A reduced SNP panel optimised for non-invasive genetic assessment of a genetically impoverished conservation icon, the European bison.

The European bison was saved from the brink of extinction due to considerable conservation efforts since the early twentieth century. The current global population of > 9500 individuals is the result of successful ex situ breeding based on a stock of only 12 founders, resulting in an extremely low level of genetic variability. Due to the low allelic diversity, traditional molecular tools, such as microsatellites, fail to provide sufficient resolution for accurate genetic assessments in European bison, let alone from non-invasive samples. Here, we present a SNP panel for accurate high-resolution genotyping of European bison, which is suitable for a wide variety of sample types. The panel accommodates 96 markers allowing for individual and parental assignment, sex determination, breeding line discrimination, and cross-species detection. Two applications were shown to be utilisable in further Bos species with potential conservation significance. The new SNP panel will allow to tackle crucial tasks in European bison conservation, including the genetic monitoring of reintroduced populations, and a molecular assessment of pedigree data documented in the world's first studbook of a threatened species.

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.

Limited historical admixture between European wildcats and domestic cats.

Domestic cats were derived from the Near Eastern wildcat (Felis lybica), after which they dispersed with people into Europe. As they did so, it is possible that they interbred with the indigenous population of European wildcats (Felis silvestris). Gene flow between incoming domestic animals and closely related indigenous wild species has been previously demonstrated in other taxa, including pigs, sheep, goats, bees, chickens, and cattle. In the case of cats, a lack of nuclear, genome-wide data, particularly from Near Eastern wildcats, has made it difficult to either detect or quantify this possibility. To address these issues, we generated 75 ancient mitochondrial genomes, 14 ancient nuclear genomes, and 31 modern nuclear genomes from European and Near Eastern wildcats. Our results demonstrate that despite cohabitating for at least 2,000 years on the European mainland and in Britain, most modern domestic cats possessed less than 10% of their ancestry from European wildcats, and ancient European wildcats possessed little to no ancestry from domestic cats. The antiquity and strength of this reproductive isolation between introduced domestic cats and local wildcats was likely the result of behavioral and ecological differences. Intriguingly, this long-lasting reproductive isolation is currently being eroded in parts of the species' distribution as a result of anthropogenic activities.

Genetic swamping of the critically endangered Scottish wildcat was recent and accelerated by disease.

The European wildcat population in Scotland is considered critically endangered as a result of hybridization with introduced domestic cats,1,2 though the time frame over which this gene flow has taken place is unknown. Here, using genome data from modern, museum, and ancient samples, we reconstructed the trajectory and dated the decline of the local wildcat population from viable to severely hybridized. We demonstrate that although domestic cats have been present in Britain for over 2,000 years,3 the onset of hybridization was only within the last 70 years. Our analyses reveal that the domestic ancestry present in modern wildcats is markedly over-represented in many parts of the genome, including the major histocompatibility complex (MHC). We hypothesize that introgression provides wildcats with protection against diseases harbored and introduced by domestic cats, and that this selection contributes to maladaptive genetic swamping through linkage drag. Using the case of the Scottish wildcat, we demonstrate the importance of local ancestry estimates to both understand the impacts of hybridization in wild populations and support conservation efforts to mitigate the consequences of anthropogenic and environmental change.

Using eDNA to understand predator-prey interactions influenced by invasive species.

Invasive predatory species may alter population dynamic processes of their prey and impact biological communities and ecosystem processes. Revealing biotic interactions, however, including the relationship between predator and prey, is a difficult task, in particular for species that are hard to monitor. Here, we present a case study that documents the utility of environmental DNA analysis (eDNA) to assess predator-prey interactions between two invasive fishes (Lepomis gibbosus, Pseudorasbora parva) and two potential amphibian prey species, (Triturus cristatus, Pelobates fuscus). We used species-specific TaqMan assays for quantitative assessment of eDNA concentrations from water samples collected from 89 sites across 31 ponds during three consecutive months from a local amphibian hotspot in Germany. We found a negative relationship between eDNA concentrations of the predators (fishes) and prey (amphibians) using Monte-Carlo tests. Our study highlights the potential of eDNA application to reveal predator-prey interactions and confirms the hypothesis that the observed local declines of amphibian species may be at least partly caused by recently introduced invasive fishes. Our findings have important consequences for local conservation management and highlight the usefulness of eDNA approaches to assess ecological interactions and guide targeted conservation action.

The extinct Sicilian wolf shows a complex history of isolation and admixture with ancient dogs.

