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

Surface mucous as a source of genomic DNA from Atlantic billfishes (Istiophoridae) and swordfish (Xiphiidae)

Procedures for sampling genomic DNA from live billfishes involve manual restraint and tissue excision that can be difficult to carry out and may produce stresses that affect fish survival. We examined the collection of surface mucous as a less invasive alternative method for sourcing genomic DNA by comparing it to autologous muscle tissue samples from Atlantic blue marlin (Makaira nigricans), white marlin (Tetrapturus albidus), sailfish (Istiophorus platypterus), and swordfish (Xiphias gladius). Purified DNA from mucous was comparable to muscle and was suitable for conventional polymerase chain reaction, random amplified polymorphic DNA analysis, and mitochondrial and nuclear locus sequencing. The nondestructive and less invasive characteristics of surface mucous collection may promote increased survival of released specimens and may be advantageous for other marine fish genetic studies, particularly those involving large live specimens destined for release

DNA Analysis of Traded Shark Fins and Mobulid Gill Plates Reveals a High Proportion of Species of Conservation Concern

Continuously increasing demand for plant and animal products causes unsustainable depletion of biological resources. It is estimated that one-quarter of sharks and rays are threatened worldwide and although the global fin trade is widely recognized as a major driver, demand for meat, liver oil, and gill plates also represents a significant threat. This study used DNA barcoding and 16 S rRNA sequencing as a method to identify shark and ray species from dried fins and gill plates, obtained in Canada, China, and Sri Lanka. 129 fins and gill plates were analysed and searches on BOLD produced matches to 20 species of sharks and five species of rays or – in two cases – to a species pair. Twelve of the species found are listed or have been approved for listing in 2017 in the appendices of the Convention on International Trade in Endangered Species of Fauna and Flora (CITES), including the whale shark (Rhincodon typus), which was surprisingly found among both shark fin and gill plate samples. More than half of identified species fall under the IUCN Red List categories ‘Endangered’ and ‘Vulnerable’, raising further concerns about the impacts of this trade on the sustainability of these low productivity species

Population Genetic Structure Of A Coral Reef Ecosystem Apex Predator, The Gray Reef Shark (Carcharhinus Amblyrhynchos)

Sharks play a major functional role as apex predators in coral reef ecosystems, raising concerns that their ongoing overexploitation will compromise the integrity and sustainability of reefs. The gray reef shark (Carcharhinus amblyrhynchos) is a strongly coral reef associated species whose populations are known to have declined substantially in some regions. There is no information on population structure in this species to aid in their management and conservation. We are assessing genetic structure in this species by using entire mitochondrial control region sequences and 15 nuclear microsatellite loci as markers. 93 gray reef shark samples were obtained from across the species’ Indo-Pacific distribution (eastern Indian Ocean [Madagascar/Seychelles], Central Pacific [Hawaii], Southwestern Pacific (eastern Australia, Palmyra, Palau, Cocos (Keeling) Islands]). Mitochondrial (AMOVA) and microsatellite (STRUCTURE) data concordantly identify the Hawaii population as a distinct genetic group relative to other sampling locations. The microsatellite data further identify 3 distinct overall gray reef shark groups (eastern Indian Ocean, Central Pacific, and Southwestern Pacific). Our current analyses do not show any evidence of population structure among islands of the Southwestern Pacific, although this question is being further addressed with additional samples from more locations. These results show strong genetic differentiation exists in gray reef shark populations separated by expanses of open ocean, and suggest proper management of this declining species will have to occur at the very least on a regional geographic scale

Genetic Connectivity of a Coral Reef Ecosystem Predator: The Population Genetic Structure and Evolutionary History of the Caribbean Reef Shark (Carcharhinus perezi)

Aim The Caribbean reef shark (Carcharhinus perezi) is one of few extant reef sharks inhabiting the Atlantic Ocean. Its variability in movements across habitat types suggests the possibility of a complex genetic population structure. Here, we use mitochondrial and nuclear DNA to investigate the genetic connectivity of the Caribbean reef shark across contemporary and evolutionary time-scales and relate our findings to the ecology of this understudied species. Location Tropical western Atlantic and Caribbean. Methods Samples were obtained from 216 individuals from six western Atlantic and Caribbean locations. Individuals were genotyped at seven nuclear microsatellite DNA loci and sequenced at two mitochondrial (control region [CR]; NADH dehydrogenase subunit 4 [ND4]) and one nuclear locus (lactate dehydrogenase [LDH]). Analyses to resolve the population genetic structure and evolutionary history of this species were adopted. Results Sequencing of the CR (1,068 bp, n = 216), ND4 (741 bp, n = 213) and LDH (258 bp, n = 165) loci, resolved 11, 8 and 13 unique haplotypes (or alleles), respectively. Overall, Caribbean reef sharks showed low levels of genetic diversity and most marker sets identified strong genetic differences (FSTand ΦST) between sharks sampled in Brazil versus all other locations (msat FST \u3e 0.017; CR-ND4 ΦST \u3e 0.013). Mitochondrial DNA showed evidence of increased genetic partitioning among western North Atlantic sampling sites, although widespread haplotype sharing (~85%–92%) and a shallow population history were found. Main Conclusions Findings of genetic differentiation are concordant with previous movement studies showing residency and/or site-fidelity to specific locations by individuals. However, similar to other reef shark studies, we found that the level of genetic connectivity among populations was context dependent—i.e., sharks occupying isolated habitats showed greater genetic differentiation compared with those sharks occupying semi-isolated or continuous reef habitats. Furthermore, low genetic diversity and a shallow mitochondrial population history were found, suggesting historical demographic fluctuations, including population collapse and more recent expansions

