THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE DEPARTMENT OF BIOLOGY SPECIES DELIMITATION AND THE EVOLUTION OF HIGHLY VARIABLE MORPHOLOGICAL TRAITS IN THE HOLARCTIC SOCIALLY PARASITIC BUMBLE BEE, BOMBUS FLAVIDUS SARAH DANIELLE WILLIAMS SPRING 2018 A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Biology with honors in Biology Reviewed and approved* by the following: Heather Hines Assistant Professor of Biology and Entomology Thesis Supervisor James Marden Professor of Biology and Associate Director, Huck Institutes of the Life Sciences Honors Adviser * Signatures are on file in the Schreyer Honors College. i ABSTRACT Bombus flavidus is a socially parasitic bumble bee with contentious species status. Multiple separate species within Bombus flavidus have been suggested, both on local and global scales. Until recently recognition of a Nearctic B. fernaldae and Palearctic B. flavidus was favored, but limited genetic data suggested that even these could be a single widespread species, B. flavidus. A combination of COI sequencing, color pattern, wing morphometric, and genitalia morphology analysis were used to resolve the species status of this lineage. In an initial analysis, male Bombus flavidus from Oregon, U.S.A. were determined to be part of one species. These individuals have high polymorphism in color but exhibit darker phenotypes in the darker Pacific mimicry zone. A broader analysis including Bombus flavidus specimens from Europe and Russia (“Old World” specimens) and North America (“New World” Bombus fernaldae), revealed that B. fernaldae is a distinct lineage, either a species or subspecies. However, B. fernaldae is not broadly Nearctic, but rather confined to the eastern Appalachian and boreal regions of the United States and far southeastern Canada, whereas B. flavidus occurs throughout the western U.S., Canada, and the Old World, a distribution broader than that achieved for any host bumble bee species. Analysis of phenotype data revealed that color polymorphisms are retained across the B. flavidus/fernaldae range and genitalic morphology is highly variable compared to other species. ii TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................... iv LIST OF TABLES ....................................................................................................... v ACKNOWLEDGEMENTS ......................................................................................... vi Chapter 1 Thesis Introduction ...................................................................................... 1 1. Taxonomy, phylogenetics, and species delimitation in bumble bees ........................... 1 2. Evolution of social parasitism in bumble bees ............................................................. 3 3. The obligately socially parasitic subgenus Psithyrus ................................................... 4 a. Taxonomy and phylogenetic relationships ................................................................... 4 b. Geographic distribution ................................................................................................ 6 4. The socially parasitic bumble bee, Bombus flavidus .................................................... 6 5. Thesis goals .................................................................................................................. 7 Chapter 2 Bombus flavidus in Oregon, USA is a single highly polymorphic species . 9 1. Abstract ........................................................................................................................ 9 2. Introduction .................................................................................................................. 9 3. Methods ........................................................................................................................ 11 4. Results .......................................................................................................................... 16 5. Discussion .................................................................................................................... 22 Chapter 3 Global analysis of Bombus flavidus species group supports the distinction of Bombus fernaldae ................................................................................................. 26 1. Abstract ........................................................................................................................ 26 2. Introduction .................................................................................................................. 26 3. Methods ........................................................................................................................ 29 4. Results .......................................................................................................................... 33 5. Discussion .................................................................................................................... 41 Appendix A Specimen Tables .................................................................................... 47 Appendix B Color Data .............................................................................................. 57 Appendix C Nexus haplotype file ............................................................................... 59 Trimmed B. flavidus haplogroup Nexus file for popART: .......................................... 59 Appendix D Genitalia Images ..................................................................................... 63 Appendix E Genitalic Data Matrices .......................................................................... 67 iii Appendix F Wing Landmark-Based Morphometric Analysis Data (.tps file) ............ 73 Appendix G R Scripts ................................................................................................. 86 BIBLIOGRAPHY ........................................................................................................ 95 iv LIST OF FIGURES Figure 1: Oregon Bombus flavidus localities ........................................................................... 11 Figure 2: (A) Template used for color pattern observation; (B) Black color B. flavidus pattern extreme from Oregon (specimen 300102); (C) Yellow color B. flavidus pattern extreme from Oregon (specimen 300304) .............................................................................................. 12 Figure 3: B. flavidus male genitalia with labeled measurements ............................................. 14 Figure 4: (A) Average individual percent yellow pile for Oregon male Bombus flavidus; (B) Boxplot of total body pile percent yellow for individuals from Western vs. Eastern Oregon localities ........................................................................................................................... 17 Figure 5: Extremes in B. flavidus gonobase shape (chosen using A:B ratio and visual observation) from each Oregon locality region (Figure 1), used for COI sequencing .......................... 19 Figure 6: Histogram of Oregon B. flavidus gonobase A:B ratios ............................................ 19 Figure 7: (A) PCA graphic of Oregon B. flavidus male genitalia measurements by locality; (B) PCA graphic of Oregon B. flavidus male genitalia measurements by region .................. 20 Figure 8: Total body pile percentage yellow vs. locality elevation (m) ................................... 22 Figure 9: Labeled measurements for sister species genitalia variation analysis ...................... 32 Figure 10: Landmarks used in global B. flavidus wing morphometric analysis ...................... 33 Figure 11: Global color pattern distribution for B. flavidus and B. fernaldae ......................... 34 Figure 12: Haplotype map for New World and Old World Bombus “flavidus” specimens .... 36 Figure 13: (A) PCA results for genitalia measurements of B. flavidus specimens from Oregon, North Carolina, and Pennsylvania, USA; (B) PCA results with eigenvectors for genitalic analysis on B. flavidus from the United States ................................................................. 36 Figure 14: Distribution of gonobase ratios for B. flavidus from the eastern United States and Oregon .............................................................................................................................. 38 Figure 15: PCA results for genitalic analysis on B. flavidus from the United States and B. californicus, B. fervidus, B. insularis, and B. melanopygus. ............................................ 39 Figure 16: 3D figure from PCA of genitalia from B. flavidus, B. fervidus, B. melanopygus, B. californicus, and B. insularis. .......................................................................................... 40 Figure 17: Wing morphometric analysis data for global B. flavidus specimens ...................... 41 v LIST OF TABLES Table 1: Results of COI analysis of Oregon Bombus flavidus males based on color pattern and genitalia morphology extremes ........................................................................................ 21 Table 2: Bombus flavidus specimen table for Oregon analysis ................................................ 47 Table 3: Bombus “flavidus” specimen table for global analysis ............................................. 50 Table 4: Oregon B. flavidus Total Body Pile Color Matrices .................................................