Vol. 396: 211–219, 2009 MARINE ECOLOGY PROGRESS SERIES Published December 9 doi: 10.3354/meps08281 Mar Ecol Prog Ser

Contribution to the Theme Section ‘Marine biodiversity: current understanding and future research’ OPENPEN ACCESSCCESS

Genetic population structures of the blue laevigata and its gastropod ectoparasite crystallina

M. Kochzius1,*,**, C. Seidel1, 2, J. Hauschild1, 3, S. Kirchhoff1, P. Mester1, I. Meyer-Wachsmuth1, A. Nuryanto1, 4, J. Timm1

1Biotechnology and Molecular Genetics, FB2-UFT, University of Bremen, Leobenerstrasse UFT, 28359 Bremen, Germany 2Present address: Insitute of Biochemistry, University of Leipzig, Brüderstrasse 34, 04103 Leipzig, Germany 3Present address: Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit, Institut für Nutztiergenetik, Höltystrasse 10, 31535 Neustadt, Germany 4Present address: Faculty of Biology, Jenderal Soedirman University, Dr. Suparno Street, Purwokerto 53122, Indonesia

ABSTRACT: Comparative analyses of the genetic population structure of hosts and parasites can be useful to elucidate factors that influence dispersal, because common ecological and evolutionary processes can lead to congruent patterns. We studied the comparative genetic population structure based on partial sequences of the mitochondrial cytochrome oxidase I gene of the blue starfish and its gastropod ectoparasite in order to elucidate evolutionary processes in the Indo-Malay Archipelago. AMOVA revealed a low fixation index but significant φ genetic population structure ( ST = 0.03) in L. laevigata, whereas T. crystallina showed panmixing φ ( ST = 0.005). According to a hierarchical AMOVA, the populations of L. laevigata could be assigned to the following groups: (1) Eastern Indian Ocean, (2) central Indo-Malay Archipelago and (3) West- ern Pacific. This pattern of a genetic break in L. laevigata between the Indian and Pacific Ocean, con- gruent to studies on other marine in the Indo-Malay Archipelago, is likely due to allopatry caused by Pliocene and Pleistocene glacial sea level low stands.

KEY WORDS: COI · Coral Triangle · · Phylogeography · Population expansion · Southeast Asia

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INTRODUCTION Warén 1980, Janssen 1985), which penetrates the radial hemal and perihemal system of L. laevigata with Comparative analyses of the genetic population its proboscis to obtain nutrients (Egloff et al. 1988). structure of a host and its parasite can be used to eluci- T. crystallina seems to be co-distributed with its host, date factors that influence dispersal (Criscione 2008), ranging from the Western Indian Ocean across the because common ecological and evolutionary pro- Indo-Malay Archipelago and northeastern Australia to cesses can lead to congruent patterns (Bermingham & Samoa and Fiji (Sloan et al. 1979, Warén 1980). Infec- Moritz 1998, Avise 2000). The blue starfish Linckia tion rates of L. laevigata with T. crystallina vary among laevigata (Ophidiasteridea; Chao 1999) is widely dis- populations, ranging from 14.3% in Fiji (Egloff et al. tributed on Indo-Pacific coral reefs, from the Western 1988), 15.7% (Troncoso & Van Goethem 1998) and Indian Ocean across the Indo-Malay Archipelago to 22.3% (Bouillon & Jangoux 1984) in eastern New southeastern Polynesia (Yamaguchi 1977). It is fre- Guinea to 62.0% in the Moluccas (Elder 1979). quently parasitised by the obligate and strictly specific Here we compare the genetic population structure ectoparasitic gastropod Thyca crystallina (; of the host Linckia laevigata and its parasite Thyca

