Evaluating the functional, sexual and seasonal variation in the chemical constituents from feces of adult Iberian wolves (Canis lupus signatus).

Chemical signals deposited in feces play an important role in intraspecific and interspecific communication of many mammals. We collected fresh feces of adult wolves from wild breeding groups. All samples visually identified as belonging to wolves were subsequently identified to species level by sequencing a small fragment of mtDNA and sexed typing DBX6 and DBY7 sex markers. Using gas chromatography-mass spectrometry (GC-MS), we identified 56 lipophilic compounds in the feces, mainly heterocyclic aromatic organic compounds, such as indole or phenol, but also steroids, such as cholesterol, carboxylic acids and their esters between n-C4 and n-C18, aldehydes, alcohols and significant quantities of squalene and α-tocopherol, which would increase the chemical stability of feces on humid substrates. There was variability in the number and proportions of compounds between sexes, which could be indicative of their function as chemical signals. We also found variability in different reproductive states, especially in odorous compounds, steroids and α-tocopherol. Feces with a presumed marking function had higher proportions of α-tocopherol and steroids than feces with non-marking function. These compounds could be involved in intragroup and intergroup communication of wolves and their levels in feces could be directly related with the wolf's sex and physiological and reproductive status.

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.

Next-generation phylogeography resolves post-glacial colonization patterns in a widespread carnivore, the red fox (Vulpes vulpes), in Europe.

Carnivores tend to exhibit a lack of (or less pronounced) genetic structure at continental scales in both a geographic and temporal sense and this can confound the identification of post-glacial colonization patterns in this group. In this study we used genome-wide data (using genotyping by sequencing [GBS]) to reconstruct the phylogeographic history of a widespread carnivore, the red fox (Vulpes vulpes), by investigating broad-scale patterns of genomic variation, differentiation and admixture amongst contemporary populations in Europe. Using 15,003 single nucleotide polymorphisms (SNPs) from 524 individuals allowed us to identify the importance of refugial regions for the red fox in terms of endemism (e.g., Iberia). In addition, we tested multiple post-glacial recolonization scenarios of previously glaciated regions during the Last Glacial Maximum using an Approximate Bayesian Computation (ABC) approach that were unresolved from previous studies. This allowed us to identify the role of admixture from multiple source population post-Younger Dryas in the case of Scandinavia and ancient land-bridges in the colonization of the British Isles. A natural colonization of Ireland was deemed more likely than an ancient human-mediated introduction as has previously been proposed and potentially points to a larger mammalian community on the island in the early post-glacial period. Using genome-wide data has allowed us to tease apart broad-scale patterns of structure and diversity in a widespread carnivore in Europe that was not evident from using more limited marker sets and provides a foundation for next-generation phylogeographic studies in other non-model species.

Strong isolation by distance among local populations of an endangered butterfly species (Euphydryas aurinia).

The marsh fritillary (Euphydryas aurinia) is a critically endangered butterfly species in Denmark known to be particularly vulnerable to habitat fragmentation due to its poor dispersal capacity. We identified and genotyped 318 novel SNP loci across 273 individuals obtained from 10 small and fragmented populations in Denmark using a genotyping-by-sequencing (GBS) approach to investigate its population genetic structure. Our results showed clear genetic substructuring and highly significant population differentiation based on genetic divergence (F ST) among the 10 populations. The populations clustered in three overall clusters, and due to further substructuring among these, it was possible to clearly distinguish six clusters in total. We found highly significant deviations from Hardy-Weinberg equilibrium due to heterozygote deficiency within every population investigated, which indicates substructuring and/or inbreeding (due to mating among closely related individuals). The stringent filtering procedure that we have applied to our genotype quality could have overestimated the heterozygote deficiency and the degree of substructuring of our clusters but is allowing relative comparisons of the genetic parameters among clusters. Genetic divergence increased significantly with geographic distance, suggesting limited gene flow at spatial scales comparable to the dispersal distance of individual butterflies and strong isolation by distance. Altogether, our results clearly indicate that the marsh fritillary populations are genetically isolated. Further, our results highlight that the relevant spatial scale for conservation of rare, low mobile species may be smaller than previously anticipated.

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.

eDNA metabarcoding for biodiversity assessment, generalist predators as sampling assistants.

With an accelerating negative impact of anthropogenic actions on natural ecosystems, non-invasive biodiversity assessments are becoming increasingly crucial. As a consequence, the interest in the application of environmental DNA (eDNA) survey techniques has increased. The use of eDNA extracted from faeces from generalist predators, have recently been described as "biodiversity capsules" and suggested as a complementary tool for improving current biodiversity assessments. In this study, using faecal samples from two generalist omnivore species, the Eurasian badger and the red fox, we evaluated the applicability of eDNA metabarcoding in determining dietary composition, compared to macroscopic diet identification techniques. Subsequently, we used the dietary information obtained to assess its contribution to biodiversity assessments. Compared to classic macroscopic techniques, we found that eDNA metabarcoding detected more taxa, at higher taxonomic resolution, and proved to be an important technique to verify the species identification of the predator from field collected faeces. Furthermore, we showed how dietary analyses complemented field observations in describing biodiversity by identifying consumed flora and fauna that went unnoticed during field observations. While diet analysis approaches could not substitute field observations entirely, we suggest that their integration with other methods might overcome intrinsic limitations of single techniques in future biodiversity surveys.

