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Diagenesis is the collective word for the physical, biological, and chemical processes that bones undergo in the post-mortem period, until their physical destruction or fossilization. In forensic anthropology, the analysis of macroscopic and microscopic bone alterations, alongside the taphonomy of the soft tissues of a body, has proven valuable for the estimation of the time-of-death, or Post-Mortem Interval (PMI), of skeletonized individuals. To date, bone alterations have been mostly researched in terrestrial settings, such as exposed or buried skeletal remains, but here the scientific literature regarding human bones submerged underwater has been reviewed. It features 20 publications in the last 42 years, of which 9 are reviews, 8 are studies on ancient material and 3 are experimental studies. Future research on analysis of microscopic diagenetic parameters of submerged bones, together with the refinement of the correlation with time of the slightly better known macroscopic underwater alterations, will prove valuable for the estimation of a Post-Mortem Submersion Interval (PMSI) in both forensic and archaeological contexts, because bones have always been and still are regularly recovered underwater. The concurrent estimation of both PMI and PMSI of bones recovered underwater will add vital information to criminal investigations. Diagenetic parameters have been identified in Histological Index, protein content, porosity and crystallinity of bioapatite. They are depicted with the analytic techniques currently available to assess their presence and magnitude, and to relate them to the diagenetic processes of bioerosion, abrasion, and encrustation, but also to the extremes of dissolution or fossilization.

A framework for the development and validation of a qPCR assay for species identification and DNA quantification for conservation and forensic purposes is presented. Elephants are commonly poached for their ivory tusks, which is the primary driving force behind their endangered status. In addition to poaching and trade, habitat loss due to logging and mining has also resulted in loss of elephants. Crimes against animals can be deterred and/or further prosecution sought through testing with forensic genetic techniques. The creation of novel genetic assays can greatly impact wildlife forensic science investigations in identifying the species. Molecular genetic techniques can help enforce conservation efforts; however, they must be properly developed and validated to be of evidentiary quality for court systems. African and Asian elephant buccal cells were used as model in this work. The assay provides a method to differentiate biological fluids of both genera of elephants simultaneously. It can be used for identification of elephant derived products and presents valuable quantification for optimized further testing, such as microsatellite detection.

© 2017 Deutsche Gesellschaft für Säugetierkunde. Published by Elsevier GmbH. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (Sept 2017) in accordance with the publisher’s archiving policy, A European Endangered Species Programme (EEP) was established in the early 1990s, in order to manage a captive population of Asian lions (Panthera leo leo) within European zoos. The founders of this population comprised of nine individuals that originated from a captive population in India. During 2007–2009, 57 lions were born in the European captive population. Of these births, 35 individuals died within 20 days, three died within two months and one individual was euthanased at four months old. Indeed, over 50% of the total historical captive population died within 30 days of birth. The ‘European Studbook for the Asian Lion’ shows that the EEP founder population contains individuals from matings of full and half siblings, including all female founders. It is probable that high levels of inbreeding within this captive population are causing high levels of stillbirths and infant mortality. Previous research has shown that there is limited genetic variation in the captive population in India. This study uses the same microsatellite markers to establish the level of genetic variation that was present when the EEP population was established in comparison with that observed in the Indian zoo population, from which it was derived. Only three of the 12 microsatellite markers, showing variation in the Indian captive population, showed bi-allelic heterozygosity in the EEP founders, indicating that most variation was not present during the establishment of the EEP population. Therefore, the future of the Asian lion EEP is compromised by lack of genetic variation and high levels of inbreeding, which can only be alleviated by importing further individuals with different genotypes from India.

The composition and diversity of bacteria forming the microbiome of parasitic organisms have implications for differential host pathogenicity and host-parasite co-evolutionary interactions. The microbiome of pathogens can therefore have consequences that are relevant for managing disease prevalence and impact in affected hosts. Here we investigate the microbiome of an invasive parasitic fly Philornis downsi, recently introduced to the Galapagos Islands, where it poses extinction threat to Darwin's finches and other land birds. Larvae infest nests of Darwin's finches and consume blood and tissue of developing nestlings, and have severe mortality impacts. Using 16s rRNA sequencing data we characterize the bacterial microbiota associated with P. downsi adults and larvae sourced from four finch host species, inhabiting two islands and representing two ecologically distinct groups. We show that larval and adult microbiomes are dominated by the phyla Proteobacteria and Firmicutes, which significantly differ between life stages in their distributions. Additionally, bacterial community structure significantly differed between larvae retrieved from strictly insectivorous warbler finches (Certhidea olivacea) and those parasitizing hosts with broader dietary preferences (ground and tree finches, Geospiza and Camarhynchus spp. respectively). Finally, we found no spatial effects on the larval microbiome, as larvae feeding on the same host (ground finches) harbored similar microbiomes across islands. Our results suggest that the microbiome of P. downsi changes during its development, according to dietary composition or nutritional needs, and is significantly affected by host-related factors during the larval stage. Unraveling the ecological significance of bacteria for this parasite will contribute to the development of novel, effective control strategies. This article is protected by copyright. All rights reserved.

