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The goals of the Earth Biogenome Project—to sequence the genomes of all eukaryotic life on earth—are as daunting as they are ambitious. The Darwin Tree of Life Project was founded to demonstrate the credibility of these goals and to deliver at-scale genome sequences of unprecedented quality for a biogeographic region: the archipelago of islands that constitute Britain and Ireland. The Darwin Tree of Life Project is a collaboration between biodiversity organizations (museums, botanical gardens, and biodiversity institutes) and genomics institutes. Together, we have built a workflow that collects specimens from the field, robustly identifies them, performs sequencing, generates high-quality, curated assemblies, and releases these openly for the global community to use to build future science and conservation efforts.

We present a complete generic‐level phylogeny of the complex thalloid liverworts, a lineage that includes the model system Marchantia polymorpha. The complex thalloids are remarkable for their slow rate of molecular evolution and for being the only extant plant lineage to differentiate gas exchange tissues in the gametophyte generation. We estimated the divergence times and analyzed the evolutionary trends of morphological traits, including air chambers, rhizoids and specialized reproductive structures. A multilocus dataset was analyzed using maximum likelihood and Bayesian approaches. Relative rates were estimated using local clocks. Our phylogeny cements the early branching in complex thalloids. Marchantia is supported in one of the earliest divergent lineages. The rate of evolution in organellar loci is slower than for other liverwort lineages, except for two annual lineages. Most genera diverged in the Cretaceous. Marchantia polymorpha diversified in the Late Miocene, giving a minimum age estimate for the evolution of its sex chromosomes. The complex thalloid ancestor, excluding Blasiales, is reconstructed as a plant with a carpocephalum, with filament‐less air chambers opening via compound pores, and without pegged rhizoids. Our comprehensive study of the group provides a temporal framework for the analysis of the evolution of critical traits essential for plants during land colonization.

Recent technological advances in long‐read high‐throughput sequencing and assembly methods have facilitated the generation of annotated chromosome‐scale whole‐genome sequence data for evolutionary studies; however, generating such data can still be difficult for many plant species. For example, obtaining high‐molecular‐weight DNA is typically impossible for samples in historical herbarium collections, which often have degraded DNA. The need to fast‐freeze newly collected living samples to conserve high‐quality DNA can be complicated when plants are only found in remote areas. Therefore, short‐read reduced‐genome representations, such as target capture and genome skimming, remain important for evolutionary studies. Here, we review the pros and cons of each technique for non‐model plant taxa. We provide guidance related to logistics, budget, the genomic resources previously available for the target clade, and the nature of the study. Furthermore, we assess the available bioinformatic analyses, detailing best practices and pitfalls, and suggest pathways to combine newly generated data with legacy data. Finally, we explore the possible downstream analyses allowed by the type of data generated using each technique. We provide a practical guide to help researchers make the best‐informed choice regarding reduced genome representation for evolutionary studies of non‐model plants in cases where whole‐genome sequencing remains impractical.

Background PacBio HiFi sequencing provides highly accurate long-read sequencing datasets which are of great advantage for whole genome sequencing projects. One limitation of the method is the requirement for high quality, high molecular weight input DNA. This can be particularly challenging for plants that frequently contain common and species-specific secondary metabolites, which often interfere with downstream processes. Cape Primroses (genus Streptocarpus ), are some of these recalcitrant plants and are selected here as material to develop a high quality, high molecular weight DNA extraction protocol for long read genome sequencing. Results We developed a DNA extraction method for PacBio HiFi sequencing for Streptocarpus grandis and Streptocarpus kentaniensis. A CTAB lysis buffer was employed to avoid guanidine, and the traditional chloroform and phenol purification steps were replaced with pre-lysis sample washes. Best cells/nucleus lysis was achieved with 4 h at 58 °C. The obtained high quality and high molecular weight DNAs were tested in PacBio SMRTBell™ library preparations, which resulted in circular consensus sequencing (CCS) reads from 17 to 27 Gb per cell, and a read length N50 from 14 to 17 kbp. To evaluate the quality of the reads for whole genome sequencing, they were assembled with HiFiasm into draft genomes, with N50 = 49 Mb and 23 Mb, and L50 = 10 and 11. The longest contigs were 95 Mb and 57 Mb respectively, showing good contiguity as these are longer than the theoretical chromosome length (genome size/chromosome number) of 78 Mb and 55 Mb, for S. grandis and S. kentaniensis respectively. Conclusions DNA extraction is a critical step towards obtaining a complete genome assembly. Our DNA extraction method here provided the required high quality, high molecular weight DNA for successful standard-input PacBio HiFi library preparation. The contigs from those reads showed a high contiguity, providing a good starting draft assembly towards obtaining a complete genome. The results obtained here were highly promising, and demonstrated that the DNA extraction method developed here is compatible with PacBio HiFi sequencing and suitable for de novo whole genome sequencing projects of plants.

