Museum genomics provide an opportunity to investigate population demographics of extinct species, especially valuable when research prior to extinction was minimal. The Bachman’s warbler (Vermivora bachmanii) is hypothesized to have gone extinct due to loss of its specialized habitat. However, little is known about other potential contributing factors such as natural rarity or changes to connectivity following habitat fragmentation. We examined mitochondrial DNA (mtDNA) and genome-wide SNPs using specimens collected from breeding and migration sites across the range of the Bachman’s warbler. We found no signals of strong population structuring across the breeding range of Bachman’s warblers in both mtDNA and genome-wide SNPs. Thus, long-term population isolation did not appear to be a significant contributor to the extinction of the Bachman’s warbler. Instead, our findings support the theory that Bachman’s warblers underwent a rapid decline likely driven by habitat destruction, which may have been exacerbated by the natural rarity, habitat specificity and low genetic diversity of the species.

Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA’s presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage—all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.

Ligilactobacillus is a diverse genus among lactobacilli with phenotypes that reflect adaptation to various hosts. CRISPR-Cas systems are highly prevalent within lactobacilli, and Ligilactobacillus salivarius, the most abundant species of Ligilactobacillus, possesses both DNA- and RNA-targeting CRISPR-Cas systems. In this study, we explore the presence and functional properties of I-B, I-C, I-E, II-A, and III-A CRISPR-Cas systems in over 500 Ligilactobacillus genomes, emphasizing systems found in L. salivarius. We examined the I-E, II-A, and III-A CRISPR-Cas systems of two L. salivarius strains and observed occurrences of split cas genes and differences in CRISPR RNA maturation in native hosts. This prompted testing of the single Cas9 and multiprotein Cascade and Csm CRISPR-Cas effector complexes in a cell-free context to demonstrate the functionality of these systems. We also predicted self-targeting spacers within L. salivarius CRISPR-Cas systems and found that nearly a third of L. salivarius genomes possess unique self-targeting spacers that generally target elements other than prophages. With these two L. salivarius strains, we performed prophage induction coupled with RNA sequencing and discovered that the prophages residing within these strains are inducible and likely active elements, despite targeting by CRISPR-Cas systems. These findings deepen our comprehension of CRISPR-Cas systems in L. salivarius, further elucidating their relationship with associated prophages and providing a functional basis for the repurposing of these Cas effectors for bacterial manipulation.

AngiospeIn the period between 5,300 and 4,900 calibrated years before present (cal. bp), populations across large parts of Europe underwent a period of demographic decline1,2. However, the cause of this so-called Neolithic decline is still debated. Some argue for an agricultural crisis resulting in the decline3, others for the spread of an early form of plague4. Here we use population-scale ancient genomics to infer ancestry, social structure and pathogen infection in 108 Scandinavian Neolithic individuals from eight megalithic graves and a stone cist. We find that the Neolithic plague was widespread, detected in at least 17% of the sampled population and across large geographical distances. We demonstrate that the disease spread within the Neolithic community in three distinct infection events within a period of around 120 years. Variant graph-based pan-genomics shows that the Neolithic plague genomes retained ancestral genomic variation present in Yersinia pseudotuberculosis, including virulence factors associated with disease outcomes. In addition, we reconstruct four multigeneration pedigrees, the largest of which consists of 38 individuals spanning six generations, showing a patrilineal social organization. Lastly, we document direct genomic evidence for Neolithic female exogamy in a woman buried in a different megalithic tomb than her brothers. Taken together, our findings provide a detailed reconstruction of plague spread within a large patrilineal kinship group and identify multiple plague infections in a population dated to the beginning of the Neolithic decline.

Genetic adaptation refers to changes in a population’s traits that enhance survival in its current environment. As environmental conditions fluctuate, allele frequencies vary, leading to genetic diversity and different phenotypes within the same species. The cooperatively breeding common mole-rats (Cryptomys hottentotus hottentotus) in South Africa serve as an example, as they inhabit areas with varying aridity. By examining mitochondrial genes and a large number of SNP loci, researchers explored the genetic diversity, population structure, and phylogenetic relationships among five mole-rat populations across this gradient. Findings revealed distinct genetic clusters for arid and semi-arid populations compared to mesic ones, with increased genetic diversity and gene flow in arid regions. The study highlighted that environmental isolation, rather than geographical distance, better explains the genetic differences observed. Further research could uncover specific genetic adaptations related to environmental factors, providing insights into how these mole-rats might respond to climate change.

