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Proceedings of the Royal Society B: Biological Sciences logoLink to Proceedings of the Royal Society B: Biological Sciences
. 2011 Jul 13;279(1729):699–708. doi: 10.1098/rspb.2011.1035

High potential for formation and persistence of chimeras following aggregated larval settlement in the broadcast spawning coral, Acropora millepora

E Puill-Stephan 1,2,3,4, M J H van Oppen 3, K Pichavant-Rafini 4, B L Willis 2,*
PMCID: PMC3248722  PMID: 21752820

Abstract

In sessile modular marine invertebrates, chimeras can originate from fusions of closely settling larvae or of colonies that come into contact through growth or movement. While it has been shown that juveniles of brooding corals fuse under experimental conditions, chimera formation in broadcast spawning corals, the most abundant group of reef corals, has not been examined. This study explores the capacity of the broadcast spawning coral Acropora millepora to form chimeras under experimental conditions and to persist as chimeras in the field. Under experimental conditions, 1.5-fold more larvae settled in aggregations than solitarily, and analyses of nine microsatellite loci revealed that 50 per cent of juveniles tested harboured different genotypes within the same colony. Significantly, some chimeric colonies persisted for 23 months post-settlement, when the study ended. Genotypes within persisting chimeric colonies all showed a high level of relatedness, whereas rejecting colonies displayed variable levels of relatedness. The nearly threefold greater sizes of chimeras compared with solitary juveniles, from settlement through to at least three months, suggest that chimerism is likely to be an important strategy for maximizing survival of vulnerable early life-history stages of corals, although longer-term studies are required to more fully explore the potential benefits of chimerism.

Keywords: chimera, microsatellites, corals, Acropora millepora

1. Introduction

Chimerism is defined as the co-habitation of more than one genetically distinct cell line originating from more than one zygote within the same individual [1], and is known to occur naturally in at least nine phyla of protists, plants and animals [2]. Chimerism is also known from humans [3,4], but it has been reported more frequently in marine environments for benthic organisms with planktonic larvae or propagules, including seaweeds and colonial marine animals such as sponges, hydroids, corals, bryozoans and ascidians [5]. Chimerism typically follows allogeneic fusions (i.e. fusions between genetically different individuals of the same species), and may be most common in species for which fragmentation and fusion are normal features of the life cycle [6]. However, the formation of chimeras in broadcast spawning corals, the spatially dominant and numerically most abundant group of reef corals, has not been investigated.

Sessile, modular marine animals have a number of life-history traits that increase the probability of prolonged contact among neighbouring colonies, which enhances opportunities for fusion, and thus the potential for chimera formation [7]. In particular, their larvae tend to settle in proximity to one another [8] and adult colonies often come into physical contact when colonies increase in size through growth or after the movement of fragments produced through asexual reproduction (e.g. sponges, cnidarians, bryozoans and ascidians). The occurrence of chimeras in natural populations [7,9–12] suggests that fusion of non-identical conspecific genotypes is sometimes permitted, despite the fact that colonial marine invertebrates generally discriminate between clone mates and non-clone mates [13–16]. Rather than representing allorecognition failure, it is possible that, by potentially increasing survival of early life-history stages, chimerism is adaptive in sessile, colonial marine animals [17]. However, this hypothesis has not been explicitly tested. In cnidarians, chimerism has been well studied for brooding corals [15,17–28]; however, there has been only one study of chimerism in a broadcast spawning coral, and it focused on adult patterns of chimerism [29]. In studies of brooding corals, kin aggregations of larvae at settlement and subsequent fusion have been shown to promote the occurrence of chimerism in coral juveniles [17,27]. In addition, a delay in maturation of the coral allorecognition system [24] has been hypothesized to give rise to a ‘window in ontogeny’ during which fusion of genetically distinct conspecifics is facilitated [30]. Chimerism challenges evolutionary theory developed for genetically homogeneous individuals [31] and the commonly held view that clonality is a mechanism for maintaining genotypes intact, particularly for well-adapted lineages [32].

Recent evidence that chimerism occurs in wild populations of the broadcast spawning coral Acropora millepora [29] indicates that chimerism is not limited to brooding species with limited larval dispersal and raises questions about mechanisms promoting chimera formation in species with dispersive larvae. For broadcast spawning corals that breed annually and release thousands to millions of gametes with high synchrony [33], closely related larvae may remain aggregated in dense spawning slicks that form during low to moderate wind conditions typical of coral spawning seasons [34]. Larvae within spawning slicks tend to reach settlement competency at the same time [35–37], further increasing the probability that closely related larvae might settle in the same area, fuse and form chimeras. Hence, broadcast spawning corals may also have life-history traits that promote chimera formation.

