Applying a Molecular Genetics Approach to Shark Conservation and Management: Assessment of DNA Barcoding in Hammerhead Sharks and Global Population Genetic Structuring in the Gray Reef Shark, Carcharhinus amblyrhynchos.

Chapter 1 DNA barcoding based on the mitochondrial cytochrome c oxidase subunit I (COI) gene sequence is emerging as a useful tool for identifying unknown, whole or partial organisms to species level. However, the application of only a single mitochondrial marker for robust species identification has also come under some criticism due to the possibility of erroneous identifications resulting from species hybridizations and/or the potential presence of nuclear-mitochondrial psuedogenes. The addition of a complementary nuclear DNA barcode has therefore been widely recommended to overcome these potential COI gene limitations, especially in wildlife law enforcement applications where greater confidence in the identifications is essential. In this study, we examined the comparative nucleotide sequence divergence and utility of the mitochondrial COI gene (N=182 animals) and nuclear ribosomal internal transcribed spacer 2 (ITS2) locus (N=190 animals) in the 8 known and 1 proposed cryptic species of globally widespread, hammerhead sharks (family Sphyrnidae). Since hammerhead sharks are under intense fishing pressure for their valuable fins with some species potentially set to receive CITES listing, tools for monitoring their fishery landings and tracking trade in their body parts is necessary to achieve effective management and conservation outcomes. Our results demonstrate that both COI and ITS2 loci function robustly as stand-alone barcodes for hammerhead shark species identification. Phylogenetic analyses of both loci independently and together accurately place each hammerhead species together in reciprocally monophyletic groups with strong bootstrap support. The two barcodes differed notably in levels of intraspecific divergence, with average intraspecific K2P distance an order of magnitude lower in the ITS2 (0.297% for COI and 0.0967% for ITS2). The COI barcode also showed phylogeographic separation in Sphyrna zygaena, S. lewini and S. tiburo, potentially providing a useful option for assigning unknown specimens (e.g. market fins) to a broad geographic origin. We suggest that COI supplemented by ITS2 DNA barcoding can be used in an integrated and robust approach for species assignment of unknown hammerhead sharks and their body parts in fisheries and international trade. Chapter 2 The gray reef shark (Carcharhinus amblyrhynchos) is an Indo-Pacific, coral reef associated species that likely plays an important role as apex predator in maintaining the integrity of coral reef ecosystems. Populations of this shark have declined substantially in some parts of its range due to over-fishing, with recent estimates suggesting a 17% decline per year on the Great Barrier Reef (GBR). Currently, there is no information on the population structure or genetic status of gray reef sharks to aid in their management and conservation. We assessed the genetic population structure and genetic diversity of this species by using complete mitochondrial control region sequences and 15 nuclear microsatellite markers. Gray reef shark samples (n=305) were obtained from 10 locations across the species’ known longitudinal Indo-Pacific range: western Indian Ocean (Madagascar), eastern Indian Ocean (Cocos [Keeling] Islands, Andaman Sea, Indonesia, and western Australia), central Pacific (Hawaii, Palmyra Atoll, and Fanning Atoll), and southwestern Pacific (eastern Australia – Great Barrier Reef). The mitochondrial and nuclear marker data were concordant in most cases with population-based analysis showing significant overall structure (FST = 0.27906 (pST = 0.071 ± 0.02), and significant pairwise genetic differentiation between nearly all of the putative populations sampled (i.e., 9 of the 10 for mitochondrial and 8 of the 10 for nuclear markers). Individual-based analysis of microsatellite genotypes identified at least 5 populations. The concordant mitochondrial and nuclear marker results are consistent with a scenario of very low to no appreciable connectivity (gene flow) among most of the sampled locations, suggesting that natural repopulation of overfished regions by sharks from distant reefs is unlikely. The results also indicate that conservation of genetic diversity in gray reef sharks will require management measures on relatively local scales. Our findings of extensive genetic structuring suggests that a high level of genetic isolation is also likely to be the case in unsampled populations of this species