Robust monitoring of wildlife populations to guide interventions is fundamental to conservation and wildlife management. Understanding how landscape characteristics are influencing predator population dynamics is often vital to inform recovery strategies, management, and policy. The pine marten Martes martes is recovering in the UK; however, population spread has occurred at different rates across the country. We investigated how spatial variation in density of recovering pine marten populations is influenced by key habitat and human-related factors. We genotyped non-invasively collected pine marten hair samples collected through standardised surveys covering four landscapes in Scotland over three seasons. We applied a multi-session, sex-structured, spatial capture-recapture model to estimate within- and between-landscapes spatial variation in density. We identify larger areas being used by individuals in recently recolonised areas compared to more established populations. We find significant differences in the spatial variation of density across landscapes ranging from 0.05 marten km- 2 (95% CI: 0.03-0.07) in the recently colonised Borders region of southern Scotland to 0.21 marten km- 2 (95% CI: 0.10-032) in the Trossachs region of central Scotland. There was substantial variation in pine marten density within landscapes. The amount of forest had a positive effect on population density, but not in the most recently recolonised population. Closer proximity to large gamebird shoots had a significant negative effect on pine marten density in one landscape suggesting that, despite being protected in law, pine martens suffer sufficiently elevated mortality close to some pheasant pens to locally depress their density. We did not find an effect of distance to nearest major road on spatial variation in population density in any landscape. While the mechanistic processes driving these patterns are not clear, our work highlights the importance of elucidating the processes underlying these effects of forest and land management.

Agricultural intensification is one of the major forces driving populations of many traditionally common native species into smaller, fragmented populations which are prone to isolation and loss of genetic diversity. Identifying the spatial extent and characteristics of rural systems that support gene flow and promote genetic diversity for these species is thus essential for their long-term conservation. Here we used asymmetric autosomal genetic structure between sexes to investigate current gene flow among four neighbouring suburban populations of hedgehogs (Erinaceus europaeus) in England, which are separated by agricultural land. Contrary to expectations, we found that individuals belonged to a single genetic population despite the populations being separated by unoccupied agricultural land. Spatial autocorrelation was significant in adult female hedgehogs, but non-significant in adult males, revealing male driven contemporary gene flow between local populations. The results suggest that male hedgehogs are capable of moving between population patches separated by at least 3 km across the agricultural matrix. This finding is crucial to aid the development of a conservation strategy for hedgehogs as, for the first time, it shows the extent that previously assumed isolated populations across a perceived inhospitable landscape are connected by current gene flow. Higher within patch relatedness, and lower allelic richness were found from smaller suburban patches, largely reflecting local population size, indicating an early stage of genetic diversity loss due to habitat loss and associated fragmentation. Our study illustrates that considering current gene flow and local genetic diversity together is important to better understand habitat effects on genetic variation and to inform future conservation management.

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.

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.

Small populations of endangered species risk losing already eroded genetic diversity, important for adaptive potential, through the effects of genetic drift. The magnitude of drift can be mitigated by maximising the effective population size, as is the goal of genetic management strategies. Different mating systems, specifically those leading to reproductive skew, exacerbate genetic drift by distorting contributions. In the absence of an active management strategy, reproductive skew will have long-term effects on the genetic composition of a population, particularly where admixture is present. Here we examine the contrasting effects of conservation management strategies in two ex situ populations of the Critically Endangered eastern black rhino (Diceros bicornis michaeli), one managed as a semi-wild population in South Africa (SAx), and one managed under a mean-kinship breeding strategy in European zoos. We use molecular data to reconstruct pedigrees for both populations and validate the method using the zoo studbook. Using the reconstructed pedigree and studbook we show there is male sex-specific skew in both populations. However, the zoo’s mean-kinship breeding strategy effectively reduces reproductive skew in comparison to a semi-wild population with little genetic management. We also show that strong male reproductive skew in SAx has resulted in extensive admixture, which may require a re-evaluation of the population’s original intended role in the black rhino meta-population. With a high potential for admixture in many ex situ populations of endangered species, molecular and pedigree data remain vital tools for populations needing to balance drift and selection.

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.

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

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.

Understanding the frequency, spatiotemporal dynamics and impacts of parasite coinfections is fundamental to developing control measures and predicting disease impacts. The European turtle dove (Streptopelia turtur) is one of Europe’s most threatened bird species. High prevalence of infection by the protozoan parasite Trichomonas gallinae has previously been identified, but the role of this and other coinfecting parasites in turtle dove declines remains unclear. Using a high-throughput sequencing approach, we identified seven strains of T. gallinae, including two novel strains, from ITS1/5.8S/ITS2 ribosomal sequences in turtle doves on breeding and wintering grounds, with further intra-strain variation and four novel sub-types revealed by the iron-hydrogenase gene. High spatiotemporal turnover was observed in T. gallinae strain composition, and infection was prevalent in all populations (89–100%). Coinfection by multiple Trichomonas strains was rarer than expected (1% observed compared to 38.6% expected), suggesting either within-host competition, or high mortality of coinfected individuals. In contrast, coinfection by multiple haemosporidians was common (43%), as was coinfection by haemosporidians and T. gallinae (90%), with positive associations between strains of T. gallinae and Leucocytozoon suggesting a mechanism such as parasite-induced immune modulation. We found no evidence for negative associations between coinfections and host body condition. We suggest that longitudinal studies involving the recapture and investigation of infection status of individuals over their lifespan are crucial to understand the epidemiology of coinfections in natural populations.

