High-throughput amplicon sequencing has transformed microsatellite (STR) genotyping by overcoming many of the limitations of fragment-length analysis, enabling more accurate, cost-effective, and standardized genotyping. Yet, protocols specifically designed for high-throughput sequencing (HTS)-based STR genotyping from low-template and degraded DNA remain scarce, despite the prevalence of these challenging sample types in ecological and conservation contexts. We present a methodology for the de novo development of robust STR multiplex panels together with a laboratory protocol for efficient and reliable STR genotyping by sequencing with low quantity and quality DNA samples. The protocol comprises (i) an automated bioinformatic pipeline to design large sets of short tetranucleotide markers optimized for multiplex amplicon sequencing of degraded and low-template DNA; (ii) guidelines for efficient in vitro optimization of multiplex amplification using directly low quantity/quality template DNA; and (iii) a library preparation procedure that improves detection of low-level allele signal while enabling quality assessment of STR amplicon sequencing under limiting DNA conditions. We demonstrate the approach by developing and validating STR panels for non-invasive genotyping of three large carnivore species: a 44-plex for the grey wolf (Canis lupus), a 41-plex for the Eurasian lynx (Lynx lynx), and a 30-plex for the brown bear (Ursus arctos). Multiplex performance was high, with ≥91% of samples successfully genotyped at ≥50% of loci (allele size range 28-110 bp across panels) and correctly assigned to known individuals, negligible levels of noise in the controls, and high discriminatory power (PIDsibs ≤2.4 x 1e-12), also owing to sequence variation among same-length alleles at 15-50% of loci. The approach is broadly applicable to animal and plant species, a wide range of sample types, and large-scale analysis such as genetic monitoring. Our study reinforces the value of STR amplicon sequencing for ecological and conservation applications while highlighting the importance of marker design and laboratory workflows tailored to HTS-based genotyping for accurate and efficient implementation.

Abstract Governments, civil society organizations and businesses often lack the biodiversity data they need for decision-making and adaptive management, impacting their planning, reporting and performance. We explored the biodiversity data needs of such actors in Colombia, Ghana and Switzerland to identify factors affecting data availability and use. Responses to questionnaire surveys showed that the data types with the biggest gaps between user needs and access were progress on conservation or sustainability actions, species populations, habitat state and ecological risk. The most frequent data blockages related to inadequate resources and organizational capacity. Obstacles significantly associated with a lack of primary data included an absence of organizational biodiversity goals and monitoring systems. Problems accessing habitat quality and species abundance data were associated with data collection methods being unknown or unavailable. Businesses were more likely than other groups to need data on threats, perhaps reflecting the increasing importance of environmental risk to the corporate sector. Businesses are less likely to collect primary data or use secondary data and are significantly more likely to be unclear on what biodiversity indicators to use. Non-business organizations are significantly more likely to be unable to access data because of a lack of funding for data collection, analysis, and use. Our results highlight the need for stakeholders across sectors to work together to find common solutions to build and invest in monitoring capacity that unblocks the flow of biodiversity data.

With increasing human domination of ecosystems, wildlife must either relocate or adapt its behaviour to anthropogenic impacts in order to survive. Vervet monkeys (Chlorocebus pygerythrus), whose natural habitats have been progressively encroached upon by urban expansion, have successfully adapted to urbanised environments because of their flexible and generalist feeding behaviour. Characterising diet composition of vervet monkeys can therefore reveal how they exploit anthropogenic resources and uncover opportunistic foraging behaviours. However, accurately determining complete diets through direct observations is challenging. In this study, we used an environmental DNA (eDNA) approach investigating the DNA mixtures present in faecal samples as a non-invasive complementary method for assessing diet and foraging strategies. We identified the dietary components of vervet monkeys through DNA metabarcoding of 447 faecal samples collected from two monkey groups over 4 months in a semi-urban neighbourhood in South Africa. We further compared the results with observational data on foraging to describe how vervet monkeys exploit anthropogenic resources. Subsequently, we evaluated whether dietary patterns can be distinguished between groups and within matrilineal levels. We found DNA metabarcoding data to be consistent with observational data, but the former revealed a broader diversity of consumed taxa. Additionally, we detected a difference in diet between the two investigated groups, and a tendency for similar dietary patterns among matrilineal pairs compared to other group members. Our results support the use of the DNA metabarcoding methodology, both to determine the complex diet of omnivorous species in urbanised ecosystems and to address interindividual foraging behaviours.

