Large-scale genotyping platforms are currently being developed for several wild species. By querying thousands of polymorphic loci, genomics can be a useful ecological tool for describing and monitoring populations. Genomics is becoming increasingly useful as a forensic tool because of its ability to identify population of origin for purposes of enforcing anti-poaching laws. Our aim was to test the new SNP chip for caribou/reindeer (Rangifer tarandus) (Illumina iSelect caribou 60 K) under recommended and non-optimal sample conditions. Impact on signal detection (call rate) and error rate were assessed using reference samples. The SNP chip was shown to be robust, highly sensitive, reliable, and accurate at more than 10-fold below the recommended DNA input. Biological source of DNA had minor impact, even with fecal pellets given sufficient amount of host DNA. Hybridization of non-Rangifer samples as well as samples bearing DNA from two Rangifer samples both showed a drop in call rate and shifted levels of heterozygosity. Based on a population-targeted subset of SNPs included in the chip design, reassignment of 981 samples to a functional group (here to a caribou ecotype) was highly accurate (99.59 %) and the relative probability of reassignment error was estimated using the logarithm of odds score. Overall, the SNP chip is suitable for analysis of caribou/reindeer genomes even with suboptimal sampling and hence useful for population management and forensics.
Despite the high density of brown bears (Ursus arctos piscator) on the Kamchatka peninsula their genetic variation has not been studied by STR analysis. Our aim was, therefore, to provide population data from the Kamchatka brown bear population applying a validated DNA profiling system. Twelve dinucleotide STRs commonly used in Western-European (WE) populations and four additional ones (G10C, G10J, G10O, G10X), were included. Template input ≥ 0.2 ng was successfully amplified. Measurements of precision, stutter and heterozygous balance showed that markers could be reliably genotyped applying the thresholds used for genotyping WE brown bears. However, locus G10X revealed an ancient allele-specific polymorphism that led to suboptimal amplification of all 174 bp alleles (Kamchatka and WE). Allele frequency estimates and forensic genetic parameters were obtained from 115 individuals successfully identified by genotyping 434 hair samples. All markers met the Hardy-Weinberg and linkage equilibrium expectations, and the power of discrimination ranged from 0.667 to 0.962. The total average probability of identity from the 15 STRs was 1.4 ×10−14 (FST = 0.05) while the total average probability of sibling identity was 6.0 ×10−6. Relationship tests revealed several parent-cub and full sibling pairs demonstrating that the marker set would be valuable for the study of family structures. The population data is the first of its kind from the Kamchatka brown bear population. Population pairwise FST`s revealed moderate genetic differentiation that mirrored the geographic distances to WE populations. The DNA profiling system, providing individual-specific profiles from non-invasive samples, will be useful for future monitoring and conservation purposes
Situations involving crimes against animals are receiving increased social attention, resulting in a high demand for complaints and the initiation of police investigations. Overall, the quality of investigations and the production of material evidence in alleged crimes against fauna is crucial. For this, there is a need for trained professionals to implement protocols and tools based on animal welfare science and Legal Veterinary Medicine (LVM). In the routine and history of Veterinary Medicine, cases of suspected intoxication in companion animals, such as dogs and cats, are common. In Brazil, the act of intoxicating animals is considered a crime of mistreatment under the Environmental Crimes Law (Article 32, n. 9.605 of February 12, 1998). Therefore, it is necessary to examine the cadaver to confirm this suspicion. In this way, Legal Veterinary Medicine is a recent specialty in Brazil, created by the Federal Council of Veterinary Medicine's resolution in 2003. In this area, the evaluation of clinical signs, characteristic anatomopathological lesions, and toxicological analysis for identification of the toxic agents allow confirmation of suspected cases. Thus, the objective of this review is to address the application of scientific knowledge to Forensic Veterinary Medicine in confirming cases of animal poisoning and to list the main toxic substances found in animal poisoning cases identified through forensic examinations in the central region of Brazil. Additionally, it is intended to demonstrate that expertise in this area ensures the production of material evidence used in judicial criminal proceedings to defend animal welfare.
