Molecular revision of Zoantharia (Anthozoa Hexacorallia) on the east Coast of South Africa Michelle Megan Risi Submitted in the fulfilment for requirements for the degree of Master of Science, in the School of Life Sciences University of KwaZulu-Natal, Westville August 2014 Abstract The order Zoantharia (Cnidaria: Anthozoa) is ubiquitous on the east coast of South Africa, and despite their widespread distribution they are poorly represented in literature. No molecular identification has been carried out on these organisms on the South African shoreline. Zoanthus sansibaricus Carlgren, 1900 has a global distribution and has been reported having numerous morphotypes in terms of polyp shape, size, colour and oral disk colour. The initial aim in this study was to examine t he molecular characteristics of three Zoanthus species; Z. sansibaricus, Z. durbanensis Carlgren, 1938 and Z. natalensis Carlgren, 1938, to determine whether they are three separate species or merely morphotypes of one another. Following on from this research, the aim was to conduct a molecular revision of all zoanthids found in the intertidal zone along the east coast of South Africa, and to identify the Symbiodinium spp. within zoanthids for comparisons with conspecifics elsewhere. S amples were collected at sites along the coast from Umgazana (31.7024° S, 29.4175° E) to Sodwana (27.6594° S, 32.6477° E) and at one site in Libanona, Madagascar (25.0421° S, 46.9952° E). Sequences of cytochrome oxidase subunit one (COI), mitochondrial 16S ribosomal DNA (mt 16S rDNA), the nuclear internal transcribed spacer region of ribosomal DNA (ITS rDNA) for zoanthids and ITS-rDNA region for Symbiodinium spp. were used in this study to run phylogenetic analyses and examine the molecular characteristics f or comparisons with zoanthids elsewhere using GenBank. Seven species were identified; Isaurus tuberculatus, Palythoa nelliae, Palythoa tuberculosa, Z. durbanensis, Z. gigantus, Z. natalensis and Z. sansibaricus. The COI sequences (for Z. sansibaricus, Z. natalensis and Z. durbanensis) had little variation between species groups, while the mt 16S rDNA tree showed that Z. sansibaricus matched with sequences of previously reported Z. sansibaricus from the Pacific. Zoanthus natalensis was identical to Z. okuroshi Reimer & Ono, 2006 and Z. durbanensis was identical to Z. vietnamensis Pax & Müller, 1957. The ITS rDNA sequences were very ii similar for these four species; Z. natalensis, Z. kuroshio, Z. durbanensis and Z. vietnamensis. Palythoa nelliae Pax, 1935 appears to match with Pacific species Palythoa mutuki Haddon & Shackleton, 1891, and this is supported by the mt 16S and ITS rDNA markers. Symbiodinium subclade A1 was most often found with Z. natalensis and subclade C15/C91 was most often found with Z. durbanensis. Subclade C1 sensu LaJeunesse (2002) was found with all Isaurus and Palythoa samples, and most of Z. sansibaricus samples. The results of this study indicate that Z. natalensis is likely conspecific to Z. kuroshio, Z. durbanensis is likely conspecific to Z. vietnamensis, and P. nelliae is likely conspecific to P. mutuki, however, this is only a tentative hypothesis as no formal morphological analyses were done on proposed conspecifics.This work highlights the importance for similar studies in the clarification of zoanthid taxonomy. Key words: COI, ITS rDNA, mt 16S rDNA, Symbiodinium, Zoantharia, South Africa, Madagascar iii Preface The following MSc thesis was undertaken at the School of Life Science, University of KwaZulu-Natal, Westville. The work described was carried out from June 2012 to July 2014 under the supervision of Dr A.H.H. Macdonald The work presented in this thesis has not been submitted previously for any degree or diploma to any other tertiary institution. The studies are the original work of the author and where the work of others has been used, the sources have been acknowledged in the text. iv Plagiarism declaration I, Michelle Risi declare that: The research reported in this thesis, except where otherwise stated, is my own original work. This thesis has not been submitted for any other degree, diploma or examination at any other tertiary institution. This thesis does not contain data, pictures, graphs or other information which are not my own, unless specifically acknowledged as being sourced from another’s work. This thesis has not been written by anyone but myself, unless specifically acknowledged as being the work of published sources. Where the work of other researchers has been quoted: a) Their words have been re-written but the conceptual ideas and general information attributed to them have been appropriately referenced. b) Where their exact words have been used, these statements have been placed inside quotation marks and