Recent increase in species-wide diversity after interspecies introgression in the highly endangered Iberian lynx.

Genetic diversity is lost in small and isolated populations, affecting many globally declining species. Interspecific admixture events can increase genetic variation in the recipient species' gene pool, but empirical examples of species-wide restoration of genetic diversity by admixture are lacking. Here we present multi-fold coverage genomic data from three ancient Iberian lynx (Lynx pardinus) approximately 2,000-4,000 years old and show a continuous or recurrent process of interspecies admixture with the Eurasian lynx (Lynx lynx) that increased modern Iberian lynx genetic diversity above that occurring millennia ago despite its recent demographic decline. Our results add to the accumulating evidence for natural admixture and introgression among closely related species and show that this can result in an increase of species-wide genetic diversity in highly genetically eroded species. The strict avoidance of interspecific sources in current genetic restoration measures needs to be carefully reconsidered, particularly in cases where no conspecific source population exists.

Monitoring of species' genetic diversity in Europe varies greatly and overlooks potential climate change impacts.

Genetic monitoring of populations currently attracts interest in the context of the Convention on Biological Diversity but needs long-term planning and investments. However, genetic diversity has been largely neglected in biodiversity monitoring, and when addressed, it is treated separately, detached from other conservation issues, such as habitat alteration due to climate change. We report an accounting of efforts to monitor population genetic diversity in Europe (genetic monitoring effort, GME), the evaluation of which can help guide future capacity building and collaboration towards areas most in need of expanded monitoring. Overlaying GME with areas where the ranges of selected species of conservation interest approach current and future climate niche limits helps identify whether GME coincides with anticipated climate change effects on biodiversity. Our analysis suggests that country area, financial resources and conservation policy influence GME, high values of which only partially match species' joint patterns of limits to suitable climatic conditions. Populations at trailing climatic niche margins probably hold genetic diversity that is important for adaptation to changing climate. Our results illuminate the need in Europe for expanded investment in genetic monitoring across climate gradients occupied by focal species, a need arguably greatest in southeastern European countries. This need could be met in part by expanding the European Union's Birds and Habitats Directives to fully address the conservation and monitoring of genetic diversity.

Genome-environment association analyses reveal geographically restricted adaptive divergence across the range of the widespread Eurasian carnivore Lynx lynx (Linnaeus, 1758).

Local adaptations to the environment are an important aspect of the diversity of a species and their discovery, description and quantification has important implications for the fields of taxonomy, evolutionary and conservation biology. In this study, we scan genomes from several populations across the distributional range of the Eurasian lynx, with the objective of finding genomic windows under positive selection which may underlie local adaptations to different environments. A total of 394 genomic windows are found to be associated to local environmental conditions, and they are enriched for genes involved in metabolism, behaviour, synaptic organization and neural development. Adaptive genetic structure, reconstructed from SNPs in candidate windows, is considerably different than the neutral genetic structure of the species. A widespread adaptively homogeneous group is recovered occupying areas of harsher snow and temperature climatic conditions in the north-western, central and eastern parts of the distribution. Adaptively divergent populations are recovered in the westernmost part of the range, especially within the Baltic population, but also predicted for different patches in the western and southern part of the range, associated with different snow and temperature regimes. Adaptive differentiation driven by climate does not correlate much with the subspecies taxonomic delimitations, suggesting that subspecific divergences are mostly driven by neutral processes of genetic drift and gene flow. Our results will aid the selection of source populations for assisted gene flow or genetic rescue programs by identifying what climatic patterns to look for as predictors of pre-adaptation of individuals. Particularly, the Carpathian population is confirmed as the best source of individuals for the genetic rescue of the endangered, isolated and genetically eroded Balkan population. Additionally, reintroductions in central and western Europe, currently based mostly on Carpathian lynxes, could consider the Baltic population as an additional source to increase adaptive variation and likely improve adaptation to their milder climate.

How genomics can help biodiversity conservation.

