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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 Author(s) 2024. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

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., Competing Interests: We declare we have no competing interests., (© 2024 The Authors.)

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., (© 2024. The Author(s).)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

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 ~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., Competing Interests: None., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

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.

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' 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 the existence of three major clades (A: Central Asia, B: SE Europe/SW Asia, C: Europe and Northern Asia) and at least five lineages, with diversification in Lynx lynx commencing at least 28kyr earlier than hitherto estimated. The subspecies L. l. isabellinus harbors the most basal matriline, consistent with the origin of Lynx lynx in this subspecies' current range. L. l. dinniki harbors 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.

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background and Aims: Small-molecule flux in tissue microdomains is essential for organ function, but knowledge of this process is scant due to the lack of suitable methods. We developed two independent techniques that allow the quantification of advection (flow) and diffusion in individual bile canaliculi and in interlobular bile ducts of intact livers in living mice, namely fluorescence loss after photoactivation and intravital arbitrary region image correlation spectroscopy., Approach and Results: The results challenge the prevailing "mechano-osmotic" theory of canalicular bile flow. After active transport across hepatocyte membranes, bile acids are transported in the canaliculi primarily by diffusion. Only in the interlobular ducts is diffusion augmented by regulatable advection. Photoactivation of fluorescein bis-(5-carboxymethoxy-2-nitrobenzyl)-ether in entire lobules demonstrated the establishment of diffusive gradients in the bile canalicular network and the sink function of interlobular ducts. In contrast to the bile canalicular network, vectorial transport was detected and quantified in the mesh of interlobular bile ducts., Conclusions: The liver consists of a diffusion-dominated canalicular domain, where hepatocytes secrete small molecules and generate a concentration gradient and a flow-augmented ductular domain, where regulated water influx creates unidirectional advection that augments the diffusive flux., (© 2020 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Homotherium was a genus of large-bodied scimitar-toothed cats, morphologically distinct from any extant felid species, that went extinct at the end of the Pleistocene [1-4]. They possessed large, saber-form serrated canine teeth, powerful forelimbs, a sloping back, and an enlarged optic bulb, all of which were key characteristics for predation on Pleistocene megafauna [5]. Previous mitochondrial DNA phylogenies suggested that it was a highly divergent sister lineage to all extant cat species [6-8]. However, mitochondrial phylogenies can be misled by hybridization [9], incomplete lineage sorting (ILS), or sex-biased dispersal patterns [10], which might be especially relevant for Homotherium since widespread mito-nuclear discrepancies have been uncovered in modern cats [10]. To examine the evolutionary history of Homotherium, we generated a ∼7x nuclear genome and a ∼38x exome from H. latidens using shotgun and target-capture sequencing approaches. Phylogenetic analyses reveal Homotherium as highly divergent (∼22.5 Ma) from living cat species, with no detectable signs of gene flow. Comparative genomic analyses found signatures of positive selection in several genes, including those involved in vision, cognitive function, and energy consumption, putatively consistent with diurnal activity, well-developed social behavior, and cursorial hunting [5]. Finally, we uncover relatively high levels of genetic diversity, suggesting that Homotherium may have been more abundant than the limited fossil record suggests [3, 4, 11-14]. Our findings complement and extend previous inferences from both the fossil record and initial molecular studies, enhancing our understanding of the evolution and ecology of this remarkable lineage., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

We use the European hedgehog ( Erinaceus europaeus ), a mammal with limited mobility, as a model species to study whether the structural matrix of the urban environment has an influence on population genetic structure of such species in the city of Berlin (Germany). Using ten established microsatellite loci we genotyped 143 hedgehogs from numerous sites throughout Berlin. Inclusion of all individuals in the cluster analysis yielded three genetic clusters, likely reflecting spatial associations of kin (larger family groups, known as gamodemes). To examine the potential bias in the cluster analysis caused by closely related individuals, we determined all pairwise relationships and excluded close relatives before repeating the cluster analysis. For this data subset ( N = 65) both clustering algorithms applied (Structure, Baps) indicated the presence of a single genetic cluster. These results suggest that the high proportion of green patches in the city of Berlin provides numerous steppingstone habitats potentially linking local subpopulations. Alternatively, translocation of individuals across the city by hedgehog rescue facilities may also explain the existence of only a single cluster. We therefore propose that information about management activities such as releases by animal rescue centres should include location data (as exactly as possible) regarding both the collection and the release site, which can then be used in population genetic studies.

Social epigenomics is a new field of research that studies how the social environment shapes the epigenome and how in turn the epigenome modulates behavior. We focus on describing known gene-environment interactions (GEIs) and epigenetic mechanisms in different mammalian social systems. To illustrate how epigenetic mechanisms integrate GEIs, we highlight examples where epigenetic mechanisms are associated with social behaviors and with their maintenance through neuroendocrine, locomotor, and metabolic responses. We discuss future research trajectories and open questions for the emerging field of social epigenomics in nonmodel and naturally occurring social systems. Finally, we outline the technological advances that aid the study of epigenetic mechanisms in the establishment of GEIs and vice versa., (© The Author(s) (2020). Published by Oxford University Press on behalf of Editorial Office, Current Zoology.)