Illegal logging is one of the largest illicit trades in the world, with high profits and generally low risks of detection and prosecution. Timber identification presents problems for law enforcement as traditionally used forensic methods such as wood anatomy and dendrochronology are often unable to confidently match wood evidence to the remains of illegally felled trees. Here we have developed and validated a set of genetic markers for individualisation in bigleaf maple (Acer macrophyllum), a high value timber species often felled illegally in the USA. Using 128 single nucleotide polymorphisms and three insertion/deletion markers developed through massively parallel sequencing, 394 individuals were genotyped on the MassARRAY® iPLEX™ platform (Agena Bio-science™, San Diego, USA) to produce a population reference database for the species. We demonstrate that the resulting DNA assay is reliable, species specific, effective at low DNA concentrations (

We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, RedSafe™, SYBR(®) Green I, and EvaGreen™ were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR(®) Green I; 99.6% for RedSafe™; 99.4% for EvaGreen™; 52.7% for Diamond™ Dye; 50.6% for GelRed™, and; could not be determined for GelGreen™. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen(TM) at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen™ (1X), although with reduced amplification products. RedSafe™ (1X), Diamond™ Dye (1X), and SYBR(®) Green I (1X) all exhibited varying degrees of locus drop-out with GelRed™ generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection.

Python snake species are often encountered in illegal activities and the question of species identity can be pertinent to such criminal investigations. Morphological identification of species of pythons can be confounded by many issues and molecular examination by DNA analysis can provide an alternative and objective means of identification. Our paper reports on the development and validation of a PCR primer pair that amplifies a segment of the mitochondrial cytochrome b gene that has been suggested previously as a good candidate locus for differentiating python species. We used this DNA region to perform species identification of pythons, even when the template DNA was of poor quality, as might be the case with forensic evidentiary items. Validation tests are presented to demonstrate the characteristics of the assay. Tests involved the cross-species amplification of this marker in non-target species, minimum amount of DNA template required, effects of degradation on product amplification and a blind trial to simulate a casework scenario that provided 100% correct identity. Our results demonstrate that this assay performs reliably and robustly on pythons and can be applied directly to forensic investigations where the presence of a species of python is in question.

Nucleic acid staining dyes are used for detecting nucleic acids in electrophoresis gels. Historically, the most common dye used for gel staining is ethidium bromide, however due to its toxicity and mutagenicity other dyes that are safer to the user and the environment are preferred. This Short Communication details the properties of dyes now available and their sensitivity for detection of DNA and their ability to permeate the cell membrane. It was found that GelRed™ was the most sensitive and safest dye to use with UV light excitation, and both GelGreen™ and Diamond™ Nucleic Acid Dye were sensitive and the safer dyes using blue light excitation.

Geographic assignment of an unknown sample is a key aim in wildlife forensic science. In the course of examining 28 hypervariable STR loci for this purpose, a locus (MsF14) was observed that routinely exhibited variable amplification efficacy, appearing heritable in a Mendelian manner and reproducible. A correlation was seen between efficacy of amplification and geographic origin using 155 samples from across the species’ native range. We propose that this STR locus shows potential to assist in the geographical assignment of an unknown python sample.

