HYBRIDIZATION OF LYTHRURUS FASCIOLARIS (COPE).AND LYTHRURUS UMBRATILIS (GIRARD) (CYPRINIFORMES: CYPRINIDAE) IN TIIE O1-IlO RIVER BASIN A Thesis Presented to the Faculty of the College of Science and Technology Morehead State University In Partial Fulfullrnent of the Requirements for the Degree Master of Science by Robert L. Hopkins, II November 2005 .• i• CAMDEN-CARROLL LIBRARY . MOREHE,AD, KY 40351 ff}~ lf 7h-fStJ (oj,. 08-;). /-1195 A Accepted by the faculty of the College of Science and Technology, Morehead State University, in partial fulfillment of the requirements for the Master of Science degree. /j~-ki~ Director o esis Master' s Committee: 14 NOJ.ffi)PH 2C05 Date II HYBRIDIZATION OF LYTHRURUS FASCIOLARJS (COPE) AND LYTHRURUS UMBRATILJS (GIRARD) (CYPRINIFORMES: CYPRINIDAE) IN THE OHIO RIVER BASIN Robert L. Hopkins, II, M.S. Morehead State University, 2005 Director ofThesis:. __rf:J=-=~==el'_,,/cc-_..~""'====------- The scarlet shiner (Lythrurusfasciolaris) and redfin shiner (L. umbratilis) are common small minnow species usually exhibiting a parapatric geographic distribution within the Ohio River basin. Historical collection records suggest several areas of possible syntopy along the periphery of distributional ranges, with suspected hybridization based upon observed intermediate coloration patterns of nuptial males. Nuptial males from nine localities of suspected hybridization were collected in June and July 2004 and analyzed using morphometric, meristic, tuberculation, coloration, and nuclear DNA data. Based on univariate and multivariate analyses and qualitative assessment of the morphological data, the Green River, Kentucky River, and Salt River drainages in Kentucky and the Scioto River drainage in Ohio all contain populations with evidence of hybrid influence. These contact areas contained nuptial males exhibiting atypical coloration and tuberculation patterns compared to parental species. Results from principal component analyses of morphometrics and meristics indicate that putative hybrid progeny exhibit either intermediate or discordant shape_ and meristic character combinations compared to parental species. Comparisons of current observations to historical collection records iii suggest hybridization may be facilitating an eastward range expansion of L. umbratilis through replacement of L. fasciolaris as indicated by the introgression of morphological characters toward those of L. umbratilis in the Green River, Salt River, and Scioto River drainages. Specimens from the Kentucky River drainage indicate that hybridization bas allowed the development of a static, morphologically and genetically distinct hybrid swarm. In each case, environmental conditions seem closely linked with the occurrence and net effects of hybridization between these two species. Accepted by: lV Dedication This thesis is dedicated to my parents, Robert and Linette Hopkins, who have given me unwavering support and encouragement. V Acknowledgements I would like to express my deepest gratitude to my major professor, Dr. David Eisenhour, for his unending patience and direction throughout this study and for developing my interest in systematic zoology. I would also like to express sincere thanks to Dr. David Peyton, for introducing me to molecular techniques and providing constant counsel with their applications. Much appreciation also goes to my other. committee members, Dr. Sean O'Keefe and Dr. Brian Reeder, for their comments and suggestions. I am also grateful to Dr. David Rudy for project funding and access to the many resources making this study possible. The following people were most helpful in field collections of specimens: Lynn Eisenhour, Jonathan Eisenhour, and Rebecca Hopkins. Collection permits were provided by the Kentucky Department of Fish and Wildlife, West Virginia Natural Resources, and the Ohio Fish and Game Departments. Morehead State University IACUC granted permission for collection of tissue samples from specimens. My most heartfelt appreciation goes to my wife, Rebecca, who assisted enormously in the editing and revision stages of the thesis and compilation of the final product. vi Table of Contents Page Introduction ....................................................................................... 1 Methods and Materials ......................................................................... I 0 Results: Reference Specimens ............................................................... .19 Results: Putative Hybrid Populations ........................................................37 Discussion ........................................................................................67 Diagnostic Characters of Reference Specimens ................................... 