University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Spring 2020 Responses of Bats to White-Nose Syndrome and Implications for Conservation Meghan Stark University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation Stark, Meghan, "Responses of Bats to White-Nose Syndrome and Implications for Conservation" (2020). Doctoral Dissertations. 2518. https://scholars.unh.edu/dissertation/2518 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. RESPONSES OF BATS TO WHITE-NOSE SYNDROME AND IMPLICATIONS FOR CONSERVATION BY MEGHAN A. STARK B.S., University of Alabama at Birmingham, 2013 DISSERTATION Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy In Genetics May 2020 i This dissertation was examined and approved in partial fulfillment of the requirements for the degree of Ph.D. in Genetics by: Dissertation Director, Matthew MacManes, Assoc. Prof. UNH MCBS Jeffrey T. Foster, Associate Professor, NAU PMI W. Kelley Thomas, Professor, UNH MCBS Rebecca Rowe, Associate Professor, UNH NREN Thomas Lee, Associate Professor Emeritus, UNH NREN On April 6, 2020 Approval signatures are on file with the University of New Hampshire Graduate School. ii DEDICATION I dedicate this work to all of the strong women in my life: Myra Michele Ange Heather Michelle Coons Kaitlyn Danielle Cagle Brindlee Michelle Coons Patricia Gail Miller Sarah Jean Lane “Here’s to strong women. May we know them. May we be them. And may we raise them.” iii ACKNOWLEDGEMENTS I want to first thank my dissertation committee for all of their support over the last five years. Matt and Jeff, you have both provided a tremendous amount of academic and professional guidance. Simply put, this degree wouldn’t have been possible without you. To Kelley Thomas, Becca Rowe, and Tom Lee, I am so thankful for every email you responded to and for every edit you provided. Your feedback—and mentorship—was instrumental. I owe a huge debt of gratitude to my husband and best friend, Randy Stark. You made me breakfast. You held my hand. You let me cry. And you did all of it without a single complaint. I love you so much. Thank you for believing in me. To my family, words cannot express how much I love you or how grateful I am for all of your continued support. You are the reason I am here today. Heather, even from a young age, I walked with confidence in the world because I felt safe and protected. That was, in large part, because I had/have the best big sister. You were a rock for me even if you didn’t realize it. Kaity, you are my biggest cheerleader. When I feel inadequate, I know to call you. Your unwavering support has meant the world to me. Mom, there aren’t enough words. To put it simply, you are my best friend. Thank you for everything. Dad, we are two peas in a pod. My sense of humor, my tenacity, and my mouth, I owe to you. You made/make me feel invincible. I love you all. While there are far too many people to individually thank, I would be remiss if I did not specifically acknowledge Andrew Lang, Nate Ennis, Erin Neff, and Toni Westbrook, who have been endless sources of support. Thank you for everything. iv TABLE OF CONTENTS DEDICATION ……………………………………………………………………………. iii ACKNOWLEDGEMENTS ……………………………………………………………...... iv LIST OF TABLES ………………………………………………………………………… viii LIST OF FIGURES ………………………………………………………………………… ix ABSTRACT ………………………………………………………………………………... xi CHAPTER PAGE INTRODUCTION …………………………………………………………………………. 1 I. White-nose syndrome restructures bat skin microbiomes ………………………..... 9 Abstract …………………………………………………………………….. 9 Introduction ………………………………………………………………… 10 Methods …………………………………………………………………….. 13 Data collection ……………………………………………………… 13 DNA extraction and testing …………………………………………. 14 Amplification and sequencing ………………………………………. 15 Data processing ………………………………………………………. 16 Microbial diversity analyses …………………………………………. 17 Linear mixed-effects modeling analyses ……………………………. 18 Taxonomic identification and differential abundance analyses …….. 18 Results ………………………………………………………………………. 19 Bacterial and fungal skin microbiome dissimilarities between Pd-negative bat species ……………………………………………… 19 v Characterization of Pd-negative bacterial and fungal microbiomes of three bat species …………………………………………………. 20 Microbial diversity analyses: microbiome differences between Pd-positive and Pd-negative bats …………………………………… 21 Does Pd influence M. lucifugus’ skin bacterial microbiome? ……… 22 Taxonomic differential abundance analyses in Pd-positive and Pd-negative M. lucifugus ……………………………………………. 