Phylogeography of the Sunda pangolin, Manis javanica: Implications for taxonomy, conservation management and wildlife forensics.

The Sunda pangolin (Manis javanica) is the most widely distributed Asian pangolin species, occurring across much of Southeast Asia and in southern China. It is classified as Critically Endangered and is one of the most trafficked mammals in the world, which not only negatively impacts wild Sunda pangolin populations but also poses a potential disease risk to other species, including humans and livestock. Here, we aimed to investigate the species' phylogeography across its distribution to improve our understanding of the species' evolutionary history, elucidate any taxonomic uncertainties and enhance the species' conservation genetic management and potential wildlife forensics applications. We sequenced mtDNA genomes from 23 wild Sunda pangolins of known provenance originating from Malaysia to fill sampling gaps in previous studies, particularly in Borneo. To conduct phylogenetic and population genetic analyses of Sunda pangolins across their range, we integrated these newly generated mitochondrial genomes with previously generated mtDNA and nuclear DNA data sets (RAD-seq SNP data). We identified an evolutionarily distinct mtDNA lineage in north Borneo, estimated to be ~1.6 million years divergent from lineages in west/south Borneo and the mainland, comparable to the divergence time from the Palawan pangolin. There appeared to be mitonuclear discordance, with no apparent genetic structure across Borneo based on analysis of nuclear SNPs. These findings are consistent with the 'out of Borneo hypothesis', whereby Sunda pangolins diversified in Borneo before subsequently migrating throughout Sundaland, and/or a secondary contact scenario between mainland and Borneo. We have elucidated possible taxonomic issues in the Sunda/Palawan pangolin complex and highlight the critical need for additional georeferenced samples to accurately apportion its range-wide genetic variation into appropriate taxonomic and conservation units. Additionally, these data have improved forensic identification testing involving these species and permit the implementation of geographic provenance testing in some scenarios.

Expediting the sampling, decalcification, and forensic DNA analysis of large elephant ivory seizures to aid investigations and prosecutions.

The illegal ivory trade continues to drive elephant poaching. Large ivory seizures in Africa and Asia are still commonplace. Wildlife forensics is recognised as a key enforcement tool to combat this trade. However, the time and resources required to effectively test large ivory seizures is often prohibitive. This limits or delays testing, which may impede investigations and/or prosecutions. Typically, DNA analysis of an ivory seizure involves pairing and sorting the tusks, sampling the tusks, powdering the sample, decalcification, then DNA extraction. Here, we optimize the most time-consuming components of this process: sampling and decalcification. Firstly, using simulations, we demonstrate that tusks do not need to be paired to ensure an adequate number of unique elephants are sampled in a large seizure. Secondly, we determined that directly powdering the ivory using a Dremel drill with a high-speed cutter bit, instead of cutting the ivory with a circular saw and subsequently powdering the sample in liquid nitrogen with a freezer mill, produces comparable results. Finally, we optimized a rapid 2 -h decalcification protocol that produces comparable results to a standard 3-day protocol. We tested/optimised the protocols on 33 raw and worked ivory samples, and demonstrated their utility on a case study, successfully identifying 94% of samples taken from 123 tusks. Using these new rapid protocols, the entire sampling and DNA extraction process takes less than one day and requires less-expensive equipment. We expect that the implementation of these rapid protocols will promote more consistent and timely testing of ivory seizures suitable for enforcement action.

Loxodonta Localizer: A Software Tool for Inferring the Provenance of African Elephants and Their Ivory Using Mitochondrial DNA.

Illegal hunting is a major threat to the elephants of Africa, with more elephants killed by poachers than die from natural causes. DNA from tusks has been used to infer the source populations for confiscated ivory, relying on nuclear genetic markers. However, mitochondrial DNA (mtDNA) sequences can also provide information on the geographic origins of elephants due to female elephant philopatry. Here, we introduce the Loxodonta Localizer (LL; www.loxodontalocalizer.org), an interactive software tool that uses a database of mtDNA sequences compiled from previously published studies to provide information on the potential provenance of confiscated ivory. A 316 bp control region sequence, which can be readily generated from DNA extracted from ivory, is used as a query. The software generates a listing of haplotypes reported among 1917 African elephants in 24 range countries, sorted in order of similarity to the query sequence. The African locations from which haplotype sequences have been previously reported are shown on a map. We demonstrate examples of haplotypes reported from only a single locality or country, examine the utility of the program in identifying elephants from countries with varying degrees of sampling, and analyze batches of confiscated ivory. The LL allows for the source of confiscated ivory to be assessed within days, using widely available molecular methods that do not depend on a particular platform or laboratory. The program enables identification of potential regions or localities from which elephants are being poached, with capacity for rapid identification of populations newly or consistently targeted by poachers.

