Reference: Biol. Bull. 215: 98 –107. (August 2008) © 2008 Marine Biological Laboratory Articulated Coralline Algae of the Genus Amphiroa Are Highly Effective Natural Inducers of Settlement in the Tropical Abalone Haliotis asinina ELIZABETH A. WILLIAMS, ALINA CRAIGIE, ALICE YEATES, AND SANDIE M. DEGNAN* School of Integrative Biology, University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia Abstract. The initiation of metamorphosis in marine invertebrates is strongly linked to the environment. Planktonic larvae typically are induced to settle and metamorphose by external cues such as coralline algae (Corallinaceae, Rhodophyta). Although coralline algae are globally abundant, invertebrate larvae of many taxa settle in response to a very limited suite of species. This specificity impacts population structure, as only locations with the appropriate coralline species can attract new recruits. Abalone (Gastropoda, Haliotidae) are among those taxa in which closely related species are known to respond to different coralline algae. Here we identify highly inductive natural cues of the tropical abalone Haliotis asinina. In contrast to reports for other abalone, the greatest proportion of H. asinina larvae are induced to settle and metamorphose (92.8% to 100% metamorphosis by 48 h postinduction) by articulated corallines of the genus Amphiroa. Comparison with field distribution data for different corallines suggests larvae are likely to be settling on the seaward side of the reef crest. We then compare the response of six different H. asinina larval families to five different coralline species to demonstrate that induction by the best inductive cue (Amphiroa spp.) effectively extinguishes substantial intraspecific variation in the timing of settlement. invertebrate phyla, from sponges to echinoderms and urochordates. It is characterized by a planktonic larval dispersal phase that ends with settlement onto the substratum and metamorphosis into the benthic adult form. Settlement and metamorphosis can take place only after two conditions are met. First, planktonic dispersing larvae must have reached a developmental state known as competency, by which time they have a capacity to respond to environmental cues that induce settlement (Degnan and Morse, 1995; Hadfield et al., 2001). Second, competent larvae must actually encounter an appropriate environmental inductive cue, likely associated with suitable juvenile habitat (Pawlik, 1992). Although freeswimming pelagic larvae can potentially disperse throughout the ocean, the location of inductive cues determines when and where larvae will ultimately settle and live their adult reproductive lives. Clearly, the specificity and distribution of these external inductive cues is crucial in determining spatial and temporal patterns of marine invertebrate population structure and evolution (Rodriguez et al., 1993; Underwood and Keough, 2000). Intraspecific variation in response to inductive cues must in turn play a crucial role in determining the capacity for species range-shifting, local adaptation, and population differentiation. Coralline algae (Rhodophyta, Corallinaceae) are commonly reported inductive cues for marine invertebrate settlement and metamorphosis. Corallines or their associated biofilm have been demonstrated to induce settlement in the larvae of echinoderms (e.g., Rowley, 1989; Johnson et al., 1991; Swanson et al., 2006), annelid worms (e.g., Gee and Knight-Jones, 1962; Gee, 1965), molluscs (e.g., Barnes and Gonor, 1973; Heslinga, 1981; Rumrill and Cameron, 1983), soft corals (e.g., Sebens, 1983; Benayahu et al., 1989; Lasker and Kim, 1996), and scleractinian corals (e.g., Harrigan, 1972; Morse et al., 1988; Heyward and Negri, 1999; Kitamura et al., 2007). Even a sponge, arguably the oldest Introduction The pelagobenthic life cycle is by far the most common life cycle in the ocean, and is shared by almost all marine Received 19 August 2007; accepted 22 February 2008. * To whom correspondence should be addressed. E-mail: s.degnan@uq.edu.au Abbreviations: CoV, coefficient of variation; FSW, filtered seawater; hpf, hours postfertilization; hpi, hours postinduction; TCD, tissue culture dish. 98 99 NATURAL SETTLEMENT CUES IN ABALONE of the extant animal phyla, has recently been found to settle and metamorphose preferentially