Conservation management strategy impacts inbreeding and mutation load in scimitar-horned oryx.

In an age of habitat loss and overexploitation, small populations, both captive and wild, are increasingly facing the effects of isolation and inbreeding. Genetic management has therefore become a vital tool for ensuring population viability. However, little is known about how the type and intensity of intervention shape the genomic landscape of inbreeding and mutation load. We address this using whole-genome sequence data of the scimitar-horned oryx (Oryx dammah), an iconic antelope that has been subject to contrasting management strategies since it was declared extinct in the wild. We show that unmanaged populations are enriched for long runs of homozygosity (ROH) and have significantly higher inbreeding coefficients than managed populations. Additionally, despite the total number of deleterious alleles being similar across management strategies, the burden of homozygous deleterious genotypes was consistently higher in unmanaged groups. These findings emphasize the risks associated with deleterious mutations through multiple generations of inbreeding. As wildlife management strategies continue to diversify, our study reinforces the importance of maintaining genome-wide variation in vulnerable populations and has direct implications for one of the largest reintroduction attempts in the world.

How admixed captive breeding populations could be rescued using local ancestry information.

This paper asks the question: can genomic information be used to recover a species that is already on the pathway to extinction due to genetic swamping from a related and more numerous population? We show that a breeding strategy in a captive breeding program can use whole genome sequencing to identify and remove segments of DNA introgressed through hybridisation. The proposed policy uses a generalized measure of kinship or heterozygosity accounting for local ancestry, that is, whether a specific genetic location was inherited from the target of conservation. We then show that optimizing these measures would minimize undesired ancestry while also controlling kinship and/or heterozygosity, in a simulated breeding population. The process is applied to real data representing the hybridized Scottish wildcat breeding population, with the result that it should be possible to breed out domestic cat ancestry. The ability to reverse introgression is a powerful tool brought about through the combination of sequencing with computational advances in ancestry estimation. Since it works best when applied early in the process, important decisions need to be made about which genetically distinct populations should benefit from it and which should be left to reform into a single population.

Locus-specific introgression in young hybrid swarms: Drift may dominate selection.

Closely related species that have previously inhabited geographically separated ranges are hybridizing at an increasing rate due to human disruptions. These human-mediated hybrid zones can be used to study reproductive isolation between species at secondary contact, including examining locus-specific rates of introgression. Introgression is expected to be heterogenous across the genome, reflecting variation in selection. Those loci that introgress especially slowly are good candidates for being involved in reproductive isolation, while those loci that introgress quickly may be involved in adaptive introgression. In the context of conservation, policy makers are especially concerned about introduced alleles moving quickly into the background of a native or endemic species, as these alleles could replace the native alleles in the population, leading to extinction via hybridization. We applied genomic cline analyses to 44,997 SNPs to identify loci introgressing more or less when compared to the genome wide expectation in a human-mediated hybridizing population of red deer and sika in Kintyre Scotland. We found 11.4% of SNPs had cline centres that were significantly different from the genome wide expectation, and 17.6% of all SNPs had excess rates of introgression. Based on simulations, we believe that many of these markers have diverged from the genome-wide average due to drift, rather than because of selection, and we suggest that these simulations can be useful as a null distribution for future studies of genomic clines. Future work on red deer and sika could determine the policy implications of allelic-replacement due to drift rather than selection, and could use replicate, geographically distinct hybrid zones to narrow down those loci that are responding to selection.

On the use of genome-wide data to model and date the time of anthropogenic hybridisation: An example from the Scottish wildcat.

While hybridisation has long been recognised as an important natural phenomenon in evolution, the conservation of taxa subject to introgressive hybridisation from domesticated forms is a subject of intense debate. Hybridisation of Scottish wildcats and domestic cats is a good example in this regard. Here, we developed a modelling framework to determine the timescale of introgression using approximate Bayesian computation (ABC). Applying the model to ddRAD-seq data from 129 individuals, genotyped at 6546 loci, we show that a population of wildcats genetically distant from domestic cats is still present in Scotland. These individuals were found almost exclusively within the captive breeding programme. Most wild-living cats sampled were introgressed to some extent. The demographic model predicts high levels of gene-flow between domestic cats and Scottish wildcats (13% migrants per generation) over a short timeframe, the posterior mean for the onset of hybridisation (T1 ) was 3.3 generations (~10 years) before present. Although the model had limited power to detect signals of ancient admixture, we found evidence that significant recent hybridisation may have occurred subsequent to the founding of the captive breeding population (T2 ). The model consistently predicts T1 after T2 , estimated here to be 19.3 generations (~60 years) ago, highlighting the importance of this population as a resource for conservation management. Additionally, we evaluate the effectiveness of current methods to classify hybrids. We show that an optimised 35 SNP panel is a better predictor of the ddRAD-based hybrid score in comparison with a morphological method.

