Individual genotypes from environmental DNA: Fingerprinting snow tracks of three large carnivore species.

Continued advancements in environmental DNA (eDNA) research have made it possible to access intraspecific variation from eDNA samples, opening new opportunities to expand non-invasive genetic studies of wildlife populations. However, the use of eDNA samples for individual genotyping, as typically performed in non-invasive genetics, still remains elusive. We present successful individual genotyping of eDNA obtained from snow tracks of three large carnivores: brown bear (Ursus arctos), European lynx (Lynx lynx) and wolf (Canis lupus). DNA was extracted using a protocol for isolating water eDNA and genotyped using amplicon sequencing of short tandem repeats (STR), and for brown bear a sex marker, on a high-throughput sequencing platform. Individual genotypes were obtained for all species, but genotyping performance differed among samples and species. The proportion of samples genotyped to individuals was higher for brown bear (5/7) and wolf (7/10) than for lynx (4/9), and locus genotyping success was greater for brown bear (0.88). The sex marker was typed in six out of seven brown bear samples. Results for three species show that reliable individual genotyping, including sex identification, is now possible from eDNA in snow tracks, underlining its vast potential to complement the non-invasive genetic methods used for wildlife. To fully leverage the application of snow track eDNA, improved understanding of the ideal species- and site-specific sampling conditions, as well as laboratory methods promoting genotyping success, is needed. This will also inform efforts to retrieve and type nuclear DNA from other eDNA samples, thereby advancing eDNA-based individual and population-level studies.

Comprehensive coverage of human last meal components revealed by a forensic DNA metabarcoding approach.

Stomach content analyses are a valuable tool in human forensic science to interpret perimortem events. While the identification of food components of plant and animal origin has traditionally been conducted by macro- and microscopical approaches in case of incomplete digestion, molecular methods provide the potential to increase sensitivity and taxonomic resolution. In particular, DNA metabarcoding (PCR-amplification and next generation sequencing of complex DNA mixtures) has seen a rapid growth in the field of wildlife ecology to assess species' diets from faecal and gastric samples. Despite clear advantages, molecular approaches have not yet been established in routine human forensics to investigate the last meal components of deceased persons. In this pilot study we applied for the first time a DNA metabarcoding approach to assess both plant and vertebrate components of 48 human stomach content samples taken during medicolegal autopsies. We obtained a final dataset with 34 vertebrate and 124 vegetal unique sequences, that were clustered to 9 and 33 operational taxonomic units (OTUs), respectively. Our results suggest that this approach can provide crucial information about circumstances preceding death, and open promising perspectives for biomedical dietary surveys based on digested food items found in the gastrointestinal tract.

Correction: Diet of the brown bear in Himalaya: Combining classical and molecular genetic techniques.

[This corrects the article DOI: 10.1371/journal.pone.0225698.].

Identification of Arabis alpina genomic regions associated with climatic variables along an elevation gradient through whole genome scan.

We performed a pooled whole-genome sequencing on samples of the alpine plant Arabis alpina, harvested in ten populations along an elevation gradient in the French Alps. A large dataset of genetic variations was produced as single nucleotide polymorphisms (SNPs). A combined genome scan approach enabled detecting genomic regions associated with a synthetic environmental variable characterizing the climate at each sampling location. Positive loci detected by two methods were retained and belong to 19 regions in the Arabis alpina genome. The most significant region harbors an ortholog of the AtNAC062 gene, encoding a membrane-bound transcription factor described as linking the cold response and pathogen resistance that may confer protection to plants under extended snow coverage at high elevations. Other genes involved in the stress response or in flowering regulation were also detected. Altogether, our results indicated that Arabis alpina represent a suitable model for studying genomic adaptation in alpine perennial plants.

Diet of the brown bear in Himalaya: Combining classical and molecular genetic techniques.