External temperature change has been shown to modify epigenetic patterns, such as DNA methylation, which regulates gene expression. DNA methylation is heritable, and as such provides a mechanism to convey environmental information to subsequent generations. Studies on epigenetic response to temperature increase are still scarce in wild mammals, even more so studies that compare tissue-specific epigenetic responses. Here, we aim to address differential epigenetic responses on a gene and gene pathway level in two organs, liver and testis. We chose these organs, because the liver is the main metabolic and thermoregulation organ, and epigenetic modifications in testis are potentially transmitted to the F2 generation. We focused on the transmission of DNA methylation changes to naive male offspring after paternal exposure to an ambient temperature increase of 10 °C, and investigated differential methylated regions of sons sired before and after the paternal exposure using Reduced Representation Bisulfite Sequencing. We detected both a highly tissue-specific epigenetic response, reflected in genes involved in organ-specific metabolic pathways, and a more general regulation of single genes epigenetically modified in both organs. We conclude that genomes are context-specifically differentially epigenetically regulated in response to temperature increase. These findings emphasize the epigenetic relevance in cell differentiation, which is essential for the specific function(s) of complex organs, and is represented in a diverse molecular regulation of genes and gene pathways. The results also emphasize the paternal contribution to adaptive processes.

Background: Many felid species are of high conservation concern, and with increasing human disturbance the situation is worsening. Small isolated populations are at risk of genetic impoverishment decreasing within-species biodiversity. Movement is known to be a key behavioural trait that shapes both demographic and genetic dynamics and affects population survival. However, we have limited knowledge on how different manifestations of movement behaviour translate to population processes. In this study, we aimed to 1) understand the potential effects of movement behaviour on the genetic diversity of small felid populations in heterogeneous landscapes, while 2) presenting a simulation tool that can help inform conservation practitioners following, or considering, population management actions targeting the risk of genetic impoverishment., Methods: We developed a spatially explicit individual-based population model including neutral genetic markers for felids and applied this to the example of Eurasian lynx. Using a neutral landscape approach, we simulated reintroductions into a three-patch system, comprising two breeding patches separated by a larger patch of differing landscape heterogeneity, and tested for the effects of various behavioural movement syndromes and founder population sizes. We explored a range of movement syndromes by simulating populations with various movement model parametrisations that range from 'shy' to 'bold' movement behaviour., Results: We find that movement syndromes can lead to a higher loss of genetic diversity and an increase in between population genetic structure for both "bold" and "shy" movement behaviours, depending on landscape conditions, with larger decreases in genetic diversity and larger increases in genetic differentiation associated with bold movement syndromes, where the first colonisers quickly reproduce and subsequently dominate the gene pool. In addition, we underline the fact that a larger founder population can offset the genetic losses associated with subpopulation isolation and gene pool dominance., Conclusions: We identified a movement syndrome trade-off for population genetic variation, whereby bold-explorers could be saviours - by connecting populations and promoting panmixia, or sinks - by increasing genetic losses via a 'founder takes all' effect, whereas shy-stayers maintain a more gradual genetic drift due to their more cautious behaviour. Simulations should incorporate movement behaviour to provide better projections of long-term population viability and within-species biodiversity, which includes genetic diversity. Simulations incorporating demographics and genetics have great potential for informing conservation management actions, such as population reintroductions or reinforcements. Here, we present such a simulation tool for solitary felids., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