Here, we investigate the potential for a broadcast spawning coral to form chimeras during early life-history stages and the fate of different genotypes following fusion when chimerism occurs. Acropora millepora, a common hermaphroditic broadcast-spawning coral, was selected as the model organism because, in addition to being abundant and widespread on the Great Barrier Reef (GBR), it is also the best-characterized coral at the molecular level, and molecular markers appropriate for exploring chimerism are readily available [38]. Nine polymorphic microsatellites were used to examine whether multiple genotypes co-occurring within the same colony persist over time, and whether persistence correlates with the level of genetic relatedness.

2. Material and methods

(a). Rearing of coral larvae

Eight mature colonies of Acropora millepora were collected from each of two field sites (Nelly Bay, Magnetic Island, and southwest Pelorus Island; both located in the central GBR and separated by 70 km) prior to the predicted spawning dates in November (Magnetic Island) and December 2006 (Pelorus Island). Colonies were transferred to 1000 l aquaria with running, temperature-controlled (28.5°C), 1 µm filtered sea water (FSW) at the Australian Institute of Marine Science for Magnetic Island corals and at Orpheus Island Research Station for Pelorus Island corals. The genotype of each coral colony collected was determined prior to spawning based on analyses of three microsatellite loci [38] to ensure colonies were genetically distinct and to avoid crosses between clone mates.

Colonies were isolated just prior to spawning in individual 70 l aquaria filled with FSW. Gametes from each of the five most prolific colonies were collected and mixed with gametes from one of the other four colonies in a new 70 l fertilization aquarium filled with FSW, according to the experimental design shown in figure 1a. Thus, three larval batches or broodstocks were produced, representing three full-sibling cultures (A, B and C). Following fertilization, embryos were cleaned in three consecutive water changes and transferred into 500 l larval culture aquaria supplied with running, temperature-controlled (28.5°C) FSW, at a density of approximately one larva per millilitre. Once swimming larvae demonstrated searching behaviour for settlement, larvae were transferred, at stocking densities of approximately 1 larva ml−1, into smaller aquaria (40 l), the bottoms of which were covered with nine settlement plates per aquarium (autoclaved 8 × 8 cm terracotta tiles).

Figure 1.

Figure 1.

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Schematic showing (a) experimental design for mixing gametes from different colonies (identified by numbers) to produce larvae that differed in kinship level (full-siblings, white square; half-siblings, grey square; non-siblings, black square), and (b) the number of replicates (indicated by numbers within each square) for combinations of broodstock larvae (A, B and C) that were mixed together and added to settlement aquaria. Overall, there were four replicate aquaria established for settlement purposes per kinship level. Note that both half-sibling and non-sibling mixtures also contained full-sibling larvae, such that any given larva had a similar probability (approx. 50%) of contacting full-sibling and either half-sibling or non-sibling larvae, respectively.

To establish mixtures of larvae with differing kinship levels for settlement purposes, two samples of approximately 20 000 larvae from the broodstock cultures were mixed into 40 l settlement aquaria. Larvae were distributed among settlement aquaria to create three kinship levels (full-sibling, half-sibling and non-sibling mixes, as illustrated in figure 1). Note that both half-sibling and non-sibling mixtures also contained full-sibling larvae, such that any given larva had a similar probability (approx. 50%) of contacting full-sibling and either half-sibling or non-sibling larvae, respectively. Each kinship level was established in four replicate settlement aquaria (figure 1b). Larval mixtures provided opportunities for larvae of varying genetic relatedness to settle on the same piece of substratum. Juveniles settling in groups of two or more recruits that were in physical contact with one another were defined as aggregations.

Twenty days after spawning, the laboratory-reared juvenile corals settled on terracotta tiles were placed in the field at Nelly Bay, Magnetic Island. Tiles were labelled, tagged and photographed prior to deployment in the field. In order to investigate the persistence of chimeras, tiles from Magnetic Island were monitored and photographed every four to eight weeks during the first year and less frequently in the second year until 23 months post-settlement.