67 Identity of Putative Hybrid Populations ............................................70 Conclusions ......................................................................................84 Future Research ................................................................................. 88 Literature Cited .................................................................................. 90 Appendix I.. .....................................................................................95 vii List of Tables Page Table I. Description of28 truss-geometric measurements for morphometric analysis ................................................................................ 14 Table 2. Sheared PCA loadings for 107 L. fasciolaris, I 02 L. umbratilis, and 102 putative hybrids ........................................................................................ 21 Table 3. Summary ofmeristic difference between two species of Lythrurus ..........26 Table 4. Frequency distribution of anal fin rays, scales above lateral line, and scales below lateral line in L. fasciolaris and L. umbratilis .................... 27 Table 5. Frequency distribution of circumferential scales and scales around caudal peduncle in L.fasciolaris and L. umbratilis .............................28 Table 6. Distribution of genotypes of parental populations .............................. 35 Table 7. Comparison of genotypes and phenotypes for Eagle Creek specimens in Grant County, KY ............................................................... .57 Table 8. Comparison of genotypes and phenotypes for Eagle Creek specimens in Owen County, KY ................................................................ 62 viii List of Figures Page Figure 1. Geographic distribution of L. fasciolaris and L. umbratilis within the Ohio River basin ......................................................... 5 Figure 2. Putative syntopic areas overlain on range map ...................................8 Figure 3. Map of collection locales for reference and putative hybrid specimens ........................................................................... 11 Figure 4. Twenty-one landmarks (white dots) shown with associated measurements used in the morphometric analysis ............................. 16 Figure 5. A) Sequences of cytoplasmic beta-actin with intron 2 ......................... 18 Figure 6. Morphometric scores for reference specimens on sheared PCA axes II and III.............................................................................. 20 Figure 7. Bivariate plots of body width (top) and D-A (bottom) versus standard length (SL) measurements for reference specimens of L.fasciolaris and L. umbratilis .................................................................... 22 Figure 8. Bivariate plots ofD-P2 (top} and Dend-A (bottom) versus standard length (SL) measurements for reference specimens of L. fasciolaris and L. umbratilis .................................................................... 23 Figure 9. Meristic scores on PCA axes I and II ............................................25 Figure IO. Combined plot of PCA scores of two separate analyses ofmeristic (x-axis) and morphometric characters (y-axis) of reference specimens .... 30 Figure I I. Composite illustrations of head tuberculation patterns for nuptial males in high condition ...........................................................31 Figure 12. Electrophoreses products of intron 2 region of cytoplasmic beta- actin with assigned genotypes labeled .......................................... 34 Figure 13. Combined plot of individual PCA scores of Lythrurus specimens from reference populations (enclosed in dashed polygon) and ix Bear Creek specimens (enclosed in solid polygon) ........................... 38 Figure 14. Composite tubercle image of seven high males captured in Bear Creek (Green River drainage) on 25 June 2004 ........................ .39 Figure 15. Combined plot of individual PCA scores of Lythrurus specimens from reference populations (enclosed in dashed polygon) and Taylor Fork specimens (enclosed in solid polygon) ......................... .41 Figure I 6. Composite head tuberculation image of 11 high males capture at Taylor Fork (Green River dr.) on 25 June 2004.............................. .42 Figure 17. Combined plot of individual PCA scores of Lythrurus specimens from reference populations (enclosed in dashed polygon) and Mott's Lick Cr. specimens (enclosed in solid polygon) ......................44 Figure 18. Composite head tuberculation image of 15 high males captured Mott's Lick Cr. (Green River dr.) on 25 June 2004 .......................... .45 Figure 19. Combined plot of individual PCA scores of Lythrurus specimens from reference populations (enclosed in dashed polygon) and Knob Creek specimens