The meerkat, Suricata suricatta , is a highly social member of the mongoose family (Herpestidae) and the only extant species of the genus Suricata . We present the first complete mitochondrial genome of the meerkat, assembled with a seed-and-extend algorithm using three closely related species as references. Phylogenetic analyses using 22 mitochondrial genome sequences confirm the position of meerkat within the Herpestidae family and the Feliformia, a suborder of Carnivora, with high support values. This position is in good agreement with formerly conducted studies based on a small number of mitochondrial and nuclear gene fragments. Our complete mitochondrial genome represents a valuable resource for further phylogenetic studies, especially of the underrepresented members of the Herpestidae family., Competing Interests: No potential conflict of interest was reported by the authors., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Immunological responses of hibernating mammals are suppressed at low body temperatures, a possible explanation for the devastating effect of the white-nose syndrome on hibernating North American bats. However, European bats seem to cope well with the fungal causative agent of the disease. To better understand the immune response of hibernating bats, especially against fungal pathogens, we challenged European greater mouse-eared bats ( Myotis myotis ) by inoculating the fungal antigen zymosan. We monitored torpor patterns, immune gene expressions, different aspects of the acute phase response and plasma oxidative status markers, and compared them with sham-injected control animals at 30 min, 48 h and 96 h after inoculation. Torpor patterns, body temperatures, body masses, white blood cell counts, expression of immune genes, reactive oxygen metabolites and non-enzymatic antioxidant capacity did not differ between groups during the experiment. However, zymosan injected bats had significantly higher levels of haptoglobin than the control animals. Our results indicate that hibernating greater mouse-eared bats mount an inflammatory response to a fungal challenge, with only mild to negligible consequences for the energy budget of hibernation. Our study gives a first hint that hibernating European bats may have evolved a hibernation-adjusted immune response in order to balance the trade-off between competent pathogen elimination and a prudent energy-saving regime., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The Caucasian lynx, Lynx lynx dinniki, has one of the southernmost distributions in the Eurasian lynx range, covering Anatolian Turkey, the Caucasus and Iran. Little is known about the biology and the genetic status of this subspecies. To collect baseline genetic, ecological and behavioural data and benefit future conservation of L. l. dinniki, we monitored 11 lynx territories (396 km2) in northwestern Anatolia. We assessed genetic diversity of this population by non-invasively collecting 171 faecal samples and trapped and sampled 12 lynx individuals using box traps. We observed high allelic variation at 11 nuclear microsatellite markers, and found no signs of inbreeding despite the potential isolation of this population. We obtained similar numbers of distinct genotypes from the two sampling sources. Our results indicated that first order female relatives occupy neighbouring territories (female philopatry) and that territorial male lynx were highly unrelated to each other and to female territorial lynx, suggesting long distance male dispersal. Particular male and female resident territorial lynx and their offspring (kittens and subadults) were more likely to be trapped than resident floaters or dispersing (unrelated) lynx. Conversely, we obtained more data for unrelated lynx and higher numbers of territorials using non-invasive sampling (faeces). When invasive and non-invasive samples were analysed separately, the spatial organisation of lynx (in terms of female philopatry and females and males occupying permanent ranges) affected measures of genetic diversity in such a way that estimates of genetic diversity were reduced if only invasive samples were considered. It appears that, at small spatial scales, invasive sampling using box traps may underestimate the genetic diversity in carnivores with permanent ranges and philopatry such as the Eurasian lynx. As non-invasive sampling can also provide additional data on diet and spatial organisation, we advocate the use of such samples for conservation genetic studies of vulnerable, endangered or data deficient territorial species., Competing Interests: The authors have declared that no competing interests exist.

Epigenetic modifications are a mechanism conveying environmental information to subsequent generations via parental germ lines. Research on epigenetic responses to environmental changes in wild mammals has been widely neglected, as well as studies that compare responses to changes in different environmental factors. Here, we focused on the transmission of DNA methylation changes to naive male offspring after paternal exposure to either diet (~40% less protein) or temperature increase (10 °C increased temperature). Because both experiments focused on the liver as the main metabolic and thermoregulation organ, we were able to decipher if epigenetic changes differed in response to different environmental changes. Reduced representation bisulfite sequencing (RRBS) revealed differentially methylated regions (DMRs) in annotated genomic regions in sons sired before (control) and after the fathers' treatments. We detected both a highly specific epigenetic response dependent on the environmental factor that had changed that was reflected in genes involved in specific metabolic pathways, and a more general response to changes in outer stimuli reflected by epigenetic modifications in a small subset of genes shared between both responses. Our results indicated that fathers prepared their offspring for specific environmental changes by paternally inherited epigenetic modifications, suggesting a strong paternal contribution to adaptive processes., Competing Interests: The authors declare no conflicts of interest.

Targeted capture coupled with high-throughput sequencing can be used to gain information about nuclear sequence variation at hundreds to thousands of loci. Divergent reference capture makes use of molecular data of one species to enrich target loci in other (related) species. This is particularly valuable for nonmodel organisms, for which often no a priori knowledge exists regarding these loci. Here, we have used targeted capture to obtain data for 809 nuclear coding DNA sequences (CDS) in a nonmodel organism, the Eurasian lynx Lynx lynx, using baits designed with the help of the published genome of a related model organism (the domestic cat Felis catus). Using this approach, we were able to survey intraspecific variation at hundreds of nuclear loci in L. lynx across the species' European range. A large set of biallelic candidate SNPs was then evaluated using a high-throughput SNP genotyping platform (Fluidigm), which we then reduced to a final 96 SNP-panel based on assay performance and reliability; validation was carried out with 100 additional Eurasian lynx samples not included in the SNP discovery phase. The 96 SNP-panel developed from CDS performed very successfully in the identification of individuals and in population genetic structure inference (including the assignment of individuals to their source population). In keeping with recent studies, our results show that genic SNPs can be valuable for genetic monitoring of wildlife species., (© 2018 John Wiley & Sons Ltd.)