Intentionally preserved biological material in natural history collections represents a vast repository of biodiversity. Advances in laboratory and sequencing technologies have made these specimens increasingly accessible for genomic analyses, offering a window into the genetic past of species and often permitting access to information that can no longer be sampled in the wild. Due to their age, preparation and storage conditions, DNA retrieved from museum and herbarium specimens is often poor in yield, heavily fragmented and biochemically modified. This not only poses methodological challenges in recovering nucleotide sequences, but also makes such investigations susceptible to environmental and laboratory contamination. In this paper, we review the practical challenges associated with making the recovery of DNA sequence data from museum collections more routine. We first review key operational principles and issues to address, to guide the decision-making process and dialogue between researchers and curators about when and how to sample museum specimens for genomic analyses. We then outline the range of steps that can be taken to reduce the likelihood of contamination including laboratory set-ups, workflows and working practices. We finish by presenting a series of case studies, each focusing on protocol practicalities for the application of different mainstream methodologies to museum specimens including: (i) shotgun sequencing of insect mitogenomes, (ii) whole genome sequencing of insects, (iii) genome skimming to recover plant plastid genomes from herbarium specimens, (iv) target capture of multi-locus nuclear sequences from herbarium specimens, (v) RAD-sequencing of bird specimens and (vi) shotgun sequencing of ancient bovid bone samples.

Various historical processes have been put forth as drivers of patterns in the spatial distribution of Amazonian trees and their population genetic variation. We tested whether five widespread tree species show congruent phylogeographic breaks and similar patterns of demographic expansion, which could be related to proposed Pleistocene refugia or the presence of geological arches in western Amazonia. We sampled Otoba parvifolia/glycycarpa (Myristicaceae), Clarisia biflora, Poulsenia armata, Ficus insipida (all Moraceae), and Jacaratia digitata (Caricaceae) across the western Amazon Basin. Plastid DNA (trnH–psbA; 674 individuals from 34 populations) and nuclear ribosomal internal transcribed spacers (ITS; 214 individuals from 30 populations) were sequenced to assess genetic diversity, genetic differentiation, population genetic structure, and demographic patterns. Overall genetic diversity for both markers varied among species, with higher values in populations of shade‐tolerant species than in pioneer species. Spatial analysis of molecular variance (SAMOVA) identified three genetically differentiated groups for the plastid marker for each species, but the areas of genetic differentiation were not concordant among species. Fewer SAMOVA groups were found for ITS, with no detectable genetic differentiation among populations in pioneers. The lack of spatially congruent phylogeographic breaks across species suggests no common biogeographic history of these Amazonian tree species. The idiosyncratic phylogeographic patterns of species could be due instead to species‐specific responses to geological and climatic changes. Population genetic patterns were similar among species with similar biological features, indicating that the ecological characteristics of species impact large‐scale phylogeography. Our study includes thorough and relatively uniform sampling of populations of five widespread western Amazonian tree species across a wide geographic area covering most of western Amazonia. As such, it is the first comparative phylogeographic study of the trees of western Amazonia, which houses the world's most species‐rich forests. By using plastid and nuclear markers sampled in 34 locations across Ecuador, Peru, and Bolivia, we studied the geographic patterns of genetic differentiation to test for shared impact of past climatic and geological events. We did not find spatially congruent phylogeographic breaks across species suggesting no common biogeographic history of these Amazonian tree species. The idiosyncratic phylogeographic patterns of species could be due instead to species‐specific responses to geological and climatic changes. Population genetic patterns were similar among species with similar biological features, indicating that the ecological characteristics of species impact large‐scale phylogeography.

