Known for its remarkable biodiversity and high levels of endemism, the Brazilian Atlantic Rainforest has been characterized as one of the most threatened biomes on the planet. Despite strong interest in recent years, we still lack a comprehensive scenario to explain the origin and maintenance of diversity in this region, partially given the relatively low power of analyses involving few independent genetic loci. In this study, we examine a phylogenomic dataset of five ant species to investigate phylogeographical patterns across the Brazilian Atlantic Forest. We sequenced ultraconserved elements to generate hundreds of loci using a bait set developed specifically for hymenopterans. We analyzed the data using Bayesian and maximum likelihood approaches of phylogenetic inference. Results were then integrated with environmental niche modeling of current and past climates, including the Last Glacial Maximum and the last interglacial period. The studied species showed differentiation patterns that were consistent with the north/south division of the Atlantic Rainforest indicated in previous studies for other taxa. However, there were differences among species, both in the location of phylogeographic breaks and in the pattern of genetic variation within these areas. Samples from southern localities tended to show recent genetic structure, although a site in Tapiraí (state of São Paulo) repeatedly showed an intriguing older history of differentiation. All species experienced shifts in areas of suitability through the time. Our study suggests that distinct groups may have responded idiosyncratically to the climatic changes that took place in the Brazilian Atlantic Forest. The amount of intraspecific genetic structure was related to the inferred geographical distribution of habitat suitability according to current and past times. Also, a parallel between the amount of Quaternary climatic suitability and the level of interspecific differentiation was detected for four species. Finally, despite strong contractions at the northeastern region of the forest, the remaining areas appear to have been able to act as refugia.

The sunflower family, Asteraceae, comprises 10% of all flowering plant species and displays an incredible diversity of form. Asteraceae are clearly monophyletic, yet resolving phylogenetic relationships within the family has proven difficult, hindering our ability to understand its origin and diversification. Recent molecular clock dating has suggested a Cretaceous origin, but the lack of deep sampling of many genes and representative taxa from across the family has impeded the resolution of migration routes and diversifications that led to its global distribution and tremendous diversity. Here we use genomic data from 256 terminals to estimate evolutionary relationships, timing of diversification(s), and biogeographic patterns. Our study places the origin of Asteraceae at ∼83 MYA in the late Cretaceous and reveals that the family underwent a series of explosive radiations during the Eocene which were accompanied by accelerations in diversification rates. The lineages that gave rise to nearly 95% of extant species originated and began diversifying during the middle Eocene, coincident with the ensuing marked cooling during this period. Phylogenetic and biogeographic analyses support a South American origin of the family with subsequent dispersals into North America and then to Asia and Africa, later followed by multiple worldwide dispersals in many directions. The rapid mid-Eocene diversification is aligned with the biogeographic range shift to Africa where many of the modern-day tribes appear to have originated. Our robust phylogeny provides a framework for future studies aimed at understanding the role of the macroevolutionary patterns and processes that generated the enormous species diversity of Asteraceae.

Development of oligonucleotide probes facilitates chromosome identification via fluorescence in situ hybridization (FISH) in many organisms.

CRISPR-Cas systems inherently multiplex through CRISPR arrays—whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis., CRISPR array generation is difficult due to reoccurring repeat sequences. Here the authors present CRATES—a modular, one-pot assembly method—and demonstrate the creation of arrays for Cas9, Cas12a and Cas13a for cell-free, bacterial, yeast and mammalian systems.

Natural history collections are increasingly valued as genomic resources. Their specimens reflect the combined efforts of collectors and curators over hundreds of years. For many rare or endangered species, specimens are the only readily available source of DNA. We leveraged specimens from a historical collection to study the evolutionary history of wood-partridges in the genus Dendrortyx. The three Dendrortyx species are found in the highlands of central Mexico and Central America south to Costa Rica. One of these species is endangered, and in general, Dendrortyx are secretive and poorly represented in tissue collections. We extracted DNA from historical museum specimens and sequenced ultraconserved elements (UCEs) and mitochondrial DNA (mtDNA) to assess their phylogeny and divergence times. Phylogenies built from hundreds to thousands of nuclear markers were well resolved and largely congruent with an mtDNA phylogeny. The divergence times revealed an unusually old avian divergence across the Isthmus of Tehuantepec in the Pliocene around 3.6 million years ago. Combined with other recent studies, our results challenge the general pattern that highland bird divergences in Mesoamerica are relatively young and influenced by the Pleistocene glacial cycles compared to the older divergences of reptiles and plants, which are thought to overlap more with periods of mountain formation. We also found evidence for monophyletic genetic lineages in mountain ranges within the widespread D. macroura, which should be investigated further with integrative taxonomic methods. Our study demonstrates the power of museum genomics to provide insight into the evolutionary histories of groups where modern samples are lacking.

