Spatial genetic structure of European wild boar, with inferences on late-Pleistocene and Holocene demographic history.

European wildlife has been subjected to intensifying levels of anthropogenic impact throughout the Holocene, yet the main genetic partitioning of many species is thought to still reflect the late-Pleistocene glacial refugia. We analyzed 26,342 nuclear SNPs of 464 wild boar (Sus scrofa) across the European continent to infer demographic history and reassess the genetic consequences of natural and anthropogenic forces. We found that population fragmentation, inbreeding and recent hybridization with domestic pigs have caused the spatial genetic structure to be heterogeneous at the local scale. Underlying local anthropogenic signatures, we found a deep genetic structure in the form of an arch-shaped cline extending from the Dinaric Alps, via Southeastern Europe and the Baltic states, to Western Europe and, finally, to the genetically diverged Iberian peninsula. These findings indicate that, despite considerable anthropogenic influence, the deeper, natural continental structure is still intact. Regarding the glacial refugia, our findings show a weaker signal than generally assumed, but are nevertheless suggestive of two main recolonization routes, with important roles for Southern France and the Balkans. Our results highlight the importance of applying genomic resources and framing genetic results within a species' demographic history and geographic distribution for a better understanding of the complex mixture of underlying processes.

A continent-wide high genetic load in African buffalo revealed by clines in the frequency of deleterious alleles, genetic hitchhiking and linkage disequilibrium.

A high genetic load can negatively affect population viability and increase susceptibility to diseases and other environmental stressors. Prior microsatellite studies of two African buffalo (Syncerus caffer) populations in South Africa indicated substantial genome-wide genetic load due to high-frequency occurrence of deleterious alleles. The occurrence of these alleles, which negatively affect male body condition and bovine tuberculosis resistance, throughout most of the buffalo's range were evaluated in this study. Using available microsatellite data (2-17 microsatellite loci) for 1676 animals from 34 localities (from 25°S to 5°N), we uncovered continent-wide frequency clines of microsatellite alleles associated with the aforementioned male traits. Frequencies decreased over a south-to-north latitude range (average per-locus Pearson r = -0.22). The frequency clines coincided with a multilocus-heterozygosity cline (adjusted R2 = 0.84), showing up to a 16% decrease in southern Africa compared to East Africa. Furthermore, continent-wide linkage disequilibrium (LD) at five linked locus pairs was detected, characterized by a high fraction of positive interlocus associations (0.66, 95% CI: 0.53, 0.77) between male-deleterious-trait-associated alleles. Our findings suggest continent-wide and genome-wide selection of male-deleterious alleles driven by an earlier observed sex-chromosomal meiotic drive system, resulting in frequency clines, reduced heterozygosity due to hitchhiking effects and extensive LD due to male-deleterious alleles co-occurring in haplotypes. The selection pressures involved must be high to prevent destruction of allele-frequency clines and haplotypes by LD decay. Since most buffalo populations are stable, these results indicate that natural mammal populations, depending on their genetic background, can withstand a high genetic load.

Wildlife roadkill patterns in a fragmented landscape of the Western Amazon.

One of the most evident and direct effects of roads on wildlife is the death of animals by vehicle collision. Understanding the spatial patterns behind roadkill helps to plan mitigation measures to reduce the impacts of roads on animal populations. However, although roadkill patterns have been extensively studied in temperate zones, the potential impacts of roads on wildlife in the Neotropics have received less attention and are particularly poorly understood in the Western Amazon. Here, we present the results of a study on roadkill in the Amazon region of Ecuador; a region that is affected by a rapidly increasing development of road infrastructure. Over the course of 50 days, in the wet season between September and November 2017, we searched for road-killed vertebrates on 15.9 km of roads near the city of Tena, Napo province, for a total of 1,590 surveyed kilometers. We recorded 593 dead specimens, predominantly reptiles (237 specimens, 40%) and amphibians (190, 32%), with birds (102, 17%) and mammals (64, 11%) being less common. Recorded species were assigned to three functional groups, based on their movement behavior and habitat use ("slow," "intermediate," and "fast"). Using Ripley's K statistical analyses and 2D HotSpot Identification Analysis, we found multiple distinct spatial clusters or hotspots, where roadkill was particularly frequent. Factors that potentially determined these clusters, and the prevalence of roadkill along road segments in general, differed between functional groups, but often included land cover variables such as native forest and waterbodies, and road characteristics such as speed limit (i.e., positive effect on roadkill frequency). Our study, which provides a first summary of species that are commonly found as roadkill in this part of the Amazon region, contributes to a better understanding of the negative impacts of roads on wildlife and is an important first step toward conservation efforts to mitigate these impacts.

