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Biological Journal of the Linnean Society, 2013, 108, 821833. With 2 gures

Very ne-scale population genetic structure of sympatric asterinid sea stars with benthic and pelagic larvae: inuence of mating system and dispersal potential
SERGIO S. BARBOSA1*, SELMA O. KLANTEN1, JONATHAN B. PURITZ2, ROBERT J. TOONEN2 and MARIA BYRNE1,3
1 2

School of Medical Science, University of Sydney, Sydney, NSW 2006, Australia Hawaii Institute of Marine Biology, University of Hawaii at Ma noa, Kaneohe, HI 96744, USA 3 Schools of Medical and Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia
Received 17 August 2012; revised 14 October 2012; accepted for publication 14 October 2012

The present study investigated the ne-scale population genetic structure of sympatric asterinid sea stars with contrasting modes of larval development (benthic versus pelagic). Parvulastra exigua lacks a dispersive life phase yet is one of the worlds most widely distributed and abundant sea stars, whereas Meridiastra calcar, a sea star with a dispersive larva, has a more limited regional scale distribution. Populations of P. exigua sampled from tide pools on three adjacent headlands showed signicant genetic substructure (mitochodrial DNA control region) at ne spatial scales (tide pools < 300 m apart: FST = 0.249, P < 0.01; headlands 515 km apart: FST = 0.125, P = 0.04). As expected, M. calcar populations sampled from the same headlands did not exhibit signicant genetic structuring (FST = 0.029, P = 0.14). The life-history traits of P. exigua, a mixed mating system (selng + outcrossing), pseudocopulation among closely-related conspecics, and an entirely benthic life cycle with a philopatric larva, undoubtedly inuence its strong genetic structure across ne spatial scales. Localized genetic structure, especially at the very ne-scale of tide pools, would not be detected in the more typical regional scale approaches adopted by most studies of marine invertebrate populations. 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 821833.

ADDITIONAL KEYWORDS: echinoderms life history Meridiastra calcar Parvulastra exigua.

INTRODUCTION
Life-history mode is a major determinant of population genetic structure in marine invertebrates, with species lacking a dispersive phase generally having greater genetic subdivision than closely-related species with a pelagic larva (Hedgecock, 1986; Hunt, 1993; Grosberg & Cunningham, 2001; Sherman, Hunt & Ayre, 2008; Shanks, 2009). Relative to species lacking such larvae, the possession of a planktonic larva facilitates genetic connectivity between populations, with habitat availability, oceanography, and historic demography also playing inuential roles

*Corresponding author. E-mail: sergio@anatomy.usyd.edu

(Roughgarden, Gaines & Possingham, 1988; Sotka et al., 2004; Banks, Piggott & Williamson, 2007; Weersing & Toonen, 2009; Hart & Marko, 2010; White et al., 2010; Marko & Hart, 2011; Puritz & Toonen, 2011; Selkoe & Toonen, 2011). Although pelagic dispersers are expected to have broader geographical distributions than holobenthic species, some species that lack a dispersive larva are among the most widely distributed marine invertebrates, including brooding and egg-laying molluscs (e.g. Lasaea, Littorina) and echinoderms (e.g. Amphipholis) (Johannesson, 1988; OFoighil, 1989; Sponer & Roy, 2002). Many invertebrates with benthic development are simultaneous hermaphrodites with the potential for self-fertilization and self-seeding of populations (e.g. Amphipholis squamata: Sponer & Roy, 2002;

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S. S. BARBOSA ET AL. 1001000 km+) spanning oceanic (Waters & Roy, 2004; Hart et al., 2006), regional (Colgan et al., 2005; Sherman et al., 2008; Ayre et al., 2009), and intraregional distances (Hunt, 1993; Ayre et al., 2009). Given its unusual life-history traits, mixed mating system (selng + outcrossing), pseudocopulation among closely-related conspecics (inbreeding), and an entirely benthic life cycle with a philopatric larva (Barbosa et al., 2012), we hypothesized that P. exigua exhibits genetic structure at the spatial scale of tide pools (< 300 m apart) and of adjacent headlands (515 km apart). A recent study of the tide pool and headland populations investigated in the present study shows that individual P. exigua can give rise to juveniles in isolation, indicating the potential to selfseed populations, although this selng ability varied among populations (Barbosa et al., 2012). We sampled populations of P. exigua from tide pools on adjacent headlands and sequenced the mitochondrial (mt)DNA control region to determine whether the life history of this species contributes to population structure at two levels of ne spatial scales. As a comparison with respect to larval type (benthic versus planktonic) and mating system (fertilization of benthic egg masses versus free spawning) (Byrne, 2006), we also determined the population genetic structure of M. calcar populations from the same headlands. This comparison allowed us to determine whether local oceanography and headland topology might inuence population genetic structure. We used the extreme divergence in life-history modes of P. exigua and M. calcar to examine whether these differences are reected in very nescale population genetic structure. Using this approach, we compliment previous larger-scale studies of these species (Hunt, 1993; Waters & Roy, 2004; Colgan et al., 2005; Sherman et al., 2008; Ayre et al., 2009). It was predicted that, despite its almost pan southern hemisphere distribution, populations of P. exigua would be highly structured at a local scale. Together with the parallel investigation of sympatric M. calcar populations, the present study provides insights into the forces shaping the population genetic structure of P. exigua, one of the worlds most widespread sea stars and considers the importance of life history and sampling scale in understanding the genetic structure of marine invertebrate populations.