on a species of articulated coralline algae (Avila and Carballo, 2006). Given that “no other group of marine algae occupies so broad a range of habitats” as the corallines (Steneck, 1986), it is not surprising that such a disparate range of invertebrates have their key life-history transition associated with these abundant and diverse algae. The widespread distribution of coralline algae might suggest that one outcome of using these algae as an inductive cue would be to maximize the potential area of settlement. However, the larvae of at least some marine invertebrate species are known to respond to only one or a few species of coralline algae (e.g., Gee and Knight-Jones, 1962; Gee, 1965; Johnson et al., 1991) from among the total species number currently described (Guiry and Guiry, 2008). A particularly good documentation of this species-specific larval response is found among abalone (Haliotidae). The primary natural inductive cues of abalone are generally described as crustose coralline algae (Daume, 2006). However, veliger larvae of different abalone species respond preferentially to different species of coralline (reviewed by Roberts, 2001) and show an ability to distinguish their algal species of choice when presented with a number of options (Daume et al., 1999; Roberts et al., 2004). Due to the species-specific relationship between abalone and coralline algae, each abalone species must be tested to identify its unique, ecologically relevant inducer or inducers of settlement. No data of this kind currently exist for the tropical abalone Haliotis asinina (Linnaeus, 1758), the type species of the genus and the target of a growing aquaculture industry (Singhagraiwan and Doi, 1993; Capinpin et al., 1998; Gallardo and Salayo, 2003). H. asinina is found in association with coral reefs throughout tropical regions of the world (Geiger, 2000; Imron et al., 2007). Like other gastropods, H. asinina develops by spiral determinate cleavage to produce a free-swimming trochophore larva that undergoes torsion to become a veliger larva (Van den Biggelaar and Haszprunar, 1996). H. asinina larvae are lecithotrophic (planktonic-dispersing larvae that subsist on yolk supplied by the egg) and remain in the water column until they reach a state of competency between 3 and 4 days after fertilization (Jackson et al., 2005). Abalone do not settle spontaneously; they must be induced by an external cue or they will remain as larvae until their yolk stores run out and they die (Degnan and Morse, 1995). In this study, we identify natural inductive species of coralline algae for Haliotis asinina larvae, collected from Heron Island Reef, part of the southern Great Barrier Reef, Queensland, Australia. Additionally, we document the distribution of different types of coralline algae across Heron Island Reef in adult H. asinina habitat. We then compare the variation in response among independent H. asinina larval families to five coralline species, to demonstrate how vari- ation in larval induction response can change according to inductive cue species. Materials and Methods Collection and identification of coralline algae A hammer and chisel were used to collect samples of coralline algae species from Heron Island Reef, Australia (23o27⬘S; 151o55⬘E). In the laboratory at the Heron Island Research Station, samples were maintained with constant aeration and rapid flow-through of seawater. A portion of each coralline sample was washed in fresh water and dried in an oven at 65 oC for identification purposes. Each coralline sample was assigned a species name according to descriptions and keys from a number of sources (Adey et al., 1982; Woelkerling, 1988; Woelkerling and Harvey, 1992; Ringeltaube and Harvey, 2000; Littler and Littler, 2003; Guiry and Guiry, 2008). Characteristics used for identification included gross morphology; crust texture and thickness; reproductive conceptacle or sori abundance, shape, and size; hypothallus and perithallus cell arrangement and size; and trichocyte field abundance, orientation, and distribution in perithallus. Cell layers were examined by light microscopy. Where information was insufficient to identify a sample to species, samples were labeled according to growth form (e.g., thin encrusting sp.1). Algae were assigned to one of four gross morphological groups: encrusting, pseudobranching, branching, or articulated (Steneck, 1986; Lipkin and Silva, 2002) (Fig. 1). Dried portions of each sample were stored in separate airtight containers at A B C D Figure 1. Corallinaceae, Rhodophyta spp. Examples of different algal gross morphologies: (A) encrusting, (B) pseudobranching, (C) branching, (D) articulated. 