Proceedings of the 12th International Coral Reef Symposium, Cairns, Australia, 9-13 July 2012 17A Science in support of the Coral Triangle Initiative Genetic structure of Culcita sp. pincushion seastar in the Coral Triangle Nina Yasuda1, Coralie Taquet2, Satoshi Nagai3, Miguel Fortes4, Suharsono5, Handoko Adi Susanto6, Niphon Phongsuwan7 and Kazuo Nadaoka2 1University of Miyazaki, Faculty of Agriculture, Department of Marine Biology and Environmental Sciences, 2Tokyo Institute of Technology 3National Research Institute of Fisheries and Environment of Inland Sea (FEIS) Fisheries Research Agency 4Marine Science Institute CS University of the Philippines 5Indonesian Institute of Sciences (LIPI) 6Wildlife Conservation Society (WCS) 7 Marine and Coastal Biology and Ecology Unit Phuket Marine Biological Center Corresponding author: [email protected] Abstract. From the Coral Triangle, which is the global centre of marine biodiversity, species richness tends to decrease eastward across the Pacific Ocean and westward across the Indian Ocean. However, this region is severely threatened by both human and natural disturbances, calling for urgent conservation efforts. Conservation and management decisions inevitably require definition of the limits of population structure of each species as well as mechanisms that produce high biodiversity in the Coral Triangle. In this study we sequenced the mitochondrial COI region of the pincushion seastar, Culcita sp., a common coral reef seastar whose life history resembles other well-studied coral reef seastars. We found a large genetic difference between Indian (Culcita schmideliana) and Pacific Ocean (Culcita novaeguineae) lineages. All the populations (except from Thailand) had Pacific mtDNA haplotypes, while central and western Indonesian populations had both Indian and Pacific haplotypes, showing a sign of sympatric distribution of these two genetic lineages around these areas. Overall gene flow of Culcita sp. in the Coral Triangle was strong and resembled that of Acanthaster planci with similar life history traits. Further analysis using nuclear marker might reveal possible hybridization between the distinct genetic lineages of the species in this region. Key words: Coral Triangle, Culcita sp, mitochondrial DNA, CO1, speciation Introduction connectivity across the Indo-Pacific (e.g. (Uthicke The Coral Triangle, bounded by the Philippines, the and Benzie 2003) while Acanthaster planci showed Malay Peninsula, and Papua New Guinea, is the clear differentiation between Indian and Pacific global centre of marine biodiversity and species lineages in the Coral Triangle (Vogler et al. 2008; richness is known to decrease from this region Yasuda et al. 2011). Linckia laevigata also shows eastward across the Pacific Ocean and westward Indian and Pacific lineages (Williams 2000), however, across the Indian Ocean. However, this region is unlike A. planci two lineages of L .laevigata are severely threatened from both human and natural mixed in the Coral Triangle (Crandall et al.2008) disturbances, calling for urgent conservation efforts. Given these discrepancies, more studies on Because most marine species have pelagic larval echinoderm species with known larval ecology for dispersal, conservation management decisions comparative phylogeography will help to infer what inevitably require defining the limits of kinds of biological and ecological traits govern the population/species structure in the Coral Triangle. patterns of gene flow in in the Coral Triangle. Hence, phylogeographic and population genetic Culcita sp. is a good candidate for examining the analysis using mitochondrial DNA sequences are phylogeography in this area because we have regarded as one of the most powerful tools (Carpenter knowledge about larval ecology (Yamaguchi 1977) et al. 2011) towards these objectives. and it has similar ecological traits with well-studied A number of genetic studies were conducted using species A. planci, the notorious coral-eating seastar mitochondrial DNAin the Coral Triangle in the last that sometimes causes population outbreaks decade and some concordant phylogenetic breaks are (Birkeland and Lucas 1990). The habitat of Culcita sp identified in some taxa (Carpernter et al. 2011). is similar to that of A. planci, Both species have a However there is still lack of concordant phylogeny in similar summer spawning period (Yasuda et al. 2010, other marine invertebrates, especially in echinoderm Ohta et al. 2011) and similar larval ecology (16 days species. Holothuria nobilis showed broad for A. planci and 18 days for Culcita novaeguineae Proceedings of the 12th International Coral Reef Symposium, Cairns, Australia, 9-13 July 2012 17A Science in support of the Coral Triangle Initiative (Yamagushi 1977). The largest ecological difference NETWORK ver 4.6.1.0 with default setting. Pairwise between the two is that A. planci has higher fecundity comparisons between Culcita novaeguineae and sometimes causes population outbreaks while populations, Wright’s FST and an analogue of Culcita sp. does not (Birkeland and Lucas 