Global Phylogeography of the Dusky Shark Carcharhinus obscurus: Implications for Fisheries Management and Monitoring the Shark Fin Trade

Genetic stock structure information is needed to delineate management units and monitor trade in sharks, many of which are heavily exploited and declining. The dusky shark Carcharhinus obscurus is a large apex predator that is sought after for its fins and is considered highly susceptible to overexploitation. The International Union for the Conservation of Nature (IUCN) classifies this species as ‘Vulnerable’ globally and ‘Endangered’ in the northwest Atlantic. We make the first assessment of global stock structure of C. obscurus by analyzing part of the mitochondrial control region (mtCR) in 255 individuals sampled from 8 geographically dispersed locations. We found 25 mtCR haplotypes and rejected a null hypothesis of panmixia (analysis of molecular variance, ΦST = 0.55, p \u3c 0.000001), detecting significant differentiation between 3 management units: US Atlantic (USATL), South Africa (SAF), and Australia (AUS). We also found preliminary evidence of population structure between the USATL and southwest Atlantic (Brazil). There were no shared haplotypes between the western Atlantic and Indo-Pacific. These analyses suggest that replenishment of the collapsed USATL management unit via immigration of females from elsewhere is unlikely. Mixed stock analysis (MSA) simulations show that reconstruction of the relative contributions of USATL, SAF, and AUS management units to the Asian fin trade is possible using these mtCR sequences. We suggest avenues for obtaining samples to conduct MSA of the shark fin trade, which could enhance management of dusky sharks and other species that are exploited for their fins

Summer 2012 Testing and Analysis of the Chemical Mixture Methodology -- Part I

This report presents the key findings made by the Chemical Mixture Methodology (CMM) project team during the first stage of their summer 2012 testing and analysis of the CMM. The study focused on answering the following questions: o What is the percentage of the chemicals in the CMM Rev 27 database associated with each Health Code Number (HCN)? How does this result influence the relative importance of acute HCNs and chronic HCNs in the CMM data set? o What is the benefit of using the HCN-based approach? Which Modes of Action and Target Organ Effects tend to be important in determining the HCN-based Hazard Index (HI) for a chemical mixture? o What are some of the potential issues associated with the current HCN-based approach? What are the opportunities for improving the performance and/or technical defensibility of the HCN-based approach? How would those improvements increase the benefit of using the HCN-based approach? o What is the Target Organ System Effect approach and how can it be used to improve upon the current HCN-based approach? How does the benefits users would derive from using the Target Organ System Approach compare to the benefits available from the current HCN-based approach

Horn&Cowley_Easypop_RunWindows

A text file containing the input parameters for the program EASYPOP used to generate the simulated datasets

Data from: Evolutionary relationships within the Triops (Notostraca: Branchiopoda) using complete mitochondrial genomes

The tadpole shrimp (Notostraca: Triops) have been called living fossils with conserved morphology, but subtle morphological variations within and between species has yielded confused taxonomic assignments. To aid in cryptic species detection of tadpole shrimp from southern New Mexico, USA, the first complete mitochondrial genomes for three putative species (T. longicaudatus “long,” T. l. “short,” T. newberryi) are reported. The genomes ranged in length from 15,058 bp to 15,060 bp with 13 coding genes, 22 tRNA genes, 2 rRNA genes and a control region. Phylogenetic trees were constructed using previously sequenced Triops-genomes to assess genetic relationships within the genus. The T. longicaudatus-genomes from Genbank were consistent with our genomes for T. newberryi and T. l. “short.” Variation in mitochondrial genes were identified that will aid future identification of cryptic lineages of tadpole shrimp. Genetic differentiation among the genomes of Triops in New Mexico support elevation to species status

Data from: Self-fertilization and the role of males in populations of tadpole shrimp (Branchiopoda: Notostraca: Triops)

Self-fertilization has both negative and positive fitness effects on species evolution. Selfing can increase inbreeding depression, thereby decreasing genetic diversity. In contrast, self-fertilization can preserve beneficial gene combinations and facilitate colonization success. Within the class of crustaceans Branchiopoda, selfing is a primary reproductive mode. Some species of Triops, in the family Notostraca, are a few of the animal species thought to have a mixed mating system between hermaphrodites and males termed androdioecy. The objective of this study is to validate the reproductive mode utilized by Triops newberryi in southern New Mexico by the use of progeny arrays and population simulations. Individuals were reared in the lab from dried soil collected from temporary ponds inhabited by T. newberryi. The adults reared and the encysted embryos contained within their brood pouches were genotyped using seven T. newberryi specific microsatellite markers to determine the relatedness between parent and offspring. Overall microsatellite diversity was low with few heterozygous individuals and limited polymorphisms. Simulated populations and allele segregation analysis suggest hermaphroditism is the primary reproductive mode for T. newberryi. In addition, based on the offspring’s alleles, there was no direct evidence that a male (ovisacless) T. newberryi outcrossed with a female. Population simulations further suggest that the rate of successful outcrossing events must be low and could explain why outcrossing was not observed in the laboratory rearing trials