MOLECULAR PHYLOGENY, BIOGEOGRAPHY, POPULATION STRUCTURE AND TAXONOMY OF LARGE BARBINE MINNOWS (LABEOBARBUS: CYPRINIDAE) by KEBEDE ALEMU BESHERA PHILLIP M. HARRIS, COMIITTEE CHAIR LESLIE RISSLER JUAN M. LOPEZ-BAUTISTA DANIEL GRAF RICHARD MAYDEN A DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biological Sciences in the Graduate School of The University of Alabama TUSCALOOSA, ALABAMA 2012 Copyright Kebede Alemu Beshera 2012 ALL RIGHTS RESERVED ABSTRACT The phylogeny, biogeography, population genetics and taxonomy of Labeobarbus (Cyprinidae), a genus of large hexaploid minnows, was estimated by a variety of phylogenetic and population genetics methods. In Chapter 2 relationships among species of large barbine minnows was examined based on analysis of variation in complete mitochondrial (mt) cytochrome b (cyt b) gene sequences. In all analyses hexaploid minnows from Saharan and Sub-Saharan Africa and the Middle East were recovered as a monophyletic taxon. This clade diverged from European tetraploid and African diploid and tetraploid lineages approximately 13 MYA. The earliest cladogewnetic event within this clade occurred ca 5.0 MYA giving rise to Sub-Saharn African and Saharan African-Levant clades. Subsequent cladodenesis within the clade took place during the Plio-Pleistocene. Chapter 3 investigated the phylogeography of Labeobarbus intermedius across its geographic distribution in Ethiopia based on analysis of complete cyt b gene sequences. Phylogenetic analysis recovered two distinct geographic lineages (northern and southern) within L. intermedius, which diverged from each other ca Late Pleistocene consistent with the timing of Pleistocene volcanic activities in East Africa. Chapter 4 developed microsatellite DNA markers for use in Chapter 5. Seventy-two microsatellite primers were developed based on a genomic library of Labeobarbus intermedius employing next generation 454 sequencing. Six polymorphic loci were examined over 35 L. intermedius specimens and tested for cross-species amplification in other Labeobarbus taxa. Genetic diversity was high with 99 alleles identified in L. intemedius with an average of 16.5 alleles per locus. Observed heterozygocity ranged from 0.8-1.0 across all loci. These loci also ii amplified successfully across all taxa. In Chapter 5, ten microsatllite DNA loci and mitochondrial cytochrome oxidase I (COI) and cyt b gene sequences were analyzed to investigate population structure and evolutionary relationships within the Labeobarbus species flock of Lake Tana. Phylogenetic analyses based on COI and cyt b gene sequences consistently rejected the monophyly of Lake Tana Labeobarbus haplotypes and recovered all haplotypes as part of a larger clade that contained haplotypes from independent drainages throughout Ethiopia. Bayesian clustering analysis of ten microsatellite markers employing STRUCTURE revealed little genetic differentiation within the Lake Tana Labeobarbus suggesting that alleles were shared among individuals and putative species in the species flock. The resolved phylogeny and lack of population differentiation in Lake Tana Labeobarbus may suggest either these evolutionary lineages are at an early stage of an ongoing adaptive radiation in Lake Tana or lack of signal in molecular markers examined. iii DEDICATION This dissertation is dedicated to my parents, Alemu Beshera and Ayelech Wodajo, for all what they have done for me including giving me the opportunity to see the light. iv LIST OF ABREVIATIONS a mean number of alleles per locus A Adenine ABI Applied Biosystems AMOVA Analysis of Molecualr Variance ANL Addis Ababa-Nekemt lineament AWMISET Agricultural Water Management Information System of Ethiopia bp base pairs BP before present C Cytocine Ca around CAR Central African Republic CBOL Barcode of Life CI Credible Interval CO1 Cytochrome Oxydease I gene CRV Central Rift Valley CTOL Cypriniformis Tree of Life Cytb cytochrome b gene o C Degree Celsius DNA Deoxyribonucleic Acid DNASP DNA Sequence Polymorphism (Software for analysis of DNA polymorphism) v DNTP Deoxy Ribonucleotide Triphosphate DRC Democratic Republic of Congo e.g. For example EtOH ethanol Et al. and others FAM Carboxyfluorescein F Forward primer FIG Figure FS Fu’s (1997) demographic statistic FIS Hardy Weinberg (Fixation Index) G Guanine GBL Goba-Bonga Lineament GBN GenBank number GIS inbreeding coefficient GTR+ Γ Generalized Time Reversible (model of DNA evolution) h haplotype diversity Hd haplotype diversity HEX Hexachloro-fluorescein HN Haplotype number Hap Haplotype HiDi Highly deionized vi H2O Water HE Expected Heterozygocity HN Haplotype number HO Observed heterozygocity HPD Highest Posterior Density Interval Ht Total heterozygocity i.e. That is to say IUCN