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Abstract Facing rapid environmental changes and anthropogenic habitat destruction, animal behavioural plasticity becomes an adaptive potential that needs to be considered in conservation strategies along with, for example, genetic diversity. Here, we evaluate to what extent non‐invasive environmental DNA (eDNA) methods may contribute to the assessment of intraspecies behavioural plasticity in terms of foraging behaviour. We analysed DNA metabarcoding data for plant components in the diet of four neighbouring groups of wild vervet monkeys Chlorocebus pygerythrus to identify intergroup variation (IGV). The faecal samples considered for the analyses were limited to the summer season to minimise the impact of seasonality. Each sample was attributed by observation to individuals with known life history data. A plant survey was conducted in each group home range during the study period to account for environmental variation. We observed mixed results when testing whether IGV in plant consumption was greater than intragroup variation, indicating that the influence of social dynamics must be considered. Intragroup variation was positively correlated with group size. We observed IGV in diet composition among all groups as well as in some pairwise comparisons. We found significant dietary differences between two group pairs when considering only adult females. Lastly, we observed IGV in foraging of specific plants that were not explained by their distribution, suggesting behavioural differences in selectivity between groups. Our study system and organism, being a highly social and non‐threatened primate species, with constant gene flow and overlapping territories between groups, provides an ideal model to evaluate the usage of eDNA‐based methods to better understand the impact of social factors on IGV. Our results highlight the need to consider social and demographic factors, the impact of which remains complicated to disentangle from environmental factors. However, we emphasise the great potential for studying social groups using eDNA and that such studies are needed to better understand intraspecific behavioural plasticity in wild populations.

Abstract Biodiversity assessments are indispensable tools for planning and monitoring conservation strategies. Camera traps (CT) are widely used to monitor wildlife and have proven their usefulness. Environmental DNA (eDNA)‐based approaches are increasingly implemented for biomonitoring, combining sensitivity, high taxonomic coverage and resolution, non‐invasiveness and easiness of sampling, but remain challenging for terrestrial fauna. However, in remote desert areas where scattered water bodies attract terrestrial species, which release their DNA into the water, this method presents a unique opportunity for their detection. In order to identify the most efficient method for a given study system, comparative studies are needed. Here, we compare CT and DNA metabarcoding of water samples collected from two desert ecosystems, the Trans‐Altai Gobi in Mongolia and the Kalahari in Botswana. We recorded with CT the visiting patterns of wildlife and studied the correlation with the biodiversity captured with the eDNA approach. The aim of the present study was threefold: (a) to investigate how well waterborne eDNA captures signals of terrestrial fauna in remote desert environments, which have been so far neglected in terms of biomonitoring efforts; (b) to compare two distinct approaches for biomonitoring in such environments; and (c) to draw recommendations for future eDNA‐based biomonitoring. We found significant correlations between the two methodologies and describe a detectability score based on variables extracted from CT data and the visiting patterns of wildlife. This supports the use of eDNA‐based biomonitoring in these ecosystems and encourages further research to integrate the methodology in the planning and monitoring of conservation strategies.

Abstract Assessing the diet of wild animals reveals valuable information about their ecology and trophic relationships that may help elucidate dynamic interactions in ecosystems and forecast responses to environmental changes. Advances in molecular biology provide valuable research tools in this field. However, comparative empirical research is still required to highlight strengths and potential biases of different approaches. Therefore, this study compares environmental DNA and observational methods for the same study population and sampling duration. We employed DNA metabarcoding assays targeting plant and arthropod diet items in 823 fecal samples collected over 12 months in a wild population of an omnivorous primate, the vervet monkey (Chlorocebus pygerythrus). DNA metabarcoding data were subsequently compared to direct observations. We observed the same seasonal patterns of plant consumption with both methods; however, DNA metabarcoding showed considerably greater taxonomic coverage and resolution compared to observations, mostly due to the construction of a local plant DNA database. We found a strong effect of season on variation in plant consumption largely shaped by the dry and wet seasons. The seasonal effect on arthropod consumption was weaker, but feeding on arthropods was more frequent in spring and summer, showing overall that vervets adapt their diet according to available resources. The DNA metabarcoding assay outperformed also direct observations of arthropod consumption in both taxonomic coverage and resolution. Combining traditional techniques and DNA metabarcoding data can therefore not only provide enhanced assessments of complex diets and trophic interactions to the benefit of wildlife conservationists and managers but also opens new perspectives for behavioral ecologists studying whether diet variation in social species is induced by environmental differences or might reflect selective foraging behaviors.

