Phuket mar. biol. Cent. Res. Bull. 67l.1,9 (2006)

PHYLOGENETIC ANALYSIS OF THAI () BASED ON PARTIAL SEQUENCES OF THE MITOCHONDRIAL 165TDNA GENE

Somchai Bussarawitl, Peter Gravlund2. Henrik Glenner2 andArne Redsted Rasmussen3

tPhuket Marine Biological Center, P.O. Box 60, Phuket, Thailand 2Department of Evolutionary Biology, Zoological Institute, ()niversity of Copenhagen, (Iniversitetsparken 15, DK-2 100, Copenhagen O, Denmark )The Royal Danish Academy of Fine Arts, School of Conservation, Esplanaden 34, DK-1263, Copenhagen K, Denmark

ABSTRACT: Ten oyster species of the family Ostreidae (Subfamilies Crassostreinae and Lophinae) from Thailand were studied using morphologic al data and mitochondrial 165 rDNA gene sequences. Additional sequence data from five specimens of Ostreidae and one specimen of Tridacna g4gas were downloaded from GenBank (7. gigas was used as outgroup). Some specimens were found to be genetically identical despite obvious morphological differences (e.g. four specimens of Crassostrea iredalei from east and west sides of the Malaysian peninsula and Saccostrea forskali and S. cf. malqbonensis, both from Thai waters). The results indicate that Striostrea mytiloides belongs to the genus ,Saccostrea. The results also suggest that none of the three subfamilies in Ostreidae is monophvletic.

INTRODUCTION postulated that the genus Crass ostrea is the only valid genus in this subfamily and that the genus Among molluscs, show great has few living species. Based on a phylogenetic plasticity of external morphology. This creates investigation of shell chambers and chomata in some problems with species identification, the family Ostreidae, Malchus (1998) doubted especially if identification is based solely upon shell Lawrence's (1 995) findings. morphology. To solve some of the taxonomic and Jozefowicz and O'Foighil (1998) used the phylogenetic problems, Harry (1985) analysed 165 rRNA gene of the southem hemisphere flat recent members ofthe superfamily Ostreacea using oysters and concluded that their results did not both shell and soft-part morphology. Harry (1985) support the separation of three subfamilies in arranged the family Ostreidae into three subfamilies: Ostreidae. Later O'Foighil and Taylor (2000) Lophinae, Crassostreinae and Ostreinae. studied the 28S nuclear rRNA genes and concluded Brock (1990) later used crossed immuno- that the family Ostreidae is robustly monophyletic electrophoresis to investigate the intergeneric but that the three subfamilies suggested by Harry distances between Ostrea, Crassostrea, and (1985) and supported by Littlewood's (1994) Saccostrea and indicated that Crassostrea and morphological analysis were paraphyletic. Saccostrea were valid genera. Harry's (1985) Oysters of the family Ostreidae are well findings were supported by Littlewood (1994) who represented in Thai waters, with about l5 made a cladistic analysis using 15 morphological recognized species (Klinbunga el a/., 2000). Nine characters selected from Harry's study (1985). of the most common species were redescribed

Littlewood (1994) also analysed 7 species of the and illustrated by Yoosukh and Duangdee ( 1 999). subfamily Crassostrinae using partial 28S nuclear Klinbunga et al. (2001) investigated the genera rRNAgene sequences and foundthat Crassostrea Crassostrea, Saccostreq and Striostrea from Thai q and S ac c o s tre w ere par aphyletic. Lawrence ( 1 995 ) waters using Randomly Amplified Polymorphic analysed the subfamily Crassostreinae and DNA data and found that the taxa fell into two Phuket mar. biol. Cent. Res. Bull.

