MARINE ECOLOGY PROGRESS SERIES Published January 26 Mar. Ecol. Prog. Ser.

Effects of contrasting patterns of larval dispersal on the genetic connectedness of local populations of two intertidal , calcar and P. exigua*

Alison Hunt

Department of Biology, University of Wollongong, Locked Bag 8844. South Coast Mail Centre. Wollongong. 2521, Australia

ABSTRACT: The closely related intertidal starfish Patirjella calcar and P exigua have markedly differ- ent dispersal capabilities. P calcar is considered to have sexually produced planktonic larvae and hence the potential for wide dispersal. If this is the case local populations should be strongly intercon- nected and there should be little or no accumulation of genetic variation. Conversely, P exigua has no known means of dispersal between local populations since development proceeds directly from egg masses laid on the rocky shore. Therefore, local populations of P exigua may be less genotypically di- verse than P calcar, but should display marked genetic heterogeneity among local populations result- ing from the accumulating effects of genetic drift and localised natural selection. To test these predic- tions genetic data were used to assess the genotypic structure and apparent connectedness of local populations of both species along 230 km of the southeast coast of Australia. Electrophoretic variation was surveyed for 5 loci. Single-locus genotype frequencies within all local populations of both species closely matched expectations for Hardy-Weinberg equilibria. This implies that local populations are maintained by recruitment of outcrossed sexually produced offspring, but as predicted, P exigua had fewer alleles at each locus and fewer heterozygous individuals than P: calcar. Furthermore, it appears that these 2 species have vastly different levels of gene flow among local populations. The 6 local pop- ulation~of P calcar were genetically homogeneous [FST= 0 f 0.001 (SE)] and these populations were thus inferred to be strongly connected by larval dispersal. In sharp contrast, the 11 local populations of the directly developing P exigua were highly genetically heterogeneous which is reflected In consi- stently high levels of genetic variance (FsT= 0.462 2 0.048).

INTRODUCTION or asexual replication by fission or autotomy (Emson & Wilkie 1980), broadcast spawning of sexually pro- are a conspicuous and important com- duced gametes (Barker & Nichols 1983) and species ponent of many marine environments (e.g. Paine 1966, which combine modes of reproduction and means of Benzie & Stoddart 1992). This has provoked enormous dispersal (Johnson & Threlfall 1987, McClary & interest in the great diversity of life histo- Mladenov 1989). ries, and these have been shown to include species Many recent studies have demonstrated the value of which are brooders (Chia 1966, Keough & Dartnall genetic data in determining local population structure 1978), species which undergo asexual replication of and inferred dispersal distances for marine organisms sexually produced planktonic larvae (Bosch et al. 1989) (e. g. Koehn et al. 1976, Burton & Feldman 1981, Grosberg 1987, Ayre & Willis 1988, Hunt & Ayre 1989, ' Publication No. 98 from the Ecology and Genetics Group of Johnson & Black 1990) and echinoderms in particular the University of Wollongong (e.g. Kwast et al. 1990, Palumbi & Kessing 1991, Benzie

