Mason Department of Entomology Approved by: Dr. Stephen L. Cameron 2 Dedicated to my family, Melba L. Soto Montañez, Melba G. Rodriguez, and Nery M. Rodriguez; three women without whom I wouldn’t be here today. 3 ACKNOWLEDGMENTS I would like to acknowledge my co-advisors Dr. Douglas S. Richmond and Dr. Laramy Enders who provided the tools that I needed in my path of becoming a scientist. I am also thankful for Dr. Linda J. Mason who provided words of kindness and extended many opportunities to me. I would also like to acknowledge Dr. Stephen S. Cameron who guided with patience in concepts otherwise unknown to me. This work would not have been possible without the funding from USDA NIFA and our collaborators Dr. Ricardo Ramirez, his graduate students from Utah State University, and Dr. Xi Xiong from University of Missouri. Additionally, I am thankful for the cooperation from Brian Bornino and gentleman at the William H. Daniel Turfgrass Research and Diagnostic Center. I would also like to thank Dr. Benjamin Goller for help with coding and all of my lab mates and post docs, but specifically Dr. Helena Avila-Arias, Dr. Elizabeth French, Dr. Mackenzie Kjeldgaard, Thorsten Hansen, Gordon Macleod, Connor Sturr, Colleen Couch, and many other who helped along the way. A special thanks to my friends for helping with ideas and writing, Dr. Julius Eason, Samuel Cody, Zachery Wolfe, Pragya Kendel, Shuangmei Ding, Katie Brill, and Sean Lewis. I would also like to thank the office of graduate diversity initiatives for all of the mentoring and support during my first year of graduate school. Lastly but not least, my family and close friends who encouraged me even in the most difficult times, thank you. 4 TABLE OF CONTENTS LIST OF TABLES .......................................................................................................................... 7 LIST OF FIGURES ........................................................................................................................ 8 ABSTRACT .................................................................................................................................. 10 DNA BARCODING OF BILLBUGS (Sphenophorus SPP.) TO TRACK THEIR SEASONAL BIOLOGY ............................................................................................................... 11 1.1 Species Complexes ........................................................................................................... 11 1.2 Billbug Species Complex .................................................................................................. 12 1.3 Billbug Damage and Management in Turfgrass ............................................................... 14 1.4 Using DNA Barcoding to Resolve Species Complexes and Improve Pest Management . 15 1.5 Understanding the Billbug Species complex using DNA Barcoding to Inform management ........................................................................................................................................... 17 1.6 References ......................................................................................................................... 19 CHARACTERIZING BILLBUG (Sphenophorus SPP.) SEASONAL BIOLOGY USING DNA BARCODES AND A SIMPLE MORPHOMETRIC ANALYSIS........................ 24 2.1 Abstract ............................................................................................................................. 24 2.2 Introduction ....................................................................................................................... 24 2.3 Materials and Methods ...................................................................................................... 27 2.3.1 Adult DNA Barcoding Reference Database .............................................................. 27 2.3.2 Larval Species Identification ..................................................................................... 30 2.3.3 Larval Seasonal Phenology Maps .............................................................................. 31 2.4 Results ............................................................................................................................... 32 2.4.1 Adult DNA Barcoding Reference Database .............................................................. 32 2.4.2 Larval Species Identification ..................................................................................... 33 2.4.3 Larval Seasonal Phenology Maps .............................................................................. 33 2.5 Discussion ......................................................................................................................... 34 2.5.1 Development of a reliable DNA Barcoding Tool for Sphenophorus in the U.S. ...... 34 2.5.2 DNA Barcoding tool provides insight into Billbug biology ...................................... 36 2.6 Conclusion ........................................................................................................................ 37 2.7 Figures and Tables ............................................................................................................ 39 5 2.8 References ......................................................................................................................... 48 2.9 Supplemental Material ...................................................................................................... 53 APPENDIX ................................................................................................................................... 55 6 LIST OF TABLES Table 2.1. Summary of DNA barcoding results used to create a reference sequence database from adult billbug (Sphenophorus spp.) species collected in Indiana, Utah, Missouri, Arizona, and two outgroup species. The total number of DNA extractions performed, successful sequencing of PCR products (% Success) and total number of sequences included in the reference database are shown for each barcoding gene (COI, 18S and ITS2). ............................................................................ 39 Table 2.2. Summary of larval specimens collected at Utah 2018 and Indiana 2020 in cool- and warm-season turfgrass. The total number of each species identified at each location and type of grass (*cool- or °warm-season) is included. .................................................................................. 40 Supplementary Tables Table S.1. COI, 18S, and ITS2 primer sequences for PCR reactions for Sphenophorus spp. Schönherr adults and larvae associated with turfgrass following Duffy et al. (2018). ................. 53 Table S.2. COI, 18S, and ITS2 primer amplification conditions for PCR reactions for Sphenophorus spp. Schönherr adults and larvae associated with turfgrass following Duffy et al. (2018). ........................................................................................................................................... 54 Appendix Table Table A.1. Association of each larval specimen with its respective species identification (Shenophorus parvulus, S. venatus, and S. minimus), state (Utah and Indiana), collection location (DTRC=William H. Daniel Turfgrass Research and Diagnostic Center, GRF=Greenville Research Farm, LCC=Logan Country Club, BPC=Bimmel Practice Complex), grass type (*cool- or °warm- season), collection date, and head capsule width diameter (mm)…………………………………55 7 LIST OF FIGURES Figure 2.1. Maximum likelihood tree of COI sequences from Sphenophorus parvulus, S. venatus, S. minimus, S. inaequalis, S. cicatristriatus, and S. phoeniciencis adults. Collection location is represented by color blocks; Utah (blue), Indiana (green), Missouri (purple), Arizona (yellow), outgroups (red). Replicate numbers are indicated to the right of the scientific name and collection state (Utah=UT, Indiana=IN, Missouri=MO, Arizona-AZ). Numbers at nodes are bootstraps values (1,000 bootstrap replicates as percentages). * indicates sequences obtained from Duffy et al. (2018). ...................................................................................................................................... 41 Figure 2.2. Maximum likelihood tree of ITS2 sequences from Sphenophorus parvulus, S. venatus, S. minimus, S. inaequalis, and S. cicatristriatus. Collection location is represented by color blocks; Utah (blue), Indiana (green), Missouri (purple), Arizona (yellow), outgroups (red). Replicate numbers are indicated to the right of the scientific name and collection state (Utah=UT, Indiana=IN, Missouri=MO, Arizona-AZ). Numbers at nodes are bootstraps values (1,000 bootstrap replicates as percentages). * indicates sequences obtained from Duffy et al. (2018). .................................. 42 Figure 2.3. Maximum likelihood tree of 18S sequences from Sphenophorus parvulus, S. venatus, S. inaequalis, and S. cicatristriatus. Collection location is represented by color blocks; Utah (blue), Indiana (green), Missouri (purple), Arizona (yellow), outgroups (red).