The Sicilian wolf remained isolated in Sicily from the end of the Pleistocene until its extermination in the 1930s-1960s. Given its long-term isolation on the island and distinctive morphology, the genetic origin of the Sicilian wolf remains debated. We sequenced four nuclear genomes and five mitogenomes from the seven existing museum specimens to investigate the Sicilian wolf ancestry, relationships with extant and extinct wolves and dogs, and diversity. Our results show that the Sicilian wolf is most closely related to the Italian wolf but carries ancestry from a lineage related to European Eneolithic and Bronze Age dogs. The average nucleotide diversity of the Sicilian wolf was half of the Italian wolf, with 37-50% of its genome contained in runs of homozygosity. Overall, we show that, by the time it went extinct, the Sicilian wolf had high inbreeding and low-genetic diversity, consistent with a population in an insular environment.

Human impact on the recent population history of the elusive European wildcat inferred from whole genome data.

BACKGROUND: The extent and impact of evolutionary change occurring in natural populations in response to rapid anthropogenic impact is still poorly understood on the genome-wide level. Here, we explore the genetic structure, demographic history, population differentiation, and domestic introgression based on whole genome data of the endangered European wildcat in Germany, to assess potential genomic consequences of the species' recent spread across human-dominated cultural landscapes. RESULTS: Reconstruction of demographic history and introgression rates based on 47 wildcat and 37 domestic cat genomes suggested late introgression between wild and domestic cat, coinciding with the introduction of domestic cat during the Roman period, but overall relatively low rates of hybridization and introgression from domestic cats. Main population divergence found between an eastern and central German wildcat clade was found to be of rather recent origin (200 y), and thus the likely consequence of anthropogenic persecution and resulting isolation in population refugia. We found similar effective population sizes and no substantial inbreeding across populations. Interestingly, highly differentiated genes between wild cat populations involved in the tryptophan-kynurenine-serotonin pathway were revealed, which plays a role in behavioral processes such as stress susceptibility and tolerance, suggesting that differential selection acted in the populations. CONCLUSIONS: We found strong evidence for substantial recent anthropogenic impact on the genetic structure of European wildcats, including recent persecution-driven population divergence, as well as potential adaptation to human-dominate environments. In contrast, the relatively low levels of domestic introgression and inbreeding found in this study indicate a substantial level of "resistance" of this elusive species towards major anthropogenic impacts, such as the omnipresence of domestic cats as well as substantial habitat fragmentation. While those findings have strong implications for ongoing conservation strategies, we demand closer inspection of selective pressures acting on this and other wildlife species in anthropogenic environments.

A reduced SNP panel to trace gene flow across southern European wolf populations and detect hybridization with other Canis taxa.

Intra- and inter-specific gene flow are natural evolutionary processes. However, human-induced hybridization is a global conservation concern across taxa, and the development of discriminant genetic markers to differentiate among gene flow processes is essential. Wolves (Canis lupus) are affected by hybridization, particularly in southern Europe, where ongoing recolonization of historic ranges is augmenting gene flow among divergent populations. Our aim was to provide diagnostic canid markers focused on the long-divergent Iberian, Italian and Dinaric wolf populations, based on existing genomic resources. We used 158 canid samples to select a panel of highly informative single nucleotide polymorphisms (SNPs) to (i) distinguish wolves in the three regions from domestic dogs (C. l. familiaris) and golden jackals (C. aureus), and (ii) identify their first two hybrid generations. The resulting 192 SNPs correctly identified the five canid groups, all simulated first-generation (F1) hybrids (0.482 ≤ Qi ≤ 0.512 between their respective parental groups) and all first backcross (BC1) individuals (0.723 ≤ Qi ≤ 0.827 to parental groups). An assay design and test with invasive and non-invasive canid samples performed successfully for 178 SNPs. By separating natural population admixture from inter-specific hybridization, our reduced panel can help advance evolutionary research, monitoring, and timely conservation management.

Do all roads lead to resistance? State road density is the main impediment to gene flow in a flagship species inhabiting a severely fragmented anthropogenic landscape.

AIM: Connectivity conservation is ideally based on empirical information on how landscape heterogeneity influences species-specific movement and gene flow. Here, we present the first large-scale evaluation of landscape impacts on genetic connectivity in the European wildcat (Felis silvestris), a flagship and umbrella species for connectivity conservation across Europe. LOCATION: The study was carried out in the core area of the distributional range of wildcats in Germany, covering about 186,000 km2 of a densely populated and highly fragmented landscape. METHODS: We used data of 975 wildcats genotyped at 14 microsatellites and an individual-based landscape genetic framework to assess the importance of twelve landscape variables for explaining observed genetic connectivity. For this, we optimized landscape resistance surfaces for all variables and compared their relative impacts using multiple regression on distance matrices and commonality analysis. RESULTS: Genetic connectivity was best explained by a synergistic combination of six landscape variables and isolation by distance. Of these variables, road density had by far the strongest individual impact followed by synergistic effects of agricultural lands and settlements. Subsequent analyses involving different road types revealed that the strong effect of road density was largely due to state roads, while highways and federal roads had a much smaller, and county roads only a negligible impact. MAIN CONCLUSIONS: Our results highlight that landscape-wide genetic connectivity in wildcats across Germany is strongly shaped by the density of roads and in particular state roads, with higher densities providing larger resistance to successful dispersal. These findings have important implications for conservation planning, as measures to mitigate fragmentation effects of roads (e.g., over- or underpasses) often focus on large, federally managed transportation infrastructures. While these major roads exert local barrier effects, other road types can be more influential on overall connectivity, as they are more abundant and more widespread across the landscape.