groupings: one included specimens from the genera evidence. Sqccostrea and Striostrea, and another included specimens from the genus Cra,r,rostrea. 1. DNA Preparation and Amplification In order to shed light on the conflicting results DNA was extracted from the tissue of ofprevious studies, we collected fifteen specimens alcohol-preserved adult specimens using QiaAmp (fourteen specimens from Thai waters and one tissue kit protocol (QIAGEN Inc. Santa Clara, from the Sydney fish market, Australia) of California). Species, sample locations, and Ostreidae and identified them to species level using GenBank accession numbers are listed in Table 1. the available literature: Faustino (1932), Iredale and Mitochondrial 165 rDNA sequences from the 15 Roughley (1933), Iredale (1939), Thomson (1954), specimens were amplified using the primers: Torigoe (1981), Harry (1985), Oliver (1992) and I650A: 5'-ccg gcc gcc ata gcg tga ggg-'3 and Yoosukh and Duangdee (1999). Using Tridacna 16508: 5'-att agc ctg tta tcc ccg gcg-'3 (Banks gigas as outgroup we tried to test whether the et al. 1993). The double stranded (ds) PCR was species in the ingroup will be separated into the carried out as one initial cycle ofdenaturation (94 three recognised subfamilies Crassostreinae, oC for 2 minutes), followed by 30 cycles (94 "C Lophinae and Ostreinae, as suggested by the for 30 sec, 50 'C for 30 sec, 72'C for 40 sec) and morphological studies of Harry (1985) and finally one cycle (94 "C for 30 sec, 50 "C for 30 Littlewood (1994), or whether one of the sec. J2 oC for l0 min). subfamilies will be paraphyletic, as suggested by Primers and unincorporated nucleotides were the molecular studies of Littlewood (1994) and removed from PCR products using QIAquik PCR Jozefowicz and O'Foighil (1998). Additionally, we Purification kit (QIAGEN Inc. Santa Clara, compared the morphological species identifications California). For the cyclic sequencing (cs) reaction with the results of the molecular phylogenetic we used the same primers as for the initial direct analysis. sequencing (ds) PCR reaction. Cs PCR conditions Although the numbers of samples and were one initial cycle (96 "C for 2 min) followed markers sequenced are small in the present study by 35 cycles (96 "C for l0 sec, 52 "C for 20 sec, the work does represent the first attempt to 60 'C for 1 min). After cyclic sequencing the compare both morphological and phylogenetic data products were purified using ethanol precipitation. on oyster from Thai waters. Cycle sequencing was conducted using a Perkin Elmer/ABI Dye Terminator Cycle MATERIALS AND METHODS Sequencing Kit. The purified dsPCRproducts were used as templates (1 pl) for a l0 pl cyclic Unless otherwise mentioned, all samples sequencing (cs) reaction using ABI prismi DNA (both shells and soft parts) are deposited in the Sequencing Kit (dRhodamine Terminator Cycle Reference Collection at the Phuket Marine Sequencing Ready Reactions) and amplified on a Biological Center (PMBC). The outgroup consists Robocycler Gradient 96, Stratagenei (ref and of Tridacna gigas.The ingroup belongs to the three the sequences were visualized using a Perkin Elmer subfamilies, Crassostreinae, Lophinae and 377 Automated Sequenceri (Applied Biosystems Ostreinae, and consists of twenty specimens. Inc. Foster City, California). Sequence editing was Fourteen of these specimens were collected in performed using Sequencer (Gene Codes Thai waters and one, S. commercialis, from the Corporation. Inc. Ann Arbor, Michigan). Sydney fish market (cultured specimen) (Table l). Sequences were assembled and aligned in Five of the twenty sequences in the ingroup have CLUSTAL X version 1.8 (Thompson et al.1997) been taken from GenBank. These five sequences using default parameters (slow multiple alignment were identified and sequenced by Jozefowicz and option: gap opening penalty: 10; gap extension O'Foighil (1998). These twenty specimens penalty : 0.20; sequences with > 30o% divergence represent fifteen species based on morphological delayed (default). Phylogenetic analysis of Thai oyster

