Linckia multifora (Echinodermata: Asteroidea) in Rarotonga, Cook Islands: Reproductive Mechanisms and Ecophenotypes1 Terry J. Crawford2 and Bruce J. Crawford3 Abstract: In Rarotonga, Linckia multifora (Lamarck) exists in two forms: a blue gray 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, as well as sexually, but the relative potential for sexual reproduction varies greatly between these different sites. In the laboratory, reciprocal crosses of the blue gray intertidal form and the red subtidal form developed as successfully as the controls and were indistinguishable in morphology. In addition, both the blue gray intertidal form and the red subtidal form contain two different classes of haplotypes of the mitochondrial gene cytochrome oxidase subunit I (COI), which exhibit 12 fixed differences. These results suggest that L. multifora of Rarotonga has a dual origin and that the two different forms seen in the two environments belong to a single interbreeding population and may represent ecophenotypes. Linckia multifora (Lamarck) is a small reef in the intertidal zone. To confirm that sea star, a member of the Ophidiasteridae, these are both L. multifora and to explore the that is found on coral reefs throughout the relationship between them we have sequenced Indo-Pacific Ocean (Clark and Rowe 1971). a mitochondria gene, cytochrome oxidase It generally has arms of uneven lengths be- subunit I (COI). cause it reproduces asexually by autotomizing To understand aspects of the ecology and arms, which can then regenerate into whole evolutionary biology of an organism, it is im- starfish (Edmondson 1935). It is identified portant to understand its mechanism(s) of re- by the arrangement of spines along the am- production (Hart 2002). Linckia multifora is bulacral grooves, the presence of two madre- generally considered to reproduce primarily porites, the arrangement of papular openings, by asexual means (Rideout 1978), and it has the granularity of the epidermis, and the gen- been referred to in the literature as an asexual eral shape of the arms (Clark and Rowe 1971). organism (Ebert 1996). However, it produces On the island of Rarotonga, Cook Islands, eggs and sperm (Mortensen 1938, Rideout South Pacific, there appear to be two differ- 1978), and viable cultures of embryos have ent types of L. multifora: a red-spotted form been produced in the laboratory (Mortensen that is found in the subtidal zone and a gray 1938). In this study we set up breeding ex- blue form that is found on the top of the periments with both types of L. multifora to see if viable embryos would result from both types and from both crosses. In addition, we studied the population size structure at sev- 1 Manuscript accepted 30 September 2006. 2 Corresponding author: Departments of Zoology eral different sites to determine whether sex- and Botany, University of British Columbia, 6270 Uni- ual as well as asexual reproduction occurs in versity Boulevard, Vancouver, British Columbia, Canada nature and to determine whether the relative V6T1Z4 (e-mail: [email protected]). importance of larval recruitment varies be- 3 Island Medical Program, University of Victoria, P.O. Box 1700, Stn CSC, Victoria, British Columbia, tween different sites. Canada V8V2Y2. materials and methods Pacific Science (2007), vol. 61, no. 3:371–381 : 2007 by University of Hawai‘i Press Specimens were collected from subtidal loca- All rights reserved tions from depths of approximately 7 to 30 m 371 372 PACIFIC SCIENCE . July 2007 and from intertidal locations by walking and Bernstein (1989) with the addition of 0.5 on the reef at low tide or by snorkeling. The mg/ml proteinase K to the extraction solution. results of these collections were recorded After two extractions with phenol:chloroform: by photography with a size marker and color isoamyl alcohol (25:24:1) and precipitation of standard. Specimens to be used as a source of DNA from the aqueous phase with ethanol, gametes for embryological studies were then the DNA pellet was washed with 70% etha- kept in an aquarium for a few days, and speci- nol, air-dried, dissolved in distilled H2O, and mens for DNA extraction were preserved in stored at –80C. 70% ethanol. In some cases specimens were PCR reactions were carried out with Plati- taken with coral rubble to Vancouver, Can- num Taq DNA polymerase (Gibco) (0.5 ml), ada, where they were kept in an aquarium 0.1 mM primers, 2.5 mM MgCl2, 0.2 mM for up to 1 yr. deoxynucleotide triphosphates, and approxi- To set up cultures of embryos, gonads mately 200 ng template DNA in a thermo- were dissected from the arms and seawater cycler (Applied Biosystems 2400) with the containing 0.1 mg/ml 1-methyl adenine