O Inter-Research 1993 180 Mar. Ecol. Prog. Ser. 92: 179-186. 1993

& Stoddart 1992) and have often overturned predic- This study examined the genotypic composition of tions based solely upon histological or ecological data. local populations of the 2 species of intertidal starfish. Consequently, the present study uses genetic data to Patiriella exigua and P calcar, in order to infer their assess the mode of reproduction and apparent scale of mode of reproduction and source of most recruitment. dispersal of 2 species of CO-occurringintertidal starfish, Gene frequencies were then used to investigate the Patiriella exigua (Lamarck) and P calcar (Lamarck). consequences of philopatry and wide dispersal for the Both specles are common and have similar geographic genetic connectedness of local populations. ranges along the southeastern coast of Australia (Dart- nall 1971). l? calcaris believed to disperse via sexually produced planktonic larvae (Lawson-Kerr & Anderson METHODS 1978, Byrne 1991) which remain in the planktonic stage for ca 10 d (Lawson-Kerr & Anderson 1978). It Collections of samples. Starfish were collected from therefore has the potential for wide dispersal. Conver- stable intertidal rock platforms, over a total distance of sely, dispersal of P exigua is thought to be severely re- 230 km, between Kurnell (34"2'S, 151" 12'E) and stricted as juveniles develop directly from egg masses Durras (350401S, 150" 18' E) on the southeast coast of deposited onto the rocky shore (Lawson-Kerr & Ander- Australia. Samples of 29 to 48 individuals of Patiriella son 1978, Byrne 1991). Although the mating system of calcar and J? exigua were collected from each of 6 and this species is unknown there is some evidence to sug- 11 separate headlands (local populations) respectively, gest that P. exiyua may be proiandric and/or sirnu!!.- inc!uding 4 sites in which they CO-occurred. Star- neously hermaphroditic (Byrne 1991). Neither P calcar fish were transported to the laboratory alive and or I? exigua is known to reproduce asexually. then stored frozen at -80 "C pending electrophoretic Philopatry, or restricted dispersal, in the absence of analysis. sdecticn, shcu!:! result ir! hnth !OW eve!^ nf variability Electrophoresis. Tissue extracts were prepared for due to a reduction in the effective population size each individual by macerating a small portion of armin (Soule 1976, Gallardo & Perron 1982), and genetic di- a 10 % sucrose solution. Electrophoresis was carried vergence of local populations, as the effects of drift and out on horizontal starch gels (12 % w/v) using methods localised selection may accumulate each generation modified from Harris & Hopkinson (1976). Each spe- due to offspring remaining within the parental cies of starfish was assayed for 5 enzyme encoding loci: (Wright 1968). Although this issue has been considered Gpi, Pgm, Hk, Mdh and Sod for Patiriella calcar, and for many years (e.g. see Gooch & Schopf 1970) few Gpi, Pgm, Hk, Lpp and Ltp for l? exigua. Gpi was studies have dealt directly with the effects of restricted apparently dimeric in both species as was Lpp in I! dispersal on the genetic structure of populations (e.g. exigua. Mdh appeared to be dimeric for 2 enzymes a!- Burton & Feldman 1981, Kwast et al. 1990, Ward 1990, though only locus l could be consistently scored. All Johnson & Black 1991, Siegismund & Miiller 1991), other enzymes in both species were assumed to be and none have looked at species from the southeast monomeric and encoded by 1 scorable locus. The elec- coast of Australia (Ayre 1990). trophoretic buffers used were No. 5 for Ltp, Lpp, Mdh, In contrast, high genetic diversity within local popu- Sod and No. 9 for Gpi, Hk, and Pgm as described and lations and genetic homogeneity among populations numbered by Selander et al. (1971).Alleles were labell- characterise a species with wlde dispersal of sexually ed alphabetically in order of decreasing mobility and produced planktonic larvae. However, the wide dis- a 5-locus genotype was inferred for each individual. persal of sexually produced planktonic larvae will typ- Analysis of intra-population variation. Regardless ically only be possible by transport via ocean currents, of whether recruits are the product of widespread dis- and a species with planktonic larvae may experience persal or direct development, the genotypic structure quite restricted dispersal if the current flow is dis- of local populations can be used to infer the mating jointed or unreliable. The unpredictable nature of the system of the species. Allozyme data were used to major current flow along the southeast coast of assess the levels and patterns of genotypic diversity Australia, the East Australian Current, may strongly present within local populations. Departures of geno- influence patterns of dispersal along this coastline. type frequencies from those expected under Hardy- This current flows in a southerly direction along the Weinberg equilibria were estimated for all loci at all eastern coast of Australia but is also composed of a sites as well as an overall estimate for each species complex set of eddies (Hamon 1965) before finally (FIs)Deficits of heterozygous individuals relative to moving offshore to the east at the southeastern corner those expected under Hardy-Weinberg equ~librium of Australia (Hamon et al. 1975). The use of genetic are reflected in positive values and may indicate sub- data is therefore essential as a means of inferring real- division of the population on some scale due to in- ised rather than potential dispersal distances. breeding and Wahlund effects, whereas asexual repro- Hunt: Patterns of larval dispersal of two starfish 181

duction can lead to both excesses and deficits. The sta- locally common allele plus all other alleles at that locus tistical significance of the departures of the observed were compared to all other loci within each local pop- number of hetel-ozygotes from those expected under ulation. Chi-square analyses were used to determine Hardy-Weinberg equilibria were calculated by chi- the statistical significance of disequilibrium in each square goodness of fit analyses with 2 categories, case. homozygotes and heterozygotes (Zar 1984, p. 42). Inter-population variation. The level of genetic dif- Although single-locus genotype frequencies may ferentiation of local populations provides an lnd~cation return to Hardy-Weinberg equilibrium after 1 genera- of the level of gene flow and can be estimated as the tion of random mating, the association between alleles standardised variance in allelic frequencies, FST. FST at different loci can persist for many generations was calculated among samples for each allele and each (Waller & Knight 1989) and may reflect past episodes locus using the formulation of Wright (1978) (BIO- of inbreeding or asexual reproduction, random drift, SYS-1 program; Swofford & Selander 1981). To provide selection, mutations or the mixing of genetically dis- a less biased estimate of the mean FsT across all loci, a tinct populations (Crow & Kimura 1970, Hedrick et al. jackknife estimate of FsT and its variance (0 -1- SE) was 1978).Therefore it was important to look at multi-locus also calculated using the method of Weir & Cockerham genotype frequencies in order to provide a more sensi- (1984). For each locus the statistical significance of the tive test of the history of patterns of mating and recruit- inter-population variation in allelic frequencies was ment. Hill's (1974) diallelic pairwise linkage disequi- calculated by heterogeneity chi-square analysis. To librium measure was used to test for the presence of ensure validity of these tests, rare alleles were pooled inter-locus associations. Although the sample sizes in with the next most common allele for each locus to this study are such that low intensities of disequilib- ensure that all chi-square cells contained expected rium will probably not be detected this test will allow values > 4. relatively intense levels of disequilibrium to be de- The overall pattern of allelic frequency variation at tected (Brown 1975). For each comparison the most polymorphic loci in Patjriella exiyua was assessed by