Sampling for genotyping was performed six times (4–10 samples collected at each time point) from about 6 months through to 2 years after settlement. Juvenile colonies were sampled haphazardly at each sampling time without knowing their origin and without regard to size; however, healthy-looking colonies were preferentially sampled. At each sampling time, one recruit was haphazardly selected from each of 4–10 tiles, which were also haphazardly selected. Because of the small size of coral juveniles (less than 1 cm2), sampling resulted in sacrificing the whole colony. Colonies were subsampled and divided into either four fragments (named 1–4) or eight fragments (named 1–8) from 15 months post-settlement (when older and larger). DNA was extracted from each subsample and was genotyped using nine microsatellite loci (table 1). Samples were named according to: (i) broodstock origin of larvae in the mixture (thus AB represented a mixture of full sibling and half sibling larvae from broodstocks A and B); (ii) tile number (up to 36 depending on broodstock mixture); (iii) recruit ID letter (a–h; used to locate recruits on photographs and trace history since settlement); and (iv) the recruit subsample number (up to eight, depending on the size of the recruit, which governed the number of subsamples).

Table 1.

Primer mixes and associated microsatellites, dyes and concentrations for genotyping juveniles of A. millepora.

primer mix name microsatellite loci repeat motif associated WellRED dye concentration in 10× primer mix
MP2 Amil2_006 (CA)4TA(CA)4 D2 0.8 µM
MP2 Amil5_028 (TCACA)7TCAC, (TCACA)4; TCACTCACTCACA D3 0.8 µM
MP2 Amil2_002 (TG)10 D4 0.28 µM
MP3 Apam3_166 (AAT)28 D2 1.5 µM
MP3 Amil2_22 (AC)10 D3 1.0 µM
MP3 Amil2_23 (AG)7 D4 0.6 µM
MP5 Amil2_010 TA(TG)11 D2 0.5 µM
MP5 Amil2_012 GA(CA)6GA(CA)2 D3 0.3 µM
MP5 Amil2_007 (TG)7AG D4 0.5 µM
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(b). DNA extraction and genotyping

DNA was extracted using ‘Wayne's method’ [39]. DNA pellets were resuspended in 200 ml of 0.1 M Tris (pH = 9.0) and stored at 4°C. Microsatellite loci were amplified in 10 µl multiplex PCR reactions, in PTC-100 Peltier thermal cyclers. Three different primer mixes (MP2, MP3 and MP5; table 1), each amplifying three microsatellite loci developed for A. millepora [38], were used. Reactions contained 1 µl DNA template, 1 µl 10× primer mix, 5 µl 2× Qiagen multiplex PCR kit and 3 µl Milli-Q water. The cycling protocol was: 1 × 95°C (15 min), 35 × (30 s at 94°C, 90 s at 50°C and 60 s at 72°C), 1 × 60°C (30 min), and 4°C. Then, 2.5 µl of the diluted (1 : 10) PCR products were loaded, together with 37.25 µl of sample loading solution (Beckman) and 0.25 µl of 400 bp size standard, into a CEQ 8800 Genetic Analysis System from Beckman Coulter, for separation and subsequent PCR product size determination.

(c). Scoring

Once samples were run through the CEQ 8800, data were analysed with fragment analysis software from Beckman Coulter (400 Fragment Analysis parameter). All results were scored manually. Based on peak values for Negative Controls, peaks under 5 000 RFU were not scored. Fragment sizes were then entered into Microsoft Excel for further analysis.

Estimates of per locus mutation rates for multi-cellular clonal organisms are rare; however, there is a considerable literature on human and yeast mutation rates [40–42]. Assuming that one can extrapolate from humans and yeast to multi-cellular clonal organisms, we took 10−7 mutations per locus per cell generation as an estimate of the rate of somatic mutations per locus per cell generation for multi-cellular clonal organisms, as suggested by Orive [43] for Goniastrea aspera, Goniastrea favulus and Platygyrus sinensis [43]. Considering that the occurrence of two independent somatic mutations within the nine loci examined was highly unlikely, we interpreted the presence of different genotypes for two or more subsamples within a single recruit at two or more alleles as evidence that the colony is chimeric. Hence, when genotypes of subsamples from a single colony displayed at least two non-shared alleles and no obvious sign of tissue rejection was detected in photographic series (figure 4), the colony is considered a chimera.

Figure 4.

Figure 4.