Myotragus balearicus was the last living representative of an extinct caprine lineage endemic to the Balearic Islands (Western Mediterranean), which became extinct following the arrival of humans around 4300 years ago. The tribal attribution of Myotragus based on morphological analyses has been complicated due to its unusual morphological characteristics, though most studies agree on its inclusion within Caprini (also including modern sheep and goats). Analyses of short fragments of ancient mitochondrial DNA have suggested Ovis (comprising extant sheep) as sister taxon to Myotragus, although other authors have suggested alternative placements within Caprini using the same data. In the present study, we present a complete high-depth mitochondrial genome of M. balearicus, which allowed us to test the previously proposed Ovis/Myotragus clade and revealed a closer relationship between Myotragus and the takin (Budorcas taxicolor). The results of our molecular clock analyses suggested that the split between Myotragus and Budorcas occurred around 7.1 Mya, which is compatible with the arrival of the ancestor of Myotragus to the Balearic Islands during the Messinian Salinity Crisis (5.97–5.33 Mya). We also conducted a preliminary phylogeographic analysis of M. balearicus in Mallorca, which revealed weak spatial and temporal structure within the population during the Holocene.

We developed an in-solution gluten exome capture system called GlutEnSeq (Gluten gene Enrichment and Sequencing), covering the sequence variation of thousands of prolamin genes from various Triticeae species and cultivars. We assessed the efficacy of this capture system in hexaploid wheat (Triticum aestivum L.) using Illumina sequencing. On-target regions were determined based on the Chinese Spring (CS) reference genome sequence. Gluten gene sequences were generally enriched around 10,000-fold. The loss of gluten genes in CS deletion line 1BS-19/6DS-4 was detected as absence of gluten gene coverage on chromosomes 1B and Un (containing the Unmapped α-gliadin genes of chromosome 6D). Two γ-irradiated lines of cultivar Paragon, shown to be affected in their gliadin protein profile, were found to contain homozygous deletions for the α-gliadins on 6A and the γ-gliadins on 1B. Four Fielder CRISPR/Cas9 gliadin gene-edited lines revealed homozygous deletions of the γ-gliadins on 1B and heterozygous deletions for the α-gliadins on 6A. We also detected a decrease of gluten gene coverage within some gluten genes. The bioinformatics pipeline developed here will be further optimised to enable characterisation of small indels within individual gluten genes, in order to fully analyse CRISPR/Cas9 mutant lines for their decrease in immunogenicity for Coeliac patients.

In the age of next-generation sequencing, the number of loci available for phylogenetic analyses has increased by orders of magnitude. But despite this dramatic increase in the amount of data, some phylogenomic studies have revealed rampant gene-tree discordance that can be caused by many historical processes, such as rapid diversification, gene duplication, or reticulate evolution. We used a target enrichment approach to sample 400 single-copy nuclear genes and estimate the phylogenetic relationships of 13 genera in the lichen-forming family Lobariaceae to address the effect of data type (nucleotides and amino acids) and phylogenetic reconstruction method (concatenation and species tree approaches). Furthermore, we examined datasets for evidence of historical processes, such as rapid diversification and reticulate evolution. We found incongruence associated with sequence data types (nucleotide vs. amino acid sequences) and with different methods of phylogenetic reconstruction (species tree vs. concatenation). The resulting phylogenetic trees provided evidence for rapid and reticulate evolution based on extremely short branches in the backbone of the phylogenies. The observed rapid and reticulate diversifications may explain conflicts among gene trees and the challenges to resolving evolutionary relationships. Based on divergence times, the diversification at the backbone occurred near the Cretaceous-Paleogene (K-Pg) boundary (65 Mya) which is consistent with other rapid diversifications in the tree of life. Although some phylogenetic relationships within the Lobariaceae family remain with low support, even with our powerful phylogenomic dataset of up to 376 genes, our use of target-capturing data allowed for the novel exploration of the mechanisms underlying phylogenetic and systematic incongruence.