The ecological requirements of brown bears are poorly known in the Himalaya region, which complicates conservation efforts. We documented the diet of the Himalayan brown bear (Ursus arctos isabellinus) by combining classical scat analysis and a newly developed molecular genetic technique (the trnL approach), in Deosai National Park, Pakistan. Brown bears consumed over 50 plant species, invertebrates, ungulates, and several rodents. Eight plant families; Poaceae, Polygonaceae, Cyperaceae, Apiaceae, Asteraceae, Caryophyllaceae, Lamiaceae, and Rubiaceae were commonly eaten with graminoids comprising the bulk of the diet. Golden marmots comprised the major mammalian biomass in the park, and were also the main meat source for bears. Animal matter, making 36% of dietary content, contributed half of the digestible energy, due to its higher nutritious value. We did not find a significant temporal pattern in diet, perhaps because the availability of the major diet (graminoids) did not change over the foraging period. Male brown bears were more carnivorous than females, probably because of their larger size, which requires higher energy and also makes them more efficient in capturing marmots. Frequencies of three plant species were also significantly higher in male brown bears; Bistorta affinis, Carex diluta, and Carex sp. Diet of the brown bear differed significantly between the park and surrounding valleys. In valleys, diet consisted predominantly of graminoids and crops, whereas the park provided more nutritious and diverse foodThe estimated digestible energy available to brown bears in Deosai was the lowest documented among brown bear populations, due to the lack of fruits and a relatively lower meat content. The low nutritious diet and high cost of metabolism in a high-altitude environment, probably explains the very low reproductive potential of this population.

Howling from the past: historical phylogeography and diversity losses in European grey wolves.

Genetic bottlenecks resulting from human-induced population declines make alarming symbols for the irreversible loss of our natural legacy worldwide. The grey wolf (Canis lupus) is an iconic example of extreme declines driven by anthropogenic factors. Here, we assessed the genetic signatures of 150 years of wolf persecution throughout the Western Palaearctic by high-throughput mitochondrial DNA sequencing of historical specimens in an unprecedented spatio-temporal framework. Despite Late Pleistocene bottlenecks, we show that historical genetic variation had remained high throughout Europe until the last several hundred years. In Western Europe, where wolves nearly got fully exterminated, diversity dramatically collapsed at the turn of the twentieth century and recolonization from few homogeneous relict populations induced drastic shifts of genetic composition. By contrast, little genetic displacement and steady levels of diversity were maintained in Eastern European regions, where human persecution had lesser effects on wolf demography. By comparing prehistoric, historic and modern patterns of genetic diversity, our study hence traces the timeframe and the active human role in the decline of the grey wolf, an emblematic yet controversial animal which symbolizes the complex relationship between human societies and nature conservation.

Upscaling the niche variation hypothesis from the intra- to the inter-specific level.

The "niche variation hypothesis" (NVH) predicts that populations with wider niches should display higher among-individual variability. This prediction originally stated at the intra-specific level may be extended to the inter-specific level: individuals of generalist species may differ to a greater extent than individuals of a specialist species. We tested the NVH at intra- and inter-specific levels based on a large diet database of three large herbivore feces collected in the field and analyzed using DNA metabarcoding. The three herbivores (roe deer Capreolus capreolus, chamois Rupicapra rupicapra and mouflon Ovis musimon) are highly contrasted in terms of sociality (solitary to highly gregarious) and diet. The NVH at the intraspecific level was tested by relating, for the same population, diet breadth and inter-individual variation across the four seasons. Compared to null models, our data supported the NVH both at the intra- and inter-specific levels. Inter-individual variation of the diet of solitary species was not larger than in social species, although social individuals feed together and could therefore have more similar diets. Hence, the NVH better explained diet breadth than other factors such as sociality. The expansion of the population niche of the three species was driven by resource availability, and achieved by an increase in inter-individual variation, and the level of inter-individual variability was larger in the generalist species (mouflon) than in the specialist one (roe deer). This mechanism at the base of the NVH appears at play at different levels of biological organization, from populations to communities.

Plant functional traits reveal the relative contribution of habitat and food preferences to the diet of grasshoppers.