The digitate-tubered clade (Dactylorhiza s.l. plus Gymnadenia s.l.) within subtribe Orchidinae is an important element of the North-temperate orchid flora and has become a model system for studying the genetic and epigenetic consequences of organism-wide ploidy change. Here, we integrate morphological phylogenetics with Sanger sequencing of nrITS and the plastid region trnL-F in order to explore phylogenetic relationships and phenotypic character evolution within the clade. The resulting morphological phylogenies are strongly incongruent with the molecular phylogenies, instead reconstructing through parsimony the genus-level boundaries recognised by traditional 20th Century taxonomy. They raise fresh doubts concerning whether Pseudorchis is sister to Platanthera or to Dactylorhiza plus Gymnadenia. Constraining the morphological matrix to the topology derived from ITS sequences increased tree length by 20%, adding considerably to the already exceptional level of phenotypic homoplasy. Both molecular and morphological trees agree that D. vindis and D. iberica are the earliestdiverging species within Dactylorhiza (emphasising the redundancy of the former genus Coeloglossum). Morphology and ITS both suggest that the former genus Nigritetta is nested within (and thus part of) Gymnadenia, the Pyrenean endemic 'N.' gabasiana apparently forming a molecular bridge between the two radically contrasting core phenotypes. Comparatively short subtending molecular branches plus widespread (though sporadic) hybridisation indicate that Dactylorhiza and Gymnadenia approximate the minimum level of molecular divergence acceptable in sister genera. They share similar tuber morphologies and base chromosome numbers, and both genera are unusually prone to polyploid speciation. Another prominent feature of multiple speciation events within Gymnadenia is floral paedomorphosis. The 'traditional' morphological and candidate-gene approaches to phylogeny reconstruction are critically appraised.

Cape Primroses (Streptocarpus, Gesneriaceae) are an ideal study system for investigating the genetics underlying species diversity in angiosperms. Streptocarpus rexii has served as a model species for plant developmental research for over five decades due to its unusual extended meristem activity present in the leaves. In this study, we sequenced and assembled the complete nuclear, chloroplast, and mitochondrial genomes of S. rexii using Oxford Nanopore Technologies long read sequencing. Two flow cells of PromethION sequencing resulted in 32 billion reads and were sufficient to generate a draft assembly including the chloroplast, mitochondrial and nuclear genomes, spanning 776 Mbp. The final nuclear genome assembly contained 5,855 contigs, spanning 766 Mbp of the 929‐Mbp haploid genome with an N50 of 3.7 Mbp and an L50 of 57 contigs. Over 70% of the draft genome was identified as repeats. A genome repeat library of Gesneriaceae was generated and used for genome annotation, with a total of 45,045 genes annotated in the S. rexii genome. Ks plots of the paranomes suggested a recent whole genome duplication event, shared between S. rexii and Primulina huaijiensis. A new chloroplast and mitochondrial genome assembly method, based on contig coverage and identification, was developed, and successfully used to assemble both organellar genomes of S. rexii. This method was developed into a pipeline and proved widely applicable. The nuclear genome of S. rexii and other datasets generated and reported here will be invaluable resources for further research to aid in the identification of genes involved in morphological variation underpinning plant diversification.

The earliest stages of osteoarthritis are characterized by peripheral pathology; however, during disease progression chronic pain emerges—a major symptom of osteoarthritis linked to neuroplasticity. Recent clinical imaging studies involving chronic pain patients, including osteoarthritis patients, have demonstrated that functional properties of the brain are altered, and these functional changes are correlated with subjective behavioral pain measures. Currently, preclinical osteoarthritis studies have not assessed if functional properties of supraspinal pain circuitry are altered, and if these functional properties can be modulated by pharmacological therapy either by direct or indirect action on brain systems. In the current study, functional connectivity was first assessed in order to characterize the functional neuroplasticity occurring in the rodent medial meniscus tear (MMT) model of osteoarthritis—a surgical model of osteoarthritis possessing peripheral joint trauma and a hypersensitive pain state. In addition to knee joint trauma at week 3 post‐MMT surgery, we observed that supraspinal networks have increased functional connectivity relative to sham animals. Importantly, we observed that early and sustained treatment with a novel, peripherally acting broad-spectrum matrix metalloproteinase (MMP) inhibitor (MMPi) significantly attenuates knee joint trauma (cartilage degradation) as well as supraspinal functional connectivity increases in MMT animals. At week 5 post‐MMT surgery, the acute pharmacodynamic effects of celecoxib (selective cyclooxygenase-2 inhibitor) on brain function were evaluated using pharmacological magnetic resonance imaging (phMRI) and functional connectivity analysis. Celecoxib was chosen as a comparator, given its clinical efficacy for alleviating pain in osteoarthritis patients and its peripheral and central pharmacological action. Relative to the vehicle condition, acute celecoxib treatment in MMT animals yielded decreased phMRI infusion responses and decreased functional connectivity, the latter observation being similar to what was detected following chronic MMPi treatment. These findings demonstrate that an assessment of brain function may provide an objective means by which to further evaluate the pathology of an osteoarthritis state as well as measure the pharmacodynamic effects of therapies with peripheral or peripheral and central pharmacological action.