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2. Analysis of the molecular data Genetic divergences between species within All substitutions were equally weighed. The genera revealed differences ranging from 0.0% distance and parsimony analyses were performed (between the specimens identified as D. rosacea in PAUP* 4.0b10 Beta version (Swofford, 1998). and D. crenulifera) to ll .7o/o, in Dendrostrea (n: For the parsimony analysis we used the heuristic 5, 3-4 species), from 0.0% (between the search option, each with 1000 random-addition specimens identified as S. forskali and S. cf. heuristic searches. In all analyses, character states malabonensis) to 12.5o/o, in Saccostrea (n : 5, 4- were unordered and weighed equally and alignment 5 species), and in Crassostrea (n : 5, 2 species) gaps were coded as missing data. The most 5.3"/o (Table 3\. parsimonious topology was evaluated by Bremer support (Felsenstein, I 993; Bremer, 1995) by the 2. Phylogenetic analysis use ofthe TreeRot program version 2C (Sorensen, Parsimony methods seek solutions that 1999). minimize the amount of evolutionary change required to explain the data. Thus, estimates of RESUXTS the amount of evolutionary change implied by parsimony will underestimate the true amount of The sequence analyses were separated into change, unless the actual rate of change is two methods; pairwise distance between the extremely small (Swofford, 1998). The parsimony samples and parsimony to resolve the phylogenetic method will be used to indicate the phylogenetic relationships between the specimens in the study. relationship between the involved specimens. Six most parsimonious trees with a length of 1. Genetic divergence 396 steps and a consistency index of 0.717 was Distance methods measure the changes that found using PAUP. A strict consensus tree was have occurred between a pair of sequences since produced based on these six most parsimonius their divergence from a common ancestor; trees (Fig. l). Using the strict consensus tree two however it is important to remember that changes sister groups could be identified in the ingroup: at a particular site along a lineage ofthe phylogeny one group (Bremer Support 6) includingSaccostrea may mask earlier changes at that site, and parallel cucullatq as sister group to a trichotomy including or convergent changes may occur at the same site Striostrea mytilioides, Saccostrea echinata and a in different lineages. The distance tree is used to second group with Saccostrea commercialis as indicate when specimens are very close to each sister group to a clade with Saccostrea forskali other (e.g. same species) or represent a different and Saccostrea cf. malabonensis. The other group taxon (e.g. not the same species). (Bremer Support 1) was separated into two sister Pairwise comparisons of the genetic groups, one (Bremer Support 1l) with Crassostrea divergence were carried out between all belcheri as sister group to a clade including the investigated specimens and the sequences adapted four specimens of Crasso strea iredalei, and another from GenBank (Table 2). In three species more (Bremer Support 6) with a quadrotomy including: than one specimen were investigated. In l) Dendrostrea folium (Thai specimen), 2) cristagalli (n: 2) a divergence of 3.3% was Dendrostrea frons (GenBank) as sister group to a observed. These two specimens came from two clade including Ostrea denselamellosa (GenBank) geographically distant locations (Guam and and Ostreola conchaphila (GenBank), 3) Phuket). D endro s tre a fo lium (n : 2) differentiated Dendrostrea folium (GenBank) as sister group to by 7.60/o, came from Cook Islands, and Phuket. a clade including Dendostrea crenulifera and Crassostrea iredalei (n:4) exhibited no genetic Dendrostrea rosqcee) and 4'1 a clade including the variation. These specimens were sampled on both two specimens of Lopha cristagalli (one from side of the Thai-Malaysian peninsula. GenBank and one from Thai waters). Phylogenetic analysis of Thai oyster

DISCUSSION specimens represent two different species or even genera and is further supported by the results of 1. Related molecular studies the phylogenetic analysis (Fig. 1), where the two Schneider and O' Foighil (1999) investigated specimens do not appear to be monophyletic. The two genera (Hippopus and Tridacna) of giant clams morphological characters of the specimen from (Tridacninae) using a fragment of the 165 gene Phuket, Thailand correspond very well to the (a62bp). A pairwise comparison revealed that the description of Dendrostrea folium according to highest level of divergence within the subfamily Thomson (1954), Torigoe (1981), and Yoosukh was 15.3% (H. hippopus and T. squamosa'). and Durangdee (1999). The type locality of D. Intrageneric divergence between the two species folium is Amboina (Ambon, Indonesia), indicating of Hippopus wasT .2o/o and between the six species that the specimen from Cook Islands may of Tridacna the divergence varied between 4o/o and represent another species. Future studies must 12.2%. investigate this conflict. Jozefowicz and O'Foighil (1998) investigated Four specimens of Crassostrea iredalei a section ofthe 165 gene 451 bp long (this section showed no genetic variation despite the fact that includes the section investigated here) in flat two specimens were collected in the Gulf of oysters. Some of their results are included in table Thailand and two from the Andaman Sea (east and 1. They observed up Io 9.23oh interspecific west sides of the Thai-Malaysian peninsula, divergence within the non-monophyletic genus respectively). However, it should be noted that Ostrea. Three species came out monophyletic (O. some degree of morphological differentiation angasi, O. chilensis, and O. edulis) with an between the two regions was observed. interspecific divergence ranging between 0.67% The specimens identified as S. forskali and 5.4 o/o. (PMBC 19521) and S. cf. malabonensis (PMBC 19530) were found to be genetically identical 2. Species level despite obvious morphological differences. The When comparing the two specimens of morphological differences in the two specimens Lopha cristagalli from the two geographically could represent plasticity within a species as also distant localities: Guam (GenBank) and Phuket indicated by allozyme data RAPD and allozlmes (present study), the observed genetic distance is are notthe same (RAPD-analysis) (Klinbunga, pers. 3.3%. This is below the observed interspecific comm.) from Thailand. The holotype of S. variation found in Tridacna (4-12.2%) (Schneider malabonensis described by Faustino (1932) from and O'Foighil, 1999) but resembles the level the Philippines was rather cup-shaped and similar observed between species in the genus Ostrea to S. forskali. However, future genetic comparison (0.67-5.41%) (Jozefowicz and O'Foighil, 1998). between S. forskali from Thailand and S. Despite the relatively great variation observed mqlabonensis from the Philippines could reveal the between the two specimens of L. cristagalli, the validity of S. malabonensis. results may represent geographical variation within the species L. cristagalli. Thus more material of 3. Generic level L. cristagalli covering its geographical range is Using Tridacna gigas as outgroup needed to evaluate this question thoroughly. Dendrostrea is found not to be monophyletic (Fig. The two specimens of Dendrostrea folium l). This is in agreement with the molecular findings from Cook Islands (GenBank) and Phuket (present of Jozefowicz and O'Foighil (1998) and O'Foighil study) revealed a very high intraspecific and Taylor (2000), but in conflict with the divergence of 7.60/o even considering the large conclusions of the morphological analysis by Harry geographical distance. This high level is ( 1985). comparable to what is observed among species The Crassostrea specimens investigated are and even among some genera within this family found to be monophyletic and supported by a very (Table 2). This result indicate that the two high Bremer Support (BS : 1l). Littlewood(1994) Phuket mar. biol. Cent. Res. Bull.