Food preferences and food availability are two major determinants of the diet of generalist herbivores and of their spatial distribution. How do these factors interact and eventually lead to diet differentiation in co-occurring herbivores? We quantified the diet of four grasshopper species co-occurring in subalpine grasslands using DNA barcoding of the plants contained in the faeces of individuals sampled in the field. The food preferences of each grasshopper species were assessed by a choice (cafeteria) experiment from among 24 plant species common in five grassland plots, in which the four grasshoppers were collected, while the habitat was described by the relative abundance of plant species in the grassland plots. Plant species were characterised by their leaf economics spectrum (LES), quantifying their nutrient vs. structural tissue content. The grasshoppers' diet, described by the mean LES of the plants eaten, could be explained by their plant preferences but not by the available plants in their habitat. The diet differed significantly across four grasshopper species pairs out of six, which validates food preferences assessed in standardised conditions as indicators for diet partitioning in nature. In contrast, variation of the functional diversity (FD) for LES in the diet was mostly correlated to the FD of the available plants in the habitat, suggesting that diet mixing depends on the environment and is not an intrinsic property of the grasshopper species. This study sheds light on the mechanisms determining the feeding niche of herbivores, showing that food preferences influence niche position whereas habitat diversity affects niche breadth.

A DNA metabarcoding study of a primate dietary diversity and plasticity across its entire fragmented range.

In tropical regions, most primary ecosystems have been replaced by mosaic landscapes in which species must cope with a large shift in the distribution of their habitat and associated food resources. Primates are particularly vulnerable to habitat modifications. Most species persist in small fragments surrounded by complex human-mediated matrices whose structure and connectivity may strongly influence their dispersal and feeding behavior. Behavioral plasticity appears to be a crucial parameter governing the ability of organisms to exploit the resources offered by new matrix habitats and thus to persist in fragmented habitats. In this study, we were interested in the dietary plasticity of the golden-crowned sifaka (Propithecus tattersalli), an endangered species of lemur, found only in the Daraina region in north-eastern Madagascar. We used a DNA-based approach combining the barcoding concept and Illumina next-generation sequencing to (i) describe the species diet across its entire range and (ii) evaluate the influence of landscape heterogeneity on diet diversity and composition. Faeces from 96 individuals were sampled across the entire species range and their contents were analyzed using the trnL metabarcoding approach. In parallel, we built a large DNA reference database based on a checklist of the plant species of the Daraina region. Our results suggest that golden-crowned sifakas exhibit remarkable dietary diversity with at least 130 plant species belonging to 80 genera and 49 different families. We highlighted an influence of both habitat type and openness on diet composition suggesting a high flexibility of foraging strategies. Moreover, we observed the presence of numerous cultivated and naturalized plants in the faeces of groups living in forest edge areas. Overall, our findings support our initial expectation that P. tattersalli is able to cope with the current level of alteration of the landscape and confirm our previous results on the distribution and the dispersal ability of this species.

Major histocompatibility complex class II compatibility, but not class I, predicts mate choice in a bird with highly developed olfaction.

Mate choice for major histocompatibility complex (MHC) compatibility has been found in several taxa, although rarely in birds. MHC is a crucial component in adaptive immunity and by choosing an MHC-dissimilar partner, heterozygosity and potentially broad pathogen resistance is maximized in the offspring. The MHC genotype influences odour cues and preferences in mammals and fish and hence olfactory-based mate choice can occur. We tested whether blue petrels, Halobaena caerulea, choose partners based on MHC compatibility. This bird is long-lived, monogamous and can discriminate between individual odours using olfaction, which makes it exceptionally well suited for this analysis. We screened MHC class I and II B alleles in blue petrels using 454-pyrosequencing and quantified the phylogenetic, functional and allele-sharing similarity between individuals. Partners were functionally more dissimilar at the MHC class II B loci than expected from random mating (p = 0.033), whereas there was no such difference at the MHC class I loci. Phylogenetic and non-sequence-based MHC allele-sharing measures detected no MHC dissimilarity between partners for either MHC class I or II B. Our study provides evidence of mate choice for MHC compatibility in a bird with a high dependency on odour cues, suggesting that MHC odour-mediated mate choice occurs in birds.

Soil sampling and isolation of extracellular DNA from large amount of starting material suitable for metabarcoding studies.