100 E. A. WILLIAMS ET AL. room temperature (⬃23 oC ) to enable comparisons with future algal collections. Field survey of algal distribution on Heron Island Reef Six 150-m transects across the southern side of Heron Island Reef were performed to assess algal distribution in the adult abalone habitat. Transects ran perpendicular to the shore from mid-reef flat to beyond the reef crest, in the same part of the reef from which the abalone were collected. A 1 ⫻ 1-m quadrat was laid at 10-m intervals along the transect, from 10 to 150 m, and used to estimate the abundance of (1) crustose coralline algae, (2) branching coralline algae, (3) articulated coralline algae, and (4) area without coralline algae (e.g., sand, live coral, bare rock). The area occupied by coralline species represented areas of potential larval settlement. Transect data were separated into three zonal positions across the reef (reef flat, coral/dead coral matrix to reef crest, beyond reef crest) and summarized as stacked histograms showing percent mean and standard deviation of coralline algae coverage within a quadrat. The distribution of different algal types was then qualitatively compared to the results of settlement assays to discern the most likely areas of larval settlement on Heron Island Reef. Production and culture of abalone larvae Gravid H. asinina were collected 2–3 days prior to the full moon from the southern side of Heron Island Reef, and were maintained in holding tanks with flowing ambient seawater (21–26 oC) and aeration. On the night of a predicted spawning (Counihan et al., 2001), male and female brood stock were placed into individual buckets and allowed to spawn freely. Eggs were collected by successive siphoning through 250-␮m and 100-␮m wet screens and subsequently fertilized for 5 min in a 1-l beaker with sperm collected by syringe from male spawning aquaria. Fertilized eggs were thoroughly washed with 0.22-␮m filtered seawater (FSW) and left to develop until 9 h postfertilization (hpf), at which time hatched trochophore larvae were transferred into 300-mm-diameter larval culture chambers with a 190-␮m screen floor and flowing 10-␮m FSW. Larvae were maintained in culture chambers until competence (96 hpf; Jackson et al., 2005), when they were transferred by 100-␮m filter and micropipette (Gilson) for use in settlement assays. For the coralline variation settlement assays, larvae were derived from a mixed fertilization combining the eggs and sperm of at least three males and three females. For the family variation settlement assays, larval families were created by combining the sperm of a single male with the eggs of a single female. Each family was created using a different male and female; that is, no two larval families shared the same mother or father. Settlement assays All settlement assays were performed in 6-well, 35-mmdiameter sterile polycarbonate tissue culture dishes (TCD) with 10 ml of 0.22-␮m FSW. Prior to use in assays, coralline shards were cleaned using a toothbrush and thoroughly washed with 0.2-␮m FSW to remove epiphytic algae and diatoms. Shards were examined under a dissecting light microscope to ensure that they were free of potential predators (e.g., errant polychaetes). Epiphytic algal and encrusting marine invertebrate species, which may have influenced abalone settlement, were also removed using small brushes, scalpel, and tweezers. Corallines were not treated with antibiotics, as previous experiments have shown that bacterial communities have negligible effects on abalone settlement and that the primary inducer of abalone metamorphosis is a component of coralline algae (Morse and Morse, 1984; Huggett et al., 2005). Shards from all species were presented to larvae as essentially the same gross morphology (flat squares), although surface topography varied with algal species. Results were plotted in Excel 11.3.3 (Microsoft Corporation), and all further statistical analyses were performed in R Statistics