Genomic signatures of climate adaptation in bank voles.

Evidence for divergent selection and adaptive variation across the landscape can provide insight into a species' ability to adapt to different environments. However, despite recent advances in genomics, it remains difficult to detect the footprints of climate-mediated selection in natural populations. Here, we analysed ddRAD sequencing data (21,892 SNPs) in conjunction with geographic climate variation to search for signatures of adaptive differentiation in twelve populations of the bank vole (Clethrionomys glareolus) distributed across Europe. To identify the loci subject to selection associated with climate variation, we applied multiple genotype-environment association methods, two univariate and one multivariate, and controlled for the effect of population structure. In total, we identified 213 candidate loci for adaptation, 74 of which were located within genes. In particular, we identified signatures of selection in candidate genes with functions related to lipid metabolism and the immune system. Using the results of redundancy analysis, we demonstrated that population history and climate have joint effects on the genetic variation in the pan-European metapopulation. Furthermore, by examining only candidate loci, we found that annual mean temperature is an important factor shaping adaptive genetic variation in the bank vole. By combining landscape genomic approaches, our study sheds light on genome-wide adaptive differentiation and the spatial distribution of variants underlying adaptive variation influenced by local climate in bank voles.

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.

Museum DNA reveals a new, potentially extinct species of rinkhals (Serpentes: Elapidae: Hemachatus) from the Eastern Highlands of Zimbabwe.

Genetic information plays a pivotal role in species recognition and delimitation, but rare or extinct animals can be difficult to obtain genetic samples from. While natural history wet collections have proven invaluable in the description of novel species, the use of these historical samples in genetic studies has been greatly impeded by DNA degradation, especially because of formalin-fixation prior to preservation. Here, we use recently developed museum genomics approaches to determine the status of an isolated population of the elapid snake genus Hemachatus from Zimbabwe. We used multiple digestion phases followed by single strand sequencing library construction and hybridisation capture to obtain 12S and 16S rDNA sequences from a poorly preserved tissue sample of this population. Phylogenetic and morphological analyses in an integrated taxonomic framework demonstrate that the Zimbabwean rinkhals population represents an old and highly distinct lineage, which we describe as a new species, Hemachatus nyangensis sp. nov. Our phylogenetic dating analysis is compatible with venom spitting having evolved in response to the threat posed by early hominins, although more data are required for a robust test of this hypothesis. This description demonstrates the power of museum genomics in revealing rare or even extinct species: Hemachatus from Zimbabwe are only known from a small area of the Eastern Highlands known for high endemism. No living specimens have been seen since the 1980s, most likely due to dramatic land-use changes in the Eastern Highlands, suggesting that the species could be extinct. In view of its recognition as a highly distinct lineage, urgent action is required to determine whether any populations survive, and to safeguard remaining habitat.

Correction: Insight into the introduction of domestic cattle and the process of Neolithization to the Spanish region Galicia by genetic evidence.

[This corrects the article DOI: 10.1371/journal.pone.0249537.].

Revisiting proboscidean phylogeny and evolution through total evidence and palaeogenetic analyses including Notiomastodon ancient DNA.

The extinct Gomphotheriidae is the only proboscidean family that colonized South America. The phylogenetic position of the endemic taxa has been through several revisions using morphological comparisons. Morphological studies are enhanced by paleogenetic analyses, a powerful tool to resolve phylogenetic relationships; however, ancient DNA (aDNA) preservation decreases in warmer regions. Despite the poor preservation conditions for aDNA in humid, sub-tropical climates, we recovered ∼3,000 bp of mtDNA of Notiomastodon platensis from the Arroyo del Vizcaíno site, Uruguay. Our calibrated phylogeny places Notiomastodon as a sister taxon to Elephantidae, with a divergence time of ∼13.5 Ma. Additionally, a total evidence analysis combining morphological and paleogenetic data shows that the three most diverse clades within Proboscidea diverged during the early Miocene, coinciding with the formation of a land passage between Africa and Eurasia. Our results are a further step toward aDNA analyses on Pleistocene samples from subtropical regions and provide a framework for proboscidean evolution.