**Email: [email protected] © Inter-Research 2009 · www.int-res.com **All authors except M.K. and C.S. are in alphabetical order 212 Mar Ecol Prog Ser 396: 211–219, 2009

crystallina in the Indo-Malay Archipelago. This highly MATERIALS AND METHODS dynamic region in terms of biodiversity, ecology, geo- logy and oceanography is the centre of marine shal- Sampling. Tissue samples from 270 specimens of the low water biodiversity (Briggs 1999, Hoeksema 2007). blue starfish Linckia laevigata and 324 specimens of its Molecular phylogenetic studies have shown a break ectoparasite, the snail Thyca crystallina, were col- between the Indian and Pacific Oceans, e.g. in lion- lected during several field trips from 2004 to 2007 at fishes (Kochzius et al. 2003), clownfishes (Timm et al. 24 sample sites across the Indo-Malay Archipelago 2008), and damselfishes (Froukh & Kochzius 2008). (Fig. 1A,C, Table 1). Several colour morphs of L. laevi- Additionally, a growing number of population genetic gata are known, such as blue, orange, green, and studies in the Indo-Malay Archipelago show a com- violet. Since genetic studies indicated that colour plex pattern of divergent lineages and restricted gene variation is congruent to genetic variation (Williams & flow, e.g. in a clownfish (Timm & Kochzius 2008), Benzie 1998) and that species boundaries in Linckia giant clams (Kochzius & Nuryanto 2008, Nuryanto & are difficult to define (Williams 2000), only blue colour Kochzius 2009), and a mushroom coral (Knittweis et morphs were analysed. Tissue samples were preserved al. 2009). in >96% ethanol and later stored at 4°C. Plate tectonic movements in the Indo-Malay Archi- DNA extraction, amplification and sequencing. pelago and global fluctuation of sea level during multi- Extraction of genomic DNA from both species was ple Pliocene and Pleistocene glaciations are the pri- done with the Chelex method, following the protocol of mary hypothesised triggers for this genetic separation Walsh et al. (1991). A fragment of the mitochondrial of the 2 ocean basins. Most of the islands in the Indo- COI gene was used for both species as molecular Malay Archipelago did not exist or were not at their marker and amplified with the primers from Folmer et current position about 30 million years ago. During al. (1994). PCR was conducted in a volume of 50 µl and that time, water masses of the Pacific South Equatorial contained 2 µl DNA template, 10 mM Tris-HCl (pH 9),

Current (SEC) entered the Indian Ocean via the so- 50 mM KCl, 4 mM MgCl2, 0.4 µM of each primer, called Indonesian seaway. This current pattern started 0.2 mM dNTPs, 2 µl BSA (2 mg/ml) and 1 U Taq poly- to change about 25 million years ago, due to the devel- merase. The following temperature profile was used opment of Sulawesi by the amalgamation of several for the PCR: 94°C for 5 min, followed by 35 cycles of fragments and the northward