Table 1 Patiriella calcar. Allelic frequencies for 5 enzyme-encoding loci for collections from local populat~onsseparated by 230 km on the southeast coast of Australia. Local populations l~stedfrom north to south. Sample slze given in parentheses - allele absent

Locus Kurnnell Woonona Bellambi Nth Beach Ulladulla Durras ~llele (29) (43) (39) (45) (41) (29)

Gpi a 0 018 0.035 0.026 0.033 0.037 - b 0.052 0.186 0.115 0.089 0.146 0.018 C 0.483 0.337 0.397 0.478 0 341 0.362 d 0.310 0.302 0.269 0.300 0.268 0.414 e 0.103 0.128 0.179 0.100 0.159 0.172 f 0.034 0.012 0.014 - 0.049 0.034

Hk a b C d e f Mdh a b C Sod a b 182 Mar. Ecol. Prog. Ser. 92: 179-186, 1993

Nei's (1978) unbiased measure of genetic identity. A found between any loci within the 6 local populations dendrogram produced by the UPGMA clustering tech- of P calcar. nique (Sokal & Sneath 1963) was used to summarise Similarly, genotype frequencies within local popula- the genetic similarity of local populations. tions of Patiriella exjgua typically provided a close fit to expectations for Hardy-Weinberg equilibria (Table 2) with only slight deficits of heterozygous individuals RESULTS (Flsweighted mean = 0.098). Only 2 of the 16 cases of deficits from Hardy-Weinberg equilibria were statisti- Intra-population variation cally significant and these were both at the Ltp locus in samples from Sandon Pt. (p<0.05) and Kioloa The genotypic composition of samples from local (p<0.001) but none of the 17 excesses. Furthermore, populations of both Patiriella calcarand l? exigua were no cases of statistically significant linkage disequilib- consistent with the genetic structure of local popula- riurn were detected (p >0.05) in any of the 11 local tions maintained by recruitment of sexually produced populations. The 5 loci examined for l? exigua were offspring. Four of the five loci examined for l? calcar less polymorphic than those of l? calcar and on average were highly polymorphic with an average of 4.0 f 0.3 (f SE) there were 1.8 f 0.1 alleles per locus and 0.8 + (f SE) alleles per locus and on average 1.6 f 0.1 (f SE) 0.0 loci heterozygous per individual. Nonetheless, all loci were heterozygous per individual (Table l), how- 5 loci could be considered polymorphic although vari- ever, the Sod locus was polymorphic in only l inclivid- ation at the Hk locus was snly found at Pt Kernbla ual at Bellambi. Almost all individuals had unique where 6 individuals contained the alternate Hka. 5-locus genotypes and genotype frequencies were in close agreement with expectations for Hardy- Weinberm eniiilihria fnr a!! !QC! 2nd $! !~ca!n~pij!;- Sn!er-population variation Y -l-"*-"- --* tions with only a slight overall deficit of heterozygotes (FIsweighted mean = 0.065). None of the 13 deficits or Local populations of Patjrjella calcar and P exigua 10 excesses of heterozygotes found proved to be signi- displayed markedly different levels of genetic diffe- ficantly different from Hardy-Weinberg expectations. rentiation. Additionally, no linkage disequilibrium (p > 0.05) was Patiriella calcar showed no appreciable allelic varia-

Table 2. As in Table 1 but for Patiriellaexigua

Locus Garie Sandon Woo- Bell- Nth Sth Port Windang Bass Kioloa Durras Allele Beach Pt nona ambi Beach Beach Kembla Is. Pt (48) (33) (47) (36) (35) (45) (40) (43) (40) (36) (46)

