Abstract Understanding evolutionary processes is greatly facilitated by high-quality data on genetic variation. We report the development of a genomic toolkit for a recently bottlenecked, long-term studied species, the Seychelles warbler (Ptimerl dezil; Acrocephalus sechellensis ). This toolkit comprises a reference genome assembled into 31 chromosomes, together with functional annotations and reference-panel-free imputation of whole-genome sequences from 1,935 individuals. The genomic data have been used to assign the sequenced individuals into a genetic pedigree. Individual genomic data are associated with a suite of phenotypic metadata, amassed from three decades of fieldwork in this closed, long-term monitored population. We compared sex and parentage assigned using the genomic data with the previously recorded sex and parentage metadata to identify and correct 41 sample DNA samples labelled with the wrong identity. This population resource enables a wide range of analyses, that include, but are not limited to phylogenetics, metabarcoding, recombination rates, linkage patterns, adaptation, heritability, demographic history, selection, and inbreeding estimates. We wish to encourage interest from researchers seeking to collaborate on future analyses and data collection. Overall, our methods demonstrate the potential of next generation sequencing and statistical tools to provide dense genomic datasets at large sample sizes for wild populations.

Sympatric speciation is defined as the formation of new species in the absence of geographic barriers, but the genomic and life history strategy mechanisms underpinning sympatric speciation are still far from clear. It has recently been discovered that the cichlid fish Astatotilapia calliptera from crater Lake Masoko in Tanzania have diverged sympatrically into littoral (shallow water) and benthic (deep water) ecotypes, which differ in head and pharyngeal jaw morphology. Carbon stable isotope analysis has also broadly indicated trophic differentiation between ecotypes. Here, we explore trophic niche divergence on a finer scale, using metabarcoding of stomach contents. A combination of the mitochondrial COI region and 18S V4 region from the eukaryotic nuclear small subunit ribosomal DNA was used to target macroinvertebrate and broader eukaryotic taxonomic diversity, respectively, revealing dietary divergence between the ecotypes. Large proportions of Arthropoda (dipterans and copepod) were found in both ecotypes, indicating some food sources common to both microhabitats. However, gut contents of benthic A. calliptera individuals were characterized by an abundance of annelids and diatoms, while Lepidoptera, mayflies, fungi, freshwater mussels, and bivalves were common in littoral ecotypes. The variation observed in the dietary contents of the ecotypes indicates the presence of resource partitioning, facilitating adaptation to unique feeding strategies.

In mammals, sexual size dimorphism often reflects the intensity of sexual selection, yet its connection to genomic evolution remains unexplored. Gene family size evolution can reflect shifts in the relative importance of different molecular functions. Here, we investigate the associate between brain development gene repertoire to sexual size dimorphism using 124 mammalian species. We reveal significant changes in gene family size associations with sexual size dimorphism. High levels of dimorphism correlate with an expansion of gene families enriched in olfactory sensory perception and a contraction of gene families associated with brain development functions, many of which exhibited particularly high expression in the human adult brain. These findings suggest a relationship between intense sexual selection and alterations in gene family size. These insights illustrate the complex interplay between sexual dimorphism, gene family size evolution, and their roles in mammalian brain development and function, offering a valuable understanding of mammalian genome evolution. Sexual size dimorphism in mammals, often linked to sexual selection, can impacts genome evolution. This study finds sexual dimorphism in body size is associated with expanded gene families for olfactory functions and contracted gene families for brain development.

ABSTRACTPollination is an ecosystem process that is crucial to maintain biodiversity and ecosystem function. Bats are important pollinators in the tropics and are an integral part of complex plant–pollinator interaction networks. However, network analysis–based approaches are still scarce at the plant species and bat community levels. We used metabarcoding to identify plant taxa present in pollen from fur and faecal samples collected across 1 year from three nectar‐feeding bat roosts in central Mexico. We calculated the frequency of occurrence of plant taxa and assembled a zoocentric network of bat–plant interactions. We constructed a year‐long network, encompassing the entire period of sampling, two seasonal networks comprising the wet and dry seasons, and six individual networks from sampling at two‐month intervals across the year. Four species of nectar‐feeding bats interacted with 36 plant species from 16 families. We found highly generalised interaction patterns across networks corresponding with opportunistic feeding behaviour by bats, with little seasonal variation in network structure. There was high resource overlap between bat species, and bats visited a diverse range of plant species even during periods with a high abundance of particular resources in the landscape. The diverse diet of nectar‐feeding bats emphasises the importance of floristically rich natural habitats in the landscape to provide reliable foraging resources year‐round in a seasonally variable system. While a generalised network structure is thought to increase robustness, further research is necessary to understand how fluctuations in pollinator abundance and diversity in the face of land use and climate change may impact bat–flower networks and the consequences to plant communities.