System Version 2.3.1 (R Foundation for Statistical Computing). Coralline variation assay. Competent larvae were induced to settle and metamorphose by transferral into 6-well TCD containing 1 ⫻ 1-cm coralline shards. Three replicate experiments for each coralline species, with 30 larvae per replicate, were created. One TCD well was considered to be an independent replicate. Shards of the coralline Mastophora pacifica were collected from Redcliffe, Queensland (27o14⬘S; 153o6⬘E), and transported to Heron Island for use as a positive control. This species is known to induce about 80% metamorphosis in H. asinina (e.g., Jackson et al., 2005; Jackson and Degnan, 2006; Lucas et al., 2006). Although M. pacifica provides a useful positive control, is it essential to use inducers from the same location as the invertebrate population, that is, Heron Island Reef, to identify ecologically relevant inducers (H. asinina does not inhabit Redcliffe). Ten milliliters of 0.22-␮m FSW was used as a negative control. At 12-h postinduction (hpi), larvae were scored as settled if they were actively crawling on a coralline shard. At that time, it is not possible to determine whether larvae have actually initiated juvenile shell growth (definitive evidence of metamorphosis). Larvae were subsequently scored as metamorphosed at 24 hpi and 48 hpi if there was visible juvenile shell growth. After this initial assay, a similar experiment was conducted in which further species of the articulated coralline genus Amphiroa, which was indicated by the first settlement assays to be a likely primary inducer, were tested. Settlement data were plotted as histograms showing mean and standard deviation. A nested ANOVA was carried out on data from NATURAL SETTLEMENT CUES IN ABALONE the initial coralline variation assay (excluding data for thin encrusting sp1, which would confound data with shell growth values of 0) to assess the variation in settlement response of a mixed larval cohort to different coralline species, nested within different gross morphologies, as described under Collection and identification of coralline algae. Family variation assay. Larvae of six different families were induced at 96 hpf by transferral to 6-well TCDs containing 10 ml of 0.22-␮m FSW with 1 cm2 of coralline algae. Family response to five different coralline species— the encrusting Hydrolithon onkodes, the branching Lithophyllum moluccense, and the articulated Amphiroa ephedraea, A. beauvoissi, and A. tribulus—was tested. Three replicates of 30 larvae each were created for each family and coralline species, and 10 ml of 0.22-␮m FSW was used as a negative control. Larvae were scored as in coralline variation assays at 12, 24, and 48 hpi. Settlement data were plotted as scatter graphs showing family and coralline interactions, with mean and standard deviation. A two-way ANOVA was carried out to assess the effect of larval family and coralline inducer species on settlement response, as well as on the interactions between these two factors (data for the coralline H. onkodes were excluded due to multiple values of 0). Coefficients of variation for family settlement data were calculated to compare the extent of family variation occurring after induction by different species of algae. Results Settlement assays— coralline species Thirteen species of coralline algae were collected from Heron Island Reef for the initial settlement assay. Of these 13, we were unable to identify 4 specimens to species level, but the information we were able to obtain suggested that these represented different species. These specimens were designated thin encrusting sp.1, thin encrusting sp. 2, thick encrusting sp., and Lithophyllum sp. (this specimen was neither L. moluccense nor L. kotschyanum, but was definitely genus Lithophyllum). The 13 species represented 6 genera—Sporolithon, Hydrolithon, Pneophyllum, Lithophyllum, Neogoniolithon, and Amphiroa—and three subfamilies—Sporolithaceae, Mastophoroideae, and Lithophylloideae (Bailey et al., 2004). A variety of morphologies were represented, sometimes within a single genus, and included 7 encrusting species, 2 pseudobranching species, 2 branching species, and 2 articulated species (Fig. 2). Amphiroa spp. were the only articulated species found. The corallines tested in the initial settlement assay induced a