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DNA metabarcoding offers a powerful, non‐invasive tool to identify dietary composition with high taxonomic resolution, yet its quantitative accuracy and bias remain a well‐recognised limitation across taxa and sample types. This universal challenge is particularly evident in herbivores, where plant material introduces additional amplification constraints. This study evaluates the accuracy of DNA metabarcoding in reconstructing the diets of sheep under controlled feeding trials involving high and low digestibility forage, using two widely used plant DNA barcodes (ITS2 and trnL). A secondary trial tested the detectability and proportional representation of a target species, Medicago sativa, when added to the diet in varying amounts (1%, 5%, 10%). ITS2 provided greater species‐level resolution, while trnL showed broader taxonomic coverage but reduced precision. Both markers distinguished diet treatments effectively; however, faecal DNA showed proportional discrepancies from vegetation input, particularly under low‐digestibility conditions. M. sativa was reliably detected even at 1% inclusion but was consistently overrepresented in sequence reads. Our findings highlight the strengths and limitations of DNA metabarcoding for herbivore diet studies and underscore the importance of marker choice and the effects of differential digestion biases. These findings demonstrate the need for multi‐marker approaches and calibration controls in dietary studies, especially when quantitative interpretation is required. Despite limitations in quantitative accuracy, faecal DNA metabarcoding provides valuable insights into herbivore diet composition and preferences, with future refinements expected to improve its resolution and reliability for ecological monitoring and grazing management. This study assessed the accuracy of DNA metabarcoding using ITS2 and trnL markers to reconstruct sheep diets under controlled feeding trials with high‐ and low‐digestibility forage, and in a secondary trial with low‐level inclusions of Medicago sativa. ITS2 offered higher species‐level resolution, while trnL provided broader taxonomic coverage but less precision when distinguishing diet treatments; moreover, faecal DNA composition diverged from vegetation inputs under low‐digestibility conditions. M. sativa was detected at 1% inclusion but overrepresented in sequence reads, highlighting the need for multi‐marker approaches, curated reference libraries, and calibration controls when interpreting herbivore dietary data quantitatively.

DNA metabarcoding provides a scalable alternative to traditional botanical surveys, which are often time‐consuming and reliant on taxonomic expertise. Here, we compare DNA metabarcoding with quadrat‐based botanical surveys to assess plant species composition in experimental grassland plots under four defoliation regimes (continuous grazing, rotational grazing, frequent cutting and conservation cutting). Botanical surveys identified 16 taxa, while metabarcoding detected 25 taxa, including the dominant species Holcus lanatus and Lolium perenne. Despite detecting more taxa, there were some discrepancies in identification, with the sequence data only able to resolve some taxa at the genus level (e.g., Agrostis spp. instead of Agrostis capillaris) and potential species misidentifications (e.g., Cardaminopsis helleri vs. Cardamine flexuosa). However, both methods provided comparable results and revealed statistically significant differences in species composition between treatments, with higher diversity in cut versus grazed plots. The semi‐quantitative nature of metabarcoding limits its capacity to accurately reflect species abundance, posing challenges for ecological interpretations where precise quantification is required. However, it provides a broader view of biodiversity and can complement traditional methods, offering new opportunities for efficient biodiversity monitoring. The findings support the integration of DNA metabarcoding into biodiversity assessments, particularly when used alongside traditional techniques. Further refinement of bioinformatics tools and reference databases will enhance their accuracy and reliability, enabling more effective monitoring of grassland biodiversity and sustainable management practices. This study highlights DNA metabarcoding as a valuable tool for understanding plant community responses to management interventions. DNA metabarcoding offers a scalable alternative to traditional botanical surveys for monitoring plant community composition under different management regimes in experimental grassland plots. Comparing quadrat surveys and DNA metabarcoding, we found both methods identified key species and showed higher species diversity in cut versus grazed plots. This study supports the integration of DNA metabarcoding as a complementary approach, enhancing the efficiency and scope of biodiversity monitoring.

