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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.

Wildlife crimes and the threats they present to elephant populations raise the need to develop and implement DNA-based methodology as an aid for wildlife forensic investigations and conservation efforts. This study describes the development of a tetra-nucleotide repeat STR multiplex, genotyping assay that will identify Asian elephant (Elephas maximus) and African elephant (Loxodonta africana) DNA. The assay targets six tetra-nucleotide STRs and two sex-typing markers simultaneously in both genera of elephants, a first for elephant genotyping assays. The developed assay has potential application in wildlife investigations to associate a biological sample to a particular individual elephant and additionally in conservation science for population management.

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.

Sexual assault samples are some of the most common samples encountered in forensic analysis. These samples can require a significant time investment due to differential extraction processes. We report on the first record of successful direct amplification of semen for STR analysis. Neat seminal fluid, dilutions ranging from 1:5 to 1:160 and GEDNAP samples were successfully amplified using a direct method. A mild differential isolation technique to enrich spermatozoa was developed and successfully implemented to separate and directly amplify a mixture of semen and female epithelial cells. Aliquots of samples subjected to the differential isolation protocol were stained with Haemotoxylin and Eosin for sperm scoring. Samples stained after PCR showed a complete lack of intact spermatozoa demonstrating that the cells are lysed during the PCR process. This paper demonstrates the potential to incorporate direct PCR in cases of sexual assault to more rapidly obtain results and achieve a higher sensitivity. (150)

Microscale microbial distributions are patchy, with abundance hotspots and coldspots that provide important microenvironments for microbial interactions. However, studies are often restricted to abundance estimates alone. At the riverbed of the Murray River, we used taxonomy to complement quantitative analysis to show that abundance hotspots, coldspots and background levels are taxonomically distinct at all taxonomic levels. Abundance hotspots varied 115- and 5.9-fold above background over 0.9 cm for viruses and bacteria, respectively. For bacteria, hotspots represent increases in particular taxa rather than in all bacteria. Genera with increased abundances—Pseudomonas, Parasporobacterium, Lachnospiraceae incertae sedis and Bacteroides—were indicative of human and animal inputs, and represented up to 14.7% of the community. Distinct dominant genera led to high taxonomic dissimilarity among hotspots. Genera exclusivity was still higher in the background and coldspots, with 54 and 48 exclusive genera compared to 7 and 4 genera in hotspots, suggesting hotspots from persistent genera, rather than introduced genera. Hotspots were more similar to coldspots than background, suggesting coldspots may represent dying hotspots. Sample category predicted taxonomic similarity better than proximity, further indicating these heterogeneities are distinct from the background at the sub-centimetre scale. Hotspots and coldspots represent distinct spatial taxonomic distributions, rather than changes of the overall community. This suggests 0.3 ml volumes are cohesive long enough for particular operational taxonomic units (OTUs) to increase and create taxonomically distinct microscale communities within the Kolmogorov and Batchelor scales.

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 (

Groundwater is increasingly used globally for domestic, industrial and agricultural production. While many studies have focused on groundwater as a resource, the diverse ecosystems within are often ignored. Here, we assess 54 Southern South Australian groundwater microbial communities from the populated part of the state to assess their status and dynamics in isolated groundwater systems. We observed a strong site-to-site individuality in groundwater bacterial communities, likely due to the isolated nature of groundwater bodies leading to unique ecosystems. Rank abundance analysis indicates bacterial diversity is maintained even at low abundances and that the distribution fits classical ecological models for strong competition in resource-limited environments. Combined, our data indicates that despite overrepresentation of pollutant-associated bacterial orders in and around the Adelaide metropolitan area, microbial communities remain diverse and show little evidence of converging on a common pollutant-effected community.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., The spatial distributions of organism abundance and diversity are often heterogeneous. This includes the sub-centimetre distributions of microbes, which have ‘hotspots’ of high abundance, and ‘coldspots’ of low abundance. Previously we showed that 300 μl abundance hotspots, coldspots and background regions were distinct at all taxonomic levels. Here we build on these results by showing taxonomic micropatches within these 300 μl microscale hotspots, coldspots and background regions at the 1 μl scale. This heterogeneity among 1 μl subsamples was driven by heightened abundance of specific genera. The micropatches were most pronounced within hotspots. Micropatches were dominated by Pseudomonas, Bacteroides, Parasporobacterium and Lachnospiraceae incertae sedis, with Pseudomonas and Bacteroides being responsible for a shift in the most dominant genera in individual hotspot subsamples, representing up to 80.6% and 47.3% average abundance, respectively. The presence of these micropatches implies the ability these groups have to create, establish themselves in, or exploit heterogeneous microenvironments. These genera are often particle-associated, from which we infer that these micropatches are evidence for sub-millimetre aggregates and the aquatic polymer matrix. These findings support the emerging paradigm that the microscale distributions of planktonic microbes are numerically and taxonomically heterogeneous at scales of millimetres and less. We show that microscale microbial hotspots have internal structure within which specific local nutrient exchanges and cellular interactions might occur., This research was supported by a CSIRO Land and Water Top-Up PhD Scholarship (LD), a CSIRO consumables grant (LD), an APA to LD and Australian Research Council grants, www.arc.gov. au, LP-130100508 and DP-150103018 to JGM.

