Benjamín J. Gómez-Moliner

A non-invasive genetic method to identify the sympatric mustelids pine marten (Martes martes) and stone marten (Martes foina): preliminary distribution survey on the northern Iberian Peninsula

The closely related mustelids European pine marten (Martes martes) and stone marten (Martes foina) sympatrically inhabit a large area of Europe. However, given our limited knowledge of their bioecological relationships, their extremely elusive behaviour and the fact that their faeces cannot be distinguished on the basis of morphology alone, it is very difficult to monitor their populations. In this study, we describe a reliable non-invasive polymerase chain reaction (PCR)–restriction fragment length polymorphism (PCR-RFLP) method for distinguishing between M. martes and M. foina based on the analysis of deoxyribonucleic acid extracted from faeces samples. The method was specifically designed to avoid possible interference from potential prey mammals and other sympatric carnivores. The procedure consists of PCR amplifying a mitochondrial D-loop region followed by digesting the resulting 276-bp-long amplicons with the restriction enzymes HaeIII and RsaI. To assess the efficiency of this technique, we conducted a preliminary field study across the potential sympatric distribution areas of both marten species in the northern Iberian Peninsula. Out of 359 faeces samples collected, we identified 80 as specimens from the stone marten and 235 from the pine marten. Unequivocal species identification was thus possible in 88% of the faeces samples collected. These findings reveal the combined use of non-invasive genetic sampling and GIS technology to be a reliable and cost-effective procedure for improving our knowledge of the spatial distributions of sympatric marten species. This protocol could also be used to identify and improve information gaps, to develop effective research and management programmes and in population and landscape genetics studies of marten species.

Genetic introgression on freshwater fish populations caused by restocking programmes

The brown trout (Salmo trutta L.) is one of the best studied native salmonids of Europe. Genetic studies on this species suggest that a large proportion of the evolutionary diversity corresponds to southern European countries, including the Iberian Peninsula, where this study is focused. Stocking activities employing non-indigenous hatchery specimens together with the destruction and fragmentation of natural habitats are major factors causing a decrease of native brown trout populations, mostly in the Mediterranean basins of the Iberian Peninsula. The main aim of the present work is to examine the genetic structure of the brown trout populations of the East Cantabrian region, studying the consequences of the restocking activities with foreign hatchery brown trout specimens into the wild trout populations. We have based our study on the Polymerase Chain Reaction and Restriction Fragment Length Polymorphism technique conducted on a mitochondrial fragment of 2700 base pairs and on the lactate dehydrogenase locus of the nuclear DNA. Our results show higher introgression rates in the Ebro (Mediterranean) basin than in the Cantabrian rivers.

PCR-RFLP identification of mustelid species: European mink (Mustela lutreola), American mink (M. vison) and polecat (M. putorius) by analysis of excremental DNA

Recent advances in molecular scatology have allowed the development of reliable and non-invasive methods that can be applied in monitoring of small carnivores, without disturbance of the animals. Here a method is described that can be used to differentiate European mink Mustela lutreola , polecat M. putorius and American mink M. vison based on the analysis of DNA extracted from faeces. It consists of a nested PCR of a region of the mitochondrial D-loop followed by digestion of the resulting 240 bp amplicons with the restriction enzymes Rsa I and Msp I. The restriction patterns of both enzymes, when used together, are found to detect species-specific sequence variation. Two different haplotypes for European mink (AA, AB), another two for the polecat (AC, AD) and one for American mink (BC) can also be discriminated by this technique. Two new haplotypes for the mitocondrial D-loop of mustelids are described after DNA sequencing.

Bayesian analysis of hybridization and introgression between the endangered european mink (Mustela lutreola) and the polecat (Mustela putorius)

Human‐mediated global change will probably increase the rates of natural hybridization and genetic introgression between closely related species, and this will have major implications for conservation of the taxa involved. In this study, we analyse both mitochondrial and nuclear data to characterize ongoing hybridization and genetic introgression between two sympatric sister species of mustelids, the endangered European mink (Mustela lutreola) and the more abundant polecat (M. putorius). A total of 317 European mink, 114 polecats and 15 putative hybrid individuals were collected from different localities in Europe and genotyped with 13 microsatellite nuclear markers. Recently developed Bayesian methods for assigning individuals to populations and identifying admixture proportions were applied to the genetic data. To identify the direction of hybridization, we additionally sequenced mtDNA and Y chromosomes from 78 individuals and 29 males respectively. We found that both hybridization and genetic introgression occurred at low levels (3% and 0.9% respectively) and indicated that hybridization is asymmetric, as only pure polecat males mate with pure European mink females. Furthermore, backcrossing and genetic introgression was detected only from female first‐generation (F1) hybrids of European mink to polecats. This latter result implies that Haldane’s rule may apply. Our results suggest that hybridization and genetic introgression between the two species should be considered a rather uncommon event. However, the current low densities of European mink might be changing this trend.

