Mylodon darwinii is the extinct giant ground sloth named after Charles Darwin, who first collected its remains in South America. We have successfully obtained a high-quality mitochondrial genome at 99-fold coverage using an Illumina shotgun sequencing of a 12 880-year-old bone fragment from Mylodon Cave in Chile. Low level of DNA damage showed that this sample was exceptionally well preserved for an ancient subfossil, probably the result of the dry and cold conditions prevailing within the cave. Accordingly, taxonomic assessment of our shotgun metagenomic data showed a very high percentage of endogenous DNA with 22% of the assembled metagenomic contigs assigned to Xenarthra. Additionally, we enriched over 15 kb of sequence data from seven nuclear exons, using target sequence capture designed against a wide xenarthran dataset. Phylogenetic and dating analyses of the mitogenomic dataset including all extant species of xenarthrans and the assembled nuclear supermatrix unambiguously place Mylodon darwinii as the sister-group of modern two-fingered sloths, from which it diverged around 22 million years ago. These congruent results from both the mitochondrial and nuclear data support the diphyly of the two modern sloth lineages, implying the convergent evolution of their unique suspensory behaviour as an adaption to arboreality. Our results offer promising perspectives for whole-genome sequencing of this emblematic extinct taxon.

CRISPR-Cas systems inherently multiplex through their CRISPR arrays–whether to confer immunity against multiple invaders or by mediating multi-target editing, regulation, imaging, and sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report an efficient, one-step scheme called CRATES to construct large CRISPR arrays through defined assembly junctions within the trimmed portion of array spacers. We show that the constructed arrays function with the single-effector nucleases Cas9, Cas12a, and Cas13a for multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. We also applied CRATES to assemble composite arrays utilized by multiple Cas nucleases, where these arrays enhanced DNA targeting specificity by blocking off-target sites. Finally, array characterization revealed context-dependent loss of spacer activity and processing of unintended guide RNAs derived from Cas12a terminal repeats. CRATES thus can facilitate diverse applications requiring CRISPR multiplexing and help elucidate critical factors influencing array function.

Several species of lizards from the megadiverse island of New Guinea have evolved green blood. An unusually high concentration of the green bile pigment biliverdin in the circulatory system of these lizards makes the blood, muscles, bones, tongue, and mucosal tissues bright green in color, eclipsing the crimson color from their red blood cells. This is a remarkable physiological feature because bile pigments are toxic physiological waste products of red blood cell catabolism and, when chronically elevated, cause jaundice in humans and all other vertebrates. Although these lizards offer a promising system to examine the evolution of extraordinary physiological characteristics, little is known about the phylogenetic relationships of green-blooded lizards or the evolutionary origins of green blood. We present the first extensive phylogeny for green-blooded lizards and closely related Australasian lizards using thousands of genomic regions to examine the evolutionary history of this unusual trait. Maximum likelihood ancestral character state reconstruction supports four independent origins of green blood. Our results lay the phylogenetic foundation necessary to determine the role, if any, of natural selection in shaping this enigmatic physiological trait as well as understanding the genetic, proteomic, and biochemical basis for the lack of jaundice in those species that have independently evolved green blood. Green blood, a remarkable physiological trait, evolved multiple times in lizards. Green blood, a remarkable physiological trait, evolved multiple times in lizards.

To expand our capacity to discover venom sequences from the genomes of venomous organisms, we applied targeted sequencing techniques to selectively recover venom gene superfamilies and nontoxin loci from the genomes of 32 cone snail species (family, Conidae), a diverse group of marine gastropods that capture their prey using a cocktail of neurotoxic peptides (conotoxins). We were able to successfully recover conotoxin gene superfamilies across all species with high confidence (> 100Â coverage) and used these data to provide new insights into conotoxin evolution. First, we found that conotoxin gene superfamilies are composed of one to six exons and are typically short in length (mean ¼ $85 bp). Second, we expanded our understanding of the following genetic features of conotoxin evolution: 1) positive selection, where exons coding the mature toxin region were often three times more divergent than their adjacent noncoding regions, 2) expression regulation, with comparisons to transcriptome data showing that cone snails only express a fraction of the genes available in their genome (24–63%), and 3) extensive gene turnover, where Conidae species varied from 120 to 859 conotoxin gene copies. Finally, using comparative phylogenetic methods, we found that while diet specificity did not predict patterns of conotoxin evolution, dietary breadth was positively correlated with total conotoxin gene diversity. Overall, the targeted sequencing technique demonstrated here has the potential to radically increase the pace at which venom gene families are sequenced and studied, reshaping our ability to understand the impact of genetic changes on ecologically relevant phenotypes and subsequent diversification.

Premise of the Study Until recently, most phylogenetic studies of ferns were based on chloroplast genes. Evolutionary inferences based on these data can be incomplete because the characters are from a single linkage group and are uniparentally inherited. These limitations are particularly acute in studies of hybridization, which is prevalent in ferns; fern hybrids are common and ferns are able to hybridize across highly diverged lineages, up to 60 million years since divergence in one documented case. However, it not yet clear what effect such hybridization has on fern evolution, in part due to a paucity of available biparentally inherited (nuclear-encoded) markers. Methods We designed oligonucleotide baits to capture 25 targeted, low-copy nuclear markers from a sample of 24 species spanning extant fern diversity. Results Most loci were successfully sequenced from most accessions. Although the baits were designed from exon (transcript) data, we successfully captured intron sequences that should be useful for more focused phylogenetic studies. We present phylogenetic analyses of the new target sequence capture data and integrate these into a previous transcript-based data set. Discussion We make our bait sequences available to the community as a resource for further studies of fern phylogeny.

Summary The sweet potato is one of the world’s most widely consumed crops, yet its evolutionary history is poorly understood. In this paper, we present a comprehensive phylogenetic study of all species closely related to the sweet potato and address several questions pertaining to the sweet potato that remained unanswered. Our research combined genome skimming and target DNA capture to sequence whole chloroplasts and 605 single-copy nuclear regions from 199 specimens representing the sweet potato and all of its crop wild relatives (CWRs). We present strongly supported nuclear and chloroplast phylogenies demonstrating that the sweet potato had an autopolyploid origin and that Ipomoea trifida is its closest relative, confirming that no other extant species were involved in its origin. Phylogenetic analysis of nuclear and chloroplast genomes shows conflicting topologies regarding the monophyly of the sweet potato. The process of chloroplast capture explains these conflicting patterns, showing that I. trifida had a dual role in the origin of the sweet potato, first as its progenitor and second as the species with which the sweet potato introgressed so one of its lineages could capture an I. trifida chloroplast. In addition, we provide evidence that the sweet potato was present in Polynesia in pre-human times. This, together with several other examples of long-distance dispersal in Ipomoea, negates the need to invoke ancient human-mediated transport as an explanation for its presence in Polynesia. These results have important implications for understanding the origin and evolution of a major global food crop and question the existence of pre-Columbian contacts between Polynesia and the American continent.