Journal of Heredity Advance Access originally published online on September 6, 2006
Journal of Heredity 2006 97(5):493-498; doi:10.1093/jhered/esl018
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Gene Diversity and Mode of Reproduction in the Brooded Larvae of the Coral Heteroxenia fuscescens
From the Department of Biology, University of Haifa–Oranim, Tivon 36006, Israel (Fuchs and Ben-Shlomo); and the National Institute of Oceanography, Israel Oceanographic and Limnological Research, Tel Shikmona, PO Box 8030, Haifa 31080, Israel (Douek and Rinkevich). Yaron Fuchs is now at the Department of Biology, Technion—Israel Institute of Technology, Haifa 32000, Israel
Address correspondence to R. Ben-Shlomo at the address above, or e-mail: ekly{at}research.haifa.ac.il.
The soft coral Heteroxenia fuscescens is a common shallow-reef brooding species in the Red Sea. By means of amplified fragment length polymorphism markers, we studied modes of reproduction of planulae (sexual vs. vegetative) and levels of gene diversity of a population residing in the northern Gulf of Eilat, Red Sea. Eighty-nine larvae were collected from 9 mother colonies at 3 locations over a distance of 5 km. Amplifications revealed 221 putative loci, of which 81.4% were polymorphic; gene diversity was 0.29, allowing good identification of individual genotypes. No 2 identical DNA samples were present, so no asexual reproduction of planulae was indicated. The sampled planulae did not exhibit any genetic structure characteristic to a specific location, indicating one large gene pool and extensive gene flow among H. fuscescens specimens inhabiting the northern Gulf of Eilat.
Coral reefs, one of the most diverse ecosystems on earth, host large numbers of cnidarians, of which the alcyonarian and the scleractinian corals are of ecological importance. Owing to the worldwide decline of coral reefs associated with anthropogenic activities (Barber et al. 2001; Bellwood et al. 2004; Cornish and DiDonato 2004; Cote et al. 2005), these anthozoans merit in-depth scientific interest. The study of population genetics of key species is of paramount conservation importance because the maintenance of large gene pools with sufficient variability is necessary for species in order to adapt efficiently to human impacted environments (Meffe and Carroll 1997; Avise 1998). The mode of reproduction is one of the factors considered in apportioning of different levels of genetic variability. Reproductive activities of alcyonarian and scleractinian corals are controlled by a variety of factors, including sexual and asexual modes of propagation (reviewed in Benayahu 1997; McFadden et al. 2001; Fautin 2002). However, the impacts of various reproductive modes on the distribution and abundance of reef cnidarians are still largely unknown (Bastidas et al. 2002).
Anthozoans vary greatly not only in their mode of reproduction but also in modes of larval development. They may broadcast gametes into the water column or brood larvae internally or externally to varying stages of development (McFadden et al. 2001). Internally brooded offspring may result from either sexual reproduction or from vegetative embryogenesis (Fautin 2002 and references therein). The latter mode produces larvae that are genetically identical to their broodparent (Ayre and Resing 1986). The diverse reproductive strategies clearly influence population genetic structures.
The brooder soft coral Heteroxenia fuscescens is a common shallow-reef xeneid species in the northern Red Sea and the Indo-Pacific Ocean (Benayahu 1985). Colonies are simultaneous hermaphrodites that bear male and female gametes in their autozooids (Achituv and Benayahu 1990). Younger colonies develop only male gametes, whereas older dimorphic colonies produce mainly eggs and relatively small quantities of sperm (Achituv and Benayahu 1990). Heteroxenia fuscescens displays year-round gametogenesis and planulation processes (Benayahu 1991), but during the summer and autumn months, the average percentage of gravid colonies is significantly higher (Ben-David-Zaslow et al. 1999).
We studied modes of reproduction (sexual vs. vegetative) and levels of gene diversity of H. fuscescens larvae collected from 9 colonies at 3 locations along the Gulf of Eilat, Red Sea. On release, the larvae lack any symbiotic algae (Yacobovitch et al. 2003) thus presenting solely coral DNA. We used amplified fragment length polymorphism (AFLP; Vos et al. 1995), highly variable DNA markers, for evaluating the possible occurrence of asexual/sexual reproduction in this species. AFLP technology generates DNA fingerprints and requires no prior sequence information or probe collections (Mickett et al. 2003). It has proved to be highly efficient in detecting asexual reproduction in various marine invertebrates including soft corals (Barki et al. 2000) and echinoderms (Uthicke and Conand 2005).
