Atlas-scale single-cell chromatin accessibility using nanowell-based combinatorial indexing.

Here we present advancements in single-cell combinatorial indexed Assay for Transposase Accessible Chromatin (sciATAC) to measure chromatin accessibility that leverage nanowell chips to achieve atlas-scale cell throughput (>105 cells) at low cost. The platform leverages the core of the sciATAC workflow where multiple indexed tagmentation reactions are performed, followed by pooling and distribution to a second set of reaction wells for polymerase chain reaction (PCR)-based indexing. In this work, we instead leverage a chip containing 5184 nanowells at the PCR stage of indexing, enabling a 52-fold improvement in scale and reduction in per-cell preparation costs. We detail three variants that balance cell throughput and depth of coverage, and apply these methods to banked mouse brain tissue, producing maps of cell types as well as neuronal subtypes that include integration with existing single-cell Assay for Transposase Accessible Chromatin (scATAC) and scRNA-seq data sets. Our optimized workflow achieves a high fraction of reads that fall within called peaks (>80%) and low cell doublet rates. The high cell coverage technique produces high unique reads per cell, while retaining high enrichment for open chromatin regions, enabling the assessment of >70,000 unique accessible loci on average for each cell profiled. When compared to current methods in the field, our technique provides similar or superior per-cell information with very low levels of cell-to-cell cross talk, and achieves this at a cost point much lower than existing assays.

Philopatry influences the genetic population structure of the blacktip shark (Carcharhinus limbatus) at multiple spatial scales.

Understanding how interactions among microevolutionary forces generate genetic population structure of exploited species is vital to the implementation of management policies that facilitate persistence. Philopatry displayed by many coastal shark species can impact gene flow and facilitate selection, and has direct implications for the spatial scales of management. Here, genetic structure of the blacktip shark (Carcharhinus limbatus) was examined using a mixed-marker approach employing mitochondrial control region sequences and 4339 SNP-containing loci generated using ddRAD-Seq. Genetic variation was assessed among young-of-the-year sampled in 11 sites in waters of the United States in the western North Atlantic Ocean, including the Gulf of Mexico. Spatial and environmental analyses detected 68 nuclear loci putatively under selection, enabling separate assessments of neutral and adaptive genetic structure. Both mitochondrial and neutral SNP data indicated three genetically distinct units-the Atlantic, eastern Gulf, and western Gulf-that align with regional stocks and suggest regional philopatry by males and females. Heterogeneity at loci putatively under selection, associated with temperature and salinity, was observed among sites within Gulf units, suggesting local adaptation. Furthermore, five pairs of siblings were identified in the same site across timescales corresponding with female reproductive cycles. This indicates that females re-used a site for parturition, which has the potential to facilitate the sorting of adaptive variation among neighbouring sites. The results demonstrate differential impacts of microevolutionary forces at varying spatial scales and highlight the importance of conserving essential habitats to maintain sources of adaptive variation that may buffer species against environmental change.

Using fisher-contributed secondary fins to fill critical shark-fisheries data gaps.

Developing-world shark fisheries are typically not assessed or actively managed for sustainability; one fundamental obstacle is the lack of species and size-composition catch data. We tested and implemented a new and potentially widely applicable approach for collecting these data: mandatory submission of low-value secondary fins (anal fins) from landed sharks by fishers and use of the fins to reconstruct catch species and size. Visual and low-cost genetic identification were used to determine species composition, and linear regression was applied to total length and anal fin base length for catch-size reconstruction. We tested the feasibility of this approach in Belize, first in a local proof-of-concept study and then scaling it up to the national level for the 2017-2018 shark-fishing season (1,786 fins analyzed). Sixteen species occurred in this fishery. The most common were the Caribbean reef (Carcharhinus perezi), blacktip (C. limbatus), sharpnose (Atlantic [Rhizoprionodon terraenovae] and Caribbean [R. porosus] considered as a group), and bonnethead (Sphyrna cf. tiburo). Sharpnose and bonnethead sharks were landed primarily above size at maturity, whereas Caribbean reef and blacktip sharks were primarily landed below size at maturity. Our approach proved effective in obtaining critical data for managing the shark fishery, and we suggest the tools developed as part of this program could be exported to other nations in this region and applied almost immediately if there were means to communicate with fishers and incentivize them to provide anal fins. Outside the tropical Western Atlantic, we recommend further investigation of the feasibility of sampling of secondary fins, including considerations of time, effort, and cost of species identification from these fins, what secondary fin type to use, and the means with which to communicate with fishers and incentivize participation. This program could be a model for collecting urgently needed data for developing-world shark fisheries globally. Article impact statement: Shark fins collected from fishers yield data critical to shark fisheries management in developing nations.