Climate warming alters the thermal environment experienced by ectotherms, whose physiological performance and fitness are constrained by temperature. Early life stages are often the temperature-sensitive phases of the life cycle, with potential consequences for population persistence, particularly in freshwater stenotherms such as the Arctic charr (Salvelinus alpinus). The persistence of populations will partly depend on the adaptive potential of critical life stages to environmental changes. In this study, we used a common garden approach to compare the response and phenotypic plasticity of four charr populations to warmer conditions. These populations inhabit thermally contrasted lakes and differ in origin (native/introduced) and management history. We reared embryos at either an optimal (5°C) temperature for larval development or a warmer but realistic (8.5°C) temperature. We tested adaptive divergence among populations in four traits (survival, incubation duration, body length and yolk sac volume), using Q_st-F_st comparisons. We report negative effects of temperature on body size, survival and earlier hatching. Thermal reaction norms differed among populations, indicating adaptive divergence. Contrary to expectations, populations originating from warmer environments did not consistently exhibit higher trait values under elevated temperatures. In contrast, the unmanaged and colder high-altitude population exhibited higher survival rates and lower yolk reserves for a given size under heat stress than the other populations. Our results suggested that evolutionary trajectories specific to each population are shaped by factors related to the population's history, including introductions, demographic fluctuations and long-term repopulation practices, which can jointly influence the potential for adaptation to heat stress.

Abstract As glaciers retreat worldwide, newly exposed terrains are rapidly colonized by plants and their associated animal communities. Although plant–animal interactions are key for biodiversity maintenance and ecosystem functioning, the ecological processes underlying the assembly and development of trophic interactions over space-time remain poorly understood. Here, we investigated the trophic niche structure of plant–arthropod interactions along a 140-year primary succession at Mont Miné glacier foreland (Switzerland). Using arthropod gut-content DNA metabarcoding, we reconstructed trophic interactions at the food web level, revealing numerous previously undetected links among 284 arthropod taxa feeding on 263 plant taxa. Trophic niche overlap among arthropods increased following glacier retreat, indicating decreasing resource partitioning and suggesting increased resource competition. Trophic niche breadth became narrower and diet species richness declined, indicating increased trophic specialization. Notably, changes in trophic interactions occurred more rapidly than shifts in species diversity or community composition of plants and arthropods. These results demonstrate that glacier retreat reorganizes trophic networks beyond simple species turnover, reshaping biotic interactions during ecosystem development. Our findings highlight trophic interactions as sensitive indicators of biodiversity change and suggest that the stability of emerging food webs may be strongly affected as glaciers vanish worldwide.

Aim: Large carnivores worldwide have experienced substantial range contractions due to human activities, though several species are recolonising parts of their historical range. We aimed to assess current and potential European brown bear (Ursus arctos arctos) habitat as well as habitat connectivity on a continental scale. Location: The extended biogeographical regions of Europe, spanning from Portugal to central Russia, longitudinally, and from Norway to T & uuml;rkiye, latitudinally. Excluding inland seas; this area covers 11,151,636 km(2). Methods: We assessed habitat suitability throughout the study area using an ensemble species distribution model with nine submodels, using data from 10 European bear populations and T & uuml;rkiye. We used the resulting habitat suitability maps to conduct a least-cost path connectivity analysis and an omnidirectional circuit connectivity analysis. Main Conclusions: Habitat suitability was strongly associated with low percentages of agricultural cover, low percentages of human development, and proximity to forest. Of our entire study area, 37% (4.09 million km(2)) is occupied or potentially suitable for bears. Connectivity analyses identified corridors that could facilitate movement among southern European bear populations, though agricultural land and human development limit connectivity between northern and southern European bear populations. Previous research estimated bears occupied 0.5 million km(2) across the European Union, while our results estimate 1.82 million km(2) of this part of our study area is potentially suitable for bears, though connectivity is limited. Our results inform conservation strategies and policy development for the future of brown bears in Europe, emphasising the need for transboundary conservation efforts.

