REGIONAL CONNECTIVITY, DIFFERENTIATION AND BIOGEOGRAPHY OF THREE SPECIES OF THE GENUS LUTJANUS IN THE WESTERN INDIAN OCEAN Submitted in fulfilment of the requirements for the degree of MASTER OF SCIENCE at RHODES UNIVERSITY By JONAS MOQEBELO MORALLANA 2013 1 DECLARATION I declare that the thesis hereby submitted to Rhodes University, Grahamstown, South Africa, for the degree of Master of Science in Ichthyology, has not previously been submitted for a degree at this or any other university. The work contained herein is my own. I am aware of the University’s policy on plagiarism, and the material (ideas, phrases and illustrations) presented from other authors have been duly acknowledged. Name: Jonas Moqebelo Morallana Student number: g09m7638 Signed: Date: 2 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to every member of the South African Institute for Aquatic Biodiversity (SAIAB) for making my stay in Grahamstown memorable and worthwhile. The Department of Science and Technology – African Coelocanth Ecosystem Project (DST- ACEP), SAIAB and Western Indian Ocean Marine Science Association (WIOMSA) provided running costs and the bursary throughout this study. Special thank you to: Dr Andy Bentley from the University of Kansas, Biodiversity Research Centre and Dr Mark McGrouther from the Australian Museum, Dr Tilman Alpermann from the Senken Museum and Dr Michael Hammer from the Northern Territory Museum and Art Gallery for loaning tissue samples, and to Dr Terrence Miller and Dr Yusong Guo for allowing the use of their data. I express my most since appreciation to my supervisors Drs Gavin Gouws and Monica Mwale for providing positive feedback, guidance and encouragement since the beginning of this study. Thank you for the opportunities you have given me throughout my study, and the support to attend the conferences, workshops and to do field surveys. Without you, it would have not been possible to complete this thesis. To Dr Gouws, special thank you for all the time you have devoted to me and help with the analyses, discussions and write-up. You have helped me academically and personally, and for that, I will forever be grateful. I would like to thank Mr Ofer Gon for the support and encouragement. Special thanks to Mr Wouter Holleman for discussions surrounding the taxonomic issues between the study families. I would also like to thank my friends: Dr Albert Chakona, Mpho Ramoejane, Thabo Maake, Tshoanelo Moloi, Nosiphiwo Springbok, Sisanda Mayekiso, Precotia Nyalungu, Poogendri Reddy, Glory Nkuna, Samella Ngxande, Khanyi Ngomane, Sherwyn Mack, Bafo Konqobe, and everyone else not mentioned who gave valuable input to this work. 3 Thank you to Nicodemus Mautsa at Rhodes University Sequencing facility and Macrogen (Korea) DNA sequencing facility for the sequencing services offered. My gratitude also goes to Maggie Shaw, Hanoria Kalimashe and Sally Schramm for making sure I get hold of those hard to obtain articles. Lastly, to my parents, Monyatsi and Mamaki Morallana for encouragement and support throughout my study, finally I will explain to you what it is I was doing. 4 TABLE OF CONTENTS Abstract……………………………………………………………………………………….iv List of figures………..………………………………………………………….………….....vi List of Tables………………………………………………………………………………....xi List of abbreviations………………………………………………………………………..xvi Chapter 1: General Introduction………………………………………….…………………1 1.1. Population structure and phylogeography………………………….…………...…1 1.2. Larval dispersal and connectivity………………………………………….……...2 1.3. Molecular data……………………………………………………………………..3 1.3.1. Mitochondrial DNA (mtDNA)…………………………………………………..4 1.3.2. Nuclear DNA (nDNA)…………………………………………………………..7 1.4. Lutjanidae (snappers)……………………………………………………...............7 1.4.1. Phylogenetic relationships………………………………………………………7 1.4.2. Study taxa….…………………………………………………………….………8 1.4.2.1. Lutjanus fulviflamma (Forsskål, 1775) – dory snapper……………………….9 1.4.2.2. Lutjanus bohar (Forsskål, 1775) – twin-spot red snapper…………………...10 1.4.2.3. Lutjanus lutjanus Bloch, 1790 – bigeye snapper……….……………………11 1.4.3. Habitat and biology…………………………………………………………….12 1.5. Study area………………………………………………………………….…….13 1.5.1. Physical oceanography of the study area………………………………………14 1.5.2. WIO fish diversity……………………………………………………………..17 1.5.3. Western Indian Ocean biogeographic regions………………………….……...18 1.5.3.1. Red Sea………………………………………………………………………18 1.5.3.2. Somali Basin…………………………………………………………………19 1.5.3.3. Mozambique Basin…………………………………………………………..20 1.5.3.4. Maldives/Chagos Laccadive Ridge………………………………………….21 1.5.3.5. Mascarene Plateau…………………………………………………….……..21 1.6. Rationale…………………………………………………………………..……..22 1.6.1. Research aims…………………………………………………………….……22 1.6.2. Key questions………………………………………………………….