Author Correction: Gene-associated markers provide tools for tackling illegal fishing and false eco-certification.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Improving cost-efficiency of faecal genotyping: New tools for elephant species.

Despite the critical need for non-invasive tools to improve monitoring of wildlife populations, especially for endangered and elusive species, faecal genetic sampling has not been adopted as regular practice, largely because of the associated technical challenges and cost. Substantial work needs to be undertaken to refine sample collection and preparation methods in order to improve sample set quality and provide cost-efficient tools that can effectively support wildlife management. In this study, we collected an extensive set of forest elephant (Loxodonta cyclotis) faecal samples throughout Gabon, Central Africa, and prepared them for genotyping using 107 single-nucleotide polymorphism assays. We developed a new quantitative polymerase chain reaction (PCR) assay targeting a 130-bp nuclear DNA fragment and demonstrated its suitability for degraded samples in all three elephant species. Using this assay to compare the efficacy of two sampling methods for faecal DNA recovery, we found that sampling the whole surface of a dung pile with a swab stored in a small tube of lysis buffer was a convenient method producing high extraction success and DNA yield. We modelled the influence of faecal quality and storage time on DNA concentration in order to provide recommendations for optimized collection and storage. The maximum storage time to ensure 75% success was two months for samples collected within 24 hours after defecation and extended to four months for samples collected within one hour. Lastly, the real-time quantitative PCR assay allowed us to predict genotyping success and pre-screen DNA samples, thus further increasing the cost-efficiency of our approach. We recommend combining the validation of an efficient sampling method, the build of in-country DNA extraction capacity for reduced storage time and the development of species-specific quantitative PCR assays in order to increase the cost-efficiency of routine non-invasive DNA analyses and expand the use of next-generation markers to non-invasive samples.

Correction: A rapid multiplex PCR assay for presumptive species identification of rhinoceros horns and its implementation in Vietnam.

[This corrects the article DOI: 10.1371/journal.pone.0198565.].

A rapid multiplex PCR assay for presumptive species identification of rhinoceros horns and its implementation in Vietnam.

Rhinoceros (rhinos) have suffered a dramatic increase in poaching over the past decade due to the growing demand for rhino horn products in Asia. One way to reverse this trend is to enhance enforcement and intelligence gathering tools used for species identification of horns, in particular making them fast, inexpensive and accurate. Traditionally, species identification tests are based on DNA sequence data, which, depending on laboratory resources, can be either time or cost prohibitive. This study presents a rapid rhino species identification test, utilizing species-specific primers within the cytochrome b gene multiplexed in a single reaction, with a presumptive species identification based on the length of the resultant amplicon. This multiplex PCR assay can provide a presumptive species identification result in less than 24 hours. Sequence-based definitive testing can be conducted if/when required (e.g. court purposes). This work also presents an actual casework scenario in which the presumptive test was successfully utlitised, in concert with sequence-based definitive testing. The test was carried out on seized suspected rhino horns tested at the Institute of Ecology and Biological Resources, the CITES mandated laboratory in Vietnam, a country that is known to be a major source of demand for rhino horns. This test represents the basis for which future 'rapid species identification tests' can be trialed.

Single-nucleotide polymorphism discovery and panel characterization in the African forest elephant.

The continuing decline in forest elephant (Loxodonta cyclotis) numbers due to poaching and habitat reduction is driving the search for new tools to inform management and conservation. For dense rainforest species, basic ecological data on populations and threats can be challenging and expensive to collect, impeding conservation action in the field. As such, genetic monitoring is being increasingly implemented to complement or replace more burdensome field techniques. Single-nucleotide polymorphisms (SNPs) are particularly cost-effective and informative markers that can be used for a range of practical applications, including population census, assessment of human impact on social and genetic structure, and investigation of the illegal wildlife trade. SNP resources for elephants are scarce, but next-generation sequencing provides the opportunity for rapid, inexpensive generation of SNP markers in nonmodel species. Here, we sourced forest elephant DNA from 23 samples collected from 10 locations within Gabon, Central Africa, and applied double-digest restriction-site-associated DNA (ddRAD) sequencing to discover 31,851 tags containing SNPs that were reduced to a set of 1,365 high-quality candidate SNP markers. A subset of 115 candidate SNPs was then selected for assay design and validation using 56 additional samples. Genotyping resulted in a high conversion rate (93%) and a low per allele error rate (0.07%). This study provides the first panel of 107 validated SNP markers for forest elephants. This resource presents great potential for new genetic tools to produce reliable data and underpin a step-change in conservation policies for this elusive species.

