PHYLOGENY OF THE FAVIIDAE (SCLERACTINIA) IN SINGAPORE BASED ON MOLECULAR AND MORPHOLOGICAL DATA HUANG DANWEI (B.Sc.(Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgements First and foremost, I thank my supervisors Dr Peter Alan Todd and Professor Chou Loke Ming for their tireless supervision and guidance, the stimulating discussions, as well as the invaluable advice and motivation for me to delve into marine science The last three years have been productive and fulfilling because of the opportunity to carry out my research in the Marine Biology Laboratory I have also been very fortunate to receive the gracious advice of A/P Rudolf Meier, who gave me permission to pursue molecular work in the Evolutionary Biology Laboratory My heartfelt gratitude goes to him for patiently coaching me in phylogenetics and scientific writing Both laboratories mentioned above have been an important part of my life as a graduate student, and I appreciate the people who have made it such To Abby Ng, Angie Seow, Farhan Ali, Gaurav Vaidya, Gwynne Lim, Hwang Wei Song, Karenne Tun, Lin Juanhui, Nalini Puniamoorthy Tay Ywee Chieh and Zhang Guanyang, for rendering assistance, constant reviews and encouragement I would like to especially mention the help of Kathy Su, Sujatha Narayanan Kutty and Zeehan Jaafar, who gave guidance on various aspects of taxonomy, phylogenetics and laboratory work This project would not have been possible without the expert advice of Professor Nancy Knowlton (Scripps Institution of Oceanography) on coral systematics, and Dr Hironobu Fukami (Seto Marine Biological Laboratory) on DNA extraction and PCR My participation in the Coral Molecular Biology Techniques Workshop conducted by the Hawaii Institute of Marine Biology helped to jumpstart my molecular work, and I appreciate Lady McNeice’s generosity that made it financially possible I thank Drs Andrew Baird (James Cook University), Wilfredo Licuanan (De La Salle University), ii Emre Turak and Lyndon DeVantier (Australian Institute of Marine Science) for their assistance with specimen identification and ideas on coral systematics I am also indebted to Professor Gregory Rouse (Scripps Institution of Oceanography), my PhD advisor, who gave me time to complete this work as I began my doctoral candidature Several staff members of the Department of Biological Sciences have been of tremendous help I thank the R.V Mudskipper crew of Salam, Rahmat and Ishak for skillful handling of the department boat, and I am also grateful to Latiff for offering help in many ways Kelvin Lim kindly facilitated my access to the coral collection at the Raffles Museum of Biodiversity Research, and I acknowledge his assistance I would like to dedicate this work to the late Yeo Keng Loo who had tirelessly curated the invertebrate collection and managed the coral specimens that I used as reference Last but not least, I am grateful to my family for supporting me through my endeavours Note on coauthorship Chapter (Slow mitochondrial COI sequence evolution at the base of the metazoan tree and its implications for DNA barcoding) has been published (Appendix II), while Chapter (Phylogenetic relationships in the Faviidae based on molecular and morphological markers) is in review In both instances, I am the first author, while A/P Rudolf Meier, Dr Peter A Todd and Professor Chou Loke Ming are coauthors Although I received substantial advice and guidance from RM, PAT and CLM as advisors, the data and thesis are my own work iii The first examining of volcanic rocks, must to a geologist be a memorable epoch, and little less so to the naturalist is the first burst of admiration at seeing corals growing on their native rock Charles Darwin iv Table of Contents Acknowledgements ii Table of Contents v Summary vii List of Tables ix List of Figures xi CHAPTER 1: GENERAL INTRODUCTION 1.1 The Scleractinia 1.2 Coral taxonomy, barcoding and phylogenetics 1.3 Faviidae in Singapore 15 1.4 Objectives of the present study 17 CHAPTER 2: SLOW MITOCHONDRIAL COI SEQUENCE EVOLUTION AT THE BASE OF THE METAZOAN TREE AND ITS IMPLICATIONS FOR DNA BARCODING 2.1 Introduction 18 2.2 