Terrestrial orchids, particularly those in the Orchidinae tribe, are vital both culturally and economically, especially due to their edible tubers used in traditional dishes like ‘salep’. However, overexploitation in the Eastern Mediterranean and Western Asia poses a significant threat to these species, highlighting the need for effective monitoring tools. To address this, a custom bait kit has been developed for the enrichment and sequencing of 205 novel genetic markers specifically designed for phylogenomic research in Orchidinae s.l. Among these, 31 markers are linked to the production of glucomannan, a key polysaccharide in salep. Testing the kit on 73 local taxa yielded high locus recovery across species, outperforming existing alternatives in terms of sequence length. The phylogenetic analysis demonstrated robust support for numerous clades, including some previously unresolved lineages. While challenges remain in resolving hybrid and recently radiated species, further analyses utilizing multiple haplotypes and non-exonic sequences could enhance our understanding of their complex evolutionary history. Overall, the Orchidinae-205 markers represent an advanced resource for studying the evolution, systematics, and trade of terrestrial orchids, surpassing traditional barcoding methods.

Conifers are vital for both ecological and economic reasons, offering valuable insights into land plant evolution. Molecular phylogenetics plays a significant role in studying evolution, but research on conifers using large-scale data from multiple nuclear genes has been limited. Target enrichment sequencing has emerged as a crucial method in phylogenomic studies. However, a specific bait set for conifers is missing. The REMcon probe set targets around 100 single-copy nuclear loci for family- and species-level phylogenetic studies of conifers. High target recovery and read coverage were observed for the REMcon when tested on 69 species, including conifers and other gymnosperm taxa. Phylogenetic analysis based on the DNA sequences generated from REMcon recovered the existing understanding of conifer relationships. The REMcon bait set will be beneficial in generating large-scale nuclear data consistently for any conifer lineage.

Angiosperms are the cornerstone of most terrestrial ecosystems and human livelihoods1,2. A robust understanding of angiosperm evolution is required to explain their rise to ecological dominance. So far, the angiosperm tree of life has been determined primarily by means of analyses of the plastid genome3,4. Many studies have drawn on this foundational work, such as classification and first insights into angiosperm diversification since their Mesozoic origins5,6,7. However, the limited and biased sampling of both taxa and genomes undermines confidence in the tree and its implications. Here, we build the tree of life for almost 8,000 (about 60%) angiosperm genera using a standardized set of 353 nuclear genes8. This 15-fold increase in genus-level sampling relative to comparable nuclear studies9 provides a critical test of earlier results and brings notable change to key groups, especially in rosids, while substantiating many previously predicted relationships. Scaling this tree to time using 200 fossils, we discovered that early angiosperm evolution was characterized by high gene tree conflict and explosive diversification, giving rise to more than 80% of extant angiosperm orders. Steady diversification ensued through the remaining Mesozoic Era until rates resurged in the Cenozoic Era, concurrent with decreasing global temperatures and tightly linked with gene tree conflict. Taken together, our extensive sampling combined with advanced phylogenomic methods shows the deep history and full complexity in the evolution of a megadiverse clade.

An excavation conducted at Harewood Cemetery to identify the unmarked grave of Samuel Washington resulted in the discovery of burials presumably belonging to George Washington’s paternal grandnephews and their mother, Lucy Payne. To confirm their identities this study examined Y-chromosomal, mitochondrial, and autosomal DNA from the burials and a living Washington descendant. The burial’s Y-STR profile was compared to FamilyTreeDNA’s database, which resulted in a one-step difference from the living descendant and an exact match to another Washington. A more complete Y-STR and Y-SNP profile from the descendant was inferred to be the Washington Y profile. Kinship comparisons performed in relation to the descendant, who is a 4th and 5th degree relative of the putative individuals, resulted in >37,000 overlapping autosomal SNPs and strong statistical support with likelihood ratios exceeding one billion. This study highlights the benefits of a multi-marker approach for kinship prediction and DNA-assisted identification of historical remains.

Parchment, the skins of animals prepared for use as writing surfaces, offers a valuable source of genetic information. Many have clearly defined provenance, allowing for the genetic findings to be evaluated in temporal and spatial context. While these documents can yield evidence of the animal sources, the DNA contained within these aged skins is often damaged and fragmented. Previously, genetic studies targeting parchment have used destructive sampling techniques and so the development and validation of non-destructive sampling methods would expand opportunities and facilitate testing of more precious documents, especially those with historical significance. Here we present genetic data obtained by non-destructive sampling of eight parchments spanning the 15th century to the modern day. We define a workflow for enriching the mitochondrial genome (mtGenome), generating next-generation sequencing reads to permit species identification, and providing interpretation guidance. Using sample replication, comparisons to destructively sampled controls, and by establishing authentication criteria, we were able to confidently assign full/near full mtGenome sequences to 56.3% of non-destructively sampled parchments, each with greater than 90% of the mtGenome reference covered. Six of eight parchments passed all four established thresholds with at least one non-destructive sample, highlighting promise for future studies.