Abstract Increasing evidence suggests that agricultural intensification is a threat to many groups of soil biota, but how the impacts of land-use intensity on soil organisms translate into changes in comprehensive soil interaction networks remains unclear. Here for the first time, we use environmental DNA to examine total soil multi-trophic diversity and food web structure for temperate agroecosystems along a gradient of land-use intensity. We tested for response patterns in key properties of the soil food webs in sixteen fields ranging from arable crops to grazed permanent grasslands as part of a long-term management experiment. We found that agricultural intensification drives reductions in trophic group diversity, although taxa richness remained unchanged. Intensification generally reduced the complexity and connectance of soil interaction networks and induced consistent changes in energy pathways, but the magnitude of management-induced changes depended on the variable considered. Average path length (an indicator of food web redundancy and resilience) did not respond to our management intensity gradient. Moreover, turnover of network structure showed little response to increasing management intensity. Our data demonstrates the importance of considering different facets of trophic networks for a clearer understanding of agriculture-biodiversity relationships, with implications for nature-based solutions and sustainable agriculture.

Abstract Leaf litter habitats shelter a great variety of organisms, which play an important role in ecosystem dynamics. However, monitoring species in leaf litter is challenging, especially in highly diverse environments such as tropical forests, because individuals may easily camouflage themselves or hide in the litter layer. Identifying species based on environmental DNA (eDNA) would allow us to assess biodiversity in this microhabitat, without the need for direct observation of individuals. We applied eDNA metabarcoding to analyze large amounts of leaf litter (1 kg per sample) collected in the Brazilian Atlantic forest. We compared two DNA extraction methods, one total and one extracellular, and amplified a fragment of the mitochondrial 18S rRNA gene common to all eukaryotes, to assess the performance of eDNA from leaf litter samples in identifying different eukaryotic taxonomic groups. We also amplified two fragments of the mitochondrial 12S rRNA gene to specifically test the power of this approach for monitoring vertebrate species, with a focus on anurans. Most of the eukaryote sequence reads obtained were classified as Fungi, followed by Metazoa, and Viridiplantae. Most vertebrate sequences were assigned to Homo sapiens; only two sequences assigned to the genus Phyllomedusa and the species Euparkerella brasiliensis can be considered true detections of anurans in our eDNA samples. The detection of taxa varied depending on the DNA extraction method applied. Our results demonstrate that the analysis of eDNA from leaf litter samples has low power for monitoring vertebrate species and should be preferentially applied to describe active and abundant taxa in terrestrial communities, such as Fungi and invertebrates.

Abstract 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.

Abstract Vertebrate animals are known to consume other species' faeces, yet the role of such coprophagy in species dynamics remains unknown, not least due to the methodological challenges of documenting it. In a large‐scale metabarcoding study of red fox and pine marten scats, we document a high occurrence of domestic dog DNA in red fox scats and investigate if it can be attributed to interspecific coprophagia. We tested whether experimental artifacts or other sources of DNA could account for dog DNA, regressed dog occurrence in the diet of fox against that of the fox’s main prey, short‐tailed field voles, and consider whether predation or scavenging could explain the presence of dog DNA. Additionally, we determined the calorific value of dog faeces through calorimetric explosion. The high occurrence of dog DNA in the diet of fox, the timing of its increase, and the negative relationship between dog and the fox's main prey, point to dog faeces as the source of DNA in fox scats. Dog faeces being highly calorific, we found that foxes, but not pine martens, regularly exploit them, seemingly as an alternative resource to fluctuating prey. Scattered accounts from the literature may suggest that interspecific coprophagia is a potentially frequent and widespread form of interaction among vertebrates. However, further work should address its prevalence in other systems and the implications for ecological communities. Tools such as metabarcoding offer a way forward.