The availability of public genomic resources can greatly assist biodiversity assessment, conservation, and restoration efforts by providing evidence for scientifically informed management decisions. Here we survey the main approaches and applications in biodiversity and conservation genomics, considering practical factors, such as cost, time, prerequisite skills, and current shortcomings of applications. Most approaches perform best in combination with reference genomes from the target species or closely related species. We review case studies to illustrate how reference genomes can facilitate biodiversity research and conservation across the tree of life. We conclude that the time is ripe to view reference genomes as fundamental resources and to integrate their use as a best practice in conservation genomics.

Purging of deleterious burden in the endangered Iberian lynx.

SignificanceThe dynamics of deleterious variation under contrasting demographic scenarios remain poorly understood in spite of their relevance in evolutionary and conservation terms. Here we apply a genomic approach to study differences in the burden of deleterious alleles between the endangered Iberian lynx (Lynx pardinus) and the widespread Eurasian lynx (Lynx lynx). Our analysis unveils a significantly lower deleterious burden in the former species that should be ascribed to genetic purging, that is, to the increased opportunities of selection against recessive homozygotes due to the inbreeding caused by its smaller population size, as illustrated by our analytical predictions. This research provides theoretical and empirical evidence on the evolutionary relevance of genetic purging under certain demographic conditions.

The era of reference genomes in conservation genomics.

Progress in genome sequencing now enables the large-scale generation of reference genomes. Various international initiatives aim to generate reference genomes representing global biodiversity. These genomes provide unique insights into genomic diversity and architecture, thereby enabling comprehensive analyses of population and functional genomics, and are expected to revolutionize conservation genomics.

Bottleneck-associated changes in the genomic landscape of genetic diversity in wild lynx populations.

Demographic bottlenecks generally reduce genetic diversity through more intense genetic drift, but their net effect may vary along the genome due to the random nature of genetic drift and to local effects of recombination, mutation, and selection. Here, we analyzed the changes in genetic diversity following a bottleneck by comparing whole-genome diversity patterns in populations with and without severe recent documented declines of Iberian (Lynx pardinus, n = 31) and Eurasian lynx (Lynx lynx, n = 29). As expected, overall genomic diversity correlated negatively with bottleneck intensity and/or duration. Correlations of genetic diversity with divergence, chromosome size, gene or functional site content, GC content, or recombination were observed in nonbottlenecked populations, but were weaker in bottlenecked populations. Also, functional features under intense purifying selection and the X chromosome showed an increase in the observed density of variants, even resulting in higher θ W diversity than in nonbottlenecked populations. Increased diversity seems to be related to both a higher mutational input in those regions creating a large collection of low-frequency variants, a few of which increase in frequency during the bottleneck to the point they become detectable with our limited sample, and the reduced efficacy of purifying selection, which affects not only protein structure and function but also the regulation of gene expression. The results of this study alert to the possible reduction of fitness and adaptive potential associated with the genomic erosion in regulatory elements. Further, the detection of a gain of diversity in ultra-conserved elements can be used as a sensitive and easy-to-apply signature of genetic erosion in wild populations.

Benign juvenile idiopathic epilepsy in captive Iberian lynx (Lynx pardinus) in the ex situ conservation program (2005-2019).

BACKGROUND: Benign juvenile idiopathic epilepsy has been described in humans but rarely in animals. The objectives of the study were to describe the clinical signs, clinical data, imaging findings, genetic examinations, treatment, long-term outcome and prognosis in Iberian lynx with juvenile epilepsy. Medical records, video recordings and diagnostic data from 2005 to 2019 were reviewed. RESULTS: Twenty lynx cubs with early onset of epileptic seizures (ES) from the conservation program were included. The average age at seizure onset was 75 days. Isolated and cluster ES were recorded. Focal ES, focal ES evolving into generalized ES with a stereotypical pattern and generalized ES were observed. All the cubs were normal between episodes, had a normal neurological examination and unremarkable investigations. Phenobarbital was used as a first line antiepileptic drug (AED). ES halted 10 days (0-34) after starting treatment in eight out of twenty cubs (40%). Treatment was discontinued in this group after a mean of 578 days and no further ES were reported (mean follow-up longer than 5 years). Eleven animals (55%) continued on AED treatment for a mean of 1306 days (70-3466). An adult-onset was observed for one lynx (5%). Polytherapy was necessary in seven lynxes (35%). The inheritance pattern observed was compatible with an autosomal recessive condition. Based on this assumption, mating between two identified carriers has been avoided since 2012, which may have contributed to the subsequent decrease in prevalence, with no further cases detected in 2018 and 2019. CONCLUSIONS: Lynx pardinus may have an early onset self-limiting ES syndrome characteristic of benign juvenile idiopathic epilepsy. Information obtained from this study strongly suggests a genetic basis for the here presented epilepsy.

