A method to generate capture baits for targeted sequencing.

Hybridization capture approaches allow targeted high-throughput sequencing analysis at reduced costs compared to shotgun sequencing. Hybridization capture is particularly useful in analyses of genomic data from ancient, environmental, and forensic samples, where target content is low, DNA is fragmented and multiplex PCR or other targeted approaches often fail. Here, we describe a DNA bait synthesis approach for hybridization capture that we call Circular Nucleic acid Enrichment Reagent, or CNER (pronounced 'snare'). The CNER method uses rolling-circle amplification followed by restriction digestion to discretize microgram quantities of hybridization probes. We demonstrate the utility of the CNER method by generating probes for a panel of 23 771 known sites of single nucleotide polymorphism in the horse genome. Using these probes, we capture and sequence from a panel of ten ancient horse DNA libraries, comparing CNER capture efficiency to a commercially available approach. With about one million read pairs per sample, CNERs captured more targets (90.5% versus 66.5%) at greater mean depth than an alternative commercial approach.

Early dispersal of domestic horses into the Great Plains and northern Rockies.

The horse is central to many Indigenous cultures across the American Southwest and the Great Plains. However, when and how horses were first integrated into Indigenous lifeways remain contentious, with extant models derived largely from colonial records. We conducted an interdisciplinary study of an assemblage of historic archaeological horse remains, integrating genomic, isotopic, radiocarbon, and paleopathological evidence. Archaeological and modern North American horses show strong Iberian genetic affinities, with later influx from British sources, but no Viking proximity. Horses rapidly spread from the south into the northern Rockies and central plains by the first half of the 17th century CE, likely through Indigenous exchange networks. They were deeply integrated into Indigenous societies before the arrival of 18th-century European observers, as reflected in herd management, ceremonial practices, and culture.

A polar bear paleogenome reveals extensive ancient gene flow from polar bears into brown bears.

Polar bears (Ursus maritimus) and brown bears (Ursus arctos) are sister species possessing distinct physiological and behavioural adaptations that evolved over the last 500,000 years. However, comparative and population genomics analyses have revealed that several extant and extinct brown bear populations have relatively recent polar bear ancestry, probably as the result of geographically localized instances of gene flow from polar bears into brown bears. Here, we generate and analyse an approximate 20X paleogenome from an approximately 100,000-year-old polar bear that reveals a massive prehistoric admixture event, which is evident in the genomes of all living brown bears. This ancient admixture event was not visible from genomic data derived from living polar bears. Like more recent events, this massive admixture event mainly involved unidirectional gene flow from polar bears into brown bears and occurred as climate changes caused overlap in the ranges of the two species. These findings highlight the complex reticulate paths that evolution can take within a regime of radically shifting climate.

Ancient horse genomes reveal the timing and extent of dispersals across the Bering Land Bridge.

The Bering Land Bridge (BLB) last connected Eurasia and North America during the Late Pleistocene. Although the BLB would have enabled transfers of terrestrial biota in both directions, it also acted as an ecological filter whose permeability varied considerably over time. Here we explore the possible impacts of this ecological corridor on genetic diversity within, and connectivity among, populations of a once wide-ranging group, the caballine horses (Equus spp.). Using a panel of 187 mitochondrial and eight nuclear genomes recovered from present-day and extinct caballine horses sampled across the Holarctic, we found that Eurasian horse populations initially diverged from those in North America, their ancestral continent, around 1.0-0.8 million years ago. Subsequent to this split our mitochondrial DNA analysis identified two bidirectional long-range dispersals across the BLB ~875-625 and ~200-50 thousand years ago, during the Middle and Late Pleistocene. Whole genome analysis indicated low levels of gene flow between North American and Eurasian horse populations, which probably occurred as a result of these inferred dispersals. Nonetheless, mitochondrial and nuclear diversity of caballine horse populations retained strong phylogeographical structuring. Our results suggest that barriers to gene flow, currently unidentified but possibly related to habitat distribution across Beringia or ongoing evolutionary divergence, played an important role in shaping the early genetic history of caballine horses, including the ancestors of living horses within Equus ferus.

The case of an arctic wild ass highlights the utility of ancient DNA for validating problematic identifications in museum collections.