Phylogenomics and species delimitation for effective conservation of manta and devil rays.

Practical biodiversity conservation relies on delineation of biologically meaningful units. Manta and devil rays (Mobulidae) are threatened worldwide, yet morphological similarities and a succession of recent taxonomic changes impede the development of an effective conservation strategy. Here, we generate genome-wide single nucleotide polymorphism (SNP) data from a geographically and taxonomically representative set of manta and devil ray samples to reconstruct phylogenetic relationships and evaluate species boundaries under the general lineage concept. We show that nominal species units supported by alternative data sources constitute independently evolving lineages, and find robust evidence for a putative new species of manta ray in the Gulf of Mexico. Additionally, we uncover substantial incomplete lineage sorting indicating that rapid speciation together with standing variation in ancestral populations has driven phylogenetic uncertainty within Mobulidae. Finally, we detect cryptic diversity in geographically distinct populations, demonstrating that management below the species level may be warranted in certain species. Overall, our study provides a framework for molecular genetic species delimitation that is relevant to wide-ranging taxa of conservation concern, and highlights the potential for genomic data to support effective management, conservation and law enforcement strategies.

First evaluation of the population structure, genetic diversity and landscape connectivity of the Endangered Arabian tahr.

The Arabian tahr (Arabitragus jayakari) occurs only in the mountains of northern Oman and the United Arab Emirates. The species is classified as Endangered due to its small declining population. In this study, we combined genetic and landscape ecology techniques in order to inform landscape scale conservation and genetic management of Arabian tahr. Using 540 base pairs of mitochondrial control region in a dataset of 53 samples, we found eight haplotypes, which fell into two haplogroups. Population genetic analysis using a panel of 14 microsatellite loci also showed a weak, but significant division. Analyses of landscape connectivity supported the genetic results showing poor connectivity between populations in the far south of the study area and those in the north. The most likely location of corridors connecting Arabian tahr populations were identified. Many corridors between tahr populations are impeded by multi-lane highways and restoration of these connections is required to maintain population viability of Arabian tahr. Owing to limited genetic samples outside of Wadi Sareen, further sampling is needed to elucidate both mtDNA and the nuclear structure of Arabian tahr more fully. Our study provides a toolkit that may be used for future genetic and connectivity monitoring of the Arabian tahr population.

The genetic legacy of the first successful reintroduction of a mammal to Britain: Founder events and attempted genetic rescue in Scotland's beaver population.

Conservation translocations often inherently involve a risk of genetic diversity loss, and thus loss of adaptive potential, but this risk is rarely quantified or monitored through time. The reintroduction of beavers to Scotland, via the Scottish Beaver Trial in Knapdale, is an example of a translocation that took place in the absence of genetic data for the founder individuals and resulted in a small and suspected to be genetically depauperate population. In this study we use a high-density SNP panel to assess the genetic impact of that initial translocation and the effect of subsequent reinforcement translocations using animals from a different genetic source to the original founders. We demonstrate that the initial translocation did, indeed, lead to low genetic diversity (H o = 0.052) and high mean kinship (KING-robust = 0.159) in the Knapdale population compared to other beaver populations. We also show that the reinforcement translocations have succeeded in increasing genetic diversity (H o = 0.196) and reducing kinship (KING robust = 0.028) in Knapdale. As yet, there is no evidence of admixture between the two genetic lineages that are now present in Knapdale and such admixture is necessary to realise the full genetic benefits of the reinforcement and for genetic reinforcement and then rescue to occur; future genetic monitoring will be required to assess whether this has happened. We note that, should admixture occur, the Knapdale population will harbour combinations of genetic diversity not currently seen elsewhere in Eurasian beavers, posing important considerations for the future management of this population. We consider our results in the wider context of beaver conservation throughout Scotland and the rest of Britain, and advocate for more proactive genetic sampling of all founders to allow the full integration of genetic data into translocation planning in general.