Parvulastra exigua: Barbosa et al., 2012). Given that isolated individuals can give rise to progeny and so have the potential to establish new populations (Sponer & Roy, 2002; Barbosa et al., 2012), founder effects and inbreeding may also contribute to genetic structure (Avise, 2000; Yund & ONeil, 2000). Studies of the population genetic structure of sexually reproducing species that lack a dispersal stage frequently report signicant genetic structure at ne spatial scales, from metres to < 10 km (Johannesson, 1988; Kyle & Boulding, 2000; Goldson, Hughes & Gliddon, 2001; Sokolova & Boulding, 2004; Grahame, Wilding & Butlin, 2006; Panova, Hollander & Johannesson, 2006). Such ne-scale genetic structure is suggested to reect life-history mode, planktonic larval duration, and local adaptation to different habitats (Grahame et al., 2006). Thus, it is important to consider life history and habitat in the geographical scale of investigations of genetic structure and connectivity of marine populations (Sotka et al., 2004; Miller, Maynard & Mundy, 2009; Pelc, Warner & Gaines, 2009; Selkoe et al., 2010). Along the coast of Australia, the great diversity of asterinid sea stars with divergent modes of development provides a unique opportunity to investigate the inuence of life-history mode on population genetic structure (Byrne, 2006). The population genetics of two species, P. exigua (Lamark, 1816; nondispersive larvae) and Meridiastra calcar (Lamark, 1816; dispersive larvae), have been investigated in several studies that show high and low genetic structure, respectively, for these species, reecting their contrasting life-history modes (Hunt, 1993; Colgan et al., 2005; Sherman et al., 2008; Ayre, Minchinton & Perrin, 2009). In eastern Australia, P. exigua and M. calcar co-occur within metres on the same rock platforms, and sometimes even in the same tide pool. Parvulastra exigua, an egg-laying species with a benthic larva (Byrne, 1992; Barbosa et al., 2012) is one of the worlds most widespread sea stars occurring from southern Australia to South Africa (Dartnall, 1971; Dartnall et al., 2003; Waters & Roy, 2004; Hart et al., 2006). Populations in Australia and South Africa are taxonomically consistent with P. exigua (Dartnall et al., 2003). This species has a signicantly greater distribution than M. calcar, an endemic species occurring in south-east Australia that has a pelagic larval stage (Dartnall, 1971; Byrne, 2006). For P. exigua, rafting of reproductive adults by west wind drift, along the subpolar gyre, may serve as an important mechanism for dispersal, as rst suggested by Fell (1962). The extensive distribution of P. exigua in the absence of a pelagic phase has been the focus of several studies of population genetic structure in this species across large spatial scales (approximately

MATERIAL AND METHODS SPECIMEN COLLECTION

Specimens of P. exigua were sampled from intertidal rock platforms at three adjacent headland sites

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GENETIC STRUCTURE OF SYMPATRIC ASTERINIDS

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Figure 1. Sampling locations of the three adjacent headlands (Cape Banks, Kurnell, and Bundeena), and the three tide pool populations (sites) sampled within each headland.

separated by 515 km: Cape Banks (335955S 1511453E) (N = 3 tide pools), Kurnell (340000S 1511319E) (N = 3 tide pools), and Bundeena (340440S 1510955E) (N = 3 tide pools), New South Wales (Fig. 1; coordinates shown in Table 1). A total of 270 P. exigua were sampled, consisting of 30 individuals from each of three tide pools within each headland (Fig. 1). Tide pools were separated by no more than 300 m. Meridiastra calcar was also collected from the three headlands (N = 46 per headland). This species forms aggregations across the rock platforms at these sites and so was not sampled at the tide pool scale. Both sea stars occur in distinct strata on the shore in a specic habitat (Stevenson, 1992) and, as much as possible, all samples for each species were collected from the same habitat, reducing the potential that habitat preference might inuence genetic structure. A nonlethal tissue collection technique was used for both species, involving the removal of a small number of tube feet (ve to ten) using forceps. Tissue samples were preserved and stored in 70% ethanol. All animals were returned to their site of origin after sampling.

