linckia multifora (echinodermata: asteroidea) in rarotonga

371

Linckia multifora (Echinodermata: Asteroidea) in Rarotonga, CookIslands: Reproductive Mechanisms and Ecophenotypes1

Terry J. Crawford2 and Bruce J. Crawford3

Abstract: In Rarotonga, Linckia multifora (Lamarck) exists in two forms: a bluegray type that is found on the reef intertidally and a red form that is found sub-tidally. Both types reproduce asexually by regeneration of autotomized arms, aswell as sexually, but the relative potential for sexual reproduction varies greatlybetween these different sites. In the laboratory, reciprocal crosses of the bluegray intertidal form and the red subtidal form developed as successfully as thecontrols and were indistinguishable in morphology. In addition, both the bluegray intertidal form and the red subtidal form contain two different classes ofhaplotypes of the mitochondrial gene cytochrome oxidase subunit I (COI),which exhibit 12 fixed differences. These results suggest that L. multifora ofRarotonga has a dual origin and that the two different forms seen in the twoenvironments belong to a single interbreeding population and may representecophenotypes.

Linckia multifora (Lamarck) is a smallsea star, a member of the Ophidiasteridae,that is found on coral reefs throughout theIndo-Pacific Ocean (Clark and Rowe 1971).It generally has arms of uneven lengths be-cause it reproduces asexually by autotomizingarms, which can then regenerate into wholestarfish (Edmondson 1935). It is identifiedby the arrangement of spines along the am-bulacral grooves, the presence of two madre-porites, the arrangement of papular openings,the granularity of the epidermis, and the gen-eral shape of the arms (Clark and Rowe 1971).

On the island of Rarotonga, Cook Islands,South Pacific, there appear to be two differ-ent types of L. multifora: a red-spotted formthat is found in the subtidal zone and a grayblue form that is found on the top of the

reef in the intertidal zone. To confirm thatthese are both L. multifora and to explore therelationship between them we have sequenceda mitochondria gene, cytochrome oxidasesubunit I (COI).

To understand aspects of the ecology andevolutionary biology of an organism, it is im-portant to understand its mechanism(s) of re-production (Hart 2002). Linckia multifora isgenerally considered to reproduce primarilyby asexual means (Rideout 1978), and it hasbeen referred to in the literature as an asexualorganism (Ebert 1996). However, it produceseggs and sperm (Mortensen 1938, Rideout1978), and viable cultures of embryos havebeen produced in the laboratory (Mortensen1938). In this study we set up breeding ex-periments with both types of L. multifora tosee if viable embryos would result from bothtypes and from both crosses. In addition, westudied the population size structure at sev-eral different sites to determine whether sex-ual as well as asexual reproduction occurs innature and to determine whether the relativeimportance of larval recruitment varies be-tween different sites.

materials and methods

Specimens were collected from subtidal loca-tions from depths of approximately 7 to 30 m

Pacific Science (2007), vol. 61, no. 3:371–381: 2007 by University of Hawai‘i PressAll rights reserved

1 Manuscript accepted 30 September 2006.2 Corresponding author: Departments of Zoology

and Botany, University of British Columbia, 6270 Uni-versity Boulevard, Vancouver, British Columbia, CanadaV6T1Z4 (e-mail: [email protected]).

3 Island Medical Program, University of Victoria,P.O. Box 1700, Stn CSC, Victoria, British Columbia,Canada V8V2Y2.

and from intertidal locations by walkingon the reef at low tide or by snorkeling. Theresults of these collections were recordedby photography with a size marker and colorstandard. Specimens to be used as a source ofgametes for embryological studies were thenkept in an aquarium for a few days, and speci-mens for DNA extraction were preserved in70% ethanol. In some cases specimens weretaken with coral rubble to Vancouver, Can-ada, where they were kept in an aquariumfor up to 1 yr.

To set up cultures of embryos, gonadswere dissected from the arms and seawatercontaining 0.1 mg/ml 1-methyl adenine wasapplied to the ovaries to cause release andmaturation of the eggs as evidenced by ger-minal vesicle breakdown (Stevens 1970, Ka-natani 1973). The mature egg suspensions ofeach type were washed in seawater, dividedinto two parts, and fertilized with dilutedsperm suspension of each type. The latterwas prepared by mixing one or two drops of‘‘dry’’ sperm from harvested testes with 50 mlof seawater. Each culture consisted of 100 ormore fertilized eggs.