Reference-free SNP discovery for the Eurasian beaver from restriction site-associated DNA paired-end data.

In this study, we used restriction site-associated DNA (RAD) sequencing to discover SNP markers suitable for population genetic and parentage analysis with the aim of using them for monitoring the reintroduction of the Eurasian beaver (Castor fibre) to Scotland. In the absence of a reference genome for beaver, we built contigs and discovered SNPs within them using paired-end RAD data, so as to have sufficient flanking region around the SNPs to conduct marker design. To do this, we used a simple pipeline which catalogued the Read 1 data in stacks and then used the assembler cortex_var to conduct de novo assembly and genotyping of multiple samples using the Read 2 data. The analysis of around 1.1 billion short reads of sequence data was reduced to a set of 2579 high-quality candidate SNP markers that were polymorphic in Norwegian and Bavarian beaver. Both laboratory validation of a subset of eight of the SNPs (1.3% error) and internal validation by confirming patterns of Mendelian inheritance in a family group (0.9% error) confirmed the success of this approach.

Genetic diversity in global populations of the critically endangered addax (Addax nasomaculatus) and its implications for conservation.

Threatened species are frequently patchily distributed across small wild populations, ex situ populations managed with varying levels of intensity and reintroduced populations. Best practice advocates for integrated management across in situ and ex situ populations. Wild addax (Addax nasomaculatus) now number fewer than 100 individuals, yet 1000 of addax remain in ex situ populations, which can provide addax for reintroductions, as has been the case in Tunisia since the mid-1980s. However, integrated management requires genetic data to ascertain the relationships between wild and ex situ populations that have incomplete knowledge of founder origins, management histories, and pedigrees. We undertook a global assessment of genetic diversity across wild, ex situ and reintroduced populations in Tunisia to assist conservation planning for this Critically Endangered species. We show that the remnant wild populations retain more mitochondrial haplotypes that are more diverse than the entirety of the ex situ populations across Europe, North America and the United Arab Emirates, and the reintroduced Tunisian population. Additionally, 1704 SNPs revealed that whilst population structure within the ex situ population is minimal, each population carries unique diversity. Finally, we show that careful selection of founders and subsequent genetic management is vital to ensure genetic diversity is provided to, and minimize drift and inbreeding within reintroductions. Our results highlight a vital need to conserve the last remaining wild addax population, and we provide a genetic foundation for determining integrated conservation strategies to prevent extinction and optimize future reintroductions.

Genetic swamping of the critically endangered Scottish wildcat was recent and accelerated by disease.

The European wildcat population in Scotland is considered critically endangered as a result of hybridization with introduced domestic cats,1,2 though the time frame over which this gene flow has taken place is unknown. Here, using genome data from modern, museum, and ancient samples, we reconstructed the trajectory and dated the decline of the local wildcat population from viable to severely hybridized. We demonstrate that although domestic cats have been present in Britain for over 2,000 years,3 the onset of hybridization was only within the last 70 years. Our analyses reveal that the domestic ancestry present in modern wildcats is markedly over-represented in many parts of the genome, including the major histocompatibility complex (MHC). We hypothesize that introgression provides wildcats with protection against diseases harbored and introduced by domestic cats, and that this selection contributes to maladaptive genetic swamping through linkage drag. Using the case of the Scottish wildcat, we demonstrate the importance of local ancestry estimates to both understand the impacts of hybridization in wild populations and support conservation efforts to mitigate the consequences of anthropogenic and environmental change.

Limited historical admixture between European wildcats and domestic cats.