Genetic studies of the Eurasian brown bear ( Ursus arctos ) have so far focused on populations from Europe and North America, although the largest distribution area of brown bears is in Asia. In this study, we reveal population genetic parameters for the brown bear population inhabiting the Grand Kaçkar Mountains (GKM) in the north east of Turkey, western Lesser Caucasus. Using both hair ( N  = 147) and tissue samples ( N  = 7) collected between 2008 and 2014, we found substantial levels of genetic variation (10 microsatellite loci). Bear samples (hair) taken from rubbing trees worked better for genotyping than those from power poles, regardless of the year collected. Genotyping also revealed that bears moved between habitat patches, despite ongoing massive habitat alterations and the creation of large water reservoirs. This population has the potential to serve as a genetic reserve for future reintroductions in the Middle East. Due to the importance of the GKM population for on-going and future conservation actions, the impacts of habitat alterations in the region ought to be minimized; e.g., by establishing green bridges or corridors over reservoirs and major roads to maintain habitat connectivity and gene flow among populations in the Lesser Caucasus., Competing Interests: The authors declare there are no competing interests.

Deciphering the genes involved in disease resistance is essential if we are to understand host-pathogen coevolutionary processes. The rabbit haemorrhagic disease virus (RHDV) was imported into Australia in 1995 as a biocontrol agent to manage one of the most successful and devastating invasive species, the European rabbit (Oryctolagus cuniculus). During the first outbreaks of the disease, RHDV caused mortality rates of up to 97%. Recently, however, increased genetic resistance to RHDV has been reported. Here, we have aimed to identify genomic differences between rabbits that survived a natural infection with RHDV and those that died in the field using a genomewide next-generation sequencing (NGS) approach. We detected 72 SNPs corresponding to 133 genes associated with survival of a RHD infection. Most of the identified genes have known functions in virus infections and replication, immune responses or apoptosis, or have previously been found to be regulated during RHD. Some of the genes identified in experimental studies, however, did not seem to play a role under natural selection regimes, highlighting the importance of field studies to complement the genomic background of wildlife diseases. Our study provides a set of candidate markers as a tool for the future scanning of wild rabbits for their resistance to RHDV. This is important both for wild rabbit populations in southern Europe where RHD is regarded as a serious problem decimating the prey of endangered predator species and for assessing the success of currently planned RHDV variant biocontrol releases in Australia., (© 2017 John Wiley & Sons Ltd.)

Southeast (SE) Asia is 1 of the most biodiverse regions in the world, and it holds approximately 20% of all mammal species. Despite this, the majority of SE Asia's genetic diversity is still poorly characterized. The growing interest in using environmental DNA to assess and monitor SE Asian species, in particular threatened mammals-has created the urgent need to expand the available reference database of mitochondrial barcode and complete mitogenome sequences. We have partially addressed this need by generating 72 new mitogenome sequences reconstructed from DNA isolated from a range of historical and modern tissue samples. Approximately 55 gigabases of raw sequence were generated. From this data, we assembled 72 complete mitogenome sequences, with an average depth of coverage of ×102.9 and ×55.2 for modern samples and historical samples, respectively. This dataset represents 52 species, of which 30 species had no previous mitogenome data available. The mitogenomes were geotagged to their sampling location, where known, to display a detailed geographical distribution of the species. Our new database of 52 taxa will strongly enhance the utility of environmental DNA approaches for monitoring mammals in SE Asia as it greatly increases the likelihoods that identification of metabarcoding sequencing reads can be assigned to reference sequences. This magnifies the confidence in species detections and thus allows more robust surveys and monitoring programmes of SE Asia's threatened mammal biodiversity. The extensive collections of historical samples from SE Asia in western and SE Asian museums should serve as additional valuable material to further enrich this reference database., (© The Author 2017. Published by Oxford University Press.)