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.

How the west was won: genetic reconstruction of rapid wolf recolonization into Germany's anthropogenic landscapes.

Following massive persecution and eradication, strict legal protection facilitated a successful reestablishment of wolf packs in Germany, which has been ongoing since 2000. Here, we describe this recolonization process by mitochondrial DNA control-region sequencing, microsatellite genotyping and sex identification based on 1341 mostly non-invasively collected samples. We reconstructed the genealogy of German wolf packs between 2005 and 2015 to provide information on trends in genetic diversity, dispersal patterns and pack dynamics during the early expansion process. Our results indicate signs of a founder effect at the start of the recolonization. Genetic diversity in German wolves is moderate compared to other European wolf populations. Although dispersal among packs is male-biased in the sense that females are more philopatric, dispersal distances are similar between males and females once only dispersers are accounted for. Breeding with close relatives is regular and none of the six male wolves originating from the Italian/Alpine population reproduced. However, moderate genetic diversity and inbreeding levels of the recolonizing population are preserved by high sociality, dispersal among packs and several immigration events. Our results demonstrate an ongoing, rapid and natural wolf population expansion in an intensively used cultural landscape in Central Europe.

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.

Combining environmental DNA and species distribution modeling to evaluate reintroduction success of a freshwater fish.

Active species reintroduction is an important conservation tool when aiming for the restoration of biological communities and ecosystems. The effective monitoring of reintroduction success is a crucial factor in this process. Here, we used a combination of environmental DNA (eDNA) techniques and species distribution models (SDMs) to evaluate the success of recent reintroductions of the freshwater fish Alburnoides bipunctatus in central Germany. We built SDMs without and with eDNA presence data to locate further suitable reintroduction sites and potentially overlooked populations of the species. We successfully detected eDNA of A. bipunctatus at all reintroduction sites, as well as several adjacent sites mostly in downstream direction, which supports the success of reintroduction efforts. eDNA-based species detection considerably improved SDMs for A. bipunctatus, which allowed to identify species presence in previously unknown localities. Our results confirm the usefulness of eDNA techniques as standard tool to monitor reintroduced fish populations. We propose that combining eDNA with SDMs is a highly effective approach for long-term monitoring of reintroduction success in aquatic species.

Conservation genetics: Linking science with practice.

Conservation genetics is a well-established scientific field. However, limited information transfer between science and practice continues to hamper successful implementation of scientific knowledge in conservation practice and management. To mitigate this challenge, we have established a conservation genetics community, which entails an international exchange-and-skills platform related to genetic methods and approaches in conservation management. First, it allows for scientific exchange between researchers during annual conferences. Second, personal contact between conservation professionals and scientists is fostered by organising workshops and by popularising knowledge on conservation genetics methods and approaches in professional journals in national languages. Third, basic information on conservation genetics has been made accessible by publishing an easy-to-read handbook on conservation genetics for practitioners. Fourth, joint projects enabled practitioners and scientists to work closely together from the start of a project in order to establish a tight link between applied questions and scientific background. Fifth, standardised workflows simplifying the implementation of genetic tools in conservation management have been developed. By establishing common language and trust between scientists and practitioners, all these measures help conservation genetics to play a more prominent role in future conservation planning and management.

Accuracy, limitations and cost efficiency of eDNA-based community survey in tropical frogs.

Rapid environmental change in highly biodiverse tropical regions demands efficient biomonitoring programmes. While existing metrics of species diversity and community composition rely on encounter-based survey data, eDNA recently emerged as alternative approach. Costs and ecological value of eDNA-based methods have rarely been evaluated in tropical regions, where high species richness is accompanied by high functional diversity (e.g., the use of different microhabitats by different species and life stages). We first tested whether estimation of tropical frogs' community structure derived from eDNA data is compatible with expert field assessments. Next, we evaluated whether eDNA is a financially viable solution for biodiversity monitoring in tropical regions. We applied eDNA metabarcoding to investigate frog species occurrence in five ponds in the Chiquitano dry forest region in Bolivia and compared our data with a simultaneous visual and audio encounter survey (VAES). We found that taxon lists and community structure generated with eDNA and VAES correspond closely, and most deviations are attributable to different species' life histories. Cost efficiency of eDNA surveys was mostly influenced by the richness of local fauna and the number of surveyed sites: VAES may be less costly in low-diversity regions, but eDNA quickly becomes more cost-efficient in high-diversity regions with many sites sampled. The results highlight that eDNA is suitable for large-scale biodiversity surveys in high-diversity areas if life history is considered, and certain precautions in sampling, genetic analyses and data interpretation are taken. We anticipate that spatially extensive, standardized eDNA biodiversity surveys will quickly emerge in the future.