Museum collections are essential for reconstructing and understanding past biodiversity. Many museum specimens are, however, challenging to identify. Museum samples may be incomplete, have an unusual morphology, or represent juvenile individuals, all of which complicate accurate identification. In some cases, inaccurate identification can lead to false biogeographic reconstructions with cascading impacts on paleontological and paleoecological research. Here, we analyzed an unusual Equid mandible found in the Far North of the Taymyr peninsula that was identified morphologically as Equus hemionus, an ancestor of present-day Asiatic wild asses. If correct, this identification represents the only finding of a putative Late Pleistocene hemione in the Arctic region, and is therefore critical to understanding wild ass evolution and paleoecology. To confirm the accuracy of this specimen's taxonomic assignment, we used ancient DNA and mitochondrial hybridization capture to identify and place this specimen in the larger equid phylogeny. We find that the specimen is actually a member of E. caballus, the ancestor of domestic horses. Our study demonstrates the utility of ancient DNA to validate morphological identification, in particular of incomplete, otherwise problematic, or taxonomically unusual museum specimens.

Erratum: Author Correction: Improved reference genome for the domestic horse increases assembly contiguity and composition.

[This corrects the article DOI: 10.1038/s42003-018-0199-z.].

Improved reference genome for the domestic horse increases assembly contiguity and composition.

Recent advances in genomic sequencing technology and computational assembly methods have allowed scientists to improve reference genome assemblies in terms of contiguity and composition. EquCab2, a reference genome for the domestic horse, was released in 2007. Although of equal or better quality compared to other first-generation Sanger assemblies, it had many of the shortcomings common to them. In 2014, the equine genomics research community began a project to improve the reference sequence for the horse, building upon the solid foundation of EquCab2 and incorporating new short-read data, long-read data, and proximity ligation data. Here, we present EquCab3. The count of non-N bases in the incorporated chromosomes is improved from 2.33 Gb in EquCab2 to 2.41 Gb in EquCab3. Contiguity has also been improved nearly 40-fold with a contig N50 of 4.5 Mb and scaffold contiguity enhanced to where all but one of the 32 chromosomes is comprised of a single scaffold.

Evolutionary mechanisms of runaway chromosome number change in Agrodiaetus butterflies.

Despite predictions of the classic, hybrid-sterility model of chromosomal speciation, some organisms demonstrate high rate of karyotype evolution. This rate is especially impressive in Agrodiaetus butterflies that rapidly evolved the greatest chromosome number diversity known in animal kingdom within a single subgenus. Here we analyzed karyotype evolution in Agrodiaetus using phylogenetic comparative methods. We found that chromosome numbers possess a strong phylogenetic signal. This disproves the chromosome megaevolution model that proposes multiple chromosome rearrangements to accumulate independently in each of closely related species. We found that Brownian motion gives a more adequate description of observed trait changes than Ornstein-Uhlenbeck model. This indicates that chromosome numbers evolve via random walk along branches of the phylogeny. We discovered a correlation between karyotype changes and phylogeny branch lengths. This gradual pattern is inconsistent with the hybrid-sterility model which, due to association of major chromosome changes with cladogenetic events, predicts a high degree of punctualism in karyotype evolution. Thus, low underdominace of chromosomal rearrangements and/or prevalence of the recombination-suppression model over the hybrid-sterility model of chromosome speciation are the most common engines of the runaway chromosome number change observed.

Ribosomal DNA clusters and telomeric (TTAGG)n repeats in blue butterflies (Lepidoptera, Lycaenidae) with low and high chromosome numbers.

Ribosomal DNA clusters and telomeric repeats are important parts of eukaryotic genome. However, little is known about their organization and localization in karyotypes of organisms with holocentric chromosomes. Here we present first cytogenetic study of these molecular structures in seven blue butterflies of the genus Polyommatus Latreille, 1804 with low and high chromosome numbers (from n=10 to n=ca.108) using fluorescence in situ hybridization (FISH) with 18S rDNA and (TTAGG) n telomeric probes. FISH with the 18S rDNA probe showed the presence of two different variants of the location of major rDNA clusters in Polyommatus species: with one or two rDNA-carrying chromosomes in haploid karyotype. We discuss evolutionary trends and possible mechanisms of changes in the number of ribosomal clusters. We also demonstrate that Polyommatus species have the classical insect (TTAGG) n telomere organization. This chromosome end protection mechanism probably originated de novo in small chromosomes that evolved via fragmentations.