Mitogenome Phylogeny Including Data from Additional Subspecies Provides New Insights into the Historical Biogeography of the Eurasian lynx Lynx lynx.

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.

Diversity and Paleodemography of the Addax (Addax nasomaculatus), a Saharan Antelope on the Verge of Extinction.

Since the 19th century, the addax (Addax nasomaculatus) has lost approximately 99% of its former range. Along with its close relatives, the blue antelope (Hippotragus leucophaeus) and the scimitar-horned oryx (Oryx dammah), the addax may be the third large African mammal species to go extinct in the wild in recent times. Despite this, the evolutionary history of this critically endangered species remains virtually unknown. To gain insight into the population history of the addax, we used hybridization capture to generate ten complete mitochondrial genomes from historical samples and assembled a nuclear genome. We found that both mitochondrial and nuclear diversity are low compared to other African bovids. Analysis of mitochondrial genomes revealed a most recent common ancestor ~32 kya (95% CI 11-58 kya) and weak phylogeographic structure, indicating that the addax likely existed as a highly mobile, panmictic population across its Sahelo-Saharan range in the past. PSMC analysis revealed a continuous decline in effective population size since ~2 Ma, with short intermediate increases at ~500 and ~44 kya. Our results suggest that the addax went through a major bottleneck in the Late Pleistocene, remaining at low population size prior to the human disturbances of the last few centuries.

Estimating the dwarfing rate of an extinct Sicilian elephant.

Evolution on islands, together with the often extreme phenotypic changes associated with it, has attracted much interest from evolutionary biologists. However, measuring the rate of change of phenotypic traits of extinct animals can be challenging, in part due to the incompleteness of the fossil record. Here, we use combined molecular and fossil evidence to define the minimum and maximum rate of dwarfing in an extinct Mediterranean dwarf elephant from Puntali Cave (Sicily).1 Despite the challenges associated with recovering ancient DNA from warm climates,2 we successfully retrieved a mitogenome from a sample with an estimated age between 175,500 and 50,000 years. Our results suggest that this specific Sicilian elephant lineage evolved from one of the largest terrestrial mammals that ever lived3 to an island species weighing less than 20% of its original mass with an estimated mass reduction between 0.74 and 200.95 kg and height reduction between 0.15 and 41.49 mm per generation. We show that combining ancient DNA with paleontological and geochronological evidence can constrain the timing of phenotypic changes with greater accuracy than could be achieved using any source of evidence in isolation.

A sliver of the past: The decimation of the genetic diversity of the Mexican wolf.

The endangered Mexican wolf (Canis lupus baileyi) is known to carry exceedingly low levels of genetic diversity. This could be (i) the result of long-term evolutionary patterns as they exist at the southernmost limit of the species distribution at a relatively reduced effective size, or (ii) due to rapid population decline caused by human persecution over the last century. If the former, purifying selection is expected to have minimized the impact of inbreeding. If the latter, rapid and recent declines in genetic diversity may have resulted in severe fitness consequences. To differentiate these hypotheses, we conducted comparative whole-genome analyses of five historical Mexican wolves (1907-1917) and 18 contemporary Mexican and grey wolves from North America and Eurasia. Based on whole-genome data, historical and modern Mexican wolves together form a discrete unit. Moreover, we found that modern Mexican wolves have reduced genetic diversity and increased inbreeding relative to the historical population, which was widespread across the southwestern United States and not restricted to Mexico as previously assumed. Finally, although Mexican wolves have evolved in sympatry with coyotes (C. latrans), we observed lower introgression between historical Mexican wolves and coyotes than with modern Mexican wolves, despite similarities in body size. Taken together, our data show that recent population declines probably caused the reduced level of genetic diversity, but not the observed differentiation of the Mexican wolves from other North American wolves.

