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

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

Abstract 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 (u) and inbreeding ( f). Accounting for these estimates, the average probability of identity (PI ave ) was 2.18 Â 10 À7 . This case study can act as a framework for others attempting to develop wildlife forensic profiling systems.

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

TigerBase:A DNA registration system to enhance enforcement and compliance testing of captive tiger facilities

The illegal trade in tigers (Panthera tigris) and their derivatives, such as bones, teeth and pelts, is a major threat to the species' long-term persistence. As wild tiger populations have dwindled, a large proportion of trafficked tiger products now derive from captive breeding facilities found throughout Asia. Moreover, wild tigers have been poached and laundered into captive facilities, then falsely designated as captive-bred. The establishment of a DNA registration system is recognized as a key tool to monitor compliance of captive facilities, support tiger trade investigations and improve prosecution outcomes. Here, we present a standardised wildlife forensic DNA profiling system for captive tigers called TigerBase. TigerBase has been developed in four South-East Asia countries with captive tiger facilities: Malaysia, Vietnam, Thailand and Lao PDR. TigerBase DNA profile data is based on 60 single nucleotide polymorphism (SNP) markers, genotyped using two different TaqMan®-based approaches: OpenArray® chip (capable of genotyping 60 SNPs for 48 samples in a single chip), and singleplex TaqMan® assays (capable of genotyping one SNP for one sample per reaction). Of the 60 SNPs, 53 are autosomal nuclear markers, suitable for individualisation and parentage applications, two are sex-linked markers, suitable for sexing, and five are mtDNA markers, suitable for maternal subspecies identification. We conducted a series of validation experiments to investigate the reliability and limitations of these SNP genotyping platforms. We found that the OpenArray® chip platform is more appropriate for generating reference data given its greater throughput, while the singleplex TaqMan® assays are more appropriate for genotyping lower quality casework samples, given their higher sensitivity and throughput flexibility. Only 19 autosomal nuclear markers were validated as singleplex TaqMan® assays, which generally provides ample power for individualisation analysis (probability of identity among siblings was &lt;6.9 ×10-4), but may lack power for specific parentage questions, such as determining parentage of an offspring when one of the parent's genotypes is missing. Further, we have developed pipelines to support standardised SNP calling and decrease the chance of genotyping errors through the use of analytical workflows and synthetic positive controls. We expect the implementation of TigerBase will enhance enforcement of tiger trafficking cases and encourage compliance among captive tiger facilities, together contributing to combatting the illegal tiger trade.</p

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

Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).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 <20 pg 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

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

DNA analyses of large pangolin scale seizures: Species identification validation and case studies

Pangolins are the mosttrafficked mammal in theworld, and all eightspecies are listed under CITESAppendix I.DNAbased wildlife forensic techniques are recognized as an important component of investigating a pangolin seizure. In particular, determining the species of pangolin in a seizure will 1) confirm the presence of pangolin to establish the legality of any trade, and 2) ensure appropriate laws are applied to theirfullest extentin a prosecution. Furthermore, valuable intelligence data, such as determining the geographic provenance of samples, can be produced through analysis of pangolin seizures. Despite the immense scale of the pangolin trade, standardized wildlife forensic techniquesfortesting pangolin seizures are in theirinfancy. To addressthis, here, we present a standardized genetic marker suitable for species identification of all eight pangolin species, and outline practical strategies for sampling large-volume pangolin scale seizures. We assessed the repeatability, reproducibility, robustness, sensitivity and phylogenetic resolution of this species identification test. Critically, the assay was tested in four wildlife forensic laboratories involved in testing pangolins. Additionally, we demonstrated the test’s utility to conduct geographic provenance analysis of Phataginus tricuspis samples. We analysed five large-volume pangolin scale seizures in Malaysia, which elucidated key targetspecies, poaching hotspots, and trafficking routes. Phataginustricuspis wasthe most commonly identified species(88.8%)from the seizure samples, and 84.3% of these P. tricuspisindividuals were likely sourced from western central Africa. We expect the im

Technical Note: A simple procedure for mimicking tissue samples from CITES controlled animal species for use in DNA proficiency tests

Proficiency tests are an important practice in forensic laboratories. However, the range of species available for wildlife forensic proficiency testing is currently limited, especially for laboratories in Africa and Asia where CITES-listed species are frequently tested. Here we present a proof of concept of a novel sample type for use in proficiency testing based on textured vegetable protein impregnated with the synthetic DNA of a target species. This sample can act as a substitute for animal tissue and can be shipped internationally without CITES and/or biosecurity permits. This simple procedure can help laboratories ensure compliance with international standards and guidelines