1. Urbanization influences ecological and evolutionary processes, including gene flow and survival. Understanding how genetic and demographic rates respond to urbanization is therefore essential for wildlife viability in human-dominated landscapes. We tested whether population genetic di-versity, genetic structure, and apparent survival of Eurasian red squirrels (Sciurus vulgaris) vary in concert along an urbanization gradient. 2. We combined a multi-year capture–mark–recapture study with microsatellite-based population genetic analyses at three sites in Berlin, Germany, representing increasing levels of urbanization. 3. Genetic diversity was broadly comparable among sites, indicating no strong genetic erosion in urban populations. However, weak heterozygote deficits and subtle differences in individual het-erozygosity suggest mild restrictions to gene flow along the urban gradient. Bayesian clustering analyses identified two genetic clusters, broadly corresponding to study sites, revealing emerging fine-scale population structure despite the small spatial extent of the study area. 4. Apparent survival did not differ clearly among sites. Several predictors received similar support, highlighting substantial uncertainty in the drivers of survival in this system. 5. By integrating genetic and demographic data, our study demonstrated that fine-scale genetic structuring can emerge in urban wildlife populations without translating into detectable short-term differences in apparent survival. These findings highlight that genetic and demographic re-sponses to urbanization can be decoupled and emphasize the importance of integrated approach-es for understanding population survival in human-modified environments.

Reference genome assemblies are essential infrastructure for investigating phylogeny and population/conservation genetics of wild organisms. Birds serve as model vertebrates in ecology and evolutionary biology due to their well-documented natural histories and extensive community science data. We release a set of 350 newly assembled avian genomes, which, when combined with 97 previously published genomes, represent 447 of the bird species recorded in Denmark, the Faroe Islands, and Greenland-the largest regional dataset of a vertebrate group to date. These genomes are published for various research activities. This data release advances the global effort to build comprehensive and accessible biodiversity genomic resources for the research community.

Advances in high-throughput sequencing (HTS) have made it a powerful resource for the conservation of threatened species, providing information at both population and individual levels to inform management decisions. In South America, however, the application of HTS in conservation has been limited, primarily due to challenges in funding and access to advanced genomic equipment and analytical expertise. Darwin’s fox (Lycalopex fulvipes), endemic to Chile’s Valdivian Temperate Rainforest, is the most endangered canid in South America with a small and declining population estimated at less than 1000 mature individuals. Despite its endangered status, significant knowledge gaps remain. Here we highlight the potential of HTS to address these challenges, such as clarifying its taxonomy, demographic history, geographic distribution, population structure, genetic diversity, and pathogen exposure. Integrating molecular data into conservation planning will be pivotal in ensuring the long-term survival of Darwin’s fox by identifying priorities for targeted management interventions, highlighting areas of critical habitat for conservation, and guiding genetic rescue efforts to enhance genetic diversity and resilience.

We studied the genetic connectivity of wrinkle-lipped free-tailed bat (Mops plicatus, formerly Chaerephon plicatus) in Thailand, where this highly mobile species is known to inhabit 18 caves. Populations of M. plicatus are experiencing a drastic decline. Understanding genetic connectivity is crucial for assessing vulnerability to reduced gene flow and inbreeding. Using non-invasive genetic sampling, we analyzed mitochondrial (cytb and D-loop) and nuclear DNA (SNPs) from 501 individuals across nine cave colonies over a 500 km range. Our analysis revealed moderately high haplotype diversity in cytb, high diversity in D-loop sequences, and low nucleotide diversity in both mitochondrial markers. FST analyses of nuclear and mitochondrial DNA showed consistently low values, indicating little to no differentiation. Additionally, FIS values for nuclear DNA were likewise low, and Bayesian clustering indicated the absence of genetic structuring, suggesting a single, connected population. These findings suggest that the M. plicatus population in Thailand maintains genetic connectivity among cave populations through high gene flow, with no signs of inbreeding. Mismatch distribution analyses of mtDNA across all studied bat individuals suggest a sudden population expansion during the Early Pleistocene, whereas stairway plots of nDNA indicate a long-term stable population with two distinct growth events, one having occurred during the Middle Pleistocene and another in the Late Holocene, respectively. In conclusion, our findings recommend managing and conserving the wrinkle-lipped free-tailed bats in Thailand as a single population, with consistent conservation measures across all colonies.