Reintroductions are a powerful tool for the recovery of endangered species. However, their long‐term success is strongly influenced by the genetic diversity of the reintroduced population. The chances of population persistence can be improved by enhancing the population's adaptive ability through the mixing of individuals from different sources. However, where source populations are too diverse the reintroduced population could also suffer from outbreeding depression or unsuccessful admixture due to behavioural or genetic barriers. For the reintroduction of Asiatic wild ass Equus hemionus ssp. in Israel, a breeding core was created from individuals of two different subspecies (E. h. onager & E. h. kulan). Today the population comprises approximately 300 individuals and displays no signs of outbreeding depression. The aim of this study was a population genomic evaluation of this conservation reintroduction protocol. We used maximum likelihood methods and genetic clustering analyses to investigate subspecies admixture and test for spatial autocorrelation based on subspecies ancestry. Further, we analysed heterozygosity and effective population sizes in the breeding core prior to release and the current wild population. We discovered high levels of subspecies admixture in the breeding core and wild population, consistent with a significant heterozygote excess in the breeding core. Furthermore, we found no signs of spatial autocorrelation associated with subspecies ancestry in the wild population. Inbreeding and variance effective population size estimates were low. Our results indicate no genetic or behavioural barriers to admixture between the subspecies and suggest that their hybridization has led to greater genetic diversity in the reintroduced population. The study provides rare empirical evidence of the successful application of subspecies hybridization in a reintroduction. It supports use of intraspecific hybridization as a tool to increase genetic diversity in conservation translocations.

Background and Aims Environmental DNA (eDNA) metabarcoding provides a highly sensitive method of surveying freshwater fish communities, although studies to date have largely been restricted to temperate ecosystems. Due to limited reference sequence availability and challenges identifying closely related and rare species in diverse tropical ecosystems, the effectiveness of metabarcoding methods for surveying tropical fish communities from eDNA samples remains uncertain. To address this, we applied an eDNA metabarcoding approach to survey Lake Tanganyika's (LT) species‐rich littoral fish communities. Materials and Methods As this system contains many closely related species, particularly cichlid fishes, we used four primer sets including a cichlid‐specific primer set (Cichlid_CR). A reference database was built for the 12s, 16s, and control region for 358 fish species including over 93% of known cichlids. Results and Discussion In silico and in situ results demonstrated wide variability in the taxonomic resolution of assignments by each primer with the cichlid‐specific marker (Cichlid_CR) enabling greater species‐level assignments for this highly diverse family. A greater number of non‐cichlid teleost species were detected at sites compared to the visual survey data. For cichlid species however, sequencing depth substantially influenced species richness estimates obtained from eDNA samples, with increased depths producing estimates comparable to that obtained from the visual survey data. Conclusions Our study highlights the importance of sequencing depth and local reference databases when undertaking metabarcoding studies within diverse ecosystems, as well as demonstrating the potential of eDNA metabarcoding for surveying diverse tropical fish communities, even those containing closely related species within evolutionary radiations. This study applies an environmental DNA metabarcoding methodology for surveying Lake Tanganyika’s highly diverse littoral fish communities. The results highlight the importance of sequencing depth and local reference database completeness when undertaking metabarcoding studies within diverse ecosystems, as well as demonstrating the potential of eDNA metabarcoding for surveying diverse tropical fish communities, even those containing closely related species within evolutionary radiations.