To determine whether specimens ofspecific sizes contained mature gonads, twoapproaches were used. Specimens collectedin late November and early December weremeasured, injected with 1-methyl adenine,and if that failed to produce spawning, thegonads were isolated and treated with 1-methyl adenine as already described. Thepresence or absence of ripe gonads was re-corded for 17 red, subtidal specimens and 9blue gray intertidal specimens. In additionseven specimens of the blue gray intertidaltype that had been collected from Town Reefand preserved in 70% ethanol were mea-sured, and about 1 cm of the proximal por-tion of the largest arm of each was sent toWax It Histology Services, Vancouver, Brit-ish Columbia, to be decalcified, embedded inparaffin, sectioned, and stained with haema-toxylin and eosin. A collection of six red, sub-tidal L. multifora was similarly treated.

For polymerase chain reaction (PCR)studies, specimens were rehydrated overnightand chopped into small pieces. DNA was ex-tracted according to the method of Zyskind

and Bernstein (1989) with the addition of 0.5mg/ml proteinase K to the extraction solution.After two extractions with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitation ofDNA from the aqueous phase with ethanol,the DNA pellet was washed with 70% etha-nol, air-dried, dissolved in distilled H2O, andstored at –80�C.

PCR reactions were carried out with Plati-num Taq DNA polymerase (Gibco) (0.5 ml),0.1 mM primers, 2.5 mM MgCl2, 0.2 mMdeoxynucleotide triphosphates, and approxi-mately 200 ng template DNA in a thermo-cycler (Applied Biosystems 2400) with thefollowing primers for COI: LINF 5 0-GCRCCR GAT ATG GCR TTY CCA-3 0 andLINR 5 0-CCT ATW GAT ACD ATRGCR TAR ACC ATT –3 0.

The primers were designed from the se-quence of LmWA3 (Genbank number AF187929). The forward primer begins fourbases from the start of that sequence and thereverse primer extends to four bases from theend of the LmWA3 sequence. The amplifica-tion conditions were as follows: denaturationfor 3 min at 95�C followed by 30 cycles of95�C for 30 sec, 45�C for 45 sec, 72�C for 60sec, followed by a final extension of 10 min.

The PCR product was electrophoresed ona 1% agarose gel in TAE. After staining withethidium bromide the approximately 600 basepair COI fragment was recovered using anUltraClean 15 DNA Purification System kit(MOBIO) and cloned into pGEM-T EasyVector (Promega) according to the suppliedprotocol. Plasmid DNA was extracted frompositive colonies with a Wizard Plus Mini-preps DNA Purification System kit (Pro-mega), and the insert was sequenced usingBig Dye3 Terminator Chemistry at the Uni-versity of British Columbia Nucleic AcidProtein Service Unit.

Sequences were aligned with the ClustalX program. The aligned sequences wereconverted to the PAUP/NEXUS formatand then analyzed with the PAUP program(Swofford 2003) using Maximum Parsimonyand Neighbor Joining with the default set-tings, except that the maximum trees limitwas set to 10,000 for Parsimony, and theNeighbor Joining distance setting was Ki-

372 PACIFIC SCIENCE . July 2007

mura 2-parameter. The robustness of thebranch points of the resulting phylogramswas determined by performance of 1,000bootstrap replicates.

results

Two morphologically different populationsof L. multifora were found at various loca-tions on and outside the fringing reef on theisland of Rarotonga, Cook Islands. One typeof specimen that taxonomic keys (Clark andRowe 1971) indicated to be L. multifora wasfound on the top of the fringing reef at lowtide. These sea stars were a dark gray blue,and some had red purple patches (Plate IA).They were found in small tide pools on peb-bles or partially buried among pebbles in thesurf. Their sizes ranged from 1.9 to 5.3 cmalong the longest arm with a median value of3.5 cm (Figure 1A). Almost all had arms ofunequal lengths, indicating that they werereproducing asexually, and comets and regen-erating individuals (counter-comets, in theterminology of Rideout [1978]) of varioussizes were present (Plate IA). Comets (regen-erating arms) were identified as individualshaving one arm at least 10% longer than theothers and represented 13.2% of the speci-mens in Figure 1A.