DNA metabarcoding refers to the DNA-based identification of multiple species from a single complex and degraded environmental sample. We developed new sampling and extraction protocols suitable for DNA metabarcoding analyses targeting soil extracellular DNA. The proposed sampling protocol has been designed to reduce, as much as possible, the influence of local heterogeneity by processing a large amount of soil resulting from the mixing of many different cores. The DNA extraction is based on the use of saturated phosphate buffer. The sampling and extraction protocols were validated first by analysing plant DNA from a set of 12 plots corresponding to four plant communities in alpine meadows, and, second, by conducting pilot experiments on fungi and earthworms. The results of the validation experiments clearly demonstrated that sound biological information can be retrieved when following these sampling and extraction procedures. Such a protocol can be implemented at any time of the year without any preliminary knowledge of specific types of organisms during the sampling. It offers the opportunity to analyse all groups of organisms using a single sampling/extraction procedure and opens the possibility to fully standardize biodiversity surveys.

Tracking earthworm communities from soil DNA.

Earthworms are known for their important role within the functioning of an ecosystem, and their diversity can be used as an indicator of ecosystem health. To date, earthworm diversity has been investigated through conventional extraction methods such as handsorting, soil washing or the application of a mustard solution. Such techniques are time consuming and often difficult to apply. We showed that combining DNA metabarcoding and next-generation sequencing facilitates the identification of earthworm species from soil samples. The first step of our experiments was to create a reference database of mitochondrial DNA (mtDNA) 16S gene for 14 earthworm species found in the French Alps. Using this database, we designed two new primer pairs targeting very short and informative DNA sequences (about 30 and 70 bp) that allow unambiguous species identification. Finally, we analysed extracellular DNA taken from soil samples in two localities (two plots per locality and eight samples per plot). The two short metabarcode regions led to the identification of a total of eight earthworm species. The earthworm communities identified by the DNA-based approach appeared to be well differentiated between the two localities and are consistent with results derived from inventories collected using the handsorting method. The possibility of assessing earthworm communities from hundreds or even thousands of localities through the use of extracellular soil DNA will undoubtedly stimulate further ecological research on these organisms. Using the same DNA extracts, our study also illustrates the potential of environmental DNA as a tool to assess the diversity of other soil-dwelling animal taxa.

Carnivore diet analysis based on next-generation sequencing: application to the leopard cat (Prionailurus bengalensis) in Pakistan.

Diet analysis is a prerequisite to fully understand the biology of a species and the functioning of ecosystems. For carnivores, traditional diet analyses mostly rely upon the morphological identification of undigested remains in the faeces. Here, we developed a methodology for carnivore diet analyses based on the next-generation sequencing. We applied this approach to the analysis of the vertebrate component of leopard cat diet in two ecologically distinct regions in northern Pakistan. Despite being a relatively common species with a wide distribution in Asia, little is known about this elusive predator. We analysed a total of 38 leopard cat faeces. After a classical DNA extraction, the DNA extracts were amplified using primers for vertebrates targeting about 100 bp of the mitochondrial 12S rRNA gene, with and without a blocking oligonucleotide specific to the predator sequence. The amplification products were then sequenced on a next-generation sequencer. We identified a total of 18 prey taxa, including eight mammals, eight birds, one amphibian and one fish. In general, our results confirmed that the leopard cat has a very eclectic diet and feeds mainly on rodents and particularly on the Muridae family. The DNA-based approach we propose here represents a valuable complement to current conventional methods. It can be applied to other carnivore species with only a slight adjustment relating to the design of the blocking oligonucleotide. It is robust and simple to implement and allows the possibility of very large-scale analyses.

Importance of accounting for detection heterogeneity when estimating abundance: the case of French wolves.

Assessing conservation strategies requires reliable estimates of abundance. Because detecting all individuals is most often impossible in free-ranging populations, estimation procedures have to account for a <1 detection probability. Capture-recapture methods allow biologists to cope with this issue of detectability. Nevertheless, capture-recapture models for open populations are built on the assumption that all individuals share the same detection probability, although detection heterogeneity among individuals has led to underestimating abundance of closed populations. We developed multievent capture-recapture models for an open population and proposed an associated estimator of population size that both account for individual detection heterogeneity (IDH). We considered a two-class mixture model with weakly and highly detectable individuals to account for IDH. In a noninvasive capture-recapture study of wolves we based on genotypes identified in feces and hairs, we found a large underestimation of population size (27% on average) occurred when IDH was ignored.