Uso de Aletas Secundarias Proporcionadas por Pescadores para Llenar Vacíos Importantes de Información sobre las Pesquerías de Tiburones Resumen Con frecuencia no se evalúan las pesquerías de tiburones del mundo en desarrollo ni cuentan con un manejo activo de sustentabilidad. Uno de los principales obstáculos para esto es la falta de información sobre las especies y la composición de los tamaños en las capturas. Probamos e implementamos una estrategia nueva y potencialmente aplicable en todas partes para la recolección de estos datos: la entrega obligatoria de las aletas secundarias de bajo valor económico (aletas anales) obtenidas de los tiburones desembarcados por parte de los pescadores y el uso de estas aletas para reconstruir las especies y tamaños en la captura. Usamos identificaciones genéticas de bajo costo e identificaciones visuales para determinar la composición de las especies y aplicamos una regresión lineal a la longitud total y a la de la base de la aleta anal para la reconstrucción del tamaño en captura. Probamos la viabilidad de esta estrategia en Belice, primero en un estudio de prueba de concepto y después subiendo al nivel nacional para la temporada de pesca de tiburón 2017-2018 (1,786 aletas analizadas). Se registraron 16 especies en esta pesquería. Las más comunes fueron Carcharhinus perezi, C. limbatus, Rhizoprionodon terraenovae y R. porosus (consideradas como un grupo) y Sphyrna cf. tiburo. Las últimas tres especies fueron desembarcadas principalmente por encima del tamaño maduro, mientras que con las dos primeras especies lo hacían por debajo del tamaño maduro. Nuestra estrategia demostró ser efectiva en la obtención de información crítica para el manejo de la pesquería de tiburones y sugerimos que las herramientas desarrolladas como parte de este programa puedan ser exportadas a otras naciones en esta región y aplicadas casi de manera inmediata si existen los medios para comunicarse con los pescadores e incentivarlos a proporcionar las aletas anales. Fuera del Atlántico Occidental tropical, recomendamos una mayor investigación de la viabilidad del muestreo de aletas secundarias, incluyendo la consideración del tiempo, esfuerzo y costo de la identificación de especies a partir de estas aletas; cuál tipo de aleta secundaria utilizar; y los medios mediante los cuales comunicarse con los pescadores e incentivarlos a participar. Este programa podría ser un modelo para la recolección de información de necesidad urgente para las pesquerías del mundo en desarrollo.

Sequencing thousands of single-cell genomes with combinatorial indexing.

Single-cell genome sequencing has proven valuable for the detection of somatic variation, particularly in the context of tumor evolution. Current technologies suffer from high library construction costs, which restrict the number of cells that can be assessed and thus impose limitations on the ability to measure heterogeneity within a tissue. Here, we present single-cell combinatorial indexed sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single-cell libraries for detection of somatic copy-number variants. We constructed libraries for 16,698 single cells from a combination of cultured cell lines, primate frontal cortex tissue and two human adenocarcinomas, and obtained a detailed assessment of subclonal variation within a pancreatic tumor.

Chromosome-scale shotgun assembly using an in vitro method for long-range linkage.