A second type of specimen that taxonomickeys also indicated to be L. multifora wasfound during dives on subtidal reefs fromdepths of 3 to 30 m. Linckia multifora wasfound at all locations, but their abundancevaried dramatically from one subtidal reef toanother. In some locations only a few individ-uals could be found in approximately 40 min,but at another (known locally as Bernie’sReef ) more than 50 could be found in thattime. Most commonly the sea stars werefound on areas of dead coral. This subtidalform of L. multifora had a cream or pale bluebackground with red blotches and had bluetips to the arms (Plate IB). The sizes rangedfrom 0.9 to 5 cm along the longest arm withmedian values of 2.7 and 2.2 cm (Figure 1Band C ). Almost all had arms of unequallengths, indicating that they were reproduc-ing asexually. There were comets (regenerat-ing arms) in various stages of regeneration as

well as counter-comets. Some of the cometswere formed from very tiny legs (less than0.7 cm) and others were regenerating fromlarger ones (Plate IB). Of the specimens inFigure 1B and C, 13.7% and 14.9%, respec-tively, were comets. Figure 1 also shows thatthe subtidal sites had the greatest frequenciesof small individuals.

We considered the possibility that theblue gray form of L. multifora might be a ju-venile form of L. laevigata or a hybrid of thered form of L. multifora with L. laevigata, aclosely related species that Williams (2000)found to be indistinguishable on the basis ofCOI sequences. Although the blue gray L.multifora and the royal blue L. laevigata couldsometimes be found only a few feet apart andwere ripe at the same time, there were nospecimens of intermediate phenotype regard-ing color or arm shape (pointed and irregularin length for L. multifora and even in lengthand rounded at the tip for L. laevigata), indi-cating that the blue gray form of L. multiforais not a hybrid of the red, subtidal form of L.multifora and L. laevigata. In addition whenwe examined 21 of the smallest L. laevigatathat we could find in early December, 6 witharm lengths of 6–8 cm had immature gonads,and 15 with arms of 10–13.5 cm in lengthwere all mature. In the case of the blue grayL. multifora, the largest we found had an arm5.3 cm in length, and all but one of the speci-mens with an arm longer than 3.0 cm thatwere checked had mature gonads. Thus thereis no overlap in the sizes of the mature adultsof the two species, which confirms that theblue gray L. multifora is not an immaturestage of L. laevigata. This is consistent withthe observation of Marsh (1974) that on thebasis of morphology L. multifora and L.laevigata are different species. Furthermore,Yamaguchi (1977) described the rarely seenpretransformation form of L. laevigata ascharacterized by distinctive brown and darkgreen blotches.

To confirm that both types of L. multiforawere capable of sexual reproduction, in earlyDecember eggs were obtained by treatingdissected gonads with 1-methyladenine. Ma-ture ovaries each produced several hundredeggs that were of the clear, nonyolky type.

Linckia multifora in Rarotonga, Cook Islands . Crawford and Crawford 373

Figure 1. Linckia multifora. Length (mm) of longest arm of specimens from (A) intertidal location (Town Reef ) andsubtidal locations, (B) Bernie’s Reef, and (C ) other subtidal reefs. Note the absence of very small specimens and thegreater frequency of large specimens in the intertidal type (A).

After fertilization the resulting cleavage stageswere typical for a planktotrophic type ofechinoderm egg and resulted in a planktotro-phic type of larva. Reciprocal crosses betweenthe blue gray and red forms of L. multiforaplus the controls were all set up at the sametime. (This was necessary because we wereunable to regulate temperature, which variedbetween 27 and 29�C [80 and 85�F]). Allcombinations of eggs and sperm resulted infertilization of more than 90% of matureeggs, as estimated by the observation of fertil-ization membranes with the dissecting micro-scope. By 15 hr after fertilization a mixture ofswimming blastulas and very early gastrulas(with the archenterons just starting to invagi-nate) was present in all cultures. By 22 hr allcultures were in the early gastrula stage (arch-enterons one-third to one-half the length ofthe blastocoel), and by 38 hr the midgastrulastage had been reached, with the tip of thearchenterons expanded but with no coelomesformed. Between 48 and 72 hr the coelomeshad formed in all cultures. Thus the recipro-cal crosses of the two types of L. multiforademonstrated the same morphology and rateof development as the cultures in which theeggs were fertilized with the same kind ofsperm. The cultures terminated at the swim-ming larval stage because we were not able toprovide adequate conditions for further de-velopment. Using different parents, we per-formed the blue gray control crosses and theblue gray female� red male crosses on an-other occasion with similar results.

