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Atlantic salmon (Salmo salar L.) populations from different river origins mix in the North Atlantic during the marine life stage. To facilitate marine stock identification, we developed a genetic baseline covering the European component of the species’ range excluding the Baltic Sea, from the Russian River Megra in the north-east, the Icelandic Ellidaar in the west, and the Spanish Ulla in the south, spanning 3737 km North to South and 2717 km East to West. The baseline encompasses data for 14 microsatellites for 26 822 individual fish from 13 countries, 282 rivers, and 467 sampling sites. A hierarchy of regional genetic assignment units was defined using a combination of distance-based and Bayesian clustering. At the top level, three assignment units were identified comprising northern, southern, and Icelandic regions. A second assignment level was also defined, comprising eighteen and twenty-nine regional units for accurate individual assignment and mixed stock estimates respectively. The baseline provides the most comprehensive geographical coverage for an Atlantic salmon genetic data-set, and a unique resource for the conservation and management of the species in Europe. It is freely available to researchers to facilitate identification of the natal origin of European salmon.

European roe deer (Capreolus capreolus L.) are the most common game species in Europe, hunted for meat and trophies. Forensic investigations involving roe deer poaching may often benefit from an individual identification method to link a suspect to a specific incident. The current paper presents a forensically validated DNA profiling system for European roe deer called “STRoe deer”. This DNA profiling system consists of 12 novel unlinked tetranucleotide short tandem repeat (STR) loci and two sex-specific markers, with an allelic ladder to facilitate accurate genotyping. Validation results demonstrate successful amplification of all 14 loci with as little as 0.05 ng of European roe deer DNA. Species-specificity tests showed that other members of the Cervidae family exhibited partial profiles and non-specific peaks, whereas most members of the Bovidae family showed just non-specific cross-species amplification products. Based on 513 European roe deer samples collected from a population from the Swiss Plateau, probability of identity was 3.1 x 10- 12 and the fixation index of 0.0275 indicated that only weak effects of genetic substructure or inbreeding were present in this population. Three different methods to calculate match probabilities for randomly sampled genotypes resulted in median match probabilities ranging from 1.4 x 10-13 to 2.5 x 10-5. These methods accounted for population structure, occurrence of null alleles or relatedness of the involved individuals. Based on these results, we conclude that STRoe deer is a robust genotyping system that should prove a valuable tool for individual identification and sexing of European roe deer to support criminal investigations.

Over recent years, mounting pressure has been placed on countries to assess their role in the ivory trade, with a view to tackling the rapidly declining numbers of elephants, due to poaching. The United Kingdom has been identified as a large re-exporter of ivory. Despite much of this trade being reported as legal or antique ivory, such provision of ivory to meet demand is known to fuel illegal markets and provide trade routes for modern ivory sales. Aside from ivory species and age, further analysis to evaluate geographic provenance, can inform where an elephant had lived, and so identify a source region or population where poaching occurred. The purpose of this study was to determine the age and species of ivory objects surrendered or seized in the UK and assess their likely geographic provenance through comparison of results from mitochondrial DNA and stable isotope analysis to publicly accessible georeferenced African elephant databases. The results demonstrated that the objects tested from an airport seizure were modern and matched existing haplotypes allowing for regional geographic inferences (supported by both techniques) to be obtained for most of these objects. In contrast, antique and modern ivory was detected amongst the amnesty objects, and several new mtDNA haplotypes were identified. Regional geographic inferences were achieved for some but not all of the objects tested. Our findings show this combination of methods provides a wealth of information which, could provide insight into targeted elephant populations and assist in disrupting international wildlife trade networks.

Aim We constructed an independent phylogeographical hypothesis (IPH) for Atlantic salmon (Salmo salar L.) in Scotland and adjacent northern England and tested if the distribution of contemporary microsatellite variation accorded with the IPH. Location NW Europe. Methods Knowledge of post-Last Glacial Maximum (LGM) landscape development and salmon biology were used to define a temporal sequence of late Quaternary ecological niche models and, combined with the central-marginal (CM) model of diversity evolution, to formulate an IPH. Observed microsatellite locus variation for salmon from 102 rivers in Scotland and northern England was tested for agreement with the IPH. Results The IPH postulated (1) initial colonization (c. 17 ka bp) of multiple isolated deglaciating coastal regions by long-distance dispersal, predominantly from SW Europe via the Atlantic, (2) intra-region colonization dominated by local range expansion, (3) differentiation by founder effects/genetic drift, in line with CM, until deglaciation complete and regions merged (c. 10 ka bp). IPH regions were weakly but significantly differentiated, showed broad agreement with genetically defined regions, and genetic diversity within them positively correlated with time post-deglaciation, conforming to the CM model. The greatest differentiation was seen for the most recently diverged populations, with evidence that accumulating homoplastic mutations muted older regional phylogeographical signatures. Main conclusions Microsatellite differentiation in Scottish Atlantic salmon is significantly but weakly conditioned by post-LGM deglaciation, with a mosaic of regional groups and a temporal cascade of within-region CM effects caused by local range expansion as patches became deglaciated. Colonization was predominantly or exclusively from the south-western European LGM refugium. Accumulating homoplastic mutations have increasingly reduced regional differentiation.