range of responses in competent larvae, from 0% metamorphosis by 48 hpi (thin encrusting sp. 1) to 100% metamorphosis by 48 hpi (Amphiroa crassa) (Fig. 2). The FSW negative control induced 0% metamorphosis by 48 101 hpi, while the positive control Mastophora pacifica (from Redcliffe, Queensland) induced a mean of 80% metamorphosis by 48 hpi, consistent with previous studies (Jackson et al., 2005). Branching and articulated species were the most effective inducers of metamorphosis. Branching species induced between 77.6% and 95% of larvae by 48 hpi, while articulated species induced between 98.4% and 100% of larvae by 48 hpi (Fig. 2). Nested ANOVA indicated that the gross morphology of the coralline inducer had a greater influence on larval settlement and metamorphosis than did the species of coralline, although both were significant (Table 1). Shell growth at 48 hpi, as a definitive indicator of metamorphosis, was the trait that showed the greatest variability across different coralline inducer species. The second settlement assay tested three more species of articulated coralline from Heron Island Reef—Amphiroa ephedraea, A. fragilissima, and A. tribulus. Once again, negative control FSW induced 0% metamorphosis by 48 hpi, and positive control M. pacifica induced a mean of 80% metamorphosis by 48 hpi. All three species of Amphiroa induced very high levels of settlement and metamorphosis, with means between 95.4% and 100% of larvae with shell growth by 48 hpi (Fig. 3). Settlement assays—family variation Six different larval families were induced at competence by five different coralline species that represented a range of inductive capacities (see Figs. 2, 3)—the encrusting Hydrolithon onkodes, the branching Lithophyllum moluccense, and the articulated Amphiroa ephedraea, A. beauvoissi, and A. tribulus. Settlement results were similar to results from the mixed larval cohort, with H. onkodes inducing metamorphosis in the lowest percentage of larvae (0 –20.95% larvae with shell growth by 48 hpi) and the three Amphiroa spp. inducing the highest percentage (94.4%–100% larvae with shell growth by 48 hpi) (Fig. 4). The greatest variation among families occurred when induced by L. moluccense, with metamorphosis ranging from 52% to 94.4% shell growth by 24 hpi, and from 75.8% to 97.8% shell growth by 48 hpi (Fig. 4). Two-way ANOVA indicated that at all time points, changing the species of inducer significantly influenced levels of larval settlement and metamorphosis (F ⫽ 15.62–32.5, P ⬍ 0.001) (Table 2). Significant variation on a smaller scale also occurred between different families, but only in percentage of larval metamorphosis by 24 hpi (F ⫽ 6.02, P ⬍ 0.001) (Table 2). Two-way ANOVA also showed that family of larvae interacted significantly with species of coralline inducer to influence levels of larval metamorphosis at 24 hpi (F ⫽ 2.2, P ⬍ 0.05). Coefficients of variation show that variation in settlement and metamorphosis between families is much higher when induced by H. onkodes or L. moluccense (CoV ⫽ 15.1%– 81.5%) than when induced by any Amphiroa spp. (CoV ⫽ 2.4%– 4.9%) 102 E. A. WILLIAMS ET AL. settlement 12hpi shell growth 24hpi shell growth 48hpi en ps ar br 100 90 % larvae / postlarvae 80 70 60 50 40 30 20 10 0 te1 the1 te2 lsp se ho pc hb hl lm nc ab ac Coralline Species Figure 2. Haliotis asinina. Induction of competent larvae by different species of coralline algae collected from Heron Island Reef for a settlement assay. Histograms show percent of larvae settled on algae at 12 hours postinduction (hpi) and percent of postlarvae with shell growth (a definitive indicator of metamorphosis) at 24 and 48 hpi (mean ⫹ SD, n ⫽ 3, 30 larvae per replicate). Algae are grouped according to gross morphology as indicated by solid lines along top of graph; en ⫽ encrusting, ps ⫽ pseudobranching, br ⫽ branching, ar ⫽ articulated. Species tested in initial assay were te1 ⫽ thin encrusting sp1, the1 ⫽ thick encrusting sp1, te2 ⫽ thin encrusting sp2, lsp ⫽ Lithophyllum sp., se ⫽ Sporolithon erythraeum, ho ⫽ Hydrolithon onkodes, pc ⫽ Pneophyllum conicum, hb ⫽ Hydrolithon breviclavium, hl ⫽ Hydrolithon laeve, lm ⫽ Lithophyllum moluccense, nc ⫽ Neogoniolithon clavacymosum, ab ⫽ Amphiroa beauvoissi, ac ⫽ Amphiroa crassa. (Table 3), although we acknowledge a potential confounding