To determine the proportion of each typeof L. multifora that was capable of sexual re-production in early December, individuals ofdifferent sizes were dissected or sectioned todetermine if gonads containing mature ga-metes were present. Nine blue gray reef-topL. multifora ranging in size from 3.5 to 5 cmalong the longest arm were dissected, and allbut one contained ripe gonads. Of the sevenblue gray specimens that were sectioned, sixthat ranged in size from 2.6 to 3.9 cm wereripe, and only one (2.9 cm) was not ripe.Thus 88% of the specimens examined wereripe in December. Although we were unableto examine any specimens from the smallestsize class (2.5 cm or less) seen in Figure 1A,

this size range makes up only 13% of the to-tal. Even if none of these was ripe, at least76% of the blue gray specimens as shown inFigure 1A would be expected to be ripe inearly December.

Seventeen of the red, subtidal specimenscollected in early December were examinedby dissection, and an additional six were sec-tioned. The smallest specimen examined (armlength 1.3 cm) was not ripe. Of the 10 speci-mens with an arm 2.3 to 3.6 cm in length,60% were ripe. The 12 specimens that hadan arm 3.7 cm or longer were all ripe. Whenthese data were compared with the size distri-bution data from Bernie’s Reef, which showed19/62 specimens with the longest arm lessthan 2.2 cm in length, 38 in the 2.3- to 3.6-cm size range, and 5 larger than 3.6 cm, itwas calculated that 45% of the specimensshown in Figure 1B could be expected to beripe. In the case of the data from otherunderwater reefs (Figure 1C ), 73/147 werein the smallest category (of which none wasexpected to be ripe), 68 were in the mediumcategory (of which 60% would be expected tobe ripe), and all 6 in the largest category wouldbe expected to be ripe, giving an estimate that32% of the entire group would be mature.

Observation of specimens held in aquar-ium tanks for up to 1 yr showed that the twodistinctive colors were maintained. Whena specimen autotomized an arm, we noticedthat it was always one of the largest armsthat was removed, confirming the observationof Rideout (1978).

Sequencing of the mitochondrial genefor cytochrome oxidase subunit I was per-formed to confirm the identifications of thetwo types of L. multifora and to examine therelationship between them. Phylogenetictrees (Figures 2 and 3) that included se-quences from L. guildingi (AF187937), Lcolumbiea (AF187935), and Fromia indica(AF187948) showed that all of the Lm Rarosequences (Genbank numbers DQ451832–DQ451839) clustered with L. multifora andL. laevigata (Genbank numbers AF187892–AF187921 plus AJ493187–AJ493190) andwere not more closely related to the sequencesof any other species of Linckia or other mem-bers of the family Ophidiasteridae.


Figure 2. Phylogenetic tree based on Maximum-Parsimony analysis of COI sequences from specimens of Linckiamultifora from Rarotonga (Lm Raro) (Genbank numbers DQ451832 to DQ451839) with sequences from Williams(2000) of Linckia laevigata (Ll) and Linckia multifora (Lm) from other localities (Genbank numbers AF187892–AF187921 plus AJ493187–AJ493190). The numbers above the branches are the base pair differences, and the boot-strap values (1,000 replicates) are shown below the branches. The Rarotonga Lm samples are identified as eitherfrom the intertidal (bg) or from the subtidal (red) locations.

Figure 3. Phylogenetic tree based on Neighbor Joining analysis of COI sequences as described in Figure 2.

PLATE I. Linckia multifora from Rarotonga. A, Specimens collected from intertidal location (reef top). B, Specimenscollected from subtidal location. Comets (arrows) and counter-comets (arrowheads) can be seen in both groups. Notethat the intertidal animals (A) are generally larger and have thicker arms relative to length than the subtidal ones (B).

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Figure 2 shows that haplotypes of COIfrom both types of L. multifora from Raro-tonga clustered within the two clades of L.multifora and L. laevigata from other local-ities. COI LmRaro sequences from the twoclades exhibit 12 fixed differences (Table 1).Both the Parsimony and Neighbor Joiningtrees (Figures 2 and 3) show that Lm Raro 1,2, 3, and 4 fall into Clade 2 of Linckia asdefined by Williams (2000). Internal to thenode that defines Clade 2, these fall into twogroups of sequences separated from the nodeby 1 and by 7 base pairs, respectively. A dis-tance of 4 base pairs separates the two clades,and a further 6 base pairs within Clade 1 leadsto an association of Lm Raro 5–8 with haplo-types from Australia, Fiji, and Guam. Se-quences from both the red, subtidal type ofL. multifora from Rarotonga and the bluegray intertidal form were found in both clades.

