Phylogeography of the American Green Treefrog Species Group
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PHYLOGEOGRAPHY OF THE AMERICAN GREEN TREEFROG SPECIES GROUP by PAUL NATHANIEL PASICHNYK Presented to the Faculty of the Graduate School of The University of Texas at Arlington In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT ARLINGTON November 2016 Copyright © by Paul N. Pasichnyk 2016 All Rights Reserved ii Acknowledgments A student’s graduate work is definitely not something that is done alone. I have received help from numerous people over the years and without their contributions, this would not have been possible. First, I would like to thank my parents for their tremendous support and motivation as I grew up and provided me with a foundation and love in education. Secondly, I would like to thank the person who has had to help me daily with tasks as simple as a pat on the back to as complex as reading a rough draft of a dissertation that might as well be in a foreign language, my wife Karen. My in-laws deserve special thanks as well, I do not believe that it is possible for others’ in-laws to be as supportive and helpful as mine. Then I would like to thank all the undergrads and friends who have helped see this to completion: Richard Hanna, Sari Mahon, Sarah Young, Nicole Lopez, Annamarie Slaven, Leslie Segovia, Pankaj BC, Matt Moseley, Christian Cox, Jeff Streicher, Claudia Marquez, Jesse Meik, Walter Schargel, Corey Roelke, Matt Watson, Rebbekah Watson, Thomas Eimermacher, Utpal Smart, David Sanchez, Jacobo Reyes-Velasco, Melissa Muenzler, Carl Franklin, Linda Taylor, Gloria Burlingham, Sherri Echols, and Paulette Williams. My committee members have been particularly supportive, encouraging, and patient. Thank you Drs. Eric Smith, Trey Fondon, and Matt Walsh for agreeing to serve on my committee, seeing this to completion, and offering whatever support possible. I would like to extend a special thanks to my co-advisors, Dr. Paul Chippindale who has guided me through a lot of difficult hurdles and tasks and has consistently given good advice, helped solve many problems, and provided the resources needed to see this project to completion and Dr. Jonathan Campbell who has consistently provided more support than I believe he realizes simply by iii setting a great example, providing kind words and advise when needed, and motivating me to push through the final stages to complete this project. I would also like to thank all the people who helped me in the field. The students and staff of the University of Georgia and the University of Florida have exhibited extraordinary kindness and help at last minute’s notice to help me collect in their regions. Also, the kind faculty and staff at the Archbold Biological Station in Florida were extremely welcoming and helpful to Matt Moseley and myself when we were in southern Florida collecting specimens. All of the herpetological societies in the southeast United States have tremendous passion for their work and exhibit great knowledge of their states’ amphibians and are willing to help locate and secure specimens; the East Texas Herpetological Society, in particular, who helped fund most of my field work. Lastly, I would like to thank the El Centro Family and all my students, current, former, and future, who prove every day that there is a passion in faculty and young people to learn, improve themselves and society, and inspire others to reach their potential. 14 August 2016 iv ABSTRACT PHYLOGEOGRAPHY OF THE AMERICAN GREEN TREEFROG SPECIES GROUP Paul Pasichnyk, PhD. The University of Texas at Arlington, 2016 Supervising Professors: Jonathan A. Campbell / Paul T. Chippindale The southeastern United States is an important temperate biodiversity hotspot, housing a multitude of endemic species and multiple large rivers fragmenting their ranges. To determine if the Riverine Barriers Hypothesis explains population divergence in this region, this study utilizes of a combination of phylogeographic methods and ecological niche modeling to investigate the role of rivers in genetic dispersal. Intra-and interspecific phylogenies for the hylid treefrogs Dryophytes cinereus, D. gratiosus and D. squirellus were estimated using nuclear and mitochondrial DNA sequences to identify relationships and genetic variation within these species and test for population structure, especially with respect to potential riverine barriers. Ecological niche models were then constructed to identify potential environmental factors affecting population structure. The results of the phylogeographic studies indicate that the Mississippi River could be a barrier to gene flow within D. cinereus; however, two samples from the western clade appeared on the eastern side of the river, indicating a limited gene exchange or colonization. Ecological niche models revealed little ecological niche variation between the