An internationally standardized species identification test for use on suspected seized rhinoceros horn in the illegal wildlife trade.

Rhinoceros (rhino) numbers have dwindled substantially over the past century. As a result, three of the five species are now considered to be critically endangered, one species is vulnerable and one species is near-threatened. Poaching has increased dramatically over the past decade due to a growing demand for rhino horn products, primarily in Asia. Improved wildlife forensic techniques, such as validated tests for species identification of seized horns, are critical to aid current enforcement and prosecution efforts and provide a deterrent to future rhino horn trafficking. Here, we present an internationally standardized species identification test based on a 230 base pair cytochrome-b region. This test improves on previous nested PCR protocols and can be used for the discrimination of samples with <20pg of template DNA, thus suitable for DNA extracted from horn products. The assay was designed to amplify water buffalo samples, a common 'rhino horn' substitute, but to exclude human DNA, a common contaminant. Phylogenetic analyses using this partial cytochrome-b region resolved the five extant rhino species. Testing successfully returned a sequence and correct identification for all of the known rhino horn samples and vouchered rhino samples from museum and zoo collections, and provided species level identification for 47 out of 52 unknown samples from seizures. Validation and standardization was carried out across five different laboratories, in four different countries, demonstrating it to be an effective and reproducible test, robust to inter laboratory variation in equipment and consumables (such as PCR reagents). This is one of the first species identification tests to be internationally standardized to produce data for evidential proceedings and the first published validated test for rhinos, one of the flagship species groups of the illegal wildlife trade and for which forensic tools are urgently required. This study serves as a model for how species identification tests should be standardized and disseminated for wildlife forensic testing.

Reference-free SNP discovery for the Eurasian beaver from restriction site-associated DNA paired-end data.

In this study, we used restriction site-associated DNA (RAD) sequencing to discover SNP markers suitable for population genetic and parentage analysis with the aim of using them for monitoring the reintroduction of the Eurasian beaver (Castor fibre) to Scotland. In the absence of a reference genome for beaver, we built contigs and discovered SNPs within them using paired-end RAD data, so as to have sufficient flanking region around the SNPs to conduct marker design. To do this, we used a simple pipeline which catalogued the Read 1 data in stacks and then used the assembler cortex_var to conduct de novo assembly and genotyping of multiple samples using the Read 2 data. The analysis of around 1.1 billion short reads of sequence data was reduced to a set of 2579 high-quality candidate SNP markers that were polymorphic in Norwegian and Bavarian beaver. Both laboratory validation of a subset of eight of the SNPs (1.3% error) and internal validation by confirming patterns of Mendelian inheritance in a family group (0.9% error) confirmed the success of this approach.

Gene-associated markers provide tools for tackling illegal fishing and false eco-certification.

Illegal, Unreported and Unregulated fishing has had a major role in the overexploitation of global fish populations. In response, international regulations have been imposed and many fisheries have been 'eco-certified' by consumer organizations, but methods for independent control of catch certificates and eco-labels are urgently needed. Here we show that, by using gene-associated single nucleotide polymorphisms, individual marine fish can be assigned back to population of origin with unprecedented high levels of precision. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, we find 93-100% of individuals could be correctly assigned to origin in policy-driven case studies. We show how case-targeted single nucleotide polymorphism assays can be created and forensically validated, using a centrally maintained and publicly available database. Our results demonstrate how application of gene-associated markers will likely revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide.

Genetic data from 15 STR loci for forensic individual identification and parentage analyses in UK domestic dogs (Canis lupus familiaris).

Eighteen STR loci and one sex determination locus present in the Finnzymes Canine 2.1 STR Multiplex Reagent Kit were screened in the UK dog population providing allele frequencies and population genetic parameters necessary for the application of STRs to forensic genetic casework. A total of 375 dogs were genotyped, including representative samples from each of twelve breeds used to evaluate Hardy-Weinberg equilibrium and calculate inter-population pairwise F(ST) values. Three loci were excluded from calculations of average random match probability due to deviations from Hardy-Weinberg Equilibrium or ambiguous genotyping. Random match probability based on fifteen STR loci and one sex locus was subsequently estimated to be 2.8 × 10(-17) for unrelated individuals across breeds.