The precious Malagasy rosewood species of the genus Dalbergia have been listed in Appendix II of CITES due to illegal logging and timber trade. Accurate identification and proper delimitation are of paramount importance for endangered species regulated by CITES. One of the most accurate methods of identifying precious woods is DNA barcoding which is a technique for rapid and accurate species identification based on a short DNA sequence or a few DNA regions. Recent studies have shown that the ITS barcode can identify Malagasy Dalbergia species, but the number of species has been limited. In this research study, a total of 136 nuclear ribosomal internal transcribed spacer (nrITS) DNA sequences representing 41 species of Malagasy Dalbergia were used to assess the effectiveness of the ITS barcode in species identification. Each sequence was evaluated by genetic distance and tree-based methods. Using clustering analyses, estimating genetic distances, and comparing sequences of 136 samples, we were able to distinguish species. The interspecific distance was greater than the intraspecific distance, but no barcoding gap was observed. It was found that ITS has a very high discriminatory power for Malagasy Dalbergia species using both NJ and TaxonDNA methods. Considering the "Best Match" and "Best Close Match" analyses, the discrimination powers of ITS were 98.54% and 93.40%, respectively. Our research indicates that the use of ITS as a barcode enables the identification of Malagasy Dalbergia species and contributes to completing the reference library for controlling the trade in precious woods. These results are essential to ensure effective enforcement of regulations and promote the long-term conservation of these species.
Despite the prevalence of elephant poaching and ivory trafficking, domestic and international ivory markets around the world are slowly closing due to increased education and enforcement efforts. This includes California’s ivory market in 2016 after the passage of Assembly Bill 96 (AB 96), which prohibits the purchase, sale, offer for sale, possession, or importation with intent to sell, of ivory from elephant, mammoth, and mastodon along with other non-proboscidean species. To assist with enforcement efforts, the California Department of Fish and Wildlife’s Wildlife Forensic Laboratory (CDFW-WFL) has created and implemented a scientific workflow to taxonomically identify, geographically assign, and age California’s seized ivory. Here we discuss the application of this scientific workflow to a 32-piece ivory chess set, which was purchased in 1969 and donated to and examined by the WFL in 2021. Genetic data revealed 11 unique haplotypes and 19 unique genotypes, suggesting a diverse set of African elephants from numerous locations in Africa were used to assemble the chess set. Stable isotope data corroborated these findings and radiocarbon dating suggested the ivory used to carve these chess pieces grew approximately 6 years prior to the chess set being purchased. Our results indicate that the use of a variety of scientific techniques provides a wide scope of information; furthermore, taxonomically identifying, geographically assigning, and aging the ivory chess set demonstrates to law enforcement officers how our ivory workflow can assist them in coordinating efforts locally, nationally, and internationally to help stop the illegal importation of ivory into California.
Historically, Boswellia resin, commonly known as frankincense, has been used for a variety of applications, including medicinal purposes and in religious ceremonies. These practices continued into the modern era, in which the trade of resin has increased dramatically. The genus Boswellia contains many species that are not currently protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), but research has found that some of these populations are predicted to decrease by 50% in the next two decades due to overexploitation (Bongers et al. 2019). This highlights the need for a method that can identify Boswellia resins. In this research, resin from nine different species of Boswellia were collected and analyzed in positive ion mode by Direct Analysis in Real Time Time-of-Flight mass spectrometry (DART TOFMS) by dissolving the resin in methanol to obtain a chemotype devoid of background noise. Chemometric analysis of the data indicated that positive ion spectra from the resins separated the species effectively, indicating that DART TOFMS can be used to identify resins in trade.
Historically, Boswellia resin, commonly known as frankincense, has been used for a variety of applications, including medicinal purposes and in religious ceremonies. These practices continued into the modern era, in which the trade of resin has increased dramatically. The genus Boswellia contains many species that are not currently protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), but research has found that some of these populations are predicted to decrease by 50% in the next two decades due to overexploitation (Bongers et al. 2019). This highlights the need for a method that can identify Boswellia resins. In this research, resin from nine different species of Boswellia were collected and analyzed in positive ion mode by Direct Analysis in Real Time Time-of-Flight mass spectrometry (DART TOFMS) by dissolving the resin in methanol to obtain a chemotype devoid of background noise. Chemometric analysis of the data indicated that positive ion spectra from the resins separated the species effectively, indicating that DART TOFMS can be used to identify resins in trade.
Forensic mitochondrial DNA analysis is a vital investigative tool for wildlife casework, but numts continue to complicate DNA analysis. A major goal of wildlife forensic DNA analysis is to identify non-human biological evidence to its taxonomic source. Species identification is accomplished by sequencing genetic markers on the mitochondrial genome and comparing evidentiary sequence data to published reference sequences. Due to the high level of sequence similarity between mitochondrial genes and numts, current sequencing methods result in co-amplification of the target gene marker and non-target numt. Co-amplification of target and non-target loci results in ambiguous nucleotide calls in sequence data. Reducing the influence of numts during sequence analysis will provide a technique that will maximize accuracy and minimize error in taxonomic identifications. To overcome the analytical burden of numts, we studied the enzymatic removal of numts using exonuclease V. To evaluate the feasibility of exonuclease V as a numt removal method, total Panthera tigris DNA was extracted from blood and liver and divided into three treatment groups: untreated, 1stdigest, and 2nddigest. For the untreated sample, 7.6% of the 620bp sequence data was classified as ambiguous. Following treatment, all samples demonstrated a reduction in ambiguous calls: liver-1st digest(48 hrs): 6.6%, liver-2nddigest(48 hrs + 16 hrs): 1.8%, and blood-1stdigest(48 hrs): 0%. Based on this preliminary study, exonuclease V treatment effectively removed numts before sequencing analysis. While exonuclease V treatment has demonstrated potential, additional studies are required to optimize the reaction and fully validate the methodology.