Food habits of genetically identified pine marten (Martes martes) expanding in agricultural lowlands (NW Italy)

We assessed the diet of pine marten (Martes martes) expanding in the heavily human-altered agricultural plain of the River Po, northern Italy. Between February 2008 and November 2009, surveys were carried out twice a week during seasonal sampling sessions of three to four consecutive weeks. To distinguish the faecal samples from those of sympatric carnivores, a polymerase chain reaction-restriction fragment length polymorphism method was applied. The availability of small mammals was assessed by the analysis of 59 barn owl (Tyto alba) pellets. A total of 109 pine marten faeces were analysed. Its diet consisted of fruit, rodents, lagomorphs and birds. Seasonal variation occurred for fruit, which prevailed in summer, and rodents, which were more preyed upon in autumn. In winter, the diet of the pine marten was almost totally based on vertebrates, and lagomorphs were the main source of protein in summer. Use of small mammals differed significantly from their availability, voles, particularly bank vole Myodes glareolus, being preferred to mice (Apodemus sp.) and rats (Rattus sp.). Medium-size mammals formed about 18% of pine marten diet, a value generally reported for high-latitude habitats. In agricultural areas, the pine marten proved to be an opportunistic predator, able to face the reduced availability of small mammals by preying upon medium-size prey and fruit.

Landscape Genetics for the Empirical Assessment of Resistance Surfaces: The European Pine Marten (Martes martes) as a Target-Species of a Regional Ecological Network

Coherent ecological networks (EN) composed of core areas linked by ecological corridors are being developed worldwide with the goal of promoting landscape connectivity and biodiversity conservation. However, empirical assessment of the performance of EN designs is critical to evaluate the utility of these networks to mitigate effects of habitat loss and fragmentation. Landscape genetics provides a particularly valuable framework to address the question of functional connectivity by providing a direct means to investigate the effects of landscape structure on gene flow. The goals of this study are (1) to evaluate the landscape features that drive gene flow of an EN target species (European pine marten), and (2) evaluate the optimality of a regional EN design in providing connectivity for this species within the Basque Country (North Spain). Using partial Mantel tests in a reciprocal causal modeling framework we competed 59 alternative models, including isolation by distance and the regional EN. Our analysis indicated that the regional EN was among the most supported resistance models for the pine marten, but was not the best supported model. Gene flow of pine marten in northern Spain is facilitated by natural vegetation, and is resisted by anthropogenic landcover types and roads. Our results suggest that the regional EN design being implemented in the Basque Country will effectively facilitate gene flow of forest dwelling species at regional scale.

Isolation by distance, resistance and/or clusters? Lessons learned from a forest‐dwelling carnivore inhabiting a heterogeneous landscape

Landscape genetics provides a valuable framework to understand how landscape features influence gene flow and to disentangle the factors that lead to discrete and/or clinal population structure. Here, we attempt to differentiate between these processes in a forest‐dwelling small carnivore [European pine marten (Martes martes)]. Specifically, we used complementary analytical approaches to quantify the spatially explicit genetic structure and diversity and analyse patterns of gene flow for 140 individuals genotyped at 15 microsatellite loci. We first used spatially explicit and nonspatial Bayesian clustering algorithms to partition the sample into discrete clusters and evaluate hypotheses of ‘isolation by barriers’ (IBB). We further characterized the relationships between genetic distance and geographical (‘isolation by distance’, IBD) and ecological distances (‘isolation by resistance’, IBR) obtained from optimized landscape models. Using a reciprocal causal modelling approach, we competed the IBD, IBR and IBB hypotheses with each other to unravel factors driving population genetic structure. Additionally, we further assessed spatially explicit indices of genetic diversity using sGD across potentially overlapping genetic neighbourhoods that matched the inferred population structure. Our results revealed a complex spatial genetic cline that appears to be driven jointly by IBD and partial barriers to gene flow (IBB) associated with poor habitat and interspecific competition. Habitat loss and fragmentation, in synergy with past overharvesting and possible interspecific competition with sympatric stone marten (Martes foina), are likely the main factors responsible for the spatial genetic structure we observed. These results emphasize the need for a more thorough evaluation of discrete and clinal hypotheses governing gene flow in landscape genetic studies, and the potential influence of different limiting factors affecting genetic structure at different spatial scales.