Gpi a 0.957 1.000 1.000 0.556 0.762 0.477 0.200 - 0.054 0.917 1.000 b 0.01 1 ------C 0.021 - - 0.444 0.238 0.523 0.788 1.000 0 946 0 083 - d 0.011 - - - - - 0.012 - - - - Pgm a 0.021 0.038 0.014 - b 0.979 0.962 0.986 1.000 C - - - - Hk a 0.100 - - b 0.900 1.000 1.000 LPP a 0.056 0.512 0.267 0.758 b - 0.058 - C 0.944 0.488 0.675 0.242 L tp a - - 0.011 - - - - b 0.638 0.875 0.985 0.989 0.988 0.779 0.725 0.917 0.870 0.552 0.652 C 0.351 0.125 0.015 0.012 0.221 0.275 0.083 0.130 0.448 0.348 d 0.011 ------Hunt: Patterns of larval dispersal of two starfish 183

Table 3. Patiriella calcar. Allelic frequency variation for 5 North Beach 5 enzyme-encoding loci among samples. FST: standardised var- Woonona 3 iance (Wright 1978). 0 k SE obta~nedby jacknife procedure (Weir & Cockerham 1984). Probability: heterogeneity chi- SandonPt. 2 square analysis for each locus. NS: not significant at p < 0.05. K~oloa 10 nt: valid chi-square could not be tested Durras 11

GarieBeach 1 LOCUS FST Probability Bellamb~ 4 Cpi 0.003 NS South Beach 6 Pgm 0.000 NS Windang Is. B Hk 0.001 NS Mdh 0.000 NS Bass Pt. 9 Sod 0.000 nt Pt. Kembla 7 8 f SE 0.000 2 0.001

tion among 6 local populations (FST = 0.000 + 0.001) Genetic Distance (Table 3). FsT values for individual alleles, weighted Fig. 1. UPGMA dendrogramm (Sokal & Sneath 1963) showing averages for each locus, and across all loci, ranged the genetic relatedness of 11 samples of Patiriella exigua col- from 0 for all alleles at 3 loci to 0.023 for the relatively lected over 230 km of the southeast coast of Australia as esti- mated by Nei's (1974) unbiased minimum genetic distance rare Gpi b. Only Gpi and Hk had measurable, although (D).Local populations are numbered from north to south not significant (p > 0.05), FSTvalues of 0.003 and 0.001 respectively (Table 3). In contrast, allelic frequencies for Patirlella exigua varied greatly among the 11 local populations (FST = DISCUSSION 0.462 +0.048) (Table 4). All alleles, except for those which were extremely rare, had high genetic variances The genetic structure of local populations of and these ranged from 0.020 for PgmC to 0.607 for GpiC. Patiriella exigua and l? calcar are consistent with the Variation at the Gpi. Lpp and Ltp loci was highly sig- predicted effects of recruitment via sexually produced nificant (p < 0.0001) and resulted in FST values of 0.601, offspring, although with very different patterns of 0.469 and 0.143 respectively. Low levels of polymor- dispersal. phism at Pgrn and Hk are reflected in the lower, but still high. FST values of 0.036 and 0.080 respectively. The statistical significance of allelic heterogeneity for Reproduction and recruitment these 2 loci could not be tested because of small ex- pected values. However, the differentiation of local In contrast with several recent studies of other spe- populations was such that 4 of the 6 loci contained at cies of echinoderms (e.g. Emson & Wilkie 1980, Bosch least 1 allele that was unique to only 1 local population et al. 1989, McClary & Mladenov 1989) there was no (Table 2). Furthermore, at both the Gpi and Lpp loci evidence to suggest asexual reproduction for either there was no allele which was the most common in all species as genotype frequencies within samples from cases, and in fact all 3 alleles at the Lpp locus were the each local population were in close agreement with most common allele in at least 1 local population. expectations for Hardy-Weinberg equilibrium and the There was no clear geographic pattern to this allelic majority of individuals had unique 5-locus genotypes. variation for P exigua (Fig. 1). The weighted average Fls value of 0.065 for Patiriella calcarand 0.098 for P exigua indicates an overall slight Table 4. As in Table 3 but for Patiriella exigua deficit of heterozygotes as compared with expections for Hardy-Weinberg equilibria. However, only 2 local Locus FST Probability populations of P exjgua had significant deficits of het- erozygotes and these were both at the Ltp locus. Such Gpi 0.601 <0.0001 isolated departures are not attributable to the mode of Pgm 0.036 nt reproduction as the breeding system of the organism Hk 0.080 nt affects all loci equally but may suggest selection LPP 0.469 <0.0001 LtP 0.143 <0.0001 favouring homozygosity at that locus. In fact, such a 0 f SE 0.462 +0.048 good fit to Hardy-Weinberg expectations is uncommon as a deficiency of heterozygous individuals tend to 184 Mar. Ecol. Prog. Ser. 92: 179-186. 1993