The Leopard cat Prionailurus bengalensis is a habitat generalist that is widely distributed across Southeast Asia. Based on morphological traits, this species has been subdivided into 12 subspecies. Thus far, there have been few molecular studies investigating intraspecific variation, and those had been limited in geographic scope. For this reason, we aimed to study the genetic structure and evolutionary history of this species across its very large distribution range in Asia. We employed both PCR-based (short mtDNA fragments, 94 samples) and high throughput sequencing based methods (whole mitochondrial genomes, 52 samples) on archival, noninvasively collected and fresh samples to investigate the distribution of intraspecific genetic variation. Our comprehensive sampling coupled with the improved resolution of a mitochondrial genome analyses provided strong support for a deep split between Mainland and Sundaic Leopard cats. Although we identified multiple haplogroups within the species' distribution, we found no matrilineal evidence for the distinction of 12 subspecies. In the context of Leopard cat biogeography, we cautiously recommend a revision of the Prionailurus bengalensis subspecific taxonomy: namely, a reduction to 4 subspecies (2 mainland and 2 Sundaic forms)., (© The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The biogeographic dynamics affecting the Indian subcontinent, East and Southeast Asia during the Plio-Pleistocene has generated complex biodiversity patterns. We assessed the molecular biogeography of the small Indian civet (Viverricula indica) through mitogenome and cytochrome b + control region sequencing of 89 historical and modern samples to (1) establish a time-calibrated phylogeography across the species' native range and (2) test introduction scenarios to western Indian Ocean islands. Bayesian phylogenetic analyses identified 3 geographic lineages (East Asia, sister-group to Southeast Asia and the Indian subcontinent + northern Indochina) diverging 3.2-2.3 million years ago (Mya), with no clear signature of past demographic expansion. Within Southeast Asia, Balinese populations separated from the rest 2.6-1.3 Mya. Western Indian Ocean populations were assigned to the Indian subcontinent + northern Indochina lineage and had the lowest mitochondrial diversity. Approximate Bayesian computation did not distinguish between single versus multiple introduction scenarios. The early diversification of the small Indian civet was likely shaped by humid periods in the Late Pliocene-Early Pleistocene that created evergreen rainforest barriers, generating areas of intra-specific endemism in the Indian subcontinent, East, and Southeast Asia. Later, Pleistocene dispersals through drier conditions in South and Southeast Asia were likely, giving rise to the species' current natural distribution. Our molecular data supported the delineation of only 4 subspecies in V. indica, including an endemic Balinese lineage. Our study also highlighted the influence of prefirst millennium AD introductions to western Indian Ocean islands, with Indian and/or Arab traders probably introducing the species for its civet oil., (© The American Genetic Association 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The members of the genus Muntiacus are of particular interest to evolutionary biologists due to their extreme chromosomal rearrangements and the ongoing discussions about the number of living species. Red muntjacs have the largest distribution of all muntjacs and were formerly considered as one species. Karyotype differences led to the provisional split between the Southern Red Muntjac (Muntiacus muntjak) and the Northern Red Muntjac (M. vaginalis), but uncertainties remain as, so far, no phylogenetic study has been conducted. Here, we analysed whole mitochondrial genomes of 59 archival and 16 contemporaneous samples to resolve uncertainties about their taxonomy and used red muntjacs as model for understanding the evolutionary history of other species in Southeast Asia., Results: We found three distinct matrilineal groups of red muntjacs: Sri Lankan red muntjacs (including the Western Ghats) diverged first from other muntjacs about 1.5 Mya; later northern red muntjacs (including North India and Indochina) and southern red muntjacs (Sundaland) split around 1.12 Mya. The diversification of red muntjacs into these three main lineages was likely promoted by two Pleistocene barriers: one through the Indian subcontinent and one separating the Indochinese and Sundaic red muntjacs. Interestingly, we found a high level of gene flow within the populations of northern and southern red muntjacs, indicating gene flow between populations in Indochina and dispersal of red muntjacs over the exposed Sunda Shelf during the Last Glacial Maximum., Conclusions: Our results provide new insights into the evolution of species in South and Southeast Asia as we found clear genetic differentiation in a widespread and generalist species, corresponding to two known biogeographical barriers: The Isthmus of Kra and the central Indian dry zone. In addition, our molecular data support either the delineation of three monotypic species or three subspecies, but more importantly these data highlight the conservation importance of the Sri Lankan/South Indian red muntjac.

Background. The bay cat Catopuma badia is endemic to Borneo, whereas its sister species the Asian golden cat Catopuma temminckii is distributed from the Himalayas and southern China through Indochina, Peninsular Malaysia and Sumatra. Based on morphological data, up to five subspecies of the Asian golden cat have been recognized, but a taxonomic assessment, including molecular data and morphological characters, is still lacking. Results. We combined molecular data (whole mitochondrial genomes), morphological data (pelage) and species distribution projections (up to the Late Pleistocene) to infer how environmental changes may have influenced the distribution of these sister species over the past 120 000 years. The molecular analysis was based on sequenced mitogenomes of 3 bay cats and 40 Asian golden cats derived mainly from archival samples. Our molecular data suggested a time of split between the two species approximately 3.16 Ma and revealed very low nucleotide diversity within the Asian golden cat population, which supports recent expansion of the population. Discussion. The low nucleotide diversity suggested a population bottleneck in the Asian golden cat, possibly caused by the eruption of the Toba volcano in Northern Sumatra (approx. 74 kya), followed by a continuous population expansion in the Late Pleistocene/Early Holocene. Species distribution projections, the reconstruction of the demographic history, a genetic isolation-by-distance pattern and a gradual variation of pelage pattern support the hypothesis of a post-Toba population expansion of the Asian golden cat from south China/Indochina to Peninsular Malaysia and Sumatra. Our findings reject the current classification of five subspecies for the Asian golden cat, but instead support either a monotypic species or one comprising two subspecies: (i) the Sunda golden cat, distributed south of the Isthmus of Kra: C. t. temminckii and (ii) Indochinese, Indian, Himalayan and Chinese golden cats, occurring north of the Isthmus: C. t. moormensis .