Abstract 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.

Abstract Assessing biodiversity loss and species extinction is necessary to warn society and raise awareness of the impacts of ongoing climate change. Prioritizing protected areas is the pragmatic and applicable management measure under the pressure of ongoing climate change and limited resources to conserve species at risk of extinction. We developed a novel conservation index (CI) to prioritize areas and populations of an endangered mountain tree species that need protection in the face of ongoing climate change, as conservation of all populations may not be realistic. This CI integrates (1) mountain topography to identify potential refugial areas with suitable microclimates, (2) genetic diversity to assess the adaptive capacity of local populations, and (3) hypothetical climate change in the species' range. We applied this CI to Atlas cedar, an endemic and threatened species whose populations are scattered throughout the Moroccan mountains. This index provided a scale for 33 populations studied and suggests that genetically diverse populations located in rugged areas where future local climate may overlap with their current climatic niche should receive a higher conservation priority. This index may also be applicable to other mountain species with scattered populations and is likely to be more accurate if more precise climate data are used at the microrefugia scale.

Primary Biogenic Organic Aerosols (PBOA) were recently shown to be produced by only a few types of microorganisms, emitted by the surrounding vegetation in the case of a regionally homogeneous field site. This study presents the first comprehensive description of the structure and main sources of airborne microbial communities associated with temporal trends in Sugar Compounds (SC) concentrations of PM10 in 3 sites under a climatic gradient in France. By combining sugar chemistry and DNA Metabarcoding approaches, we intended to identify PM10-associated microbial communities and their main sources at three sampling-sites in France, under different climates, during the summer of 2018. This study accounted also for the interannual variability in summer airborne microbial community structure (bacteria and fungi only) associated with PM10-SC concentrations during a 2 consecutive years’ survey at one site. Our results showed that temporal changes in PM10-SC in the three sites are associated with the abundance of only a few specific taxa of airborne fungi and bacterial. These taxa differ significantly between the 3 climatic regions studied. The microbial communities structure associated with SC concentrations of PM10 during a consecutive 2-year study remained stable in the rural area. Atmospheric concentration levels of PM10-SC species varied significantly between the 3 study sites, but with no clear difference according to site typology (rural vs. urban), suggesting that SC emissions are related to regional rather than local climatic characteristics. The overall microbial beta diversity in PM10 samples is significantly different from that of the main vegetation around the urban sites studied. This indicates that the airborne microorganisms at these urban sites are not solely from the immediate surrounding vegetation, which contrasts with observations at the scale of a regionally homogeneous rural site in 2017. These results improve our understanding of the spatial behavior of tracers of PBOA emission sources, which need to be better characterized to further implement this important mass fraction of Organic Matter (OM) in Chemical Transport models (CTM).

The sheep and goat were domesticated ~10,500 years ago in the same region of the Middle-East. Here, Alberto et al compare the genomes of wild Asiatic mouflon and Bezoar ibex with that of domestics from local, traditional and improved breeds and find common targets of selection related to domestication and improvement in sheep and goats.

The domestication of plants and animals causes genomic changes underlying various morphologic, physiologic and behavioral changes. Here, Naval-Sanchez et al. provide a ChIP-Seq validated comparative functional annotation of the sheep genome, and show widespread evolution of proximal regulatory elements.

Recently, an eDNA metabarcoding data set was used to describe northern high-latitude vegetation during the past 50,000 years. Here, Zobel et al. use the data set to examine how the abundance of key plant mutualistic traits changed during this period and discuss possible environmental drivers.

