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Biodiversity genomics research requires reliable organismal identification, which can be difficult based on morphology alone. DNA-based identification using DNA barcoding can provide confirmation of species identity and resolve taxonomic issues but is rarely used in studies generating reference genomes. Here, we describe the development and implementation of DNA barcoding for the Darwin Tree of Life Project (DToL), which aims to sequence and assemble high quality reference genomes for all eukaryotic species in Britain and Ireland. We present a standardised framework for DNA barcode sequencing and data interpretation that is then adapted for diverse organismal groups. DNA barcoding data from over 12,000 DToL specimens has identified up to 20% of samples requiring additional verification, with 2% of seed plants and 3.5% of animal specimens subsequently having their names changed. We also make recommendations for future developments using new sequencing approaches and streamlined bioinformatic approaches., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Twyford AD et al.)

We present a genome assembly from an individual Trifolium dubium (lesser trefoil; Tracheophyta; Magnoliopsida; Fabales; Fabaceae) as part of a collaboration between the Darwin Tree of Life and the European Reference Genome Atlas. The genome sequence is 679.1 megabases in span. Most of the assembly is scaffolded into 15 chromosomal pseudomolecules. The two mitochondrial genomes have lengths of 133.86 kb and 182.32 kb, and the plastid genome assembly has a length of 126.22 kilobases., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Ruhsam M et al.)

We present a genome assembly from an individual specimen of Luzula sylvatica (great wood-rush; Tracheophyta; Magnoliopsida; Poales; Juncaceae). The genome sequence is 444.5 megabases in span. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules. The mitochondrial and plastid genome assemblies have lengths of 633.36 kilobases and 201.32 kilobases in length, respectively., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Goodwin ZA et al.)

We present a genome assembly from an individual Polygonum aviculare (common knotgrass; Eudicot; Magnoliopsida; Caryophyllales; Polygonaceae). The genome sequence is 351.6 megabases in span. Most of the assembly is scaffolded into 10 chromosomal pseudomolecules. The mitochondrial and plastid genome assemblies have lengths of 333.39 kilobases and 163.28 kilobases in length, respectively., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Christenhusz MJM et al.)

Understanding the effects of cash crop expansion on natural forest is of fundamental importance. However, for most crops there are no remotely sensed global maps 1 , and global deforestation impacts are estimated using models and extrapolations. Natural rubber is an example of a principal commodity for which deforestation impacts have been highly uncertain, with estimates differing more than fivefold 1-4 . Here we harnessed Earth observation satellite data and cloud computing 5 to produce high-resolution maps of rubber (10 m pixel size) and associated deforestation (30 m pixel size) for Southeast Asia. Our maps indicate that rubber-related forest loss has been substantially underestimated in policy, by the public and in recent reports 6-8 . Our direct remotely sensed observations show that deforestation for rubber is at least twofold to threefold higher than suggested by figures now widely used for setting policy 4 . With more than 4 million hectares of forest loss for rubber since 1993 (at least 2 million hectares since 2000) and more than 1 million hectares of rubber plantations established in Key Biodiversity Areas, the effects of rubber on biodiversity and ecosystem services in Southeast Asia could be extensive. Thus, rubber deserves more attention in domestic policy, within trade agreements and in incoming due-diligence legislation., (© 2023. The Author(s).)

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., Competing Interests: No conflict of interest to declare Disclaimer: This article is (co-)authored by any of the Editors-in-Chief, Managing Editors or their deputies in this journal., (Giada Ferrari, Lore Esselens, Michelle L Hart, Steven Janssens, Catherine Kidner, Maurizio Mascarello, Joshua V Peñalba, Flávia Pezzini, Thomas von Rintelen, Gontran Sonet, Carl Vangestel, Massimiliano Virgilio, Peter M Hollingsworth.)