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Summary of four different outcomes following settlement and growth of juveniles of A. millepora, showing (a) rejection reaction between three distinct genotypes (colony AC3d), (b) chimera originating from the fusion of two isolated juveniles that came into contact through growth (colony AC8e), (c) chimera originating from gregarious larval settlement (colony AB7c), and (d) a single isolated recruit, remaining as a single genotype from settlement to collection at 15 months (colony AC7e). Genotypes were determined based on analyses of nine microsatellite loci.

(d). Surface area and survival

To compare the size of colonies originating from solitary versus aggregated larval settlement, the surface area of coral juveniles was measured from photographs, using the software package Canvas (ACD systems), for 30 randomly selected colonies from each of the solitary and aggregated categories of juveniles raised at Magnetic Island. Colonies measured did not show signs of rejection among interacting genotypes. Measurements of each colony were taken at settlement and at monthly intervals until four months post-settlement. To compare survival of Magnetic Island juveniles in each category, the number of colonies originating from solitary versus aggregated larval settlement on each tile was counted at settlement and at monthly intervals until four months post-settlement.

(e). Analysis

Statistical tests were performed with Statistica v. 6.0. Normality of settlement, surface area and survival data were investigated with the Shapiro–Wilk test (if n < 50) or with the Kolmogorov–Smirnov test (with Lillefors significance correction). As the normality assumption was not met, a non-parametric Mann–Whitney U-test was performed for comparisons of the mean number and mean surface area of juveniles per tile originating from solitary versus aggregated larval settlement.

Relatedness between genotypes within chimeras and among genotypes of rejecting colonies was calculated with the Queller and Goodnight estimator in GenAlEx v. 6.1 [44]. Queller and Goodnight's pairwise relatedness estimator (QG) values are expected to be equal or higher than 0.5 (i.e. QG ≥ 0.5) for full-siblings. Half-siblings are expected to have values around 0.25, and QGs of unrelated individuals (i.e. non-siblings) are expected to be close to zero [45].

3. Results

(a). Gregarious versus solitary larval settlement

Under the experimental conditions of this study, more than 47 per cent of Acropora millepora juveniles monitored originated from gregarious larval settlement (n = 2168 juveniles examined; figure 2a). In the Magnetic Island study, the mean number of colonies per tile originating from gregarious settlement (14.3 ± 1.7) was similar to the mean number originating from solitary larval settlement (14.5 ± 1.5; U = 1473, n1 = n2 = 56, p = 0.580; figure 2a). However, significantly more (approx. 1.5-fold more) colonies originated from solitary polyps (10.2 ± 2.0) than from aggregations (7.0 ± 2.8) in the Pelorus Island study (U = 326.5, n1 = n2 = 32, p = 0.012; figure 2a). Nevertheless, because each aggregation represented a minimum of two fused recruits, overall, the majority of larvae settled in aggregations. Indeed, at least a twofold greater number of larvae settled gregariously in the Magnetic Island study (i.e. a minimum of 1600 larvae assuming that aggregations originated from only two larvae) and at least a 1.3-fold greater number settled gregariously in the Pelorus Island study (i.e. a minimum of 456 larvae).

Figure 2.

Figure 2.

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Mean (±s.e.) number of Acropora millepora juveniles per tile that settled in aggregations (grey bar) or solitarily (white bar) for (a) juveniles originating from spawning at Magnetic Island (n = 56 tiles) or Pelorus Island (n = 32 tiles) and (b) juveniles from the combined Magnetic and Pelorus Island studies originating from full-sibling cultures or from a mix of full-, half- and non-sibling larvae. Asterisk denotes a significant (p < 0.05) difference in the number of recruits originating from aggregations versus solitary juveniles in the Pelorus Island study. Numbers above each histogram represent the total number of juveniles counted in each category (i.e. numbers of recruits originating from aggregated versus solitary larval settlement).

To evaluate whether genetic relatedness was a driving factor behind aggregated settlement, the numbers of juveniles with aggregated versus solitary origins were compared between larval mixtures representing full-siblings only (i.e. AA, BB and CC larval mixtures in figure 1b) and larval mixtures including all relatedness groups (full-siblings, half-siblings and non-siblings; i.e. AB, AC, BC larval mixtures in figure 1b). No significant difference was found between the mean number (11.8 ± 2.9) of juveniles originating from aggregated settlement in full-sibling larval mixtures versus the mean number (11.6 ± 1.6) of juveniles originating from aggregated settlement in larval mixtures with the full range of kinship types (U = 860.5, n1 = 54, n2 = 34, p = 0.622; figure 2b).