Successful application of ancient DNA extraction and library construction protocols to museum wet collection specimens.

Millions of scientific specimens are housed in museum collections, a large part of which are fluid preserved. The use of formaldehyde as fixative and subsequent storage in ethanol is especially common in ichthyology and herpetology. This type of preservation damages DNA and reduces the chance of successful retrieval of genetic data. We applied ancient DNA extraction and single stranded library construction protocols to a variety of vertebrate samples obtained from wet collections and of different ages. Our results show that almost all samples tested yielded endogenous DNA. Archival DNA extraction was successful across different tissue types as well as using small amounts of tissue. Conversion of archival DNA fragments into single-stranded libraries resulted in usable data even for samples with initially undetectable DNA amounts. Subsequent target capture approaches for mitochondrial DNA using homemade baits on a subset of 30 samples resulted in almost complete mitochondrial genome sequences in several instances. Thus, application of ancient DNA methodology makes wet collection specimens, including type material as well as rare, old or extinct species, accessible for genetic and genomic analyses. Our results, accompanied by detailed step-by-step protocols, are a large step forward to open the DNA archive of museum wet collections for scientific studies.

African and Asian leopards are highly differentiated at the genomic level.

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

Middle Pleistocene genome calibrates a revised evolutionary history of extinct cave bears.

Palaeogenomes provide the potential to study evolutionary processes in real time, but this potential is limited by our ability to recover genetic data over extended timescales.1 As a consequence, most studies so far have focused on samples of Late Pleistocene or Holocene age, which covers only a small part of the history of many clades and species. Here, we report the recovery of a low coverage palaeogenome from the petrous bone of a ∼360,000 year old cave bear from Kudaro 1 cave in the Caucasus Mountains. Analysis of this genome alongside those of several Late Pleistocene cave bears reveals widespread mito-nuclear discordance in this group. Using the time interval between Middle and Late Pleistocene cave bear genomes, we directly estimate ursid nuclear and mitochondrial substitution rates to calibrate their respective phylogenies. This reveals post-divergence mitochondrial transfer as the dominant factor explaining their mito-nuclear discordance. Interestingly, these transfer events were not accompanied by large-scale nuclear introgression. However, we do detect additional instances of nuclear admixture among other cave bear lineages, and between cave bears and brown bears, which are not associated with mitochondrial exchange. Genomic data obtained from the Middle Pleistocene cave bear petrous bone has thus facilitated a revised evolutionary history of this extinct megafaunal group. Moreover, it suggests that petrous bones may provide a means of extending both the magnitude and time depth of palaeogenome retrieval over substantial portions of the evolutionary histories of many mammalian clades.

Ancient DNA from the Asiatic Wild Dog (Cuon alpinus) from Europe.

The Asiatic wild dog (Cuon alpinus), restricted today largely to South and Southeast Asia, was widespread throughout Eurasia and even reached North America during the Pleistocene. Like many other species, it suffered from a huge range loss towards the end of the Pleistocene and went extinct in most of its former distribution. The fossil record of the dhole is scattered and the identification of fossils can be complicated by an overlap in size and a high morphological similarity between dholes and other canid species. We generated almost complete mitochondrial genomes for six putative dhole fossils from Europe. By using three lines of evidence, i.e., the number of reads mapping to various canid mitochondrial genomes, the evaluation and quantification of the mapping evenness along the reference genomes and phylogenetic analysis, we were able to identify two out of six samples as dhole, whereas four samples represent wolf fossils. This highlights the contribution genetic data can make when trying to identify the species affiliation of fossil specimens. The ancient dhole sequences are highly divergent when compared to modern dhole sequences, but the scarcity of dhole data for comparison impedes a more extensive analysis.

Identifying the true number of specimens of the extinct blue antelope (Hippotragus leucophaeus).