Materials and Methods 20 2.3 Results 22 2.4 Discussion 26 v CHAPTER 3: PHYLOGENETIC RELATIONSHIPS IN THE FAVIIDAE BASED ON MOLECULAR AND MORPHOLOGICAL MARKERS 3.1 Introduction 32 3.2 Materials and Methods 35 3.3 Results 47 3.4 Discussion 60 GENERAL CONCLUSIONS 68 References 70 Appendix I 104 Appendix II 128 Appendix III 137 vi Summary The Faviidae constitutes one of the most important families of hermatypic corals on Indo-Pacific reefs Several species in this group are taxonomically difficult and little is known about their phylogenetic relationships at the species level DNA barcoding holds enormous potential for species identification and subsequent resolution of evolutionary relationships among faviid corals However, the efficacy of this technique for non-bilaterians, including the Scleractinia (hard corals), has not been empirically assessed Here, I present a comprehensive analysis of intra- and interspecific COI variabilities in Porifera (sponges) and Cnidaria (corals, jellyfish and hydrozoans) using a dataset of 685 sequences from 283 species Variation within and among species was found to be much lower in Porifera and Anthozoa (containing Scleractinia) compared to the Medusozoa (Hydrozoa and jellyfish, i.e Scyphozoa), which has divergences similar to typical metazoans Given that recent evidence has shown that fungi also exhibit limited COI divergence, slow-evolving mtDNA is likely to be plesiomorphic for the Metazoa Higher rates of evolution could have originated independently in Medusozoa and Bilateria, or acquired in the Cnidaria + Bilateria clade and lost in the Anthozoa Low identification success and substantial overlap between intra- and interspecific COI distances render the Anthozoa, and hence the Scleractinia, unsuitable for DNA barcoding Caution is advised for Porifera and Hydrozoa because of relatively low identification success rates It has been suggested previously that the barcoding limitation generally exist for Cnidaria, but here, I confirm that it is restricted to the Anthozoa and caution against the use of COI for species delimitation in this taxon vii Due to the likely futility of coral DNA barcoding using COI, reconstructing evolutionary relationships within the Faviidae remains as difficult as before Relying solely on conventional taxonomic traits used in this family, I collected 84 colonies from 42 ingroup species as well as two outgroup specimens (Acanthastrea echinata and Scapophyllia cylindrica) These were sampled for two mitochondrial genes (COI and a mitochondrial noncoding region adjacent to COI) GenBank sequences were also extracted for four Caribbean faviid species and an Acropora outgroup A morphological dataset for monocentric species was generated with 12 descriptive and eight morphometric characters Maximum parsimony analysis was carried out separately on the molecular and morphological data using both dynamic (optimisation alignment) and static (ClustalX) homologies Both data types were also combined for total analysis I found that phylogenies based on both data types are incongruent and did not recover traditional taxonomic classification Of the eight genera with more than one species examined using molecular data, only two are monophyletic Furthermore, the outgroup Scapophyllia cylindrica is deeply nested within Faviidae, while the Indo-Pacific Montastrea spp are distinct from the Atlantic M annularis complex Data show clearly that conventional taxonomy has masked morphological convergence and reticulate evolution within this family Results also support the hypothesis that some species within genera spanning both the Indo-Pacific and Caribbean are less closely related to one another than to taxa from other families viii List of Tables Table 1.1 List of publications since Romano & Palumbi (1996) that use DNA sequence data to build phylogenies among scleractinian species Number of species analysed, molecular markers employed and the citation for each work are included 12S and 16S are mitochondrial rRNA genes; 5.8S and 28S are nuclear rDNA genes; ITS = nuclear internal transcribed spacer regions, including 5.8S (ITS1-5.8S-ITS2); cytB = cytochrome b (mitochondrial); COI = cytochrome oxidase c subunit (mitochondrial);