Ocean currents, driven by gravity, wind, and water density, disperse marine biota worldwide, often leading species to shorelines alive or as carcasses. These carcasses provide vital information about species' health conditions and threats within their habitats. Marine animal strandings thus offer crucial insights into the ecological implications of population mortality. This research is instrumental for conservation efforts and identifying trends and threats. Scientists use human and animal forensics approaches to trace the origins of beached bodies. The capability to backtrack carcass drift and estimate death sites helps evaluate anthropogenic impacts. This information also forms the basis for legal applications and gives ecological indicators for marine megafauna conservation. Using backtracking in forensic ecology for conservation research presents expansive investigative opportunities. This paper offers a comprehensive review of: 1) Physical and environmental processes; 2) Drift applications; 3) Marine megafauna examples; 4) Forensic principles; 5) Postmortem intervals; 6) Marine megafauna backtracking. We further discuss these findings' potential conservation applications for endangered species. Our review aims to enhance understanding of coastal animal distribution, estimate mortality rates from strandings, explore seasonal variations for beach monitoring programs, and investigate anthropogenic impacts.
Trade of wild-caught animals is illegal for many taxa and in many countries. Common regulatory procedures involve documentation and marking techniques. However, these procedures are subject to fraud and thus should be complemented by routine genetic testing in order to authenticate the captive-bred origin of animals intended for trade. A suitable class of genetic markers are SNPSTRs that combine a short tandem repeat (STR) and single nucleotide polymorphisms (SNPs) within one amplicon. This combined marker type can be used for genetic identification and for parentage analyses and in addition, provides insight into haplotype history. As a proof of principle, this study establishes a set of 20 SNPSTR markers for Athene noctua, one of the most trafficked owls in CITES Appendix II. These markers can be coamplified in a single multiplex reaction. Based on population data, the percentage of observed and expected heterozygosities of the markers ranged from 0.400 to 1.000 and 0.545 to 0.850, respectively. A combined probability of identity of 5.3 * 10−23 was achieved with the whole set, and combined parentage exclusion probabilities reached over 99.99%, even if the genotype of one parent was missing. A direct comparison of an owl family and an unrelated owl demonstrated the applicability of the SNPSTR set in parentage testing. The established SNPSTR set thus proved to be highly useful for identifying individuals and analysing parentage to determine wild or captive origin. We propose to implement SNPSTR-based routine certification in wildlife trade as a way to reveal animal laundering and misdeclaration of wild-caught animals.
Because of the rich omega-3 fatty acids content, harp seal (Pagophilus groenlandicus) oil is a popular supplement that is packaged as pills in Canada and sold for medicinal purposes, although this practice is banned in the United States. Due to US regulations, it is important to be able to distinguish between fish oil and seal oil, but the taxonomic determination of oils provenance has been a difficult problem to solve. In this study, Direct Analysis in Real Time time-of-flight mass spectrometry (DART TOFMS) was used to analyze the chemotypes of blubber samples collected from seven species of marine mammals, including seals, sea lions, and a porpoise. Results indicated that the chemotype profiles found in negative-ion mode could be used to separate all of the species using Discriminant Analysis of Principal Components (DAPC). Consequently, this study suggests that it may be possible to identify the taxonomic source of marine mammal oils based on chemical chemotypes.
Estimating the postmortem interval (PMI) in cetaceans is challenging. These mammals often sink after death, later floating and traveling considerable distances before stranding, complicating decomposition stage analysis. Our study investigates decomposition patterns in humpback whale, guiana dolphin, and franciscana dolphin. We analyzed decomposition stages using photographic data from Instituto Baleia Jubarte (IBJ) and conducted a controlled guiana dolphin carcass decomposition study in a laboratory to establish a reliable PMI baseline for carcasses found along Brazil's Abrolhos Bank region. Our findings reveal species-specific decomposition timelines: humpback whale carcasses typically beach within 14 days post-death maximum, while franciscana and guiana dolphins strand within seven to eight days. The most common PMI for whales was five to six days, guiana dolphins four days, and franciscana dolphins four to five days. We used the five decomposition codes: I alive animals, II fresh carcass, III moderate decomposition, IV advanced decomposition, V skeletal remains. For small cetaceans, code II indicates a PMI of two days, III four days, and IV seven days. For large whales, code II signifies a PMI of one day, III three days, and IV seven days. PMI estimation is vital for identifying the period a carcass floated from death site to shoreline. This information supports forensic approaches in understanding anthropogenic impacts on cetacean mortality and aids ecological and conservation studies regarding cetacean strandings, using PMI and backtracking techniques.
