Aletta E. Bester-van der Merwe

Historical isolation and hydrodynamically constrained gene flow in declining populations of the South-African abalone, Haliotis midae

Over the past two decades, the South African abalone (Haliotis midae), has been under serious threat mainly due to overexploitation. To assure successful management and conservation of wild stocks, the consideration of species-specific evolutionary and population dynamic aspects is critical. In this study, eight microsatellites and 12 single nucleotide polymorphic loci (SNPs) were applied to determine genetic structure in nine populations sampled throughout the species’ natural distribution range. It spans along three biogeographical regions of the South African coastline: temperate in the West coast, warm temperate in the South coast and subtropical in the East coast. Data analysis applying frequentist and Bayesian-based clustering methods indicated weak genetic differentiation between populations of the West, South and East coast. Spatial Bayesian inference further revealed clinal variation along a longitudinal gradient and a transitional zone in the South coast. Coalescent analysis of long-term migration showed restricted interchange among the sampling locations of the South coast while estimates of effective population size were comparable between coastal regions. Furthermore demographic analysis of microsatellite data suggested population expansion, probably reflecting range expansion that occurred following glacial retreat during the Pleistocene. Overall, population structure analysis suggested contemporary (hydrographical conditions) as well as historical (Pleistocene contraction of habitat) restrictions to gene flow. This study provides the foundation for the establishment of an integrated management policy for preserving the natural diversity and adaptive potential of H. midae.

Comparison of population genetic estimates amongst wild, F1 and F2 cultured abalone (Haliotis midae)

Haliotis midae is South Africas most important aquaculture species. The reproduction cycle is currently not closed as many farms rely on wild-caught broodstock for seed production. However, there is an increasing interest in genetic improvement in commercial stocks, with a growing number of producers implementing selective breeding strategies. High throughput commercial production and mass spawning make it difficult to maintain breeding records; therefore, mostly mass selection is practised. The high fecundity and unequal parental contributions also often lead to increased levels of inbreeding. This study therefore aimed to assess the genetic effects of such breeding practices on commercial populations of H. midae. Using microsatellite loci, the genetic properties of a wild, an F1 and an F2 population were estimated and compared. Although there was no significant loss of genetic diversity amongst the cultured populations in comparison with the wild progenitor population, there was low-to-moderate genetic differentiation between populations. Relatedness amongst the F2 population was significant, and the rate of inbreeding was high. The effective population size for the F2 (±50) was also comparatively small with respect to the wild (∞) and F1 (±470) populations. These results suggest that farms need to give caution to breeding practices beyond the first (F1) generation and aim to increase effective population sizes and minimise inbreeding to ensure long-term genetic gain and productivity. This study also confirms the usefulness of population genetic analyses for commercial breeding and stock management in the absence of extensive pedigree records.

Microsatellite cross-species amplification and utility in southern African elasmobranchs: A valuable resource for fisheries management and conservation

BackgroundSimilarly to the rest of the world, southern Africa’s diverse chondrichthyan fauna is currently experiencing high fishing pressures from direct and non-direct fisheries to satisfy market demands for shark products such as fins and meat. In this study, the development of microsatellite markers through cross-species amplification of primer sets previously developed for closely related species is reported as an alternative approach to de novo marker development. This included the design of four microsatellite multiplex assays and their cross-species utility in genetic diversity analysis of southern African elasmobranchs. As this study forms part of a larger project on the development of genetic resources for commercially important and endemic southern African species, Mustelus mustelus was used as a candidate species for testing these multiplex assays in down-stream applications.ResultsThirty five microsatellite primer sets previously developed for five elasmobranch species were selected from literature for testing cross-species amplification in 16 elasmobranch species occurring in southern Africa. Cross-species amplification success rates ranged from 28.6%-71.4%. From the successfully amplified microsatellites, 22 loci were selected and evaluated for levels of polymorphism, and four multiplex assays comprising of the 22 microsatellites were successfully constructed, optimised and characterised in a panel of 87 Mustelus mustelus individuals. A total of 125 alleles were observed across all loci, with the number of alleles ranging from 3–12 alleles. Cross-species amplification of the four optimised multiplex assays was further tested on 11 commercially important and endemic southern African elasmobranch species. Percentage of polymorphism ranged from 31.8%-95.5% in these species with polymorphic information content decreasing exponentially with evolutionary distance from the source species.ConclusionsCross-species amplification of the 35 microsatellites proved to be a time- and cost-effective approach to marker development in elasmobranchs and enabled the construction of four novel multiplex assays for characterising genetic diversity in a number of southern African elasmobranch species. This study successfully demonstrated the usefulness of these markers in down-stream applications such as genetic diversity assessment and species identification which could potentially aid in a more integrative, multidisciplinary approach to management and conservation of commercially important cosmopolitan and endemic elasmobranch species occurring in southern Africa.