after which they have been appropriately referenced. This thesis does not contain text, graphs or tables copied and pasted from the Internet, unless specifically acknowledged. The relevant sources are detailed in the text of the thesis and in the References section. v Publication declaration The following details the contribution to the publication which forms part of the research presented in this thesis. Publication 1: Risi, M., Macdonald, A. (2015) Possible synonymies of Zoanthus (Anthozoa: Hexacorallia) species on the east coast of South Africa with Pacific congeners. Systematics and Biodiversity, 13:1, 93-103, DOI: 10.1080/14772000.2014.968233 Author contributions: Conception and design: Michelle Risi and Angus Macdonald. Collection of data: Michelle Risi. Analysis of data: Michelle Risi and Angus Macdonald. Wrote the draft paper: Michelle Risi. Angus Macdonald and anonymous reviewers contributed comments on the manuscript. Publication 2 (accepted, 2 March 2015): Risi, M., Macdonald, A. Molecular examination of rocky shore brachycnemic zoantharians (Anthozoa: Hexacorallia) and their Symbiodinium symbionts (Dinophyceae) in the southwest Indian Ocean. Marine Biodiversity, DOI: 10.1007/s12526-015-0331-y Author contributions: Conception and design: Michelle Risi and Angus Macdonald. Collection of data: Michelle Risi. Analysis of data: Michelle Risi and Angus Macdonald. Wrote the draft paper: Michelle Risi. Angus Macdonald contributed comments on the manuscript. vi Other research outputs Oral presentation: Risi, M., Macdonald, A. November 2013. Molecular characterisation of rocky-shore Zoanthus species (Anthozoa: Hexacorallia) on the KwaZulu-Natal coast, South Africa. Symposium of Contemporary Conservation Practice, Howick, South Africa. Poster presentations: Risi, M., Macdonald, A. October 2013. Molecular characterisation of rocky-shore Zoanthus species (Anthozoa: Hexacorallia) on the KwaZulu-Natal coast, South Africa. Eighth Western Inidian Ocean Marine Science Association (WIOMSA) scientific symposium, Maputo, Mozambique. Risi, M., Macdonald, A. July 2014. Zoanthid phylogenetics: molecular revision of zoanthids in South Africa. 15th Southern African Marine Science Symposium (SAMSS), Stellenbosch, South Africa. vii Acknowledgements I would like to acknowledge my supervisor Dr. Macdonald, as this research was inspired by him and would not have taken place if he had not wondered “what the deal is with all of these zoanthids”. His guidance and support have been unfaltering. I am grateful to those that assisted with sampling; Charles Smith, Jyothi Kara, Brent Chiazzari, Emily Chemoiwa, Lindile Cele, Ryan van Rooyen and Christopher Jones. Thank you to Justin Hart for his willingness to assist in the morphological aspect of this study. I would like to thank Assoc. Prof. James Reimer for his readiness to give advice and assistance to the direction of this research. A special thank you to Prof. Michael Schleyer, the team at Oceanographic Research Institute and the African Coelacanth Ecosystem Project for collecting zoanthids in Madagascar. Lastly, thank you to the all members of the Marine Evolution Lab (MAREVOL), with which I am affiliated, for the endless advice received during presentation of results and the discussion of this project. This work was supported by the National Research Foundation under Grant 83704, and the Western Indian Ocean Marine Science Association under Grant MARG I Contract 7. viii Table of Contents Abstract ..................................................................................................................................... ii Preface ...................................................................................................................................... iv Plagiarism declaration ............................................................................................................. v Publication declaration ........................................................................................................... vi Other research outputs .......................................................................................................... vii Oral presentation: ................................................................................................................. vii Poster presentations: ............................................................................................................. vii Acknowledgements .............................................................................................................. viii Chapter 1: General introduction ............................................................................................ 1 Chapter 2: Possible synonymies of Zoanthus (Anthozoa: Hexacorallia) species on the east