DNA barcoding reveals the coral ‘‘laboratory-rat’’, Stylophora pistillata SUBJECT AREAS: ADAPTIVE RADIATION encompasses multiple identities EVOLUTIONARY BIOLOGY Shashank Keshavmurthy1*, Sung-Yin Yang1,2*, Ada Alamaru3*, Yao-Yang Chuang1,23, Michel Pichon4,5, EVOLUTIONARY ECOLOGY David Obura6, Silvia Fontana1,7,8, Stephane De Palmas1, Fabrizio Stefani9, Francesca Benzoni7, MARINE BIOLOGY Angus MacDonald10, Annika M. E. Noreen11, Chienshun Chen12, Carden C. Wallace4, Ruby Moothein Pillay13, Vianney Denis1, Affendi Yang Amri14,15, James D. Reimer16, Takuma Mezaki17, Received Charles Sheppard18, Yossi Loya3, Avidor Abelson3, Mohammed Suleiman Mohammed19, 5 September 2012 Andrew C. Baker20, Pargol Ghavam Mostafavi21, Budiyanto A. Suharsono22 & Chaolun Allen Chen1,8,23 Accepted 5 March 2013 1Biodiversity Research Centre, Academia Sinica, Nangang, Taipei 115, Taiwan, 2Graduate school of Engineering and Science, 3 Published University of the Ryukyus, Okinawa 903-0213, Japan, Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv 4 5 22 March 2013 University, Tel Aviv 69978, Israel, Museum of Queensland, Townsville 4811, Australia, James Cook University, Australia, 6Coastal Ocean Research and Development of Indian Ocean (CORDIO), Mombasa, Kenya, 7Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza, 2, 20126 Milan, Italy, 8Taiwan International Graduate Program (TIGP)-Biodiversity, Academia Sinica, Nangang, Taipei 115, Taiwan, 9Water Research Institute, National Research Council Correspondence and (IRSA-CNR), Via del Mulino 19, 20861 Brugherio (MB), Italy, 10School of Life Sciences, University of KwaZulu-Natal, Private Bag 11 requests for materials X54001, Durban, 4001, South Africa, Coral Reef Research Centre, School of Environmental Science and Management, Southern Cross University, PO Box 157, Lismore, New South Wales 2480, Australia, 12Taiwan Ocean Research Institute, National Applied should be addressed to Research Laboratories, Kaohsiung 852, Taiwan, 13Mauritius Oceanography Institute, France Centre, Victoria Avenue Quatre C.A.C. (cac@gate. Bornes, Mauritius, 14Institute of Biological Sciences, Faculty of Science, Universiti Malaya, 50603 Kuala Lumpur, Malaysia and sinica.edu.tw) School of Plant Biology, , 15The University of Western Australia, Crawley, 6009 Western Australia, 16Rising Star Program, Transdisciplinary Research Organization for Subtropical Island Studies (TRO-SIS), University of the Ryukyus, Okinawa 903-0213, Japan, 17Biological Institute on Kuroshio, Otuski, Kochi, Japan, 18School of Life Sciences, University of Warwick, CV4 7AL, United of * These authors Kingdom, 19Institute of Marine Science, University of Dar es Salaam, Zanzibar, Tanzania, 20Division of Marine Biology and contributed equally to Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami 4600 Rickenbacker Causeway, Miami, FL 21 this work. 33149, USA, Department of Marine Biology, Graduate school of Marine Science and Technology, Science and Research Branch, 3 Islamic Azad University, Iran, 22Research Center for Oceanography, Indonesian Institute of Sciences (LIPI), Jl. Pasir Putih I, Ancol Timur Jakarta, Indonesia, 23Institute of Oceanography, National Taiwan University, Taipei 106, Taiwan. Stylophora pistillata is a widely used coral ‘‘lab-rat’’ species with highly variable morphology and a broad biogeographic range (Red Sea to western central Pacific). Here we show, by analysing Cytochorme Oxidase I sequences, from 241 samples across this range, that this taxon in fact comprises four deeply divergent clades corresponding to the Pacific-Western Australia, Chagos-Madagascar-South Africa, Gulf of Aden-Zanzibar-Madagascar, and Red Sea-Persian/Arabian Gulf-Kenya. On the basis of the fossil record of Stylophora, these four clades diverged from one another 51.5-29.6 Mya, i.e., long before the closure of the Tethyan connection between the tropical Indo-West Pacific and Atlantic in the early Miocene (16–24 Mya) and should be recognised as four distinct species. These findings have implications for comparative ecological and/or physiological studies carried out using Stylophora pistillata as a model species, and highlight the fact that phenotypic plasticity, thought to be common in scleractinian corals, can mask significant genetic variation. NA barcoding, usually based on the mitochondrial COI fragment1, has been extensively used to discrim- inate between closely related species, to identify new, cryptic or invasive species, and to assess biodiversity across many animal phyla1. The rule-of-thumb in DNA barcoding is that interspecific COI divergence is D 1 generally . 