ABSTRACT Designing effective conservation plans to protect species from extinction requires a comprehensive understanding of their ecology. Conventional methods used to investigate habitat use are time‐consuming, and the detectability of cryptic species is often insufficient. Environmental DNA (eDNA)‐based approaches provide a complementary tool to traditional monitoring methods for ecosystem monitoring and assessment. Nevertheless, to our knowledge, such methods have rarely been applied to investigate habitat use at a fine scale in a continuous wetland environment. Here, we used an eDNA metabarcoding approach to characterize the breeding habitat use of local amphibian species in a wet meadow expanse along the southern shore of Lake Neuchâtel, Switzerland. We retrieved DNA from six out of the seven species expected to be present. We tested the influence of six abiotic environmental variables on overall species assemblages and individual species occurrences. We showed that the main factor structuring species assemblages was water temperature and that the distribution of three amphibian species was associated with several environmental variables. Our results indicate that the eDNA detection approaches are promising tools to study species' ecology at a small scale in continuous wetland habitats.

In tropical grazer assemblies with abundant large predators, smaller herbivores have been shown to be limited by predation and food quality, while the larger species are regulated by food abundance. Much less is known about herbivore resource partitioning in temperate grazing ecosystems, where humans typically regulate large animal abundances. The Oostvaardersplassen ecosystem in the Netherlands is a unique multispecies assemblage of cattle, horses, red deer, and geese developed after the initial introduction of a few individuals in 1983. During the first 35 years, this herbivore assemblage without predation or human regulation gradually changed into an increasing dominance of the smaller herbivore species. Carrying capacity was reached around 2008, after which numbers started fluctuating depending on winter conditions. From 2018, management changed and population numbers became regulated for biodiversity and animal welfare reasons; however, population numbers still remained close to carrying capacity for several years. We used eDNA metabarcoding of dung to quantify the diet composition of cattle, horses, red deer, and geese, annually in early winter from 2018 to 2021 and calculated their niche overlap. We found strong interspecific diet overlap. Horse and cattle diets remained mostly stable with fluctuating densities of the different species, while only red deer diet showed density dependence. Interspecific niche overlap decreased with increasing red deer population size, the most abundant species. When calculated as total energy expenditure, we found that niche overlap was more linked to red deer abundance than to total herbivore energy intake. We suggest that red deer changed their diet mainly in response to their own population size, reducing their niche overlap in relation to their population increase. In this case, resource competition reduced resource availability and forced herbivores to consume different plant taxa. We conclude that in this predator-free temperate ecosystem, inter- and intraspecific resource competition are key factors structuring this assemblage of different size herbivores. We find a general competitive advantage of the more diet-flexible red deer over horses and cattle, but with also clear signs of resource partitioning.

Accurate detection and identification of vector-host-parasite systems are key to understanding their evolutionary dynamics and to design effective disease prevention strategies. Traditionally, microscopical and serological techniques were employed to analyse arthropod blood meals for host/parasite detection, but these were limited in taxonomic resolution and only to pre-selected taxa. In recent years, molecular techniques have emerged as a promising alternative, offering enhanced resolution and taxonomic range. While singleplex polymerase chain reaction (PCR) assays were used at first to identify host, vector and parasite components in separate reactions, today multiple primer pairs can be combined in a single reaction, i.e., multiplex, offering substantial time and cost savings. Nonetheless, despite the potential benefits of multiplex PCR, studies quantifying its efficacy compared to singleplex reactions are scarce. In this study, we used partially digested mosquito blood meals within an avian malaria framework to jointly identify the host, vector and parasite using multiplex DNA metabarcoding, and to compare it with separate singleplex PCRs. We aimed to compare the detection probabilities and taxonomic assignments between both approaches. We found both to have similar performances in terms of detection for the host and the vector, but singleplex clearly outperformed multiplex for the parasite component. We suggest adjusting the relative concentrations of the PCR primers used in the multiplex assay could increase the efficiency of multiplex in detecting all the components of the studied multi-species system. Overall, the results show that multiplex DNA metabarcoding can be an effective approach that could be applied to any vector-borne interaction involving blood-feeding arthropods. Our insights from this proof-of-concept study will help improve laboratory procedures for accurate and cost-efficient medical diagnosis of vector-borne diseases, the spread of which is globally exacerbated by current climate change.