Chromosomal Differentiation in Genetically Isolated Populations of the Marsh-Specialist Crocidura suaveolens (Mammalia: Soricidae).

The genus Crocidura represents a remarkable model for the study of chromosome evolution. This is the case of the lesser white-toothed shrew (Crocidura suaveolens), a representative of the Palearctic group. Although continuously distributed from Siberia to Central Europe, C. suaveolens is a rare, habitat-specialist species in the southwesternmost limit of its distributional range, in the Gulf of Cádiz (Iberian Peninsula). In this area, C. suaveolens is restricted to genetically isolated populations associated to the tidal marches of five rivers (Guadiana, Piedras, Odiel, Tinto and Guadalquivir). This particular distributional range provides a unique opportunity to investigate whether genetic differentiation and habitat specialization was accompanied by chromosomal variation. In this context, the main objective of this study was to determinate the chromosomal characteristics of the habitat-specialist C. suaveolens in Southwestern Iberia, as a way to understand the evolutionary history of this species in the Iberian Peninsula. A total of 41 individuals from six different populations across the Gulf of Cádiz were collected and cytogenetically characterized. We detected four different karyotypes, with diploid numbers (2n) ranging from 2n = 40 to 2n = 43. Two of them (2n = 41 and 2n = 43) were characterized by the presence of B-chromosomes. The analysis of karyotype distribution across lineages and populations revealed an association between mtDNA population divergence and chromosomal differentiation. C. suaveolens populations in the Gulf of Cádiz provide a rare example of true karyotypic polymorphism potentially associated to genetic isolation and habitat specialization in which to investigate the evolutionary significance of chromosomal variation in mammals and their contribution to phenotypic and ecological divergence.

Genomic patterns in the widespread Eurasian lynx shaped by Late Quaternary climatic fluctuations and anthropogenic impacts.

Disentangling the contribution of long-term evolutionary processes and recent anthropogenic impacts to current genetic patterns of wildlife species is key to assessing genetic risks and designing conservation strategies. Here, we used 80 whole nuclear genomes and 96 mitogenomes from populations of the Eurasian lynx covering a range of conservation statuses, climatic zones and subspecies across Eurasia to infer the demographic history, reconstruct genetic patterns, and discuss the influence of long-term isolation and/or more recent human-driven changes. Our results show that Eurasian lynx populations shared a common history until 100,000 years ago, when Asian and European populations started to diverge and both entered a period of continuous and widespread decline, with western populations, except Kirov, maintaining lower effective sizes than eastern populations. Population declines and increased isolation in more recent times probably drove the genetic differentiation between geographically and ecologically close westernmost European populations. By contrast, and despite the wide range of habitats covered, populations are quite homogeneous genetically across the Asian range, showing a pattern of isolation by distance and providing little genetic support for the several proposed subspecies. Mitogenomic and nuclear divergences and population declines starting during the Late Pleistocene can be mostly attributed to climatic fluctuations and early human influence, but the widespread and sustained decline since the Holocene is more probably the consequence of anthropogenic impacts which intensified in recent centuries, especially in western Europe. Genetic erosion in isolated European populations and lack of evidence for long-term isolation argue for the restoration of lost population connectivity.

Genetic rescue by distant trees mitigates qualitative pollen limitation imposed by fine-scale spatial genetic structure.