Body condition, as a sign of animal welfare, is of management concern in rewilding projects where fenced animals are subject to winter starvation, which may conflict with animal welfare legislation. Investigating the relationship between body condition, age, sex, diet quality and diet composition is therefore relevant to increase understanding of herbivores' ecosystem function and to inform management. In this study, we focused on red deer, Cervus elaphus, in a fenced nature reserve in Denmark, where the deer are managed as ecosystem engineers to contribute to biodiversity conservation. We measured body mass and body size of 91 culled red deer, and determined diet composition using DNA metabarcoding and diet quality using fecal nitrogen on 246 fecal samples. We found that body condition was predicted by age and diet composition, but not diet quality. We also found that individuals of different body condition had different diets, i.e., the fecal samples of red deer in poorer body condition contained significantly more Ericaceae sequences than red deer in good body condition. This may imply that certain functions of red deer in ecosystems, such as regeneration of heather by grazing, may depend on variation in body condition within the population. Our findings call for the need to consider the consequences of management practices, including culling or supplemental feeding, on the outcomes of habitat restoration, and more broadly underline the importance of preserving the overall breath of herbivore ecosystem functions for effective biodiversity conservation.

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

The protection, preservation and restoration of aquatic ecosystems and their functions are of global importance. For European states it became legally binding mainly through the EU-Water Framework Directive (WFD). In order to assess the ecological status of a given water body, aquatic biodiversity data are obtained and compared to a reference water body. The quantified mismatch obtained determines the extent of potential management actions. The current approach to biodiversity assessment is based on morpho-taxonomy. This approach has many drawbacks such as being time consuming, limited in temporal and spatial resolution, and error-prone due to the varying individual taxonomic expertise of the analysts. Novel genomic tools can overcome many of the aforementioned problems and could complement or even replace traditional bioassessment. Yet, a plethora of approaches are independently developed in different institutions, thereby hampering any concerted routine application. The goal of this Action is to nucleate a group of researchers across disciplines with the task to identify gold-standard genomic tools and novel eco-genomic indices for routine application in biodiversity assessments of European fresh- and marine water bodies. Furthermore, DNAqua-Net will provide a platform for training of the next generation of European researchers preparing them for the new technologies. Jointly with water managers, politicians, and other stakeholders, the group will develop a conceptual framework for the standard application of eco-genomic tools as part of legally binding assessments.

The use of lake sedimentary DNA to track the long-term changes in both terrestrial and aquatic biota is a rapidly advancing field in paleoecological research. Although largely applied nowadays, knowledge gaps remain in this field and there is therefore still research to be conducted to ensure the reliability of the sedimentary DNA signal. Building on the most recent literature and seven original case studies, we synthesize the state-of-the-art analytical procedures for effective sampling, extraction, amplification, quantification and/or generation of DNA inventories from sedimentary ancient DNA (sedaDNA) via high-throughput sequencing technologies. We provide recommendations based on current knowledge and best practises.

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.

This study reconstructs and interprets the changing range of Atlas cedar in northern Morocco over the last 9,000 years. A synthesis of fossil pollen records indicated that Atlas cedars occupied a wider range at lower elevations during the mid-Holocene than today. The mid-Holocene geographical expansion reflected low winter temperatures and higher water availability over the whole range of the Rif Mountains relative to modern conditions. A trend of increasing aridity observed after 6,000 years BP progressively reduced the range of Atlas cedar and prompted its migration toward elevations above 1,400 masl. To assess the impact of climate change on cedar populations over the last decades, we performed a transient model simulation for the period between 1960 and 2010. Our simulation showed that the range of Atlas cedar decreased by about 75% over the last 50 years and that the eastern populations of the range in the Rif Mountains were even more threatened by the overall lack of water availability than the western ones. Today, Atlas cedar populations in the Rif Mountains are persisting in restricted and isolated areas (Jbel Kelti, Talassemtane, Jbel Tiziren, Oursane, Tidighine) that we consider to be modern microrefugia. Conservation of these isolated populations is essential for the future survival of the species, preserving polymorphisms and the potential for population recovery under different climatic conditions.