movement of New 1 min at 94°C, 1.5 min at 45°C and 1 min at 72°C. Final Guinea, the Bird’s Head Peninsula, and Australia (Hall extension was conducted at 72°C for 5 min. The PCR 1998). The Indonesian seaway was finally closed about products were purified using the QIAquick spin col- 5 million years ago by the northward displacement of umn PCR purification kit (Qiagen), following the New Guinea (Cane & Molnar 2001). Since then, the manufacturer’s protocol. Sequencing was done using major exchange of water masses between the 2 oceans the DyeDeoxy terminator chemistry (PE Biosystem) has been facilitated by the Indonesian throughflow and an automated sequencer (ABI PRISM 310 and (ITF), which originates from the northern Pacific (Gor- 3100, Applied Biosystems). New primers were de- don & Fine 1996, Gordon 2005). signed for cycle sequencing in Linckia laevigata Additionally, multiple glaciations in the Pliocene and (Linckia Seq Frw [forward]: 5’-AAA ATC AGA ATA Pleistocene caused global fluctuations in sea level with AGT GCT GGA-3’; Linckia Seq Rev [reverse]: 5’-TTT low stands of up to 120 m below present sea level GGA GCT TGA GCT GGA ATG-3’) and Thyca crystal- (Krantz 1991, Rohling et al. 1998, Siddall et al. 2003). lina (Thyca Seq Frw [forward]: 5’-TAT TGT AAC TGC Shallow shelf areas such as the Sunda shelf were TCA TGC TTT TG-3’). exposed, and ocean basins were separated (Voris 2000; Genetic diversity. Sequences were edited with the Fig. 1). Molecular clock estimates support the view of programmes Sequence Navigator (version 1.0.1, allopatric speciation in separate ocean basins during Applied Biosystems) or Seqman (version 4.05, DNAS- the Pliocene and Pleistocene in some species (Kochzius tar). They were translated to amino acids with the et al. 2003, Timm et al. 2008). program Bioedit (version 7.0.9.0, Hall 1999) in order The present study aims to elucidate if common eco- to exclude mistakes in sequencing and to verify if a logical and evolutionary processes lead to congruence functional mitochondrial DNA sequence was obtained in the genetic structure of Linckia laevigata and Thyca and not a nuclear pseudogene. Multiple alignment crystallina populations from the Indo-Malay Archipel- was done using Clustal W (Thompson et al. 1994) as ago. The genetic marker used for both species in the implemented in the software Bioedit. Haplotype present study is the cytochrome c oxidase I gene (COI), diversity h (Nei 1987) and nucleotide diversity (Nei & which is suitable to investigate the genetic population Jin 1989) were calculated with the programme structure of (e.g. Harley et al. 2006) and Arlequin (http://cmpg.unibe.ch/software/arlequin3, snails (e.g. Reid et al. 2006). version 3.11; Excoffier et al. 2005). Kochzius et al.: Genetic structure of host and ectoparasite 213 Bk New Guinea SEC SSP NECC Mi Eddy Halmahera Eddy Sea Mindanao Banda LS ITF Sea Maluku