Targeted resequencing of coding DNA sequences for SNP discovery in nonmodel species.

Targeted capture coupled with high-throughput sequencing can be used to gain information about nuclear sequence variation at hundreds to thousands of loci. Divergent reference capture makes use of molecular data of one species to enrich target loci in other (related) species. This is particularly valuable for nonmodel organisms, for which often no a priori knowledge exists regarding these loci. Here, we have used targeted capture to obtain data for 809 nuclear coding DNA sequences (CDS) in a nonmodel organism, the Eurasian lynx Lynx lynx, using baits designed with the help of the published genome of a related model organism (the domestic cat Felis catus). Using this approach, we were able to survey intraspecific variation at hundreds of nuclear loci in L. lynx across the species' European range. A large set of biallelic candidate SNPs was then evaluated using a high-throughput SNP genotyping platform (Fluidigm), which we then reduced to a final 96 SNP-panel based on assay performance and reliability; validation was carried out with 100 additional Eurasian lynx samples not included in the SNP discovery phase. The 96 SNP-panel developed from CDS performed very successfully in the identification of individuals and in population genetic structure inference (including the assignment of individuals to their source population). In keeping with recent studies, our results show that genic SNPs can be valuable for genetic monitoring of wildlife species.

Low rates of hybridization between European wildcats and domestic cats in a human-dominated landscape.

Hybridization between wild species and their domestic congeners is considered a major threat for wildlife conservation. Genetic integrity of the European wildcat, for instance, is a concern as they are outnumbered by domestic cats by several orders of magnitude throughout its range. We genotyped 1,071 individual wildcat samples obtained from hair traps and roadkills collected across the highly fragmented forests of western Central Europe, in Germany and Luxembourg, to assess domestic cat introgression in wildcats in human-dominated landscapes. Analyses using a panel of 75 autosomal SNPs suggested a low hybridization rate, with 3.5% of wildcat individuals being categorized as F1, F2, or backcrosses to either parental taxon. We report that results based on a set of SNPs were more consistent than on a set of 14 microsatellite markers, showed higher accuracy to detect hybrids and their class in simulation analyses, and were less affected by underlying population structure. Our results strongly suggest that very high hybridization rates previously reported for Central Europe may be partly due to inadequate choice of markers and/or sampling design. Our study documents that an adequately selected SNP panel for hybrid detection may be used as an alternative to commonly applied microsatellite markers, including studies relying on noninvasively collected samples. In addition, our finding of overall low hybridization rates in Central European wildcats provides an example of successful wildlife coexistence in human-dominated, fragmented landscapes.

Fast and cost-effective single nucleotide polymorphism (SNP) detection in the absence of a reference genome using semideep next-generation Random Amplicon Sequencing (RAMseq).

Biodiversity has suffered a dramatic global decline during the past decades, and monitoring tools are urgently needed providing data for the development and evaluation of conservation efforts both on a species and on a genetic level. However, in wild species, the assessment of genetic diversity is often hampered by the lack of suitable genetic markers. In this article, we present Random Amplicon Sequencing (RAMseq), a novel approach for fast and cost-effective detection of single nucleotide polymorphisms (SNPs) in nonmodel species by semideep sequencing of random amplicons. By applying RAMseq to the Eurasian otter (Lutra lutra), we identified 238 putative SNPs after quality filtering of all candidate loci and were able to validate 32 of 77 loci tested. In a second step, we evaluated the genotyping performance of these SNP loci in noninvasive samples, one of the most challenging genotyping applications, by comparing it with genotyping results of the same faecal samples at microsatellite markers. We compared (i) polymerase chain reaction (PCR) success rate, (ii) genotyping errors and (iii) Mendelian inheritance (population parameters). SNPs produced a significantly higher PCR success rate (75.5% vs. 65.1%) and lower mean allelic error rate (8.8% vs. 13.3%) than microsatellites, but showed a higher allelic dropout rate (29.7% vs. 19.8%). Genotyping results showed no deviations from Mendelian inheritance in any of the SNP loci. Hence, RAMseq appears to be a valuable tool for the detection of genetic markers in nonmodel species, which is a common challenge in conservation genetic studies.