Organic Learning Gardens in Higher Education: Do They Improve Kindergarten Pre-service Teachers' Connectedness to and Conception of Nature?

Studies have shed light on the idea that people who have experiences in natural settings might be more aware of the environment. Learning gardens, as outdoor contexts, might contribute to the development of students' affective relations toward nature, pro-environmental attitudes, and protective actions; neverthless, these aspects begging to be explored. This preliminary research investigates the impact that the use of organic gardens to teach natural sciences at university has on kindergarten pre-service teachers' (KPST) connectedness to and conceptions of nature. The research follows a pre-/post-design and it uses a mixed methods approach. A total of 74 students completed four quantitative scales (INS, CCC, LCN, and NR-6), and 66 of them an open question about the concept of nature. After the garden experience, students scored higher in all the scales, nevertheless the change was significant only for INS and CCC. The phenomenographic analysis evidenced an initial predominant static and non-social concept of nature, biased toward the most obvious biological elements. After the garden-based learning experience, more informed conceptions of nature - including notions of complexity and systemic character - increased from 7 to 19%; however, statistical comparison was not significant. In spite of the absence of concluding results, further research is required to assess the role that learning gardens may play regarding connectedness to nature and pro-environmental behaviors.

Integrated genome-wide investigations of the housefly, a global vector of diseases reveal unique dispersal patterns and bacterial communities across farms.

BACKGROUND: Houseflies (Musca domestica L.) live in intimate association with numerous microorganisms and is a vector of human pathogens. In temperate areas, houseflies will overwinter in environments constructed by humans and recolonize surrounding areas in early summer. However, the dispersal patterns and associated bacteria across season and location are unclear. We used genotyping-by-sequencing (GBS) for the simultaneous identification and genotyping of thousands of Single Nucleotide Polymorphisms (SNPs) to establish dispersal patterns of houseflies across farms. Secondly, we used 16S rRNA gene amplicon sequencing to establish the variation and association between bacterial communities and the housefly across farms. RESULTS: Using GBS we identified 18,000 SNPs across 400 individuals sampled within and between 11 dairy farms in Denmark. There was evidence for sub-structuring of Danish housefly populations and with genetic structure that differed across season and sex. Further, there was a strong isolation by distance (IBD) effect, but with large variation suggesting that other hidden geographic barriers are important. Large individual variations were observed in the community structure of the microbiome and it was found to be dependent on location, sex, and collection time. Furthermore, the relative prevalence of putative pathogens was highly dependent on location and collection time. CONCLUSION: We were able to identify SNPs for the determination of the spatiotemporal housefly genetic structure, and to establish the variation and association between bacterial communities and the housefly across farms using novel next-generation sequencing (NGS) techniques. These results are important for disease prevention given the fine-scale population structure and IBD for the housefly, and that individual houseflies carry location specific bacteria including putative pathogens.

Novel Graphical Analyses of Runs of Homozygosity among Species and Livestock Breeds.

Runs of homozygosity (ROH), uninterrupted stretches of homozygous genotypes resulting from parents transmitting identical haplotypes to their offspring, have emerged as informative genome-wide estimates of autozygosity (inbreeding). We used genomic profiles based on 698 K single nucleotide polymorphisms (SNPs) from nine breeds of domestic cattle (Bos taurus) and the European bison (Bison bonasus) to investigate how ROH distributions can be compared within and among species. We focused on two length classes: 0.5-15 Mb to investigate ancient events and >15 Mb to address recent events (approximately three generations). For each length class, we chose a few chromosomes with a high number of ROH, calculated the percentage of times a SNP appeared in a ROH, and plotted the results. We selected areas with distinct patterns including regions where (1) all groups revealed an increase or decrease of ROH, (2) bison differed from cattle, (3) one cattle breed or groups of breeds differed (e.g., dairy versus meat cattle). Examination of these regions in the cattle genome showed genes potentially important for natural and human-induced selection, concerning, for example, meat and milk quality, metabolism, growth, and immune function. The comparative methodology presented here permits visual identification of regions of interest for selection, breeding programs, and conservation.

Modelling the Distribution of Forest-Dependent Species in Human-Dominated Landscapes: Patterns for the Pine Marten in Intensively Cultivated Lowlands.