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Materials and Methods |
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Collection and Maintenance of Colonies
Colonies of H. fuscescens were collected during summer (July, August) 2003 from the coral reef at the northern Gulf of Eilat, at 3 locations, over a 5-km stretch from south to north (Figure 1). Field collection was conducted using SCUBA gear, at depths of 3–12 m. As coral fragmentation and clumping are common phenomena, to avoid a biased estimate of gene diversity, minimal distance between colonies (sampling points) was determined as 25 m. Each collected colony was placed separately in a plastic bag, marked, and taken to a shady ex-situ secluded flow-through seawater tank. Planulae were collected at sunset, and each planula was individually tagged. Altogether, 89 planulae originating from 9 maternal colonies were scored (Table 1).
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DNA Extraction and AFLP Protocol
AFLP is one of the best genetic techniques recently developed and has proved to be highly efficient in individual-based population assignment tests (Campbell et al. 2003). We isolated genomic DNA by Graham's (1978) method. The AFLP procedure applied was that described by Vos et al. (1995). High-quality genomic DNA (0.5 µg) was digested with a pair of restriction enzymes (EcoRI/MseI) at 37 °C for 3 h and then ligated to double-stranded EcoRI and MseI adaptors. The resulting fragments were preamplified with nonselective primers, the ligated adaptors serving as target sites for primer annealing. Four selective primer combinations were used for AFLP amplification as follows: E-ACG/M-CAA, E-ACA/M-CAC, E-ACT/M-CTT, and E-ACT/M-CAC. Selective EcoRI (E-) primers were end-labeled with [
-33P]ATP. Polymerase chain reactions (PCRs) were carried out in a total volume of 20 µl. PCR amplification cycles started at annealing temperature of 65 °C, after which the annealing temperature was lowered by 0.7 °C per cycle for 12 cycles (a touch down phase of 13 cycles), and then followed 23 cycles at annealing temperature of 56 °C. The products were separated on 6% denaturing polyacrylamide gels and exposed to Kodak BioMax film.
Data Analysis
Amplification products were scored as discrete character states (present/absent) and transformed into band frequencies. DNA of several samples (
5%) were amplified and run in duplicates to validate repeatability. The similarities between duplicated fingerprints were found to be higher than 98%. Samples that exhibited unclear band formations (<5% of all amplification products), suggesting contamination, were excluded from the analysis. Diversity values were based on phenotype frequency (phenotypes being the band patterns produced by individual primer pairs). Data were analyzed by POPGENE software version 1.31 (Yeh et al. 1999) or Tools for Population Genetic Analyses software version 1.3 (Miller 1997). These programs consider AFLP bands as diploid-dominant markers, in which the estimated allele frequencies are based on the square root of the frequency of the null (recessive) genotype. Population differentiation was tested by exact tests (1000 dememorization steps, 10 batches, 2000 permutations per batch: Raymond and Rousset 1995).
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Results |
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Eighty-nine planulae originating from 9 mother colonies were scored and analyzed. The 4 primer combinations, E-ACG/M-CAA, E-ACA/M-CAC, E-ACT/M-CTT, and E-ACT/M-CAC, revealed 53, 53, 53, and 62 loci, respectively, yielding a total of 221 putative loci (bands); fragment size ranged from 100 to 400 bp. Levels of polymorphism and gene diversity for planulae collected from each colony and for each location are summarized in Table 1. Most loci, 180 (81.4%), were polymorphic at the stringent 95% criterion. Polymorphism levels of planulae from the 3 locations did not differ significantly, ranging from 47% to 63%. Polymorphism levels of sibling planulae (originating from the same mother colony) showed lower variation, as expected; yet polymorphism was high, ranging from 16.7% to 43.9%, and gene diversity was from 0.08 to 0.20. Polymorphism level allows good identification of individual genotypes. All 89 tested planulae, from 9 mother colonies, were found to be genetically distinct (Figure 2); no 2 identical samples were present, so no planulae asexual reproduction was indicated. The genetic identities (0.855 ± 0.052, mean ± SD) and distances (0.159 ± 0.062) between H. fuscescens colonies are summarized in Table 2. Pairwise analysis (exact tests, Raymond and Rousset 1995) showed no colony differentiation (P = 0.9745–1.000). A detailed fingerprinting analysis of each individual planula sampled allows an estimation of the genetic identity of sibling planulae within colonies, similarities between colonies at each location, and similarities between locations. The resultant dendrogram provides a visual tool that expresses these distinctions (Figure 2).
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Discussion |
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Are Planulae the Product of Sexual Reproduction?
Anthozoans show complex asexual and sexual modes of reproduction, including meiotic and ameiotic processes (reviewed by Blackwelder and Shepherd 1981; McFadden et al. 2001; Fautin 2002). In some species, brooding is the outcome of sexual reproduction; others brood asexual offspring that are morphologically indistinguishable (Fautin 2002). Heteroxenia fuscescens, in contrast to the gonochoric nature of the Xeniidae family, is a hermaphrodite brooding species (Benayahu 1997) that displays gametogenesis and planulation throughout the year (Benayahu 1991). It is not known whether planulae are also the product of asexual reproduction.