tub =
-tubulin (nuclear); mt-genome = complete mitochondrial genome sequence; ATP6 = ATPase (mitochondrial); h2ab = partial histone 2A and 2B (nuclear); MC2 = mini-collagen intron (nuclear); Pax-C = DNA 46/47 Pax-C intron (nuclear); MIR = mtDNA intergenic/control region; MNC = mtDNA noncoding region (adjacent to COI) (pp 13–14) Table 2.1 COI distances, pairwise Mann-Whitney U-statistics, significance tests and the rank of each taxon (largest to smallest distances) among Porifera, Anthozoa, Hydrozoa and Scyphozoa for intraspecific (a) and closest congeneric interspecific distances (b) Distance values denote means and standard errors (p 25) Table 2.2 Frequencies of sequences (percentages in parentheses) with accuracy of species attribution using three threshold values: 3.0%, 10
mean intraspecific distance (10
) and point of minimum overlap (MO) An accurate identification of a sequence is classified as ‘correct’; sequence with best matches to the correct barcode and at least an incorrect one is ambiguous; erroneous species attribution is ‘incorrect’; and ‘unmatched’ sequence does not have any match closer than the threshold (p 26) ix Table 3.1 List of 86 specimens from 44 species sampled in this study (asterisk denotes taxon designated as outgroup) Successful PCR amplifications of two genes, including the presence of an intron embedded within COI, are shown Analyses carried out for each taxon are also indicated ALL represents 91-taxon molecular analysis; ALN is reconstruction with all taxa for which the gene fragment MNC can be aligned; MOR is the morphological analysis of monocentric taxa (pp 37–41) Table 3.2 List of genera with long segments of T repeats that cannot be fully amplified with MNC1f and MNC1r Internal primer sequences designed for these taxa and melting temperatures employed for PCR are shown (p 41) Table 3.3 List and synopses of morphological characters, including descriptive and morphometric parameters, used to analyse the monocentric species Character states and corresponding codes are indicated (pp 43–45) x Shoo, J H H., 2005 Investigating the Biodiversity of Coral Reefs in the Southern Islands of Singapore Honours thesis submitted to the Department of Biological Sciences, National University of Singapore (unpublished) 144 pp Spalding, M D., C Ravilious & E P Green, 2001 World Atlas of Coral Reefs University of California Press, California 424 pp Titlyanov, E A & Y Y Latypov, 1991 Light-dependence in scleractinian distribution in the sublittoral zone of South China Sea Islands Coral Reefs, 10: 133-138 Todd, P A., P G Sanderson & L M Chou, 2001 Morphological variation in the polyps of the scleractinian coral Favia speciosa (Dana) around Singapore Hydrobiologia, 444: 227-235 Todd, P.A., R.C Sidle & L.M Chou, 2002a Plastic corals from Singapore Coral Reefs, 21: 391-392 Todd, P.A., R.C Sidle & L.M Chou, 2002b Plastic corals from Singapore Coral Reefs, 21: 407-408 Todd, P A., R J Ladle, N J I Lewin-Koh & L M Chou, 2004 Genotype
environment interactions in transplanted clones of the massive corals Favia speciosa and Diploastrea heliopora Marine Ecology Progress Series, 271: 167-182 Veron, J E N., 2000 Corals of the World Australian Institute of Marine Science, Townsville Veron, J E N & M Pichon, 1976 Scleractinia of Eastern Australia Part I: Families Thamnasteriidae, Astrocoeniidae, Pocilloporidae Australian Government Publishing Service, Australia 208 pp 126 Veron, J E N & M Pichon, 1980 Scleractinia of Eastern Australia Part III: Families Agariciidae, Siderastreidae, Fungiidae, Oculinidae, Merulinudae, Mussidae, Pectiniidae, Caryophylliidae, Dendrophylliidae Australian Institute of Marine Science and Australian National University Press, Australia 471 pp Veron, J E N & M Pichon, 1982 Scleractinia of Eastern Australia Part IV: Family Poritidae Australian Institute of Marine Science and Australian National University Press, Australia 210 pp Veron, J E N., M Pichon & M Wijsman-Best, 1977 Scleractinia of Eastern Australia Part II: Families Faviidae, Trachyphlliidae Australian Government