History of Diversification and Adaptation from North to South Revealed by Genomic Data: Guanacos from the Desert to Sub-Antarctica.

The increased availability of quality genomic data has greatly improved the scope and resolution of our understanding of the recent evolutionary history of wild species adapted to extreme environments and their susceptibility to anthropogenic impacts. The guanaco (Lama guanicoe), the largest wild ungulate in South America, is a good example. The guanaco is well adapted to a wide range of habitats, including the Sechura Desert, the high Andes Mountains to the north, and the extreme temperatures and conditions of Navarino Island to the south. Guanacos also have a long history of overexploitation by humans. To assess the evolutionary impact of these challenging habitats on the genomic diversity, we analyzed 38 genomes (∼10 to 16×) throughout their extensive latitudinal distribution from the Sechura and Atacama Desert to southward into Tierra del Fuego Island. These included analyses of patterns of unique differentiation in the north and geographic region further south with admixture among L. g. cacsilensis and L. g. guanicoe. Our findings provide new insights on the divergence of the subspecies ∼800,000 yr BP and document two divergent demographic trajectories and to the initial expansion of guanaco into the more southern portions of the Atacama Desert. Patagonian guanacos have experienced contemporary reductions in effective population sizes, likely the consequence of anthropogenic impacts. The lowest levels of genetic diversity corresponded to their northern and western limits of distribution and some varying degrees of genetic differentiation. Adaptive genomic diversity was strongly linked with environmental variables and was linked with colonization toward the south followed by adaptation.

Chromosome-length genome assembly and karyotype of the endangered black-footed ferret (Mustela nigripes).

The black-footed ferret (Mustela nigripes) narrowly avoided extinction to become an oft-cited example of the benefits of intensive management, research, and collaboration to save a species through ex situ conservation breeding and reintroduction into its former range. However, the species remains at risk due to possible inbreeding, disease susceptibility, and multiple fertility challenges. Here, we report the de novo genome assembly of a male black-footed ferret generated through a combination of linked-read sequencing, optical mapping, and Hi-C proximity ligation. In addition, we report the karyotype for this species, which was used to anchor and assign chromosome numbers to the chromosome-length scaffolds. The draft assembly was ~2.5 Gb in length, with 95.6% of it anchored to 19 chromosome-length scaffolds, corresponding to the 2n = 38 chromosomes revealed by the karyotype. The assembly has contig and scaffold N50 values of 148.8 kbp and 145.4 Mbp, respectively, and is up to 96% complete based on BUSCO analyses. Annotation of the assembly, including evidence from RNA-seq data, identified 21,406 protein-coding genes and a repeat content of 37.35%. Phylogenomic analyses indicated that the black-footed ferret diverged from the European polecat/domestic ferret lineage 1.6 million yr ago. This assembly will enable research on the conservation genomics of black-footed ferrets and thereby aid in the further restoration of this endangered species.

Comparative genomics uncovers the evolutionary history, demography, and molecular adaptations of South American canids.

The remarkable radiation of South American (SA) canids produced 10 extant species distributed across diverse habitats, including disparate forms such as the short-legged, hypercarnivorous bush dog and the long-legged, largely frugivorous maned wolf. Despite considerable research spanning nearly two centuries, many aspects of their evolutionary history remain unknown. Here, we analyzed 31 whole genomes encompassing all extant SA canid species to assess phylogenetic relationships, interspecific hybridization, historical demography, current genetic diversity, and the molecular bases of adaptations in the bush dog and maned wolf. We found that SA canids originated from a single ancestor that colonized South America 3.9 to 3.5 Mya, followed by diversification east of the Andes and then a single colonization event and radiation of Lycalopex species west of the Andes. We detected extensive historical gene flow between recently diverged lineages and observed distinct patterns of genomic diversity and demographic history in SA canids, likely induced by past climatic cycles compounded by human-induced population declines. Genome-wide scans of selection showed that disparate limb proportions in the bush dog and maned wolf may derive from mutations in genes regulating chondrocyte proliferation and enlargement. Further, frugivory in the maned wolf may have been enabled by variants in genes associated with energy intake from short-chain fatty acids. In contrast, unique genetic variants detected in the bush dog may underlie interdigital webbing and dental adaptations for hypercarnivory. Our analyses shed light on the evolution of a unique carnivoran radiation and how it was shaped by South American topography and climate change.