Genetic analyses reveal declining trends and low effective population size in an overfished South African sciaenid species, the dusky kob (Argyrosomus japonicus)

Overfishing has led to the collapse of many marine fish stocks along the South African coast, particularly species characterised by predictable distribution patterns and vulnerable life-history traits. Dusky kob (Argyrosomus japonicus) is an estuarine-dependent sciaenid fish that has been severely depleted by overfishing and is currently managed by suboptimal management measures aggravated by poor enforcement and compliance to regulations. The present study utilised microsatellite markers to evaluate levels of genetic diversity and population structuring of dusky kob along the South African coast, including five estuarine areas. Results showed signatures of bottlenecking, low and declining trends of effective population size and weak differentiation among samples collected along 2000km of coastline, indicating that dusky kob continues to be subject to the detrimental influence of fishing pressure. Additionally, because dusky kob is a key species in the emerging South African aquaculture sector, parentage analyses were used to successfully identify the origin of first-generation (F1) individuals from a commercial hatchery, hence allowing traceability of farmed products and discrimination between wild and farmed fish. Findings from the present study provided essential information to aid future management of wild populations, as well as to establish sustainable fish farming.

Species identification and comparative population genetics of four coastal houndsharks based on novel NGS-mined microsatellites

Abstract The common smooth‐hound (Mustelus mustelus) is the topmost bio‐economically and recreationally important shark species in southern Africa, western Africa, and Mediterranean Sea. Here, we used the Illumina HiSeq™ 2000 next‐generation sequencing (NGS) technology to develop novel microsatellite markers for Mustelus mustelus. Two microsatellite multiplex panels were constructed from 11 polymorphic loci and characterized in two populations of Mustelus mustelus representative of its South African distribution. The markers were then tested for cross‐species utility in Galeorhinus galeus, Mustelus palumbes, and Triakis megalopterus, three other demersal coastal sharks also subjected to recreational and/or commercial fishery pressures in South Africa. We assessed genetic diversity (N A, A R, H O, H E, and PIC) and differentiation (F ST and D est) for each species and also examined the potential use of these markers in species assignment. In each of the four species, all 11 microsatellites were variable with up to a mean N A of 8, A R up to 7.5, H E and PIC as high as 0.842. We were able to reject genetic homogeneity for all species investigated here except for T. megalopterus. We found that the panel of the microsatellite markers developed in this study could discriminate between the study species, particularly for those that are morphologically very similar. Our study provides molecular tools to address ecological and evolutionary questions vital to the conservation and management of these locally and globally exploited shark species.

Molecular species identification and population genetics of chondrichthyans in South Africa: current challenges, priorities and progress

Molecular genetic techniques, such as DNA barcoding and genotyping, are increasingly being used to assist with the conservation and management of chondrichthyans worldwide. Southern Africa is a shark biodiversity hotspot, with a large number of endemic species. According to the IUCN Red List, a quarter of South Africas chondrichthyans are threatened with extinction. South Africas commercial shark fisheries have increased over the last decade and there is a need to ensure sustainable utilisation and conservation of this fisheries resource. Here, we give an overview of the molecular techniques that are used to assist in the identification of species, cryptic speciation and possible interspecific hybridisation, as well as the assessment of population structure and reproductive behaviour of endemic and commercially important chondrichthyan species of southern Africa. We discuss the potential application of these techniques for management and conservation of several species affected by South African fisheries. Acquiring baseline barcode data of all chondrichthyans in southern African waters and assessing the population structure of exploited species on a local and greater regional scale are recommended as research priorities. Future prospects should also include high-throughput molecular marker development and investigation of intraspecies functional variation using next generation sequencing technology.

