University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Masters Theses Graduate School 12-2006 Insights into the Etheostoma spectabile species complex: Incongruence between mitochondrial and nuclear gene sequence data Christen M. Bossu University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_gradthes Part of the Ecology and Evolutionary Biology Commons Recommended Citation Bossu, Christen M., "Insights into the Etheostoma spectabile species complex: Incongruence between mitochondrial and nuclear gene sequence data. " Master's Thesis, University of Tennessee, 2006. https://trace.tennessee.edu/utk_gradthes/1509 This Thesis is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a thesis written by Christen M. Bossu entitled "Insights into the Etheostoma spectabile species complex: Incongruence between mitochondrial and nuclear gene sequence data." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Master of Science, with a major in Ecology and Evolutionary Biology. Thomas J. Near, Major Professor We have read this thesis and recommend its acceptance: James A. Fordyce, Christine R. Boake Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a thesis written by Christen M. Bossu entitled “Insights into the Etheostoma spectabile species complex: Incongruence between mitochondrial and nuclear gene sequence data.” I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Ecology and Evolutionary Biology. ________Thomas J. Near_________ Thomas Near, Major Professor We have read this thesis and recommend its acceptance: ___ James A. Fordyce____________ ______Christine R. Boake___________ Accepted for the Council: __ ___Anne Mayhew _______ Vice Chancellor and Dean Of Graduate Studies (Original signatures are on file with official student records.) Insights into the Etheostoma spectabile species complex: Incongruence between the mitochondrial and nuclear gene sequence data. A Thesis Presented for the Master of Science Degree The University of Tennessee, Knoxville Christen M. Bossu December 2006 Dedication I would like to dedicate my thesis to my parents, Frank P. Bossu and Nancy Uridil. I experienced the passion they would exude in every endeavor, and that dedication and passion I inherited made this research possible. ii Acknowledgements I would like to express my sincere appreciation to my faculty advisor, Dr. Thomas J. Near, for his guidance and support throughout the course of this thesis effort, making it clear that distance was never an issue. The insight and experience was certainly appreciated. I would, also, like to thank my other committee members Dr. Christine Boake and Dr. James Fordyce for both the guidance and conversations that helped focus and shape this thesis. I am also indebted to my labmates whose presence in the field and the molecular lab made my thesis a joy to do. So to Ben Keck, Phillip Hollingsworth, Jacob Kendrick and Richard Harrington, I thank you for the conversations around the fire, your enthusiasm for the field and the fish and the constant laughter. I would also like to express my sincere thanks to the Ecology and Evolutionary department at the University of Tennessee, Knoxville. From the summer research funding that made my field collections and lab work possible to the afternoon patio time conversations that provided insight and spirits to my research and to my life. iii Abstract Hybridization is recognized as an evolutionary process that can provide a significant source of genetic variation and whose genetic consequences have been investigated across a wide taxonomic range of plants and animals. Darters (Percidae: Etheostomatinae) are a clade with documented interspecific hybridization and many species with a recent evolutionary origin, yet most molecular phylogenetic analyses of darters to date have relied primarily on mitochondrial DNA (mtDNA) sequences. Inferring relationships within and between closely related species using a single locus gene tree is potentially confounded by introgression as well as retention of ancestral polymorphisms. This can lead to incongruence between the gene tree and the species tree, and confound interpretations of phylogeography and species relationships. Considering these limitations, I utilized both mtDNA and six nuclear genes to reconstruct the phylogeny of the E. spectabile species complex, a hypothesized reciprocally monophyletic group with known instances of intergradation and hybridization. My objectives were twofold: 1) to determine if the molecular evidence supported the recent species delimitations based on meristics and breeding male coloration and 2) to determine the phylogenetic utility and congruence of mtDNA and nuclear DNA data to address possible hybridization in the species complex. I found concordance between distinct genetic signals, meristics and geographic distributions that supported many, but not all of the recognized species. I also found that introgression is prevalent throughout the history of the E. spectabile species complex, and confounds the monophyly of the complex, specifically with E. fragi and E. uniporum mtDNA haplotypes grouping outside of the iv complex. Understanding the prevalence of introgression is crucial for future investigation of the evolution of these fishes. v Table of Contents Section Page 1. Introduction....................................................................................................1 2. Materials and Methods ..................................................................................8 2.1 Specimen sampling and DNA sequencing...............................................8 2.2 Phylogenetic analysi............................................................................21 2.3 Tests of incongruence ..........................................................................23 3. Results...........................................................................................................25 3.1 Mitochondrial and nuclear DNA sequencing of the Etheostoma spectabile complex .........................................................25 3.2 Nuclear phylogenetic analyses..........................................................32 3.3 Incongruence among the mitochondrial and nuclear genes of the Etheostoma spectabile complex ........................................................41 3.4 Total combined data analysi .............................................................43 3.5 Repeatability of clade .......................................................................46 4. Discussion .....................................................................................................51 4.1 Phylogeography of the Etheostoma spectabile complex........................51 4.1.1 Species relationships...................................................................51 4.1.2 Status of species..........................................................................54 4.2 Hybridization and introgression...........................................................55 Literature Cited................................................................................................60 Vita ...................................................................................................................72 vi List of Tables Table Page 1. Specimen examined, tissue voucher number with clone names, and collection localities ......................................................................... 10-17 2. Nuclear Primers used for amplification and sequencing ..........................19 3. Models of evolution selected by AIC in Modeltest 3.06 for each character partition of each gene ....................................................................23 4. Summary of the overall resolution power of individual nuclear genes and the presence and absence of particular nodes for each individual nuclear analysis ............................................................................................39 5. Shimodaira-Hasegawa (SH) test of incongruence between the mtDNA and nuclear DNA topologies of the E. spectabile species complex................43 6. Shimodaira-Hasegawa (SH) test of incongruence between the Bayesian individual nuclear gene and mtDNA tree topologies .....................................44 7. Shimodaira-Hasegawa (SH) test of incongruence between the Bayesian tree topologies of the total combined data and the nuclear DNA and mtDNA data partitions...............................................................................................47 8. Repeatability of clades (A-O) in the total combined data set looking at independent individual data sets ...................................................................48 vii List of Figures Figure Page 1. Etheostoma spectabile species complex range map...................................9