Epigenetic modifications, of which DNA methylation is the best studied one, can convey environmental information through generations via parental germ lines. Past studies have focused on the maternal transmission of epigenetic information to the offspring of isogenic mice and rats in response to external changes, whereas heterogeneous wild mammals as well as paternal epigenetic effects have been widely neglected. In most wild mammal species, males are the dispersing sex and have to cope with differing habitats and thermal changes. As temperature is a major environmental factor we investigated if genetically heterogeneous Wild guinea pig (Cavia aperea) males can adapt epigenetically to an increase in temperature and if that response will be transmitted to the next generation(s). Five adult male guinea pigs (F0) were exposed to an increased ambient temperature for 2 months, i.e. the duration of spermatogenesis. We studied the liver (as the main thermoregulatory organ) of F0 fathers and F1 sons, and testes of F1 sons for paternal transmission of epigenetic modifications across generation(s). Reduced representation bisulphite sequencing revealed shared differentially methylated regions in annotated areas between F0 livers before and after heat treatment, and their sons' livers and testes, which indicated a general response with ecological relevance. Thus, paternal exposure to a temporally limited increased ambient temperature led to an 'immediate' and 'heritable' epigenetic response that may even be transmitted to the F2 generation. In the context of globally rising temperatures epigenetic mechanisms may become increasingly relevant for the survival of species., (© 2015 John Wiley & Sons Ltd.)

Although significantly more money is spent on the conservation of tigers than on any other threatened species, today only 3200 to 3600 tigers roam the forests of Asia, occupying only 7% of their historical range. Despite the global significance of and interest in tiger conservation, global approaches to plan tiger recovery are partly impeded by the lack of a consensus on the number of tiger subspecies or management units, because a comprehensive analysis of tiger variation is lacking. We analyzed variation among all nine putative tiger subspecies, using extensive data sets of several traits [morphological (craniodental and pelage), ecological, molecular]. Our analyses revealed little variation and large overlaps in each trait among putative subspecies, and molecular data showed extremely low diversity because of a severe Late Pleistocene population decline. Our results support recognition of only two subspecies: the Sunda tiger, Panthera tigris sondaica, and the continental tiger, Panthera tigris tigris, which consists of two (northern and southern) management units. Conservation management programs, such as captive breeding, reintroduction initiatives, or trans-boundary projects, rely on a durable, consistent characterization of subspecies as taxonomic units, defined by robust multiple lines of scientific evidence rather than single traits or ad hoc descriptions of one or few specimens. Our multiple-trait data set supports a fundamental rethinking of the conventional tiger taxonomy paradigm, which will have profound implications for the management of in situ and ex situ tiger populations and boost conservation efforts by facilitating a pragmatic approach to tiger conservation management worldwide.

Noninvasive genetics based on microsatellite markers has become an indispensable tool for wildlife monitoring and conservation research over the past decades. However, microsatellites have several drawbacks, such as the lack of standardisation between laboratories and high error rates. Here, we propose an alternative single-nucleotide polymorphism (SNP)-based marker system for noninvasively collected samples, which promises to solve these problems. Using nanofluidic SNP genotyping technology (Fluidigm), we genotyped 158 wolf samples (tissue, scats, hairs, urine) for 192 SNP loci selected from the Affymetrix v2 Canine SNP Array. We carefully selected an optimised final set of 96 SNPs (and discarded the worse half), based on assay performance and reliability. We found rates of missing data in this SNP set of <10% and genotyping error of ~1%, which improves genotyping accuracy by nearly an order of magnitude when compared to published data for other marker types. Our approach provides a tool for rapid and cost-effective genotyping of noninvasively collected wildlife samples. The ability to standardise genotype scoring combined with low error rates promises to constitute a major technological advancement and could establish SNPs as a standard marker for future wildlife monitoring., (© 2014 John Wiley & Sons Ltd.)

Knowledge of coronaviruses in wild carnivores is limited. This report describes coronavirus genetic diversity, species specificity and infection prevalence in three wild African carnivores. Coronavirus RNA was recovered from fresh feces from spotted hyena and silver-backed jackal, but not bat-eared fox. Analysis of sequences of membrane (M) and spike (S) gene fragments revealed strains in the genus Alphacoronavirus, including three distinct strains in hyenas and one distinct strain in a jackal. Coronavirus RNA prevalence was higher in feces from younger (17 %) than older (3 %) hyenas, highlighting the importance of young animals for coronavirus transmission in wild carnivores.

Zoo animal husbandry aims at constantly improving husbandry, reproductive success and ultimately animal welfare. Nevertheless, analyses to determine factors influencing husbandry of different species are rare. The relative life expectancy (rLE; life expectancy (LE) as proportion of longevity) describes husbandry success of captive populations. Correlating rLE with biological characteristics of different species, reasons for variation in rLE can be detected. We analysed data of 166 901 animals representing 78 ruminant species kept in 850 facilities. The rLE of females correlated with the percentage of grass in a species' natural diet, suggesting that needs of species adapted to grass can be more easily accommodated than the needs of those adapted to browse. Males of monogamous species demonstrate higher rLE than polygamous males, which matches observed differences of sexual bias in LE in free-living populations and thus supports the ecological theory that the mating system influences LE. The third interesting finding was that rLE was higher in species managed by international studbooks when compared with species not managed in this way. Our method facilitates the identification of biological characteristics of species that are relevant for their husbandry success, and they also support ecological theory. Translating these findings into feeding recommendations, our approach can help to improve animal husbandry.