ITF Timor Ke TI Ma Se Bi Lu Ku ITF icated by the light grey area (Voris Sulawesi ). ITF: Indonesian throughflow; SEC: Sumba Do Sp Ko al to haplotype frequency. Small circles al to haplotype frequency. Sa BI ITF itochondrial cytochrome c oxidase I haplotypes. Ba Thyca crystallina Borneo Ka Java PS Sumatra N 0 C D Bk New Guinea SEC NB SSP Pi NECC Mi Eddy Halmahera indicate missing intermediate haplotypes Eddy Sea Banda Mindanao LS ITF Sea

Maluku imor

ITF Ce T Ke TI (C, D). (A, C) Indo-Malay Archipelago 1) as well oceanographic pat- with sample sites (for abbreviations see Table Ma Bi Lu Ku ITF Sulawesi Sumba Do Sp Ko BI Sa ITF KK Thyca crystallina Borneo Ka Linckia laevigata Java PS (A, B) and Sumatra Linckia laevigata AS N 0 A B Large circles represent haplotypes and connecting lines between them one mutational step. The size of the circles is proportion Southern Equatorial Current; NECC: Northern Equatorial Counter Current. Pleistocene maximum sea level low stand of 120 m is ind 2000). Pie charts represent the proportion of clades defined in the network at different sample sites. (B, D) Networks of m terns with dominant (solid lines) and seasonally changing (dashed currents (Wyrtki 1961, Gordon & Fine 1996, Gordon 2005 Fig. 1. 214 Mar Ecol Prog Ser 396: 211–219, 2009 ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 0.03 0.13 0.03 0.56 0.02 0.06 0.03 0.03 0.15 0.04 0.04 0.06 0.05 0.03 0.04 0.09 0.001; ***: 0.001; ***: ns ns ≥ ns ns ns ns ns ns ns ns ns ns ns ns ns ns –– SSD HRI 0.01 0.03 0.09 0.01 0.01 0.04 0.02 0.02 ), haplotype diversity ns ns ns ns ns ns ns ns ns Fs hp 0.20 1.39 –2.43 –1.99 –2.01 –0.75 –9.44*** 0.01 –4.69** 0.01 –3.86** 0.18*–5.04** 0.01 0.73* Fu’s Fu’s –11.2*** 0.01 –10.2*** 0.002 –10.1*** 0.003 –12.4*** 0.01 D 0.01; **: 0.01 > p 0.01; **: ns ns ns ns ns ns ns ns ns ns ns ns ns ns ≥ p ≥ (%) Tajima’s π h hp ––– – – – –––– – – – –––– – – – – – – – ––– –––– – – – – – – – – – 3 22 0.67 0.2 2 1.00 0.00 1.1 0.00 7 7 1.00 0.9 –0.61 1429 6 17 0.80 0.9523 0.6 0.9 1317 1.33 –0.72 0.91 921 2.821 0.89 13 –2.01*11 0.7 12 0.9314 –1.71 8 0.90 2.8 –0.57ns 1019 0.8 0.95 –3.00* –2.08** 0.95 1.1 12 –0.49 0.02 –2.18 3.9 0.94 –0.29 –1.83*40 0.9 –0.76 21 –1.39 19 0.9340 15 0.9 20 0.9710 –1.44 0.91 0.9 7 0.8 –0.76 0.93 –1.48 0.8 –0.61 ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns 1.11 0.04 0.02 0.07 0.03 0.11 0.06 0.06 0.31 0.05 0.11 0.02 0.18 0.03 0.06 0.02 0.07 0.02 0.01 0.08 0.06 ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ––– – –– ––––––– SSD HRI n N 0.37 0.03 0.01 0.02 0.05 0.10 0.02 0.09 0.02 0.03 0.01 0.05 0.01 0.04 0.02 0.03 0.01 0.03 ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns Fs 0.59 0.06 1.39 Fu’s Fu’s –1.00 –1.07 –1.04 –2.16 –1.16 –1.65 –2.26 –2.88 –1.62 –1.09 –1.13 –1.45 –2.10 –3.44* 0.05 –0.24 –3.12 –3.12 –4.30** 0.04 –11.88** 0.01 D ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns p < 0.001; ns: not significant, –: determined (%) Tajima’s π Linckia laevigata Thyca crystallina Genetic Neutrality tests Mismatch Genetic Neutrality tests Mismatch diversity distribution diversity distribution h . Sample sites in the Indo-Malay Archipelago, number of sequences (n), haplotypes (N , sum of square deviation (SSD) and Harpending’s raggedness index (HRI). *: 0.05 , sum of square deviation (SSD) and Harpending’s hp Fs , Fu’s , Fu’s D Thyca crystallina and ), Tajima’s ), Tajima’s π Linckia laevigata ), nucleotide diversity ( BaliDonggala, SulawesiKomodo Do Ba 11 – 6 0.84 – 0.8Pulau Seribu, Java – –0.89 KoSangalaki, Borneo – 9 PS 8 – Sa 0.97 9 11 1.8 8 – –0.50 9 0.97 0.96 2.3 – 1.8 0.67 –0.32 – 15 8 0.88 0.6 1.05 Misool, Moluccas Mi 16 13 0.97 2.1 –0.51 Pisang, New Guinea (NG) Pi 13 11 0.96 1.7 –0.14 Andaman Sea AS 3 3 1.00 1.4 0.00 Sample siteBira, SulawesiBanggi Islands, BorneoBiak, New GuineaCebu, Visayas Code BI nKarimunjava, Java 15 N BiKota Kinabalu, Borneo Bk 10 13 9Kupang, Timor 0.92 Ce KaLembeh Strait, Sulawesi KK 9 1.6 9Luwuk, Sulawesi 10 10 6Manado, Sulawesi 0.94 0.30 1.00 LS 8 9 1.5 1.8New Britain, Guinea 6 12 0.96 0.98 Ku 0.27 1.00 NB 0.10 10 1.7 1.9 Lu Ma 8 2.1 0.97 2 –0.40Sembilan, Sulawesi 0.38 13 9 –0.27 1.4Spermonde, Sulawesi 7 2Sebakor/Sanggala/Papisol, 10 –0.98 8 0.96New Guinea SSP 0.95 1.00 Islands, SulawesiTogian 0.97 0.9 Se 10 Sp 2.2 1.0 1.8 –0.33 52 – TI 9 0.44 0.00 –0.23 30 20 0.98 – 0.95 0.9 13 1.7 – –1.49 0.91 –1.05 1.7 – –0.31 – – – – 19 14 0.96 0.7 –1.29 Kendari, Sulawesi Ke 11 8 0.93 1.9 –0.55 h ( Table 1. Table Kochzius et al.: Genetic structure of host and ectoparasite 215