Restricted seed dispersal frequently leads to fine-scale spatial genetic structure (i.e., FSGS) within plant populations. Depending on its spatial extent and the mobility of pollinators, this inflated kinship at the immediate neighbourhood can critically impoverish pollen quality. Despite the common occurrence of positive FSGS within plant populations, our knowledge regarding the role of long-distance pollination preventing reproductive failure is still limited. Using microsatellite markers, we examined the existence of positive FSGS in two low-density populations of the tree Pyrus bourgaeana. We also designed controlled crosses among trees differing in their kinship to investigate the effects of increased local kinship on plant reproduction. We used six pollination treatments and fully monitored fruit production, fruit and seed weight, proportion of mature seeds per fruit, and seed germination. Our results revealed positive FSGS in both study populations and lower fruit initiation in flowers pollinated with pollen from highly-genetically related individuals within the neighbourhood, with this trend intensifying as the fruit development progressed. Besides, open-pollinated flowers exhibited lower performance compared to those pollinated by distant pollen donors, suggesting intense qualitative pollen limitation in natural populations. We found positive fine-scale spatial genetic structure is translated into impoverished pollen quality from nearby pollen donors which negatively impacts the reproductive success of trees in low-density populations. Under this scenario of intrapopulation genetic rescue by distant pollen donors, the relevance of highly-mobile pollinators for connecting spatially and genetically distant patches of trees may be crucial to safeguarding population recruitment.

Genetic evaluation of the Iberian lynx ex situ conservation programme.

Ex situ programmes have become critical for improving the conservation of many threatened species, as they establish backup populations and provide individuals for reintroduction and reinforcement of wild populations. The Iberian lynx was considered the most threatened felid species in the world in the wake of a dramatic decline during the second half of the 20th century that reduced its numbers to around only 100 individuals. An ex situ conservation programme was established in 2003 with individuals from the two well-differentiated, remnant populations, with great success from a demographic point of view. Here, we evaluate the genetic status of the Iberian lynx captive population based on molecular data from 36 microsatellites, including patterns of relatedness and representativeness of the two remnant genetic backgrounds among founders, the evolution of diversity and inbreeding over the years, and genetic differentiation among breeding facilities. In general terms, the ex situ population harbours most of the genetic variability found in the two wild populations and has been able to maintain reasonably low levels of inbreeding and high diversity, thus validating the applied management measures and potentially representing a model for other species in need of conservation.

Spatiotemporal Dynamics of Genetic Variation in the Iberian Lynx along Its Path to Extinction Reconstructed with Ancient DNA.

There is the tendency to assume that endangered species have been both genetically and demographically healthier in the past, so that any genetic erosion observed today was caused by their recent decline. The Iberian lynx (Lynx pardinus) suffered a dramatic and continuous decline during the 20th century, and now shows extremely low genome- and species-wide genetic diversity among other signs of genomic erosion. We analyze ancient (N = 10), historical (N = 245), and contemporary (N = 172) samples with microsatellite and mitogenome data to reconstruct the species' demography and investigate patterns of genetic variation across space and time. Iberian lynx populations transitioned from low but significantly higher genetic diversity than today and shallow geographical differentiation millennia ago, through a structured metapopulation with varying levels of diversity during the last centuries, to two extremely genetically depauperate and differentiated remnant populations by 2002. The historical subpopulations show varying extents of genetic drift in relation to their recent size and time in isolation, but these do not predict whether the populations persisted or went finally extinct. In conclusion, current genetic patterns were mainly shaped by genetic drift, supporting the current admixture of the two genetic pools and calling for a comprehensive genetic management of the ongoing conservation program. This study illustrates how a retrospective analysis of demographic and genetic patterns of endangered species can shed light onto their evolutionary history and this, in turn, can inform conservation actions.

Novel efficient genome-wide SNP panels for the conservation of the highly endangered Iberian lynx.

BACKGROUND: The Iberian lynx (Lynx pardinus) has been acknowledged as the most endangered felid species in the world. An intense contraction and fragmentation during the twentieth century left less than 100 individuals split in two isolated and genetically eroded populations by 2002. Genetic monitoring and management so far have been based on 36 STRs, but their limited variability and the more complex situation of current populations demand more efficient molecular markers. The recent characterization of the Iberian lynx genome identified more than 1.6 million SNPs, of which 1536 were selected and genotyped in an extended Iberian lynx sample. METHODS: We validated 1492 SNPs and analysed their heterozygosity, Hardy-Weinberg equilibrium, and linkage disequilibrium. We then selected a panel of 343 minimally linked autosomal SNPs from which we extracted subsets optimized for four different typical tasks in conservation applications: individual identification, parentage assignment, relatedness estimation, and admixture classification, and compared their power to currently used STR panels. RESULTS: We ascribed 21 SNPs to chromosome X based on their segregation patterns, and identified one additional marker that showed significant differentiation between sexes. For all applications considered, panels of autosomal SNPs showed higher power than the currently used STR set with only a very modest increase in the number of markers. CONCLUSIONS: These novel panels of highly informative genome-wide SNPs provide more powerful, efficient, and flexible tools for the genetic management and non-invasive monitoring of Iberian lynx populations. This example highlights an important outcome of whole-genome studies in genetically threatened species.