effect of boundedness constraining the latter. Table 1 Haliotis asinina: nested ANOVA of the response of larvae to different gross morphologies and species nested within gross morphologies of coralline algae at settlement at 12 hours postinduction (A) and shell growth (metamorphosis) at 24 (B) and 48 (C) hours postinduction Factor A. Settlement 12 hpi Morph Morph (Spp) B. Shell growth 24 hpi Morph Morph (Spp) C. Shell growth 48 hpi Morph Morph (Spp) Df Sum Sq Mean Sq F-value Pr (⬎F) 2 9 17157.9 5011.8 8579.0 556.9 70.05 9.6E-11*** 4.55 1.4E-03** 2 9 17579.7 4804.6 8789.9 533.8 48.67 3.6E-09*** 2.96 1.6E-02* 2 9 38026 8745 19013 972 121.94 2.67E-13*** 6.23 1.6E-04*** Significance codes: *** ⫽ 0; ** ⫽ 0.001; * ⫽ 0.01. Algal distribution Coralline algae community composition differs between zones across the reef (Fig. 5). One factor common to all zones is the abundance of crustose coralline algae, which inhabit almost all potential substrate, defined as any hard substrate—that is, any substrate other than sand (Dethier, 1994). Amphiroa spp. were the only genus of articulated coralline found; therefore the articulated corallines group is hereafter referred to as Amphiroa spp. Transect data indicate that the zone closest to shore (Zone 1) has the highest percentage of substrate unoccupied by corallines (mean coverage 47.4%, uninhabitable by corallines), while crustose coralline algae dominate on the rocks, rubble, and coral heads that are scattered in this area (mean coverage 52%). Branching and articulated Amphiroa spp. corallines are very 103 NATURAL SETTLEMENT CUES IN ABALONE 100 A 90 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 % larvae / postlarvae B Figure 3. Haliotis asinina. Induction of competent larvae by different Amphiroa spp. collected from Heron Island Reef for a follow-up settlement assay. Histograms show percent of larvae settled on algae at 12 hours postinduction (hpi) and percent of postlarvae with shell growth (a definitive indicator of metamorphosis) at 24 and 48 hpi (mean ⫹ SD, n ⫽ 3, 30 larvae per replicate). Species tested in follow-up assay were ae ⫽ Amphiroa ephedraea, af ⫽ Amphiroa fragilissima, at ⫽ Amphiroa tribulus. Mastophora pacifica (mean 80% shell growth by 48 hpi) and 0.22 ␮m FSW (0% shell growth by 48 hpi) were included as positive and negative controls (not shown). 100 90 80 70 60 50 40 30 20 10 0 C 100 90 80 70 60 50 40 30 20 rare in this zone (mean coverage 0.48% and 0.29%, respectively). The second zone, an area dominated by living and dead coral matrix substrate, comprises almost solely coralline algae (mean coverage 73%), with branching corallines only slightly more abundant than in the first zone, and Amphiroa spp. almost nonexistent. In the third zone, beyond the live/dead coral matrix and over the reef crest, algal composition changes such that while encrusting corallines are still the most abundant algae (mean coverage 86.5%), the presence of Amphiroa spp. (mean coverage 29.17%) is much higher than in the inshore areas, with the articulated species Amphiroa fragilissima often growing among fleshy Laurencia intricata (S. D., pers. obs.). All mean coverage values have high standard deviations due to the patchy nature of both potential substrate and corallines (Fig. 5). 10 0 Family A. ephedraea A. beauvoissi A. tribulus L. moluccense H. onkodes Figure 4. Haliotis asinina. Induction of 6 families of competent larvae by 5 species of coralline algae. Interaction plots show (A) settlement on algae at 12 hours postinduction (hpi), (B) postlarvae with shell growth at 24 hpi, (C) postlarvae with shell growth at 48 hpi. FSW (0% shell growth by 48 hpi, not shown) was included as a negative control for each family. 