Temporal Dynamics of Cloacal Microbiota in Adult Laying Chickens With and Without Access to an Outdoor Range.

Associations between animal health and performance, and the host's microbiota have been recently established. In poultry, changes in the intestinal microbiota have been linked to housing conditions and host development, but how the intestinal microbiota respond to environmental changes under farm conditions is less well understood. To gain insight into the microbial responses following a change in the host's immediate environment, we monitored four indoor flocks of adult laying chickens three times over 16 weeks, during which two flocks were given access to an outdoor range, and two were kept indoors. To assess changes in the chickens' microbiota over time, we collected cloacal swabs of 10 hens per flock and performed 16S rRNA gene amplicon sequencing. The poultry house (i.e., the stable in which flocks were housed) and sampling time explained 9.2 and 4.4% of the variation in the microbial community composition of the flocks, respectively. Remarkably, access to an outdoor range had no detectable effect on microbial community composition, the variability of microbiota among chickens of the same flock, or microbiota richness, but the microbiota of outdoor flocks became more even over time. Fluctuations in the composition of the microbiota over time within each poultry house were mainly driven by turnover in rare, rather than dominant, taxa and were unique for each flock. We identified 16 amplicon sequence variants that were differentially abundant over time between indoor and outdoor housed chickens, however none were consistently higher or lower across all chickens of one housing type over time. Our study shows that cloacal microbiota community composition in adult layers is stable following a sudden change in environment, and that temporal fluctuations are unique to each flock. By exploring microbiota of adult poultry flocks within commercial settings, our study sheds light on how the chickens' immediate environment affects the microbiota composition.

Limited changes in the fecal microbiome composition of laying hens after oral inoculation with wild duck feces.

Interspecies transmission of fecal microbiota can serve as an indicator for (indirect) contact between domestic and wild animals to assess risks of pathogen transmission, e.g., avian influenza. Here, we investigated whether oral inoculation of laying hens with feces of wild ducks (mallards, Anas platyrhynchos) resulted in a hen fecal microbiome that was detectably altered on community parameters or relative abundances of individual genera. To distinguish between effects of the duck inoculum and effects of the inoculation procedure, we compared the fecal microbiomes of adult laying hens resulting from 3 treatments: inoculation with wild duck feces (duck), inoculation with chicken feces (auto), and a negative control group with no treatment. We collected cloacal swabs from 7 hens per treatment before (day 0), and 2 and 7 D after inoculation, and performed 16S rRNA amplicon sequencing. No distinguishable effect of inoculation with duck feces on microbiome community (alpha and beta diversity) was found compared to auto or control treatments. At the individual taxonomic level, the relative abundance of the genus Alistipes (phylum Bacteroidetes) was significantly higher in the inoculated treatments (auto and duck) compared to the control 2 D after inoculation. Seven days after inoculation, the relative abundance of Alistipes had increased in the control and no effect was found anymore across treatments. These effects might be explained by the perturbation of the hen's microbiome caused by the inoculation procedure itself, or by intrinsic temporal variation in the hen's microbiome. This experiment shows that a single inoculation of fecal microbiota from duck feces to laying hens did not cause a measurable alteration of the gut microbiome community. Furthermore, the temporary change in relative abundance for Alistipes could not be attributed to the duck feces inoculation. These outcomes suggest that the fecal microbiome of adult laying hens may not be a useful indicator for detection of single oral exposure to wild duck feces.

