Global-Scale Genetic Identification of Hammerhead Sharks: Application to Assessment of the International Fin Trade and Law Enforcement

The future status of sharks is an issue of widespread conservation concern due to declines in many species in the face of high levels of exploitation to satisfy market demands for products, especially fins. Substantial declines in the large-bodied hammerhead sharks, Sphyrna lewini, S. mokarran and S. zygaena, even in regions where some management occurs, indicate that informed conservation measures are warranted for these circumglobally distributed species. Despite the importance of assessing shark catch and trade on a species-specific basis to detect potential overexploitation of individual species, achieving this goal for hammerheads has proven elusive due to difficulties in identification of their products. Here, we present the development and application of a diagnostic, streamlined, five-primer multiplex polymerase chain reaction assay utilizing species-specific primers based on nuclear ribosomal ITS2 for the three hammerhead species throughout their global distribution. Application of this assay to investigations of the fin market confirmed the presence of hammerhead fins in the international trade. A study of the world’s largest fin market in Hong Kong revealed a high concordance between specific Chinese-name trade categories and fins from these three species (‘‘Bai Chun’’ with S. lewini, ‘‘Gui Chun’’ with S. zygaena and ‘‘Gu Pian’’ with S. mokarran), and clear species preferences. This concordance information allows the use of market records for monitoring species-specific trends in trade and exploitation rates. The assay is also proving useful for identification of shark body parts in U.S. fisheries law-enforcement activities. Screening of morphologically identified ‘‘S. lewini’’ from globally distributed areas using this assay with subsequent whole ITS2 sequencing suggests a cryptic species closely related to S. lewini occurs off the SE USA coast

Sexual conflicts in sand tiger sharks Carcharias taurus (Rafinesque, 1810) in an artificial environment

Understanding reproductive behaviours and the environmental conditions that facilitate reproduction is important for successful reproduction in managed care. Complex reproductive behaviours have been observed in both aquarium and free-ranging elasmobranchs. Sexual conflicts, including pre-copulatory behaviours in sand tiger sharks Carcharias taurus in managed care have been previously documented. This study reports observations that corroborate the previous accounts of reproductive behaviours as well as describing novel consexual conflicts between males and detailing the male dominance hierarchy in six sand tigers at the National Aquarium. The observations suggest that male behaviours are not strictly pre-copulatory, as consexual male conflicts occurred to the exclusion of attempted copulation, and in the absence of females. Physical separation of either the dominant or most subordinate male decreased the occurrence of sexual conflicts, suggesting that these individuals are the key sharks in hierarchical settings and that conflicts among sand tigers are not strictly related to reproduction, but may be driven by social, physiological, olfactory and/or electrosensory cues

Isolation and Characterization of Polymorphic Microsatellite Markers from a Globally Distributed Marine Apex Predator, the Tiger Shark (Galeocerdo cuvier)

High levels of fishing have resulted in declines among many of the oceans top predators, including the globally distributed tiger shark (Galeocerdo cuvier). Overexploitation of this species has led to declines in parts of its distribution, which may have important ecological consequences given the tiger shark’s trophic position as a large bodied, generalist predator. To assess the population genetics of this species, an enrichment protocol was used to isolate a suite of nine di- and tetra-nucleotide microsatellite loci within the tiger shark, which were subsequently characterized using genomic DNA derived from 40 individuals sampled from Hawaiian waters. The number of alleles per locus ranged from 3 to 23, and the average expected heterozygosities across loci ranged from 0.30 to 0.93. Analyses suggested a low frequency of null alleles across markers, and all loci conformed to both Hardy–Weinberg and linkage equilibrium at P \u3e 0.01

Identification of Species Composition in the Hong Kong Shark Fin Trade using Genetic Techniques and Trader Records

Trade in shark fins represents one of the most serious threats to shark populations worldwide. Previous studies have indicated that certain types of fins are more valued than others, but due to the largely unregulated and often covert nature of the trade, information on actual species composition has been anecdotal and unverified. In order to examine the potential impacts of the shark fin trade on the abundance of various shark species, a study of the species composition in the world’s largest shark fin trading center, Hong Kong, was initiated. Several approaches for distinguishing the species identity of dried fins were evaluated including visual differentiation (shape, color and morphometrics), denticle recognition, and DNA-based methods. This assessment found that genetic analyses were necessary to reliably determine species identity, and a technique involving application of polymerase chain reactions (PCR) with species-specific primers was selected. A sampling program was developed based on the requirements of the PCR technique, the practicalities of accessing samples, and the ability to draw statistically robust conclusions. Shark fins from twelve market categories were sampled and analyzed across a broad range of traders to investigate the concordance between trader names for fins and the associated species identity. Preliminary results indicating an initial matching of trade names and species identities will be presented. These data will subsequently be used in combination with daily shark fin auction records to estimate verified, speciesspecific proportions and quantities of shark fins in the trade

Development and Characterization of 11 Novel Microsatellite Loci for the Roundscale Spearfish Tetrapturus georgii and Their Cross-Species Amplification Among Other Istiophorid Species

Eleven novel polymorphic microsatellite loci were developed and characterized for the recently validated roundscale spearfish Tetrapturus georgii. Characterization of these markers, based on 35 roundscale spearfish from the western North Atlantic, revealed two to 21 alleles per locus with an average expected heterozygosity (HE) of 0·09–0·94, and all loci conformed to Hardy–Weinberg expectations. Cross-amplification of these 11 loci against all other eight known istiophorid species indicates promising prospects for the utility of these markers for istiophorids in general

A Molecular Assessment of Speciation and Evolutionary History of the Globally Distributed Spotted Eagle Ray (Aetobatus narinari)