Significant differences in the mean size of recruits derived from solitary versus gregarious larval settlement were detected both at settlement (U = 18, n1 = n2 = 30, p < 0.001; figure 3a) and at three months post-settlement (U = 185.5, n1 = n2 = 30, p < 0.001; figure 3a). Mean surface area of recruits originating from gregarious settlement was approximately threefold greater than mean surface area of recruits originating from solitary larvae at settlement and 2.5-fold greater at three months post-settlement.

Figure 3.

Figure 3.

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Comparisons among means (±s.e.) from the Magnetic Island study for (a) surface area of juvenile colonies of Acropora millepora that had originated from solitary (white bar, n = 30) versus aggregated (grey bar, n = 30) larval settlement at both settlement and three months post-settlement, and (b) survival of A. millepora juveniles originating from aggregated (grey squares) versus solitary (white squares) settlement in the four months post-settlement. Asterisk denotes significant (p < 0.05) differences in surface areas between colonies originating from solitary versus aggregated larvae.

No significant difference was observed in the mean number of recruits per tile originating from solitary versus aggregated settlement at any time in the first four months post-settlement (e.g. at three months: 4.6 ± 0.7 solitary recruits per tile versus 4.3 ± 0.7 aggregated recruits per tile; n.s., U = 1496, n1 = n2 = 56, p = 0.675; figure 3b). Consequently, mortality of both aggregated and solitary recruits was similar. The observed rates of approximately 50 per cent mortality after three months in the field are normal for Nelly Bay, Magnetic Island (B. Willis 2005, personal observation). Sources of mortality are mainly due to natural death through fouling by other organisms such as bryozoans and ascidians, Drupella sp. predation, and overgrowth by algae.

(b). Persistence of chimerism in coral juveniles

A total of 42 of the experimental juvenile colonies of A. millepora raised at Magnetic Island were sampled for genetic analysis over a period of 2 years (between settlement in November 2006 and termination of the study in October 2008). Half of the corals sampled were chimeras (i.e. 21 of the 42 colonies; table 2). Chimeras originated from fusion of recruits at settlement (i.e. 10 of the chimeric juveniles detected; figure 4c), from fusion of colonies coming into contact through growth (six cases; figure 4b) or potentially from both types of fusions (five cases, including AB4g). The origin of colony AB4g could not be determined as it was not observed at settlement because of its location on the underside of the tile.

Table 2.

Summary of fused versus isolated status of A. millepora juveniles raised at Magnetic Island and collected at six different sampling times, denoted as sampling periods: 1 (May 2007), 2 (July 2007), 3 (Sep 2007), 4 (Feb 2008), 5 (Mar 2008) and 6 (Oct 2008). Chimeras were identified based on nine microsatellites and are indicated in bold. Asterisk denotes cultures comprising only full-sibling larvae.