Efficient and scalable methods for monitoring marine biodiversity are critical for understanding ecological change in coastal environments, given the limited resources available. Environmental DNA (eDNA) metabarcoding shows promise for monitoring coastal taxa, but its ability to differentiate communities from different locations remains insufficiently understood, particularly in dynamic marine environments. Here, we evaluate the effectiveness and resolution capacity of eDNA metabarcoding in detecting rocky intertidal taxa across three spatial scales—national, regional, and local—in the United Kingdom. Onshore surface‐water samples were collected from 32 sites across five UK Regional Seas from rockpools in high and low shore zones, as well as directly from the sea. We detected 1026 target taxa within 442 families and 19 phyla using two established markers targeting invertebrates (COI) and macroalgae (18S). Distinct eDNA signals were found at all spatial scales, indicating local discreteness even between vertical shore heights within the same sites. Communities were more discrete at larger scales (i.e., between regions) than at smaller scales (i.e., between shore heights). eDNA signals were more strongly structured by geographical location than by vertical shore height as a probable consequence of greater DNA homogenization over the tidal cycle at smaller spatial scales. Established ecological zonation patterns were reflected in eDNA signals, with higher richness at lower shore heights, reflecting abiotic stress gradients. Detections of cold‐affinity boreal species increased with latitude, while warm‐affinity lusitanian species declined with latitude. Our work supports the utility of eDNA metabarcoding for multiscale biodiversity monitoring in dynamic marine environments and for detections beyond this study's target taxa. We recommend the adoption of scale‐appropriate sampling protocols to optimize the benefits of eDNA, such as prioritizing open water sampling at high tide for broad‐scale assessments and rockpool sampling at low tide for capturing local‐scale patterns. Future work should validate detections through direct visual comparisons. We assess the resolution of eDNA metabarcoding for detecting rocky intertidal taxa across three spatial scales—national, regional, and local. Distinct eDNA signals were observed at each scale, with community differences more pronounced at broader scales. Detections aligned with known ecological patterns and we offer scale‐appropriate sampling recommendations to optimize the utility of eDNA.

When and where animals breed can shape the genetic structure and diversity of animal populations. The importance of drivers of genetic diversity is amplified in island populations that tend to have more delineated gene pools compared to continental populations. Studies of relatedness as a function of the spatial distribution of individuals have demonstrated the importance of spatial organisation for individual fitness with outcomes that are conditional on the overall genetic diversity of the population. However, few studies have investigated the impact of breeding timing on genetic structure. We characterise the fine-scale genetic structure of a geographically-isolated population of seabirds. Microsatellite markers provide evidence for largely transient within-breeding season temporal processes and limited spatial processes, affecting genetic structure in an otherwise panmictic population of sooty terns Onychoprion fuscatus . Earliest breeders had significantly different genetic structure from the latest breeders. Limited evidence was found for localised spatial structure, with a small number of individuals being more related to their nearest neighbours than the rest of the population. Therefore, population genetic structure is shaped by heterogeneities in collective movement in time and to a lesser extent space, that result in low levels of spatio-temporal genetic structure and the maintenance of genetic diversity.

Background Understanding the structure and variability of adaptive loci such as the major histocompatibility complex (MHC) genes is a primary research goal for evolutionary and conservation genetics. Typically, classical MHC genes show high polymorphism and are under strong balancing selection, as their products trigger the adaptive immune response in vertebrates. Here, we assess the allelic diversity and patterns of selection for MHC class I and class II loci in a threatened shorebird with highly flexible mating and parental care behaviour, the Snowy Plover (Charadrius nivosus) across its broad geographic range. Results We determined the allelic and nucleotide diversity for MHC class I and class II genes using samples of 250 individuals from eight breeding population of Snowy Plovers. We found 40 alleles at MHC class I and six alleles at MHC class II, with individuals carrying two to seven different alleles (mean 3.70) at MHC class I and up to two alleles (mean 1.45) at MHC class II. Diversity was higher in the peptide-binding region, which suggests balancing selection. The MHC class I locus showed stronger signatures of both positive and negative selection than the MHC class II locus. Most alleles were present in more than one population. If present, private alleles generally occurred at very low frequencies in each population, except for the private alleles of MHC class I in one island population (Puerto Rico, lineage tenuirostris). Conclusion Snowy Plovers exhibited an intermediate level of diversity at the MHC, similar to that reported in other Charadriiformes. The differences found in the patterns of selection between the class I and II loci are consistent with the hypothesis that different mechanisms shape the sequence evolution of MHC class I and class II genes. The rarity of private alleles across populations is consistent with high natal and breeding dispersal and the low genetic structure previously observed at neutral genetic markers in this species.