The spotted eagle ray (Aetobatus narinari), a species of conservation concern (Near Threatened IUCN category) is commonly associated with coral reef ecosystems worldwide where it is likely to play an important predatory role. Currently described as a single, circumglobally distributed species, geographic differences in parasite diversity have led to suggestions that A. narinari may constitute a species complex. There has been no systematic evidence to support this suggestion, however. If multiple species of spotted eagle ray exist, each will likely posses decreased geographic ranges and population sizes, altering the impacts of threats and requiring separate assessments of conservation needs. We assessed the validity of A. narinari as a single cosmopolitan species using 1570bp of sequence data from two mitochondrial genes (cytochrome b and COI) and the nuclear ribosomal ITS2 locus. Individuals from four major geographic regions were examined: western north Atlantic, and western, central, and eastern Pacific. Phylogenies for each locus concordantly described three distinct lineages (western north Atlantic, western/central Pacific, and eastern Pacific) with no genetic exchange among regions. Genetic distances among the most divergent lineages were comparable to taxonomically uncontroversial batoid and teleost congener pairs. Using combined genealogical concordance and genetic distance results, we recommend 1) that the western/central Pacific lineage be recognized as a distinct species from the western north Atlantic and eastern Pacific lineages, and 2) the western north Atlantic and eastern Pacific lineages, separated by the Isthmus of Panama, be given subspecies status. Dramatically higher nucleotide diversity and sequence divergence coupled with a basal position in multiple phylogenetic analyses support an Indo-West Pacific origin for the A. narinari species complex, with subsequent migration into the Atlantic. Evolutionary relationships among lineages suggest a westerly migration around the southern tip of Africa, with intensification of the Benguela coldwater upwelling system a possible vicariant mechanism underlying speciation

Population Structure in The Brown Tube Sponge (agelas Conifera) in The Florida Reef Tract And Caribbean

Sponges are broadly distributed, occur in a wide range of habitats, and comprise a substantial amount of the biomass and macro-biodiversity on coral reefs. Despite their ubiquity in reef ecosystems, their dispersal dynamics are largely unknown. Here we report on gene flow and population structure for the common brown tube sponge, Agelas conifera (Demospongiae, Poecilosclerida) in the Florida reef tract and Caribbean. Over 300 samples collected from ten geographically distinct locations throughout Florida and the Caribbean are being analyzed for variation at eight microsatellite loci. Preliminary results from screening four loci in 295 individuals from eight locations indicated significant population structure overall (FST = 0.072; P = 0.0018) with a significant heterozygosity deficit (FIS = 0.310; P = 0.001). Lower FST values occurred within the Florida reef tract sites compared to those between Caribbean island locations suggesting relatively greater gene flow within the Florida reef system. Significant isolation by distance was not found to occur Caribbean-wide (r = 0.25; P = 0.14), nor within the Florida reef tract (r = 0.39; P = 0.10). Fine scale population structure occurred among sites separated by \u3c1 km around the islands of Utila (Honduras) and St. Croix (USVI) suggesting A. conifera recruitment in these areas is highly local source dependant. In contrast, no structure was observed among sites around Grand Cayman (Cayman Islands). Only populations within St. Croix showed a significant isolation by distance pattern (r = 0.74; P \u3c 0.001). The strong overall and in some instances even highly localized genetic structure observed is consistent with the typically short larval durations known for sponges generally; suggesting recruitment in these important reef biomass components may be driven mainly from local sources

Characterization of the Heart Transcriptome of the White Shark (Carcharodon carcharias)

Background: The white shark (Carcharodon carcharias) is a globally distributed, apex predator possessing physical, physiological, and behavioral traits that have garnered it significant public attention. In addition to interest in the genetic basis of its form and function, as a representative of the oldest extant jawed vertebrate lineage, white sharks are also of conservation concern due to their small population size and threat from overfishing. Despite this, surprisingly little is known about the biology of white sharks, and genomic resources are unavailable. To address this deficit, we combined Roche-454 and Illumina sequencing technologies to characterize the first transcriptome of any tissue for this species. Results: From white shark heart cDNA we generated 665,399 Roche 454 reads (median length 387-bp) that were assembled into 141,626 contigs (mean length 503-bp). We also generated 78,566,588 Illumina reads, which we aligned to the 454 contigs producing 105,014 454/Illumina consensus sequences. To these, we added 3,432 non-singleton 454 contigs. By comparing these sequences to the UniProtKB/Swiss-Prot database we were able to annotate 21,019 translated open reading frames (ORFs) of ≥ 20 amino acids. Of these, 19,277 were additionally assigned Gene Ontology (GO) functional annotations. While acknowledging the limitations of our single tissue transcriptome, Fisher tests showed the white shark transcriptome to be significantly enriched for numerous metabolic GO terms compared to the zebra fish and human transcriptomes, with white shark showing more similarity to human than to zebra fish (i.e. fewer terms were significantly different). We also compared the transcriptome to other available elasmobranch sequences, for signatures of positive selection and identified several genes of putative adaptive significance on the white shark lineage. The white shark transcriptome also contained 8,404 microsatellites (dinucleotide, trinucleotide, or tetranucleotide motifs ≥ five perfect repeats). Detailed characterization of these microsatellites showed that ORFs with trinucleotide repeats, were significantly enriched for transcription regulatory roles and that trinucleotide frequency within ORFs was lower than for a wide range of taxonomic groups including other vertebrates. Conclusion: The white shark heart transcriptome represents a valuable resource for future elasmobranch functional and comparative genomic studies, as well as for population and other biological studies vital for effective conservation of this globally vulnerable species