Analysing diet of small herbivores: the efficiency of DNA barcoding coupled with high-throughput pyrosequencing for deciphering the composition of complex plant mixtures.

BACKGROUND: In order to understand the role of herbivores in trophic webs, it is essential to know what they feed on. Diet analysis is, however, a challenge in many small herbivores with a secretive life style. In this paper, we compare novel (high-throughput pyrosequencing) DNA barcoding technology for plant mixture with traditional microhistological method. We analysed stomach contents of two ecologically important subarctic vole species, Microtus oeconomus and Myodes rufocanus, with the two methods. DNA barcoding was conducted using the P6-loop of the chloroplast trnL (UAA) intron. RESULTS: Although the identified plant taxa in the diets matched relatively well between the two methods, DNA barcoding gave by far taxonomically more detailed results. Quantitative comparison of results was difficult, mainly due to low taxonomic resolution of the microhistological method, which also in part explained discrepancies between the methods. Other discrepancies were likely due to biases mostly in the microhistological analysis. CONCLUSION: We conclude that DNA barcoding opens up for new possibilities in the study of plant-herbivore interactions, giving a detailed and relatively unbiased picture of food utilization of herbivores.

Universal DNA-based methods for assessing the diet of grazing livestock and wildlife from feces.

Because of the demand for controlling livestock diets, two methods that characterize the DNA of plants present in feces were developed. After DNA extraction from fecal samples, a short fragment of the chloroplastic trnL intron was amplified by PCR using a universal primer pair for plants. The first method generates a signature that is the electrophoretic migration pattern of the PCR product. The second method consists of sequencing several hundred DNA fragments from the PCR product through pyrosequencing. These methods were validated with a blind analysis of feces from concentrate- and pasture-fed lambs. The signature method allowed differentiation of the two diets and confirmed the presence of concentrate in one of them. The pyrosequencing method allowed the identification of up to 25 taxa in a diet. These methods are complementary to the chemical methods already used. They could be applied to the control of diets and the study of food preferences.

New perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing: the trnL approach.

The development of DNA barcoding (species identification using a standardized DNA sequence), and the availability of recent DNA sequencing techniques offer new possibilities in diet analysis. DNA fragments shorter than 100-150 bp remain in a much higher proportion in degraded DNA samples and can be recovered from faeces. As a consequence, by using universal primers that amplify a very short but informative DNA fragment, it is possible to reliably identify the plant taxon that has been eaten. According to our experience and using this identification system, about 50% of the taxa can be identified to species using the trnL approach, that is, using the P6 loop of the chloroplast trnL (UAA) intron. We demonstrated that this new method is fast, simple to implement, and very robust. It can be applied for diet analyses of a wide range of phytophagous species at large scales. We also demonstrated that our approach is efficient for mammals, birds, insects and molluscs. This method opens new perspectives in ecology, not only by allowing large-scale studies on diet, but also by enhancing studies on resource partitioning among competing species, and describing food webs in ecosystems.

Long-term evolutionary stability of bacterial endosymbiosis in curculionoidea: additional evidence of symbiont replacement in the dryophthoridae family.