In the European Union, all bat species are strictly protected and member states must ensure their conservation. However, if populations are genetically structured, conservation units that correspond to whole countries may be too large, putting small populations with specific conservation requirements at risk. Geoffroy’s bat ( Myotis emarginatus ) has undergone well-documented declines at its north-western European range edge between the 1960 and 1990s and is considered to be negatively affected by habitat fragmentation. Here we analysed the species’ genetic population structure and diversity to identify subpopulations with reduced genetic diversity and to scientifically inform conservation management. We generated 811 microsatellite-based genetic profiles obtained from 42 European nursery colonies and analysed a total of 932 sequences of the hypervariable region II of the mitochondrial control region sampled from across Europe. While two geographically widespread genetic populations were inferred to be present in north-western Europe, both nuclear and mitochondrial genetic diversity were lowest in the areas that had experienced a decline during the last century. A microsatellite-based analysis of demographic history did not permit, however, to unequivocally link that reduced genetic diversity to the population contraction event. Given the large geographic extent of the genetic populations, preserving the connectivity of mating sites requires concerted conservation efforts across multiple political jurisdictions. Genetic monitoring ought to be done on a regular basis to ensure that large-scale connectivity is maintained and further loss of genetic diversity is prevented.

Background Solitary bees, such as the red mason bee (Osmia bicornis), provide important ecosystem services including pollination. In the face of global declines of pollinator abundance, such haplodiploid Hymenopterans have a compounded extinction risk due to the potential for limited genetic diversity. In order to assess the genetic diversity of Osmia bicornis populations, we developed microsatellite markers and characterised them in two populations. Methods and results Microsatellite sequences were mined from the recently published Osmia bicornis genome, which was assembled from DNA extracted from a single male bee originating from the United Kingdom. Sequences were identified that contained dinucleotide, trinucleotide, and tetranucleotide repeat regions. Seventeen polymorphic microsatellite markers were designed and tested, sixteen of which were developed into four multiplex PCR sets to facilitate cheap, fast and efficient genotyping and were characterised in unrelated females from Germany (n = 19) and England (n = 14). Conclusions The microsatellite markers are highly informative, with a combined exclusion probability of 0.997 (first parent), which will enable studies of genetic structure and diversity to inform conservation efforts in this bee.

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.

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.

Segmentina nitida Müller 1774 is a rare European freshwater snail of drainage ditches and marshland, which has seen a marked decrease in range (~ 80%) over the last 100 years in the UK. This has been attributed to over-dredging of drainage ditches for land management, conversion of grazing marshes to arable farmland, as well as eutrophication. Segmentina nitida is identified as a priority species in the UK Biodiversity Action Plan (UKBAP) that recommends further research to inform reintroduction and translocation for its conservation. We used nuclear markers (microsatellites and ITS2) and a mitochondrial (COI) marker to investigate population structure in S. nitida individuals sampled from Poland, Germany, Sweden, and the UK to identify differences within and between populations. Data based on 2D landmark-based geometric morphometrics of S. nitida shells was used to determine if phenotypic variation followed genetic differentiation. Two distinct genetic lineages of S. nitida were identified in ITS and COI phylogenies as well as cluster analysis of microsatellite markers, one of these lineages was present in eastern Europe (Poland, Sweden- Lineage 2), and one in western Europe (UK, Germany- Lineage 1), with lineages co-occurring in German populations. No genetic admixture was observed in German populations containing both lineages. These two lineages were also distinct in shape, with lineage 2 individuals having significantly wider shells and taller and wider apertures than those in Lineage 1. ~ 85% of shells assigned to the predicted lineage in a discriminant analysis of Procrustes shape coordinates. We infer that S. nitida includes at least one sympatric cryptic species. We discuss the implications of these findings on the conservation status of S. nitida in the UK and Europe.