Despite their wide use, the limits of presumptive tests can be poorly understood. The aim of this study was to investigate the specificity and sensitivity of conventional, as well as innovative, presumptive tests for blood, semen and saliva. We investigated Kastle–Meyer (KM) and leucomalachite green (LMG) tests for blood with regard to their sensitivity and specificity in the presence of oxidizing (hypochlorite) and anti-oxidizing (ascorbic acid) agents. The suitability and specificity of the red starch paper (RSP) test for saliva was assessed. Finally, the inhibitory effect of detergent on the acid phosphatase (AP) test for semen was investigated along with possible cross reactions to tea stains. Our results confirm previous findings of higher sensitivity and specificity of the KM test compared to LMG test for blood. Contrary to previous studies, no statistically significant difference was observed in the sensitivity of the tests between dry and wet stains. The novel RSP test was found to successfully detect saliva. We demonstrated that acid phosphatase (AP) testing for semen is possible on used RSP. A common multipurpose detergent had an inhibitory effect on AP tests. False positive results were obtained from tea stains. Testing different sorts of tea (black, green and herbal teas) revealed that only Camellia varieties produce positive result with the AP test, due to AP being present in the plants. From our results we conclude that specific knowledge of each test, including substances that may affect the test outcome, is imperative to ensure correct interpretation of presumptive test results.

An identification assay has been developed that allows accurate detection of 19 of the most common terrestrial mammals present in New Zealand (cow, red deer, goat, dog, horse, hedgehog, cat, tammar wallaby, mouse, weasel, ferret, stoat, sheep, rabbit, Pacific rat, Norway rat, ship rat, pig, and brushtail possum). This technique utilizes species-specific primers that, combined in a multiplex PCR, target small fragments of the mitochondrial cytochrome b gene. Each species, except hedgehog, produces two distinctive species-specific fragments, making the assay self-confirmatory and enabling the identification of multiple species simultaneously in DNA mixtures. The multiplex assay detects as little as 100 copies of mitochondrial DNA, which makes it a very reliable tool for degraded and trace samples. Reliability, accuracy, reproducibility, and sensitivity tests to validate the technique were performed. The technique featured here enabled a prompt response in a predation specific event, but can also be useful for wildlife management and conservation, pest incursions detection, forensic, and industrial purposes in a very simple and cost-effective manner.

Poaching is a crime that occurs worldwide and can be extremely difficult to investigate and prosecute due to the nature of the evidence available. If a species is protected by international legislation such as the Convention on International Trade in Endangered Species of Wild Fauna and Flora then simply possessing any part of that species is illegal. Previous studies have focused on the identification of endangered species in cases of potential poaching. Difficulties arise if the poached animal is not endangered. Species such as deer have hunting seasons whereby they can legally be hunted however poaching is the illegal take of deer, irrespective of season. Therefore, identification of deer alone has little probative value as samples could have originated from legal hunting activities in season. After a deer is hunted it is usual to remove the innards, head and lower limbs. The limbs are removed through manual force and represent a potential source of human touch DNA. We investigate the potential to recover and profile human autosomal DNA from poached deer remains. Samples from the legs of ten culled deer were obtained (40 in total) using minitapes. DNA from samples was extracted, quantified and amplified to determine if it would be possible to recover human STR profiles. Low quantification data led to the use of an extended PCR cycling protocol of 34 cycles. Samples from seven deer amplified, however some samples were excluded from further analysis due to ‘drop in’ alleles or the low level of successfully amplified loci. Samples from five deer could be further analysed and gave match probabilities ranging from 6.37 × 10− 3 to 9.53 × 10− 11. This study demonstrates the potential of recovering human touch DNA from poached animal remains. There is the potential for this test to be used in relation to other species of poached remains or other types of wildlife crimes. This is the first time, to our knowledge, that human STR profiling has been successfully applied to touch DNA in regards to simulated wildlife crime.

DNA profiles can be obtained from fabrics where a person has made direct contact with clothing. A standard approach is to cut out a section of the fabric and then use a commercially available method to extract and isolate the DNA. Alternative methods to isolate DNA include the use of adhesive tape to remove traces of cellular material from the fabric prior to extraction. We report on a process to obtain full DNA profiles using direct amplification from a range of fabrics. The absence of an extraction step both reduces the opportunity for contamination and reduces the loss of DNA during the extraction process, increasing the sensitivity of the process of generating a DNA profile. The process does not require the use of commercially available extraction kits thus reducing the cost of generating a DNA profile from trace amounts of starting material. The results are in part dependent upon the nature of the fabric used to which the DNA has been transferred.