Reference genomes for conservation.

High-quality reference genomes for non-model species can benefit conservation.

Genomic Signatures of Divergent Ecological Strategies in a Recent Radiation of Neotropical Wild Cats.

Ecological differentiation among diverging species is an important component of the evolutionary process and can be investigated in rapid and recent radiations. Here, we use whole genome sequences of five species from the genus Leopardus, a recently diversified Neotropical lineage with species bearing distinctive morphological, ecological, and behavioral features, to investigate genome-wide diversity, comparative demographic history and signatures of positive selection. Our results show that divergent ecological strategies are reflected in genomic features, for example a generalist species shows historically larger effective population size and higher heterozygosity than habitat specialists. The demographic history of these cats seems to have been jointly driven by climate fluctuations and habitat specialization, with different ecological adaptations leading to distinct trajectories. Finally, a gene involved in vertebrate retinal neurogenesis (POU4F2) was found to be under positive selection in the margay, a cat with notoriously large eyes that are likely associated with its nocturnal and arboreal specializations.

Incomplete lineage sorting and phenotypic evolution in marsupials.

Incomplete lineage sorting (ILS) makes ancestral genetic polymorphisms persist during rapid speciation events, inducing incongruences between gene trees and species trees. ILS has complicated phylogenetic inference in many lineages, including hominids. However, we lack empirical evidence that ILS leads to incongruent phenotypic variation. Here, we performed phylogenomic analyses to show that the South American monito del monte is the sister lineage of all Australian marsupials, although over 31% of its genome is closer to the Diprotodontia than to other Australian groups due to ILS during ancient radiation. Pervasive conflicting phylogenetic signals across the whole genome are consistent with some of the morphological variation among extant marsupials. We detected hundreds of genes that experienced stochastic fixation during ILS, encoding the same amino acids in non-sister species. Using functional experiments, we confirm how ILS may have directly contributed to hemiplasy in morphological traits that were established during rapid marsupial speciation ca. 60 mya.

Standards recommendations for the Earth BioGenome Project.

A global international initiative, such as the Earth BioGenome Project (EBP), requires both agreement and coordination on standards to ensure that the collective effort generates rapid progress toward its goals. To this end, the EBP initiated five technical standards committees comprising volunteer members from the global genomics scientific community: Sample Collection and Processing, Sequencing and Assembly, Annotation, Analysis, and IT and Informatics. The current versions of the resulting standards documents are available on the EBP website, with the recognition that opportunities, technologies, and challenges may improve or change in the future, requiring flexibility for the EBP to meet its goals. Here, we describe some highlights from the proposed standards, and areas where additional challenges will need to be met.

Whole genome analysis of clouded leopard species reveals an ancient divergence and distinct demographic histories.

Similar to other apex predator species, populations of mainland (Neofelis nebulosa) and Sunda (Neofelis diardi) clouded leopards are declining. Understanding their patterns of genetic variation can provide critical insights on past genetic erosion and a baseline for understanding their long-term conservation needs. As a step toward this goal, we present draft genome assemblies for the two clouded leopard species to quantify their phylogenetic divergence, genome-wide diversity, and historical population trends. We estimate that the two species diverged 5.1 Mya, much earlier than previous estimates of 1.41 Mya and 2.86 Mya, suggesting they separated when Sundaland was becoming increasingly isolated from mainland Southeast Asia. The Sunda clouded leopard displays a distinct and reduced effective population size trajectory, consistent with a lower genome-wide heterozygosity and SNP density, relative to the mainland clouded leopard. Our results provide new insights into the evolutionary history and genetic health of this unique lineage of felids.

Cross-species transmission of retroviruses among domestic and wild felids in human-occupied landscapes in Chile.