Natural hybridization can have a profound evolutionary impact, with consequences ranging from the extinction of rare taxa to the origin of new species. Natural hybridization is particularly common in plants; however, our understanding of the general factors that promote or prevent hybridization is hampered by the highly variable outcomes in different lineages. Here, we quantify the influence of different predictors on hybrid formation across species from an entire flora. We combine estimates of hybridization with ecological attributes and a new species-level phylogeny for over 1,100 UK flowering plant species. Our results show that genetic factors, particularly parental genetic distance, as well as phylogenetic position and ploidy, are key determinants of hybrid formation, whereas many other factors such as range overlap and genus size explain much less variation in hybrid formation. Overall, intrinsic genetic factors shape the evolutionary and ecological consequences of natural hybridization across species in a flora.

Background and Aims: Rhododendron is a species-rich and taxonomically challenging genus due to recent adaptive radiation and frequent hybridization. A well-resolved phylogenetic tree would help to understand the diverse history of Rhododendron in the Himalaya-Hengduan Mountains where the genus is most diverse., Methods: We reconstructed the phylogeny based on plastid genomes with broad taxon sampling, covering 161 species representing all eight subgenera and all 12 sections, including ~45 % of the Rhododendron species native to the Himalaya-Hengduan Mountains. We compared this phylogeny with nuclear phylogenies to elucidate reticulate evolutionary events and clarify relationships at all levels within the genus. We also estimated the timing and diversification history of Rhododendron, especially the two species-rich subgenera Rhododendron and Hymenanthes that comprise >90 % of Rhododendron species in the Himalaya-Hengduan Mountains., Key Results: The full plastid dataset produced a well-resolved and supported phylogeny of Rhododendron. We identified 13 clades that were almost always monophyletic across all published phylogenies. The conflicts between nuclear and plastid phylogenies suggested strongly that reticulation events may have occurred in the deep lineage history of the genus. Within Rhododendron, subgenus Therorhodion diverged first at 56 Mya, then a burst of diversification occurred from 23.8 to 17.6 Mya, generating ten lineages among the component 12 clades of core Rhododendron. Diversification in subgenus Rhododendron accelerated c. 16.6 Mya and then became fairly continuous. Conversely, Hymenanthes diversification was slow at first, then accelerated very rapidly around 5 Mya. In the Himalaya-Hengduan Mountains, subgenus Rhododendron contained one major clade adapted to high altitudes and another to low altitudes, whereas most clades in Hymenanthes contained both low- and high-altitude species, indicating greater ecological plasticity during its diversification., Conclusions: The 13 clades proposed here may help to identify specific ancient hybridization events. This study will help to establish a stable and reliable taxonomic framework for Rhododendron, and provides insight into what drove its diversification and ecological adaption. Denser sampling of taxa, examining both organelle and nuclear genomes, is needed to better understand the divergence and diversification history of Rhododendron., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Understanding the evolutionary and ecological processes driving population differentiation and speciation can provide critical insights into the formation of biodiversity. Here, we examine the link between population genetic processes and biogeographic history underlying the generation of diversity in the Hengduan Mountains (HM), a region harboring a rich and dynamic flora. We used restriction site-associated DNA sequencing to generate 1,907 single-nucleotide polymorphisms (SNPs) and four-kb of plastid sequence in species of the Gentiana hexaphylla complex (Gentianaceae). We performed genetic clustering with spatial and non-spatial models, phylogenetic reconstructions, and ancestral range estimation, with the aim of addressing the processes influencing diversification of G. hexaphylla in the HM. We find the G. hexaphylla complex is characterized by geographic genetic structure with clusters corresponding to the South, North and the central HM. Phylogenetic reconstruction and pairwise F ST analyses showed deep differentiation between Southern and Northern populations in the HM. The population in Mount Taibai exhibited the highest genetic similarity to the North HM. Ancestral range estimation indicated that the G. hexaphylla complex originated in the central HM and then diverged in the Pliocene and the Early Pleistocene, before dispersing widely, resulting in the current distinct lineages. Overall, we found deep genomic differentiation in the G. hexaphylla complex corresponds to geographic barriers to dispersal in the HM and highlights a critical role of the uplift of the Daxue Mountains and subsequent climatic fluctuations underlying diversification. The colonization of G. hexaphylla in the Mount Taibai region suggests directional dispersal between the alpine flora of the Qinling Mountains and the HM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fu, Sun, Hollingsworth, Chen, Favre and Twyford.)