was following primers for COI: LINF 50-GCR applied to the ovaries to cause release and CCR GAT ATG GCR TTY CCA-3 0 and maturation of the eggs as evidenced by ger- LINR 50-CCT ATW GAT ACD ATR minal vesicle breakdown (Stevens 1970, Ka- GCR TAR ACC ATT –30. natani 1973). The mature egg suspensions of The primers were designed from the se- each type were washed in seawater, divided quence of LmWA3 (Genbank number AF into two parts, and fertilized with diluted 187929). The forward primer begins four sperm suspension of each type. The latter bases from the start of that sequence and the was prepared by mixing one or two drops of reverse primer extends to four bases from the ‘‘dry’’ sperm from harvested testes with 50 ml end of the LmWA3 sequence. The amplifica- of seawater. Each culture consisted of 100 or tion conditions were as follows: denaturation more fertilized eggs. for 3 min at 95C followed by 30 cycles of To determine whether specimens of 95C for 30 sec, 45C for 45 sec, 72C for 60 specific sizes contained mature gonads, two sec, followed by a final extension of 10 min. approaches were used. Specimens collected The PCR product was electrophoresed on in late November and early December were a 1% agarose gel in TAE. After staining with measured, injected with 1-methyl adenine, ethidium bromide the approximately 600 base and if that failed to produce spawning, the pair COI fragment was recovered using an gonads were isolated and treated with 1- UltraClean 15 DNA Purification System kit methyl adenine as already described. The (MOBIO) and cloned into pGEM-T Easy presence or absence of ripe gonads was re- Vector (Promega) according to the supplied corded for 17 red, subtidal specimens and 9 protocol. Plasmid DNA was extracted from blue gray intertidal specimens. In addition positive colonies with a Wizard Plus Mini- seven specimens of the blue gray intertidal preps DNA Purification System kit (Pro- type that had been collected from Town Reef mega), and the insert was sequenced using and preserved in 70% ethanol were mea- Big Dye3 Terminator Chemistry at the Uni- sured, and about 1 cm of the proximal por- versity of British Columbia Nucleic Acid tion of the largest arm of each was sent to Protein Service Unit. Wax It Histology Services, Vancouver, Brit- Sequences were aligned with the Clustal ish Columbia, to be decalcified, embedded in X program. The aligned sequences were paraffin, sectioned, and stained with haema- converted to the PAUP/NEXUS format toxylin and eosin. A collection of six red, sub- and then analyzed with the PAUP program tidal L. multifora was similarly treated. (Swofford 2003) using Maximum Parsimony For polymerase chain reaction (PCR) and Neighbor Joining with the default set- studies, specimens were rehydrated overnight tings, except that the maximum trees limit and chopped into small pieces. DNA was ex- was set to 10,000 for Parsimony, and the tracted according to the method of Zyskind Neighbor Joining distance setting was Ki- Linckia multifora in Rarotonga, Cook Islands . Crawford and Crawford 373 mura 2-parameter. The robustness of the well as counter-comets. Some of the comets branch points of the resulting phylograms were formed from very tiny legs (less than was determined by performance of 1,000 0.7 cm) and others were regenerating from bootstrap replicates. larger ones (Plate IB). Of the specimens in Figure 1B and C, 13.7% and 14.9%, respec- results tively, were comets. Figure 1 also shows that the subtidal sites had the greatest frequencies Two morphologically different populations of small individuals. of L. multifora were found at various loca- We considered the possibility that the tions on and outside the fringing reef on the blue gray form of L. multifora might be a ju- island of Rarotonga, Cook Islands. One type venile form of L. laevigata or a hybrid of the of specimen that taxonomic keys (Clark and red form of L. multifora with L. laevigata, a Rowe 1971) indicated to be L. multifora was closely related species that Williams (2000) found on the top of the fringing reef at low found to be indistinguishable on the basis of tide. These sea stars were a dark gray blue, COI sequences. Although the blue gray L. and some had red purple patches (Plate IA). multifora and the royal blue L. laevigata could They were found in small tide pools on peb- sometimes be found only a few feet apart and bles or partially buried among pebbles in the were ripe at the same time, there were no surf. Their sizes ranged from 1.9 to 5.3 cm specimens of intermediate phenotype regard- along the longest arm with a median value of ing color or arm shape (pointed and irregular 3.5 cm (Figure 1A). Almost all had arms of in length for L.