discussion

In this study we have described the presenceof two morphotypes of Linckia multifora thatare found in different environments on Raro-tonga, and we have studied the mechanismsof reproduction that they use. Linckia multi-fora has often been assumed to reproduce pri-

marily or only by asexual means through theprocess of autotomy. This occurs when a par-ent animal autotomizes an arm that then re-generates four arms (comet stage) while theparent (counter-comet) regenerates the autot-omized arm. Rideout (1978) felt that sexualreproduction probably was a sporadic phe-nomenon in this species and that populationson Guam successfully maintain themselvesthrough asexual reproduction. However, pho-tographs of samples taken at various sites inRarotonga suggested to us that here, at least,sexual reproduction must be common. If onlyasexual reproduction occurred, we would ex-pect that all of the comets would be regener-ating from arms of the largest size. Instead wefound that the individuals covered a wholerange of sizes and that comets were regener-ating from both very tiny (as small as 0.7 cm)as well as larger arms (up to ca. 5 cm) (PlateI). The very small arms come from very small(young) parents, because individuals alwayslose their largest arm to autotomy. This typeof size distribution suggests that the popula-tion consists of individuals of a whole varietyof ages since metamorphosis from the larvalstage. Although some variation in size canoccur in starfish by shrinkage, individuals inthese populations did not appear to be starv-

TABLE 1

Comparison of Mitochondrial COI Haplotypes of Linckia multifora (Lm) of Rarotonga with Haplotypes of Lm and Ll(L. laevigata) from Australia (WA) and Fiji

56 182 185 194 206 227 230 298 352 408 430 508Haplotype Base Positions Phenotype

HaplotypeCategory

GenBankNo.

Lm WA3 T T A A C T C G A C A T AF187929Lm Raro 1 (bg) T T A A C T C G A C A T gray, reef top Clade 2 DQ451832Lm Raro 2 (bg) T T A A C T C G A C A T gray, reef top Clade 2 DQ451833Lm Raro 3 (bg) T T A A C T C G A C A T gray, reef top Clade 2 DQ451834Lm Raro 4 (red) T T A A C T C G A C A T red, subtidal Clade 2 DQ451835Lm Raro 10 (red) T T A A C T C G A C A T red, subtidal Clade 2Lm Raro 5 (red) C C G G A C T A G T C C red, subtidal Clade 1 DQ451836Lm Raro 6 (red) C C G G A C T A G T G C red, subtidal Clade 1 DQ451837Lm Raro 7 (red) C C G G A C T A G T G C red, subtidal Clade 1 DQ451838Lm Raro 8 (bg) C C G G A C T A G T G C gray, reef top Clade 1 DQ451839Lm Raro 9 (red) C C G G A C T ? G T G C red, subtidal Clade 1Ll Fiji 2 C C G G A C T A A T G C AF187895

Note: The base position is given relative to Lm WA2. Underlining indicates the bases shared by the Clade 1 haplotype class and theLl Fiji 2 haplotype. Lm Raro 9 and Lm Raro 10 sequences were derived from a single clone each and therefore were not used for thephylogenetic tree or submitted to GenBank.


ing because they were turgid in appearance;in our experience, individuals that did not eatwhile being kept in the aquarium becamesomewhat flacid. As shown by dissection andsectioning of specimens, there is a high pro-portion of sexually mature individuals in thered, subtidal group (32–45%) and the bluegray intertidal group (76%). At the subtidalsites there is a greater abundance of smallspecimens (Figure 1), and the median size ofthe smallest specimens is 1 cm less than thoseat the intertidal site. At the intertidal site theindividuals are larger, and therefore a greaterproportion of the individuals are capable ofreproducing sexually as well as asexually.

The sequencing data strongly suggest thatthe two morphological types of L. multiforain Rarotonga form a single interbreedingpopulation. Each group contains the sametwo classes of haplotypes of COI (Table 1)with no consistent differences between them.If these two types of L. multifora were repro-ductively isolated we would expect that theywould have accumulated different sets of ran-dom mutations in the COI gene (Avise 2000).The occurrence of equally successful fertil-ization and early development for the fourcombinations of eggs and sperm from thesetwo types also suggests that the two popula-tions are capable of interbreeding. However,due to lack of facilities and methods to allowlater development and metamorphosis wecannot be certain that development of allcombinations can continue to adulthood.