Historical demography. The null hypothesis of neu- lina. The sequences of these haplotypes are available tral evolution of the marker was tested using Tajima’s at the EMBL sequence database under the accession D-test (Tajima 1989) and Fu’s Fs-test (Fu 1997). Nega- numbers FN392698-FN392798 (L. laevigata) and tive Tajima’s D-values can indicate selection, but FN392799-FN392866 (T. crystallina). also population bottlenecks or population expansions Genetic diversity within each population of Linckia (Tajima 1989). The historical demography was ana- laevigata showed high levels of haplotype and nucleo- lysed by mismatch distribution (Schneider & Excoffier tide diversity, ranging from 0.84 in the population from 1999) of the sum of square deviation (Rogers & Harp- Donggala (Sulawesi) to 1.00 in Kota Kinabalu (Borneo), ending 1992) and Harpending’s raggedeness index New Britain, and Biak (both New Guinea). Nucleotide (Harpending 1994), thus testing the model of sudden diversity was between 0.8% in Donggala and 2.3% in population expansion (Rogers 1995). The mismatch Pulau Seribu (Java Sea) (Table 1). distribution, which is the distribution of the observed In Thyca crystalina, haplotype diversity was similar to differences between haplotypes, is multimodal in pop- Linckia laevigata, but nucleotide diversity was generally ulations under a demographic equilibrium and uni- lower in most populations. Haplotype diversity in T. crys- modal if a recent and fast demographic expansion of tallina ranged between 0.80 in Bira (Sulawesi) to 1.00 in the population has taken place. All tests were con- Karimunjava (Java Sea) and Manado (Sulawesi). Nu- ducted with 10 000 permutations as implemented in cleotide diversity was between 0.2 in Donggala and the programme Arlequin. 3.9% in Luwuk (Sulawesi) (Table 1). Genetic population structure and connectivity. Analysis of molecular variance (AMOVA; Excoffier φ et al. 1992) and pairwise ST-values were used to test for Historical demography significance of population structure. Both statistical cal- culations were carried out using the software Arlequin, The null hypothesis of neutral evolution of the COI applying the Tamura-Nei substitution model and marker in Linckia laevigata was not rejected for all gamma shape parameters of 0.3 (Linckia laevigata) and sample sites based on Tajima’s D-test, whereas the 0.2 (Thyca crystallina), based on the result obtained results of Fu’s Fs-test rejected the null hypothesis for 3 with the programmes PAUP (version 4.0b10; Swofford sites. In Thyca crystallina, Tajima’s D-test rejected the 1998) and Modeltest (version 3.7; Posada & Crandall null hypothesis of neutral evolution in 3 cases; Fu’s Fs- 1998). Several groupings of L. laevigata and T. crystalli- test rejected the null hypothesis in half of the cases. na populations were tested in a hierarchical AMOVA However, this could indicate population expansion in φ CT, considering the geography of the region. both species, which is supported by the mismatch Since the coverage of sample sites for the 2 species distribution analysis and Rogers’ test of sudden pop- was different, AMOVA was also conducted with ulation expansion, except in the population of T. crys- reduced data sets, containing only sites for which sam- tallina from Manado (Sulawesi) (Rogers 1995; Table 1). ples of both species were available. This was done in order to test if differences in the genetic population structure were due to unequal sampling. Based on an Genetic population structure and connectivity analysis with Modeltest, a gamma shape parameter of 0.2 and the Tamura-Nei substitution model were The evolutionary relationships among 101 Linckia lae- applied for both species. vigata haplotypes and 68 Thyca crystallina haplotypes Haplotype networks were calculated with the pro- are presented in networks, showing 2 clades in both gramme TCS (version 1.21; Clement et al. 2000). L. laevigata (Fig. 1B) and T. crystallina (Fig. 1D). The Clades were defined by the highest number of muta- distribution of clades across the Indo-Malay Archipelago tional steps found in a network. is presented in Fig. 1A, C, respectively. In Linckia laevigata, the 2 clades are separated by 6 mutational steps. The samples from the sites in the RESULTS Andaman Sea and in Timor consisted only of haplo- types from the black clade, whereas the samples from Genetic diversity New Britain and the southwestern coast of New Guinea were presented only by haplotypes of the Sequence alignments of the COI fragment of 473 and white clade. All other sites showed haplotypes from 401 bp lengths were obtained from 270 individuals of both clades, >75 % of them dominated by the black Linckia laevigata and 324 individuals of Thyca crystal- clade (Fig. 1A). AMOVA revealed a low fixation index, φ lina, respectively (Table 1). The observed number of but significant genetic population structure ( ST = 0.03, haplotypes was 101 in L. laevigata and 68 in T. crystal- p = 0.029) across the Indo-Malay Archipelago. How- 216 Mar Ecol Prog Ser 396: 211–219, 2009