Human transformation of natural habitats facilitates pathogen transmission between domestic and wild species. The guigna (Leopardus guigna), a small felid found in Chile, has experienced habitat loss and an increased probability of contact with domestic cats. Here, we describe the interspecific transmission of feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) between domestic cats and guignas and assess its correlation with human landscape perturbation. Blood and tissue samples from 102 free-ranging guignas and 262 domestic cats were collected and analyzed by PCR and sequencing. Guigna and domestic cat FeLV and FIV prevalence were very similar. Phylogenetic analysis showed guigna FeLV and FIV sequences are positioned within worldwide domestic cat virus clades with high nucleotide similarity. Guigna FeLV infection was significantly associated with fragmented landscapes with resident domestic cats. There was little evidence of clinical signs of disease in guignas. Our results contribute to the understanding of the implications of landscape perturbation and emerging diseases.

Past and Recent Effects of Livestock Activity on the Genetic Diversity and Population Structure of Native Guanaco Populations of Arid Patagonia.

Extensive livestock production and urbanization entail modifications of natural landscapes, including installation of fences, development of agriculture, urbanization of natural areas, and construction of roads and infrastructure that, together, impact native fauna. Here, we evaluate the diversity and genetic structure of endemic guanacos (Lama guanicoe) of the Monte and Patagonian Steppe of central Argentina, which have been reduced and displaced by sheep ranching and other impacts of human activities. Analyses of genetic variation of microsatellite loci and d-loop revealed high levels of genetic variation and latitudinal segregation of mitochondrial haplotypes. There were indications of at least two historical populations in the Monte and the Patagonian Steppe based on shared haplotypes and shared demographic history among localities. Currently, guanacos are structured into three groups that were probably reconnected relatively recently, possibly facilitated by a reduction of sheep and livestock in recent decades and a recovery of the guanaco populations. These results provide evidence of the genetic effects of livestock activity and urbanization on wild herbivore populations, which were possibly exacerbated by an arid environment with limited productive areas. The results highlight the importance of enacting conservation management plans to ensure the persistence of ancestral and ecologically functional populations of guanacos.

Genomic Adaptations and Evolutionary History of the Extinct Scimitar-Toothed Cat, Homotherium latidens.

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

The evolutionary history of extinct and living lions.

Lions are one of the world's most iconic megafauna, yet little is known about their temporal and spatial demographic history and population differentiation. We analyzed a genomic dataset of 20 specimens: two ca. 30,000-y-old cave lions (Panthera leo spelaea), 12 historic lions (Panthera leo leo/Panthera leo melanochaita) that lived between the 15th and 20th centuries outside the current geographic distribution of lions, and 6 present-day lions from Africa and India. We found that cave and modern lions shared an ancestor ca. 500,000 y ago and that the 2 lineages likely did not hybridize following their divergence. Within modern lions, we found 2 main lineages that diverged ca. 70,000 y ago, with clear evidence of subsequent gene flow. Our data also reveal a nearly complete absence of genetic diversity within Indian lions, probably due to well-documented extremely low effective population sizes in the recent past. Our results contribute toward the understanding of the evolutionary history of lions and complement conservation efforts to protect the diversity of this vulnerable species.

Utility of genetic variation in coat color genes to distinguish wild, domestic and hybrid South American camelids for forensic and judicial applications.

A molecular genetic protocol for distinguishing pure and hybrid South American camelids was developed to provide strong, quantifiable, and unbiased species identification. We detail the application of the approach in the context of a criminal case in the Andes Mountains of central Chile where the defendants were alleged to have illegally hunted three wild guanacos (Lama guanicoe), as opposed to hybrid domestic llama (Lama glama)/wild guanaco crosses, which are unregulated. We describe a workflow that differentiates among wild, domestic and hybrid South American camelids (Lama versus Vicugna) based on mitochondrial cytochrome b genetic variation (to distinguish between Lama and Vicugna), and MC1R and exon 4 variation of the ASIP gene (to differentiate wild from domestic species). Additionally, we infer the population origin and sex of each of the three individuals from a panel of 15 autosomal microsatellite loci and the presence or absence of the SRY gene. Our analyses strongly supported the inference that the confiscated carcasses corresponded with 2 male and 1 female guanacos that were hunted illegally. Statistical power analyses suggested that there was an extremely low probability of misidentifying domestic camelids as wild camelids (an estimated 0 % Type I error rate), or using more conservative approached a 1.17 % chance of misidentification of wild species as domestic camelids (Type II error). Our case report and methodological and analytical protocols demonstrate the power of genetic variation in coat color genes to identify hybrids between wild and domestic camelid species and highlight the utility of the approach to help combat illegal wildlife hunting and trafficking.