A natural gene drive system influences bovine tuberculosis susceptibility in African buffalo: Possible implications for disease management.

Bovine tuberculosis (BTB) is endemic to the African buffalo (Syncerus caffer) of Hluhluwe-iMfolozi Park (HiP) and Kruger National Park, South Africa. In HiP, the disease has been actively managed since 1999 through a test-and-cull procedure targeting BTB-positive buffalo. Prior studies in Kruger showed associations between microsatellite alleles, BTB and body condition. A sex chromosomal meiotic drive, a form of natural gene drive, was hypothesized to be ultimately responsible. These associations indicate high-frequency occurrence of two types of male-deleterious alleles (or multiple-allele haplotypes). One type negatively affects body condition and BTB resistance in both sexes. The other type has sexually antagonistic effects: negative in males but positive in females. Here, we investigate whether a similar gene drive system is present in HiP buffalo, using 17 autosomal microsatellites and microsatellite-derived Y-chromosomal haplotypes from 401 individuals, culled in 2002-2004. We show that the association between autosomal microsatellite alleles and BTB susceptibility detected in Kruger, is also present in HiP. Further, Y-haplotype frequency dynamics indicated that a sex chromosomal meiotic drive also occurred in HiP. BTB was associated with negative selection of male-deleterious alleles in HiP, unlike positive selection in Kruger. Birth sex ratios were female-biased. We attribute negative selection and female-biased sex ratios in HiP to the absence of a Y-chromosomal sex-ratio distorter. This distorter has been hypothesized to contribute to positive selection of male-deleterious alleles and male-biased birth sex ratios in Kruger. As previously shown in Kruger, microsatellite alleles were only associated with male-deleterious effects in individuals born after wet pre-birth years; a phenomenon attributed to epigenetic modification. We identified two additional allele types: male-specific deleterious and beneficial alleles, with no discernible effect on females. Finally, we discuss how our findings may be used for breeding disease-free buffalo and implementing BTB test-and-cull programs.

Determining Mhc-DRB profiles in wild populations of three congeneric true lemur species by noninvasive methods.

The major histocompatibility complex (MHC) is a highly polymorphic and polygenic genomic region that plays a crucial role in immune-related diseases. Given the need for comparative studies on the variability of immunologically important genes among wild populations and species, we investigated the allelic variation of MHC class II DRB among three congeneric true lemur species: the red-fronted lemur (Eulemur rufifrons), red-bellied lemur (Eulemur rubriventer), and black lemur (Eulemur macaco). We noninvasively collected hair and faecal samples from these species across different regions in Madagascar. We assessed DRB exon 2 polymorphism with a newly developed primer set, amplifying nearly all non-synonymous codons of the antigen-binding sites. We defined 26 DRB alleles from 45 individuals (17 alleles from E. rufifrons (N = 18); 5 from E. rubriventer (N = 7); and 4 from E. macaco (N = 20). All detected alleles are novel and show high levels of nucleotide (26.8%) and non-synonymous codon polymorphism (39.4%). In these lemur species, we found neither evidence of a duplication of DRB genes nor a sharing of alleles among sympatric groups or allopatric populations of the same species. The non-sharing of alleles may be the result of a geographical separation over a long time span and/or different pathogen selection pressures. We found dN/dS rates > 1 in the functionally important antigen recognition sites, providing evidence for balancing selection. Especially for small and isolated populations, quantifying and monitoring DRB variation are recommended to establish successful conservation plans that mitigate the possible loss of immunogenetic diversity in lemurs.

Genetic insights into dispersal distance and disperser fitness of African lions (Panthera leo) from the latitudinal extremes of the Kruger National Park, South Africa.

