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Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UM I directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Arm Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 MOLECULAR POPULATION GENETIC ANALYSES OF LAKE VICTORIA CICHLID FISHES USING MICRO SATELLITE DNA MARKERS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Lizhao Wu, B. S., M. S. ***** The Ohio State University 1999 Dissertation Committee: Approved by Dr. Paul A. Fuerst, Advisor Dr. Arthur H. M. Burghes Advisor Dr. Thomas J. Byers Molecular Genetics Graduate Program Dr. Thomas W. Prior DMI Number: 9931702 UMI Microform 9931702 Copyright 1999, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT The cichlid species flocks of East Afiica offer powerful models of explosive spéciation and reiterative adaptive radiation. However, the phylogenetic history and population structure of the fishes of the Lake Victoria Region (LYR) have been problematic, due to a lack of sufficiently variable genetic markers. Previous studies of the Lake Victoria cichlids using mitochondrial DNA sequences showed little divergence among species. To determine whether low mtDNA variation was due to small sample sizes, or is a characteristic of the cichlids of Lake Victoria, a 432 base pair mtDNA region was sequenced from 35 individuals of a LVR widespread species, Astatoreochromis alluaudi, from six localities. Very little phylogenetic information was detected, and no significant relationship existed between either the frequency or the occurrence of mtDNA haplotype and the geographical source of samples. The results suggest that the low mtDNA diversity in the LVR cichlid species reported previously was not solely a function of small sample sizes, and rule out the usefulness of mtDNA sequence data for phylogenetic inferences of the LVR cichlids. To develop markers that can be used to study the problematic phylogeny of haplochromine cichlids in the LVR, both dinucleotide and trinucleotide microsatellite ii DNA probes were used to screen a partial genomic library constructed &om A alluaudi. The (GT)^ motifs were estimated to occur at an average interval of 24kb in the A. alluaudi genome. Like several other teleost genomes, the A. alluaudi genome seems to have a higher percentage of long dinucleotide microsatellites than mammalian genomes. Unlike mtDNA sequencing data, nine DNA microsatellite markers revealed significant regional differentiation among the same six population samples of A. alluaudi that were used for mtDNA studies, coupled with very high levels of intra-population genetic variability. Furthermore, measures of genetic differentiation based on microsatellites are consistent with patterns predicted by both the biogeography and the jaw morphology of the six populations. In sharp contrast to both allozyme markers and mtDNA sequences, 14 microsatellite markers detected substantial amounts of genetic variation within each of 24 cichlid species from the LVR, as well as a single representative species from Lake Malawi. Phylogenetic analyses indicated that microsatellite markers, as a group, contain informative phylogenetic signals that not only confirmed taxonomic relationships of some, but not all, morphologically defined congeners, but also suggested regional spéciation and differentiation in the LVR. Moreover, the microsatellite based phylogeny is consistent with an ancient invasion of Lake Edward species into the Lake Victoria basin after Lake Victoria dried up and refilled in the Late Pleistocene. Microsatellites appear to evolve rapidly enough to reveal population structure and phylogeny of the tightly knit members of the Lake Victoria haplochromine cichlid species flock. However, 111 a robust and reliable phylogeny of these cichlids may still require additional data from more microsatellite markers, as weU as the inclusion of additional genera and additional sets of congeners from different lakes. IV Dedicated to my parents for their faith in education and science ACKNOWLEDGMENTS I would like to sincerely thank my advisor. Dr. Paul A. Fuerst, for his constant stimulation, guidance, support, and patience throughout the course of my dissertation research. I would also like to thank the other members of my committee. Dr. Arthur H. M. Burghes, Dr. Thomas J. Byers, and Dr. Thomas W. Prior, for their helpful advice on my research and valuable comments on my dissertation. I am especially indebted to Drs. Thomas J. Byers and Paul A. Fuerst for suggesting numerous improvements, both grammatically and scientifically, on an earlier draft of my dissertation. Special thanks go to Dr. Les Kaufinan at Boston University, for his broad knowledge of the Lake Victoria cichlid system and intellectual support. I thank my friends and colleagues in the Fuerst laboratory, Greg Booton, Wenrui Duan, Jeannette Kreiger, Brian Mark, Godfrey Mbahinzireki, Wilsom Mwanja, Brady Porter, Malcolm Schug, and Diane Stothard, for providing technical support, stimulating ideas, making jokes, and admitting the ignorance of the number of beetle species. Special thanks go to Greg Booton and Wilson Mwanja, for their insights into the Lake Victoria cichlid problems, and to Malcolm Schug, for his understanding, encouragement, vi and intellectual support. Data analysis and figure preparation were assisted by Greg Booton, Jeannette Kreiger, and Braian Mark. This project would have been impossible without the samples collected by numerous researchers: Wilson Mwanja; Les Kaufinan; Collins Chapman and Lauren Chapman at the University of Florida; Audrey Armoudlian at the Michigan State University; Researchers at the Fisheries Research Institute of Uganda, the Kenyan Marine and Fisheries Research Institute, and the Columbus Zoo. All the samples were morphologically identified by Les Kaufinan. These studies were supported by grants firom the National Sciences Foundation, the U.S. Department of Agriculture, the Columbus Zoo, the Sigma Xi Grant-In-Aid Research Award, the Ohio State University Graduate Student Alumni Research Award, as well as the Presidential Fellowship from the Ohio State University. Finally, I would like to deeply thank my wife, Judy, and my daughters, Janet and Jessica, for their love, support, sacrifices, and patience throughout my career at the Ohio State University. I may never repay them for their understanding of my absence from their lives during many evening hours and weekends when they need me the most. VII VITA November 28, 1963 ....................Bom in Ezhou, Hubei, P. R. China 1982 ............................................. B. S. Biology, The Central China Normal University 1985 .............................................M. S. Genetics, The Chinese Academy of Sciences 1985 — 1987 .................................Research Assistant, The Chinese Academy of Sciences 1987 — 1992 .................................Research Associate, The Chinese Academy of Sciences 1993-present ................................Graduate Teaching and Research Associate The Ohio State University 1997............................................. President Fellow, Graduate School at the Ohio State University PUBLICATIONS 1. Wu, L-, L. Kaufinan, and P. Fuerst, “Isolation of microsatellite markers in Astatoreochromis alluaudi and their cross species amplification in other Afiican cichlids.” Mol. EcoL, in press (1999). viii 2. Wilson, G. A., C. Strobeck, L. Wu, and J. W. Coffin, “Characterization of microsatellite loci in caribou Rangifer tarandus, and their use in other artiodactyls.” Mol. EcoL 6: 697-699 (1997). 3. Wu, L.and Z. Wang, “Studies on the expressions and regulations of isozymic genes in silver carp {Hypophthalmicythys molitrix) during their ontogenesis.” Acta Hydrobiol. Sin., 21: 49-58 (1997a). 4. Wu, L. & Z. Wang, “Biochemical genetic structure and variation in a natural population of silver carp from the middle reaches of the Yangtze River.” Acta Hydrobiol. Sin., 21: 157-162 (1997b). 5. Wu, L., G. Booton, M. Chandler, L. Kaufinan, and P. Fuerst, “Use of DNA microsatellite loci to identify