Long-range and highly accurate de novo assembly from short-read data is one of the most pressing challenges in genomics. Recently, it has been shown that read pairs generated by proximity ligation of DNA in chromatin of living tissue can address this problem, dramatically increasing the scaffold contiguity of assemblies. Here, we describe a simpler approach ("Chicago") based on in vitro reconstituted chromatin. We generated two Chicago data sets with human DNA and developed a statistical model and a new software pipeline ("HiRise") that can identify poor quality joins and produce accurate, long-range sequence scaffolds. We used these to construct a highly accurate de novo assembly and scaffolding of a human genome with scaffold N50 of 20 Mbp. We also demonstrated the utility of Chicago for improving existing assemblies by reassembling and scaffolding the genome of the American alligator. With a single library and one lane of Illumina HiSeq sequencing, we increased the scaffold N50 of the American alligator from 508 kbp to 10 Mbp.

Species composition of the international shark fin trade assessed through a retail-market survey in Hong Kong.

The shark fin trade is a major driver of shark exploitation in fisheries all over the world, most of which are not managed on a species-specific basis. Species-specific trade information highlights taxa of particular concern and can be used to assess the efficacy of management measures and anticipate emerging threats. The species composition of the Hong Kong Special Administrative Region of China, one of the world's largest fin trading hubs, was partially assessed in 1999-2001. We randomly selected and genetically identified fin trimmings (n = 4800), produced during fin processing, from the retail market of Hong Kong in 2014-2015 to assess contemporary species composition of the fin trade. We used nonparametric species estimators to determine that at least 76 species of sharks, batoids, and chimaeras supplied the fin trade and a Bayesian model to determine their relative proportion in the market. The diversity of traded species suggests species substitution could mask depletion of vulnerable species; one-third of identified species are threatened with extinction. The Bayesian model suggested that 8 species each comprised >1% of the fin trimmings (34.1-64.2% for blue [Prionace glauca], 0.2-1.2% for bull [Carcharhinus leucas] and shortfin mako [Isurus oxyrinchus]); thus, trade was skewed to a few globally distributed species. Several other coastal sharks, batoids, and chimaeras are in the trade but poorly managed. Fewer than 10 of the species we modeled have sustainably managed fisheries anywhere in their range, and the most common species in trade, the blue shark, was not among them. Our study and approach serve as a baseline to track changes in composition of species in the fin trade over time to better understand patterns of exploitation and assess the effects of emerging management actions for these animals.

Genetic Diversity of White Sharks, Carcharodon carcharias, in the Northwest Atlantic and Southern Africa.

The white shark, Carcharodon carcharias, is both one of the largest apex predators in the world and among the most heavily protected marine fish. Population genetic diversity is in part shaped by recent demographic history and can thus provide information complementary to more traditional population assessments, which are difficult to obtain for white sharks and have at times been controversial. Here, we use the mitochondrial control region and 14 nuclear-encoded microsatellite loci to assess white shark genetic diversity in 2 regions: the Northwest Atlantic (NWA, N = 35) and southern Africa (SA, N = 131). We find that these 2 regions harbor genetically distinct white shark populations (Φ ST = 0.10, P < 0.00001; microsatellite F ST = 0.1057, P < 0.021). M-ratios were low and indicative of a genetic bottleneck in the NWA (M-ratio = 0.71, P < 0.004) but not SA (M-ratio = 0.85, P = 0.39). This is consistent with other evidence showing a steep population decline occurring in the mid to late 20th century in the NWA, whereas the SA population appears to have been relatively stable. Estimates of effective population size ranged from 22.6 to 66.3 (NWA) and 188 to 1998.3 (SA) and evidence of inbreeding was found (primarily in NWA). Overall, our findings indicate that white population dynamics within NWA and SA are determined more by intrinsic reproduction than immigration and there is genetic evidence of a population decline in the NWA, further justifying the strong domestic protective measures that have been taken for this species in this region. Our study also highlights how assessment of genetic diversity can complement other sources of information to better understand the status of threatened marine fish populations.

A metagenomic assessment of the bacteria associated with Lucilia sericata and Lucilia cuprina (Diptera: Calliphoridae).