In wildlife populations, parasites often go unnoticed, as infected animals appear asymptomatic. However, these infections can subtly alter behaviour. Field evidence of how these subclinical infections induce changes in movement behaviour is scarce in free-ranging animals, yet it may be crucial for zoonotic disease surveillance. We used an ultra-high-resolution tracking system (ATLAS) to monitor the movements of 60 free-ranging swallows every 8 seconds across four breeding seasons, resulting in over 1 million localizations. About 40% of these swallows were naturally infected with haemosporidian parasites. Here, we show that infected individuals had reduced foraging ranges, foraged in lower quality habitats, and faced a lowered survival probability, with an average reduction of 7.4%, albeit with some variation between species and years. This study highlights the impact of subclinical infections on movement behaviour and survival, emphasizing the importance of considering infection status in movement ecology. Our findings provide insights into individual variations in behaviour and previously unobservable local parasite transmission dynamics. Subclinical infections in wild swallows reduce foraging ranges, alter habitat use, and decrease survival rates. A study using ultra-high-resolution tracking reveals overlooked impacts of infection on movement behaviour and links them to demography.

Females mainly increase their reproductive success by improving the quality of their mates and need to be discriminative in their mate choices. Here, we investigate whether female mammals can trade up sire quality in sequential mate choice during already progressed pregnancies. A male-induced pregnancy termination (functional ‘Bruce effect’) could thus have an adaptive function in mate choice as a functional part of a pregnancy replacement. We used bank voles (Myodes glareolus) as a model system and exchanged the breeding male in the early second trimester of a potential pregnancy. Male quality was determined using urine marking values. Females were offered a sequence of either high- then low-quality male (HL) or a low- then high-quality male (LH). The majority of females bred with high-quality males independent of their position in the sequence, which may indicate a pregnancy replacement in LH but not in HL. The body size of the second male, which could have been related to the coercion of females by males into remating, did not explain late pregnancies. Thus, pregnancy replacement, often discussed as a counterstrategy to infanticide, may constitute adaptive mate choice in female mammals, and female choice may induce pregnancy replacement in mammals.

Lions are widely known as charismatic predators that once roamed across the globe, but their populations have been greatly affected by environmental factors and human activities over the last 150 yr. Of particular interest is the Addis Ababa lion population, which has been maintained in captivity at around 20 individuals for over 75 yr, while many wild African lion populations have become extinct. In order to understand the molecular features of this unique population, we conducted a whole-genome sequencing study on 15 Addis Ababa lions and detected 4.5 million distinct genomic variants compared with the reference African lion genome. Using functional annotation, we identified several genes with mutations that potentially impact various traits such as mane color, body size, reproduction, gastrointestinal functions, cardiovascular processes, and sensory perception. These findings offer valuable insights into the genetics of this threatened lion population.

In mammalian societies, dominance hierarchies translate into inequalities in health, reproductive performance and survival. DNA methylation is thought to mediate the effects of social status on gene expression and phenotypic outcomes, yet a study of social status-specific DNA methylation profiles in different age classes in a wild social mammal is missing. We tested for social status signatures in DNA methylation profiles in wild female spotted hyenas (Crocuta crocuta), cubs and adults, using non-invasively collected gut epithelium samples. In spotted hyena clans, female social status influences access to resources, foraging behavior, health, reproductive performance and survival. We identified 149 differentially methylated regions between 42 high- and low-ranking female spotted hyenas (cubs and adults). Differentially methylated genes were associated with energy conversion, immune function, glutamate receptor signalling and ion transport. Our results provide evidence that socio-environmental inequalities are reflected at the molecular level in cubs and adults in a wild social mammal.

We present a complete, chromosome-scale reference genome for the long-distance migratory bat Pipistrellus nathusii. The genome encompasses both haplotypic sets of autosomes and the separation of both sex chromosomes by utilizing highly accurate long-reads and preserving long-range phasing information through the use of three-dimensional chromatin conformation capture sequencing (Hi-C). This genome, accompanied by a comprehensive protein-coding sequence annotation, provides a valuable genomic resource for future investigations into the genomic bases of long-distance migratory flight in bats as well as uncovering the genetic architecture, population structure and evolutionary history of Pipistrellus nathusii. The reference-quality genome presented here gives a fundamental resource to further our understanding of bat genetics and evolution, adding to the growing number of high-quality genetic resources in this field. Here, we demonstrate its use in the phylogenetic reconstruction of the order Chiroptera, and in particular, we present the resources to allow detailed investigations into the genetic drivers and adaptations related to long-distance migration.