The long-term success of species reintroductions is strongly dependent on the availability of large areas of suitable habitat and the genetic make-up of the population. If available habitat is poorly connected this can hinder gene flow and lead to genetic fragmentation of the population, potentially increasing its extinction risk. We employed a conservation genomics approach in which we combined analyses of genetic structure with testing for potential landscape effects on habitat selection and gene flow in reintroduced Asiatic wild ass Equus hemionus ssp. in the Israeli Negev desert. Genetic structure and pairwise relatedness were first investigated followed by examination of landscape effects on individual habitat selection using records of GPS collared individuals. We then built habitat resistance surfaces and used electrical circuit theory to test for landscape effects on genetic relatedness. We detected weak genetic structuring, yet low spatial coherence among individuals from the same genetic cluster. Landscape variables had a significant impact on individual habitat selection, with wild ass avoiding steep slopes and habitats of low suitability as predicted by a species distribution model. However, the landscape genetic analysis revealed no effect of habitat resistance on genetic relatedness. These results suggest that gene flow in the reintroduced population is not impacted by landscape resistance. Indeed, the high mobility of the species may increase its resistance to the genetic effects of habitat fragmentation, at least over a small number of generations. We discuss other potential causes for the observed genetic structure including a behavioural effect. Our study highlights the importance of understanding species-habitat interactions for the long-term success of reintroductions.

AbstractBackground and AimsEnvironmental 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 MethodsAs 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 DiscussionIn 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. ConclusionsOur 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.

Invasive species can have wide-ranging negative impacts, and an understanding of the process and success of invasions can be vital to determine management strategies, mitigate impacts and predict range expansions of such species. Monk parakeets (Myiopsitta monachus) and ring-necked parakeets (Psittacula krameri) are both widespread invasive species, but there has been little research into the genetic and social structure of these two species despite the potential links with invasion success. The aim of this study was to isolate novel microsatellite loci from the monk parakeet and characterise them in both monk and ring-necked parakeets in order to facilitate future investigations into their behaviour and population ecology. Sex-typing markers were also tested in both species. Of the 20 microsatellite loci assessed in 24 unrelated monk parakeets, 16 successfully amplified and were polymorphic displaying between 2 and 14 alleles (mean=8.06). Expected heterozygosity ranged from 0.43 to 0.93 and observed heterozygosity ranged from 0.23 to 0.96. Nine of the 20 loci also successfully amplified and were polymorphic in the ring-necked parakeet, displaying between 2 and 10 alleles. Suitable markers to sex both species and a Z-linked microsatellite locus were identified. A multiplex marker set was validated for monk parakeets. These novel microsatellite loci will facilitate fine and broad-scale population genetic analyses of these two widespread invasive species.

Animal parasitic nematodes can cause serious diseases and their emergence in new areas can be an issue of major concern for biodiversity conservation and human health. Their ability to adapt to new environments and hosts is likely to be affected by their degree of genetic diversity, with gene flow between distinct populations counteracting genetic drift and increasing effective population size. The raccoon roundworm (Baylisascaris procyonis), a gastrointestinal parasite of the raccoon (Procyon lotor), has increased its global geographic range after being translocated with its host. The raccoon has been introduced multiple times to Germany, but not all its populations are infected with the parasite. While fewer introduced individuals may have led to reduced diversity in the parasite, admixture between different founder populations may have counteracted genetic drift and bottlenecks. Here, we analyse the population genetic structure of the roundworm and its raccoon host at the intersection of distinct raccoon populations infected with B. procyonis. We found evidence for two parasite clusters resulting from independent introductions. Both clusters exhibited an extremely low genetic diversity, suggesting small founding populations subjected to inbreeding and genetic drift with no, or very limited, genetic influx from population admixture. Comparison of the population genetic structures of both host and parasite suggested that the parasite spread to an uninfected raccoon founder population. On the other hand, an almost perfect match between cluster boundaries also suggested that the population genetic structure of B. procyonis has remained stable since its introduction, mirroring that of its raccoon host.

Island species provide excellent models for investigating how selection and drift operate in wild populations, and for determining how these processes act to influence local adaptation and speciation. Here, we examine the role of selection and drift in shaping genomic and phenotypic variation across recently separated populations of Berthelot's pipit (Anthus berthelotii), a passerine bird endemic to three archipelagos in the Atlantic. We first characterized genetic diversity and population structuring that supported previous inferences of a history of recent colonizations and bottlenecks. We then tested for regions of the genome associated with the ecologically important traits of bill length and malaria infection, both of which vary substantially across populations in this species. We identified a SNP associated with variation in bill length among individuals, islands, and archipelagos; patterns of variation at this SNP suggest that both phenotypic and genotypic variation in bill length is largely shaped by founder effects. Malaria was associated with SNPs near/within genes involved in the immune response, but this relationship was not consistent among archipelagos, supporting the view that disease resistance is complex and rapidly evolving. Although we found little evidence for divergent selection at candidate loci for bill length and malaria resistance, genome scan analyses pointed to several genes related to immunity and metabolism as having important roles in divergence and adaptation. Our findings highlight the utility and challenges involved with combining association mapping and population genetic analysis in nonequilibrium populations, to disentangle the effects of drift and selection on shaping genotypes and phenotypes.