Lucilia Robineau-Desvoidy (Diptera: Calliphoridae) is a blow fly genus of forensic, medical, veterinary, and agricultural importance. This genus is also famous because of its beneficial uses in maggot debridement therapy (MDT). Although the genus is of considerable economic importance, our knowledge about microbes associated with these flies and how these bacteria are horizontally and trans-generationally transmitted is limited. In this study, we characterized bacteria associated with different life stages of Lucilia sericata (Meigen) and Lucilia cuprina (Wiedemann) and in the salivary gland of L. sericata by using 16S rDNA 454 pyrosequencing. Bacteria associated with the salivary gland of L. sericata were also characterized using light and transmission electron microscopy (TEM). Results from this study suggest that the majority of bacteria associated with these flies belong to phyla Proteobacteria, Firmicutes, and Bacteroidetes, and most bacteria are maintained intragenerationally, with a considerable degree of turnover from generation to generation. In both species, second-generation eggs exhibited the highest bacterial phylum diversity (20 % genetic distance) than other life stages. The Lucilia sister species shared the majority of their classified genera. Of the shared bacterial genera, Providencia, Ignatzschineria, Lactobacillus, Lactococcus, Vagococcus, Morganella, and Myroides were present at relatively high abundances. Lactobacillus, Proteus, Diaphorobacter, and Morganella were the dominant bacterial genera associated with a survey of the salivary gland of L. sericata. TEM analysis showed a sparse distribution of both Gram-positive and Gram-negative bacteria in the salivary gland of L. sericata. There was more evidence for horizontal transmission of bacteria than there was for trans-generational inheritance. Several pathogenic genera were either amplified or reduced by the larval feeding on decomposing liver as a resource. Overall, this study provides information on bacterial communities associated with different life stages of Lucilia and their horizontal and trans-generational transmission, which may help in the development of better vector-borne disease management and MDT methods.

Population-based resequencing of experimentally evolved populations reveals the genetic basis of body size variation in Drosophila melanogaster.

Body size is a classic quantitative trait with evolutionarily significant variation within many species. Locating the alleles responsible for this variation would help understand the maintenance of variation in body size in particular, as well as quantitative traits in general. However, successful genome-wide association of genotype and phenotype may require very large sample sizes if alleles have low population frequencies or modest effects. As a complementary approach, we propose that population-based resequencing of experimentally evolved populations allows for considerable power to map functional variation. Here, we use this technique to investigate the genetic basis of natural variation in body size in Drosophila melanogaster. Significant differentiation of hundreds of loci in replicate selection populations supports the hypothesis that the genetic basis of body size variation is very polygenic in D. melanogaster. Significantly differentiated variants are limited to single genes at some loci, allowing precise hypotheses to be formed regarding causal polymorphisms, while other significant regions are large and contain many genes. By using significantly associated polymorphisms as a priori candidates in follow-up studies, these data are expected to provide considerable power to determine the genetic basis of natural variation in body size.

Complex patterns of genetic population structure in the mouthbrooding marine catfish, Bagre marinus, in the Gulf of Mexico and U.S. Atlantic.

Patterns of genetic variation reflect interactions among microevolutionary forces that vary in strength with changing demography. Here, patterns of variation within and among samples of the mouthbrooding gafftopsail catfish (Bagre marinus, Family Ariidae) captured in the U.S. Atlantic and throughout the Gulf of Mexico were analyzed using genomics to generate neutral and non-neutral SNP data sets. Because genomic resources are lacking for ariids, linkage disequilibrium network analysis was used to examine patterns of putatively adaptive variation. Finally, historical demographic parameters were estimated from site frequency spectra. The results show four differentiated groups, corresponding to the (1) U.S. Atlantic, and the (2) northeastern, (3) northwestern, and (4) southern Gulf of Mexico. The non-neutral data presented two contrasting signals of structure, one due to increases in diversity moving west to east and north to south, and another to increased heterozygosity in the Atlantic. Demographic analysis suggested that recently reduced long-term effective population size in the Atlantic is likely an important driver of patterns of genetic variation and is consistent with a known reduction in population size potentially due to an epizootic. Overall, patterns of genetic variation resemble that of other fishes that use the same estuarine habitats as nurseries, regardless of the presence/absence of a larval phase, supporting the idea that adult/juvenile behavior and habitat are important predictors of contemporary patterns of genetic structure.