Domestic cats were derived from the Near Eastern wildcat (Felis lybica), after which they dispersed with people into Europe. As they did so, it is possible that they interbred with the indigenous population of European wildcats (Felis silvestris). Gene flow between incoming domestic animals and closely related indigenous wild species has been previously demonstrated in other taxa, including pigs, sheep, goats, bees, chickens, and cattle. In the case of cats, a lack of nuclear, genome-wide data, particularly from Near Eastern wildcats, has made it difficult to either detect or quantify this possibility. To address these issues, we generated 75 ancient mitochondrial genomes, 14 ancient nuclear genomes, and 31 modern nuclear genomes from European and Near Eastern wildcats. Our results demonstrate that despite cohabitating for at least 2,000 years on the European mainland and in Britain, most modern domestic cats possessed less than 10% of their ancestry from European wildcats, and ancient European wildcats possessed little to no ancestry from domestic cats. The antiquity and strength of this reproductive isolation between introduced domestic cats and local wildcats was likely the result of behavioral and ecological differences. Intriguingly, this long-lasting reproductive isolation is currently being eroded in parts of the species' distribution as a result of anthropogenic activities.

Rescued back from extinction in the wild: past, present and future of the genetics of the Arabian oryx in Oman.

The Arabian oryx was the first species to be rescued from extinction in the wild by the concerted efforts of captive programmes in zoos and private collections around the world. Reintroduction efforts have used two main sources: the 'World Herd', established at the Phoenix Zoo, and private collections in Saudi Arabia. The breeding programme at the Al-Wusta Wildlife Reserve (WWR) in Oman has played a central role in the rescue of the oryx. Individuals from the 'World Herd' and the United Arab Emirates have been the main source for the WWR programme. However, no breeding strategies accounting for genetic diversity have been implemented. To address this, we investigated the diversity of the WWR population and historical samples using mitochondrial DNA (mtDNA) and single nucleotide polymorphisms (SNPs). We found individuals at WWR contain 58% of the total mtDNA diversity observed globally. Inference of ancestry and spatial patterns of SNP variation shows the presence of three ancestral sources and three different groups of individuals. Similar levels of diversity and low inbreeding were observed between groups. We identified individuals and groups that could most effectively contribute to maximizing genetic diversity. Our results will be valuable to guide breeding and reintroduction programmes at WWR.

Phylogeographical analysis shows the need to protect the wild yaks' last refuge in Nepal.

The wild yak Bos mutus was believed to be regionally extinct in Nepal for decades until our team documented two individuals from Upper Humla, north-western Nepal, in 2014. The International Union for Conservation of Nature (IUCN) seeks further evidence for the conclusive confirmation of that sighting. We conducted line transects and opportunistic sign surveys in the potential wild yak habitats of Humla, Dolpa, and Mustang districts between 2015 and 2017 and collected genetic samples (present and historic) of wild and domestic yaks Bos grunniens. We also sighted another wild yak in Upper Humla in 2015. Phylogenetic and haplotype network analyses based on mitochondrial D-loop sequences (~450 bp) revealed that wild yaks in Humla share the haplotype with wild yaks from the north-western region of the Qinghai-Tibetan Plateau in China. While hybridization with domestic yaks is a major long-term threat, illegal hunting for meat and trophy put the very small populations of wild yaks in Nepal at risk. Our study indicates that the unprotected habitat of Upper Humla is the last refuge for wild yaks in Nepal. We recommend wild yak conservation efforts in the country to focus on Upper Humla by (i) assigning a formal status of protected area to the region, (ii) raising awareness in the local communities for wild yak conservation, and (iii) providing support for adaptation of herding practice and pastureland use to ensure the viability of the population.

Genetic diversity of the Nubian ibex in Oman as revealed by mitochondrial DNA.

The Nubian ibex (Capra nubiana) is patchily distributed across parts of Africa and Arabia. In Oman, it is one of the few free-ranging wild mammals found in the central and southern regions. Its population is declining due to habitat degradation, human expansion, poaching and fragmentation. Here, we investigated the population's genetic diversity using mitochondrial DNA (D-loop 186 bp and cytochrome b 487 bp). We found that the Nubian ibex in the southern region of Oman was more diverse (D-loop HD; 0.838) compared with the central region (0.511) and gene flow between them was restricted. We compared the genetic profiles of wild Nubian ibex from Oman with captive ibex. A Bayesian phylogenetic tree showed that wild Nubian ibex form a distinct clade independent from captive animals. This divergence was supported by high mean distances (D-loop 0.126, cytochrome b 0.0528) and high F ST statistics (D-loop 0.725, cytochrome b 0.968). These results indicate that captive ibex are highly unlikely to have originated from the wild population in Oman and the considerable divergence suggests that the wild population in Oman should be treated as a distinct taxonomic unit. Further nuclear genetic work will be required to fully elucidate the degree of global taxonomic divergence of Nubian ibex populations.