DNA

EXTRACTION AND POLYMERASE CHAIN REACTION

(PCR)

REACTIONS

Total DNA was extracted from tissues using a standard salt technique sensu Meeker et al. (2007). For P. exigua, the forward and reverse primers used were E12Sa (5-ACACATCGCCCGTCACTCTC-3) and 16Sr (5-CGGTACCCCTGCTTAAAGGT-3), respectively. For M. calcar, the forward and reverse primers used were Cal_12sL1 (5-GGAAATGCGCCTGGTACTAA-3) and Cal_16sR2 (5-AAAGGCAATACCCCTGCTTT-3), respectively (Smith et al., 1993; Williams & Benzie, 1997; Evans, White & Ward, 1998; Waters, OLoughlin & Roy, 2004). These primers were used to amplify an approximately 1000-bp fragment that includes the 3 end of 12S rRNA, tRNAGlu, and tRNAThr, the putative control region, and the 5 end of 16S rRNA. Each 20-ml PCR reaction volume contained 1 ml of 2 ng ml-1 template DNA, 10ml of BioMix Red (Bioline USA), 0.4 ml of 10 mmol l-1 forward and reverse primers, and 8.2 ml of milliQ water. Amplications followed the same basic cycling protocol: an initial denaturing step of 3 min at 95 C, followed by 35 cycles of 95 C for 30 s, an annealing temperature

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S. S. BARBOSA ET AL.

Table 1. Population diversity measures for all Parvulastra exigua tide pool populations and Meridiastra calcar headland populations; indicating the number of individuals (N), total number of haplotypes (Nh) with private haplotypes (u) in parenthesis, and the percentage of private haplotypes (% u) Population Parvulastra exigua Cape Banks Tide pool Coordinates N Nh (u) %u h p

Kurnell

Bundeena

All locations Meridiastra calcar Cape Banks Kurnell Bundeena All locations

1 2 3 Mean 1 2 3 Mean 1 2 3 Mean Mean N/A N/A N/A Mean

335955S 1511453E 335952S 1511445E 340000S 1511432E 340000S 1511319E 340012S 1511337E 340040S 1511350E 340440S 1510955E 340430S 1511006E 340434S 1511009E

29 26 26 81 29 29 27 85 25 29 28 82 248 46 46 46 138

4 4 3 6 5 6 3 8 7 3 3 8 20 14 16 19 37

(1) (1) (1) (3) (1) (2) (1) (4) (5) (1) (1) (7) (14) (8) (10) (12) (30)

25.0 25.0 33.3 50.0 20.0 33.2 33.3 50.0 71.4 33.3 33.3 87.0 70 57 63 63 81

0.613 0.649 0.676 0.646 0.421 0.320 0.552 0.431 0.693 0.197 0.404 0.431 0.650 0.857 0.846 0.872 0.865

0.00487 0.00505 0.00563 0.00518 0.00191 0.00242 0.00422 0.00285 0.00212 0.00036 0.00081 0.00109 0.00459 0.00412 0.00315 0.00488 0.00724

335952S 1511445E 340012S 1511325E 340434S 1511009E

Haplotype (h) and nucleotide (p) diversity indices are shown. Coordinates for all locations are also provided.

of 54 C for 30 s, and 30 s at 72 C, with a nal extension of 10 min at 72 C. PCR products were checked visually on 1% agarose gels and puried using the standard protocol of Exosap-it (USB Corporation). PCR products were sequenced directly using the E12Sa primer on an ABI 3730 Automated Sequencer. Sequences were aligned and edited manually using GENEIOUS PRO, version 5.1.6. (Drummond et al., 2010). After an initial alignment, sequences were trimmed resulting in 569-bp (P. exigua) and 622-bp (M. calcar) fragments, including gaps, which were treated as point mutations during the analysis.