Spine patient care with wearable medical technology: state-of-the-art, opportunities, and challenges: a systematic review.

BACKGROUND CONTEXT: Healthcare reforms that demand quantitative outcomes and technical innovations have emphasized the use of Disability and Functional Outcome Measurements (DFOMs) to spinal conditions and interventions. Virtual healthcare has become increasingly important following the COVID-19 pandemic and wearable medical devices have proven to be a useful adjunct. Thus, given the advancement of wearable technology, broad adoption of commercial devices (ie, smartwatches, phone applications, and wearable monitors) by the general public, and the growing demand from consumers to take control of their health, the medical industry is now primed to formally incorporate evidence-based wearable device-mediated telehealth into standards of care. PURPOSE: To (1) identify all wearable devices in the peer-reviewed literature that were used to assess DFOMs in Spine, (2) analyze clinical studies implementing such devices in spine care, and (3) provide clinical commentary on how such devices might be integrated into standards of care. STUDY DESIGN/SETTING: A systematic review. METHODS: A comprehensive systematic review was conducted in adherence to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Guidelines (PRISMA) across the following databases: PubMed; MEDLINE; EMBASE (Elsevier); and Scopus. Articles related to wearables systems in spine healthcare were selected. Extracted data was collected as per a predetermined checklist including wearable device type, study design, and clinical indices studied. RESULTS: Of the 2,646 publications that were initially screened, 55 were extensively analyzed and selected for retrieval. Ultimately 39 publications were identified as being suitable for inclusion based on the relevance of their content to the core objectives of this systematic review. The most relevant studies were included, with a focus on wearables technologies that can be used in patients' home environments. CONCLUSIONS: Wearable technologies mentioned in this paper have the potential to revolutionize spine healthcare through their ability to collect data continuously and in any environment. In this paper, the vast majority of wearable spine devices rely exclusively on accelerometers. Thus, these metrics provide information about general health rather than specific impairments caused by spinal conditions. As wearable technology becomes more prevalent in orthopedics, healthcare costs may be reduced and patient outcomes will improve. A combination of DFOMs gathered using a wearable device in conjunction with patient-reported outcomes and radiographic measurements will provide a comprehensive evaluation of a spine patient's health and assist the physician with patient-specific treatment decision-making. Establishing these ubiquitous diagnostic capabilities will allow improvement in patient monitoring and help us learn about postoperative recovery and the impact of our interventions.

Epigenetic loss of heterogeneity from low to high grade localized prostate tumours.

Identifying precise molecular subtypes attributable to specific stages of localized prostate cancer has proven difficult due to high levels of heterogeneity. Bulk assays represent a population-average, which mask the heterogeneity that exists at the single-cell level. In this work, we sequence the accessible chromatin regions of 14,424 single-cells from 18 flash-frozen prostate tumours. We observe shared chromatin features among low-grade prostate cancer cells are lost in high-grade tumours. Despite this loss, high-grade tumours exhibit an enrichment for FOXA1, HOXB13 and CDX2 transcription factor binding sites, indicating a shared trans-regulatory programme. We identify two unique genes encoding neuronal adhesion molecules that are highly accessible in high-grade prostate tumours. We show NRXN1 and NLGN1 expression in epithelial, endothelial, immune and neuronal cells in prostate cancer using cyclic immunofluorescence. Our results provide a deeper understanding of the active gene regulatory networks in primary prostate tumours, critical for molecular stratification of the disease.

Spatially mapped single-cell chromatin accessibility.

High-throughput single-cell epigenomic assays can resolve cell type heterogeneity in complex tissues, however, spatial orientation is lost. Here, we present single-cell combinatorial indexing on Microbiopsies Assigned to Positions for the Assay for Transposase Accessible Chromatin, or sciMAP-ATAC, as a method for highly scalable, spatially resolved, single-cell profiling of chromatin states. sciMAP-ATAC produces data of equivalent quality to non-spatial sci-ATAC and retains the positional information of each cell within a 214 micron cubic region, with up to hundreds of tracked positions in a single experiment. We apply sciMAP-ATAC to assess cortical lamination in the adult mouse primary somatosensory cortex and in the human primary visual cortex, where we produce spatial trajectories and integrate our data with non-spatial single-nucleus RNA and other chromatin accessibility single-cell datasets. Finally, we characterize the spatially progressive nature of cerebral ischemic infarction in the mouse brain using a model of transient middle cerebral artery occlusion.