We assessed spatio-temporal patterns of diversity in deep-sea sediment communities using metabarcoding. We chose a recently developed eukaryotic marker based on the v7 region of the 18S rRNA gene. Our study was performed in a submarine canyon and its adjacent slope in the Northwestern Mediterranean Sea, sampled along a depth gradient at two different seasons. We found a total of 5,569 molecular operational taxonomic units (MOTUs), dominated by Metazoa, Alveolata and Rhizaria. Among metazoans, Nematoda, Arthropoda and Annelida were the most diverse. We found a marked heterogeneity at all scales, with important differences between layers of sediment and significant changes in community composition with zone (canyon vs slope), depth, and season. We compared the information obtained from metabarcoding DNA and RNA and found more total MOTUs and more MOTUs per sample with DNA (ca. 20% and 40% increase, respectively). Both datasets showed overall similar spatial trends, but most groups had higher MOTU richness with the DNA template, while others, such as nematodes, were more diverse in the RNA dataset. We provide metabarcoding protocols and guidelines for biomonitoring of these key communities in order to generate information applicable to management efforts.

Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate.

Honey is produced by honeybees from nectar and from secretions of living plants. It reflects the honeybees’ diet and the local plant communities. Honey also shows different plant compositions in different geographical locations. We propose a new method for studying the plant diversity and the geographical origin of honey using a DNA barcoding approach that combines universal primers and massive parallel pyrosequencing. To test this method we use two commercial honeys, one from a regional origin and one composed of a worldwide mix of different honeys. We demonstrate that the method proposed here is fast, simple to implement, more robust than classical methods, and therefore suitable for analyzing plant diversity in honey.

Microbial community analysis was carried out on ruminal digesta obtained directly via rumen fistula and buccal fluid, regurgitated digesta (bolus) and faeces of dairy cattle to assess if non-invasive samples could be used as proxies for ruminal digesta. Samples were collected from five cows receiving grass silage based diets containing no additional lipid or four different lipid supplements in a 5 x 5 Latin square design. Extracted DNA was analysed by qPCR and by sequencing 16S and 18S rRNA genes or the fungal ITS1 amplicons. Faeces contained few protozoa, and bacterial, fungal and archaeal communities were substantially different to ruminal digesta. Buccal and bolus samples gave much more similar profiles to ruminal digesta, although fewer archaea were detected in buccal and bolus samples. Bolus samples overall were most similar to ruminal samples. The differences between both buccal and bolus samples and ruminal digesta were consistent across all treatments. It can be concluded that either proxy sample type could be used as a predictor of the rumen microbial community, thereby enabling more convenient large-scale animal sampling for phenotyping and possible use in future animal breeding programs aimed at selecting cattle with a lower environmental footprint.

In the last few years, the study of environmental DNA (eDNA) has drawn attention for many reasons, including its advantages for monitoring and conservation purposes. So far, in aquatic environments, most of eDNA research has focused on the detection of single species using species-specific markers. Recently, species inventories based on the analysis of a single generalist marker targeting a larger taxonomic group (eDNA metabarcoding) have proven useful for bony fish and amphibian biodiversity surveys. This approach involves in situ filtering of large volumes of water followed by amplification and sequencing of a short discriminative fragment from the 12S rDNA mitochondrial gene. In this study, we went one step further by investigating the spatial representativeness (i.e. ecological reliability and signal variability in space) of eDNA metabarcoding for large-scale fish biodiversity assessment in a freshwater system including lentic and lotic environments. We tested the ability of this approach to characterize large-scale organization of fish communities along a longitudinal gradient, from a lake to the outflowing river. First, our results confirm that eDNA metabarcoding is more efficient than a single traditional sampling campaign to detect species presence, especially in rivers. Second, the species list obtained using this approach is comparable to the one obtained when cumulating all traditional sampling sessions since 1995 and 1988 for the lake and the river, respectively. In conclusion, eDNA metabarcoding gives a faithful description of local fish biodiversity in the study system, more specifically within a range of a few kilometers along the river in our study conditions, i.e. longer than a traditional fish sampling site.