Aim: Climate change is expected to impact mountain biodiversity by shifting species ranges and the biomes they shape. The extent and regional variation in these impacts are still poorly understood, particularly in the highly biodiverse Andes. Regional syntheses of climate change impacts on vegetation are pivotal to identify and guide research priorities. Here we review current data, knowledge and uncertainties in past, present and future climate change impacts on vegetation in the Andes. Location: Andes. Taxon: Plants., Methods: We (i) conducted a literature review on Andean vegetation responses to past and contemporary climatic change, (ii) analysed future climate projections for different elevations and slope orientations at 19 Andean locations using an ensemble of model outputs from the Coupled Model Intercomparison Project 5, and (iii) calculated changes in the suitable climate envelope area of Andean biomes and compared these results to studies that used species distribution models., Results: Future climatic changes (2040-2070) are projected to be stronger at high-elevation areas in the tropical Andes (up to 4°C under RCP 8.5), while in the temperate Andes temperature increases are projected to be up to 2°C. Under this worst-case scenario, temperate deciduous forests and the grasslands/steppes from the Central and Southern Andes are predicted to show the greatest losses of suitable climatic space (30% and 17%-23%, respectively). The high vulnerability of these biomes contrasts with the low attention from researchers modelling Andean species distributions. Critical knowledge gaps include a lack of an Andean wide plant checklist, insufficient density of weather stations at high-elevation areas, a lack of high-resolution climatologies that accommodates the Andes' complex topography and climatic processes, insufficient data to model demographic and ecological processes, and low use of palaeo data for distribution modelling., Main Conclusions: Climate change is likely to profoundly affect the extent and composition of Andean biomes. Temperate Andean biomes in particular are susceptible to substantial area contractions. There are, however, considerable challenges and uncertainties in modelling species and biome responses and a pressing need for a region-wide approach to address knowledge gaps and improve understanding and monitoring of climate change impacts in these globally important biomes., Competing Interests: The authors have no conflict of interest to declare., (© 2022 The Authors. Journal of Biogeography published by John Wiley & Sons Ltd.)

The vascular flora of Britain and Ireland is among the most extensively studied in the world, but the current knowledge base is fragmentary, with taxonomic, ecological and genetic information scattered across different resources. Here we present the first comprehensive data repository of native and alien species optimized for fast and easy online access for ecological, evolutionary and conservation analyses. The inventory is based on the most recent reference flora of Britain and Ireland, with taxon names linked to unique Kew taxon identifiers and DNA barcode data. Our data resource for 3,227 species and 26 traits includes existing and unpublished genome sizes, chromosome numbers and life strategy and life-form assessments, along with existing data on functional traits, species distribution metrics, hybrid propensity, associated biomes, realized niche description, native status and geographic origin of alien species. This resource will facilitate both fundamental and applied research and enhance our understanding of the flora's composition and temporal changes to inform conservation efforts in the face of ongoing climate change and biodiversity loss., (© 2022. The Author(s).)

Standard plant DNA barcodes based on 2-3 plastid regions, and nrDNA ITS show variable levels of resolution, and fail to discriminate among species in many plant groups. Genome skimming to recover complete plastid genome sequences and nrDNA arrays has been proposed as a solution to address these resolution limitations. However, few studies have empirically tested what gains are achieved in practice. Of particular interest is whether adding substantially more plastid and nrDNA characters will lead to an increase in discriminatory power, or whether the resolution limitations of standard plant barcodes are fundamentally due to plastid genomes and nrDNA not tracking species boundaries. To address this, we used genome skimming to recover near-complete plastid genomes and nuclear ribosomal DNA from Rhododendron species and compared discrimination success with standard plant barcodes. We sampled 218 individuals representing 145 species of this species-rich and taxonomically difficult genus, focusing on the global biodiversity hotspots of the Himalaya-Hengduan Mountains. Only 33% of species were distinguished using ITS+matK+rbcL+trnH-psbA. In contrast, 55% of species were distinguished using plastid genome and nrDNA sequences. The vast majority of this increase is due to the additional plastid characters. Thus, despite previous studies showing an asymptote in discrimination success beyond 3-4 plastid regions, these results show that a demonstrable increase in discriminatory power is possible with extensive plastid genome data. However, despite these gains, many species remain unresolved, and these results also reinforce the need to access multiple unlinked nuclear loci to obtain transformative gains in species discrimination in plants., (© 2021 John Wiley & Sons Ltd.)