104 E. A. WILLIAMS ET AL. Table 2 Haliotis asinina: two-way ANOVA of the response of 6 larval families to 4 coralline species: (A) settlement 12 hours postinduction; (B) shell growth (metamorphosis) 24 hours postinduction; (C) shell growth (metamorphosis) 48 hours postinduction Factor A. Settlement 12 hpi Family Coralline Family X Coralline Residuals B. Shell growth 24 hpi Family Coralline sp. Family X Coralline Residuals C. Shell growth 48 hpi Family Corraline sp. Family X Coralline Residuals Df Sum Sq Mean Sq F-value Pr (⬎F) 5 3 15 48 1204.5 7247.2 1197.8 3568.0 240.9 2415.7 79.9 74.3 3.2408 32.4991 1.0743 0.01336* 1.282e-11*** 0.40352 5 3 15 48 2925.8 8052.8 3190.3 4667.8 585.2 2684.3 212.7 97.2 6.0173 27.6029 2.1871 0.0002124*** 1.605e-10*** 0.0206388* 5 3 15 48 517.93 2536.62 968.93 2597.58 1.9141 15.6245 1.1936 0.1094 3.185e-07*** 0.3089 103.59 845.54 64.6 54.12 Data for Hydrolithon onkodes were not included due to confounding values of 0. Signifance codes: *** ⫽ 0; ** ⫽ 0.001; * ⫽ 0.01 Discussion Because of their ability to induce settlement, metamorphosis, or both, of larvae, coralline algae play a crucial role in the life cycle of diverse pelagobenthic marine invertebrates. The specificity and location of this cue in the benthic environment determines where larvae can settle and thus can significantly influence population structure. In the tropical abalone Haliotis asinina, competent larvae settle and metamorphose in response to articulated Amphiroa spp. significantly more than to crustose species. Amphiroa spp. have not been described as a primary natural inducer for any other abalone species. Most studies of the natural inducers of abalone focus on crustose coralline algae, although often this algae is not identified to species level, making comparisons difficult. Fewer studies include articulated algae in their tests of species-specific response (Morse and Morse, 1984; Roberts, 2001; Huggett et al., 2005). In these studies, larvae always responded in higher numbers to crustose coralline algae. It is unusual for an abalone species to respond as poorly to crustose coralline algae as Haliotis asinina does. For example, all encrusting coralline species induced metamorphosis in H. iris larvae, if presented individually, with more than 88% of larvae settling within 1 day and more than 80% metamorphosing within 3 days (Roberts et al., 2004). The close relationship between specific abalone and coralline species may reflect an adaptive coevolution between these species (Morse et al., 1979). One possible explanation for the disparity in settlement response between Haliotis asinina and other abalone species is that H. asinina is a tropical species, while species previously examined were temperate species. Other differences in induction of settlement and metamorphosis have previously been reported between H. asinina and temperate abalone species. Most notably, temperate species and the semitropical species H. diversicolor require significantly higher concentrations, compared to H. asinina, of gamma-aminobutyric acid (GABA)—a neurotransmitter thought to mimic the inductive peptides found in coralline algae (Morse et al., 1979)— or of potassium chloride (KCl) to induce larval Table 3 Haliotis asinina: coefficients of variation (mean % ⫾ SD) of the response of six larval families to five coralline species at settlement 12 hours postinduction and shell growth (metamorphosis) 24 and 48 hours postinduction Time Hydrolithon onkodes Lithophyllum moluccense Amphiroa ephedraea Amphiroa beauvoissi Amphiroa tribulus 12 hpi 24 hpi 48 hpi 81.5 ⫾ 5.8 28.9 ⫾ 7.1 45.1 ⫾ 49.5 20.9 ⫾ 1.2 26.6 ⫾ 17.0 15.1 ⫾ 9.9 3.5 ⫾ 3.0 3.7 ⫾ 2.3 3.4 ⫾ 1.5 3.1 ⫾ 2.0 3.0 ⫾ 4.2 2.4 ⫾ 1.5 4.9 ⫾ 4.0 4.3 ⫾ 3.2 2.6 ⫾ 1.9 NATURAL SETTLEMENT CUES IN ABALONE 100% % coverage within quadrat 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% CCA BrCA Amphi No CA mid reef flat outer reef flat (52 ± 35) (0.48 ± 1.5) (0.29 ± 0.94) (47.4 ± 35.1) (0.58 ± 1.34) (0.03 ± 0.09) (86.5 ± 20.98) (0.37 ± 1.15) (29.17 ± 38.58) (26.9 ± 35.93) (13.47 ± 20.97) (73 ± 36) reef slope Figure 5. Rhodophyta, Corallinaceae spp. Algal distribution in adult Haliotis asinina habitat. Distribution of key algae species on south side of Heron Island Reef. Values represent percent coverage of each type of algae within a quadrat (mean ⫾ SD) on mid-reef flat, outer reef flat, and reef slope. CCA ⫽ crustose coralline algae, BrCA ⫽ branching coralline algae, Amphi ⫽ articulated Amphiroa spp., No CA ⫽ area not occupied by corallines. Six 150-m transects were carried out during 2006/07, measuring algal abundance in a 1 ⫻ 1-m quadrat every 10 m. Amphiroa spp. were the only genus of articulated coralline present. Amphiroa spp. and crustose coralline distribution overlapped on the reef slope. Standard deviations were high due to the patchy distribution of algal species. metamorphosis (Gapasin and Polohan, 2004). These findings, and the results of our