POPULATION

STRUCTURE

Haplotype networks were generated for both species using the median-joining algorithm of NETWORK, version 4.5.1.6 (http://www.uxus-engineering.com; Bandelt, Forster & Rhl, 1999), and statistical parsimony in TCS, version 1.21 (Clement, Posada & Crandall, 2000). Because both methods generated the same haplotype network, only one is shown (medianjoining). DNASP, version 4.5 (Rozas et al., 2003) was used to estimate measures of intra-population variability, such as numbers of haplotypes (Nh), haploypte diversity (h; Nei, 1987), and nucleotide diversity (p; Tajima, 1983) for both species. In addition, two neu-

trality tests, Tajimas D and Fu and Lis F, were undertaken, using DNASP, version 4.5, to test for departures from demographic equilibrium and neutral mtDNA sequence variation in allelic diversity relative to the number of segregating sites (Tajima, 1989) or the number of alleles (Fu & Li, 1993). If these test statistics are signicantly negative, the populations under question may have undergone expansion or positive selection, whereas a signicantly positive value may indicate a population contraction or balancing selection (Wares, 2010). Fixation indices (F-statistics; Weir & Cockerham, 1984) were calculated using ARLEQUIN, version 3.01 (Excoffier, Laval & Schneider, 2005) to estimate the genetic structure among and within pairs of P. exigua tide pool populations and among M. calcar headland populations. A sequential Bonferroni correction (Rice, 1989) was used to determine signicance levels for pairwise FST tests after applying 10 000 permutations. For P. exigua, genetic partitioning of the total haplotype variance was estimated using a hierarchical analysis of molecular variance (AMOVA) (Excofer, Smouse & Quattro, 1992) as implemented in ARLEQUIN. To identify measures of genetic structure (f) that were signicantly different from zero, 15 000 permutations of the data were used. Genetic variance was partitioned into differences between headland locations (fCT), among tide pool populations

2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 821833

GENETIC STRUCTURE OF SYMPATRIC ASTERINIDS within each headland location (fST), and among individuals within tide pool populations (fSC). The Akaike information criterion in JMODELTEST, version 0.1.1 (Posada, 2008) was used to select the Tamura and Nei corrected distances model (Tamura & Nei, 1993) with a gamma correction of 0.01 for P. exigua. To infer migration rates and effective population sizes for both species, coalescence analyses were carried out using the Bayesian method in both MIGRATE-N, version 3.1.6.2 (Beerle & Felsenstein, 2001; Beerli, 2006) and IMA (Hey & Nielsen, 2007; Hart & Marko, 2010). However, the mtDNA data did not converge with either method, despite several heated Markov chain Monte Carlo searches of millions of generations each. For P. exigua, Mantels tests were used in IBDWS, version 3.16 (Jensen, Bohonak & Kelley, 2005) to characterize isolation-by-distance as the correlation of genetic distances (FST/1 FST) with log-transformed geographical distances (Rousset, 1997). Isolation-bydistance was tested for all pairs of populations, and 10 000 permutations were executed for each analysis.

825

RESULTS
PARVULASTRA
EXIGUA

The haplotype network revealed that most P. exigua individuals (79%) shared one of two haplotypes that differed from each other by four substitutions (Fig. 2). The most common haplotype, from a total of 20 identied haplotypes, occurred in 127 individuals from all three locations and all populations within each location. The second most common haplotype was only present in the 68 individuals from Cape Banks (i.e. all three tide pool populations) and Kurnell (two of the three tide pool populations) (Fig. 2). Private haplotypes made up 70% of the total number of haplotypes (Table 1). Three out of the six haplotypes recorded at