ever, no significant genetic structure was found among tween selection and changes in population size. Demo- φ sites in the Spermonde Archipelago ( ST = 0.01, p = graphic growth was indicated by mismatch distribution φ 0.39). Pairwise ST-values between sample sites re- analysis and Rogers’ test of sudden population expan- vealed homogeneity among most of them; only 13% sion (Rogers & Harpending 1992, Rogers 1995). Since showed significant differences (data not shown). A hi- large shelf areas, such as the Sunda shelf between Java erarchical AMOVA with several groupings of sample and Borneo, fell dry during several Pliocene and Pleis- sites was carried out, but only the following rejected tocene glacial sea level low stands, the extent of coral φ the hypothesis of panmixing ( CT = 0.07, p = 0.003): (1) reef habitats in the Indo-Malay Archipelago and, con- Eastern Indian Ocean (Andaman Sea and Kupang), (2) sequently, population sizes were reduced (Fig. 1). Be- Western Pacific (Biak, New Britain) and (3) all remain- cause of rising sea level in interglacial periods, new ing sites in the central Indo-Malay Archipelago. habitats could be colonised, resulting in a demographic The 2 clades in Thyca crystallina were separated by and spatial population expansion. Signals of population 35 mutational steps (Fig. 1D). All sample sites were dom- expansion in the Indo-Malay Archipelago have also inated by the white clade, and haplotypes of the black been observed in another study on Linckia laevigata clade were only present at sample sites from the central and Thyca crystallina (Crandall et al. 2008a), as well as Indo-Malay Archipelago (Komodo, Donggala and giant clams (Kochzius & Nuryanto 2008, Nuryanto & Luwuk) (Fig. 1C). AMOVA revealed a very low and non- Kochzius 2009) and the clownfish Amphiprion ocellaris φ significant fixation index ( ST = 0.005, p = 0.24) across the (Timm & Kochzius 2008). Indo-Malay Archipelago. Similar to Linckia laevigata, φ most pairwise ST-values were not significant in T. crys- tallina and only 10% showed restricted gene flow (data Genetic population structure and connectivity not shown). A hierarchical AMOVA did not show any significant groupings of sample sites. Linckia laevigata showed a rather weak, but signifi- φ AMOVA of data sets reduced to sites for which sam- cant population structure ( ST = 0.03) across the Indo- ples of both species were available did not show signif- Malay Archipelago. However, analysis of the reduced φ φ icant population structures for Linckia laevigata ( ST = data set indicated panmixing ( ST = 0.02). This is prob- φ 0.02, p = 0.09) or Thyca crystallina ( ST = 0.002, p = 0.33). ably due to the fact that populations from the margins of the study area, such as the Eastern Indian Ocean, Eastern New Guinea and the Philippines, have been DISCUSSION removed. This indicates that there are high levels of gene flow in the central part of the Indo-Malay Archi- Genetic diversity pelago and that populations at the margin are less well connected to the centre. Several studies based on Most populations of the starfish Linckia laevigata and allozyme data also showed low levels of genetic het- its ectoparasite Thyca crystallina showed high levels of erogeneity in L. laevigata on different geographic haplotype diversity. These values are comparable to scales, ranging from panmixing (Williams & Benzie other studies on invertebrate species in the Indo-Malay 1993, 1996) to significant but shallow genetic struc- Archipelago using COI as a marker, such as the giant tures (Williams & Benzie 1998, Magsino et al. 2000, clams Tridacna crocea (DeBoer et al. 2008, Kochzius & Juinio-Meñez et al. 2003). On large geographic scales Nuryanto 2008) and Tridacna maxima (Nuryanto & in the Indo-West Pacific, PCR-restriction fragment Kochzius 2009), as well as the mantis shrimp Hap- length polymorphism analysis of mitochondrial DNA tosquilla pulchella (Barber et al. 2002). Nucelotide revealed a more prominent genetic population struc- diversity was generally >1% in L. laevigata, but <1% in ture in L. laevigata (Williams & Benzie 1997, 1998) T. crystallina, a pattern also observed in another study compared to the present study. Other invertebrate spe- (Crandall et al. 2008a). Levels of nucleotide diversity cies, such as giant clams (DeBoer et al. 2008, Kochzius <1% were also detected in giant clams (DeBoer et al. & Nuryanto 2008, Nuryanto & Kochzius 2009) and 2008, Kochzius & Nuryanto 2008, Nuryanto & Kochzius mantis shrimp (Barber et al. 2002), showed much φ 2009) and mantis shrimp (Barber et al. 2002). higher ST-values for the COI marker than L. laevigata in the present study. The less prominent genetic popu- lation structure in L. laevigata could be due to substan- Historical demography tial gene flow resulting from its high dispersal poten- tial (pelagic larval duration [PLD] = 22 d; Yamaguchi The null hypothesis of neutral evolution was rejected 1973). In contrast, the PLD for giant clams is only 9 d by Tajima’s D and Fu’s Fs neutrality tests at some sam- (Lucas 1988). A much stronger genetic population ple sites (Table 1), but these tests cannot distinguish be- structure was also revealed based on other genetic Kochzius et al.: Genetic structure of host and ectoparasite 217

