Assessment of genetic diversity, population structure and wolf-dog hybridisation in the Eastern Romanian Carpathian wolf population.

The Carpathian Mountains have been constantly inhabited by grey wolves and present one of the largest distribution areas in Europe, comprising between 2300 and 2700 individuals in Romania. To date, however, relatively little is known about the Romanian wolf population. We aimed to provide a first assessment of genetic diversity, population structure and wolf-dog hybridisation based on 444 mostly non-invasively collected samples in the Eastern Romanian Carpathians. Pack reconstruction and analysis of population genetic parameters were performed with mitochondrial DNA control-region sequencing and microsatellite genotyping. We found relatively high levels of genetic diversity, which is similar to values found in previous studies on Carpathian wolves from Poland and Slovakia, as well as to the long-lasting Dinaric-Balkan wolf population. We found no significant population structure in our study region, suggesting effective dispersal and admixture. Analysis of wolf-dog hybridisation using a Single Nucleotide Polymorphism panel optimised for hybrid detection revealed low rates of admixture between wolves and domestic dogs. Our results provide evidence for the existence of a genetically viable wolf population in the Romanian Carpathians. The genetic data obtained in this study may serve as valuable baseline information for the elaboration of monitoring standards and management plans for wolves in Romania.

Reliable wolf-dog hybrid detection in Europe using a reduced SNP panel developed for non-invasively collected samples.

BACKGROUND: Understanding the processes that lead to hybridization of wolves and dogs is of scientific and management importance, particularly over large geographical scales, as wolves can disperse great distances. However, a method to efficiently detect hybrids in routine wolf monitoring is lacking. Microsatellites offer only limited resolution due to the low number of markers showing distinctive allele frequencies between wolves and dogs. Moreover, calibration across laboratories is time-consuming and costly. In this study, we selected a panel of 96 ancestry informative markers for wolves and dogs, derived from the Illumina CanineHD Whole-Genome BeadChip (174 K). We designed very short amplicons for genotyping on a microfluidic array, thus making the method suitable also for non-invasively collected samples. RESULTS: Genotypes based on 93 SNPs from wolves sampled throughout Europe, purebred and non-pedigree dogs, and suspected hybrids showed that the new panel accurately identifies parental individuals, first-generation hybrids and first-generation backcrosses to wolves, while second- and third-generation backcrosses to wolves were identified as advanced hybrids in almost all cases. Our results support the hybrid identity of suspect individuals and the non-hybrid status of individuals regarded as wolves. We also show the adequacy of these markers to assess hybridization at a European-wide scale and the importance of including samples from reference populations. CONCLUSIONS: We showed that the proposed SNP panel is an efficient tool for detecting hybrids up to the third-generation backcrosses to wolves across Europe. Notably, the proposed genotyping method is suitable for a variety of samples, including non-invasive and museum samples, making this panel useful for wolf-dog hybrid assessments and wolf monitoring at both continental and different temporal scales.

Informing conservation strategies with museum genomics: Long-term effects of past anthropogenic persecution on the elusive European wildcat.

Like many carnivore species, European wildcats (Felis silvestris) have suffered severe anthropogenic population declines in the past, resulting in a strong population bottleneck at the beginning of the 20th century. In Germany, the species has managed to survive its near extinction in small isolated areas and is currently recolonizing former habitats owing to legal protection and concerted conservation efforts. Here, we SNP-genotyped and mtDNA-sequenced 56 historical and 650 contemporary samples to assess the impact of massive persecution on genetic diversity, population structure, and hybridization dynamics of wildcats. Spatiotemporal analyses suggest that the presumed postglacial differentiation between two genetically distinct metapopulations in Germany is in fact the result of the anthropogenic bottleneck followed by re-expansion from few secluded refugia. We found that, despite the bottleneck, populations experienced no severe genetic erosion, nor suffered from elevated inbreeding or showed signs of increased hybridization with domestic cats. Our findings have significant implications for current wildcat conservation strategies, as the data analyses show that the two presently recognized wildcat population clusters should be treated as a single conservation unit. Although current populations appear under no imminent threat from genetic factors, fostering connectivity through the implementation of forest corridors will facilitate the preservation of genetic diversity and promote long-term viability. The present study documents how museum collections can be used as essential resource for assessing long-term anthropogenic effects on natural populations, for example, regarding population structure and the delineation of appropriate conservation units, potentially informing todays' species conservation.

Applying genomic data in wildlife monitoring: Development guidelines for genotyping degraded samples with reduced single nucleotide polymorphism panels.

The genomic era has led to an unprecedented increase in the availability of genome-wide data for a broad range of taxa. Wildlife management strives to make use of these vast resources to enable refined genetic assessments that enhance biodiversity conservation. However, as new genomic platforms emerge, problems remain in adapting the usually complex approaches for genotyping of noninvasively collected wildlife samples. Here, we provide practical guidelines for the standardized development of reduced single nucleotide polymorphism (SNP) panels applicable for microfluidic genotyping of degraded DNA samples, such as faeces or hairs. We demonstrate how microfluidic SNP panels can be optimized to efficiently monitor European wildcat (Felis silvestris S.) populations. We show how panels can be set up in a modular fashion to accommodate informative markers for relevant population genetics questions, such as individual identification, hybridization assessment and the detection of population structure. We discuss various aspects regarding the implementation of reduced SNP panels and provide a framework that will allow both molecular ecologists and practitioners to help bridge the gap between genomics and applied wildlife conservation.

Assessing SNP genotyping of noninvasively collected wildlife samples using microfluidic arrays.

Noninvasively collected samples are a common source of DNA in wildlife genetic studies. Currently, single nucleotide polymorphism (SNP) genotyping using microfluidic arrays is emerging as an easy-to-use and cost-effective methodology. Here we assessed the performance of microfluidic SNP arrays in genotyping noninvasive samples from grey wolves, European wildcats and brown bears, and we compared results with traditional microsatellite genotyping. We successfully SNP-genotyped 87%, 80% and 97% of the wolf, cat and bear samples, respectively. Genotype recovery was higher based on SNPs, while both marker types identified the same individuals and provided almost identical estimates of pairwise differentiation. We found that samples for which all SNP loci were scored had no disagreements across the three replicates (except one locus in a wolf sample). Thus, we argue that call rate (amplification success) can be used as a proxy for genotype quality, allowing the reduction of replication effort when call rate is high. Furthermore, we used cycle threshold values of real-time PCR to guide the choice of protocols for SNP amplification. Finally, we provide general guidelines for successful SNP genotyping of degraded DNA using microfluidic technology.