Cape Banks were both unique to this headland and to each of the tide pool populations. For Kurnell, six private haplotypes out of nine were evident, with four of the six belonging to a single tide pool population. Bundeena contained the highest number of private haplotypes, with eight out of nine in total, and ve of these private haplotypes belonged to a single tide pool population. Haplotype diversities (h) ranged from 0.190.69 with a mean of 0.65, and nucleotide diversities (p) ranged from 0.00030.0056 with a mean of 0.0045 (Table 1). The two neutrality analyses were not signicantly different from zero for any of the tide pool populations within the three locations, suggesting neutral or random population expansion (Tajimas D = -0.86, range = -0.10 to -2.44; Fu & Lis F = -0.68, range = +0.85 to -2.97: Table S1). Population pairwise FST comparisons revealed signicant levels of genetic subdivision between tide pool populations (FST ranges from 0.095 to 0.733; 75% of comparisons; Table 2). High levels of structure were observed between all three headlands, as well as between rock pool populations within the Kurnell (FST ranges from 0.095 to 0.618; Table 2) and Bundeena (FST ranges from 0.113 to 0.259; Table 2) headlands. No signicant structure was observed within the Cape Banks headland (FST ranges from -0.023 to 0.017; Table 2). AMOVA revealed signicant levels of genetic partitioning among tide pool populations within the three locations (FST = 0.249, P < 0.01), accounting for 25 % of the total variation. The AMOVA in which tide pool populations were partitioned into the three locations (Table 3) also demonstrated signicant genetic structure among these populations (fST = 0.305, P < 0.01, 27% of variance), and among headland locations (fCT = 0.125, P = 0.04, 12% of total variance). Most of the genetic variation was partitioned within tide pool populations between individuals (fSC = 0.392, P < 0.01). Mantel tests

Table 2. Parvulastra exigua pairwise FST comparisons between all tide pool subpopulations Cape Banks Location Cape Banks Sub 1 2 3 1 2 3 1 2 3 1 0.023 0.015 0.213 0.239 0.011 0.170 0.322 0.232 2 3 Kurnell 1 2 3 Bundeena 1 2

0.017 0.238 0.202 0.036 0.157 0.341 0.242

0.227 0.258 0.048 0.157 0.341 0.242 0.618 0.095 0.163 0.033 0.041 0.392 0.465 0.733 0.628

Kurnell

0.132 0.179 0.118 0.259 0.061 0.113

Bundeena

Bold values indicate signicant pairwise comparisons (P < 0.05).

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Figure 2. Haplotype network for all Parvulastra exigua tide pool populations (sites 13) located within each headland (top) and all Meridiastra calcar headland populations (bottom). Solid lines connect haplotypes by a single mutation and crossbars represent additional mutations.

demonstrated no correlation between genetic differentiation and geographical distances (r = 0.272, P = 0.12), and therefore the genetic variation present in P. exigua does not t an isolation-by-distance model.

MERIDIASTRA

CALCAR

The haplotype network revealed that most individuals (N = 103; 77%) shared one of the ve major hap-

lotypes that differed from each other by no more than ve substitutions (Fig. 2). These ve haplotypes, from a total of 37 identied haplotypes, occurred at all three headland locations. Unique haplotypes made up 81% of the total number of haplotypes and were present in all headlands (ranging from eight to 12; Table 1). Haplotype diversities (h) ranged from 0.84 0.87 with a mean of 0.86, and nucleotide diversities (p) ranged from 0.00310.0048 with a mean of 0.0072 (Table 1). As with tests for P. exigua, the two neu-

2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 821833

GENETIC STRUCTURE OF SYMPATRIC ASTERINIDS

Table 3. Analyses of molecular variance (AMOVA) for Parvulastra exigua, including all tide pool populations and populations grouped by headland Source of variation Parvulastra exigua Tide pool populations Among sites Within sites Headland locations Among headlands Among tide pool populations Within tide pool populations Meridiastra calcar Among headlands % Variance F P

827

25 75 12 27 61

FST = 0.249 < 0.01 FIS = 0.260 < 0.01 fCT = 0.125 0.04 fST = 0.305 < 0.01 fSC = 0.392 < 0.01

FST = 0.029

0.14

AMOVA results for Meridiastra calcar at the headland locations are also shown.

trality analyses were not signicantly different from zero for any of the headland populations (Tajimas D = -0.98, range = -0.96 to -1.61; Fu & Lis F = -2.92, range = -1.98 to -2.16; Table S1). Pairwise FST comparisons did not reveal signicant levels of genetic subdivision between headland populations (FST ranges from 0.019 to 0.056). Similarly, the analysis of molecular variance did not detect signicant genetic partitioning among headland populations (FST = 0.029, P = 0.14; Table 3).