The promotion of responsible and sustainable trade in biological resources is widely proposed as one solution to mitigate current high levels of global biodiversity loss. Various molecular identification methods have been proposed as appropriate tools for monitoring global supply chains of commercialized animals and plants. Here, we demonstrate the efficacy of target capture genomic barcoding in identifying and establishing the geographic origin of samples traded as Anacyclus pyrethrum, a medicinal plant assessed as globally vulnerable in the IUCN Red List of Threatened Species. Samples collected from national and international supply chains were identified through target capture sequencing of 443 low-copy nuclear makers and compared to results derived from genome skimming of plastome and DNA barcoding of standard plastid regions and ITS. Both target capture and genome skimming provided approximately 3.4 million reads per sample, but target capture largely outperformed standard plant barcodes and entire plastid genome sequences. We were able to discern the geographical origin of Anacyclus samples collected in Moroccan, Indian and Sri Lankan markets, differentiating between plant materials originally harvested from diverse populations in Algeria and Morocco. Dropping costs of analysing samples enables the potential of target capture to routinely identify commercialized plant species and determine their geographic origin. It promises to play an important role in monitoring and regulation of plant species in trade, supporting biodiversity conservation efforts, and in ensuring that plant products are unadulterated, contributing to consumer protection., (© 2021 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Background: Flowering plants (angiosperms) are dominant components of global terrestrial ecosystems, but phylogenetic relationships at the familial level and above remain only partially resolved, greatly impeding our full understanding of their evolution and early diversification. The plastome, typically mapped as a circular genome, has been the most important molecular data source for plant phylogeny reconstruction for decades., Results: Here, we assembled by far the largest plastid dataset of angiosperms, composed of 80 genes from 4792 plastomes of 4660 species in 2024 genera representing all currently recognized families. Our phylogenetic tree (PPA II) is essentially congruent with those of previous plastid phylogenomic analyses but generally provides greater clade support. In the PPA II tree, 75% of nodes at or above the ordinal level and 78% at or above the familial level were resolved with high bootstrap support (BP ≥ 90). We obtained strong support for many interordinal and interfamilial relationships that were poorly resolved previously within the core eudicots, such as Dilleniales, Saxifragales, and Vitales being resolved as successive sisters to the remaining rosids, and Santalales, Berberidopsidales, and Caryophyllales as successive sisters to the asterids. However, the placement of magnoliids, although resolved as sister to all other Mesangiospermae, is not well supported and disagrees with topologies inferred from nuclear data. Relationships among the five major clades of Mesangiospermae remain intractable despite increased sampling, probably due to an ancient rapid radiation., Conclusions: We provide the most comprehensive dataset of plastomes to date and a well-resolved phylogenetic tree, which together provide a strong foundation for future evolutionary studies of flowering plants., (© 2021. The Author(s).)