Publishing Service, Australia 231 pp Wallace, C C., 1999 Staghorn Corals of the World CSIRO Publishing, Australia 421 pp Wong, L S., 2001 Assessing the Dynamics between Sedimentation, Sediment Load and Sediment Removal by Scleractinian Corals in a Singapore Fringing Reef Honours thesis submitted to the Department of Biological Sciences, National University of Singapore (unpublished) 51 pp 127 APPENDIX II: SLOW MITOCHONDRIAL COI SEQUENCE EVOLUTION AT THE BASE OF THE METAZOAN TREE AND ITS IMPLICATIONS FOR DNA BARCODING (Published, Journal of Molecular Evolution, 66: 167–174) D Huang, R Meier, P A Todd & L M Chou Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543 128 129 130 131 132 133 134 135 136 APPENDIX III: SPECIMENS EXAMINED AND RAFFLES MUSEUM OF BIODIVERSITY RESEARCH CATALOGUE NUMBERS Acanthastrea echinata ZRC.CNI.0130 Barabattoia amicorum ZRC.CNI.0174 Barabattoia amicorum ZRC.CNI.0196 Caulastrea echinulata ZRC.CNI.0171 Cyphastrea chalcidicum ZRC.CNI.0188 Cyphastrea microphthalma ZRC.CNI.0181 Cyphastrea serailia ZRC.CNI.0123 Cyphastrea serailia ZRC.CNI.0177 Diploastrea heliopora ZRC.CNI.0167 Diploastrea heliopora ZRC.CNI.0168 Echinopora gemmacea ZRC.CNI.0203 Echinopora lamellosa ZRC.CNI.0193 Echinopora pacificus ZRC.CNI.0194 Favia aff favus ZRC.CNI.0183 Favia aff favus ZRC.CNI.0184 Favia danae ZRC.CNI.0159 Favia danae ZRC.CNI.0190 Favia favus ZRC.CNI.0124 Favia favus ZRC.CNI.0136 Favia favus ZRC.CNI.0178 Favia favus ZRC.CNI.0179 Favia helianthoides ZRC.CNI.0139 Favia helianthoides ZRC.CNI.0140 137 Favia lizardensis ZRC.CNI.0148 Favia matthaii ZRC.CNI.0107 Favia matthaii ZRC.CNI.0114 Favia matthaii ZRC.CNI.0128 Favia matthaii ZRC.CNI.0137 Favia matthaii ZRC.CNI.0166 Favia maxima ZRC.CNI.0142 Favia maxima ZRC.CNI.0182 Favia pallida ZRC.CNI.0133 Favia pallida ZRC.CNI.0144 Favia rotumana ZRC.CNI.0180 Favia rotumana ZRC.CNI.0197 Favia speciosa ZRC.CNI.0103 Favia speciosa ZRC.CNI.0125 Favia speciosa ZRC.CNI.0126 Favia speciosa ZRC.CNI.0132 Favia speciosa ZRC.CNI.0158 Favites abdita ZRC.CNI.0104 Favites abdita ZRC.CNI.0185 Favites abdita ZRC.CNI.0189 Favites complanata ZRC.CNI.0109 Favites complanata ZRC.CNI.0149 Favites complanata ZRC.CNI.0161 Favites flexuosa ZRC.CNI.0131 Favites paraflexuosa ZRC.CNI.0165 138 Favites pentagona ZRC.CNI.0157 Favites pentagona ZRC.CNI.0195 Goniastrea aspera ZRC.CNI.0191 Goniastrea australensis ZRC.CNI.0186 Goniastrea australensis ZRC.CNI.0164 Goniastrea edwardsi ZRC.CNI.0138 Goniastrea edwardsi ZRC.CNI.0200 Goniastrea favulus ZRC.CNI.0122 Goniastrea favulus ZRC.CNI.0187 Goniastrea palauensis ZRC.CNI.0121 Goniastrea pectinata ZRC.CNI.0173 Goniastrea retiformis ZRC.CNI.0155 Goniastrea retiformis ZRC.CNI.0160 Leptoria phrygia ZRC.CNI.0153 Montastrea curta ZRC.CNI.0115 Montastrea curta ZRC.CNI.0116 Montastrea curta ZRC.CNI.0119 Montastrea magnistellata ZRC.CNI.0105 Montastrea magnistellata ZRC.CNI.0150 Montastrea magnistellata ZRC.CNI.0151 Montastrea valenciennesi ZRC.CNI.0108 Montastrea valenciennesi ZRC.CNI.0110 Montastrea valenciennesi ZRC.CNI.0113 Montastrea valenciennesi ZRC.CNI.0120 Montastrea valenciennesi ZRC.CNI.0135 139 Oulastrea crispata ZRC.CNI.0192 Oulophyllia bennettae ZRC.CNI.0169 Oulophyllia bennettae ZRC.CNI.0172 Oulophyllia bennettae ZRC.CNI.0175 Oulophyllia crispa ZRC.CNI.0145 Oulophyllia crispa ZRC.CNI.0202 Platygyra daedalea ZRC.CNI.0199 Platygyra lamellina ZRC.CNI.0198 Platygyra pini ZRC.CNI.0134 Platygyra pini ZRC.CNI.0152 Platygyra sinensis ZRC.CNI.0201 Platygyra verweyi ZRC.CNI.0170 Scapophyllia cylindrica ZRC.CNI.0176 140 [...]