DISCUSSION
Several recent studies have documented a strongly bimodal trend in dispersal estimates of marine species in which juveniles are highly philopatric, or tend to disperse on the order of tens to hundreds of kilometres, even with relatively short planktonic larval durations (Bradbury et al., 2008; Shanks, 2009; Weersing & Toonen, 2009; Selkoe & Toonen, 2011). The present study of the population genetic structure of two asterinid species with divergent mating strategies and larval types supports the paradigm that the life-history traits for these, and other marine invertebrates with contrasting dispersal capabilities, can be a major driver of genetic structure (Shanks, Grantham & Carr, 2003; Bradbury & Bentzen, 2007; Ross et al., 2009; Riginos et al., 2011; Selkoe & Toonen, 2011). Our hypothesis that populations of P. exigua would be genetically partitioned at a very ne spatial scale was supported. The life-history traits of this egg laying species, with pseudocopulation among conspecics, is very unusual in the

Echinodermata and is only known for four other species, all of which are small asterinid sea stars (Emson & Crump, 1979; Komatsu et al., 1979; Byrne, 2006; Barbosa et al., 2012). Of these sea stars, only Asterina gibbosa has been investigated with respect to its population genetics, and similar to P. exigua, strong levels of genetic structure (using amplied fragment length polymorphisms) were detected across spatial scales ranging from 1001000 km in a regional investigation of Atlantic and Mediterranean populations (Baus, Darrock & Bruford, 2005). Most studies of echinoderm genetic structure have investigated broadcast spawning species with planktonic larvae (Benzie & Stoddart, 1992; Arndt & Smith, 1998; Addison & Hart, 2004; Duran et al., 2004; Banks et al., 2007; Calderon, Giribet & Turon, 2008; Hunter & Halanych, 2008; Keever et al., 2009; Zulliger et al., 2009; Banks et al., 2010; Maltagliati et al., 2010; Prez-Portela, Villamor & Almada, 2010; Borrero-Perez et al., 2011; Janosik, Mahon & Halanych, 2011; Puritz & Toonen, 2011; Hemery et al., 2012; Timmers et al., 2012) in populations sampled across large spatial scales (kms) and incorporating a variety of molecular markers (e.g. allozymes, microsatellites). For benthic developers, studies have incorporated sampling across regional spatial scales or greater and have involved three asterinids (A. gibbosa, P. exigua, Cryptasterina hystera) and a cosmopolitan ophiuroid (A. squamata) that broods its young (Hunt, 1993; Sponer & Roy, 2002; Colgan et al., 2005; Boissin et al., 2008; Sherman et al., 2008; Ayre et al., 2009; Puritz et al., 2012). The present study appears to be the rst to examine a benthic developing echinoderm with a mixed mating reproductive strategy at such a ne spatial scale (metres). The ne-scale (< 100 m) approach to population genetic structure in sexually reproducing marine invertebrates, within the same habitat type, has been applied to studies of ascidians, abalones, sponges, and corals (Grosberg, 1987, 1991; Hellberg, 1994; Yund & ONeil, 2000; Miller et al., 2009; Blanquer & Uriz, 2010). In these studies, it is suggested that strong population genetic structure is inuenced by short planktonic larval duration times. Other studies, predominantly involving molluscs, show habitat-driven (e.g. salinity and temperature gradients) genetic structure (Johnson & Black, 1984; Schmidt & Rand, 1999; Sokolova & Boulding, 2004; Grahame et al., 2006) at the scale of metres to kilometres. At the scale of centimetres to metres, the contribution of sexual and asexual reproduction to population genetic structure has been determined for cnidarians and sponges (Ayre, 1991; Ayre & Dufty, 1994; Billingham & Ayre, 1996; McFadden, 1997; Adjeroud & Tsuchiya, 1999; Yund & ONeil, 2000; Calderon et al., 2007). Many of these studies report signicant levels of genetic

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S. S. BARBOSA ET AL. differences in life histories and mating systems between them (Hart & Marko, 2010). This suggestion would need to be addressed through use of multiple additional genetic markers with much faster coalescent times than the mtDNA loci used in the present study, and with many populations across the broad distribution of these species to tease apart with coalescent analyses (Hart & Marko, 2010; Marko & Hart, 2011). Species that rely on rafting to disperse are known to display strong genetic partitioning between populations, as seen with P. exigua, and typically exhibit very weak isolation by distance signals (Waters et al., 2004; Thiel & Haye, 2006). This unusual life history, combined with the unpredictability of rare dispersal by rafting, may translate to colonization and founder effects also contributing to the strong population structuring observed for P. exigua (Hart & Marko, 2010). Whatever the underlying cause of the genetic differentiation, the results obtained among the studies performed to date are consistent with extremely limited gene ow among populations of P. exigua at all spatial scales, and all studies point to the role of the unusual benthic life history in the resultant population structuring. Localized genetic structure, especially at the very ne-scale of tide pools, would not be detected in the more typical regional scale approaches adopted by most studies when investigating the genetic structure of marine invertebrate populations. Pairwise comparisons of FST values between the P. exigua tide pool populations within the three headlands revealed signicant genetic partitioning within the Kurnell and Bundeena populations. There are no obvious physical barriers to adult movement within the intertidal rock platform at either of these locations that might explain the structure observed. Interestingly, despite the strong partitioning detected at Kurnell (FST = 0.618), FST levels were one order of magnitude lower when comparing Kurnell and Bundeena tide pool populations located 10 km away. This pattern may be more a result of stochastic drift processes, or the idiosyncratic history of colonization via a few rare rafting events that might cause neighbouring populations to be different from one another, rather than enhanced connectivity between the headland populations. By contrast to these populations, no signicant population structure was evident between the Cape Banks tide pool populations. Although it is possible that local currents or circulation patterns may facilitate movement of individuals among the Cape Banks populations, they would have to operate over an extremely ne spatial scale. Alternatively, the lower population structure at Cape Banks may be inuenced by human-mediated movement of adults, in association with the areas use as a scientic research