A novel species-specific multiplex to identify 18 common European mammalian species (badger, cat, cow, dog, donkey, fox, goat, guinea pig, harvest mouse, hedgehog, horse, house mouse, human, pig, rabbit, rat, red deer and sheep), many of which are often associated with forensic investigations, has been developed. The assay is based on the mitochondrial cytochrome b gene, which is commonly used in species identification and phylogeny studies. Areas of homology and variation were identified and were used to create universal and species-specific primers. The species-specific primers were designed such that they will only react with the species for which they were designed. Two primer sets were designed for each species making the test self-confirmatory. All primer sets produced the expected results. The multiplex was balanced at template concentration of 40 000 copies (approximately 1.36 pg). Validation was accomplished by analysing the same sample ten times to determine run variation and several samples for each species to determine between-sample variation. Twenty-eight additional mammalian species were reacted with the multiplex. The multiplex provides, for the first time, a definitive method for identification of species in a forensic context.

Species identification using mitochondrial DNA (mtDNA) loci is a standard method for mammalian species testing. Less is understood about the conservation and variability in the avian mitochondrial genome, yet many exotic bird species are threatened with extinction and are traded illegally. In this study 80 different avian species were chosen from 22 different Orders and their gene sequences for the cytochrome b, cytochrome oxidase I and the ND2 genes were obtained from the NCBI web site. Alignments of the sequence determined the areas of greatest variation and conservation. The alignment result of DNA sequence showed that the cytochrome b gene placed the most number of avian species into their appropriate Orders, ND2 was next closest and COI the poorest of the three loci. These data support the use of cytochrome b over the other two mitochondrial loci for avian species identification.

The trade in samples of tiger (Panthera tigris), or parts derived from tiger, is controlled through the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), which lists all subspecies as protected at the highest level. Tiger has been used as a component in traditional medicines for centuries, often as powder thus making its presence difficult to identify. It is therefore necessary to use a molecular approach for the unambiguous identification of the species. Some countries require knowledge of the exact subspecies present in order to prosecute anyone alleged to trade in tiger products. To this end, mitochondrial DNA from two individuals of four of the five subspecies of tiger were sequenced to determine if subspecies-specific variation could be identified that could be the basis for a molecular test. We report on the determination of a total of 7891 bp of the tiger mitochondrial genome spanning 16S rRNA through ND4.

There is no standard test to identify the species of origin of a sample. A general method is to amplify part of the mitochondrial genome, generally the 12S, 16S or cytochrome b gene, and sequence it for comparison with known sequences on GenBank. Highly degraded samples and mixtures make this technique unsuitable. As a functioning protein, cytochrome b cannot mutate unconditionally. Detrimental changes in the amino acid sequence or composition will result in cell death and would not be passed on to offspring. By examining the cytochrome b sequences non-detrimental variation can be found which can be used for specific-species identification. Areas of high homology can also be identified for universal amplification sites. Species-specific primers have been developed based on these SNPs in the cytochrome b gene such that they will only react for a particular species. By combining universal priming sites with species-specific sites, a simple yet effect test has been constructed for the identification of species. This test will produce a product of a particular size for each species. It will work on mixtures and has sensitivity to the femtogramme (10−15 g) level.

There are currently no tests available to quantify total non-human mammalian mtDNA. Standard universal DNA quantification tests are unsuitable due to the large size difference between nuclear and mitochondrial genomes and the ubiquity of human mtDNA. A method has therefore been developed to quantify total mammalian mtDNA and total human mtDNA present in a sample using SYBR Green. Mammalian primers designed to react with all mammals were designed on the 12S and human specific primers were designed on the cytochrome b gene. Each primer set was reacted separately with sample and SYBR Green and detected using RT-PCR. A standard curve was developed using dilutions ranging from 1 billion copies to 100 copies of mtDNA. Twenty-four human samples were analysed and an average log (copy number) human/universal ratio of 1.00 was obtained. Samples falling below this ratio will contain some non-human mtDNA while samples falling above this ratio contain human mtDNA only. Twenty-nine mammal samples were also tested. 96.6% of these showed human contamination to some extent. This test is able to quantify mtDNA down to the femtogramme (10E−15g) level.

Nuclei within anagen hair follicles can be viewed using fluorescent microscopy after staining with a nucleic acid binding dye. Anagen hairs are still in the growth phase and generally only removed by force such as pulling, plucking or trauma and can be important evidentiary items at a crime scene. Staining of the hairs can be a fast, easy, method to determine if hair samples have DNA present making it worthwhile to attempt to obtain a DNA profile for evidentiary purposes. SYBR® Green I and Diamond™ Nucleic Acid Dye are two such dyes. The duration of the in situ fluorescent signal of both these nucleic acid dyes was studied to determine how long the samples can be kept in storage after staining yet still be capable of producing a fluorescent signal. Our results show that when stained with Diamond™ Nucleic Acid Dye the fluorescent signal could be viewed months after initial staining period. However when the hair was stained with SYBR® Green I there was a significant reduction in the fluorescent signal within 7 days of initial staining. Our conclusion is that Diamond™ Nucleic acid dye was a viable dye for staining hair and keeping the samples in storage for later analysis.