ABSTRACT 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.

Forced copulation is common, presumably because it can increase male reproductive success. Forced extra-pair copulation (FEPC) occurs in birds, even though most species lack a penis and are widely thought to require female cooperation for fertilization. How FEPC persists, despite a presumed lack of siring success and likely non-negligible costs to the male, is unknown. Using the jackdaw (Corvus monedula) as a case study, we use SNPs to quantify the extra-pair paternity rate through FEPC and evaluate explanations for the persistence of FEPC in species without a penis. We then collate evidence for FEPC across penis-lacking birds. Combining genetic and behavioural analyses, our study suggests that the most likely explanations for the maintenance of FEPC in jackdaws are that it provides a selective advantage to males or it is a relic. Our literature review shows that across birds lacking a penis, FEPC is taxonomically widespread, and yet, little is known about its evolution. A broader implementation of the approach used here, combining both genetic and behavioural data, may shed light on why this widespread sexual behaviour persists. Additional work is necessary to understand whether a penis is needed for paternity through forced copulation and to quantify the costs of FEPC.

Pollinating insects are declining due to habitat loss and climate change, and cities with limited habitat and floral resources may be particularly vulnerable. The effects of urban landscapes on pollination networks remain poorly understood, and comparative studies of taxa with divergent niches are lacking. Here, for the first time, we simultaneously compare nocturnal moth and diurnal bee pollen-transport networks using DNA metabarcoding and ask how pollination networks are affected by increasing urbanisation. Bees and moths exhibited substantial divergence in the communities of plants they interact with. Increasing urbanisation had comparable negative effects on pollen-transport networks of both taxa, with significant declines in pollen species richness. We show that moths are an important, but overlooked, component of urban pollen-transport networks for wild flowering plants, horticultural crops, and trees. Our findings highlight the need to include both bee and non-bee taxa when assessing the status of critical plant-insect interactions in urbanised landscapes.

Restoring and maintaining soil biodiversity is important for the sustainability of our food systems, particularly as the health of agricultural soils continues to degrade at alarming rates. Earthworms are key components of soil biodiversity and provide many benefits to agricultural soil functioning, but effective and standardised approaches to monitor their populations are lacking. In this study, the feasibility of using soil environmental DNA (eDNA) metabarcoding for surveying earthworm populations in agroecosystems was explored. Soil samples were collected from arable fields with temporary grass-clover ley strips sown into them, to investigate the effects of different land use treatments on earthworm communities. Extracellular earthworm eDNA was extracted, amplified and sequenced using next-generation sequencing, and earthworm populations were also sampled using a traditional hand-sorting approach. This allowed the performance of the eDNA method to be assessed and quantitative comparisons to be made between the different methods. The eDNA method detected significantly higher local species richness than the traditional hand-sorting approach, with the site occupancy proportions indicating that eDNA was markedly better at detecting anecic (deep burrowing) earthworms. The high sensitivity of eDNA sampling enabled the detection of differences in the earthworm communities resulting from different land-use treatments in the same field, with significantly higher earthworm diversity detected in the 33-month old ley strips than in the adjacent arable soils. Both relative abundances and site occupancy proportions were used as potential proxies for abundance, and the performance of each of these measures and the implications for further work are discussed. eDNA sampling has potential as a powerful tool for monitoring agricultural earthworm diversity and soil health, and could be utilised on farms to promote better management and soil care.

When backcrosses are fertile, interbreeding between endangered taxa can lead to the admixture of gene pools under threat. One such case pertains to the Mesoamerican crocodile Crocodylus moreletii , a species which shows strong signatures of both recent hybridisation and historic intogression with the American crocodile C. acutus across large parts of its range . In the present paper, we use RAD-seq derived SNPs (4980 nuclear and seven mtDNA loci) to demonstrate that C. moreletii populations inhabiting the region of Calakmul in central Yucatan (Mexico) are rather unaffected by hybridization, despite being surrounded by coastal areas where pervasive admixture has previously been documented. All (based on fastSTRUCTURE) and 96% (based on NGSadmix) of 84 genotyped individuals from 18 sampled waterbodies (locally termed aguadas) were free from nuclear introgression of C . acutus DNA at at threshold of 0.95. Seven individuals (8%) possessed a C. acutus mtDNA haplotype, five of which were derived from two adjacent, rather peripheral aguadas. Spatial inferences based on a DAPC and fineRADstructure further showed that the region of Calakmul is inhabited by three genetic clusters spanning across a set of distinct aguadas each. Taken together, our findings reveal that central Yucatan contains the currently largest documented stronghold of C. moreletii populations only marginally affected by introgression, which has major implications for the conservation management of this important flagship species.