Understanding predator-prey interactions is crucial for wildlife management and human-wildlife coexistence, particularly in multi-use landscapes such as western Europe. As wolves Canis lupus recolonize their former habitats, knowledge of their diet is essential for conservation, management and public acceptance. However, data from these regions is scarce, in particular for the Alpine region and the Jura Mountains. As opportunistic hunters, wolves adapt their diet to the local prey structure, suggesting that abundant wild ungulates are the main prey source. We also expect diet composition to be influenced by region, season and social status. DNA metabarcoding has emerged as a powerful tool in ecological research, offering high-resolution insights into dietary composition, yet its application in wolves remains limited. Using a DNA metabarcoding approach, we analyzed 698 wolf scat samples collected in Switzerland from 2017 to 2024. We found red deer Cervus elaphus was the dominant prey in most areas, and together with roe deer Capreolus capreolus and chamois Rupicapra rupicapra, accounted for 80.8% of the retrieved sequences. We found similarities in prey selectivity between the Alps and the Jura Mountains, but found significant differences across seasonal periods and between pack and non-pack wolves. This study provides the first detailed dietary analysis of wolves in the northern Alps and Jura Mountains, offering critical insights for wildlife management. Our findings highlight the importance of wild ungulates in wolf diet and underscore the value of non-invasive DNA approaches as a reliable conservation and biomonitoring tool.

The selective pressure from pathogens on individuals can have direct consequences on reproduction. Genes from the major histocompatibility complex (MHC) are central to the vertebrate adaptive immune system and pathogen resistance. In species with biparental care, each sex has distinct reproductive roles and levels of investment, and due to a trade-off with immunity, one can expect different selective regimes acting upon the MHC of each parent. Here, we addressed whether couples combine each other's variation at MHC loci to increase their breeding success. Specifically, we used a 23-year dataset from a barn owl population (Tyto alba) to understand how MHC class I alpha and II beta functional divergence and supertypes of each parent were associated with clutch size and fledging success. We did not detect associations between MHC diversity and supertypes with the clutch size or with the fledging success. In addition, to understand the relative contribution from the MHC of the genetic parents and the social parents, we analyzed the fledging success using only a cross-fostered dataset. We found several associations of weak-to-moderate effect sizes between the father's MHC and fledging success: (i) lower MHC-I alpha divergence in the genetic father increases fledging success, which might improve paternal care during incubation, and (ii) one and two MHC-II beta DAB2 supertypes in the social father decrease and increase, respectively, fledging success, which may affect the paternal care after hatching. Furthermore, fledging success increased when both parents did not carry MHC-II beta DAB1 supertype 2, which could suggest conditional effects of this supertype. Although our study relied on a substantial dataset, we showed that the associations between MHC diversity and reproductive success remain scarce and of complex interpretation in the barn owl. Moreover, our results highlighted the need to incorporate more than one proxy of reproductive success and several MHC classes to capture more complex associations.

Necrophagous Diptera are the most important group of insects used for the purposes of forensic entomology. While the most utilized fly family in this context is the family Calliphoridae, there are several other families that can be of great importance during real-case investigations. This article analyzes the necrophagous flies of all families recorded from 160 real cases in Switzerland between 1993 and 2007. A total of 56 species belonging to 16 families was identified with Calliphoridae being the most dominant family (90.63% of all cases), followed by Muscidae (26.25%), Sarcophagidae (19.38%), Phoridae (14.38%), and Fanniidae (12.50%). For specimens that were difficult to identify morphologically, a new PCR primer has been specifically designed for the amplification of a short, informative COI barcode in degraded museum samples of forensically important Diptera taxa. The richest family in terms of species was the family Muscidae with 16 species. Fannia fuscula (Fallen) and Fannia monilis (Haliday) were recorded from human cadavers for the first time. The study highlights the importance of different fly families in forensic investigation, enhancing our comprehension of their prevalence and dispersion in real cases in Central Europe. The results pave the way for additional exploration, especially regarding the involvement of less frequently observed species in forensic entomology.