Retention of functional variation despite extreme genomic erosion: MHC allelic repertoires in the Lynx genus.

BACKGROUND: Demographic bottlenecks erode genetic diversity and may increase endangered species' extinction risk via decreased fitness and adaptive potential. The genetic status of species is generally assessed using neutral markers, whose dynamic can differ from that of functional variation due to selection. The MHC is a multigene family described as the most important genetic component of the mammalian immune system, with broad implications in ecology and evolution. The genus Lynx includes four species differing immensely in demographic history and population size, which provides a suitable model to study the genetic consequences of demographic declines: the Iberian lynx being an extremely bottlenecked species and the three remaining ones representing common and widely distributed species. We compared variation in the most variable exon of the MHCI and MHCII-DRB loci among the four species of the Lynx genus. RESULTS: The Iberian lynx was characterised by lower number of MHC alleles than its sister species (the Eurasian lynx). However, it maintained most of the functional genetic variation at MHC loci present in the remaining and genetically healthier lynx species at all nucleotide, amino acid, and supertype levels. CONCLUSIONS: Species-wide functional genetic diversity can be maintained even in the face of severe population bottlenecks, which caused devastating whole genome genetic erosion. This could be the consequence of divergent alleles being retained across paralogous loci, an outcome that, in the face of frequent gene conversion, may have been favoured by balancing selection.

Extreme genomic erosion after recurrent demographic bottlenecks in the highly endangered Iberian lynx.

BACKGROUND: Genomic studies of endangered species provide insights into their evolution and demographic history, reveal patterns of genomic erosion that might limit their viability, and offer tools for their effective conservation. The Iberian lynx (Lynx pardinus) is the most endangered felid and a unique example of a species on the brink of extinction. RESULTS: We generate the first annotated draft of the Iberian lynx genome and carry out genome-based analyses of lynx demography, evolution, and population genetics. We identify a series of severe population bottlenecks in the history of the Iberian lynx that predate its known demographic decline during the 20th century and have greatly impacted its genome evolution. We observe drastically reduced rates of weak-to-strong substitutions associated with GC-biased gene conversion and increased rates of fixation of transposable elements. We also find multiple signatures of genetic erosion in the two remnant Iberian lynx populations, including a high frequency of potentially deleterious variants and substitutions, as well as the lowest genome-wide genetic diversity reported so far in any species. CONCLUSIONS: The genomic features observed in the Iberian lynx genome may hamper short- and long-term viability through reduced fitness and adaptive potential. The knowledge and resources developed in this study will boost the research on felid evolution and conservation genomics and will benefit the ongoing conservation and management of this emblematic species.

PCR Strategies for Complete Allele Calling in Multigene Families Using High-Throughput Sequencing Approaches.

The characterization of multigene families with high copy number variation is often approached through PCR amplification with highly degenerate primers to account for all expected variants flanking the region of interest. Such an approach often introduces PCR biases that result in an unbalanced representation of targets in high-throughput sequencing libraries that eventually results in incomplete detection of the targeted alleles. Here we confirm this result and propose two different amplification strategies to alleviate this problem. The first strategy (called pooled-PCRs) targets different subsets of alleles in multiple independent PCRs using different moderately degenerate primer pairs, whereas the second approach (called pooled-primers) uses a custom-made pool of non-degenerate primers in a single PCR. We compare their performance to the common use of a single PCR with highly degenerate primers using the MHC class I of the Iberian lynx as a model. We found both novel approaches to work similarly well and better than the conventional approach. They significantly scored more alleles per individual (11.33 ± 1.38 and 11.72 ± 0.89 vs 7.94 ± 1.95), yielded more complete allelic profiles (96.28 ± 8.46 and 99.50 ± 2.12 vs 63.76 ± 15.43), and revealed more alleles at a population level (13 vs 12). Finally, we could link each allele's amplification efficiency with the primer-mismatches in its flanking sequences and show that ultra-deep coverage offered by high-throughput technologies does not fully compensate for such biases, especially as real alleles may reach lower coverage than artefacts. Adopting either of the proposed amplification methods provides the opportunity to attain more complete allelic profiles at lower coverages, improving confidence over the downstream analyses and subsequent applications.