In recent years, the "forest-specialist" pine marten Martes martes has been reported to also occur also in largely fragmented, lowland landscapes of north-western Italy. The colonization of such an apparently unsuitable area provided the opportunity for investigating pine marten ecological requirements and predicting its potential south- and eastwards expansion. We collected available pine marten occurrence data in the flood plain of the River Po (N Italy) and relate them to 11 environmental variables by developing nine Species Distribution Models. To account for inter-model variability we used average ensemble predictions (EP). EP predicted a total of 482 suitable patches (8.31% of the total study area) for the pine marten. The main factors driving pine marten occurrence in the western River Po plain were the distance from watercourses and the distance from woods. EP suggested that the pine marten may further expand in the western lowland, whilst the negligible residual wood cover of large areas in the central and eastern plain makes the habitat unsuitable for the pine marten, except for some riparian corridors and the pine wood patches bordering the Adriatic coast. Based on our results, conservation strategies should seek to preserve remnant forest patches and enhance the functional connectivity provided by riparian corridors.

Recovering full-length viral genomes from metagenomes.

Infectious disease metagenomics is driven by the question: "what is causing the disease?" in contrast to classical metagenome studies which are guided by "what is out there?" In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral genome from a metagenomic sample. However, retrieval of a full-length genome of a divergent virus is technically challenging and can be time-consuming and costly. Here we discuss different assembly and fragment linkage strategies such as iterative assembly, motif searches, k-mer frequency profiling, coverage profile binning, and other strategies used to recover genomes of potential viral pathogens in a timely and cost-effective manner.

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.

Inferring Population Genetic Structure in Widely and Continuously Distributed Carnivores: The Stone Marten (Martes foina) as a Case Study.

The stone marten is a widely distributed mustelid in the Palaearctic region that exhibits variable habitat preferences in different parts of its range. The species is a Holocene immigrant from southwest Asia which, according to fossil remains, followed the expansion of the Neolithic farming cultures into Europe and possibly colonized the Iberian Peninsula during the Early Neolithic (ca. 7,000 years BP). However, the population genetic structure and historical biogeography of this generalist carnivore remains essentially unknown. In this study we have combined mitochondrial DNA (mtDNA) sequencing (621 bp) and microsatellite genotyping (23 polymorphic markers) to infer the population genetic structure of the stone marten within the Iberian Peninsula. The mtDNA data revealed low haplotype and nucleotide diversities and a lack of phylogeographic structure, most likely due to a recent colonization of the Iberian Peninsula by a few mtDNA lineages during the Early Neolithic. The microsatellite data set was analysed with a) spatial and non-spatial Bayesian individual-based clustering (IBC) approaches (STRUCTURE, TESS, BAPS and GENELAND), and b) multivariate methods [discriminant analysis of principal components (DAPC) and spatial principal component analysis (sPCA)]. Additionally, because isolation by distance (IBD) is a common spatial genetic pattern in mobile and continuously distributed species and it may represent a challenge to the performance of the above methods, the microsatellite data set was tested for its presence. Overall, the genetic structure of the stone marten in the Iberian Peninsula was characterized by a NE-SW spatial pattern of IBD, and this may explain the observed disagreement between clustering solutions obtained by the different IBC methods. However, there was significant indication for contemporary genetic structuring, albeit weak, into at least three different subpopulations. The detected subdivision could be attributed to the influence of the rivers Ebro, Tagus and Guadiana, suggesting that main watercourses in the Iberian Peninsula may act as semi-permeable barriers to gene flow in stone martens. To our knowledge, this is the first phylogeographic and population genetic study of the species at a broad regional scale. We also wanted to make the case for the importance and benefits of using and comparing multiple different clustering and multivariate methods in spatial genetic analyses of mobile and continuously distributed species.

Genomic resources notes accepted 1 October 2014-30 November 2014.

This article documents the public availability of (i) RAD sequencing data and validated SNPs for the American mink Neovison vison and (ii) Transcriptome resources for two nonmodel freshwater crustacean species, the copepod Eucyclops serrulatus and the amphipod Echinogammarus veneris.

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.

Viral metagenomic analysis of feces of wild small carnivores.

BACKGROUND: Recent studies have clearly demonstrated the enormous virus diversity that exists among wild animals. This exemplifies the required expansion of our knowledge of the virus diversity present in wildlife, as well as the potential transmission of these viruses to domestic animals or humans. METHODS: In the present study we evaluated the viral diversity of fecal samples (n = 42) collected from 10 different species of wild small carnivores inhabiting the northern part of Spain using random PCR in combination with next-generation sequencing. Samples were collected from American mink (Neovison vison), European mink (Mustela lutreola), European polecat (Mustela putorius), European pine marten (Martes martes), stone marten (Martes foina), Eurasian otter (Lutra lutra) and Eurasian badger (Meles meles) of the family of Mustelidae; common genet (Genetta genetta) of the family of Viverridae; red fox (Vulpes vulpes) of the family of Canidae and European wild cat (Felis silvestris) of the family of Felidae. RESULTS: A number of sequences of possible novel viruses or virus variants were detected, including a theilovirus, phleboviruses, an amdovirus, a kobuvirus and picobirnaviruses. CONCLUSIONS: Using random PCR in combination with next generation sequencing, sequences of various novel viruses or virus variants were detected in fecal samples collected from Spanish carnivores. Detected novel viruses highlight the viral diversity that is present in fecal material of wild carnivores.

Assembly of viral genomes from metagenomes.

Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity are, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.