DNA barcoding and metabarcoding provide new avenues for investigating biological systems. These techniques require well-curated reference libraries with extensive coverage. Generating an exhaustive national DNA barcode reference library can open up new avenues of research in ecology, evolution and conservation, yet few studies to date have created such a resource. In plant DNA barcoding, herbarium collections provide taxonomically robust material but also pose challenges in lab processing. Here, we present a national DNA barcoding resource covering all of the native flowering plants and conifers of the United Kingdom. This represents 1,482 plant species, with the majority of specimens (81%) sourced from herbaria. Using Sanger sequencing of the plant DNA barcode markers, rbcL, matK, and ITS2, at least one DNA barcode was retrieved from 98% of the UK flora. We sampled from multiple individuals, resulting in a species coverage for rbcL of 96% (4,477 sequences), 90% for matK (3,259 sequences) and 75% for ITS2 (2,585 sequences). Sequence recovery was lower for herbarium material compared to fresh collections, with the age of the specimen having a significant effect on the success of sequence recovery. Species level discrimination was highest with ITS2, however, the ability to successfully retrieve a sequence was lowest for this region. Analyses of the genetic distinctiveness of species across a complete flora showed DNA barcoding to be informative for all but the most taxonomically complex groups. The UK flora DNA barcode reference library provides an important resource for many applications that require plant identification from DNA., (© 2021 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Some animals fashion tools or constructions out of plant materials to aid foraging, reproduction, self-maintenance, or protection. Their choice of raw materials can affect the structure and properties of the resulting artifacts, with considerable fitness consequences. Documenting animals' material preferences is challenging, however, as manufacture behavior is often difficult to observe directly, and materials may be processed so heavily that they lack identifying features. Here, we use DNA barcoding to identify, from just a few recovered tool specimens, the plant species New Caledonian crows ( Corvus moneduloides ) use for crafting elaborate hooked stick tools in one of our long-term study populations. The method succeeded where extensive fieldwork using an array of conventional approaches-including targeted observations, camera traps, radio-tracking, bird-mounted video cameras, and behavioral experiments with wild and temporarily captive subjects-had failed. We believe that DNA barcoding will prove useful for investigating many other tool and construction behaviors, helping to unlock significant research potential across a wide range of study systems., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Genome skimming has the potential for generating large data sets for DNA barcoding and wider biodiversity genomic studies, particularly via the assembly and annotation of full chloroplast (cpDNA) and nuclear ribosomal DNA (nrDNA) sequences. We compare the success of genome skims of 2051 herbarium specimens from Norway/Polar regions with 4604 freshly collected, silica gel dried specimens mainly from the European Alps and the Carpathians. Overall, we were able to assemble the full chloroplast genome for 67% of the samples and the full nrDNA cluster for 86%. Average insert length, cover and full cpDNA and rDNA assembly were considerably higher for silica gel dried than herbarium-preserved material. However, complete plastid genomes were still assembled for 54% of herbarium samples compared to 70% of silica dried samples. Moreover, there was comparable recovery of coding genes from both tissue sources (121 for silica gel dried and 118 for herbarium material) and only minor differences in assembly success of standard barcodes between silica dried (89% ITS2, 96% matK and rbcL ) and herbarium material (87% ITS2, 98% matK and rbcL ). The success rate was > 90% for all three markers in 1034 of 1036 genera in 160 families, and only Boraginaceae worked poorly, with 7 genera failing. Our study shows that large-scale genome skims are feasible and work well across most of the land plant families and genera we tested, independently of material type. It is therefore an efficient method for increasing the availability of plant biodiversity genomic data to support a multitude of downstream applications.

Microsatellite markers were developed for the tree peony Paeonia delavayi to investigate fine scale population genetics of this species. Using ddRAD-seq data from twenty individuals of P. delavayi, we identified 529 polymorphic microsatellite loci, of which 195 were suitable for designing microsatellite primers. Of the 120 microsatellite loci selected for validation, 20 were successfully amplified with clear peaks and displayed polymorphism. Three populations were genotyped using the 20 polymorphic microsatellites. The number of alleles per locus ranged from two to thirteen. Observed and expected heterozygosity ranged from 0 to 0.941 and 0 to 0.834 respectively. The cross-species amplification test using five individuals from a population of P. ludlowii showed that 15 of the 20 polymorphic loci were successfully amplified, and four loci showed polymorphism. Among the 22 alleles occurring in P. ludlowii across fifteen loci, eight alleles across five loci were exclusive to P. ludlowii. The results demonstrate that ddRAD-seq is an efficient method for the development of microsatellite markers for non-model organisms with large genomes. The newly developed markers will be valuable tools to investigate the genetic diversity, genetic structure, and gene flow of P. delavayi from local to regional spatial scales.