Repeated introductions and spread of invasive mosquito species (IMS) have been recorded on a large scale these last decades worldwide. In this context, members of the mosquito genus Aedes can present serious risks to public health as they have or may develop vector competence for various viral diseases. While the Tiger mosquito (Aedes albopictus) is a well-known vector for e.g. dengue and chikungunya viruses, the Asian bush mosquito (Ae. j. japonicus) and Ae. koreicus have shown vector competence in the field and the laboratory for a number of viruses including dengue, West Nile fever and Japanese encephalitis. Early detection and identification is therefore crucial for successful eradication or control strategies. Traditional specific identification and monitoring of different and/or cryptic life stages of the invasive Aedes species based on morphological grounds may lead to misidentifications, and are problematic when extensive surveillance is needed. In this study, we developed, tested and applied an environmental DNA (eDNA) approach for the detection of three IMS, based on water samples collected in the field in several European countries. We compared real-time quantitative PCR (qPCR) assays specific for these three species and an eDNA metabarcoding approach with traditional sampling, and discussed the advantages and limitations of these methods. Detection probabilities for eDNA-based approaches were in most of the specific comparisons higher than for traditional survey and the results were congruent between both molecular methods, confirming the reliability and efficiency of alternative eDNA-based techniques for the early and unambiguous detection and surveillance of invasive mosquito vectors. The ease of water sampling procedures in the eDNA approach tested here allows the development of large-scale monitoring and surveillance programs of IMS, especially using citizen science projects.

Sympatric species are expected to minimize competition by partitioning resources, especially when these are limited. Herbivores inhabiting the High Arctic in winter are a prime example of a situation where food availability is anticipated to be low, and thus reduced diet overlap is expected. We present here the first assessment of diet overlap of high arctic lemmings during winter based on DNA metabarcoding of feces. In contrast to previous analyses based on microhistology, we found that the diets of both collared (Dicrostonyx groenlandicus) and brown lemmings (Lemmus trimucronatus) on Bylot Island were dominated by Salix while mosses, which were significantly consumed only by the brown lemming, were a relatively minor food item. The most abundant plant taxon, Cassiope tetragona, which alone composes more than 50% of the available plant biomass, was not detected in feces and can thus be considered to be non-food. Most plant taxa that were identified as food items were consumed in proportion to their availability and none were clearly selected for. The resulting high diet overlap, together with a lack of habitat segregation, indicates a high potential for resource competition between the two lemming species. However, Salix is abundant in the winter habitats of lemmings on Bylot Island and the non-Salix portion of the diets differed between the two species. Also, lemming grazing impact on vegetation during winter in the study area is negligible. Hence, it seems likely that the high potential for resource competition predicted between these two species did not translate into actual competition. This illustrates that even in environments with low primary productivity food resources do not necessarily generate strong competition among herbivores.

Marine sediments are home to one of the richest species pools on Earth, but logistics and a dearth of taxonomic work-force hinders the knowledge of their biodiversity. We characterized α- and β-diversity of deep-sea assemblages from submarine canyons in the western Mediterranean using an environmental DNA metabarcoding. We used a new primer set targeting a short eukaryotic 18S sequence (ca. 110 bp). We applied a protocol designed to obtain extractions enriched in extracellular DNA from replicated sediment corers. With this strategy we captured information from DNA (local or deposited from the water column) that persists adsorbed to inorganic particles and buffered short-term spatial and temporal heterogeneity. We analysed replicated samples from 20 localities including 2 deep-sea canyons, 1 shallower canal, and two open slopes (depth range 100-2,250 m). We identified 1,629 MOTUs, among which the dominant groups were Metazoa (with representatives of 19 phyla), Alveolata, Stramenopiles, and Rhizaria. There was a marked small-scale heterogeneity as shown by differences in replicates within corers and within localities. The spatial variability between canyons was significant, as was the depth component in one of the canyons where it was tested. Likewise, the composition of the first layer (1 cm) of sediment was significantly different from deeper layers. We found that qualitative (presence-absence) and quantitative (relative number of reads) data showed consistent trends of differentiation between samples and geographic areas. The subset of exclusively benthic MOTUs showed similar patterns of β-diversity and community structure as the whole dataset. Separate analyses of the main metazoan phyla (in number of MOTUs) showed some differences in distribution attributable to different lifestyles. Our results highlight the differentiation that can be found even between geographically close assemblages, and sets the ground for future monitoring and conservation efforts on these bottoms of ecological and economic importance.