study, corroborate phylogenetic analyses showing that tropical Indo-Pacific abalone species form a clade separate from temperate Australian species (Geiger, 1999; Estes et al., 2005; Degnan et al., 2006). Perhaps tropical abalone such as H. asinina have evolved a system of algal detection and subsequent induction distinct from that of temperate species, the result of a coevolution with certain branching or articulated coralline species. However, studies on a greater number of tropical abalone species, in addition to past and present distribution data for different coralline species, are required before any clear trends can emerge. Articulated Amphiroa spp. were the best inducers of H. asinina larvae, with in some cases an astonishing 100% of larvae metamorphosing by 48 h postinduction. Our transect data on distribution of different coralline morphologies indicate that Amphiroa spp. are abundant only down the reef slope, suggesting that H. asinina larvae transported from the open ocean are settling immediately upon the first sign of Heron Island Reef, on the seaward side of the reef crest. Our transects on Heron Island Reef did not reveal any other genera of articulated coralline; however, it would be of interest to test the effect of some of these other algae to 105 determine whether H. asinina has a high settlement response to more than one genus of articulated coralline. In Haliotis asinina, settlement response is not affected solely by species of algal inducer. Differences in larval genotype can also result in variation in timing of settlement (Jackson et al., 2005). Our study showed that significant variations between different families induced by the same coralline species occurred in shell growth at 24 h postinduction, which we consider to reflect the rate of initiation of metamorphosis. Different families also showed variable induction responses when exposed to different coralline species. Variation in timing of metamorphosis within a species has also been recorded in other molluscs and is likely to be maintained within a population (e.g., Hadfield, 1984; Krug, 2001). This temporal variation results in larvae from a single cohort dispersing over a range of distances, thereby increasing the chances of encountering appropriate inductive cues. Our results indicate that although variation within a population can be significant, the composition of the algal community will play a more substantial role in shaping the extent of this variation. Coefficients of variation indicate that when induced with the best natural inductive cue, Amphiroa spp., any variation in timing of metamorphosis between families is effectively extinguished. We therefore predict that in the field, algal communities dominated by Amphiroa spp. would be likely to support a highly genetically diverse adult H. asinina population. The encrusting Hydrolithon onkodes represents an interesting inductive cue, considering that in family experiments, only 3 of 6 families contained any larvae that were able to metamorphose in response to this algae. In the initial settlement assay for H. onkodes, in which a genetically mixed cohort of larvae was used, levels of metamorphosis were higher overall than in single families, suggesting that only certain families or individuals possess the ability to respond to this algae. An algal community dominated by H. onkodes would therefore be likely to support a genetically limited adult H. asinina population, with implications for population divergence and potential speciation. Studies of variation in settlement and metamorphosis at the level of the individual would likely be an informative addition to current knowledge, as would data on the distribution, both current and past, of all types of coralline algae in abalone habitat. Overall, Haliotis asinina larvae showed a broad range of responses across the 16 coralline species tested. The results of our initial settlement assays suggest that the induction response of H. asinina increases with increased algal branching morphology (from encrusting to branching to articulated). In settlement assays with H. laevigata, the surface characteristics of coralline species were found to influence settlement, but they were not considered the main factors of variation (Daume et al., 1999). 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