characterise marine invertebrate populations (e.g. It would appear that local populations of Patiriella Koehn et al. 1976, Johnson & Black 1984, Foltz 1986, exigua do not regularly receive recruits from outside Kwast et al. 1990, Grant & Lang 1991) (but see Grant & sources. Furthermore, the absence of intense linkage Lang 1991). disequ~libriumwithin populations suggests that they have not recently been subject to population bottle- necks or close inbreeding (Waller & Knight 1989) as Genetic connectedness has been demonstrated for some species with re- stricted dispersal (e.g. Foltz et al. 1982). Instead it Differences in the dispersal capabilities of Patiriella would seem that these samples were taken from ran- calcar and l? exigua are reflected in the inter-popula- domly mating, isolated local populations; however, fur- tion allelic variation. As was predicted, due to the pres- ther fine-scale sampling is required to confirm this. ence of a planktonic larvae, local populations of l? cal- Conversely, local populations of P calcar sampled in car appear to be strongly interconnected. There was this study appear to form a large and undifferentiated little detectable variation in allelic frequencies for pop- panmictic group in which genetic exchange between ulation~separated by up to 230 km. The overall FST local populations would prevent the effects of drift value of 0 f 0.001 was well within the range expected or selection from accumulating. Such strong inter- for a sexually reproducing species with widespread connectedness must be a consequence of the southerly dispersal (Levinton & Suchanek 1978, Johnson & Black flow of the East Australian Current despite its unpre- 1984). Low levels of genetic variatioii among local pop- dictaS:!ity; this has been showr? to be the case for 2 ulation~have been demonstrated for other marine spe- other marine species on the southeast coast of Aus- cies (e.g.Koehn et al. 1976, Levinton & Suchanek 1978, tralia, Pyura stolonifera (Killingly 1976) and Oulactis Johnson & Black 1984, Hunt & Ayre 1989, Benzie & nluscosa (Hunt & Ayre 1989). The East Australian St=dd-rt 1992) acd thnl~nht.---3-*- he the re~ij!t. of di.z- Cl~rrentmay have important consequences for the persal of sexually produced planktonic larvae. genetic composit.ion of southern and eastern popula- In sharp contrast to the apparently panmictic breed- tions of species along this coastline as there is some ev- ing unit of Patiriella exigua, local populations of l? exi- idence to suggest that movement off the coast near the gua appear to be, at most, only weakly connected with southeastern corner of Australia may effectively divide the majority of recruits coming from within local popu- species into eastern and southern populations (Ayre et lation~.There was marked inter-population variation al. 1991). Further sampling will be required to assess in allelic frequencies at all loci for P exigua (FST = the potential impact of the divergence of the East 0.462 f 0.048), and unlike l? calcar in which only those Australian Current on more southerly local popula- alleles which were shared by all sites reached high fre- tions of l? calcar and P exigua. quencies, patchily distributed alleles reached high fre- quencies in a few localities of l? exigua. However, Acknowledgements. I am especially grateful to my super- visor. David Ayre, for his advice and encouragement through- there was no apparent geographic pattern to this alle- out this study and invaluable criticism of thls manuscnpt I lic variation. Such haphazard allelic variation and high thank K.Ayre, S. Schibeci, R. Standish and C. Styan for pro- frequencies of patchily distributed alleles are a pre- v~d~nguseful comments onthis m.anuscriptand D J Ayre and dictable consequence of limited gene flow and in- K. Ayre for collection of Ulladulla specimens. T Holtsford's program (Version2) was used to calculatejackkn~fe estlrnates creased genetic drift (Slatkin 1981, Grant & Utter 1988) of FsT. I gratefully acknowledge the financial support pro- and although there have been no comparable studies v~dedby the University of Wollongong. on the east coast of Australia, a few studies elsewhere have revealed similar patterns of variation for species with restricted dispersal (e.g. intertidal gastropods: LITERATURE CITED & Ward 1990, Johnson Black 1991; starfish: Kwast et al. Ayre, D. J. (1990).Population subdivisionin Australian tem- 1990; freshwater amphipod: Siegismund & Muller perate marine invertebrates:larval connections versushis- 1991; marine copepod: Burton & Feldman 1981). torical factors. Aust.J. Ecol. 15: 403-412 A further consequence of restricted gene flow is that Ayre, D. J., Read. J., Wishart. J. 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