Phenotypic correlates of hybridisation between red and sika deer (genus Cervus).

1. Hybridisation with an invasive species has the potential to alter the phenotype and hence the ecology of a native counterpart. 2. Here data from populations of native red deer Cervus elaphus and invasive sika deer Cervus nippon in Scotland is used to assess the extent to which hybridisation between them is causing phenotypic change. This is done by regression of phenotypic traits against genetic hybrid scores. 3. Hybridisation is causing increases in the body weight of sika-like deer and decreases in the body weight of red-like females. Hybridisation is causing increases in jaw length and increases in incisor arcade breadth in sika-like females. Hybridisation is also causing decreases in incisor arcade breadth in red-like females. 4. There is currently no evidence that hybridisation is causing changes in the kidney fat weight or pregnancy rates of either population. 5. Increased phenotypic similarity between the two species is likely to lead to further hybridisation. The ecological consequences of this are difficult to predict.

Increased genetic marker density reveals high levels of admixture between red deer and introduced Japanese sika in Kintyre, Scotland.

Hybridization is a natural process at species range boundaries, but increasing numbers of species are hybridizing due to direct or indirect human activities. In such cases of anthropogenic hybridization, subsequent introgression can threaten the survival of native species. To date, many such systems have been studied with too few genetic markers to assess the level of threat resulting from advanced backcrossing. Here, we use 44,999 single nucleotide polymorphisms (SNPs) and the ADMIXTURE program to study two areas of Scotland where a panel of 22 diagnostic microsatellites previously identified introgression between native red deer (Cervus elaphus) and introduced Japanese sika (Cervus nippon). In Kintyre, we reclassify 26% of deer from the pure species categories to the hybrid category whereas in the NW Highlands we only reclassify 2%. As expected, the reclassified individuals are mostly advanced backcrosses. We also investigate the ability of marker panels selected on different posterior allele frequency criteria to find hybrids assigned by the full marker set and show that in our data, ancestry informative markers (i.e. those that are highly differentiated between the species, but not fixed) are better than diagnostic markers (those markers that are fixed between the species) because they are more evenly distributed in the genome. Diagnostic loci are concentrated on the X chromosome to the detriment of autosomal coverage.

Chromosomal-level genome assembly of the scimitar-horned oryx: Insights into diversity and demography of a species extinct in the wild.

Captive populations provide a valuable insurance against extinctions in the wild. However, they are also vulnerable to the negative impacts of inbreeding, selection and drift. Genetic information is therefore considered a critical aspect of conservation management. Recent developments in sequencing technologies have the potential to improve the outcomes of management programmes; however, the transfer of these approaches to applied conservation has been slow. The scimitar-horned oryx (Oryx dammah) is a North African antelope that has been extinct in the wild since the early 1980s and is the focus of a large-scale and long-term reintroduction project. To enable the selection of suitable founder individuals, facilitate post-release monitoring and improve captive breeding management, comprehensive genomic resources are required. Here, we used 10X Chromium sequencing together with Hi-C contact mapping to develop a chromosomal-level genome assembly for the species. The resulting assembly contained 29 chromosomes with a scaffold N50 of 100.4 Mb, and displayed strong chromosomal synteny with the cattle genome. Using resequencing data from six additional individuals, we demonstrated relatively high genetic diversity in the scimitar-horned oryx compared to other mammals, despite it having experienced a strong founding event in captivity. Additionally, the level of diversity across populations varied according to management strategy. Finally, we uncovered a dynamic demographic history that coincided with periods of climate variation during the Pleistocene. Overall, our study provides a clear example of how genomic data can uncover valuable insights into captive populations and contributes important resources to guide future management decisions of an endangered species.

Variable extent of hybridization between invasive sika (Cervus nippon) and native red deer (C. elaphus) in a small geographical area.