It is known that DNA can be deposited onto a surface by touch yet few means have been developed for its in situ detection. Collecting touch-DNA samples can be difficult as likely locations rather than the DNA is targeted leading to many samples that are submitted to a forensic laboratory containing little or no DNA. A range of dyes are available that bind to DNA at high specificity for application within the laboratory and here we report on the use of these dyes to detect latent DNA on various substrates and within biological samples. Six common nucleic acid-binding dyes were selected due to their increase in fluorescence when in the presence of double stranded-DNA; four of the six dyes are permeable to cell membranes. The fluorescence from dye/DNA complex was detected using a high intensity light source, the Polilight® (PL500), an excitation wavelength of 490 nm and emission observed/recorded through interference filters centred at 530 nm or 550 nm depending on the dye emission. The samples were visualised under a fluorescent microscope (Nikon Optiphot) using a B2A filter cube. The detection limit of DNA was determined for the selected dyes along with the optimal conditions, such as buffer composition and dye concentration for a range of surfaces. The ability for the dyes to detect DNA within biological samples such as saliva, hair, skin, fingermarks, and hair follicles was also determined.

Hairs are commonly encountered at a crime scene through natural shedding (telogen) or trauma such as during an assault (anagen). The forcible removal of anagen hairs results in retention of the root sheath and therefore the potential for DNA evidence. Telogen hairs lack a root sheath and do not provide a good source of DNA. Microscopy may be performed on all hair samples to detect whether there are cells adhering to the proximal tip to determine if there is a chance of success from subsequent DNA profiling. To aid in improving the microscopy, we report of the staining of hair roots using a range of dyes (SYBR® Green I, Diamond™ Nucleic Acid Dye, GelGreen™, EvaGreen™ and Redsafe™) and subsequent results from DNA profiling. Results showed that nuclei were visualized using all the dyes except for GelGreen™. The hairs were then directly amplified and all samples produced an STR profile that met requirements for uploading to a DNA database. The staining procedure conducted before direct amplification had no or little effect on the PCR and electrophoresis of the STR fragments. These results show that these nucleic acid binding dyes can be used as a preliminary assessment as to the viability of the sample (number of nuclei present) for STR analysis.

An identification assay has been developed that allows accurate detection of 19 of the most common terrestrial mammals present in New Zealand (cow, red deer, goat, dog, horse, hedgehog, cat, tammar wallaby, mouse, weasel, ferret, stoat, sheep, rabbit, Pacific rat, Norway rat, ship rat, pig, and brushtail possum). This technique utilizes species-specific primers that, combined in a multiplex PCR, target small fragments of the mitochondrial cytochrome b gene. Each species, except hedgehog, produces two distinctive species-specific fragments, making the assay self-confirmatory and enabling the identification of multiple species simultaneously in DNA mixtures. The multiplex assay detects as little as 100 copies of mitochondrial DNA, which makes it a very reliable tool for degraded and trace samples. Reliability, accuracy, reproducibility, and sensitivity tests to validate the technique were performed. The technique featured here enabled a prompt response in a predation specific event, but can also be useful for wildlife management and conservation, pest incursions detection, forensic, and industrial purposes in a very simple and cost-effective manner.

DNA is deposited onto a surface by touch yet few means have been developed for its in situ detection. A range of dyes are available that bind to DNA at high specificity and here we report on the use of two of these dyes to detect latent DNA. SYBR® Green I and GelGreen were used to detect DNA within fingermarks after fingers and thumbs were pressed onto a range of substrates such as Parafilm®. A solution of dye was then pipetted onto the mark and allowed to dry briefly. There was a high level of fluorescence where the fingermark was present indicating the dye had bound to DNA however a low level of fluorescence was present in the negative controls. To determine whether this background fluorescence was due to bacteria present on the substrates the dyes were pipetted onto a bacterial culture and the level of fluorescence was observed. It was found that SYBR® Green I had a higher level of fluorescence compared with GelGreen™ and that both dyes fluoresce when in the presence of bacterial cells. By altering the volume and concentration of dye, ridge detail within the fingermark may be observed allowing for the possibility of not only detecting latent DNA but also using this method for human identification and fingermark comparison.