Summary Seafood is one of the most traded food commodities in the world with demand steadily increasing [1] . There is, however, a rising concern over the vulnerability of seafood supply chains to species mislabelling and fraud 1 , 2 . DNA methods have been widely used to detect species mislabelling and a recent meta-analysis of 4500 seafood product tests from 51 publications found an average of 30 percent were not the species stated on the label or menu [3] . This high rate poses a serious threat to consumer trust, reputations of seafood businesses and the sustainability of fishery resources. Seafood certification schemes may help reduce this problem. Here, we use DNA barcoding [4] to validate the species identity of 1402 certified seafood products derived from 27 species across 18 countries and find that in over 99% of cases species labelling was correct.

Wildlife crime continues unabated contributing to the extinction or near extinction of many plant and animal species. Species identification is a key tool in the enforcement of national legislation. If no morphology exists, comparison of DNA sequences generated from a mitochondrial gene are compared to those on a reference database, commonly GenBank. Sequences up-loaded to GenBank are unregulated and can lead to uncertainty with the adequacy of this DNA sequence repository for identification in a forensic context. We propose the establishment of ForCyt as a fully-regulated database of species that are commonly encountered in forensic investigations. The establishment of ForCyt will allow confidence in future species identification; something that is an absolute requirement to ensure high quality forensic science.

Much remains to be discovered about the population genetic structure of parasites, despite the importance of such knowledge to understanding the processes involved in the spread of drug resistance through populations. Here we present a study of population genetic diversity in Trichostrongylus tenuis, an avian parasitic nematode infecting both poultry and game birds, where anthelmintic use is common. We examined diversity of nicotinamide dehydrogenase subunit 4 (nad4) mtDNA sequences within and between seven locations: five in the UK (red grouse hosts), one in Iceland (domestic goose) and one in Norway (willow grouse). Within-UK comparisons showed high nucleotide diversity (π = 0.015, n = 23) but no structure between locations (ΦST = 0.022, P = 0.27), with over 97% of variation distributed within-hosts. The highest diversity was found in Iceland (π = 0.043, n = 4), and the lowest in Norway (π = 0.003, n = 4). Differentiation between countries was considerable (ΦCT = 0.44, P < 0.05), in spite of the potential mixing effects of gene flow via migrating wild hosts and the poultry trade. However, significant pairwise FST values were found only between Norway and the other locations. Phylogenetic analysis provided statistical support for a separate clade for Norwegian samples only, with unresolved diversity leading to a star-shaped relationship between Icelandic and UK haplotypes. These results suggest that Norwegian T. tenuis are isolated, but that there is some connectivity between UK and Icelandic populations. Although anthelmintic resistance has not yet been reported for T. tenuis, the population structure is such that emerging resistance has the potential to spread by gene flow over a large geographic scale.

Many studies have addressed the latitudinal gradients in intraspecific genetic diversity of European taxa generated during postglacial range expansion from southern refugia. Although Asia Minor is known to be a centre of diversity for many taxa, relatively few studies have considered its potential role as a Pleistocene refugium or a potential source for more ancient westward range expansion into Europe. Here we address these issues for an oak gallwasp, Andricus quercustozae (Hymenoptera: Cynipidae), whose distribution extends from Morocco along the northern coast of the Mediterranean through Turkey to Iran. We use sequence data for a fragment of the mitochondrial gene cytochrome b and allele frequency data for 12 polymorphic allozyme loci to answer the following questions: (1). which regions represent current centres of genetic diversity for A. quercustozae? Do eastern populations represent one refuge or several discrete glacial refugia? (2). Can we infer the timescale and sequence of the colonization processes linking current centres of diversity? Our results suggest that A. quercustozae was present in five distinct refugia (Iberia, Italy, the Balkans, southwestern Turkey and northeastern Turkey) with recent genetic exchange between Italy and Hungary. Genetic diversity is greatest in the Turkish refugia, suggesting that European populations are either (a). derived from Asia Minor, or (b). subject to more frequent population bottlenecks. Although Iberian populations show the lowest diversity for putatively selectively neutral markers, they have colonized a new oak host and represent a genetically and biologically discrete entity within the species.

The development of microsatellite markers for parasitic nematodes has been hampered by technical difficulties in isolation and PCR amplification. We have investigated the potential for circumventing these problems using microsatellites from 3 trichostrongyloid species on a panel of 7 species. Ten of the 22 PCR primer pairs tested amplified in species other than the target species, usually in closely related species, and 2 new variable loci were discovered in the sheep parasite Trichostrongylus vitrinus. This study provides evidence that cross-species testing of microsatellite primers can be an effective alternative to isolation de novo.