Forensic point of origin testing is a key tool in identifying the provenance of a sample, whether it be for human remains, the food industry, or illegal wildlife trade. A variety of techniques – morphological, genetic and isotopic – have been developed to provide resolution on the origin of an individual or sample. Herein, we employed all three approaches to identify a reported case of food contamination. A consumer in New Jersey, USA reported finding a rodent in a beer can, which was bottled in Dallas-Fort Worth (DFW), Texas, USA. Photographs of the organism as well as biological material (muscle and hair) were evaluated to determine the species, location and ultimate source of the contamination. The rodent removed from the can was surprisingly intact and was identified belonging to the family Cricetidae and subfamily Neotominae, and likely of the genus Peromyscus spp. Sequencing of the COIII mitochondrial gene confirmed a species identification of P. leucopus. The analysis of δ2H and δ18O and subsequent probability assignment showed little support that the sample did not originate from the bottling facility (PDFW = 0.02 ± 0.02) but very high support that the mouse originated from New Jersey (PNew Jersey = 0.98 ± 0.02). Together, these results provide clear and consistent results that the mouse did not enter the food system at the bottling facility. This complimentary approach of morphological and molecular identification as well as point-of-origin assignment using stable isotope analysis yielded a highly cost-effective and probabilistic approach to assign origin of species that can be used by future forensic scientists.
Method validation is an essential step ahead of applying a method in forensic casework, to ensure the results will be admissible in court. However, unlike mainstream forensic disciplines, wildlife forensic labs often evolve from conservation-oriented units and may not have a strong foundation in generating data within a legal context. As such, the processes and principles of method validation may not be familiar or fully understood. In this paper we describe the process of method validation in a wildlife forensic science context. We provide guidance on the documentation required to take a DNA based method, which has been developed to identify a specific target species, through the validation process so that it is fit for use in forensic casework. This process has been agreed upon among members of the Society for Wildlife Forensic Sciences (SWFS) Technical Working Group (TWG) to illuminate the requirements for both practitioners and academics.
This study introduces an innovative method using DNA barcodes to accurately identify species, addressing the critical issue of trafficking invaluable medicinal plants like Trillium govanianum. The success rate in amplification and sequencing of barcoding regions was an impressive 100%. The Internal Transcribed Spacer (ITS) region proved most effective in distinguishing closely related Trillium species. By testing herbal products from markets, the study revealed extensive illegal trade, with 50% confirmed as Trillium govanianum and 30% adulterated with Dioscorea villosa. This research contributes new DNA sequences to identification databases, enhancing protocols. Genetic variations within Trillium govanianum were narrower than to the nearest non-conspecific neighbor in barcoding regions (matK: 0.006, rbcL: 0.003, ITS: 0.043). Combining coding and non-coding regions (matK + ITS) achieved 100% species discrimination. Market samples showed 30% product substitution, with varying success rates for amplification (rbcL: 70%, matK: 60%, ITS: 40%, improved with ITS2 primers). Half the sequences were Trillium govanianum, 30% were Dioscorea villosa, revealing covert substitution. ITS and matK sequences were effective, with a genetic distance gap, while rbcL had limited divergence (0.003), making it less suitable for accurate identification compared to matK and ITS.
Tortoiseshell, traditionally made from Hawksbill Turtle (Eretmochelys imbricata) shell, has long been a popular material for the production of coveted ornamental items. Hawksbill Turtles are critically endangered and like all sea turtles the trade in their products (e.g., tortoiseshell) is illegal. Tortoiseshell objects are also produced from other species and plastics, so the identification of the tortoiseshell source is important for distinguishing illegally and legally traded items. Distinguishing faux and real tortoiseshell visually can be challenging, so a screening method using infrared spectroscopy has been developed to provide a rapid means of discriminating the source of objects. A non-destructive attenuated total reflectance sampling technique has been employed. Marine turtle, horn, casein, cellulose nitrate, cellulose acetate and polyester were identified as the materials used in tortoiseshell production by employing a visual comparison of their spectra. A simple method for the discrimination of the protein-based spectra produced by marine turtle, horn and casein objects is provided, enabling the source of such objects to be differentiated.