Heterogeneous road networks have no apparent effect on the genetic structure of small mammal populations.

Roads are widely recognized to represent a barrier to individual movements and, conversely, verges can act as potential corridors for the dispersal of many small mammals. Both barrier and corridor effects should generate a clear spatial pattern in genetic structure. Nevertheless, the effect of roads on the genetic structure of small mammal populations still remains unclear. In this study, we examine the barrier effect that different road types (4-lane highway, 2-lane roads and single-lane unpaved roads) may have on the population genetic structure of three species differing in relevant life history traits: southern water vole Arvicola sapidus, the Mediterranean pine vole Microtus duodecimcostatus and the Algerian mouse Mus spretus. We also examine the corridor effect of highway verges on the Mediterranean pine vole and the Algerian mouse. We analysed the population structure through pairwise estimates of FST among subpopulations bisected by roads, identified genetic clusters through Bayesian assignment approaches, and used simple and partial Mantel tests to evaluate the relative barrier or corridor effect of roads. No strong evidences were found for an effect of roads on population structure of these three species. The barrier effect of roads seems to be site-specific and no corridor effect of verges was found for the pine vole and Algerian mouse populations. The lack of consistent results among species and for each road type lead us to believe that the ability of individual dispersers to use those crossing structures or the habitat quality in the highway verges may have a relatively higher influence on gene flow among populations than the presence of crossing structures per se. Further research should include microhabitat analysis and the estimates of species abundance to understand the mechanisms that underlie the genetic structure observed at some sites.

Individual identification of endangered species using mosquito blood meals: a proof-of-concept study in Iberian lynx.

Host identification from mosquito blood meals has been routinely used to identify the feeding preferences of insects in studies on transmission of vector-borne pathogens. Here, we identified for the first time the susceptibility of the endangered Iberian lynx (Lynx pardinus) to the attack of a wild mosquito female, the mosquito Anopheles atroparvus. Furthermore, we used 11 microsatellite markers to test for the utility of vertebrate DNA isolated from insect blood meals for individual identification of wildlife. Only the three smallest markers were successfully amplified; however, this genotype did not match with any of the previously genotyped individuals in southern Spain. These results support the use of DNA from mosquito blood meals as a non-invasive source of DNA and a powerful tool on epidemiological and conservation biology studies. However, as may be the case of other non-invasive sampling methods, the utility of this technique is probably limited by the quantity and quality of vertebrate DNA.

Joint effects of population size and isolation on genetic erosion in fragmented populations: finding fragmentation thresholds for management.

Size and isolation of local populations are main parameters of interest when assessing the genetic consequences of habitat fragmentation. However, their relative influence on the genetic erosion of local populations remains unclear. In this study, we first analysed how size and isolation of habitat patches influence the genetic variation of local populations of the Dupont's lark (Chersophilus duponti), an endangered songbird. An information-theoretic approach to model selection allowed us to address the importance of interactions between habitat variables, an aspect seldom considered in fragmentation studies, but which explained up to 65% of the variance in genetic parameters. Genetic diversity and inbreeding were influenced by the size of local populations depending on their degree of isolation, and genetic differentiation was positively related to isolation. We then identified a minimum local population of 19 male territories and a maximum distance of 30 km to the nearest population as thresholds from which genetic erosion becomes apparent. Our results alert on possibly misleading conclusions and suboptimal management recommendations when only additive effects are taken into account and encourage the use of most explanatory but easy-to-measure variables for the evaluation of genetic risks in conservation programmes.