2%, whereas intraspecific variation is , 1% . However, based on 90 species from 44 genera belonging to 14 families, this criterion has been suggested as not being appropriate for most scleractinian corals2 (or anthozoans in general) because this region evolves very slowly in these organisms and consequently both SCIENTIFIC REPORTS | 3 : 1520 | DOI: 10.1038/srep01520 1 www.nature.com/scientificreports inter- and intra-specific variation are extremely low. We re-evaluated Phylogenetic construction based on COI sequences found 4 dis- this conclusion by examining COI divergence and phylogeny of the tinct clades of S. pistillata (Fig. 2A). Clade 1 is comprised of speci- ‘‘lab-rat’’ scleractinian coral, Stylophora pistillata, a species, which mens from the entire Pacific region and East Indian Ocean, ranging has been the focus of coral research over the last four decades. S. from New Caledonia in the southwest Pacific, Shikoku (Japan) in the pistillata is a widely-distributed coral species in the Indo-Pacific, with northwest Pacific, to western Australia in the Indian Ocean (Pacific- numerous morphological variations (morphotypes) across different Western Australia clade, PWA). Clade 2 includes specimens from habitats, depths, and geographic regions3. Revisiting relationships Chagos, St. Brandon’s Island (Mauritius), La Re´union, Zanzibar, between these morphotypes is necessary in order to ensure the cor- South Africa, and Madagascar (Chagos-Madagascar-South Africa rect taxonomy for comparative studies based on this common coral clade, CMSA). Clade 3 includes specimens from the east African species4. coast, the west coast of Madagascar and the Gulf of Aden (Gulf of Aden-Zanzibar-Madagascar clade, AGZM). Clade 4 includes the northwest Indian Ocean (Red Sea-Persian/Arabian Gulf and Results Kenya, RSPAGK). The genus Seriatopora formed a fifth clade We obtained COI sequences from 241 S. pistillata colonies collected embedded at the base of clades 1 and 2. from 34 locations across most of its range (Western Pacific Ocean, Intra-clade p-distance based on COI (Table S1) was considerably Indian Ocean, Red Sea and Persian/Arabian Gulf, Fig. 1). Interspe- low (clade 1: 0.00017 6 0.00009, clade 2: 0.00000 6 0.00000, clade 3: cific variation in COI sequences was one of the highest documented 0.00460 6 0.00193, and clade 4: 0.00017 6 0.00012). Higher intra- among scleractinian genera (sequence diversity (p-distance) 5 clade distance for clade 3 was due to the presence of specimens from 0.01245 6 0.00434), only surpassed by Porites (0.02686 6 0.0030) Yemen that made a separate sub-clade into clade 3 (Fig. 2A). Inter- and Siderastrea (0.0141 6 0.0027), which are globally distributed clade p-distance (Table S1) was always higher than intra-clade p- genera consisting of multiple morphologically divergent species distance and ranged from 0.01274 6 0.00436 (clade 3 vs clade 4) to (Fig. S1). High variation of the COI gene among these 3 genera sug- 0.01592 6 0.00492 (clade 1 vs clade 3), with the exception for clade 1 gests very early divergence of species within these genera5. Porites vs clade 2 (0.00378 6 0.00248) and this could be due to the recent and Siderastrea are extant in the Atlantic, Pacific and Indian Oceans, split between clade 1 and 2 compared to that between other clades whereas Stylophora is extinct in the Atlantic and only survived in the (Fig. 3). The results form p-distance analyses also suggested the Indo-Pacific after the Plio-Pleistocene5. presence of 4 clades of Stylophora (Table S1). Figure 1 | Map showing the locations from which Stylophora pistillata samples were obtained (shown as red dots). The division of four regions based on the four clades of Stylophora is marked in colours. Clade 1 - Pacific-Western Australia clade (Pink), Clade 2 - Chagos-Madagascar-South Africa clade (Green), Clade 3 - Gulf of Aden-Zanzibar-Madagascar clade (Blue) and Clade 4 - Red Sea-Persian/Arabian Gulf-Kenya clade (Brown). The samples were obtained from following locations; Japan (Kochi, Okinawa), Taiwan (Beitou, Penghu, Green Island, Kenting), Taiping Island, Tioman, Indonesia, Australia (Lord Howe, Lizard Island, Western Australia), New Caledonia, The Chagos Archipelago, Re Union, Mauritius (St. Brandon’s), Madagascar (North and South), South Africa, Kenya (Kanamai), Zanzibar, Yemen, Dijibouti, Saudi Arabia (Arabian Gulf and Gulf of Aqaba), Oman, Iran and Red Sea (Eilat). The dotted green lines represent hypothetical extension of clade 4 and clade 2 to Pakistan-North India (Gulf of Kachhch) and Laccadives (west coast of India), south India and Sri Lanka. Area covered by blue line in the inset map shows the distribution of Stylophora pistillata (modified from the map in www.coralgeographic.com). The maps were drawn using the software Magic Maps Ver. 1.4.3 and Adobe Illustrator CS5 (Macintosh version). SCIENTIFIC REPORTS