A species' demographic history gives important context to contemporary population genetics and a possible insight into past responses to climate change; with an individual's genome providing a window into the evolutionary history of contemporary populations. Pairwise sequentially Markovian coalescent (PSMC) analysis uses information from a single genome to derive fluctuations in effective population size change over the last ~5 million years. Here, we apply PSMC analysis to two European nightjar (Caprimulgus europaeus) genomes, sampled in Northwest and Southern Europe, with the aim of revealing the demographic history of nightjar in Europe. We successfully reconstructed effective population size over the last 5 million years. Our analysis shows that in response to global climate change, the effective population size of nightjar broadly increased under stable warm periods and decreased during cooler spans and prolonged glacial periods. PSMC analysis on the pseudo‐diploid combination of the two genomes revealed fluctuations in gene flow between ancestral populations over time, with gene flow ceasing by the last‐glacial period. Our results are tentatively suggestive of divergence in the European nightjar population, with timings consistent with differentiation being driven by restriction to different refugia during periods of glaciation. Finally, our results suggest that migratory behaviour in nightjar likely evolved prior to the last‐glacial period, with long‐distance migration seemingly persisting throughout the Pleistocene. However, further genetic structure analysis of individuals from known breeding sites across the species' contemporary range is needed to understand the extent and origins of range‐wide differentiation in nightjar. We used pairwise sequentially Markovian coalescent analysis to reveal the ancient demographic history of two contemporary European nightjar (Caprimulgus europaeus) populations. We show that nightjar effective population size has fluctuated over the last 5 million years in relation to changes in global climate. We further show that restriction to glacial refugia likely led to divergence within the European population, with long‐distance migration seemingly persisting throughout the Pleistocene.

DNA metabarcoding is a rapidly growing technique for obtaining detailed dietary information. Current metabarcoding methods for herbivory, using a single locus, can lack taxonomic resolution for some applications. We present novel primers for the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) designed for dietary studies in Mauritius and the UK, which have the potential to give unrivalled taxonomic coverage and resolution from a short-amplicon barcode. In silico testing used three databases of plant ITS2 sequences from UK and Mauritian floras (native and introduced) totalling 6561 sequences from 1790 species across 174 families. Our primers were well-matched in silico to 88% of species, providing taxonomic resolution of 86.1%, 99.4% and 99.9% at the species, genus and family levels, respectively. In vitro , the primers amplified 99% of Mauritian (n = 169) and 100% of UK (n = 33) species, and co-amplified multiple plant species from degraded faecal DNA from reptiles and birds in two case studies. For the ITS2 region, we advocate taxonomic assignment based on best sequence match instead of a clustering approach. With short amplicons of 187–387 bp, these primers are suitable for metabarcoding plant DNA from faecal samples, across a broad geographic range, whilst delivering unparalleled taxonomic resolution.

Phenotypic plasticity is essential for the persistence of organisms under changing environmental conditions but the control of the relevant cellular mechanisms including which genes are involved and the regulation of those genes remains unclear. One way to address this issue is to evaluate links between gene expression, methylation and phenotype using transplantation and common garden experiments within genetically homogeneous populations. This approach was taken using the Antarctic limpet Nacella concinna. In this species, two distinct phenotypes are associated with the intertidal and subtidal zones. The in situ gene expression and methylation profiles of intertidal and subtidal cohorts were directly compared before and after reciprocal transplantation as well as after a common garden acclimation to aquarium conditions for 9 months. Expression profiles showed significant modulation of cellular metabolism to habitat zone with the intertidal profile characterised by transcription modules for antioxidant production, DNA repair and the cytoskeleton reflecting the need to cope with continually fluctuating and stressful conditions including wave action, UV irradiation and desiccation. Transplantation had an effect on gene expression. The subtidal animals transplanted to the intertidal zone modified their gene expression patterns towards that of an intertidal profile. In contrast, many of the antioxidant genes were still differentially expressed in the intertidal animals several weeks after transplantation into the relatively benign subtidal zone. Furthermore, a core of genes involved in antioxidation was still preferentially expressed in intertidal animals at the end of the common garden experiment. Thus, acclimation in an aquarium tank for 9 months did not completely erase the intertidal gene expression profile. Significant methylation differences were measured between intertidal and subtidal animals from the wild and after transplantation, which were reduced on common garden acclimation. This suggests that epigenetic factors play an important role in physiological flexibility associated with environmental niche. A plain language summary is available for this article. Plain Language Summary