Scleractinian corals are among the most widely distributed organisms. Despite this, little is known about their population structure, gene flow or population connectivity. While previous studies have analysed allozyme expression and have revealed some important patterns, the power of this method is limited as far as allowing an effective investigation of local scale population variance. The latter is important to understand a range of issues on coral reefs, including the genetic variability and gene flow between populations of corals separated by one to several hundred kilometres. These questions have become crucially important as we head into a century of climate change in which understanding the connectivity of populations is a critically important question in management. As compared to methods based on allozymes, methods that directly use molecular variance in the DNA offer greater power in detecting differences among individuals and populations.
Only a limited number of DNA markers, however, have been identified and developed for scleractinian coral genetic studies. This thesis addressed the search for coral molecular markers, and investigated their applicability to the assessment of the intraspecific variability of the scleractinian coral Pocillopora verrucosa along the southeast African (South Africa and Mozambique) coastline.
The genetic structure of six local South African populations of P verrucosa from two different reef complexes was examined using allozyme electrophoresis in order to establish the basis for comparing new molecular markers to established allozyme methods. Of the five polymorphic loci (Gpi-1, Gdh-1, Lgg-2, Lpp-1, Est-1) used, no
significant differences in allelic frequencies were detected among the six sites. All local collections were genotypically diverse, with evidence of only very limited clonal replication. The ratio of observed to expected genotypic diversity (mean Go.Ge = 0.64 ± 0.05), the ratio of observed number of genotypes to the number of individuals (mean Ng:N = 0.65 ± 0.04), and deviations from Hardy-Weinberg equilibrium indicate that sexual reproduction plays a major role in the maintenance of the populations. No genetic differentiation was found either within (FSR = 0.026 ± 0.003) or between (FRT = 0.000 ± 0.001) reef complexes, indicating strong genetic connectivity and maintenance by high levels of regional self-seeding.
Three DNA-based methods were tested to assess
intraspecific genetic variation within the southern African populations of P. verrucosa: the cytochrome c oxidase subunit I (COI), the internal transcribed spacer region 1 (ITSl), and the pocilloporin gene. The COI fragment was 710 bp long and no differences were detected either within P. verrucosa samples, or between P verrucosa and P. damicornis from the Great Barrier Reef, thereby confirming that the coral mitochondrial genome evolves at a very slow rate. Despite the complete ITS1-5.8S-ITS2 being the largest reported to date (956 bp) for any scleractinian coral species, the ITSl fragment showed only two sequence types (one containing a 13 bp insert). More importantly, 24% of the ITSl sequences were heterogenous, indicating the existence of different multiple copies of the sequence. Pocilloporin turned out to be an intronless gene (514 bp) that was conserved (100% homology) throughout all southern African
populations of P verrucosa. The three DNA regions therefore appear unsuitable for the assessment of intraspecific variability of P. verrucosa at local scales.
The apparent unsuitability of COI, ITS and pocilloporin to population analyses of P. verrucosa, indicated that sequencing individual genes may be less efficient than the use of approaches that sidestep primer design and screen broadly across the genome. Preliminary results using intersimple sequence repeats (ISSR) highlighted the possible applicability of a random amplification technique. DNA amplification fingerprinting (DAF) offered greater resolution than ISSRs, and was therefore applied to rapidly screen the P. verrucosa genome in an attempt to locate potential novel "coral specific' regions of DNA. Of the 16 DAF primers screened, 15 yielded positive amplification results. From these amplifications, five markers (ranging in size from
183 bp to 366 bp) were developed and sequence characterised amplified region (SCAR) analysis revealed no variation in the P verrucosa populations using these five newly developed 'coral specific' markers. All possible sequence alignments showed 100% sequence homology. Even though no variability was detected, the combination of DAF, SCAR, and single-stranded conformation polymorphism (SSCP) methodologies appears to be a potentially valuable tool for 'coral specific' marker discovery, and the nearly infinite number of DAF primer sequences available offers great opportunities to test the applicability of this new approach to both species and population level analyses.
It is imperative that effort and resources continue to be channelled into the search for suitable population genetic molecular markers for scleractinian corals. Rates of hybridisation both within and between scleractinian coral species remain poorly understood, and the
variability of species within reef systems and the connectivity of populations between reef systems largely remain unanswered and untested for most scleractinian coral species.