Real-time PCR (qPCR) is widely used in the life sciences. For quantifying DNA, a standard curve is required. Common methods for standard development are time consuming, costly, necessitate a specific skill set, and pose a contamination risk. Using a targeted synthetic oligonucleotide, such as a gBlocks® Gene Fragment, overcomes these drawbacks and provides researchers an accurate and quick solution to standard development. Here, we demonstrate that using a gBlocks fragment as a standard provides comparable sensitivity, reliability, and assay performance to a purified amplicon standard.

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.

Mitochondrial DNA (mtDNA) can provide a means for forensic identity testing when genotyping of nuclear DNA (nuDNA) targets is not possible due to degradation or lack of template. For degraded samples, an indication of the quantity and quality of mtDNA is essential to allow selection of appropriately sized targets for hypervariable region (HVR) analysis, which may conserve sample and resources. Three human-specific mtDNA targets of increasing length (86, 190 and 452 base pairs) were amplified by singleplex quantitative real-time PCR (qPCR), capable of providing an index of mtDNA degradation from fragment length information. Quantification was achieved by preparation of a standard curve for each target, using a purified mtDNA standard containing all three targets of interest, which produced a linear, accurate and precise result from 1×108 to 10 copies. These novel assays demonstrated excellent sensitivity, specificity and reproducibility in line with the minimum information for qPCR experiments (MIQE) guidelines. Further, a separate inhibition control reaction was included to guide sample clean-up and ensure the validity of degradation assays. This protocol assists the selection and analysis of appropriately sized targets to maximize the chance of obtaining an informative result in downstream assays like sequencing.

The complete genome sequences of two endophytic Streptomyces sp. strains, LUP30 and LUP47B, were analyzed. These strains were isolated from surface-sterilized roots of lucerne plants from South Australia and were found to promote the growth of the rhizobial partner in vitro and significantly increased nodulation and nitrogen fixation in lucerne plants.

The complete genome sequences of two endophytic Streptomyces sp. strains, LUP30 and LUP47B, were analyzed. These strains were isolated from surface-sterilized roots of lucerne plants from South Australia and were found to promote the growth of the rhizobial partner in vitro and significantly increased nodulation and nitrogen fixation in lucerne plants.

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.

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.

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.

The complete genome sequences of three endophytic Streptomyces species were compared. Strains EN16, EN23, and EN27 were isolated from surface-sterilized roots of wheat plants from South Australia. In field trials, these strains are effective in suppressing fungal root diseases of wheat when added as spore coatings to wheat seed.

Copyright © 2016, The Author(s) This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/, More than 97% of the world’s freshwater reserves are found in aquifers, making groundwater one of the most important resources on the planet. Prokaryotic communities in groundwater underpin the turnover of energy and matter while also maintaining groundwater purity. Thus, knowledge of microbial transport in the subsurface is crucial for maintaining groundwater health. Here, we describe for the first time the importance of stygofauna as vectors for prokaryotes. The “hitch-hiking” prokaryotes associated with stygofauna may be up to 5 orders of magnitude higher in abundance and transported up to 34× faster than bulk groundwater flow. We also demonstrate that prokaryotic diversity associated with stygofauna may be higher than that of the surrounding groundwater. Stygofauna are a newly recognized prokaryotic niche in groundwater ecosystems that have the potential to transport remediating, water purifying and pathogenic prokaryotes. Therefore, stygofauna may influence ecosystem dynamics and health at a microbial level, and at a larger scale could be a new source of prokaryotic diversity in groundwater ecosystems.

The complete mitochondrial genomes of five tiger samples from three subspecies (P. t. sumatrae, P. t. altica, and P. t. tigris) were successfully obtained by using 26 specifically designed Panthera-specific primer sets. The genome organization and gene arrangement of the five tiger samples were similar to each other; however polymorphic tandem repeat sequences were observed in the control region (CR). This led to a difference in the genome lengths obtained from these five samples with an average size of 16,994 bp for the five tiger mitochondrial genomes. The nucleotide base composition was on average as follows: A, 31.8%; T, 27.0%; C, 26.6%; G, 14.6% and exhibited compositional asymmetry. Most of tiger mitochondrial genome characteristics are similar to those of other common vertebrate species; however, some distinctive features were observed in the CR. First, the repetitive sequence 2 (RS 2) contained two repeat units of 80 bp and the first 15 bp of what would be the third repeat motif. The repetitive sequence 3 (RS 3) contained 47-50 repeat motifs of a shorter 8 bp (ACGTAYAC)(n). Second, length heteroplasmy polycystosine (poly-C) stretches was observed at the end of the HV I locus in all tiger samples.

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.

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.

