Molecular Systematics of the Greater Fritillary Butterflies (Nymphalidae: Speyeria): Reduced Representation Sequencing, Phylogeny, and Incipient Speciation
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Molecular systematics of the greater fritillary butterflies (Nymphalidae: Speyeria): reduced representation sequencing, phylogeny, and incipient speciation by Erin O’dell Campbell A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Systematics and Evolution Department of Biological Sciences University of Alberta © Erin O’dell Campbell, 2019 Abstract Species delimitation and phylogenetic reconstruction are essential for understanding and categorizing lineage divergences, and ultimately species. Yet, because of the complexities underlying speciation, inferring these patterns and characterizing their sources has been a consistent challenge for systematists. Recent advances in genomics have increased our understanding of how speciation may be influenced by ecology, biology, geography, and demography. However, the expansion of methods for producing genomic data warrants continued assessment to ensure they produce robust systematic inferences. Here, I conceptually and empirically assess the technical compatibility of some of the most commonly-used reduced representation sequencing (RRS) approaches for producing genomic single nucleotide polymorphism (SNP) data for systematic and population genomic inference in non-model taxa. I then apply these RRS approaches to re-assess the molecular systematic relationships of species in the butterfly genus Speyeria Scudder, 1872. These butterflies have a long history of taxonomic instability that is largely due to morphological variability and insufficient taxon sampling in previous systematic studies, which has impeded the assessment of species limits despite growing conservation concern for several taxa. In a literature review of studies that describe new RRS techniques, I found that several published methods are only subtly different from one another, and often have convergent technical features despite being published with distinct names. I then empirically assessed the compatibility of two commonly used RRS techniques using several Speyeria species. Despite large intial differences between methods in sequencing depth and coverage, the genotyping consistency between methods was high and improved the recovery of monophyletic clades of Speyeria. In an expanded taxonomic and geographic phylogenetic study of Speyeria, I recovered ! ii extensive mito-nuclear discordance between the SNP-based species phylogeny and the mitochondrial COI gene phylogeny, and recovered genomic divergences in two species that likely warrant taxonomic revision. Finally, I re-assessed the species limits in the S. atlantis- hesperis species complex, which has become infamous for its morphological variability. My results uphold S. atlantis (Edwards, 1862) as a species distinct from S. hesperis (Edwards, 1864), and additionally indicate that S. hesperis should be taxonomically recognized as two species. This work presents the most geographically comprehensive taxonomic sampling of Speyeria to date, and the first use of genomic SNPs for elucidating the systematics of these enigmatic butterflies. These studies build conceptually on methodological advances to investigate their impact on species detection, and adds to a growing body of research that has demonstrated the utility of genomic SNPs for clarifying long-standing evolutionary problems. ! iii Preface This thesis represents original work by Erin O Campbell, however it would not have been possible without the collaboration and support of several other people. As such, I use the term “we” to signify their contributions throughout. A version of Chapter 2 has been published as Campbell EO, Brunet BTM, Dupuis JR, and Sperling FAH (2018) “Would an RRS by any other name sound as RAD?”, Methods in Ecology and Evolution volume 9, pages 1920-1927. EOC and BTMB conducted the meta-analysis and literature review, and drafted the manuscript. All authors contributed to the study design and to manuscript draft revisions. A version of Chapter 3 has been published as Campbell EO, Davis CS, Dupuis JR, Muirhead K, and Sperling FAH (2017) “Cross-platform compatibility of de novo-aligned SNPs in a non- model butterfly genus”, Molecular Ecology Resources volume 17, pages e84-e93. EOC conducted data analysis, and drafted the manuscript. KM provided custom bioinformatic scripts. EOC, CSD, JRD, and FAHS contributed to the study design and to manuscript draft revisions. A version of Chapter 4 has been published as Campbell EO, Gage EV, Gage RV, and Sperling FAH (2019) “Single nucleotide polymorphism-based species phylogeny