Pasteurella are an important cause of fatal infections in free-ranging bats, but the genetic diversity of bat-derived strains is unclear. In the current study, 81 Pasteurella strains associated with pneumonia, severe organ necroses and systemic infection in free-ranging European vespertilionid bats were characterized by biochemical and molecular typing methods. Genetic relationships and subspecies status of Pasteurella multocida strains were determined by comparative 16S rDNA and rpoB gene sequence analysis. In addition, 30 representatives of the bat-derived P. multocida strains were selected based on phenotypic and genotypic tests to be compared by pulsed-field gel electrophoresis using SmaI. Most (85%) of the Pasteurella strains obtained from free-ranging bats in this study represented P. multocida ssp. septica. P. multocida ssp. multocida and Pasteurella species B were also identified in a small number of isolates. PFGE analysis correlated well with the sequencing results and revealed a high genetic diversity among bat-derived strains of P. multocida ssp. septica. Strains sharing identical or closely related SmaI fragment patterns were cultured from bats of different species, geographic origins, and years of isolation. The presence of numerous different P. multocida strains allows the assumption that Pasteurella infections in vespertilionid bats are not solely based on intra- but also on inter-species transmission. And indeed, our results present evidence of P. multocida infections in bats following cat predation., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Recent morphological and molecular studies led to the recognition of two extant species of clouded leopards; Neofelis nebulosa from mainland southeast Asia and Neofelis diardi from the Sunda Islands of Borneo and Sumatra, including the Batu Islands. In addition to these new species-level distinctions, preliminary molecular data suggested a genetic substructure that separates Bornean and Sumatran clouded leopards, indicating the possibility of two subspecies of N. diardi. This suggestion was based on an analysis of only three Sumatran and seven Bornean individuals. Accordingly, in this study we re-evaluated this proposed subspecies differentiation using additional molecular (mainly historical) samples of eight Bornean and 13 Sumatran clouded leopards; a craniometric analysis of 28 specimens; and examination of pelage morphology of 20 museum specimens and of photographs of 12 wild camera-trapped animals. Molecular (mtDNA and microsatellite loci), craniomandibular and dental analyses strongly support the differentiation of Bornean and Sumatran clouded leopards, but pelage characteristics fail to separate them completely, most probably owing to small sample sizes, but it may also reflect habitat similarities between the two islands and their recent divergence. However, some provisional discriminating pelage characters are presented that need further testing. According to our estimates both populations diverged from each other during the Middle to Late Pleistocene (between 400 and 120 kyr). We present a discussion on the evolutionary history of Neofelis diardi sspp. on the Sunda Shelf, a revised taxonomy for the Sunda clouded leopard, N. diardi, and formally describe the Bornean subspecies, Neofelis diardi borneensis, including the designation of a holotype (BM.3.4.9.2 from Baram, Sarawak) in accordance with the rules of the International Code of Zoological Nomenclature., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Vascular endothelial growth factor A (VEGFA) influences spermatogenesis, but its impact on seasonally regulated sperm production is still not fully understood. Thus, we investigated both expression levels and localisation of VEGFA and its receptors VEGFR1 and 2 in roe buck testis via real-time reverse transcription polymerase chain reaction and immunohistochemistry in relation to seasonal changes in the cellular composition of the testis. VEGFA was expressed by interstitial cells while its receptors were found on endothelial and perivascular cells. Inside the tubules, VEGFA was located in spermatogonia and spermatocytes, VEGFR1 was present on elongating spermatids and VEGFR2 on Sertoli cells. VEGFR1 mRNA was expressed tenfold lower than VEGFR2 and VEGF mRNAs. Relative VEGF and VEGFR2 expression (divided by the number of VEGFA and VEGFR2 expressing cells) showed an increase towards the rut (July/August) and a decrease thereafter. The results suggest involvement of VEGFA in the adjustment of vascular permeability as well as in spermiogenesis and the proliferation of spermatogonia.

Adult roe deer males show seasonal cycles of testicular growth and involution. The exact timing of these cycles requires endocrine regulation and local testicular control by autocrine/paracrine factors. Recent findings suggest that the vascular endothelial growth factor (VEGF) might have effects on both vascular and germinative cells in testis. Thus, we studied the expression pattern of vascular endothelial growth factor (VEGF) in roe deer testis using quantitative RT-PCR. The strength of VEGF mRNA expression depended on season. It reached its highest level at the peak of spermatogenesis during the pre-rutting period and had its nadir at the end of the rut when involution already began. The results suggested that VEGF may directly affect the regulation of spermatogenesis but may not be involved predominantly in testicular microvasculature as initially expected.