Volatilomics is the study of the total biogenic volatile organic compounds (BVOCs) produced by an organism. This field has been used to assess organism and ecosystem health, as well as determine BVOC biomarkers for forensic purposes, including the detection of human remains, ignitable liquid residues and illicit drugs. For volatilomics to be applied in wildlife-victim casework (e.g. the illegal wildlife trade) a large reference database must be collected across each targeted species range. Adequate sample sizes must be collected from different habitats spanning across the species range of the targeted species to evaluate volatilome variability associated with different environmental and dietary characteristics. This will allow for assessment of chemical diversity and the determination of BVOC biomarkers that are relevant to wildlife forensic cases (e.g. detection, species identification, geographic origin assessment). This study collected the first live animal volatilome database, using the highly trafficked and widely distributed Australian shingleback lizard (Tiliqua rugosa). Optimised thermal desorption and analysis methods were used to examine 127 wild shingleback volatilome samples collected from sites across New South Wales, South Australia and Western Australia and 28 volatilome samples from captive shinglebacks. The results demonstrated that volatilome profiles and chemical diversity differed across each sampling region, potentially related to habitat and diet changes. At least 7 volatilome samples were required to capture chemical diversity in a sampling region. Forty-four tentatively identified BVOCs were shared across all sampling regions and captive animals which may aid in detection purposes. Bioregion-specific BVOCs were also identified, which will also aid in geographic assignment of confiscated individuals. This work demonstrates the importance of sample sizes in capturing chemical diversity within Bioregions prior to downstream volatilome analysis for the establishment of wildlife forensic databases and biomarker selection.
Efficient tools for the identification and discrimination of species are imperative in wildlife conservation since they can endow with information of species exploitation and also abet in solving problems related to forensic science. Herein, a non-destructive and rapid analytical method (ATR-FTIR Spectroscopy) coupled with PCA and PLS-DA was employed to analyze the dry blood samples for the species discrimination. Asian Elephant (Elephas maximus), Indian Leopard (Panthera pardus fusca), and Royal Bengal Tiger (Panthera tigris tigris) species were used to construct the chemometric models. Additionally, Domestic Pig (Sus scrofa domesticus) and Human (Homo sapiens) blood were taken for the external validation study. The evaluation results illustrate that the ATR FT-IR Spectroscopy in combination with PLS-DA model showed statistically confident discrimination among selected species from dry blood traces. ATR-FTIR spectroscopy supported with predictive models has been a robust, ideal, and suitable tool for species discrimination from dry blood traces recovered in wildlife investigations.
Illegal Wildlife Trade (IWT) is among the most lucrative illegal industries in the world. Its consequences go far beyond direct effects on the species in trade. In this review, we outline the basics of IWT and discuss its cascading consequences on environments, human lives and communities, national stability, and the economy. In addition, we outline structures used in IWT, from subsistence and local use to more complicated configurations, which can include multiple players. Furthermore, while a small fraction of poaching is opportunistic, most of the international IWT is run by organised crime groups. We outline how IWT can be associated with many different crimes like drug trafficking, corruption, or whitewashing. Additionally, many studies have observed a rapidly increasing trend of online trade with endangered and protected species. Moreover, this review gives a short overview of the situation in the European Union (EU) regarding laws and implementation of CITES and highlights that the EU acts as a major source, transit hub, and consumer in IWT. To address the highly dynamic and complicated problem of IWT, research, knowledge exchange, funding, and collaborations in all fields are necessary.
Genetic parentage tests have great relevance in breed registration processes in equine specimens. In addition, these tests are used to verify progeny genetic accuracy, using microsatellite molecular markers (STR) as a method for individual identification and kinship between individuals. Specifically, we evaluated a Colombian Creole breed equine population, using blood, saliva, and hair samples as sources of DNA. The samples were extracted using Chelex chelating resins. DNA quantification was performed by NanoDrop and amplified by PCR using the 17 STRs included in the Equine Genotypes panel 1.1 kit. Subsequently, electrophoresis was performed and stained with SYBR safe, and genotypes were determined using capillary electrophoresis. Additionally, the allelic and genotypic frequencies were calculated using the GENEPOP software and the previously obtained genetic profiles. Also, the HWE, heterozygosity per locus, inbreeding, and gametic imbalance were estimated. As a result, some genetic markers such as ASB23, ASB17, and LEX3 were not suitable for executing the tests since they biased the population parameters. However, we recommend expanding the equine population and using more STRs to obtain better results in these genetic tests in future population studies.