Angiosperms are by far the most species-rich clade of land plants, but their origin and early evolutionary history remain poorly understood. We reconstructed angiosperm phylogeny based on 80 genes from 2,881 plastid genomes representing 85% of extant families and all orders. With a well-resolved plastid tree and 62 fossil calibrations, we dated the origin of the crown angiosperms to the Upper Triassic, with major angiosperm radiations occurring in the Jurassic and Lower Cretaceous. This estimated crown age is substantially earlier than that of unequivocal angiosperm fossils, and the difference is here termed the 'Jurassic angiosperm gap'. Our time-calibrated plastid phylogenomic tree provides a highly relevant framework for future comparative studies of flowering plant evolution.

Background and Aims: Rapid evolutionary divergence and reticulate evolution may result in phylogenetic relationships that are difficult to resolve using small nucleotide sequence data sets. Next-generation sequencing methods can generate larger data sets that are better suited to solving these puzzles. One major and long-standing controversy in conifers concerns generic relationships within the subfamily Cupressoideae (105 species, approx. 1/6 of all conifers) of Cupressaceae, in particular the relationship between Juniperus, Cupressus and the Hesperocyparis-Callitropsis-Xanthocyparis (HCX) clade. Here we attempt to resolve this question using transcriptome-derived data., Methods: Transcriptome sequences of 20 species from Cupressoideae were collected. Using MarkerMiner, single-copy nuclear (SCN) genes were extracted. These were applied to estimate phylogenies based on concatenated data, species trees and a phylogenetic network. We further examined the effect of alternative backbone topologies on downstream analyses, including biogeographic inference and dating analysis., Results: Based on the 73 SCN genes (>200 000 bp total alignment length) we considered, all tree-building methods lent strong support for the relationship (HCX, (Juniperus, Cupressus)); however, strongly supported conflicts among individual gene trees were also detected. Molecular dating suggests that these three lineages shared a most recent common ancestor approx. 60 million years ago (Mya), and that Juniperus and Cupressus diverged about 56 Mya. Ancestral area reconstructions (AARs) suggest an Asian origin for the entire clade, with subsequent dispersal to North America, Europe and Africa., Conclusions: Our analysis of SCN genes resolves a controversial phylogenetic relationship in the Cupressoideae, a major clade of conifers, and suggests that rapid evolutionary divergence and incomplete lineage sorting probably acted together as the source for conflicting phylogenetic inferences between gene trees and between our robust results and recently published studies. Our updated backbone topology has not substantially altered molecular dating estimates relative to previous studies; however, application of the latest AAR approaches has yielded a clearer picture of the biogeographic history of Cupressoideae.

Although DNA barcoding has been widely used to identify plant species composition in temperate and tropical ecosystems, relatively few studies have used DNA barcodes to document both herbaceous and woody components of forest plot. A total of 201 species (72 woody species and 129 herbaceous species) representing 135 genera distributed across 64 families of seed plants were collected in a 25 ha CForBio subalpine forest dynamics plot. In total, 491 specimens were screened for three DNA regions of the chloroplast genome ( rbcL , matK , and trnH - psbA ) as well as the internal transcribed spacers (ITS) of nuclear ribosomal DNA. We quantified species resolution for each barcode separately or in combination using a ML tree-based method. Amplification and sequencing success were highest for rbcL , followed by trnH-psbA , which performed better than ITS and matK . The rbcL  + ITS barcode had slightly higher species resolution rates (88.60%) compared with rbcL  +  matK (86.60%) and rbcL + trnH-psbA (86.01%). The addition of trnH-psbA or ITS to the rbcL + matK barcode only marginally increased species resolution rates, although in combination the four barcodes had the highest discriminatory power (90.21%). The situations where DNA barcodes did not discriminate among species were typically associated with higher numbers of co-occurring con-generic species. In addition, herbaceous species were much better resolved than woody species. Our study represents one of the first applications of DNA barcodes in a subalpine forest dynamics plot and contributes to our understanding of patterns of genetic divergence among woody and herbaceous plant species.