... present study The purpose of the present study is to first examine mitochondrial COI sequence evolution and DNA barcoding efficacy at the base of the metazoan tree, and then reconstruct the first broad-based species-level phylogeny of the Faviidae using molecular and morphological data The following hypotheses are tested: (1) COI sequence evolution in non-bilaterian taxa is slow relative to the Bilateria;... symbiotic dinoflagellate protists of the genus Symbiodinium (Wells 1956; Veron 2000) These organisms, commonly known as zooxanthellae, are ubiquitous members of the coral reef (Taylor 1974; Trench 1993; Rowan 1998) They are the source of up to 50% of the host’s nutrient intake, provided in the form of nitrogen and carbon, while obtaining metabolic products such as PO4, NH3 and CO2 from the coral host... 2004) The order is also distinguished by a calcareous exoskeleton made up of radial septa between the mesenteries (developed in multiples of six) as well as a complex array of surrounding and supporting structures (Wells 1956) The taxon includes over 700 recorded zooxanthellate species, i.e those containing symbiotic dinoflagellates, and these form the basis of coral reefs along the tropical and subtropical... (Dawson 2005), and 10.9–23.4% in Cassiopea (Holland et al 2004) The evolution of divergence rates in COI is not only interesting from an evolutionary point of view, but also important for the prospects of DNA barcoding (see Hebert et al 19 2003a) The success of this species identification tool is often thought to depend on the presence of a barcoding ‘gap’; i.e., the separation between intra- and interspecific... comprising the sponges, possesses limited speeds akin to the Anthozoa (e.g Watkins & Beckenbach 1999; Lavrov et al 2005) Evidently, a better understanding of the evolutionary processes determining mitochondrial sequence variation is desirable before use of the COI barcode becomes a standard practice among coral scientists The increasing ease and falling cost of DNA sequencing has not only generated interest... success using DNA barcodes varies among lineages in Cnidaria and Porifera; (3) Phylogeny of the Faviidae is incompatible with conventional taxonomy of this group; and (4) Phylogenetic recontructions based on mitochondrial sequence data and morphological characters are incongruent The results of this study will provide crucial information on the evolutionary history of the Faviidae and fuel the impetus... 1.9% of species pairs in other metazoan groups had such distances However, their conclusion was based on only 17 species pairs of Cnidaria, while an average of 950 pairs were included for the other major animal taxa Furthermore, all three Cnidaria genera (10 spp.) in the survey (Corallium, Narella and Urticina) belonged to the Anthozoa Thus, the status of the remaining Cnidaria remained uncertain Subsequently,... from the mainland, likely due to a declining gradient of sedimentation However, no clarification of species limits in these marginal habitats was attempted In the recent update of Singapore s zooxanthellate Scleractinia, Huang et al (manuscript accepted; Appendix I) extracted all Faviidae specimens from the coral reference collection at Raffles Museum of Biodiversity Research (RMBR) and reidentified them... explain the rate variation in ‘basal’ Metazoa Firstly, sequence evolution was slow in the metazoan ancestor and later gathered speed in the Bilateria The second hypothesis was fast-evolving mitochondrial DNA in the metazoan ancestor and a secondary slow down in Anthozoa Here, we use COI as a model to present a consolidated analysis of our own and GenBank data pertaining to this question We furthermore... and fuel the impetus for taxonomic revisions of the Scleractinia Findings will also be useful for future forecasts of reef evolution in the region and may contribute to the designation of conservation priorities in Singapore 17 CHAPTER 2: SLOW MITOCHONDRIAL COI SEQUENCE EVOLUTION AT THE BASE OF THE METAZOAN TREE AND ITS IMPLICATIONS FOR DNA BARCODING 2.1 Introduction Most biologists assume that metazoan ... use of the COI barcode becomes a standard practice among coral scientists The increasing ease and falling cost of DNA sequencing has not only generated interest in the barcoding of the Scleractinia—reconstructions... zooxanthellate species, i.e those containing symbiotic dinoflagellates, and these form the basis of coral reefs along the tropical and subtropical coasts of the Indo-Pacific and Atlantic Azooxanthellate... further away from the mainland, likely due to a declining gradient of sedimentation However, no clarification of species limits in these marginal habitats was attempted In the recent update of Singapore s