The sun bear Helarctos malayanus is one of the most endangered ursids, and to date classification of sun bear populations has been based almost exclusively on geographic distribution and morphology. The very few molecular studies focussing on this species were limited in geographic scope. Using archival and non-invasively collected sample material, we have added a substantial number of complete or near-complete mitochondrial genome sequences from sun bears of several range countries of the species' distribution. We here report 32 new mitogenome sequences representing sun bears from Cambodia, Thailand, Peninsular Malaysia, Sumatra, and Borneo. Reconstruction of phylogenetic relationships revealed two matrilines that diverged similar to 295 thousand years ago: one restricted to portions of mainland Indochina (China, Cambodia, Thailand; "Mainland clade"), and one comprising bears from Borneo, Sumatra, Peninsular Malaysia but also Thailand ("Sunda clade"). Generally recent coalescence times in the mitochondrial phylogeny suggest that recent or historical demographic processes have resulted in a loss of mtDNA variation. Additionally, analysis of our data in conjunction with shorter mtDNA sequences revealed that the Bornean sun bear, classified as a distinct subspecies (H. m. euryspilus), does not harbor a distinctive matriline. Further molecular studies of H. malayanus are needed, which should ideally include data from nuclear loci.

Free-ranging cheetahs (Acinonyx jubatus) are generally healthy, whereas cheetahs under human care, such as those in zoological gardens, suffer from ill-defined infectious and degenerative pathologies. These differences are only partially explained by husbandry management programs because both groups share low genetic diversity. However, mounting evidence suggests that physiological differences between populations in different environments can be tracked down to differences in epigenetic signatures. Here, we identified differentially methylated regions (DMRs) between free-ranging cheetahs and conspecifics in zoological gardens and prospect putative links to pathways relevant to immunity, energy balance and homeostasis. Comparing epigenomic DNA methylation profiles obtained from peripheral blood mononuclear cells (PBMCs) from eight free-ranging female cheetahs from Namibia and seven female cheetahs living in zoological gardens within Europe, we identified DMRs of which 22 were hypermethylated and 23 hypomethylated. Hypermethylated regions in cheetahs under human care were located in the promoter region of a gene involved in host-pathogen interactions (KLC1) and in an intron of a transcription factor relevant for the development of pancreatic β-cells, liver, and kidney (GLIS3). The most canonical mechanism of DNA methylation in promoter regions is assumed to repress gene transcription. Taken together, this could indicate that hypermethylation at the promoter region of KLC1 is involved in the reduced immunity in cheetahs under human care. This approach can be generalized to characterize DNA methylation profiles in larger cheetah populations under human care with a more granular longitudinal data collection, which, in the future, could be used to monitor the early onset of pathologies, and ultimately translate into the development of biomarkers with prophylactic and/or therapeutic potential.

The Asian elephant population is continuously declining due to several extrinsic reasons in their range countries, but also due to diseases in captive populations worldwide. One of these diseases, the elephant endotheliotropic herpesvirus (EEHV) hemorrhagic disease, is very impactful because it particularly affects Asian elephant calves. It is commonly fatal and presents as an acute and generalized hemorrhagic syndrome. Therefore, having reference values of coagulation parameters, and obtaining such values for diseased animals in a very short time, is of great importance. We analyzed prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen concentrations using a portable and fast point-of-care analyzer (VetScan Pro) in 127 Asian elephants from Thai camps and European captive herds. We found significantly different PT and aPTT coagulation times between elephants from the two regions, as well as clear differences in fibrinogen concentration. Nevertheless, these alterations were not expected to have biological or clinical implications. We have also sequenced the coagulation factor VII gene of 141 animals to assess the presence of a previously reported hereditary coagulation disorder in Asian elephants and to investigate the presence of other mutations. We did not find the previously reported mutation in our study population. Instead, we discovered the presence of several new single nucleotide polymorphisms, two of them being considered as deleterious by effect prediction software.

Runs of Homozygosity (ROHs) are extensively found in the genomes of farm animals. The genes contained in these regions are important for the selected traits, having reached fixation through selective breeding, but also as a result of inbreeding. Two essential goals of breeding programs are to increase reproductive fitness and muscle growth, both of which are largely determined by ROHs and the genes therein. In this study, the genomes of pigs selected primarily for reproductive (Large White (LW), n=10) or growth (Pietrain (PI), n=10) traits were sequenced, and ROHs were detected. In both breeds, approximately one third of the genome was found to be homozygous. Although breed-specific genes overlapping ROHs were not significantly enriched for biological functions related to reproduction or growth, five genes were associated to muscle biology. Furthermore, these genes were affected by non-synonymous mutations, which may result from selection.