Native to southern Africa, the blue antelope (Hippotragus leucophaeus) is the only large African mammal species known to have become extinct in historical times. However, it was poorly documented prior to its extinction ~ 1800 AD, and many of the small number of museum specimens attributed to it are taxonomically contentious. This places limitations on our understanding of its morphology, ecology, and the mechanisms responsible for its demise. We retrieved genetic information from ten of the sixteen putative blue antelope museum specimens using both shotgun sequencing and mitochondrial genome target capture in an attempt to resolve the uncertainty surrounding the identification of these specimens. We found that only four of the ten investigated specimens, and not a single skull, represent the blue antelope. This indicates that the true number of historical museum specimens of the blue antelope is even smaller than previously thought, and therefore hardly any reference material is available for morphometric, comparative and genetic studies. Our study highlights how genetics can be used to identify rare species in natural history collections where other methods may fail or when records are scarce. Additionally, we present an improved mitochondrial reference genome for the blue antelope as well as one complete and two partial mitochondrial genomes. A first analysis of these mitochondrial genomes indicates low levels of maternal genetic diversity in the 'museum population', possibly confirming previous results that blue antelope population size was already low at the time of the European colonization of South Africa.

Ancestral mitogenome capture of the Southeast Asian banded linsang.

Utilising a reconstructed ancestral mitochondrial genome of a clade to design hybridisation capture baits can provide the opportunity for recovering mitochondrial sequences from all its descendent and even sister lineages. This approach is useful for taxa with no extant close relatives, as is often the case for rare or extinct species, and is a viable approach for the analysis of historical museum specimens. Asiatic linsangs (genus Prionodon) exemplify this situation, being rare Southeast Asian carnivores for which little molecular data is available. Using ancestral capture we recover partial mitochondrial genome sequences for seven banded linsangs (P. linsang) from historical specimens, representing the first intraspecific genetic dataset for this species. We additionally assemble a high quality mitogenome for the banded linsang using shotgun sequencing for time-calibrated phylogenetic analysis. This reveals a deep divergence between the two Asiatic linsang species (P. linsang, P. pardicolor), with an estimated divergence of ~12 million years (Ma). Although our sample size precludes any robust interpretation of the population structure of the banded linsang, we recover two distinct matrilines with an estimated tMRCA of ~1 Ma. Our results can be used as a basis for further investigation of the Asiatic linsangs, and further demonstrate the utility of ancestral capture for studying divergent taxa without close relatives.

Ancient DNA reveals twenty million years of aquatic life in beavers.

Xenikoudakis et al. report a partial mitochondrial genome of the extinct giant beaver Castoroides and estimate the origin of aquatic behavior in beavers to approximately 20 million years. This time estimate coincides with the extinction of terrestrial beavers and raises the question whether the two events had a common cause.

Consensify: A Method for Generating Pseudohaploid Genome Sequences from Palaeogenomic Datasets with Reduced Error Rates.

A standard practise in palaeogenome analysis is the conversion of mapped short read data into pseudohaploid sequences, frequently by selecting a single high-quality nucleotide at random from the stack of mapped reads. This controls for biases due to differential sequencing coverage, but it does not control for differential rates and types of sequencing error, which are frequently large and variable in datasets obtained from ancient samples. These errors have the potential to distort phylogenetic and population clustering analyses, and to mislead tests of admixture using D statistics. We introduce Consensify, a method for generating pseudohaploid sequences, which controls for biases resulting from differential sequencing coverage while greatly reducing error rates. The error correction is derived directly from the data itself, without the requirement for additional genomic resources or simplifying assumptions such as contemporaneous sampling. For phylogenetic and population clustering analysis, we find that Consensify is less affected by artefacts than methods based on single read sampling. For D statistics, Consensify is more resistant to false positives and appears to be less affected by biases resulting from different laboratory protocols than other frequently used methods. Although Consensify is developed with palaeogenomic data in mind, it is applicable for any low to medium coverage short read datasets. We predict that Consensify will be a useful tool for future studies of palaeogenomes.