Non-invasive genetic sampling of sympatric marten species (Martes martes and Martes foina): assessing species and individual identification success rates on faecal DNA genotyping

The use of non-invasive genetic sampling (NGS) has become increasingly popular in wildlife research but needs well-planned sampling strategies and reliable laboratory protocols. In this study, we planned to assess the reliability and success of species and individual identifications of sympatric martens (European pine marten Martes martes and stone marten Martes foina) by genotyping non-invasively collected faecal samples. First, we developed a novel and accurate multiplex panel of 15 microsatellite loci, selected by cross species amplification of 41 loci. The application of this panel facilitated species distinction, discarding the presence of putative hybrids. Then, we assessed the impact of sample collector skills on the lab protocol performances. The faecal DNA quality was evaluated by (a) the success of polymerase chain reaction–restriction fragment length polymorphism identification of the two Martes species and (b) the genotyping success and error rates of individual pine marten identifications. The survey was conducted over all the sympatric range of the two species in the Iberian Peninsula by three groups of sample collectors with different experience: expert wildlife biologist, trained volunteers and technical staff from natural parks. Results show that the different expertise between sample collectors significantly influences the success rate of pine marten individual genotyping, but not the species identification success rate. Based on our results, we recommend conducting sampling by experienced field biologist to maximise the quality of NGS and ensure accurate genotyping success. Application of our methods to field collected scats can be used in a cost-effective way to investigate distribution, patterns of genetic diversity and structure as well as to estimate population abundance for sympatric martens.

Genetic distinction of American and European mink (Mustela vison and M. lutreola) and European polecat (M. putorius) hair samples by detection of a species‐specific SINE and a RFLP assay

The European mink Mustela lutreola is the focus of extensive conservation programmes whereas the American mink M. vison is exotic in Europe and a possible cause of European mink decline. Since both species, together with the European polecat M. putorius have similar hair and produce similar faeces, molecular techniques are essential for reliable species identification when disturbance is not appropriate. In this report a simple, cheap and reliable molecular method for the genetic distinction of mink is provided based on the amplification of the microsatellite locus Mel 08 that results in a longer PCR product (436 bp) in American mink than in other mustelid species, such as the European mink (221 bp) and the European polecat (221 bp). Sequencing these PCR products revealed, in the microsatellite-flanking region of American mink, a CAN-SINE insertion of 213 bp that was absent in 22 species of the Canoidea superfamily tested. When the Mel 08 PCR product is cut with restriction enzyme Aci I and then run in an agarose gel, the restriction pattern observed is different in the three Mustela spp. The method was successfully tested with DNA amplified from a large sample of hair roots, thus making it suitable for non-invasive sampling.

Species limits, interspecific hybridization and phylogeny in the cryptic land snail complex Pyramidula: The power of RADseq data

Restriction site-associated DNA sequencing (RADseq) was used to jointly assess phylogenetic relationships, interspecific hybridization and species delimitation in the cryptic, non-model land snail complex Pyramidula. A robust phylogeny was inferred using a matrix of concatenated sequences of almost 1,500,000bp long, containing >97,000 polymorphic sites. Maximum likelihood analyses fully resolved the phylogenetic relationships among species and drastically improved phylogenetic trees obtained from mtDNA and nDNA gene trees (COI, 16S rRNA, 5.8S rRNA, ITS2 and 28S rRNA sequence data). The best species delimitation scenario was selected on the basis of 875 unlinked single nucleotide polymorphisms, showing that nine Pyramidula species should be distinguished in Europe. Applying D-statistics provided no or weak evidence of interspecific hybridization among Pyramidula, except for some evidence of gene flow between two species.