Author Correction: Puma genomes from North and South America provide insights into the genomic consequences of inbreeding.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Puma genomes from North and South America provide insights into the genomic consequences of inbreeding.

Pumas are the most widely distributed felid in the Western Hemisphere. Increasingly, however, human persecution and habitat loss are isolating puma populations. To explore the genomic consequences of this isolation, we assemble a draft puma genome and a geographically broad panel of resequenced individuals. We estimate that the lineage leading to present-day North American pumas diverged from South American lineages 300-100 thousand years ago. We find signatures of close inbreeding in geographically isolated North American populations, but also that tracts of homozygosity are rarely shared among these populations, suggesting that assisted gene flow would restore local genetic diversity. The genome of a Florida panther descended from translocated Central American individuals has long tracts of homozygosity despite recent outbreeding. This suggests that while translocations may introduce diversity, sustaining diversity in small and isolated populations will require either repeated translocations or restoration of landscape connectivity. Our approach provides a framework for genome-wide analyses that can be applied to the management of similarly small and isolated populations.

Avian Binocularity and Adaptation to Nocturnal Environments: Genomic Insights from a Highly Derived Visual Phenotype.

Typical avian eyes are phenotypically engineered for photopic vision (daylight). In contrast, the highly derived eyes of the barn owl (Tyto alba) are adapted for scotopic vision (dim light). The dramatic modifications distinguishing barn owl eyes from other birds include: 1) shifts in frontal orientation to improve binocularity, 2) rod-dominated retina, and 3) enlarged corneas and lenses. Some of these features parallel mammalian eye patterns, which are hypothesized to have initially evolved in nocturnal environments. Here, we used an integrative approach combining phylogenomics and functional phenotypes of 211 eye-development genes across 48 avian genomes representing most avian orders, including the stem lineage of the scotopic-adapted barn owl. Overall, we identified 25 eye-development genes that coevolved under intensified or relaxed selection in the retina, lens, cornea, and optic nerves of the barn owl. The agtpbp1 gene, which is associated with the survival of photoreceptor populations, was pseudogenized in the barn owl genome. Our results further revealed that barn owl retinal genes responsible for the maintenance, proliferation, and differentiation of photoreceptors experienced an evolutionary relaxation. Signatures of relaxed selection were also observed in the lens and cornea morphology-associated genes, suggesting that adaptive evolution in these structures was essentially structural. Four eye-development genes (ephb1, phactr4, prph2, and rs1) evolved in positive association with the orbit convergence in birds and under relaxed selection in the barn owl lineage, likely contributing to an increased reliance on binocular vision in the barn owl. Moreover, we found evidence of coevolutionary interactions among genes that are expressed in the retina, lens, and optic nerve, suggesting synergetic adaptive events. Our study disentangles the genomic changes governing the binocularity and low-light perception adaptations of barn owls to nocturnal environments while revealing the molecular mechanisms contributing to the shift from the typical avian photopic vision to the more-novel scotopic-adapted eye.

Whole Genome Sequencing and Re-sequencing of the Sable Antelope (Hippotragus niger): A Resource for Monitoring Diversity in ex Situ and in Situ Populations.