Our analysis revealed that all 89 tested planulae, from 9 mother colonies, were genetically distinct (Figure 2). From the high level of gene diversity among brooded planulae within a colony, we infer that planulae in Eilat's population of this soft coral are primarily produced sexually.
Genetic Diversity of Heteroxenia fuscescens in Eilat
Heteroxenia fuscescens is a common shallow-reef soft coral in the Gulf of Eilat, northern Red Sea (Benayahu 1985). We documented here an extensive polymorphism and high level of gene diversity among sibling planulae as mentioned above, among colonies in each location, or between locations. As expected, the mean genetic distance between sibling planulae was lower that that between planulae originating from different mother colonies (Figure 2). Nevertheless, sibling planulae differed, from which we infer that the eggs in a single colony may be fertilized by sperm from multiple colonies. Genetic similarities in colonies within any specific location were not closer than those between colonies at all 3 locations. The sampled colonies did not exhibit genetic structure by location (Table 1). Planulae collected from colonies at different locations are clustered closer to each other than to planulae from geographically neigboring colonies (Figure 2). These results indicate extensive gene flow among the H. fuscescens inhabiting the north part of the Gulf of Eilat.
Larval dispersal is an important factor in determining the distribution of adult corals. Heteroxenia fuscescens is a brooding species, a reproductive mode suggested to be associated with limited distance dispersal of propagules (Harrison and Wallace 1990; Bastidas et al. 2002). Restricted dispersal of brooding corals was in fact found in the soft coral Clavularia koellikeri, the blue coral Heliopora coerulea, and the scleractinian coral Balanophyllia europaea (Bastidas et al. 2002; Harii and Kayanne 2003; Goffredo et al. 2004).
Genetic studies revealed local recruitment in 9 common brooder and spawner species from Australia's Great Barrier Reef (Ayre and Hughes 2000). However, for all these species, long-distance gene flow was sufficient to prevent alternative fixation of alleles between reefs 500–1200 km apart. The results for H. fuscescens genetic diversity reveal an extensive gene flow across the 5-km sampling distance. Heteroxenia fuscescens planulae competency period can reach 49 planktonic days (Ben-David-Zaslow and Benayahu 1996), so they can disperse widely by currents. The currents in the Eilat area vary significantly in space and time, the direction being mainly from north to south (Berman et al. 2000). However, a clear reversal of flow occurs in February of each year (Genin and Paldor 1998; Berman et al. 2000; Figure 1). This phenomenon may enable H. fuscescens larvae originating in southern Red Sea sites to reach Eilat within a period of few weeks after release, while still competent (Ben-David-Zaslow and Benayahu 2000), thus making possible interaction between distant populations.
Only few studies on population genetics of coral reefs have used molecular fingerprinting techniques. Symbiosis of many coral species with algae exposes nonspecific molecular biology techniques to DNA contamination. Nonetheless, several other studies have found high levels of genetic diversity in coral species around the world, as well as in Eilat. High levels of microsatellite variation were found in the gorgonian coral Pseudopterogorgia elisabethae across the Bahamas (Gutierrez-Rodriguez and Lasker 2004) and in the scleractinian coral Seriatopora hystrix from the Red Sea (Meir et al. 2005). Elevated level of random amplified polymorphic DNA was found in brooding Caribbean corals Porites astreoides and Favia fragum (77% and 83%, respectively) (Brazeau et al. 1998). A similar tendency to high polymorphism (AFLP, 77.8%) was found in the soft coral species Parerythropodium fulvum fulvum from Eilat (Barki et al. 2000).
The reef of Eilat is facing an accelerating process of decline in coral biodiversity. Although molecular genetic variability was tested only in few species (Barki et al. 2000; Meir et al. 2005; this study), all studies reveal high levels of polymorphism. This outcome suggests that decrease in gene diversity of corals in Eilat is not intimately associated with the observed phenomenon of the declining reef. It also conveys that active restoration measures of nursery-grown colony transplantation into denuded reef sites and forest restoration principles for reef rehabilitation (Epstein et al. 2003; Rinkevich 2005) may not significantly affect genetic structures of existing populations.
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Acknowledgments |
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We would like to thank S. Shafir for helping in animal collection and G. Paz for technical assistance. We express our appreciation to D. Fautin and to the anonymous referees for their helpful suggestions that contributed significantly to this study. This work was supported by a grant from the Israel Science Foundation (408/03) to B.R.
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Footnotes |
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Corresponding Editor: Martin Tracey
Received February 7, 2006
Accepted July 6, 2006
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