1990). Wright’s FST (ΦST), which incorporates the model of In order to gain further insights on gene flow in the sequence evolution, were calculated only for Coral Triangle, we conducted a phylogeographic populations with N > 9 in ARLEQUIN. study of Culcita sp. using partial mitochondrial Cytochrome Oxidase subunit I (COI) region collected Results from the Coral Triangle as well as in its neighboring We obtained a 650 bp segment of mitochondrial COI countries including Thailand, Japan and Palau. in 273 individuals yielding 97 haplotypes with a total of 110 segregating sites. The basic polymorphism Material and Methods parameters for each population, including, haplotype We sampled a total of 273 individuals of Culcita sp. diversity (h) and nucleotide diversity (π) are provided by diving from 15 locations in Southeast Asia and in Table 1. Overall nucleotide diversity in Culcita sp. West Pacific regions collaborating with 5 different was 0.012 and corresponding haplotype diversity was countries between 2009 and 2011 (Fig.1). 0.880. The highest nucleotide diversity 0.03 was Abbreviations of sampling sites are shown in Table 1. found in KES, whereas the lowest value 0.0008 was There are two species in this region, Pacific species, found in CAL. The highest haplotype diversity 1.0 C. novaeguineae and Indian Ocean species, Culcita was found in THI and BOT and the lowest value schmideliana. In this paper, we use the term Culcita 0.464 was found in BOL. sp. including both two species because this study does Clade-B not include any detailed morphological analysis. All specimens were photo-vouchered and were returned to their original habitats after collecting a few tube 3 feet per individual. The tube feet were preserved in 39 2 micro tubes filled with 99.5% ethanol until they are used for genetic analysis. We amplified and Clade-C sequenced partial cytochrome c oxidase subunit I Clade-A gene (COI) from genomic DNA extracted using 5 % Chelex (Bio-rad, Hercules, CA) (Walsh et al. 1991) via polymerase chain reaction (PCR) using CO1EF AMM and CO1ER primers (Arndt et al. 1996) with thermocycling parameters of 35 cycles of 94 oC/30 o o sec, 45 C/30 sec, 72 C/30 sec. One microliter of BOL PCR product were cleaned using one unit of o exonuclease, and then incubated 37 C for 15 min and PAC Verde 80 oC for 15 min. Cleaned PCR products were sequenced on an ABI 3130xl automated sequencer THI PAU using BigDye ver.3 (Applied Biosystems, Foster DER BUN City,CA) terminator chemistry. Forward and Reverse BON sequences were proofread and subsequently aligned on GENETIX version 10 and then trimmed to 650 PAL base pairs. KES SPE We calculated haplotype diversity h and nucleotide diversity π (Nei 1987) with the program Arlequin ver. Figure 1: Haplotype network and geographic distribution of three 3.5. The null hypothesis of neutral evolution of the genetic clades marker was tested using Tajima’s D test (Tajima The median-joining network revealed three genetic 1989) and Fu’s Fs test (Fu 1997). Mismatch clusters of haplotypes (Fig. 1). The most distinct clade distribution and the model of sudden population C was diverged from clades A and B by more than 40 expansion (Rogers 1995) were also analyzed. We substitutions. Clade C was only found in Thailand and conducted all the sequence divergence and neutrality two sites in western and central Indonesia (KES and tests with 20,000 permutations as implemented in the SPE), corresponding to Culcita schmideliana Alrequin 3.5 (Excoffier and Lischer 2010). To sequences (Gustav Paulay unpublished data) while investigate the genetic structure of C. novaeguineae in clade A and B were distributed throughout central the Coral Triangle region, we first draw median- Indonesia and the Pacific populations, corresponding joining haplotype network (Bandelt et al. 1999) using Proceedings of the 12th International Coral Reef Symposium, Cairns, Australia, 9-13 July 2012 17A Science in support of the Coral Triangle Initiative to C. novaeguineae (Fig. 1, Table 1). Pairwise ΦST coastal marine species between the Oceans (Voris and FST values showed PAU and KES are 2000). significantly differentiated (p<0.05) from the other Neutrality tests showed either selection in form of populations in the Coral Triangle region (Table 2). genetic hitchhiking, or sudden population expansion Neutrality tests showed both Fu’s Fs (-78.33, p have occurred in recent history. Given the scenario of <0.0001) and Tajima’s D (-1.82, p <0.01). a population reduction due to sea level fluctuations, following population expansion might cause this Sampling sit es Abbr. Nind Nhaplo π h FS Tajima's D excess of low-frequency haplotypes. Similar Amami Ohshima AMM 5 4 0.0077 0.900 0.49 - 0.95 Verde Island Passage Anilao BAT 4 4 0.0041 1.000 - 1.41 - 0.21 population expansion is seen in other reef organisms Verde Island Passage Caban CAB 10 7 0.0030 0.867 - 3.35 - 1.69 such as reef fish and invertebrates in the Coral Verde Island Passage Calat agan CAL 4 2 0.0008 0.500 0.17 - 0.61 Verde Island Passage Maricaban MAR 6 5 0.0142 0.933 0.68 0.66 Triangle (Crandall et al.