Species composition of the largest shark fin retail-market in mainland China.

Species-specific monitoring through large shark fin market surveys has been a valuable data source to estimate global catches and international shark fin trade dynamics. Hong Kong and Guangzhou, mainland China, are the largest shark fin markets and consumption centers in the world. We used molecular identification protocols on randomly collected processed fin trimmings (n = 2000) and non-parametric species estimators to investigate the species composition of the Guangzhou retail market and compare the species diversity between the Guangzhou and Hong Kong shark fin retail markets. Species diversity was similar between both trade hubs with a small subset of species dominating the composition. The blue shark (Prionace glauca) was the most common species overall followed by the CITES-listed silky shark (Carcharhinus falciformis), scalloped hammerhead shark (Sphyrna lewini), smooth hammerhead shark (S. zygaena) and shortfin mako shark (Isurus oxyrinchus). Our results support previous indications of high connectivity between the shark fin markets of Hong Kong and mainland China and suggest that systematic studies of other fin trade hubs within Mainland China and stronger law-enforcement protocols and capacity building are needed.

Structural and thermodynamic characterization of T4 lysozyme mutants and the contribution of internal cavities to pressure denaturation.

Using small-angle X-ray scattering (SAXS) and tryptophan fluorescence spectroscopy, we have identified multiple compact denatured states of a series of T4 lysozyme mutants that are stabilized by high pressures. Recent studies imply that the mechanism of pressure denaturation is the penetration of water into the protein rather than the transfer of hydrophobic residues into water. To investigate water penetration and the volume change associated with pressure denaturation, we studied the solution behavior of four T4 lysozyme mutants having different cavity volumes at low and neutral pH up to a pressure of 400 MPa (0.1 MPa = 0.9869 atm). At low pH, L99A T4 lysozyme expanded from a compact folded state to a partially unfolded state with a corresponding change in radius of gyration from 17 to 32 A. The volume change upon denaturation correlated well with the total cavity volume, indicating that all of the molecule's major cavities are hydrated with pressure. As a direct comparison to high-pressure crystal structures of L99A T4 lysozyme solved at neutral pH [Collins, M. D., Hummer, G., Quillin, M. L., Matthews, B. W., and Gruner, S. M. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 16668-16671], pressure denaturation of L99A and the structurally similar L99G/E108V mutant was studied at neutral pH. The pressure-denatured state at neutral pH is even more compact than at low pH, and the small volume changes associated with denaturation suggest that the preferential filling of large cavities is responsible for the compactness of the pressure-denatured state. These results confirm that pressure denaturation is characteristically distinct from thermal or chemical denaturation.

Highly scalable generation of DNA methylation profiles in single cells.

We present a highly scalable assay for whole-genome methylation profiling of single cells. We use our approach, single-cell combinatorial indexing for methylation analysis (sci-MET), to produce 3,282 single-cell bisulfite sequencing libraries and achieve read alignment rates of 68 ± 8%. We apply sci-MET to discriminate the cellular identity of a mixture of three human cell lines and to identify excitatory and inhibitory neuronal populations from mouse cortical tissue.

Differentiation-state plasticity is a targetable resistance mechanism in basal-like breast cancer.

Intratumoral heterogeneity in cancers arises from genomic instability and epigenomic plasticity and is associated with resistance to cytotoxic and targeted therapies. We show here that cell-state heterogeneity, defined by differentiation-state marker expression, is high in triple-negative and basal-like breast cancer subtypes, and that drug tolerant persister (DTP) cell populations with altered marker expression emerge during treatment with a wide range of pathway-targeted therapeutic compounds. We show that MEK and PI3K/mTOR inhibitor-driven DTP states arise through distinct cell-state transitions rather than by Darwinian selection of preexisting subpopulations, and that these transitions involve dynamic remodeling of open chromatin architecture. Increased activity of many chromatin modifier enzymes, including BRD4, is observed in DTP cells. Co-treatment with the PI3K/mTOR inhibitor BEZ235 and the BET inhibitor JQ1 prevents changes to the open chromatin architecture, inhibits the acquisition of a DTP state, and results in robust cell death in vitro and xenograft regression in vivo.