Instances of hybridization between endemic and alien species pose a threat to species integrity but also provide us with an opportunity to study the dynamics of gene flow between two species as they first meet. Here, we used variation at 22 highly differentiated microsatellite loci and one mitochondrial DNA (mtDNA) marker in a sample of 735 individuals, to investigate the genetic consequences of an introduction of Japanese sika deer (Cervus nippon) for native red deer (C. elaphus) on the Kintyre Peninsula in Scotland. We investigated population structure, estimated null-allele frequency and assigned individual hybrid scores using a Bayesian clustering algorithm implemented in structure 2.2. The dataset clearly divided into two clusters and generally, introgression into red and sika was low. However at one site, West Loch Awe, 43% of individuals were hybrids. MtDNA introgression indicated that hybridization was occurring between red-deer hinds and sika-deer stags. We argue that the pattern of differential introgression across the study area is primarily due to the rarity of hybridization events between the two species and the limited time the two species have been in contact (< 120 years). This contrasts with the causes of classic mosaic hybrid zones (selection induced by habitat variability). Currently, it seems possible that, in time, the level of hybridization found at West Loch Awe could also be found across the whole of the peninsula.

Distinguishing the victim from the threat: SNP-based methods reveal the extent of introgressive hybridization between wildcats and domestic cats in Scotland and inform future in situ and ex situ management options for species restoration.

The degree of introgressive hybridization between the Scottish wildcat and domestic cat has long been suspected to be advanced. Here, we use a 35-SNP-marker test, designed to assess hybridization between wildcat and domestic cat populations in Scotland, to assess a database of 295 wild-living and captive cat samples, and test the assumptions of the test using 3,097 SNP markers generated independently in a subset of the data using ddRAD. We discovered that despite increased genetic resolution provided by these methods, wild-living cats in Scotland show a complete genetic continuum or hybrid swarm structure when judged against reference data. The historical population of wildcats, although hybridized, clearly groups at one end of this continuum, as does the captive population of wildcats. The interpretation of pelage scores against nuclear genetic data continues to be problematic. This is probably because of a breakdown in linkage equilibrium between wildcat pelage genes as the two populations have become increasingly mixed, meaning that pelage score or SNP score alone is poor diagnostic predictors of hybrid status. Until better tools become available, both should be used jointly, where possible, when making management decisions about individual cats. We recommend that the conservation community in Scotland must now define clearly what measures are to be used to diagnose a wildcat in the wild in Scotland, if future conservation action is to be effective.

Improving cost-efficiency of faecal genotyping: New tools for elephant species.

Despite the critical need for non-invasive tools to improve monitoring of wildlife populations, especially for endangered and elusive species, faecal genetic sampling has not been adopted as regular practice, largely because of the associated technical challenges and cost. Substantial work needs to be undertaken to refine sample collection and preparation methods in order to improve sample set quality and provide cost-efficient tools that can effectively support wildlife management. In this study, we collected an extensive set of forest elephant (Loxodonta cyclotis) faecal samples throughout Gabon, Central Africa, and prepared them for genotyping using 107 single-nucleotide polymorphism assays. We developed a new quantitative polymerase chain reaction (PCR) assay targeting a 130-bp nuclear DNA fragment and demonstrated its suitability for degraded samples in all three elephant species. Using this assay to compare the efficacy of two sampling methods for faecal DNA recovery, we found that sampling the whole surface of a dung pile with a swab stored in a small tube of lysis buffer was a convenient method producing high extraction success and DNA yield. We modelled the influence of faecal quality and storage time on DNA concentration in order to provide recommendations for optimized collection and storage. The maximum storage time to ensure 75% success was two months for samples collected within 24 hours after defecation and extended to four months for samples collected within one hour. Lastly, the real-time quantitative PCR assay allowed us to predict genotyping success and pre-screen DNA samples, thus further increasing the cost-efficiency of our approach. We recommend combining the validation of an efficient sampling method, the build of in-country DNA extraction capacity for reduced storage time and the development of species-specific quantitative PCR assays in order to increase the cost-efficiency of routine non-invasive DNA analyses and expand the use of next-generation markers to non-invasive samples.