sample state at settlement (16 Nov 2006) genotype state at sampling (sampling period) comments during growth
CC8a* 1 juvenile 1 genotype 1 colony (1)
AC6a 1 juvenile 1 genotype 1 colony (1)
CC3c* 1 juvenile 1 genotype 1 colony (2)
AB14c 1 juvenile 1 genotype 1 colony (2)
AA1d* 1 juvenile 1 genotype 1 colony (3)
AC7e 1 juvenile 1 genotype 1 colony (4)
AC4g 1 juvenile 1 genotype 1 colony (5)
BC9k 1 juvenile 1 genotype 1 colony (6)
AC7k 1 juvenile 1 genotype 1 colony (6)
AC4d 2 isolated juveniles 2 genotypes 2 colonies fusing (3)
BB3a* 2 isolated juveniles chimera 1 colony (1) Apr 2007: contact and fusion
AC8c 2 isolated juveniles chimera 1 colony (2) Apr–May 2007: contact and fusion
BB4c* 2 isolated juveniles chimera 1 colony (2) June 2007: contact and fusion
BB4g 2 isolated juveniles chimera 1 colony rejecting BB4h (5) Aug 2007: contact and fusion, contact with BB4h and rejection (Jan 2008)
BB4h* 2 isolated juveniles chimera 1 colony rejecting BB4g (5) Aug 2007: contact and fusion, contact with BB4g and rejection (Jan 2008)
AC3d 3 isolated juveniles 2 genotypes 2 rejecting colonies (3) contact and fusion between two juveniles (Apr 2007), contact and rejection with 3rd juvenile in Aug 2007
BB4k = BB4g&h* 4 isolated juveniles 2 genotypes 2 colonies Aug 2007: contact and fusion by pair, contact and rejection of pairs (Jan 2008)
no obvious rejection (6)
BC8a 2 fused juveniles 1 genotype 1 colony (2)
AA5d* 2 fused juveniles 1 genotype 1 colony (3)
AB16a 2 fused juveniles chimera 1 colony (1)
BC9a 2 fused juveniles chimera 1 colony (1)
AC3c 3 fused juveniles 1 genotype 1 colony (2)
BC9b 3 fused juveniles chimera 1 colony (1)
AA5e* 3 fused juveniles chimera 1 colony (4)
AB4e 3 fused juveniles chimera 1 colony (4)
AC3e 3 fused juveniles chimera 1 colony (4)
AB1e 4 fused juveniles 1 genotype 1 colony (4)
AC1a 4 fused juveniles chimera 1 colony (1) Apr 2007: contact and fusion with another small colony
AA4c* 4 fused juveniles chimera 1 colony (2)
AB7c 6 fused juveniles chimera 1 colony (3)
BB6c* juvenile aggregation 1 genotype 1 colony (3)
AB1f juvenile aggregation 1 genotype 1 colony (4)
AB2a juvenile aggregation chimera 1 colony (1)
AB2b juvenile aggregation chimera 1 colony (1)
BB3c* juvenile aggregation chimera 1 colony (2) 2 separated colonies surviving, contact and fusion (June 2007)
AB2k juvenile aggregation + 2 juveniles chimera 1 colony
no sign of rejection (6)
BC7k 1 juvenile + aggregation 1 genotype 1 colony (6) contact and rejection with solitary juvenile, death of solitary (May 2007)
BC4c 1 juvenile and 4 fused juveniles 2 genotypes 2 rejecting colonies (2) May 2007: contact and rejection
AA3e* 3 fused juveniles + 1 isolated juvenile 1 genotype 1 colony (4) Sep 2007: contact and fusion
AC4e 2 fused juveniles + 2 isolated juveniles chimera 1 colony (4) Sep 2007: contact and fusion
AC8e 3 fused juveniles + 2 fused juveniles chimera 1 colony (4) Aug 2007: contact and fusion
AB4g unknown … chimera 1 colony (5)
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Fusion events were observed from the time of settlement (November 2006) until about 10–11 months post-settlement (September 2007, i.e. sampling period 3 in table 2). Fusion was assumed when no discontinuity in tissues could be observed along the zone of contact, and newly formed polyps appeared in the contact area (figure 4b, July 2007). Histological confirmation of fusion (and rejection reactions) could not be performed because juveniles were sacrificed for DNA analysis. Chimeras were found at every sampling time, even 23 months post-settlement (table 2).

A slight majority of chimeras (12 out of 21) were multi-partner chimeras, resulting from the association of three or more initial recruits (table 2). Eight of the remaining chimeras resulted from the fusion of two initial recruits (table 2), and were defined as bi-partner chimeras. As colony AB4g was not observed at settlement, it could not be defined as either a bi- or multi-partner chimera.

Colonies BC4c, AC3d, BC7k and the contact interaction between colonies BB4g and BB4h showed clear rejection reactions, characterized by a white line along tissue margins where the two colonies met in the contact zone, and clear tissue discontinuity (figure 4a). All rejections detected (four in total) originated from the rejection of juveniles coming into contact during growth. The first observations of a rejection reaction were noted approximately six months post-settlement (May 2007, sampling period 1 in table 2).

(c). Relatedness

Relatedness within chimeric colonies was high, with an average QG of 0.54 ± 0.01 (table 3). All genotypes within chimeric colonies were compared with each other (n = 58 paired genotype combinations). A large majority of genotypes within chimeric colonies were either full-siblings (n = 36 genotype comparisons with QG > 0.5) or half-siblings (n = 11 genotype comparisons with 0.25 < QG < 0.5). Hence, more than 62 per cent of the chimeras detected were full-sibling associations, rising to more than 81 per cent when half-siblings are included. Rejecting colonies (n = 3 paired genotypes) displayed variable levels of relatedness (QG = 0.20 ± 0.29; table 3). Through time, the percentage of fused colonies originating from full-sibling larval cultures and persisting as chimeras increased (figure 5). In the second year of the study, the percentage of full-sibling chimeras was nearly twofold greater than chimeras consisting of mixes of sibling and non-sibling offspring (figure 5).