Sequencing of SARS-CoV-2 in wastewater provides a key opportunity to monitor the prevalence of variants spatiotemporally, potentially facilitating their detection simultaneously with, or even prior to, observation through clinical testing. However, there are multiple sequencing methodologies available. This study aimed to evaluate the performance of alternative protocols for detecting SARS-CoV-2 variants. We tested the detection of two synthetic RNA SARS-CoV-2 genomes in a wide range of ratios and at two concentrations representative of those found in wastewater using whole-genome and Spike -gene-only protocols utilising Illumina and Oxford Nanopore platforms. We developed a Bayesian hierarchical model to determine the predicted frequencies of variants and the error surrounding our predictions. We found that most of the sequencing protocols detected polymorphic nucleotide frequencies at a level that would allow accurate determination of the variants present at higher concentrations. Most methodologies, including the Spike -only approach, could also predict variant frequencies with a degree of accuracy in low-concentration samples but, as expected, with higher error around the estimates. All methods were additionally confirmed to detect the same prevalent variants in a set of wastewater samples. Our results provide the first quantitative statistical comparison of a range of alternative methods that can be used successfully in the surveillance of SARS-CoV-2 variant frequencies from wastewater.Impact Genetic sequencing of SARS-CoV-2 in wastewater provides an ideal system for monitoring variant frequencies in the general population. The advantages over clinical data are that it is more cost efficient and has the potential to identify new variants before clinical testing. However, to date, there has been no direct comparison to determine which sequencing methodologies perform best at identifying the presence and prevalence of variants. Our study compares seven sequencing methods to determine which performs best. We also develop a Bayesian statistical methodology to estimate the confidence around variant frequency estimates. Our results will help monitor SARS-CoV-2 variants in wastewater, and the methodology could be adapted for other disease monitoring, including future pandemics.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementNatural Environment Research Council (NERC) (N-WESP, NE/V010441/1 grant to TB) ### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Males and females often exhibit differences in behaviour, life histories, and ecology, many of which are typically reflected in their brains. Neuronal protection and maintenance include complex processes led by the microglia, which also interacts with metabolites such as hormones or immune components. Despite increasing interest in sex-specific brain function in laboratory animals, the significance of sex-specific immune activation in the brain of wild animals along with the variables that could affect it is widely lacking. Here, we use the Kentish plover (Charadrius alexandrinus) to study sex differences in expression of immune genes in the brain of adult males and females, in two wild populations breeding in contrasting habitats: a coastal sea-level population and a high-altitude inland population in China. Our analysis yielded 379 genes associated with immune function. We show a significant male-biased immune gene upregulation. Immune gene expression in the brain did not differ in upregulation between the coastal and inland populations. We discuss the role of dosage compensation in our findings and their evolutionary significance mediated by sex-specific survival and neuronal deterioration. Similar expression profiles in the coastal and inland populations suggest comparable genetic control by the microglia and possible similarities in pathogen pressures between habitats. We call for further studies on gene expression of males and females in wild population to understand the implications of immune function for life-histories and demography in natural systems.

The gut microbiome (GM) can influence many biological processes in the host, impacting its health and survival, but the GM can also be influenced by the host’s traits. In vertebrates, Major Histocompatibility Complex (MHC) genes play a pivotal role in combatting pathogens and are thought to shape the host’s GM. Despite this—and the documented importance of both GM and MHC variation to individual fitness—few studies have investigated the association between the GM and MHC in the wild. We characterised MHC class I (MHC-I), MHC class II (MHC-II) and GM variation in individuals within a natural population of the Seychelles warbler (Acrocephalus sechellensis). We determined how the diversity and composition of the GM varied with MHC characteristics, in addition to environmental factors and other host traits. Our results show that the presence of specific MHC alleles, but not MHC diversity, influences both the diversity and composition of the GM in this population. MHC-I alleles, rather than MHC-II alleles, had the greatest impact on the GM. GM diversity was negatively associated with the presence of three MHC-I alleles (Ase-ua3, Ase-ua4, Ase-ua5), and one MHC-II allele (Ase-dab4), while changes in GM composition were associated with the presence of four different MHC-I alleles (Ase-ua1, Ase-ua7, Ase-ua10, Ase-ua11). There were no associations between GM diversity and TLR3 genotype, but GM diversity was positively correlated with genome-wide heterozygosity and varied with host age and field period. These results suggest that components of the host’s immune system play a role in shaping the GM of wild animals. Host genotype—specifically MHC-I and to a lesser degree MHC-II variation—can modulate the GM, although whether this occurs directly, or indirectly through effects on host health, is unclear. Importantly, if immune genes can regulate host health through modulation of the microbiome, then it is plausible that the microbiome could also influence selection on immune genes. As such, host–microbiome coevolution may play a role in maintaining functional immunogenetic variation within natural vertebrate populations.