Species of the mustelid subfamily Guloninae inhabit diverse habitats on multiple continents, and occupy a variety of ecological niches. They differ in feeding ecologies, reproductive strategies and morphological adaptations. To identify candidate loci associated with adaptations to their respective environments, we generated a de novo assembly of the tayra (Eira barbara), the earliest diverging species in the subfamily, and compared this with the genomes available for the wolverine (Gulo gulo) and the sable (Martes zibellina). Our comparative genomic analyses included searching for signs of positive selection, examining changes in gene family sizes and searching for species-specific structural variants. Among candidate loci associated with phenotypic traits, we observed many related to diet, body condition and reproduction. For example, for the tayra, which has an atypical gulonine reproductive strategy of aseasonal breeding, we observed species-specific changes in many pregnancy-related genes. For the wolverine, a circumpolar hypercarnivore that must cope with seasonal food scarcity, we observed many changes in genes associated with diet and body condition. All types of genomic variation examined (single nucleotide polymorphisms, gene family expansions, structural variants) contributed substantially to the identification of candidate loci. This argues strongly for consideration of variation other than single nucleotide polymorphisms in comparative genomics studies aiming to identify loci of adaptive significance.

AbstractSampling genetic material from rare and often secretive mammals can be difficult in challenging environments such as tropical rainforests. Large‐scale sampling is important however for resolving species' taxonomic uncertainties, as well as to help provide genetic material for reference databases that can be used with DNA forensics to combat the illegal wildlife trade. Environmental DNA (eDNA) and invertebrate‐derived DNA (iDNA) offer a promising way to overcome this sampling bottleneck, though to date e/iDNA studies have been primarily used to obtain information about species presence using barcoding or metabarcoding approaches. In this study, we collected 4,710 terrestrial leeches across six protected areas in the Annamite Mountains of Viet Nam and Laos and sequenced fragments (223–694 bp) of mt‐Cytb and/or mt‐Dcr of the mitogenomes from six species that are frequently found in the illegal wildlife trade. Although amplification success was generally low (<50%) because DNA was highly degraded, we showed that e/iDNA samples can be an important source of genetic material for difficult to sample species and show promise as a way to fill existing sampling gaps. Because e/iDNA sampling has the potential to generate much‐needed genetic data to improve taxonomic assessments, this approach can be used to support conservation management by contributing to genetic databases that are necessary to determine the origin of wildlife products or to inform the reintroduction of confiscated animals.

BACKGROUND:Long-term selection experiments are a powerful tool to understand the genetic background of complex traits. The longest of such experiments has been conducted in the Research Institute for Farm Animal Biology (FBN), generating extreme mouse lines with increased fertility, body mass, protein mass and endurance. For >140 generations, these lines have been maintained alongside an unselected control line, representing a valuable resource for understanding the genetic basis of polygenic traits. However, their history and genomes have not been reported in a comprehensive manner yet. Therefore, the aim of this study is to provide a summary of the breeding history and phenotypic traits of these lines along with their genomic characteristics. We further attempt to decipher the effects of the observed line-specific patterns of genetic variation on each of the selected traits. RESULTS:Over the course of >140 generations, selection on the control line has given rise to two extremely fertile lines (>20 pups per litter each), two giant growth lines (one lean, one obese) and one long-distance running line. Whole genome sequencing analysis on 25 animals per line revealed line-specific patterns of genetic variation among lines, as well as high levels of homozygosity within lines. This high degree of distinctiveness results from the combined effects of long-term continuous selection, genetic drift, population bottleneck and isolation. Detection of line-specific patterns of genetic differentiation and structural variation revealed multiple candidate genes behind the improvement of the selected traits. CONCLUSIONS:The genomes of the Dummerstorf trait-selected mouse lines display distinct patterns of genomic variation harbouring multiple trait-relevant genes. Low levels of within-line genetic diversity indicate that many of the beneficial alleles have arrived to fixation alongside with neutral alleles. This study represents the first step in deciphering the influence of selection and neutral evolutionary forces on the genomes of these extreme mouse lines and depicts the genetic complexity underlying polygenic traits.