φ markers in clownfish ( ST = 0.24; Timm & Kochzius the Indo-Malay Archipelago. This species produces in 2008) with a PLD of 8 to 12 d (Fautin & Allen 1994), and extended breeding periods probably planktonic larvae φ a mushroom coral ( ST = 0.26; Knittweis et al. 2009) (Elder 1979) with a high dispersal potential, which is with a PLD of 3 d (Abe 1937). supported by the shallow genetic population structure. The significant genetic structure was more pro- This lack of a genetic break between the Indian and φ nounced in the hierarchical analysis ( CT = 0.07) with Pacific Oceans has also been shown for other species, the following grouping: (1) Eastern Indian Ocean, such as the bigeye tuna Thunnus obesus (Alvarado (2) central Indo-Malay Archipelago and (3) Western Pa- Bremer et al. 1998, Chow et al. 2000), the swordfish cific. This pattern of a discontinuity between the Indian Xiphias gladius (Chow et al. 1997), the tasslefish and Pacific Oceans was more or less similar in the giant Polynemus sheridani (Chenoweth & Hughes 2003), the clams Tridacna crocea (DeBoer et al. 2008, Kochzius & snails Echinolittorina reticulata (Reid et al. 2006) and Nuryanto 2008) and Tridacna maxima (Nuryanto & Nerita plicata (Crandall et al. 2008b), as well as the sea Kochzius 2009), the clownfish Amphiprion ocellaris urchin Diadema savignyi (Lessios et al. 2001). How- (Timm & Kochzius 2008) and the mushroom coral He- ever, mechanisms that lead to genetic homogeneity liofungia actiniformis (Knittweis et al. 2009). In another across the Indo-Malay Archipelago are expected to be study (Crandall et al. 2008a) on Linckia laevigata, a ge- different among species. netic break between grouped sites from the (1) Eastern Even though no significant genetic population struc- Indian Ocean and (2) the central Indo-Malay Archipel- ture was observed, Thyca crystallina showed 2 highly ago, as well as the Western Pacific, showed the same divergent clades that were separated by 35 mutational φ CT-value as the present analysis. Earlier large-scale steps (Fig. 1D). This deep divergence might indicate a studies using COI sequences also detected a genetic cryptic species, but this hypothesis should be verified differentiation between the 2 ocean basins (Williams in an integrative approach, including 2000, Williams et al. 2002). Such a break was also de- genetic, morphological and ecological data. Such an tected in the crown-of-thorns starfish Acanthaster approach revealed, for instance, a new species of giant planci based on allozymes (Benzie 1999) and COI se- clam Tridacna costata in the Red Sea (Richter et al. quences (Vogler et al. 2008). 2008). Similar deep divergences have been observed This genetic break between the 2 ocean basins was in the giant clams Tridacna crocea (DeBoer et al. 2008, also apparent in the distribution of the black and white Kochzius & Nuryanto 2008) and Tridacna maxima clades in Linckia laevigata. Two populations in the In- (Nuryanto & Kochzius 2009) across the Indo-Malay dian Ocean (Andaman Sea and Kupang) harboured only Archipelago. It is possible that these 2 divergent clades individuals carrying haplotypes of the black clade, in T. crystallina were affiliated to the Indian and Pacific whereas the populations in the Western Pacific either Oceans, respectively, caused by sea level low stands. only consisted of white clade haplotypes (New Britain) or After the rise in sea level, the 2 clades came into con- were dominated by them (Biak and Cebu). These clades tact again and the black clade was replaced by the showed a strong mixing in the central Indo-Malay Arch- white clade. Even though Linckia laevigata and Thyca ipelago, but the black clade showed a higher frequency crystallina are codistributed due to a host–parasite in most of the populations (Fig. 1). In contrast to other relationship, they showed differences in their genetic species that show a main route of dispersal from the population structure. This is probably caused by differ- Western Pacific into the Eastern Indian Ocean along the ences in their mode of reproduction, showing that it is path of the ITF (Kochzius & Nuryanto 2008, Timm & not only common ecological and evolutionary pro- Kochzius 2008, Knittweis et al. 2009, Nuryanto & Kochz- cesses that are important in shaping the genetic popu- ius 2009), L. laevigata seems to enter the Western Pacific lation structure of the 2 species. from the Eastern Indian Ocean against the ITF. This pat- tern can also be observed in other studies on L. laevigata Acknowledgements. We thank the institutions and individuals (Williams et al. 2002, Crandall et al. 2008a). Dispersal that have made this study possible: German Federal Ministry of against the strong ITF that transports up to 19 million m3 Education and Research (BMBF, grant nos. 03F0390B and of water per second from the Pacific to the Indian Oceans 03F0427B), which funded this study in the framework of the (Gordon & Fine 1996, Gordon 2005) might be facilitated joint German-Indonesian project SPICE (Science for the Protec- tion of Indonesian Coastal Marine Ecosystems); German by seasonally changing currents in Makassar Strait Academic Exchange Service (DAAD) for supporting A.N.; along the coast of Borneo, which are directed to the north Centre for Tropical Marine Ecology (Bremen, Germany) for in October (Wyrtki 1961; Fig. 1). This coincides with the project coordination, especially C. Richter; GEO Magazine spawning period of L. laevigata in October on Heron Is- (Hamburg, Germany) for financing the research to Misool and New Guinea; G. Heiss and U. Krumme for providing samples; land, Great Barrier Reef (Laxton 1974). colleagues from Universitas Hasanuddin (Makassar, Indonesia) Neither an AMOVA nor hierarchical AMOVA indi- for logistical support in Spermonde, especially J. Jompa; and cated restricted gene flow in Thyca crystallina across anonymous reviewers for constructive comments. The SPICE 218 Mar Ecol Prog Ser 396: 211–219, 2009

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Submitted: May 18, 2009; Accepted: August 24, 2009 Proofs received from author(s): November 29, 2009