structure and, in some cases, at levels similar to that determined for the same species at much larger spatial scales (Hellberg, 1994). This highlights the importance of considering both geographical scale and life history when investigating population genetics. In the present study, tide pool and headland populations of P. exigua showed signicant genetic structure. The partitioning detected between tide pools (FST = 0.249, P < 0.01) and adjacent headland populations (fCT = 0.125, P = 0.04), is lower but comparable to the results of previous studies for this species along the south-east coast of Australia (1001000 km+) using a range of molecular markers (FST ranges from 0.462 to 0.755; Hunt, 1993; Waters & Roy, 2004; Colgan et al., 2005; Hart et al., 2006; Sherman et al., 2008; Ayre et al., 2009). The headland populations of M. calcar exhibited no signicant genetic structure (FST = 0.029, P = 0.14), similar to the ndings of previous studies (Hunt, 1993; Sherman et al., 2008; Ayre et al., 2009). The lack of genetic structure among M. calcar populations, sampled from the same headlands as P. exigua, indicates that neither local oceanography, nor headland topology inuence genetic structure for these species at the spatial scale investigated. The similarity of tide pool habitats occupied by P. exigua among locations makes differing environmental conditions an unlikely explanation for population structuring for this species. In addition, cross headland reproductive and larval rearing (to juvenile) studies using gametes from all locations in all combinations of males and females show complete compatibility, indicating that the partitioning detected in P. exigua is also not a result of reproductive barriers between headland populations (Barbosa et al., 2012). Given that P. exigua appears to rely on stochastic rafting events and other idiosyncratic methods for dispersal, it is likely that gene ow occurs over varied distances and between random populations, thereby diminishing signicant associations between geography and genetic distance (Waters & Roy, 2004). Because FST is a summary statistic, signicant structure among populations may be a result of differences in effective population size, demographic or colonization history, migration, or some combination of these factors, especially for populations that may not have reached migration drift equilibrium, and thus direct interpretation of population structure in the context of gene ow can sometimes be problematic (Hart & Marko, 2010; Lowe & Allendorf, 2010; Marko & Hart, 2011; Karl et al., 2012). A previous coalescent analysis at regional spatial scales estimated migration rates for both P. exigua and M. calcar that were essentially zero, concluding that differences in spatial differentiation between the species were likely a result of