Current histological investigation of vaginal swabs after alleged sexual assault includes the scoring of spermatozoa (0, + to ++++) and the recording of visible tails. It is a method that is universally employed. Despite this method being used for 40 years, there has never been a study investigating its suitability for forensic science. Here, we investigate the reproducibility and subjectivity of sperm scoring among different investigators. Dilutions of seminal fluid were randomly distributed onto 20 slides, stained with haematoxylin/eosin and assessed by 37 investigators, over 2 years. Slides were assessed for levels of spermatozoa and the presence of tails. Each slide was scored by a minimum of 25 investigators. On no slide was there a consensus between all scores. Standard deviation remained below 1, but relative standard deviation (RSD) ranged from 6 to 105% in a positive correlation as the average score decreased. Spermatozoa were not observed 56 times (9.6%) and 27 investigators (73%) did not observe spermatozoa on at least one slide. Spermatozoa with tails were observed on every slide by at least 10 examiners, but as the average score of the slide decreased, so did the observation of tails. The current sperm scoring method is highly subjective with a particularly high %RSD in slides with low overall sperm counts. Moreover, the recording of tails does not add value to the current technique of sperm scoring. Further research might improve the objectivity of sperm scoring and the reliability of recording of tails.

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.

The tiger (Panthera tigris) is currently listed on Appendix I of the Convention on the International Trade in Endangered Species of Wild Fauna and Flora; this affords it the highest level of international protection. To aid in the investigation of alleged illegal trade in tiger body parts and derivatives, molecular approaches have been developed to identify biological material as being of tiger in origin. Some countries also require knowledge of the exact tiger subspecies present in order to prosecute anyone alleged to be trading in tiger products. In this study we aimed to develop and validate a reliable single assay to identify tiger species and subspecies simultaneously; this test is based on identification of single nucleotide polymorphisms (SNPs) within the tiger mitochondrial genome. The mitochondrial DNA sequence from four of the five extant putative tiger subspecies that currently exist in the wild were obtained and combined with DNA sequence data from 492 tiger and 349 other mammalian species available on GenBank. From the sequence data a total of 11 SNP loci were identified as suitable for further analyses. Five SNPs were species-specific for tiger and six amplify one of the tiger subspecies-specific SNPs, three of which were specific to P. t. sumatrae and the other three were 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 (approximately 0.26 pg), indicating that it will work on trace amounts of tissue, bone or hair samples. This simple test will add to the DNA-based methods currently being used to identify the presence of tiger within mixed samples.

Although poaching is a common wildlife crime, the high and prohibitive cost of specialised animal testing means that many cases are left un-investigated. We previously described a novel approach to wildlife crime investigation that looked at the identification of human DNA on poached animal remains (Tobe, Govan and Welch, 2011). Human DNA was successfully isolated and amplified from simulated poaching incidents, however a low template protocol was required which made this method unsuitable for use in many laboratories. We now report on an optimised recovery and amplification protocol which removes the need for low template analysis. Samples from 10 deer (40 samples total — one from each leg) analysed in the original study were re-analysed in the current study with an additional 11 deer samples. Four samples analysed using Chelex did not show any results and a new method was devised whereby the available DNA was concentrated. By combining the DNA extracts from all tapings of the same deer remains followed by concentration, the recovered quantity of human DNA was found to be 29.5 pg ± 43.2 pg, 31 × greater than the previous study. The use of the Investigator Decaplex SE (QIAGEN) STR kit provided better results in the form of more complete profiles than did the AmpFlSTR® SGM Plus® kit at 30 cycles (Applied Biosystems). Re-analysis of the samples from the initial study using the new, optimised protocol resulted in an average increase of 18% of recovered alleles. Over 17 samples, 71% of the samples analysed using the optimised protocol showed sufficient amplification for comparison to a reference profile and gave match probabilities ranging from 7.7690 × 10− 05 to 2.2706 × 10− 14. The removal of low template analysis means this optimised method provides evidence of high probative value and is suitable for immediate use in forensic laboratories. All methods and techniques used are standard and are compatible with current SOPs. As no high cost non-human DNA analysis is required the overall process is no more expensive than the investigation of other volume crime samples. The technique is suitable for immediate use in poaching incidents.

The phylogeny and taxonomy of mammalian species were originally based upon shared or derived morphological characteristics. However, genetic analyses have more recently played an increasingly important role in confirming existing or establishing often radically different mammalian groupings and phylogenies. The two most commonly used genetic loci in species identification are the cytochrome oxidase I gene (COI) and the cytochrome b gene (cyt b). For the first time this study provides a detailed comparison of the effectiveness of these two loci in reconstructing the phylogeny of mammals at different levels of the taxonomic hierarchy in order to provide a basis for standardizing methodologies in the future. Interspecific and intraspecific variation is assessed and for the first time, to our knowledge, statistical confidence is applied to sequence comparisons. Comparison of the DNA sequences of 217 mammalian species reveals that cyt b more accurately reconstructs their phylogeny and known relationships between species based on other molecular and morphological analyses at Super Order, Order, Family and generic levels. Cyt b correctly assigned 95.85% of mammal species to Super Order, 94.31% to Order and 98.16% to Family compared to 78.34%, 93.36% and 96.93% respectively for COI. Cyt b also gives better resolution when separating species based on sequence data. Using a Kimura 2-parameter p-distance (x100) threshold of 1.5–2.5, cyt b gives a better resolution for separating species with a lower false positive rate and higher positive predictive value than those of COI.