International Union for Conservation of Nature K The number of genetically differentiated clusters ΔK rate of change in the log probability of data between successive K values KU Kansas Universisty µ Micro µL Microliter µM Micromolar mtDNA mitochondrial DNA ncDNA nuclear DNA MCMC Markov chain MgCl2 Magnesium Chloride MHC Major Histocompatibility Complex ML Maximum Likelihood MY million years vii MYA million years Ago MP Maximum Parsimony Ng Nanogram NA Number of alleles NS Number of samples NSF National Science Foundation OTU Operational Taxonomic Unit P p-value PAUP Phylogenetic Analysis Using Parsimony (Computational phylogenetics program) PCR Polymerase Chain Reaction PEG Polyethylene Glycol PMY Percent per million years R Reverse primer R2 Statistical test (developed by by Ramos-Onsins and Rozas, 2002) for detecting population growth RNAse Ribonuclease (type of nuclease that catalyzes the degradation of RNA) RV Rift Valley SA Allele size range SAIAB South African Institute of Aquatic Biodiversity SL Standard length SLC Sampling locality coordinates viii SRV Southern Rift Valley ФST statistical measure of population differentiation R2 statistics used to detect departures from constant population size RAxML Randomized Accelerated Maximum Likelihood T Thymine TAMRA One of four Fluorescent dyes used to label DNA templates TCS Phylogenetic network estimation software using Statistical Parsimony TBR Tree-Bisection-Reconnection Tm Annealing temperature TMRCA time to Most Common Ancestor UAIC University of Alabama Ichthyological Collections YA years ago ix ACKNOWLEDGMENTS Thanks are especially due to Dr. Phillip M. Harris, my advisor, for his support and guidance from developing to completion of this dissertation. I couldn’t have done this work without his help. I am also grateful to my Graduate Committee members, Drs. Leslie Rissler, Daniel Graf, Juan Lopez-Bautista, and Richard Mayden, for their support throughout this dissertation and constructive comments on the manuscript. Financial support of the Cypriniforms Tree of Life Project was invaluable in allowing me to conduct fieldwork in Ethiopia and covering all costs associated with my dissertation. My appreciation also goes to the Department of Biological Sciences and Graduate School, The University of Alabama, for providing additional financial assistance. I express my thanks to South African Institute of Aquatic Biodiversity (SAIAB) and University of Kansas Fish Collection for providng some samples used in this dissertation, Jimma University for logistic support during field work, local fisheries departments and personnel in Ethiopia for their assistance in field collections. I can’t say enough to thank my friend, Brook Fluker, for the love, respect and support he showed me, taking time out of no time to share information, help in data analysis and reviewing some papers. I thank, my friend, Berhanu Tekle, who gave me constant support through this long, adventurous and sometimes arduous journey and helped me endure at times when the going was tough. I am grateful to Lindsay Clark, Yonathan Berhanu, and Yonas Goiton for their technical support. I also thank my friends Michael Sandel, Justin Bagley, Grey Hubbard, Jenjit x Khudamrongsawat, Nathan Whelan, Alex Teoh, and John Pffefier for their helpful discussions and comments and the late Wally Holznagel for sharing his vast knowledge on Molecular techniques and his assistance in the lab. Finally, I thank my kids, Ruth, Candor, and Yanet, whose presence inspires me and gives me purpose to keep on going and my wife Tigist for assisting and supporting me through this process. Without them on my side nothing of this would have been possible. My parents deserve special thanks for their love and encouragement along with my brothers (Dagne, Shiferaw, Eyob, Getnet, and Yibeltal) who supported me all the way. xi TABLE OF CONTENTS ABSTRACT………………….……..…………………………………………………………….ii DEDICATION..………………………………..…………………………………………………iv LIST OF ABREVIATIONS ………………...……………………………………………………v ACKNOWLEDGMENTS..……………………………………………………………………….x LIST OF TABLES ……………………………………………………………………………..xiii LIST OF FIGURES….…………………………………………………..……………………...xiv CHAPTER 1: GENERAL INTRODUCTION….………...………………………………………1 CHAPTER 2: MOLECULAR PHYLOGENEY OF LARGE HEXAPLOID BARBINE MINNOWS (LABEOBARBUS: CYPRINIDAE)…..……………………………….…….7 CHAPTER 3: MITOCHONDRIAL DNA PHYLOGEOGRAPHY OF LABEOBARBUS INTERMEDIUS (CYPRINIDAE), EAST AFRICA…………………….…………….37 CHAPTER 4: ISOLATION AND CHARACTERIZATION OF SIX NOVEL MICROSATELLITE LOCI IN LABEOBARBUS INTERMEDIUS (CYPRINIDAE) AND THEIR CROSS-SPECIES UTILITY IN THE LAKE TANA LABEOBARBUS SPECIES FLOCK………..………………………………………….60 CHAPTER 5: NUCLEAR MICROSATELLITE AND MITOCHONDRIAL DNA MARKERSREVEAL EXTREMELY