Mate choice is thought to contribute to the maintenance of the spectacularly high polymorphism of the Major Histocompatibility Complex (MHC) genes, along with balancing selection from parasites, but the relative contribution of the former mechanism is debated. Here, we investigated the association between male MHC genotype and mating success in the brown bear. We analysed fragments of sequences coding for the peptide-binding region of the highly polymorphic MHC class I and class II DRB genes, while controlling for genome-wide effects using a panel of 18 microsatellite markers. Male mating success did not depend on the number of alleles shared with the female or amino-acid distance between potential mates at either locus. Furthermore, we found no indication of female mating preferences for MHC similarity being contingent on the number of alleles the females carried. Finally, we found no significant association between the number of MHC alleles a male carried and his mating success. Thus, our results provided no support for the role of mate choice in shaping MHC polymorphism in the brown bear.

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.

Characterizing the trophic relationships between large herbivores and the outstanding plant diversity in rainforest is a major challenge because of their elusiveness. This is crucial to understand the role of these herbivores in the functioning of the rainforest ecosystems. We tested a non-invasive approach based on the high-throughput sequencing of environmental samples using small plant plastid sequences (the trnL P6 loop) and ribosomal ITS1 primers, referred to as DNA metabarcoding, to investigate the diet of the largest neotropical herbivore, the lowland tapir. Sequencing was performed on plant DNA extracted from tapir faeces collected at the Nouragues station, a protected area of French Guiana. In spite of a limited sampling, our approach reliably provided information about the lowland tapir's diet at this site. Indeed, 95.1% and 74.4% of the plant families and genera identified thanks to the trnL P6 loop, respectively, matched with taxa already known to be consumed by tapirs. With this approach we were able to show that two families and eight new genera are also consumed by the lowland tapir. The taxonomic resolution of this method is limited to the plant family and genera. Complementary barcodes, such as a small portion of ITS1, can be used to efficiently narrow identifications down to the species in some problematic families. We will discuss the remaining limitations of this approach and how useful it is at this stage to unravel the diet of elusive rainforest herbivores and better understand their role as engineers of the ecosystem.

Food habits of the Moroccan dorcas gazelle, Gazella dorcas massaesyla, previously investigated in the 1980s using microhistological fecal analysis, in the M'Sabih Talaa Reserve, west central Morocco, were re-evaluated over three seasons (spring, summer and autumn 2009) using the trnL approach to determine the diet composition and its seasonal variation from fecal samples. Taxonomic identification was carried out using the identification originating from the database built from EMBL and the list of plant species within the reserve. The total taxonomic richness in the reserve was 130 instead of 171 species in the 1980s. The diet composition revealed to be much more diversified (71 plant taxa belonging to 57 genus and 29 families) than it was 22 years ago (29 identified taxa). Thirty-four taxa were newly identified in the diet while 13 reported in 1986-87 were not found. Moroccan dorcas gazelle showed a high preference to Acacia gummifera, Anagallis arvensis, Glebionis coronaria, Cladanthus arabicus, Diplotaxis tenuisiliqua, Erodium salzmannii, Limonium thouini, Lotus arenarius and Zizyphus lotus. Seasonal variations occurred in both number (40-41 taxa in spring-summer and 49 taxa in autumn vs. respectively 23-22 and 26 in 1986-1987) and taxonomic type of eaten plant taxa. This dietary diversification could be attributed either to the difference in methods of analysis, trnL approach having a higher taxonomic resolution, or a potential change in nutritional quality of plants over time.

Accurate information about the diet of large carnivores that are elusive and inhabit inaccessible terrain, is required to properly design conservation strategies. Predation on livestock and retaliatory killing of predators have become serious issues throughout the range of the snow leopard. Several feeding ecology studies of snow leopards have been conducted using classical approaches. These techniques have inherent limitations in their ability to properly identify both snow leopard feces and prey taxa. To examine the frequency of livestock prey and nearly-threatened argali in the diet of the snow leopard, we employed the recently developed DNA-based diet approach to study a snow leopard population located in the Tost Mountains, South Gobi, Mongolia. After DNA was extracted from the feces, a region of ∼100 bp long from mitochondrial 12S rRNA gene was amplified, making use of universal primers for vertebrates and a blocking oligonucleotide specific to snow leopard DNA. The amplicons were then sequenced using a next-generation sequencing platform. We observed a total of five different prey items from 81 fecal samples. Siberian ibex predominated the diet (in 70.4% of the feces), followed by domestic goat (17.3%) and argali sheep (8.6%). The major part of the diet was comprised of large ungulates (in 98.8% of the feces) including wild ungulates (79%) and domestic livestock (19.7%). The findings of the present study will help to understand the feeding ecology of the snow leopard, as well as to address the conservation and management issues pertaining to this wild cat.