This work was based upon earlier studies which exploited indels located in the mitochondrial genome to discriminate between different varieties of grass. The nad7 and nad5 indels were amplified from the DNA of nine different varieties of amenity grass species typical of lawn and recreational use. Discrimination was achieved by differences in Tm values as shown by dissociation curve and supported by gel electrophoresis. Experiments were also implemented to determine the applicability of a genetic test using samples that were exposed to different environmental conditions. These were designed to include simulated conditions experienced by real forensic samples and consisted of grass leaves, stored dry inside paper envelopes at room temperature, desiccated grass leaves contaminated with fungal growth, and also stains made from grass leaves on cotton cloth to simulate grass marks that might be found on clothing. These stains were stored in a variety of conditions including, contact with soil, water logged, dry and exposed to sunlight and also stored dry in the dark and at 4 °C for comparison. All samples except the soil and the fungus contaminated samples gave amplification products that could be distinguished by means of both gel electrophoresis and by Tm determined by a dissociation curve.

Species identification is a tool used extensively in forensic science; particularly in the investigation of wildlife crime. The two most commonly used genetic loci in species identification are the cytochrome oxidase I gene (COI) and the cytochrome b gene (cyt b), and identification is generally carried out through the use of DNA sequencing. However, there is currently no standard method to quantify the data from sequence comparisons for presentation in reports and to courts as there have been no detailed studies of the expected levels of inter- and intraspecific variation. For the first time this study provides a detailed comparison of the effectiveness of these two loci. Interspecific and intraspecific variation are assessed and statistical confidence is applied to sequence comparisons. Comparison of 217 different mammalian species revealed that cyt b more accurately reconstructed their phylogeny and known relationships, and gave better resolution when separating species based on sequence data. Intraspecific variation was assessed using three model species and showed variation ranging from 0 to 1.16% (Kimura 2-parameter p-distance (K2P) × 100%), indicating that some level of variation should be expected. Interspecific variation was greater in cyt b than in COI. Using a K2P (×100) threshold of 1.5, cyt b gives a better resolution for separating species with a lower false positive rate and higher positive predictive value than those of COI. This study allows, for the first time, application of statistical confidence to sequences comparisons for species identification.

Poaching is a worldwide crime that can be difficult to investigate due to the nature of the evidence. Previous studies have focused on the identification of endangered species in cases of poaching. Difficulties arise if the poached animal is not endangered. In the UK deer have hunting seasons whereby they can legally be hunted. Therefore, identification of deer alone has little probative value as samples could have originated from legal hunting activities in season. After a deer is hunted it is usual to remove the innards, head and lower limbs. The limbs are removed through manual force and represent a potential source of human ‘touch DNA’. We investigate the potential to recover and profile human autosomal DNA from poached deer remains. Samples from the legs of ten culled deer were obtained (40 in total) using minitapes. DNA from samples was extracted, quantified and amplified to determine if it would be possible to recover human STR profiles. Low quantification data led to the use of an extended PCR cycling protocol of 34 cycles. Samples from five deer gave match probabilities of varying quality. This study demonstrates the recovery of human touch DNA from poached animal remains. This is the first time that human STR profiling has been successfully applied to touch DNA in regards to wildlife crime.

The Convention on the International Trade in Endangered Species of Wild Fauna and Flora (CITES) monitors the international trade in endangered animal and plant species; a high profile example is the tiger, Panthera tigris. We report on the application of a SNaPshot multiplex technique to simultaneously identify tiger species and subspecies; this test is based on identification of SNPs within the tiger mitochondrial genome. Mitochondrial DNA sequences from four of the five extant putative tiger subspecies were obtained and combined with DNA sequence data from 492 tiger and 349 other mammalian species. A total of 11 SNP loci were identified: five specific for tiger; three specific to Panthera tigris sumatrae and; three specific to P. t. tigris. The multiplex assay was able to reliably identify 15 voucher tiger samples. The sensitivity of the test was 15,000 mitochondrial DNA copies, indicating that it will work on trace amounts of tissue, bone or hair.