BACKGROUND: Female lions generally do not disperse far beyond their natal range, while males can disperse distances of over 200 km. However, in bush-like ecosystems dispersal distances less than 25 km are reported. Here, we investigate dispersal in lions sampled from the northern and southern extremes of Kruger National Park, a bush-like ecosystem in South Africa where bovine tuberculosis prevalence ranges from low to high across a north-south gradient. RESULTS: A total of 109 individuals sampled from 1998 to 2004 were typed using 11 microsatellite markers, and mitochondrial RS-3 gene sequences were generated for 28 of these individuals. Considerable north-south genetic differentiation was observed in both datasets. Dispersal was male-biased and generally further than 25 km, with long-distance male gene flow (75-200 km, detected for two individuals) confirming that male lions can travel large distances, even in bush-like ecosystems. In contrast, females generally did not disperse further than 20 km, with two distinctive RS-3 gene clusters for northern and southern females indicating no or rare long-distance female dispersal. However, dispersal rate for the predominantly non-territorial females from southern Kruger (fraction dispersers ≥0.68) was higher than previously reported. Of relevance was the below-average body condition of dispersers and their low presence in prides, suggesting low fitness. CONCLUSIONS: Large genetic differences between the two sampling localities, and low relatedness among males and high dispersal rates among females in the south, suggestive of unstable territory structure and high pride turnover, have potential implications for spread of diseases and the management of the Kruger lion population.

Genetic responsiveness of African buffalo to environmental stressors: A role for epigenetics in balancing autosomal and sex chromosome interactions?

In the African buffalo (Syncerus caffer) population of the Kruger National Park (South Africa) a primary sex-ratio distorter and a primary sex-ratio suppressor have been shown to occur on the Y chromosome. A subsequent autosomal microsatellite study indicated that two types of deleterious alleles with a negative effect on male body condition, but a positive effect on relative fitness when averaged across sexes and generations, occur genome-wide and at high frequencies in the same population. One type negatively affects body condition of both sexes, while the other acts antagonistically: it negatively affects male but positively affects female body condition. Here we show that high frequencies of male-deleterious alleles are attributable to Y-chromosomal distorter-suppressor pair activity and that these alleles are suppressed in individuals born after three dry pre-birth years, likely through epigenetic modification. Epigenetic suppression was indicated by statistical interactions between pre-birth rainfall, a proxy for parental body condition, and the phenotypic effect of homozygosity/heterozygosity status of microsatellites linked to male-deleterious alleles, while a role for the Y-chromosomal distorter-suppressor pair was indicated by between-sex genetic differences among pre-dispersal calves. We argue that suppression of male-deleterious alleles results in negative frequency-dependent selection of the Y distorter and suppressor; a prerequisite for a stable polymorphism of the Y distorter-suppressor pair. The Y distorter seems to be responsible for positive selection of male-deleterious alleles during resource-rich periods and the Y suppressor for positive selection of these alleles during resource-poor periods. Male-deleterious alleles were also associated with susceptibility to bovine tuberculosis, indicating that Kruger buffalo are sensitive to stressors such as diseases and droughts. We anticipate that future genetic studies on African buffalo will provide important new insights into gene fitness and epigenetic modification in the context of sex-ratio distortion and infectious disease dynamics.

Occupancy strongly influences faecal microbial composition of wild lemurs.

The microbiota of the mammalian gut is a complex ecosystem, the composition of which is greatly influenced by host genetics and environmental factors. In this study, we aim to investigate the influence of occupancy (a geographical area of habitation), species, age and sex on intestinal microbiota composition of the three lemur species: Eulemur fulvus, E. rubriventer and E. rufifrons. Faecal samples were collected from a total of 138 wild lemurs across Madagascar, and microbial composition was determined using next-generation sequencing of PCR-amplified 16S rRNA gene fragments. Consistent with reports from other primate species, the predominant phyla were Firmicutes (43 ± 6.4% [s.d.]) and Bacteroidetes (30.3 ± 5.3%). The microbial composition was strongly associated with occupancy in the E. fulvus population, with up to 19.9% of the total variation in microbial composition being explained by this factor. In turn, geographical differences observed in faecal microbiota of sympatric lemur species were less pronounced, as was the impact of the factors sex and age. Our findings showed that among the studied factors occupancy had the strongest influence on intestinal microbiota of congeneric lemur species. This suggests adaptation of microbiota to differences in forest composition, climate variations and correspondingly available diet in different geographical locations of Madagascar.