Understanding the relationship between a species feeding strategy and its environment (trophic ecology) is critical to assess environmental requirements and improve management policies. However, measuring trophic interactions remains challenging. Among the available methods, quantifying the plant composition of a species' diet indicates how species use their environment and their associated niche overlap. Nevertheless, most studies focusing on herbivore trophic ecology ignore the influence that landscape variability may have. Here, we explored how landscape variability influences diet composition through niche overlap. We used eDNA metabarcoding to quantify the diet composition of two large herbivores of the Bialowieza Forest, red deer (Cervus elaphus) and European bison (Bison bonasus) to investigate how increasing habitat quality (i.e. higher abundance of deciduous forage species) and predation risk (i.e. density of wolf in the area) influence their diet composition and niche partitioning. Our findings indicate diet composition is non-homogeneous across the landscape, both within and between species. Red deer showed greater diet variability and lower niche overlap within species compared to bison. We detected a reduction of niche overlap for red deer with increasing predation risk, leading to more dissimilar diets, suggesting their feeding behaviour is affected by wolf presence. This correlation was not found for bison, which are rarely predated by wolf. Higher habitat quality was associated with higher niche overlap only within bison, probably due to their suboptimal feeding strategy as browsers. These results show the importance of integrating environment-induced diet variation in studies aimed at determining the landscape usage or niche overlap of a species.

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.

Understanding the status and abundance of species is essential for effective conservation decision-making. However, the availability of species data varies across space, taxonomic groups and data types. A case study was therefore conducted in a high biodiversity region—East Africa—to evaluate data biases, the factors influencing data availability, and the consequences for conservation. In each of the eleven target countries, priority animal species were identified as threatened species that are protected by national governments, international conventions or conservation NGOs. We assessed data gaps and biases in the IUCN Red List of Threatened Species, the Global Biodiversity Information Facility and the Living Planet Index. A survey of practitioners and decision makers was conducted to confirm and assess consequences of these biases on biodiversity conservation efforts. Our results showed data on species occurrence and population trends were available for a significantly higher proportion of vertebrates than invertebrates. We observed a geographical bias, with higher tourism income countries having more priority species and more species with data than lower tourism income countries. Conservationists surveyed felt that, of the 40 types of data investigated, those data that are most important to conservation projects are the most difficult to access. The main challenges to data accessibility are excessive expense, technological challenges, and a lack of resources to process and analyse data. With this information, practitioners and decision makers can prioritise how and where to fill gaps to improve data availability and use, and ensure biodiversity monitoring is improved and conservation impacts enhanced.

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.

Many stakeholders, from governments to civil society to businesses, lack the data they need to make informed decisions on biodiversity, jeopardising efforts to conserve, restore and sustainably manage nature. Here we review the importance of enhancing biodiversity monitoring, assess the challenges involved and identify potential solutions. Capacity for biodiversity monitoring needs to be enhanced urgently, especially in poorer, high-biodiversity countries where data gaps are disproportionately high. Modern tools and technologies, including remote sensing, bioacoustics and environmental DNA, should be used at larger scales to fill taxonomic and geographic data gaps, especially in the tropics, in marine and freshwater biomes, and for plants, fungi and invertebrates. Stakeholders need to follow best monitoring practices, adopting appropriate indicators and using counterfactual approaches to measure and attribute outcomes and impacts. Data should be made openly and freely available. Companies need to invest in collecting the data required to enhance sustainability in their operations and supply chains. With governments soon to commit to the post-2020 global biodiversity framework, the time is right to make a concerted push on monitoring. However, action at scale is needed now if we are to enhance results-based management adequately to conserve the biodiversity and ecosystem services we all depend on.

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.