Table 3.

Average (±s.e.) pairwise relatedness for chimeras versus all other paired genotypes, calculated according to the Queller & Goodnight [45] pairwise relatedness estimator.

samples average relatedness s.e.
chimeras (n = 58 paired genotypes) 0.54 0.04
rejections (n = 3 paired genotypes) 0.20 0.29
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Figure 5.

Figure 5.

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Percentage of chimeras detected from six months post-settlement (sampling period 1) up to 2 years post-settlement (sampling period 6), according to the kinship of the juveniles mixed together. Recruit numbers correspond to the total number of recruits genotyped (chimeras and non-chimeras) in each category.

4. Discussion

This study demonstrates that, under experimental conditions, larvae of the broadcast spawning coral A. millepora tend to settle in aggregations that enhance the likelihood of chimera formation. At least 1.5-fold more larvae settled gregariously than solitarily in the combined Magnetic Island and Pelorus Island experiments, and overall 47 per cent of juveniles originated from gregarious larval settlement. Amar et al. [17] recorded that 67 per cent of all newly settled juvenile colonies of the brooding coral Stylophora pistillata arose from aggregated larval settlement in experimental conditions. Further, aggregated larval settlement is commonly observed in the field and in experimental conditions in coral recruitment studies [17,27,46–48], and is considered to be a characteristic settlement pattern for a number of coral species [26,49]. This suggests that while larval densities in our experimental studies (approx. 1 larva ml−1) were likely to be substantially higher than densities occurring in the field, aggregated larval settlement is a naturally occurring phenomenon in A. millepora.

Fifty per cent of experimental juvenile colonies tested (n = 42 colonies) were chimeras. All chimeric colonies formed as a consequence of aggregated settlement, providing further support for our suggestion that aggregated larval settlement of broadcast spawning corals contributes to chimera formation in the field. Nevertheless, the extent of chimera formation and persistence was probably due to the high relatedness of the larvae used here.

Chimeric juveniles persisted for up to 2 years, indicating that allogeneic fusions can be stable for extended periods of time. The recent discovery that a minimum of 2 to 5 per cent of adult colonies are chimeric in populations of A. millepora on these same reefs [29] provides corroborative evidence that aggregated larval settlement gives rise to persistent allogeneic fusions. The field estimates for adult corals are likely to be underestimates because only eight fragments per colony were genotyped. The alternative explanation (i.e. that naturally occurring adult chimeras represent fusions between allogeneic fragments) is less likely, given the low likelihood of successful survival of fragments in this species because its morphology is dependent on an intact stalk (sensu [50]). The persistence of chimeric colonies of A. millepora for almost 2 years post-settlement, combined with evidence of chimerism in adult field populations, suggests that two or more genotypes are able to cohabit within the same colony indefinitely, with compatibility potentially arising from allelic similarities at currently unknown allorecognition loci.

Evidence that over 60 per cent of persistent chimeras detected formed as a consequence of full-sibling associations indicates that relatedness of larvae also plays an important role in chimera formation in this species. Although it is possible that non-related conspecific larvae form chimeras but only closely related individuals survive and maintain a chimeric state, the high relatedness we found between genotypes within chimeric colonies from the earliest sampling at six months suggests that the probability of allogeneic fusion and chimera formation is increased if coral planulae settle in kin aggregations. The limited pool of parents (n = 5 colonies per site; figure 1a) and subsequent relatedness of a large number of larvae in this study are likely to have contributed to the gregarious larval behaviour observed at settlement. Such an interpretation is consistent with studies that have shown that larvae of other colonial marine invertebrates tend to aggregate with closely related larvae [8,17,26,51], the net result being to minimize the frequency of rejections attributed to recognition of ‘non-self’. For example, in distribution studies of the ascidian Botryllus schlosseri, sibling colonies aggregated strongly, whereas unrelated colonies were significantly over-dispersed [51]. Taken together, our results suggest that the frequency of chimera formation in early life-history stages of corals is related to intrinsic gregarious settling behaviour of larvae and their level of kin relatedness. For broadcast spawning corals on the GBR, highly synchronized breeding [33,52], combined with larval development in dense spawning slicks [34] and comparatively short pre-competent periods [53,54] that promote synchronous settlement, would all favour aggregated settlement of closely related larvae.