A multiplex PCR mini-barcode assay to identify processed shark products in the global trade.

Protecting sharks from overexploitation has become global priority after widespread population declines have occurred. Tracking catches and trade on a species-specific basis has proven challenging, in part due to difficulties in identifying processed shark products such as fins, meat, and liver oil. This has hindered efforts to implement regulations aimed at promoting sustainable use of commercially important species and protection of imperiled species. Genetic approaches to identify shark products exist but are typically based on sequencing or amplifying large DNA regions and may fail to work on heavily processed products in which DNA is degraded. Here, we describe a novel multiplex PCR mini-barcode assay based on two short fragments of the cytochrome oxidase I (COI) gene. This assay can identify to species all sharks currently listed on the Convention of International Trade of Endangered Species (CITES) and most shark species present in the international trade. It achieves species diagnosis based on a single PCR and one to two downstream DNA sequencing reactions. The assay is capable of identifying highly processed shark products including fins, cooked shark fin soup, and skin-care products containing liver oil. This is a straightforward and reliable identification method for data collection and enforcement of regulations implemented for certain species at all governance levels.

Facultative parthenogenesis in a critically endangered wild vertebrate.

Facultative parthenogenesis - the ability of sexually reproducing species to sometimes produce offspring asexually - is known from a wide range of ordinarily sexually reproducing vertebrates in captivity, including some birds, reptiles and sharks [1-3]. Despite this, free-living parthenogens have never been observed in any of these taxa in the wild, although two free-living snakes were recently discovered each gestating a single parthenogen - one copperhead (Agkistrodon contortrix) and one cottonmouth (Agkistrodon piscivorus) [1]. Vertebrate parthenogens are characterized as being of the homogametic sex (e.g., females in sharks, males in birds) and by having elevated homozygosity compared to their mother [1-3], which may reduce their viability [4]. Although it is unknown if either of the parthenogenetic snakes would have been carried to term or survived in the wild, facultative parthenogenesis might have adaptive significance [1]. If this is true, it is reasonable to hypothesize that parthenogenesis would be found most often at low population density, when females risk reproductive failure because finding mates is difficult [5]. Here, we document the first examples of viable parthenogens living in a normally sexually reproducing wild vertebrate, the smalltooth sawfish (Pristis pectinata). We also provide a simple approach to screen any microsatellite DNA database for parthenogens, which will enable hypothesis-driven research on the significance of vertebrate parthenogenesis in the wild.

A novel mini-DNA barcoding assay to identify processed fins from internationally protected shark species.

There is a growing need to identify shark products in trade, in part due to the recent listing of five commercially important species on the Appendices of the Convention on International Trade in Endangered Species (CITES; porbeagle, Lamna nasus, oceanic whitetip, Carcharhinus longimanus scalloped hammerhead, Sphyrna lewini, smooth hammerhead, S. zygaena and great hammerhead S. mokarran) in addition to three species listed in the early part of this century (whale, Rhincodon typus, basking, Cetorhinus maximus, and white, Carcharodon carcharias). Shark fins are traded internationally to supply the Asian dried seafood market, in which they are used to make the luxury dish shark fin soup. Shark fins usually enter international trade with their skin still intact and can be identified using morphological characters or standard DNA-barcoding approaches. Once they reach Asia and are traded in this region the skin is removed and they are treated with chemicals that eliminate many key diagnostic characters and degrade their DNA ("processed fins"). Here, we present a validated mini-barcode assay based on partial sequences of the cytochrome oxidase I gene that can reliably identify the processed fins of seven of the eight CITES listed shark species. We also demonstrate that the assay can even frequently identify the species or genus of origin of shark fin soup (31 out of 50 samples).