Background: The world's herbaria contain millions of specimens, collected and named by thousands of researchers, over hundreds of years. However, this treasure has remained largely inaccessible to genetic studies, because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today's next-generation sequencing world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates., Results: As a practical test of routine recovery of rDNA and plastid genome sequences from herbarium specimens, we sequenced 25 herbarium specimens up to 80 years old from 16 different Angiosperm families. Paired-end reads were generated, yielding successful plastid genome assemblies for 23 species and nuclear rDNAs for 24 species, respectively. These data showed that genome skimming can be used to generate genomic information from herbarium specimens as old as 80 years and using as little as 500 pg of degraded starting DNA., Conclusions: The routine plastome sequencing from herbarium specimens is feasible and cost-effective (compare with Sanger sequencing or plastome-enrichment approaches), and can be performed with limited sample destruction.

DNA barcoding is emerging as a useful tool not only for species identification but also for studying evolutionary and ecological processes. Although plant DNA barcodes do not always provide species-level resolution, the generation of large DNA barcode data sets can provide insights into the mechanisms underlying the generation of species diversity. Here, we study evolutionary processes in taxonomically complex British Euphrasia (Orobanchaceae), a group with multiple ploidy levels, frequent self-fertilization, young species divergence and widespread hybridization. We use a phylogenetic approach to investigate the colonization history of British Euphrasia , followed by a DNA barcoding survey and population genetic analyses to reveal the causes of shared sequence variation. Phylogenetic analysis shows Euphrasia have colonized Britain from mainland Europe on multiple occasions. DNA barcoding reveals that no British Euphrasia species has a consistent diagnostic sequence profile, and instead, plastid haplotypes are either widespread across species, or are population specific. The partitioning of nuclear genetic variation suggests differences in ploidy act as a barrier to gene exchange, while the divergence between diploid and tetraploid ITS sequences supports the polyploids being allotetraploid in origin. Overall, these results show that even when lacking species-level resolution, analyses of DNA barcoding data can reveal evolutionary patterns in taxonomically complex genera.

Driven by limited resources and a sense of urgency, the prioritization of species for conservation has been a persistent concern in conservation science. Gymnosperms (comprising ginkgo, conifers, cycads, and gnetophytes) are one of the most threatened groups of living organisms, with 40% of the species at high risk of extinction, about twice as many as the most recent estimates for all plants (i.e. 21.4%). This high proportion of species facing extinction highlights the urgent action required to secure their future through an objective prioritization approach. The Evolutionary Distinct and Globally Endangered (EDGE) method rapidly ranks species based on their evolutionary distinctiveness and the extinction risks they face. EDGE is applied to gymnosperms using a phylogenetic tree comprising DNA sequence data for 85% of gymnosperm species (923 out of 1090 species), to which the 167 missing species were added, and IUCN Red List assessments available for 92% of species. The effect of different extinction probability transformations and the handling of IUCN data deficient species on the resulting rankings is investigated. Although top entries in our ranking comprise species that were expected to score well (e.g. Wollemia nobilis, Ginkgo biloba), many were unexpected (e.g. Araucaria araucana). These results highlight the necessity of using approaches that integrate evolutionary information in conservation science.