On red grouse estates in the UK the nematode parasite Trichostrongylus tenuis is often controlled by application of grit medicated with the anthelmintic fenbendazole (FBZ). To date, assessment of the efficacy has been inhibited by the inability to quantify uptake of FBZ by the birds. We have developed a simple and sensitive HPLC-MS-MS method for detecting and quantifying FBZ and its metabolites from a 300 mg sample of red grouse liver. This method could be used to improve the efficacy of medicated grit treatment by allowing the identification of conditions and application methods that optimize the uptake of FBZ. With the necessary modifications, our method will also be applicable to other wildlife species where self-medication is used for parasite control.

Mating behaviour is a fundamental aspect of the evolutionary ecology of sexually reproducing species, but one that has been under-researched in parasitic nematodes. We analysed mating behaviour in the parasitic nematode Trichostrongylus tenuis by performing a paternity analysis in a population from a single red grouse host. Paternity of the 150 larval offspring of 25 mothers (sampled from one of the two host caeca) was assigned among 294 candidate fathers (sampled from both caeca). Each candidate father's probability of paternity of each offspring was estimated from 10-locus microsatellite genotypes. Seventy-six (51%) offspring were assigned a father with a probability of >0.8, and the estimated number of unsampled males was 136 (95% credible interval (CI) 77-219). The probability of a male from one caecum fathering an offspring in the other caecum was estimated as 0.024 (95% CI 0.003-0.077), indicating that the junction of the caeca is a strong barrier to dispersal. Levels of promiscuity (defined as the probability of two of an adult's offspring sharing only one parent) were high for both sexes. Variance in male reproductive success was moderately high, possibly because of a combination of random mating and high variance in post-copulatory reproductive success. These results provide the first data on individual mating behaviour among parasitic nematodes.

Benzimidazole (BZ) resistance is widespread in nematode parasites of livestock, but very little is known about the levels of BZ resistance in parasites with avian hosts. We investigated BZ resistance in Trichostrongylus tenuis, a nematode parasite of red grouse, Lagopus lagopus scotica. BZ anthelmintics had been in use in this system for up to 15 years, yet existing phenotypic evidence for resistance was inconclusive. We screened 1530 individuals from 14 populations at the principal beta-tubulin locus for BZ resistance (isotype 1, residue 200) and 940 of these at two further resistance sites (isotype 1, residue 167; isotype 2, residue 200). No BZ resistant genotypes were found. Alternative mechanisms may be responsible for BZ resistance in this system, or the method and timing of treatments may reduce selection pressure for BZ resistance by creating substantial refugia for susceptible genotypes.

An understanding of how genes move between and within populations of parasitic nematodes is important in combating the evolution and spread of anthelmintic resistance. Much has been learned by studying mitochondrial DNA markers, but autosomal markers such as microsatellites have been applied to only a few nematode species, despite their many advantages for studying gene flow in eukaryotes. Here, we describe the isolation of 307 microsatellites from Trichostrongylus tenuis, an intestinal nematode of red grouse. High levels of variation were revealed at sixteen microsatellite loci (including three sex-lined loci) in 111 male T. tenuis nematodes collected from four hosts at a single grouse estate in Scotland (average He = 0.708; mean number of alleles = 12.2). A population genetic analysis detected no deviation from panmixia either between (F(ST) = 0.00) or within hosts (F(IS) = 0.015). We discuss the feasibility of developing microsatellites in parasitic nematodes and the problem of null alleles. We also describe a novel 146-bp repeat element, TteREP1, which is linked to two-thirds of the microsatellites sequenced and is associated with marker development failure. The sequence of TteREP1 is related to the TcREP-class of repeats found in several other trichostrongyloid species including Trichostrongylus colubriformis and Haemonchus contortus.

The astigmatid mite Acarus siro (Linnaeus 1758) is an important agricultural pest and environmental allergen. However, it is likely that many mites described in the literature as A. siro, collected from both outdoor and stored product habitats, may belong to one of its sibling species, A. farris [Ent. Ber. Amst. 2 (26) (1905) 20] or A. immobilis [Bull. Br. Mus. Nat. Hist. 11 (1964a) 413; Acarologia. 6 (Suppl) (1964) 101]. The three species are difficult to separate morphologically, gene exchange between some of them is possible and, although each species displays environmental preferences, they occur together in some environments. This raises a question about their separate species status. In a pilot study, we investigated whether genetic data supported the separate species status of these forms. Both nuclear (the second internal transcribed spacer region [ITS-2] of the ribosomal cistron) and mitochondrial (cytochrome oxidase subunit I, mtcoxI hereafter) loci were employed for this purpose. Mtcox1 data does not conflict the differentiation into three separate species and while the ITS2 data were problematic for this group of mites it suggested that a congener, Acarus gracilis [Ann. Mag. Nat. Hist. 10 (1957) 753], is basal to the A. siro species complex.