Despite renewed public interest, education, research and legislation, Traditional East Asian Medication (TEAM) continues to incorporate animal parts from endangered species and can be obtained openly in many countries. Products sold as having medicinal properties include plasters, pills, ointments, tonic wine and others. Due to the low population levels of some endangered species other more common species may be substituted or very small amounts may be used. This results in low levels of DNA which can be difficult to identify. Most species identification tests rely on sequence comparison with a known reference sample. Most TEAM contains mixtures of several different species making this technique unsuitable. We describe a novel single step assay to rapidly and simultaneously identify rhino, tiger, bear, leopard, pangolin, musk deer and several non-endangered mammals often substituted in TEAM. The test targets the mitochondrial genome to amplify species-specific fragments that can be separated easily using a genetic analyzer. Each fragment is of a different size so that none can be confused. The specificity of each primer pair allows for species identification to be made even if a mixture of several species is present. Further, if more than one of the target species is present then all the species will be amplified and identified simultaneously. The test is sensitive to very low levels of DNA equating to several hundred mitochondrial copies (a fraction of single cell).

Species identification for forensic purposes is being increasingly used, as the value of non-human evidence is realized. This requires the identification of the species before individual analysis can take place. Traditionally the cytochrome b (cyt b) gene was used for species identification, but in 2003 the cytochrome c oxidase subunit 1 (CO1) gene was introduced under the terminology ‘barcoding’. This started an ongoing debate as to which gene offers the best template for species identification (high inter-species variability and low intra-species variation). Sequence data from 236 mammals were compared with multiple sequence alignments for a large number of human, cow and dog samples. Comparisons were made based on the number of inter-species variations between the different species and the intra-species variation between members of the same species.

Although endangered species are afforded protection under national and international laws, trade in products containing endangered species is still one of the most lucrative criminal enterprises in the world. Forensic science is employed to determine if endangered species are present in commercial products but runs into problems due to the extremely low levels of DNA and degraded nature of the majority of samples. Therefore a section of the mitochondrial genome is generally amplified and sequenced to determine if endangered species are present. This technique is sensitive and accurate, but cannot identify components of a DNA mixture due to the use of universal primers and can fail with subcellular levels of DNA. A multiplex PCR was therefore developed, based on the cytochrome b and 12S rRNA genes, to identify the presence of endangered mammalian species, if present, in commercial products. Four species [tiger (Panthera tigris ssp.), leopard (Panthera pardus), musk deer (Moschus sp.) and Asiatic black bear (Ursus thibetanus ssp.)] in addition to several common non-protected mammalian species can be identified using species-specific primers. A specific PCR product is produced depending on which species is/are present. The test is accurate and sensitive to low levels of DNA. It has been tested using traditional East Asian Medicine claiming to contain one or more endangered mammalian species. The test can be performed using standard DNA analysis techniques and implemented by any laboratory with these facilities.

The five extant sub-species of tiger are all protected by international agreement in an effort to protect the few tigers that remain. The remaining sub-species are threatened by poaching as many parts of the tiger are used in Traditional East Asian Medicines (TEAM). Products can be bought in many countries that openly indicate that they contain tiger parts and contravene international laws. This presentation describes how DNA testing can be used to unambiguously identify the presence of tiger and other Panthera species in TEAM bought in the UK.

The extent of wildlife crime is unknown but it is on the increase and has observable effects with the dramatic decline in many species of flora and fauna. The growing awareness of this area of criminal activity is reflected in the increase in research papers on animal DNA testing, either for the identification of species or for the genetic linkage of a sample to a particular organism. This review focuses on the use of species testing in wildlife crime investigations. Species identification relies primarily on genetic loci within the mitochondrial genome; focusing on the cytochrome b and cytochrome oxidase 1 genes. The use of cytochrome b gained early prominence in species identification through its use in taxonomic and phylogenetic studies, while the gene sequence for cytochrome oxidase was adopted by the Barcode for Life research group. This review compares how these two loci are used in species identification with respect to wildlife crime investigations. As more forensic science laboratories undertake work in the wildlife area, it is important that the quality of work is of the highest standard and that the conclusions reached are based on scientific principles. A key issue in reporting on the identification of a particular species is a knowledge of both the intraspecies variation and the possible overlap of sequence variation from one species to that of a closely related species. Recent data showing this degree of genetic separation in mammalian species will allow greater confidence when preparing a report on an alleged event where the identification of the species is of prime importance. The aim of this review is to illustrate aspects of species testing in wildlife forensic science and to explain how a knowledge of genetic variation at the genus and species level can aid in the reporting of results.