……….23 1.7. Conservation and management…………………………………………………..24 i Chapter 2: Material and Methods……………………………………………………...…..26 2.1. Sampling and processing…………………………………………………...……26 2.2. Genetic data generation…………………………………………………………..26 2.2.1. DNA extraction……………………………………………………………...…26 2.2.2. Polymerase Chain Reaction (PCR)…………………………………………….27 2.2.3. DNA sequencing……………………………………………………….………28 2.2.4. Statistical analyses……………………………………………………………..28 2.2.4.1. Sequence alignment………………………………………………………….28 2.2.4.2. Nucleotide and haplotype diversity estimates………………………………..29 2.2.4.3. Population structure and Analyses of Molecular Variance (AMOVA)……...29 2.2.4.4. Isolation by distance………………………………………………………….30 2.2.4.5. Historical demographic analyses…………………………………………….30 2.2.4.6. Phylogenetic analyses………………………………………………..............31 2.3. Meristic and morphological variation (Lutjanus fulviflamma)…………………..32 Chapter 3: Regional differentiation in the dory snapper (Lutjanus fulviflamma Forsskål, 1775) in the Western Indian Ocean………………………………………………………...33 3.1. Introduction……………………………………………………………..………..33 3.2. Material and Methods….………………………………………………………...37 3.2.1. Sampling…………………………………………………………...…….…….37 3.2.2. Genetic data…………………………………………………………………….39 3.2.3. Morphometric analyses………………………………………………………...41 3.3. Results……………………………………………………………………………44 3.3.1. Genetic data……………………………………………………………………44 3.3.1.1. Cytochrome b analyses………………………………………………………44 3.3.1.2. NADH-2 analyses……………………………………………………………53 3.3.1.3. S7 intron 1 analyses………………………………………………………….58 3.4. Morphometric analyses…………………………………………………………..63 3.5. Discussion……………………………………………………………………..…70 ii Chapter 4: A comparison of patterns of differentiation in Lutjanus bohar (Forsskål, 1775) and Lutjanus lutjanus Bloch 1790 across the Western Indian Ocean as inferred from three DNA markers.…………………………………………………………………..77 4.1. Introduction………………………………………………………………………77 4.2. Material and Methods………….………………………………………………...79 4.3. Results……………………………………………………………………………80 4.3.1. Lutjanus bohar (Forsskål, 1775)………………………………………………80 4.3.2. Lutjanus lutjanus Bloch, 1790...……………………………………………….90 4.4. Discussion………………………………………………………………………..97 Chapter 5: Phylogenetic relationships in the genus Lutjanus with a focus on the position of the Western Indian Ocean snappers in relation to the Indo-Pacific…………………101 5.1. Introduction………………………………………………………….………….101 5.2. Material and Methods…………….………………………………..…………...103 5.2.1. Sampling and data analysis………………………………………….………..103 5.3. Results…………………………………………………………………………..105 5.3.1. Cytochrome c oxidase I (COI)………………………………………………..105 5.3.2. Cytochrome c oxidase II (COII)…………………………………….………..108 5.3.3. 16S ribosomal-RNA (16S rDNA)…………………………………………….111 5.4. Discussion…………………………………………………………….………...114 Chapter 6: General discussion………………………………………………….………....118 References……………………………………………………………………..………..…..124 Appendices……………………………………………………………………………...…..152 iii ABSTRACT Snappers of the genus Lutjanus are small to large predatory fishes occurring in inshore circumtropical and subtropical waters throughout the world. These fishes support fisheries across their distribution range. Within the Western Indian Ocean (WIO), previous studies on Lutjanus kasmira revealed limited spatial genetic differentiation, whereas Lutjanus fulviflamma showed high genetic connectivity. The phylogenetic relationships among WIO snappers are unknown. Previous studies in the Indo-Pacific (IP) did not include any WIO representatives. This study examined (1) the phylogeographic patterns in Lutjanus bohar, L. fulviflamma and L. lutjanus to understand the origins and factors influencing the distribution of diversity in the region, (2) how the physical environment, biological, and ecological factors influence genetic diversity, (3) the placement of WIO snappers in context to those from the IP, as well as the placement of taxa not included previously, (4) extent of differentiation among conspecifics from the two regions, and (5) the relationship of the Caesionidae to the Lutjanidae. Samples were sourced from across the WIO and from peripheral localities, where possible. DNA sequence data were generated from two mitochondrial gene regions (cyt-b and NADH- 2) and a nuclear gene region (S7 intron 1). Data were analysed under a phylogeographic framework to examine genetic structure, diversity and differentiation among identified regions for each of the three species. Other sequence data were generated from two mitochondrial gene regions (COII and 16S rDNA) to examine the phylogenetic placement of WIO snappers in context of the IP snappers and the relationship of the Caesionidae to the Lutjanidae. Lutjanus bohar and L. fulviflamma displayed high genetic diversity, but lower diversities were observed for L. lutjanus. Genetic