Many bird species are vulnerable to environmental change, so knowledge of their diet and its variation can help understand population status and flexibility to respond to change. Insectivorous species are predicted to have a flexible diet within and between individuals, which can respond to naturally fluctuating prey abundance, thus allowing opportunistic exploitation of available resources. We analysed the diet of a nocturnal, aerial insectivore, the European NightjarCaprimulgus europaeus, using high‐throughput metabarcoding. We quantified diet diversity and composition of 130 faecal samples from nests and roosts on a northern breeding site in the UK from 2015 to 2018, and compared differences among individuals and years. Although dominated by moths, diet varied significantly between individual faecal samples and between years and months. Prey species composition varied between years, and was more variable between samples in 2017 than in other years. Faecal samples were significantly more likely to contain moth species with a wingspan of > 60 mm and less likely to contain moth species of < 25 mm wingspan. This indicated size‐selective foraging, which also varied between months and years. Diet was driven by interindividual variation, indicating population‐level flexibility in prey choice. Metabarcoding provided a valuable tool in the exploration of insectivorous diets, but efforts are needed to build comprehensive reference libraries in order to compile full prey species lists.

SARS-CoV-2 and the resulting COVID-19 pandemic represents one of the greatest recent threats to human health, wellbeing and economic growth. Wastewater-based epidemiology (WBE) of human viruses can be a useful tool for population-scale monitoring of SARS-CoV-2 prevalence and epidemiology to help prevent further spread of the disease, particularly within urban centres. Here, we present a longitudinal analysis (March-July 2020) of SARS-CoV-2 RNA prevalence in sewage across six major urban centres in the UK (total population equivalent 3 million) by q(RT-)PCR and viral genome sequencing. Our results demonstrate that levels of SARS-CoV-2 RNA generally correlated with the abundance of clinical cases recorded within the community in large urban centres, with a marked decline in SARS-CoV-2 RNA abundance following the implementation of lockdown measures. The strength of this association was weaker in areas with lower confirmed COVID-19 case numbers. Further, sequence analysis of SARS-CoV-2 from wastewater suggested that multiple genetically distinct clusters were co-circulating in the local populations covered by our sample sites, and that the genetic variants observed in wastewater reflected similar SNPs observed in contemporaneous samples from cases tested in clinical diagnostic laboratories. We demonstrate how WBE can be used for both community-level detection and tracking of SARS-CoV-2 and other virus' prevalence, and can inform public health policy decisions. Although, greater understanding of the factors that affect SARS-CoV-2 RNA concentration in wastewater are needed for the full integration of WBE data into outbreak surveillance. In conclusion, our results lend support to the use of routine WBE for monitoring of SARS-CoV-2 and other human pathogenic viruses circulating in the population and assessment of the effectiveness of disease control measures.

This protocol describes a procedure for sequencing the Spike region of SARS-CoV-2 using short amplicons (146-208bp). The method has proved to successful with both clinical RNA samples and degraded wastewater samples. The primers are unique to this method. The library prep procedure has been heavily adapted from the ncov-2019 sequencing v3 (ARTIC) protocol by Josh Quick (https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye) and the "low cost" method from the NEOF Liverpool Illumina ARTIC protocol.