of greater fritillary butterflies (Lepidoptera: Nymphalidae: Speyeria) demonstrates widespread mito-nuclear discordance”, Systematic Entomology, DOI:10.1111/syen.12393. EOC, EVG, RVG, and FAHS contributed to specimen collection. EOC conceived the study design, with input from EVG, RVG, and FAHS, and EOC conducted analyses, and drafted the manuscript. FAHS provided conceptual guidance. All others contributed to manuscript draft revisions. ! iv Acknowledgements First, thank you Felix, for taking a chance on a student with an English degree and no prior entomological, wet lab, or bioinformatic experience. Your leap of faith and mentorship made this thesis possible. Thank you also to Heather Proctor and Paul Stothard for your support and input as committee members, to Yan Boucher and Jocelyn Hall for participating in my candidacy examination, and to Toby Spribille and Niklas Wahlberg for acting as my arms-length and external defense examiners. This work was facilitated by so many people - thanks to Lien Luong for your early support and for helping me through my first experience with the academic publishing process. Thanks to Sophie Dang and Cheryl Nargang from the MBSU, and Brian Boyle from IBIS for their sequencing support, and to the many avid Speyeria enthusiasts I’ve met along the way that have contributed specimens or insight to this work: Thomas Simonsen, one of the first friendly faces I met at LepSoc, Chris Schmidt, Ted Pike, Bob Parsons, Dave Lawrie, Gary Anweiler, Norbert Kondla, and John Acorn. And of course, a huge thanks to Ed and Randy Gage - your cross-country butterfly-hunting expeditions over the past 5 years have been an amazing contribution and commitment to this work, and the reason this project was able to grow from a regionally-focused M.Sc into a Ph.D. Thanks to Corey Davis for your ongoing advice and support - our chats helped me to navigate the deep waters of molecular biology and NGS. Thanks also to the many fellow students and lab members I’ve had the privilege of crossing paths with over the years - Giovanny Fagua, Christianne McDonald, Stephen Trevoy, Gwylim Blackburn, Fabien Condamine, Kurt Yakimovich, Emily Dong, Marnie Wright, Zac MacDonald, Federico Riva, Kyle Snape, Phil Batista, Lisa Lumley, Caroline Whitehouse, Rowan French, Janet Sperling, Tiago Simões, Bryan ! v Brunet, Jasmine Janes, and Oksana Vernygora. And to the “core four” and the “elite” - Tyler, Victor, Brittany, Ronald, and Julian - your friendship and support over the years has meant so much. Enormous thanks are also due to my family, who have put up with my incessant talking about work, manuscript writing on Christmas Eve, and the co-opting of family vacations to collect butterflies: Mom, Dad, Sheila, Grandma, Kevin, Danny, Hunter, William, Daniel, Kirby, Guy, and Luisa - I won the jackpot with all of you. Mom: thank you for your unwavering support and your cowboy attitude towards fieldwork and international travel. Kirby and Rusty, thank you for braving ornery cattle and murderous grouse with me. And to the triplets, a veritable gang of ant-stompers and moth-catchers - thank you for reminding me of the magic of “bug science” seen through the eyes of a child (or three). And James, none of this would have been possible without you. Your unconditional love and support during grad school and over the past fourteen years has brought so much to my life that words just don’t do it justice. Funding for this research was provided by an Alberta Conservation Association Grant in Biodiversity to myself, and by a Natural Sciences and Engineering Research Council of Canada Discovery Grant to Felix Sperling. ! vi Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Acknowledgements ........................................................................................................................v List of Figures ............................................................................................................................... xi List of Tables ............................................................................................................................... xii List of Abbreviations ................................................................................................................. xiii List of Appendices ...................................................................................................................... xiv ! Introduction and thesis objectives ................................................................................................1 1.1 General introduction ..............................................................................................................1 1.2 A shifting perspective of the “species