Transcriptome-Wide Single Nucleotide Polymorphisms (SNPs) for Abalone (Haliotis midae): Validation and Application Using GoldenGate Medium-Throughput Genotyping Assays

Haliotis midae is one of the most valuable commercial abalone species in the world, but is highly vulnerable, due to exploitation, habitat destruction and predation. In order to preserve wild and cultured stocks, genetic management and improvement of the species has become crucial. Fundamental to this is the availability and employment of molecular markers, such as microsatellites and single nucleotide (SNPs). Transcriptome sequences generated through sequencing-by-synthesis technology were utilized for the in vitro and in silico identification of 505 putative SNPs from a total of 316 selected contigs. A subset of 234 SNPs were further validated and characterized in wild and cultured abalone using two Illumina GoldenGate genotyping assays. Combined with VeraCode technology, this genotyping platform yielded a 65%–69% conversion rate (percentage polymorphic markers) with a global genotyping success rate of 76%–85% and provided a viable means for validating SNP markers in a non-model species. The utility of 31 of the validated SNPs in population structure analysis was confirmed, while a large number of SNPs (174) were shown to be informative and are, thus, good candidates for linkage map construction. The non-synonymous SNPs (50) located in coding regions of genes that showed similarities with known proteins will also be useful for genetic applications, such as the marker-assisted selection of genes of relevance to abalone aquaculture.

Detection of molecular signatures of selection at microsatellite loci in the South African abalone (Haliotis midae) using a population genomic approach

Identifying genomic regions that may be under selection is important for elucidating the genetic architecture of complex phenotypes underlying adaptation to heterogeneous environments. A population genomic approach, using a classical neutrality test and various Fst-outlier detection methods was employed to evaluate genome-wide polymorphism data in order to identify loci that may be candidates for selection amongst six populations (three cultured and three wild) of the South African abalone, Haliotis midae. Approximately 9% of the genome-wide microsatellite markers were putatively subject to directional selection, whilst 6-18% of the genome is thought to be influenced by balancing selection. Genetic diversity estimates for candidate loci under directional selection was significantly reduced in comparison to candidate neutral loci, whilst candidate balancing selection loci demonstrated significantly higher levels of genetic diversity (Kruskal-Wallis test, P<0.05). Pairwise Fst estimates based on candidate directional selection loci also demonstrated increased levels of differentiation between study populations. Various candidate loci under selection showed significant inter-chromosomal linkage disequilibrium, suggesting possible gene-networks underling adaptive phenotypes. Furthermore, several loci had significant hits to known genes when performing BLAST searches to NCBIs non-redundant databases, whilst others are known to be derived from expressed sequences even though homology to a known gene could not be established. A number of loci also demonstrated relatively high similarity to transposable elements. The association of these loci to functional and genomically active sequences could in part explain the observed signatures of selection.

Molecular systematics and biogeography of the circumglobally distributed genus Seriola (Pisces: Carangidae).

The genus Seriola includes several important commercially exploited species and has a disjunct distribution globally; yet phylogenetic relationships within this genus have not been thoroughly investigated. This study reports the first comprehensive molecular phylogeny for this genus based on mitochondrial (Cytb) and nuclear gene (RAG1 and Rhod) DNA sequence data for all extant Seriola species (nine species, n=27). All species were found to be monophyletic based on Maximum parsimony, Maximum likelihood and Bayesian inference. The closure of the Tethys Sea (12-20 MYA) coincides with the divergence of a clade containing ((S. fasciata and S. peruana), S. carpenteri) from the rest of the Seriola species, while the formation of the Isthmus of Panama (±3 MYA) played an important role in the divergence of S. fasciata and S. peruana. Furthermore, factors such as climate and water temperature fluctuations during the Pliocene played important roles during the divergence of the remaining Seriola species.

Molecular phylogeny of South African abalone, its origin and evolution as revealed by two genes

Abstract The marine family Haliotidae (Mollusca: Gastropoda) includes approximately 56 extant abalone species found worldwide. None of these species are globally distributed while four areas of endemism (temperate Australia, South Africa, New Zealand and North Pacific) have been recognized. Phylogenetic relationships of 18 abalone species including five South African species were reconstructed using a combined data set containing sequence data of the mitochondrial NADH-dehydrogenase subunit 1 (ND1) gene and the nuclear hemocyanin gene. The molecular topologies confirmed former findings of two major abalone lineages with Northern and Southern Pacific distribution within the Haliotidae family. The phylogeny revealed all five South African species as a monophyletic group with a sister relationship to the Australian endemics clade. It further suggested a relatively recent radiation of the South African species and places it within the same evolutionary context as the Mediterranean/Atlantic and Australasian clades. Molecular phylogeny also revealed a split within the South African group as well as further speciation within one of the two subclades. Possible place of origin for South African abalone are discussed and ecological specialization, e.g. differential adaptation to environmental conditions, is proposed as the most likely scenario describing divergence within the South African clade.