Paleogenome Reveals Genetic Contribution of Extinct Giant Panda to Extant Populations.

Historically, the giant panda was widely distributed from northern China to southwestern Asia [1]. As a result of range contraction and fragmentation, extant individuals are currently restricted to fragmented mountain ranges on the eastern margin of the Qinghai-Tibet plateau, where they are distributed among three major population clusters [2]. However, little is known about the genetic consequences of this dramatic range contraction. For example, were regions where giant pandas previously existed occupied by ancestors of present-day populations, or were these regions occupied by genetically distinct populations that are now extinct? If so, is there any contribution of these extinct populations to the genomes of giant pandas living today? To investigate these questions, we sequenced the nuclear genome of an ∼5,000-year-old giant panda from Jiangdongshan, Tengchong County in Yunnan Province, China. We find that this individual represents a genetically distinct population that diverged prior to the diversification of modern giant panda populations. We find evidence of differential admixture with this ancient population among modern individuals originating from different populations as well as within the same population. We also find evidence for directional gene flow, which transferred alleles from the ancient population into the modern giant panda lineages. A variable proportion of the genomes of extant individuals is therefore likely derived from the ancient population represented by our sequenced individual. Although extant giant panda populations retain reasonable genetic diversity, our results suggest that this represents only part of the genetic diversity this species harbored prior to its recent range contractions.

Emergence of a Chimeric Globin Pseudogene and Increased Hemoglobin Oxygen Affinity Underlie the Evolution of Aquatic Specializations in Sirenia.

As limits on O2 availability during submergence impose severe constraints on aerobic respiration, the oxygen binding globin proteins of marine mammals are expected to have evolved under strong evolutionary pressures during their land-to-sea transition. Here, we address this question for the order Sirenia by retrieving, annotating, and performing detailed selection analyses on the globin repertoire of the extinct Steller's sea cow (Hydrodamalis gigas), dugong (Dugong dugon), and Florida manatee (Trichechus manatus latirostris) in relation to their closest living terrestrial relatives (elephants and hyraxes). These analyses indicate most loci experienced elevated nucleotide substitution rates during their transition to a fully aquatic lifestyle. While most of these genes evolved under neutrality or strong purifying selection, the rate of nonsynonymous/synonymous replacements increased in two genes (Hbz-T1 and Hba-T1) that encode the α-type chains of hemoglobin (Hb) during each stage of life. Notably, the relaxed evolution of Hba-T1 is temporally coupled with the emergence of a chimeric pseudogene (Hba-T2/Hbq-ps) that contributed to the tandemly linked Hba-T1 of stem sirenians via interparalog gene conversion. Functional tests on recombinant Hb proteins from extant and ancestral sirenians further revealed that the molecular remodeling of Hba-T1 coincided with increased Hb-O2 affinity in early sirenians. Available evidence suggests that this trait evolved to maximize O2 extraction from finite lung stores and suppress tissue O2 offloading, thereby facilitating the low metabolic intensities of extant sirenians. In contrast, the derived reduction in Hb-O2 affinity in (sub)Arctic Steller's sea cows is consistent with fueling increased thermogenesis by these once colossal marine herbivores.

Double-Stranded Library Preparation for Ancient and Other Degraded Samples.

High-throughput sequencing (HTS) allows fast and cost-efficient sequencing of ancient DNA (aDNA) without prior information about what sequences should be targeted. One necessary step for HTS is the preparation of a sequencing library. Commercial kits are available for this purpose, but many of these are not suitable for aDNA or other types of damaged DNA. Here, we outline a protocol for HTS library preparation that is optimized for ancient DNA. We report the library conversion rate for a range of input template and adapter concentrations. Our results show that the protocol performs at a high efficiency.