Marine genomics meets ecology: Diversity and divergence in South African sea stars of the genus Parvulastra Katherine Dunbar Thesis submitted for the degree of Doctor of Philosophy Biodiversity and Ecological Processes Research Group School of Biosciences Cardiff University December 2006 UMI Number: U584961 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U584961 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 DECLARATION This work has not previously been substance for any degree and is not being concurrently submitted in c y degree. Signed ................................(candidate) Date.... 3 l . ™ MW. ... ..... STATEMENT 1 This thesis is the result of my own M ent work/investigation, except where otherwise stated. Other source* edged by footnotes giving explicit references. Signed (candidate) S.**: Q tife : ...... STATEMENT 2 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the tJfJSJa^^prrmqary to be made available to outside organisations Signed ................................................................... (candidate) Date............................. Abstract The coast of South Africa is situated between the warm Indian and the cold Atlantic Oceans, resulting in an extreme intertidal temperature gradient and potentially strong opposing selection pressures between the east and west coasts. Several intertidal biogeographic divides have been identified, including one at Cape Point between the cold west coast and the temperate south coast provinces. However, few studies have investigated the effects of these opposing environments on phylogeography or gene flow in intertidal organisms. A small intertidal sea star, Parvulastra exigua , was chosen as a model organism to investigate these issues. Taxonomic confusion in this species and its systematic relationship with a related South African Parvulastra species, Parvulastra dyscrita, was resolved using nuclear (Actin intron sequences and AFLP) and mtDNA molecular markers. At least one cryptic species was identified within Parvulastra in South Africa, which occupied an extremely restricted geographic distribution and therefore may be a candidate for conservation. Molecular and morphological evidence confirmed that P. exigua and P. dyscrita are separate species. An ecological survey was conducted on P. exigua at 19 locations in South Africa covering a distance of 2500 km. P. exigua samples from each location were sequenced for mtDNA and screened for 421 AFLP loci. AFLP was also used to identify outlier loci that were potentially under selection. An ‘unmottled’ colour morph was distributed from the Namibian border to Cape Point and a ‘mottled’ morph was distributed from Cape Point to the Mozambique border, with an area of sympatry around Cape Point. The unmottled morphs were positively influenced by under boulder and bare rock habitats, but negatively affected by canopy, coralline algae and sand. Mottled morphs were positively influenced by under boulder, protected habitats, encrusting algae and bare rock, and negatively affected by algal tufts and sand. MtDNA revealed two divergent, reciprocally monophyletic clades, one comprising the east coast samples and the other encompassing the west coast samples. Both clades showed evidence for a recent, rapid population expansion. The genetic break-point was located on the south coast, but did not coincide with the divergence in colour morphs, being approximately 500 km to the east. AFLP indicated a strong isolation by distance pattern of genetic structure among sampling locations and did not recapitulate the mtDNA genetic divide. Such incongruence among data sets might be caused by a vicarance event if sea level changes separated the east and west coast populations, which expanded in isolation, followed by secondary contact, restoring present day gene flow between the coasts. Population genomic analysis revealed approximately 7% of the genome to potentially be under divergent selection, and the phenotype frequencies of the ‘diverging outlier loci’ revealed high directionality (spatial correlation). This suggests strong selection pressures between the east and west coasts may be acting on these loci, which could have arisen when the populations were in allopatry. The habitat and colour morph differences of P. exigua between the two coasts are potentially also influenced by selection. However, the isolation by distance pattern indicates that divergent selection pressures are not strong enough to cause reproductive isolation, or disrupt gene flow between the east and west coast populations. Acknowledgements This work was funded by the Natural Environment Research Council (NERC). I am extremely indebted to my supervisor Prof Mike Bruford for enabling me to undertake this research, and his support, patience, enthusiasm and encouragement throughout my Ph.D. My thanks also goes to Mike and Dr. David Lees for inspiring me to study molecular ecology and animal speciation. I would also like to sincerely thank my second supervisor Dr. Jo Cable for being extremely supportive and helpful throughout. She was both a great supervisor and friend. My thanks goes to my other supervisor Prof. Charlie Griffiths for providing me with lab space both times I visited South Africa, as well as drawing my attention to the little seastars that are the subject of this work. I would also like to very much thank Dr. Jo Lello for her invaluable statistical help and advice. My thanks goes to all the people who helped me in South Africa with my fieldwork, specifically Tom Peschak for making my first trip to South Africa so amazing, and inspiring me to go back, Ben Pizii, who kept me laughing all the way round the coast, and remains a good friend back in the UK, Grahame and Shane who kept me safe and provided me with their fantastic vehicles and experience, Bronwen Hobbs and her family for being kind to me and providing me with a place to live and the girls in Charlie’s lab who made me laugh and kept me sane (just!). A massive thank you goes to all my friends and colleagues from G10, Jo’s lab and Bill’s lab who have given me advice and help with lab work and kept me laughing throughout good times and frustrating times. They are too numerous to name. My deepest thanks also go to my parents and my brother who have been supportive and encouraging throughout. My sincerest thanks also goes to my surrogate Cardiff family, Matt, Nicky, Jo, Justin, Penny, Stewart, Rebecca and Matt L, for their friendship and laughter for which I am truly grateful. Finally, but most importantly I would like to dedicate this PhD to Jon Russill, He has been supportive and patient every step of the way. Without his quiet steady love and encouragement I would not have been able to complete this work. He is my strength and my sunshine. Chapter 1: General Introduction.............................................................................6 1.0. Introduction ...................................................................................................... 6 1.1. Asterinid systematics ......................................................................................6 1.1.1 The genus Parvulastra ..............................................................................9 1.1.2 Taxonomic confusion of P. exigua and P. dyscrita .................................9 1.2. Parvulastra: The study species .................................................................... 12 1.2.1. Parvulastra exigua .................................................................................. 12 1.2.2 Parvulastra dyscrita .................................................................................15 1.3. South Africa: The study site ..........................................................................15 1.3.1 Intertidal environment and geological history ........................................15 1.3.2 Biogeography.......................................................................................... 20 1.4. Asterinid life histories ....................................................................................21 1.5. Parvulastra phylogeography........................................................................ 29 1.6. Molecular approaches ................................................................................... 34 1.6.1 Single Gene approaches ........................................................................ 35 1.6.1.1 Mitochondrial sequence markers .................................................... 35 1.6.1.1.1. Mitochondrial heteroplasmy .................................................... 39 1.6.1.1.2. Paternal inheritance of the mitochondrial genome ................39 1.6.1.1.3. Recombination in mitochondrial DNA ..................................... 40 1.6.1.1.4. Mitochondrial DNA as a neutral marker .................................. 40 1.6.1.1.5. Mitochondrial gene genealogies not species genealogies ... 41 1.6.1.1.6. Nuclear copies