Tridacna gigas (AF 1 2291 5) Saccostrea cuculata (PMBC 19524) Saccostrea echinata (PMBC 19527)

Saccostrea mytiloides (PMBC 1 9533)

Saccostrea commercialzs (PMBC 1 9536)

S ac c o s t re a fosfali (PMB C 19 521) Saccostrea malabonensjs (PMBC 19499) Crassastrea belcheri (PMBC 19507) Crassostrea iredalei (PMBC 19519) Crassostrea iredalei (PMBC 19516) Crassostrea iredalei (PMBC 19513) Crussostrea iredalei (PMBC 19510) D endt ostrea.folium (PMBC 19496)

Lopha cr ist agal I i (AF052066) Lopha cristagalli (PMBC 19493)

D endro s tre a fo I ium (AFO5 2069) Dendrostrea rosacea (PMBC 19499) Dendrostrea crenulifera (PMBC I 9500)

D e ndro s treay'ons (AF05 207 0)

O strea dens elamellosa (AF052067) Os treol a c onc haphil a (AF05207 I )

Figure 1. Most parsimonious phylogeny resulting from the PAUP analysis ofthe 165 rRNA data. Tree length : 260 steps, CI : 0.65. Branch support is estimated by Bremer Support over the branches of the topology. Numbers in brackets are collection reference numbers from Phuket Marine Biological Center (PMBC) or GenBankreference numbers (AF).

also investigated the relationships among following Asian oyster species: C. belcheri, C. crassostreinid oysters and found Crassostrea to gigas, C. rivularis, C. ariakensis, and C. iredalei be paraphyletic. However, Littlewood's Crassostrea (Littlewood, 1994; O'Foighil and Taylor 2000). species from Asia formed a monophyletic clade, Conceming the genus Saccostrea, the present whereas the Crassostrea species from the Atlantic study strongly supports (BS : 6) monophyly grouped together with species of Saccostrea from except for the inclusion of the specimen identified Asia (Littlewood, 1994). Also O'Foighil and Taylor as Str io stre a mytil o ides (PMBC 1 95 3 3 ). Again, the (2000) found a monophyletic clade of Asian studies of Littlewood (1994) suggested that the

Cr as s o s tre a species. This biogeographical pattem western species of Crassostrea grouped with the suggests that the Asian species of the genus Asian species of Saccostrea. However, this is not Crassostrea are monophyletic and indicates the supported by the findings of O' Foighil and Taylor existence of a new senus includins at least the (2000), which agree with the present study in Phylogenetic analysis of Thai oyster

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finding support for a clade of Asian Saccostrea ACKNOWLEDGEMENTS species. None of the two above-mentioned studies We wish to thank the PMBC and DANIDA includes Striostrea mytiloides, which is placed in this study through Scientific this study within the genus Sacco strea'The finding for supporting Progtamme, Dr. Tomas Cedhagen, that mytiloides belongs to the genus Saccostrea is Cooperation Marine Ecology' University of also suggested by Klinbvnga et a/. (2001) using Department of for his general supervision on the tropical RAPD data. The three sub-families Crassostreinae, Aarhus, and Dr. Mary E. Petersen, Lophinae and Ostreinae are not, in this study, oyster research Copenhagen' for proof monophyletic as proposed by the morphological Zoological Museum, draft manuscript. We also thank studies by Harry (1985), but polyphyletic as reading an early referees whose valuable suggestions suggested by the molecular studies by Littlewood anonymous the manuscript. Henrik Glenner was Og9o and Jozefowiczand O'Foighil (I998)' improved supported by the Danish National Science Research Council (No' 51-00-0457) and the Velux Research Foundation.

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Manuscript received: March 2005 Accepted: May 2005