22 Comparison of skin microbiome between bats and substrate ……….. 23 Discussion …………………………………………………………………… 23 II. Population structure of northern long-eared bats and adaptive responses to white-nose syndrome ……………………………………………………………….. 33 Abstract ……………………………………………………………………... 33 Introduction …………………………………………………………………. 34 Methods ……………………………………………………………………... 37 Data collection and RAD library preparation ………………………. 37 Sequence quality control …………………………………………….. 39 Variant calling and genetic diversity statistics ………………………. 39 Clustering analyses …………………………………………………... 41 Effective population size estimation …………………………………. 41 Selection analyses ……………………………………………………. 41 Results ………………………………………………………………………… 43 RAD sequence data quality and processing ………………………….. 43 vi Pre-WNS population structure ……………………………………….. 43 Pre-WNS effective population size estimation ……………………….. 44 Pre-WNS genetic variation …………………………………………… 44 Changes in genetic diversity …………………………………………. 44 Loci under selection ………………………………………………….. 45 Discussion ……………………………………………………………………. 46 III. Species status assessment for Perimyotis subflavus: taxonomy, genetics, and stressors to genetic diversity …………………………………………………….. 59 Species taxonomy ……………………………………………………………. 59 Species genetics ……………………………………………………………… 60 Genetic structure and sex-biased dispersal ………………………….. 60 Effective population size and population size change ………………. 61 Stressors to genetic diversity ………………………………………… 62 Loss of genetic variation and genetic drift …………………………… 65 Inbreeding, genetic load, and inbreeding depression ………………… 67 Reduction in fitness/adaptive capacity ………………………………. 69 Extinction vortex …………………………………………………….. 70 Maintaining genetic variation ……………………………………….. 71 Monitoring Perimyotis subflavus: genetic signals of necessary intervention ………………………………………………………….. 74 References …………………………………………………………………………………… 76 Appendix …………………………………………………………………………………….. 89 vii LIST OF TABLES TABLE PAGE Table 1. Genetic statistics summary for pre-WNS M. septentrionalis by location ………… 51 Table 2. Pre-WNS location pairwise FST values ……………………………………………. 51 Table 3. Spread zone sample information …………………………………………………... 52 Table S1. Microbiome sample list …………………………………………………………... 89 Table S2. Complete list of skin bacterial orders per species ………………………………... 114 Table S3. Complete list of skin fungal orders per species ………………………………….. 122 Table S4. Table of SNP-associated genes under a model of positive selection ……………. 128 viii LIST OF FIGURES FIGURE PAGE Figure 1. Bat sample collection distribution map ………………………………………….. 29 Figure 2. Bacterial and fungal microbiome taxonomy characterization ……………………. 30 Figure 3. Measures of bacterial and fungal evenness, richness, and Shannon indices between Pd- positive and Pd-negative bats ……………………………………………………………….. 31 Figure 4. Relative abundances of bacterial epidermal communities of Pd-positive and Pd- negative Myotis lucifugus ……………………………………………………………………. 32 Figure 5. Bacterial and fungal differences between bat epidermal and substrate microbiome composition ………………………………………………………………………………….. 32 Figure 6. Northern long-eared bat (Myotis septentrionalis) range map; White-nose syndrome spread map as of July 2019 ………………………………………………………………….. 53 Figure 7. Map of locations from which Myotis septentrionalis samples were collected prior to the arrival of Pseudogymnoascus destructans …………………………………………………... 53 Figure 8. Structure bar plot results …………………………………………………………... 54 Figure 9. Principal component analysis results with individuals from western Canada removed ……………………………………………………………………………………… 54 Figure 10. White-nose syndrome spread zone map …………………………………………. 55 Figure 11. Temporal changes in nucleotide diversity (π) in four spread zones ……………... 55 Figure 12. Temporal changes in inbreeding coefficients (FIS) in four spread zones ………... 56 Figure 13. Biological process gene ontology terms from PANTHER ………………………. 56 Figure 14. Molecular function gene ontology terms from PANTHER ……………………… 57 Figure 15. Cellular component gene ontology terms from PANTHER ……………………… 58 ix Figure S1. 16S rRNA alpha rarefaction plot ………………………………………………… 208 Figure S2. ITS alpha rarefaction plot ………………………………………………………... 208 Figure S3. Bacterial beta-diversity analyses between Pd-negative Eptesicus fuscus, Myotis lucifugus, and Perimyotis subflavus …………………………………………………………. 209 Figure S4. Bacterial principal coordinate plots between Pd-negative Eptesicus fuscus, Myotis lucifugus, and Perimyotis subflavus …………………………………………………………. 209 Figure S5. Fungal