two clades of D. cinereus which supports the presence of a geographic barrier to gene flow in a relatively homogeneous environment. However, the individuals belonging to clades associated v with opposing sides of the river suggest that the river might not serve as an absolute barrier to dispersal. Evidence of hybridization between D. cinereus and D. gratiosus based on the phylogenies indicates that the ultimate cause of the variation in D. cinereus might not be entirely geographic. This study provides new insight into a long-debated question regarding the presence of reproductive character displacement in D. cinereus versus the possibility that intraspecific variation resulted from isolation during historic climate fluctuations, and it enlightens previous studies that indicate sex-biased hybridization in this group. vi Table of Contents Acknowledgments iii Abstract v List of figures vii List of tables x 1. Introduction to the project 1 1.1. Introduction 1 1.2. Introduction to the study system 3 2. Phylogeography 11 2.1. Introduction 11 2.2. Materials and methods 11 2.3. Results 31 2.4. Discussion 52 3. Ecological niche modeling 59 3.1. Introduction 59 3.2. Materials and methods 60 3.3. Results 62 3.4. Discussion 67 4. Conclusions and future directions 69 Appendix 1 76 Appendix 2 150 References 153 Biographical information 173 vii List of Figures Page 1.1. Phylogenetic relationships of the species group 7 1.2. Distribution of the species group 8 2.1. Samples of Dryophytes cinereus 22 2.2. Samples of Dryophytes gratiosus 23 2.3. Samples of Dryophytes squirellus 24 2.4. Bayesian tree of concatenated dataset 36 2.5. Bayesian tree of nuclear dataset 37 2.6. Bayesian tree of mitochondrial dataset 38 2.7. Maximum likelihood tree of concatenated dataset 39 2.8. Maximum likelihood tree of nuclear dataset 40 2.9. Maximum likelihood tree of mitochondrial dataset 41 2.10. *BEAST species tree 42 2.11. Divergence estimates using HKY model of nucleotide evolution 43 2.12. Divergence estimates using GTR + G model of nucleotide evolution 44 2.13. Haplotype network for mitochondrial dataset for Dryophytes cinereus 45 2.14. Haplotype network for nuclear dataset for Dryophytes cinereus 47 2.15. Distribution map for Dryophytes cinereus clades 49 3.1. Maxent predictive map of range of eastern clade of Dryophytes cinereus 63 3.2. GARP predictive map of range of eastern clade of Dryophytes cinereus 64 3.3. Maxent predictive map of range of western clade of Dryophytes cinereus 65 3.4. GARP predictive map of range of western clade of Dryophytes cinereus 66 A.1.1. Bayesian tree of 12S mitochondrial gene 76 viii A.1.2. Bayesian tree of 16S mitochondrial gene 77 A.1.3. Bayesian tree of RAG-1 nuclear gene and 16S mitochondrial gene 78 A.1.4. Bayesian tree of RAG-1- nuclear gene and 12S mitochondrial gene 79 A.1.5. Maximum likelihood tree of RAG-1 nuclear and 16S mitochondrial genes 80 A.1.6. Maximum likelihood tree of RAG-1 nuclear and 12S mitochondrial genes 81 A.1.7. Divergence estimates using HKY model of nucleotide substitution with 95% HPD 82 A.1.8. Divergence estimates using GTR + G model of nucleotide substitution with 95% HPD 83 A.2.1. Maxent predictive map of range of eastern clade of Dryophytes cinereus using 75% of the samples to test the model 150 A.2.2. Maxent predictive map of range of eastern clade of Dryophytes cinereus using the jackknife method to test the model 151 A.2.3. Maxent predictive map of range of western clade of Dryophytes cinereus using the jackknife method to test the model 152 ix List of Tables Page 2.1. Specimens used in study 14 2.2. Sequences obtained from GenBank 21 2.3. Standard PCR reaction 25 2.4. Master Mix PCR reaction 26 2.5. Primers used in study 26 2.6. Thermal cycler protocol for nuclear PCR reactions 27 2.7. Thermal cycler protocol for mitochondrial PCR reactions 27 2.8. Sequencing reaction mix 28 2.9. Thermal cycler protocol for sequencing reaction 28 2.10. List of specimens in mitochondrial haplotype clusters 46 2.11. List of specimens in nuclear haplotype clusters 48 2.12. FST for mitochondrial dataset 50 2.13.FST for nuclear dataset 50 2.14. FST for concatenated dataset 50 2.15. Indices of genetic polymorphisms 51 3.1. Definition of Bioclim variables 61 A.1.1. Pairwise differences in nuclear sequences for all samples 84 A.1.2. Pairwise differences in mitochondrial 16S sequences for all samples 97 A.1.3. Pairwise differences in mitochondrial 12S sequences for all samples 126 x Chapter 1: Introduction to the project 1.1 Introduction Understanding the processes underlying the origin and maintenance of biodiversity is critical for many fields within biology, including speciation and conservation biology (Amano & Sutherland, 2013). The loss of biodiversity can impact agriculture, waste management, tourism, medicine, and can have devastating effects on ecological systems (Pimentel et al., 1997; Wu & David, 2002).