Urban wildlife ecology is gaining relevance as metropolitan areas grow throughout the world, reducing natural habitats and creating new ecological niches. However, knowledge is still scarce about the colonisation processes of such urban niches, the establishment of new communities, populations and/or species, and the related changes in behaviour and life histories of urban wildlife. Wild boar (Sus scrofa) has successfully colonised urban niches throughout Europe. The aim of this study is to unveil the processes driving the establishment and maintenance of an urban wild boar population by analysing its genetic structure. A set of 19 microsatellite loci was used to test whether urban wild boars in Barcelona, Spain, are an isolated population or if gene flow prevents genetic differentiation between rural and urban wild boars. This knowledge will contribute to the understanding of the effects of synurbisation and the associated management measures on the genetic change of large mammals in urban ecosystems. Despite the unidirectional gene flow from rural to urban areas, the urban wild boars in Barcelona form an island population genotypically differentiated from the surrounding rural ones. The comparison with previous genetic studies of urban wild boar populations suggests that forest patches act as suitable islands for wild boar genetic differentiation. Previous results and the genetic structure of the urban wild boar population in Barcelona classify wild boar as an urban exploiter species. These wild boar peri-urban island populations are responsible for conflict with humans and thus should be managed by reducing the attractiveness of urban areas. The management of peri-urban wild boar populations should aim at reducing migration into urban areas and preventing phenotypic changes (either genetic or plastic) causing habituation of wild boars to humans and urban environments.

Phylogenetic and phylogeographic assessments of species should ideally include individuals of all known populations. However, this is difficult for species with large distribution areas. Previous molecular studies of the wide-ranging Eurasian lynx Lynx lynx focused mainly on its northern Palearctic populations, with the consequence that the reconstruction of this species&rsquo; evolutionary history did not include genetic variation present in its southern Palearctic distribution. We sampled a previously not considered Asian subspecies (L. l. dinniki), added published data from another Asian subspecies (L. l. isabellinus), and reassessed the Eurasian lynx mtDNA phylogeny along with previously published data from northern Palearctic populations. Our mitogenome-based analyses revealed that the subspecies L. l. isabellinus harbours the most basal matriline, consistent with the origin of Lynx lynx in this subspecies&rsquo; current range. L. l. dinniki harbours the second-most basal matriline, which is related to, and may be the source of, the mtDNA diversity of the critically endangered Balkan lynx L. l. balcanicus. Our results suggest that the Anatolian peninsula was a glacial refugium for Eurasian lynx, with previously unconsidered implications for the colonization of Europe by this species. Genetic variation present in southern Palearctic populations of Lynx lynx is clearly important for elucidating the evolutionary history of this species.

Leopards are the only big cats still widely distributed across the continents of Africa and Asia. They occur in a wide range of habitats and are often found in close proximity to humans. But despite their ubiquity, leopard phylogeography and population history have not yet been studied with genomic tools. Here, we present population-genomic data from 26 modern and historical samples encompassing the vast geographical distribution of this species. We find that Asian leopards are broadly monophyletic with respect to African leopards across almost their entire nuclear genomes. This profound genetic pattern persists despite the animals' high potential mobility, and despite evidence of transfer of African alleles into Middle Eastern and Central Asian leopard populations within the last 100,000 years. Our results further suggest that Asian leopards originated from a single out-of-Africa dispersal event 500-600 thousand years ago and are characterized by higher population structuring, stronger isolation by distance, and lower heterozygosity than African leopards. Taxonomic categories do not take into account the variability in depth of divergence among subspecies. The deep divergence between the African subspecies and Asian populations contrasts with the much shallower divergence among putative Asian subspecies. Reconciling genomic variation and taxonomy is likely to be a growing challenge in the genomics era.

Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome in North America, has decimated bat populations within a decade. The fungus impacts bats during hibernation when physiological functions, including immune responses, are down-regulated. Studies have shown that Pd is native to Europe, where it is not associated with mass mortalities. Moreover, genomic and proteomic studies indicated that European bats may have evolved an effective immune defence, which is lacking in North American bats. However, it is still unclear which defence strategy enables European bats to cope with the pathogen. Here, we analyzed selected physiological and immunological parameters in torpid, Pd infected European greater mouse-eared bats (Myotis myotis) showing three different levels of infection (asymptomatic, mild and severe symptoms). From a subset of the studied bats we tracked skin temperatures during one month of hibernation. Contrasting North American bats, arousal patterns remained unaffected by Pd infections in M. myotis. In general, heavier M. myotis aroused more often from hibernation and showed less severe disease symptoms than lean individuals; most likely because heavy bats were capable of reducing the Pd load more effectively than lean individuals. In the blood of severely infected bats, we found higher gene expression levels of an inflammatory cytokine (IL-1β), but lower levels of an acute phase protein (haptoglobin), reactive oxygen metabolites (ROMs) and plasma non-enzymatic antioxidant capacity (OXY) compared to conspecifics with lower levels of infection. We conclude that M. myotis, and possibly also other European bat species, tolerate Pd infections during torpor by using selected acute phase response parameters at baseline levels, yet without arousing from torpor and without synthesizing additional immune molecules.