Genetic data from 28 STR loci for forensic individual identification and parentage analyses in 6 bird of prey species.

Twenty-eight STR loci were screened in wild populations of six bird of prey species providing allele frequencies and population genetic parameters necessary for the application of STRs in wildlife forensic genetic casework. Individual STR loci were validated according to forensic recommendations in specimens of golden eagle (Aquila chrysaetos), goshawk (Accipiter gentilis), merlin (Falco columbarius), peregrine falcon (Falco peregrinus), gyr falcon (Falco rusticolus) and saker falcon (Falco cherrug). Deviations from Hardy-Weinberg expectations and linkage disequilibrium between locus pairs were examined. The average probability of identity (PI(ave)) and power of exclusion (PE) suggest the profiling systems of golden eagle, goshawk, merlin and peregrine falcons are capable of providing robust and highly discriminatory forensic evidence for legal proceedings. Due to low sample numbers the allele frequency data for gyr and saker falcons is not currently capable of providing an effective probability of identity. Further work should focus on increasing the size of these data sets.

A forensic STR profiling system for the Eurasian badger: a framework for developing profiling systems for wildlife species.

Developing short tandem repeat (STR) profiling systems for forensic identification is complicated in animal species. Obtaining a representative number of individuals from populations, limited access to family groups and a lack of developed STR markers can make adhering to human forensic guidelines difficult. Furthermore, a lack of animal specific guidelines may explain why many wildlife forensic STR profiling systems developed to date have not appropriately addressed areas such as marker validation or the publication and analysis of population data necessary for the application of these tools to forensic science. Here we present a methodology used to develop an STR profiling system for a legally protected wildlife species, the Eurasian badger Meles meles. Ten previously isolated STR loci were selected based on their level of polymorphism, adherence to Hardy-Weinberg expectations and their fragment size. Each locus was individually validated with respect to its reproducibility, inheritance, species specificity, DNA template concentration and thermocycling parameters. The effects of chemical, substrate and environmental exposure were also investigated. All ten STR loci provided reliable and reproducible results, and optimal amplification conditions were defined. Allele frequencies from 20 representative populations in England and Wales are presented and used to calculate the level of population substructure (theta) and inbreeding (f). Accounting for these estimates, the average probability of identity (PI(ave)) was 2.18 x 10(-7). This case study can act as a framework for others attempting to develop wildlife forensic profiling systems.

A DNA-based approach for the forensic identification of Asiatic black bear (Ursus thibetanus) in a traditional Asian medicine.

Attempts to prevent illegal trade in bile and gallbladders from Asiatic black bears, Ursus thibetanus, are hampered by difficulties associated with identifying such items. We extracted DNA from bile crystals of unknown species origin and generated partial cytochrome b (cyt b) sequences using either universal primers (positioned in conserved regions of cyt b), or primers designed on existing U. thibetanus sequences (UT). Species origin was determined by aligning resolved sequences to reference sequence data. The universal primers were unsuitable for U. thibetanus identification when multiple species templates were present in the samples. The UT primers amplified U. thibetanus DNA from all sample extracts, including those containing mixed species templates. The amplified fragment can distinguish U. thibetanus from the most closely related species, U. americanus, a distinct advantage of DNA sequencing over the methods currently used to analyze suspected U. thibetanus bile.

Validation of the barcoding gene COI for use in forensic genetic species identification.

The application of forensics to wildlife crime investigation routinely involves genetic species identification based on DNA sequence similarity. This work can be hindered by a lack of authenticated reference DNA sequence data resulting in weak matches between evidence and reference samples. The introduction of DNA barcoding has highlighted the expanding use of the mtDNA gene, cytochrome c oxidase I (COI), as a genetic marker for species identification. Here, we assess the COI gene for use in forensic analysis following published human validation guidelines. Validation experiments investigated reproducibility, heteroplasmy, mixed DNA, DNA template concentration, chemical treatments, substrate variation, environmental conditions and thermocycling parameters. Sequence similarity searches using both GenBank BLASTn and BOLD search engines indicated that the COI gene consistently identifies species where authenticated reference sequence data exists. Where misidentification occurred the cause was attributable to either erroneous reference sequences from published data, or lack of primer specificity. Although amplification failure was observed under certain sample treatments, there was no evidence of environmentally induced sequence mutation in those sequences that were generated. A simulated case study compared the performance of COI and cytochrome b mtDNA genes. Findings are discussed in relation to the utility of the COI gene in forensic species identification.