Bacterial intracellular symbiosis (endosymbiosis) is well documented in the insect world where it is believed to play a crucial role in adaptation and evolution. However, although Coleopteran insects are of huge ecological and economical interest, endosymbiont molecular analysis is limited to the Dryophthoridae family. Here, we have analyzed the intracellular symbiotic bacteria in 2 Hylobius species belonging to the Molytinae subfamily (Curculionoidea superfamily) that exhibit different features from the Dryophthoridae insects in terms of their ecology and geographical spanning. Fluorescence in situ hybridization has shown that both Hylobius species harbor rod-shaped pleiomorphic symbiotic bacteria in the oocyte and in the bacteria-bearing organ (the bacteriome), with a shape and location similar to those of the Dryophthoridae bacteriome. Phylogenetic analysis of the 16S ribosomal DNA gene sequences, using the heterogeneous model of DNA evolution, has placed the Hylobius spp. endosymbionts (H-group) at the basal position of the ancestral R-clade of Dryophthoridae endosymbionts named Candidatus Nardonella but relatively distant from the S-clade of Sitophilus spp. endosymbionts. Endosymbionts from the H-group and the R-clade evolved more quickly compared with free-living enteric bacteria and endosymbionts from the S- and D-clades of Dryophthoridae. They are AT biased (58.3% A + T), and they exhibit AT-rich insertions at the same position as previously described in the Candidatus Nardonella 16S rDNA sequence. Moreover, the host phylogenetic tree based on the mitochondrial COI gene was shown to be highly congruent with the H-group and the R-clade, the divergence of which was estimated to be around 125 MYA. These new molecular data show that endosymbiosis is old in Curculionids, going back at least to the common ancestor of Molytinae and Dryophthoridae, and is evolutionary stable, except in 2 Dryophthoridae clades, providing additional and independent supplementary evidence for endosymbiont replacement in these taxa.

From the Apennines to the Alps: colonization genetics of the naturally expanding Italian wolf (Canis lupus) population.

Wolves in Italy strongly declined in the past and were confined south of the Alps since the turn of the last century, reduced in the 1970s to approximately 100 individuals surviving in two fragmented subpopulations in the central-southern Apennines. The Italian wolves are presently expanding in the Apennines, and started to recolonize the western Alps in Italy, France and Switzerland about 16 years ago. In this study, we used a population genetic approach to elucidate some aspects of the wolf recolonization process. DNA extracted from 3068 tissue and scat samples collected in the Apennines (the source populations) and in the Alps (the colony), were genotyped at 12 microsatellite loci aiming to assess (i) the strength of the bottleneck and founder effects during the onset of colonization; (ii) the rates of gene flow between source and colony; and (iii) the minimum number of colonizers that are needed to explain the genetic variability observed in the colony. We identified a total of 435 distinct wolf genotypes, which showed that wolves in the Alps: (i) have significantly lower genetic diversity (heterozygosity, allelic richness, number of private alleles) than wolves in the Apennines; (ii) are genetically distinct using pairwise F(ST) values, population assignment test and Bayesian clustering; (iii) are not in genetic equilibrium (significant bottleneck test). Spatial autocorrelations are significant among samples separated up to c. 230 km, roughly correspondent to the apparent gap in permanent wolf presence between the Alps and north Apennines. The estimated number of first-generation migrants indicates that migration has been unidirectional and male-biased, from the Apennines to the Alps, and that wolves in southern Italy did not contribute to the Alpine population. These results suggest that: (i) the Alps were colonized by a few long-range migrating wolves originating in the north Apennine subpopulation; (ii) during the colonization process there has been a moderate bottleneck; and (iii) gene flow between sources and colonies was moderate (corresponding to 1.25-2.50 wolves per generation), despite high potential for dispersal. Bottleneck simulations showed that a total of c. 8-16 effective founders are needed to explain the genetic diversity observed in the Alps. Levels of genetic diversity in the expanding Alpine wolf population, and the permanence of genetic structuring, will depend on the future rates of gene flow among distinct wolf subpopulation fragments.

Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding.

DNA barcoding should provide rapid, accurate and automatable species identifications by using a standardized DNA region as a tag. Based on sequences available in GenBank and sequences produced for this study, we evaluated the resolution power of the whole chloroplast trnL (UAA) intron (254-767 bp) and of a shorter fragment of this intron (the P6 loop, 10-143 bp) amplified with highly conserved primers. The main limitation of the whole trnL intron for DNA barcoding remains its relatively low resolution (67.3% of the species from GenBank unambiguously identified). The resolution of the P6 loop is lower (19.5% identified) but remains higher than those of existing alternative systems. The resolution is much higher in specific contexts such as species originating from a single ecosystem, or commonly eaten plants. Despite the relatively low resolution, the whole trnL intron and its P6 loop have many advantages: the primers are highly conserved, and the amplification system is very robust. The P6 loop can even be amplified when using highly degraded DNA from processed food or from permafrost samples, and has the potential to be extensively used in food industry, in forensic science, in diet analyses based on feces and in ancient DNA studies.