Early embryonic resorption or fetal loss is known to occur occasionally in captive elephants; however, this has mostly been reported anecdotally. The present study documents the case of a 24-year-old, multiparous Asian elephant cow that suffered embryonic death and resorption at around 18 weeks of gestation. From ovulation onwards, this female was sonographically examined 58 times. Blood was collected twice weekly for progestagen determination via enzyme immunoassay. On Day 42 after ovulation, a small quantity of fluid was detected in the uterine horn, which typically indicates the presence of a developing conceptus. Repeated inspections followed what appeared to be a normal pregnancy until Day 116. However, on Day 124, signs of embryonic life were absent. Progestagen concentrations started declining two weeks later, reaching baseline levels one month after embryonic death. Retrospectively, ultrasound examination revealed several abnormalities in the uterine horn. Besides an existing leiomyoma, multiple small cystic structures had formed in the endometrium at the implantation site and later in the placenta. These pathological findings were considered as possible contributors to the early pregnancy failure. PCR for endotheliotropic elephant herpes virus (EEHV) (which had occurred previously in the herd) as well as serology for other infectious organisms known to cause abortion in domestic animals did not yield any positive results. Although no definitive reason was found for this pregnancy to abort, this ultrasonographically and endocrinologically documented study of an early pregnancy loss provides important insights into the resorption process in Asian elephants.

Browsing and grazing ruminants are thought to differ in the degree their rumen contents are stratified-which may be due to different characteristics of their respective forages, to particular adaptations of the animals, or both. However, this stratification is difficult to measure in live animals. The papillation of the rumen has been suggested as an anatomical proxy for stratification-with even papillation indicating homogenous contents, and uneven papillation (with few and small dorsal and ventral papillae, and prominent papillae in the atrium ruminis) stratified contents. Using the surface enlargement factor (SEF, indicating how basal mucosa surface is increased by papillae) of over 55 ruminant species, we demonstrate that differences between the SEF(dorsal) or SEF(ventral) and the SEF(atrium) are significantly related to the percentage of grass in the natural diet. The more a species is adapted to grass, the more distinct this difference, with extreme grazers having unpapillated dorsal and ventral mucosa. The relative SEF(dorsal) as anatomical proxy for stratification, and the difference in particle and fluid retention in the rumen as physiological proxy for stratification, are highly correlated in species (n = 9) for which both kind of data are available. The results support the concept that the stratification of rumen contents varies among ruminants, with more homogenous contents in the more browsing and more stratified contents in the more grazing species., ((c) 2009 Wiley-Liss, Inc.)

Using cranioskeletal measurements, several studies have generated evidence that grazing ruminants have a more pronounced mastication apparatus, in terms of muscle insertion areas and protuberances, than browsing ruminants, with the resulting hypothesis that grazers should have larger, heavier chewing muscles than browsers. However, the only investigation of this so far [Axmacher and Hofmann (J Zool 215:463-473, 1988)] did not find differences between ruminant feeding types in the masseter muscle mass of 22 species. Here, we expand the dataset to 48 ruminant species. Regardless of phylogenetic control in the statistical treatment, there was a significant positive correlation of body mass and masseter mass, and also a significant association between percent grass in the natural diet and masseter mass. The results support the concept that ruminant species that ingest more grass have relatively larger masseter muscles, possibly indicating an increased requirement to overcome the resistance of grass forage. The comparative chewing resistance of different forage classes may represent a rewarding field of ecophysiological research.

Thirteen strains of Streptococcus equi subsp. ruminatorum from free-ranging spotted hyenas (Crocuta crocuta) and plains zebras (Equus burchelli) in Tanzania were characterized by biochemical and molecular-biological methods. Although the colony appearance of the S.e. ruminatorum wildlife strains differed from that of the S.e. ruminatorum type strain CECT 5772(T), all biochemical reactions of the wildlife strains were similar to those of the type strain. In addition, all wildlife strains produced hyaluronidase and were capable of hydrolysing arginine, three strains (23%) synthesized acetoin, but only eight strains (62%) produced acid from ribose. rep-PCR indicated that different clones of S.e. ruminatorum were distributed among the hyena and zebra populations in the study area. Identical rep-PCR patterns in hyena and zebra strains suggest that a direct transmission of S.e. ruminatorum between these species may occur. The presence of a M-like protein (SrM) gene was demonstrated in all S.e. ruminatorum strains including the type strain. Sequencing of the M-like protein gene revealed a hypervariable region within the deduced amino acid sequence. Most of the strains clustered with previously described strains based on the hypervariable region of the S.e. zooepidemicus SzP protein. Sequencing also demonstrated that identical SrM protein sequences were shared among S.e. ruminatorum strains from different host species.