Genome-wide assessment of genetic diversity has the potential to increase the ability to understand admixture, inbreeding, kinship and erosion of genetic diversity affecting both captive (ex situ) and wild (in situ) populations of threatened species. The sable antelope (Hippotragus niger), native to the savannah woodlands of sub-Saharan Africa, is a species that is being managed ex situ in both public (zoo) and private (ranch) collections in the United States. Our objective was to develop whole genome sequence resources that will serve as a foundation for characterizing the genetic status of ex situ populations of sable antelope relative to populations in the wild. Here we report the draft genome assembly of a male sable antelope, a member of the subfamily Hippotraginae (Bovidae, Cetartiodactyla, Mammalia). The 2.596 Gb draft genome consists of 136,528 contigs with an N50 of 45.5 Kbp and 16,927 scaffolds with an N50 of 4.59 Mbp. De novo annotation identified 18,828 protein-coding genes and repetitive sequences encompassing 46.97% of the genome. The discovery of single nucleotide variants (SNVs) was assisted by the re-sequencing of seven additional captive and wild individuals, representing two different subspecies, leading to the identification of 1,987,710 bi-allelic SNVs. Assembly of the mitochondrial genomes revealed that each individual was defined by a unique haplotype and these data were used to infer the mitochondrial gene tree relative to other hippotragine species. The sable antelope genome constitutes a valuable resource for assessing genome-wide diversity and evolutionary potential, thereby facilitating long-term conservation of this charismatic species.

Improving Illumina assemblies with Hi-C and long reads: An example with the North African dromedary.

Researchers have assembled thousands of eukaryotic genomes using Illumina reads, but traditional mate-pair libraries cannot span all repetitive elements, resulting in highly fragmented assemblies. However, both chromosome conformation capture techniques, such as Hi-C and Dovetail Genomics Chicago libraries and long-read sequencing, such as Pacific Biosciences and Oxford Nanopore, help span and resolve repetitive regions and therefore improve genome assemblies. One important livestock species of arid regions that does not have a high-quality contiguous reference genome is the dromedary (Camelus dromedarius). Draft genomes exist but are highly fragmented, and a high-quality reference genome is needed to understand adaptation to desert environments and artificial selection during domestication. Dromedaries are among the last livestock species to have been domesticated, and together with wild and domestic Bactrian camels, they are the only representatives of the Camelini tribe, which highlights their evolutionary significance. Here we describe our efforts to improve the North African dromedary genome. We used Chicago and Hi-C sequencing libraries from Dovetail Genomics to resolve the order of previously assembled contigs, producing almost chromosome-level scaffolds. Remaining gaps were filled with Pacific Biosciences long reads, and then scaffolds were comparatively mapped to chromosomes. Long reads added 99.32 Mbp to the total length of the new assembly. Dovetail Chicago and Hi-C libraries increased the longest scaffold over 12-fold, from 9.71 Mbp to 124.99 Mbp and the scaffold N50 over 50-fold, from 1.48 Mbp to 75.02 Mbp. We demonstrate that Illumina de novo assemblies can be substantially upgraded by combining chromosome conformation capture and long-read sequencing.

Evolution of gene regulation in ruminants differs between evolutionary breakpoint regions and homologous synteny blocks.

The role of chromosome rearrangements in driving evolution has been a long-standing question of evolutionary biology. Here we focused on ruminants as a model to assess how rearrangements may have contributed to the evolution of gene regulation. Using reconstructed ancestral karyotypes of Cetartiodactyls, Ruminants, Pecorans, and Bovids, we traced patterns of gross chromosome changes. We found that the lineage leading to the ruminant ancestor after the split from other cetartiodactyls was characterized by mostly intrachromosomal changes, whereas the lineage leading to the pecoran ancestor (including all livestock ruminants) included multiple interchromosomal changes. We observed that the liver cell putative enhancers in the ruminant evolutionary breakpoint regions are highly enriched for DNA sequences under selective constraint acting on lineage-specific transposable elements (TEs) and a set of 25 specific transcription factor (TF) binding motifs associated with recently active TEs. Coupled with gene expression data, we found that genes near ruminant breakpoint regions exhibit more divergent expression profiles among species, particularly in cattle, which is consistent with the phylogenetic origin of these breakpoint regions. This divergence was significantly greater in genes with enhancers that contain at least one of the 25 specific TF binding motifs and located near bovidae-to-cattle lineage breakpoint regions. Taken together, by combining ancestral karyotype reconstructions with analysis of cis regulatory element and gene expression evolution, our work demonstrated that lineage-specific regulatory elements colocalized with gross chromosome rearrangements may have provided valuable functional modifications that helped to shape ruminant evolution.