2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 821833

GENETIC STRUCTURE OF SYMPATRIC ASTERINIDS zone since 1940. Translocation of P. exigua within Cape Banks has occurred in several studies (Arrontes & Underwood, 1991; Jackson, Murphy & Underwood, 2009) and a plethora of student projects. Thus, it is likely that anthropogenic redistribution of P. exigua individuals across the Cape Banks headland has occurred many times over the last 50 years. As a facultative hermaphrodite with an entirely benthic life cycle, it is highly probable that the developmental mode of P. exigua in combination with its mixed mating strategy, shapes its population genetic structure by promoting localized inbreeding and genetic differentiation. In the present study, levels of inbreeding were not estimated from our haplotypic data; however, it was evident that most of the genetic variation present in P. exigua was retained within tide pool sites (FIS = 0.26, P < 0.05, 75% of variation). It has been suggested that mating system is not necessarily a reliable predictor of the degree of genetic differentiation within populations because selective pressure on the molecular marker used may contribute to the levels detected (Addison & Hart, 2004). However, considering the nonsignicant results of our neutrality tests (Tajimas D, Fu & Lis F), it appears that, for the present study, the combination of selng potential, pseudocopulation among relatives, and low mobility at all life stages comprises the major driver of genetic structure in P. exigua populations. Additionally, the presence of low haplotype and nucleotide diversities for P. exigua may indicate that the populations under investigation have experienced a recent founder event, demographic bottleneck or selective sweep (Avise, 2000). A molecular study of another asterinid sea star, A. gibbosa, provided evidence that selng potentially accounted for the reduced genetic diversity observed in a laboratory population; however, it was not considered to be a major factor in shaping population genetic structure in natural populations (Baus et al., 2005). To better characterize this phenomenon for the P. exigua populations, application of additional markers, which show less spatial subdivision (e.g. microsatellites; McGovern et al., 2010), is required. The detection of genetic structure in P. exigua populations, as seen in the present study and in other benthic developing asterinids (Puritz et al., 2012) is consistent with the ndings of previous studies of the same species using different molecular markers (e.g. allozymes: Hunt, 1993; Colgan et al., 2005; Sherman et al., 2008; mtDNA: Ayre et al., 2009), albeit at larger spatial scales. The strong genetic partitioning exhibited by P. exigua at ne spatial scales, with 70% of haplotypes being private to a given population, suggests that they are predominantly maintained through selfrecruitment. However, sufficient gene ow must occur

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through extrinsic dispersal mechanisms to explain the shared common haplotypes and the lack of extensive cryptic speciation across this sea stars broad geographical distribution in Australia. Philopatrydriven high genetic structure, as apparent in the present study for P. exigua populations, should accelerate evolutionary change and species divergence (Hellberg, 1994) and this is well illustrated by the Parvulastra clade (Hart, Byrne & Smith, 1997; Byrne, 2006). A P. exigua ancestor gave rise to two viviparous Parvulastra species in Tasmania and South Australia (Hart et al., 1997). These species are small range endemics located at the southern Parvulastra vivipara (Tasmania) and western P. parvivipara (South Australia) range edges of P. exigua (Dartnall, 1971; Hart et al., 2006). Their divergence is likely to have been facilitated by the minimal dispersal of P. exigua and parapatric speciation, as well as periods of geographical isolation (exposure of Bassian Isthmus) in association with late Pliocene and Pleistocene glacial cycles, a driving force underlying evolution in the region (OHara & Poore, 2000; Waters et al., 2004). Rapid range edge speciation (approximately 6000 years) is recently reported for intertidal asterinids (Puritz et al., 2012). The present study and previous research on Australian asterinids (Hunt, 1993; Waters & Roy, 2004; Colgan et al., 2005; Hart et al., 2006; Sherman et al., 2008; Ayre et al., 2009; Hart & Marko, 2010) comprise the small number of investigations of the relationship between life history and population genetic structure in congeneric or confamilial marine species with extremely divergent life histories: egg laying (P. exigua: nondispersive, potential inbreeding) versus free spawning (M. calcar: dispersive, full out crossing). Comparisons of other closely-related species with these life-history traits have involved littorinid and muricid snails (Hoskin, 1997; Kyle & Boulding, 2000; Lee & Boulding, 2009) shes (Waples, 1987), and congeneric shrimps (Duffy, 1993). Similar to the present study, most of these studies indicated that reproductive mode and dispersal capacity were strong predictors of the level of population genetic structure. A comparison of the genetic structure of P. exigua and M. calcar populations at the ne spatial scale of tide pools (< 300 m) and headlands (515 km) supports the hypothesis that mating strategy, life-history differences, and dispersal capacity are major drivers of population genetic structure in these sea stars and provides novel insights into the spatial levels over which these factors operate. This emphasizes the importance of considering both life history and spatial scale when investigating genetic structure and connectivity between marine populations (Sotka et al., 2004; Pelc et al., 2009; Selkoe et al., 2010; Riginos et al., 2011).

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ACKNOWLEDGEMENTS
The present study was supported by a grant from the New South Wales Marine Parks Authority. This project was funded by two grants from the National Science Foundation (Bio-OCE 0623699 awarded to RJT and OISE-EAPSI 1108582 to JBP). Contribution number 81 of the Sydney Institute of Marine Sciences. We thank Team Asterina: Rick Grosberg, Mike Hart, and Carson Keever for their support and advice that assisted in improving this manuscript. Sampling complied with a NSW Marine Parks Authority Collecting Permit.

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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article at the publishers web-site: Table S1. Neutrality tests for Parvulastra exigua headland and tide pool populations (A) and for Meridiastra calcar headland populations (B).

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