Delineating conservation units is a complex and often controversial process that is particularly challenging for highly vagile species. Here, we reassess population genetic structure and identify those populations of highest conservation value in the threatened snowy plover (Charadrius nivosus, Cassin, 1858), a partial migrant shorebird endemic to the Americas. We use four categories of genetic data-mitochondrial DNA (mtDNA), microsatellites, Z-linked and autosomal single nucleotide polymorphisms (SNPs)-to: (1) assess subspecies delineation and examine population structure (2) compare the sensitivity of the different types of genetic data to detect spatial genetic patterns, and (3) reconstruct demographic history of the populations analysed. Delineation of two traditionally recognised subspecies was broadly supported by all data. In addition, microsatellite and SNPs but not mtDNA supported the recognition of Caribbean snowy plovers (C. n. tenuirostris) and Floridian populations (eastern C. n. nivosus) as distinct genetic lineage and deme, respectively. Low migration rates estimated from autosomal SNPs (m < 0.03) reflect a general paucity of exchange between genetic lineages. In contrast, we detected strong unidirectional migration (m = 0.26) from the western into the eastern nivosus deme. Within western nivosus, we found no genetic differentiation between coastal Pacific and inland populations. The correlation between geographic and genetic distances was weak but significant for all genetic data sets. All demes showed signatures of bottlenecks occurring during the past 1000 years. We conclude that at least four snowy plover conservation units are warranted: in addition to subspecies nivosus and occidentalis, a third unit comprises the Caribbean tenuirostris lineage and a fourth unit the distinct eastern nivosus deme.

Understanding how incipient species are maintained with gene flow is a fundamental question in evolutionary biology. Whole genome sequencing of multiple individuals holds great potential to illustrate patterns of genomic differentiation as well as the associated evolutionary histories. Kentish ( Charadrius alexandrinus ) and the white-faced ( C. dealbatus ) plovers, which differ in their phenotype, ecology and behaviour, are two incipient species and parapatrically distributed in East Asia. Previous studies show evidence of genetic diversification with gene flow between the two plovers. Under this scenario, it is of great importance to explore the patterns of divergence at the genomic level and to determine whether specific regions are involved in reproductive isolation and local adaptation. Here we present the first population genomic analysis of the two incipient species based on the de novo Kentish plover reference genome and resequenced populations. We show that the two plover lineages are distinct in both nuclear and mitochondrial genomes. Using model-based coalescence analysis, we found that population sizes of Kentish plover increased whereas white-faced plovers declined during the Last Glaciation Period. Moreover, the two plovers diverged allopatrically, with gene flow occurring after secondary contact. This has resulted in low levels of genome-wide differentiation, although we found evidence of a few highly differentiated genomic regions in both the autosomes and the Z-chromosome. This study illustrates that incipient shorebird species with gene flow after secondary contact can exhibit discrete divergence at specific genomic regions and provides basis to further exploration on the genetic basis of relevant phenotypic traits.

Adult sex ratio (ASR) is a central concept in population biology and a key factor in sexual selection, but why do most demographic models ignore sex biases? Vital rates often vary between the sexes and across life history, but their relative contributions to ASR variation remain poorly understood-an essential step to evaluate sex ratio theories in the wild and inform conservation. Here, we combine structured two-sex population models with individual-based mark-recapture data from an intensively monitored polygamous population of snowy plovers. We show that a strongly male-biased ASR (0.63) is primarily driven by sex-specific survival of juveniles rather than adults or dependent offspring. This finding provides empirical support for theories of unbiased sex allocation when sex differences in survival arise after the period of parental investment. Importantly, a conventional model ignoring sex biases significantly overestimated population viability. We suggest that sex-specific population models are essential to understand the population dynamics of sexual organisms: reproduction and population growth are most sensitive to perturbations in survival of the limiting sex. Overall, our study suggests that sex-biased early survival may contribute toward mating system evolution and population persistence, with implications for both sexual selection theory and biodiversity conservation.

Extra-pair paternity is a common reproductive strategy in many bird species. However, it remains unclear why extra-pair paternity occurs and why it varies among species and populations. Plovers (Charadrius spp.) exhibit considerable variation in reproductive behaviour and ecology, making them excellent models to investigate the evolution of social and genetic mating systems. We investigated inter- and intra-specific patterns of extra-pair parentage and evaluated three major hypotheses explaining extra-pair paternity using a comparative approach based on the microsatellite genotypes of 2049 individuals from 510 plover families sampled from twelve populations that constituted eight species. Extra-pair paternity rates were very low (0 to 4.1% of chicks per population). No evidence was found in support of the sexual conflict or genetic compatibility hypotheses, and there was no seasonal pattern of extra-pair paternity (EPP). The low prevalence of EPP is consistent with a number of alternative hypotheses, including the parental investment hypothesis, which suggests that high contribution to care by males restricts female plovers from engaging in extra-pair copulations. Further studies are needed to critically test the importance of this hypothesis for mate choice in plovers.