A third factor probably contributing to the high potential for chimerism in A. millepora is delayed maturation of allorecognition mechanisms required to discriminate genetically distinct conspecifics. The first rejection reactions were observed approximately six months post-settlement, but fusions between allogeneic juveniles continued to occur for up to 10–11 months post-settlement (table 2). Thus, maturation of allorecognition mechanisms appears to take at least six months in A. millepora and may not be fully established even by 11 months post-settlement. Fusions among both aggregated conspecific larvae at settlement and conspecific juveniles coming into contact during their early growth may be facilitated by a ‘window in ontogeny’, during which self-recognition responses have not matured, as proposed by Rinkevich [27]. It is known that the period of time required for many marine invertebrates to acquire a mature state of allorecognition varies from less than two weeks after metamorphosis in the hydrozoan Hydractinia symbiolongicarpus [55] to more than two weeks in the bryozoan Celleporella hyalina [56], and up to four months post-settlement for the corals Stylophora pistillata [24] and Seriatopora spp. [28]. Interestingly, maturation of allorecognition in the brooding coral S. pistillata occurs in a step-wise manner, culminating in full allogeneic incompatibility at four months post-settlement [24]. In general, lack of an efficient allorecognition system in the early stages of ontogeny in scleractinian and soft corals is probably universal. However, rather than chimera formation representing allorecognition ‘failure’, such a universal pattern suggests that lack of precision in self-recognition might be adaptive. Consistent with the possibility that delayed allorecognition is adaptive, the non-selectivity of Symbiodinium uptake in the first few months post-settlement in acroporid juveniles [57] provides corroborative evidence that acroporid corals lack a mature allorecognition system early in life.

A major advantage of multi-partner kin aggregations at settlement is thought to reside in ensuing immediate and long-term increases in colony size [58]. Size-related benefits may overcome costs associated with interacting genotypes (for example, intra-colony competition resulting in colony fission if incompatible genotypes reject each other, and potentially absorption or mortality of one or more genotypes within chimeric colonies [17]). In our study, 12 out of a total of 21 chimeras were formed from the fusion of three or more juveniles (table 2); thus, chimeras were significantly larger in size (by almost threefold) than juveniles settling solitarily, even at three months post-settlement. Our results support hypotheses suggesting that chimera formation represents an important opportunity for substantial increases in the size of recruits, as well as in surface area occupied at settlement, both of which would be much greater than those possible through growth alone. Although escape in size is widely accepted as an important survival strategy for early life-history stages of sessile marine invertebrates [59], the lack of difference found for survival rates between chimeras and single-genotype juveniles in the first four months post-settlement raises questions as to whether the benefits of allogeneic fusion relate primarily to rapid increase in colony size. Longer-term studies of comparative mortality rates of chimeras and single-genotype colonies are needed to more fully evaluate potential adaptive benefits of chimerism, and may reveal differential size-related survival after four months post-settlement. Alternatively, benefits of chimerism may relate to the presence of increased genetic variation within colonies, which might have implications for the survival of later life-history stages. Higher stress tolerance of one genotype within a chimera could play an important role in ensuring at least partial colony survival following stress, and could explain observations of partially bleached corals following exposure to thermal stress.

In summary, juveniles of the broadcast spawning coral A. millepora show a high potential to form bi- and multi-partner chimeras following gregarious larval settlement. High potential for co-settlement of closely related larvae and delayed maturation of the allorecognition system also contribute to the propensity for fusion between genetically different individuals in early life-history stages of this species. While there may be costs associated with chimerism, its occurrence and persistence for up to 2 years in the present study and also in wild adult populations of this species [29] indicate that, at least in some cases, there are net benefits associated with chimerism. Further investigations of the benefits and costs of chimerism in modular marine invertebrates should provide important evolutionary insights into this little-studied aspect of coral life histories.

Acknowledgements

We thank David Abrego, Andrew Muirhead, Lesa Peplow, Francois Seneca and Andrew Negri for their very precious and enthusiastic help in the field and in the laboratory. Funding was provided by an Australian Research Council DP grant to B.L.W. and M.J.H.V.O.

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