The most likely explanation of these ob-servations is that sexual reproduction of L.multifora in Rarotonga occurs in the breedingseason (which includes the period from lateNovember to early December) and, afterentering the plankton, the larvae may prefer-entially settle at the Bernie’s Reef site orother subtidal sites. This would account forthe larger proportion of individuals of thesmaller sizes found at the subtidal sites aswell as the very large total number found atthe Bernie’s Reef site. From here the smallestadults may be dispersed to various other sitesby the waves and currents, or they may mi-grate toward the shallower areas, and thosearriving on the reef top may be triggered todevelop additional blue gray pigment as a

protection from excessive sunlight, which hasalso been suggested to occur in L. laevigatathat are found in shallow waters (Williams2000). In Rarotonga larval recruitment of L.multifora may also occur directly at all of thesites, and our lack of observation of verysmall specimens (less than 1.9 cm in thelength of the longest arm) at the intertidalsite may be due to the difficulty of seeingthem in this environment. In addition, atboth the intertidal and subtidal sites, autot-omy of arms followed by regeneration mustbe very important in maintaining the popula-tions, as shown by the substantial proportionof individuals that can be classified as comets(having one arm larger than the others) of10–15% at all sites. The presence of cometsshows that autotomized arms have success-fully developed into new individuals.

Sexual reproduction in L. multifora hadnot been examined since Mortenson (1938)described fertilization and development of avery few eggs in culture. It is now knownthat to obtain a high proportion of fertilizablestarfish eggs it is necessary to treat them witha maturation factor such as nerve extract or1-methyl adenine (Stevens 1970, Kanatani1973). Rideout (1978), who studied the L.multifora population in Guam, found thateggs were released from one specimen wheninjected with 1-methyl adenine but concludedthat the population in Guam was maintainedprimarily by asexual reproduction. Our find-ings that a high proportion of both male andfemale specimens collected from the field hadmature gonads during late November andearly December and that fertilization andearly development could be accomplished inthe laboratory suggest that sexual reproduc-tion probably occurs in nature. In addition,the presence of two distinctive types of COIsequences in both the reef-top and the subti-dal forms of L. multifora strongly suggeststhat sexual reproduction, which would resultin gene flow between different areas, hasoccurred.

Many observations have been made ofcryptic sympatric species or subspecies ofcoral reef organisms (reviewed by Knowlton1993). The two types of COI sequencesfound in the L. multifora of Rarotonga differ


in 12 base positions, amounting to a 2.3% dif-ference in the 524 base pair sequence. This isclose to the lower limit 2.5–24.2 differencefor COI found between nominal Asterinidaespecies (Hart et al. 1997), suggesting thatthere may be two cryptic subspecies or spe-cies within the L. multifora in Rarotonga.However, the accumulating evidence for spo-radic gene flow rather than continuous genemixing throughout the Pacific (Benzie 1999)can explain the apparent existence of crypticsympatric species or subspecies in some coralreef organisms, such as in L. guildingi(Williams 2000) as well as in L. multifora inRarotonga. A dual origin of L. multifora ofRarotonga is suggested by the COI sequenc-ing data, which did not reveal any intermedi-ates between the two categories of COIsequences having the 12 fixed differences.

The existence of the two differentmorphological types of Linckia multifora onRarotonga cannot be explained solely by theexistence of two founding groups becausethe two classes of haplotypes of COI arefound in both morphological types. One pos-sible explanation is that each morphologicaltype had a different geographical origin, butmitochondrial introgression has blurred the(sub)species boundaries. Another possible ex-planation for the two morphological typesis that they are ecophenotypes (i.e., theirpermanent differences have been triggeredby environmental effects during develop-ment). This phenomenon has been reportedin plants and colonial invertebrates (reviewedby Appleton and Palmer 1988) as well as insome solitary mollusks (Appleton and Palmer1988, Espoz et al. 2004). Williams (2000)invoked this mechanism to explain the obser-vation that L. multifora and L. laevigata can-not be distinguished on the bases of COIsequences and therefore may actually beecophenotypes of the same species. The exis-tence of ecophenotypes is probably the sim-plest explanation for the morphologicaldifferences between the intertidal and sub-tidal groups of L. multifora on Rarotonga,because the formation of additional darkpigment may protect against the effects ofsunlight on the intertidal organisms. In futureit may be possible to determine if the mor-

phological differences between the two typesof L. multifora (and also L. laevigata) are a re-sult of genetic differences or environmentaleffects if their development can be followedin the laboratory through metamorphosisand beyond.

acknowledgments

We thank Barry Hill and Eric Bateman ofDive Rarotonga for their assistance with col-lections and Michael Chan for assistance withPCR. We thank three anonymous reviewersfor their comments, which have greatly im-proved this work.

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linckia multifora (echinodermata: asteroidea) in rarotonga

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