The goat (Capra hircus) is one of the earliest domesticated species ca. 10,500 years ago in the Middle-East where its wild ancestor, the bezoar (Capra aegagrus), still occurs. During the Neolithic dispersal, the domestic goat was then introduced in Europe, including the main Mediterranean islands. Islands are interesting models as they maintain traces of ancient colonization, historical exchanges or of peculiar systems of husbandry. Here, we compare the mitochondrial genetic diversity of both medieval and extant goats in the Island of Corsica that presents an original and ancient model of breeding with free-ranging animals. We amplified a fragment of the Control Region for 21 medieval and 28 current goats. Most of them belonged to the A haplogroup, the most worldwide spread and frequent today, but the C haplogroup is also detected at low frequency in the current population. Present Corsican goats appeared more similar to medieval goats than to other European goat populations. Moreover, 16 out of the 26 haplotypes observed were endemic to Corsica and the inferred demographic history suggests that the population has remained constant since the Middle Ages. Implications of these results on management and conservation of endangered Corsican goats currently decimated by a disease are addressed.

The precise knowledge of species distribution is a key step in conservation biology. However, species detection can be extremely difficult in many environments, specific life stages and in populations at very low density. The aim of this study was to improve the knowledge on DNA persistence in water in order to confirm the presence of the focus species in freshwater ecosystems. Aquatic vertebrates (fish: Siberian sturgeon and amphibian: Bullfrog tadpoles) were used as target species. In control conditions (tanks) and in the field (ponds), the DNA detectability decreases with time after the removal of the species source of DNA. DNA was detectable for less than one month in both conditions. The density of individuals also influences the dynamics of DNA detectability in water samples. The dynamics of detectability reflects the persistence of DNA fragments in freshwater ecosystems. The short time persistence of detectable amounts of DNA opens perspectives in conservation biology, by allowing access to the presence or absence of species e.g. rare, secretive, potentially invasive, or at low density. This knowledge of DNA persistence will greatly influence planning of biodiversity inventories and biosecurity surveys.

BackgroundFrom the beginning of domestication, the transportation of domestic animals resulted in genetic and demographic processes that explain their present distribution and genetic structure. Thus studying the present genetic diversity helps to better understand the history of domestic species.Methodology/principal findingsThe genetic diversity of domestic goats has been characterized with 2430 individuals from all over the old world, including 946 new individuals from regions poorly studied until now (mainly the Fertile Crescent). These individuals represented 1540 haplotypes for the HVI segment of the mitochondrial DNA (mtDNA) control region. This large-scale study allowed the establishment of a clear nomenclature of the goat maternal haplogroups. Only five of the six previously defined groups of haplotypes were divergent enough to be considered as different haplogroups. Moreover a new mitochondrial group has been localized around the Fertile Crescent. All groups showed very high haplotype diversity. Most of this diversity was distributed among groups and within geographic regions. The weak geographic structure may result from the worldwide distribution of the dominant A haplogroup (more than 90% of the individuals). The large-scale distribution of other haplogroups (except one), may be related to human migration. The recent fragmentation of local goat populations into discrete breeds is not detectable with mitochondrial markers. The estimation of demographic parameters from mismatch analyses showed that all groups had a recent demographic expansion corresponding roughly to the period when domestication took place. But even with a large data set it remains difficult to give relative dates of expansion for different haplogroups because of large confidence intervals.Conclusions/significanceWe propose standard criteria for the definition of the different haplogroups based on the result of mismatch analysis and on the use of sequences of reference. Such a method could be also applied for clarifying the nomenclature of mitochondrial haplogroups in other domestic species.