Siberian ginseng (Eleutherococcus senticosus, Araliaceae) and roseroot (Rhodiola rosea, Rosaceae) are popular herbal supplements which have been shown to improve resilience to conditions such as stress and exhaustion. Using DNA barcoding methods we tested 25 Siberian ginseng and 14 roseroot products which are widely available to UK customers to test whether the herbal ingredient stated on the label is also in the product. All Siberian ginseng supplements contained E. senticosus, however, 36% also contained an Eleutherococcus species other than E. senticosus. In three out of the 13 roseroot products which produced amplifiable DNA, we could only retrieve sequences matching alfalfa (declared on the product label) and fenugreek (not declared). In the other 10 supplements Rhodiola was detected but only five matched the target species R. rosea. As DNA can get severely degraded during the manufacturing process we did not take the absence of Rhodiola DNA as proof for a compromised product. Contamination could explain the presence of non-target species such as fenugreek but is unlikely to be account for the detection of congeneric Rhodiola species in roseroot preparations. Our results therefore suggest that the substitution or mixing of the target medicinal ingredient in these two popular supplements with other species is common., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Participants in the 7th International Barcode of Life Conference (Kruger National Park, South Africa, 20-24 November 2017) share the latest findings in DNA barcoding research and its increasingly diversified applications. Here, we review prevailing trends synthesized from among 429 invited and contributed abstracts, which are collated in this open-access special issue of Genome. Hosted for the first time on the African continent, the 7th Conference places special emphasis on the evolutionary origins, biogeography, and conservation of African flora and fauna. Within Africa and elsewhere, DNA barcoding and related techniques are being increasingly used for wildlife forensics and for the validation of commercial products, such as medicinal plants and seafood species. A striking trend of the conference is the dramatic rise of studies on environmental DNA (eDNA) and on diverse uses of high-throughput sequencing techniques. Emerging techniques in these areas are opening new avenues for environmental biomonitoring, managing species-at-risk and invasive species, and revealing species interaction networks in unprecedented detail. Contributors call for the development of validated community standards for high-throughput sequence data generation and analysis, to enable the full potential of these methods to be realized for understanding and managing biodiversity on a global scale.

China is investing immense resources for planting trees, totalling more than US$ 100 billion in the past decade alone. Every year, China reports more afforestation than the rest of the world combined. Here, we show that China's forest cover gains are highly definition-dependent. If the definition of 'forest' follows FAO criteria (including immature and temporarily unstocked areas), China has gained 434 000 km 2 between 2000 and 2010. However, remotely detectable gains of vegetation that non-specialists would view as forest (tree cover higher than 5 m and minimum 50% crown cover) are an order of magnitude less (33 000 km 2 ). Using high-resolution maps and environmental modelling, we estimate that approximately 50% of the world's forest with minimum 50% crown cover has been lost in the past approximately 10 000 years. China historically lost 1.9-2.7 million km 2 (59-67%), and substantial losses continue. At the same time, most of China's afforestation investment targets environments that our model classes as unsuitable for trees. Here, gains detectable via satellite imagery are limited. Conversely, the regions where modest gains are detected are environmentally suitable but have received little afforestation investment due to conflicting land-use demands for agriculture and urbanization. This highlights the need for refined forest monitoring, and greater consideration of environmental suitability in afforestation programmes., (© 2017 The Authors.)

Intraspecific variation is a major component of biodiversity, yet it has received relatively little attention from governmental and nongovernmental organizations, especially with regard to conservation plans and the management of wild species. This omission is ill-advised because phenotypic and genetic variations within and among populations can have dramatic effects on ecological and evolutionary processes, including responses to environmental change, the maintenance of species diversity, and ecological stability and resilience. At the same time, environmental changes associated with many human activities, such as land use and climate change, have dramatic and often negative impacts on intraspecific variation. We argue for the need for local, regional, and global programs to monitor intraspecific genetic variation. We suggest that such monitoring should include two main strategies: (i) intensive monitoring of multiple types of genetic variation in selected species and (ii) broad-brush modeling for representative species for predicting changes in variation as a function of changes in population size and range extent. Overall, we call for collaborative efforts to initiate the urgently needed monitoring of intraspecific variation.

Land plants underpin a multitude of ecosystem functions, support human livelihoods and represent a critically important component of terrestrial biodiversity-yet many tens of thousands of species await discovery, and plant identification remains a substantial challenge, especially where material is juvenile, fragmented or processed. In this opinion article, we tackle two main topics. Firstly, we provide a short summary of the strengths and limitations of plant DNA barcoding for addressing these issues. Secondly, we discuss options for enhancing current plant barcodes, focusing on increasing discriminatory power via either gene capture of nuclear markers or genome skimming. The former has the advantage of establishing a defined set of target loci maximizing efficiency of sequencing effort, data storage and analysis. The challenge is developing a probe set for large numbers of nuclear markers that works over sufficient phylogenetic breadth. Genome skimming has the advantage of using existing protocols and being backward compatible with existing barcodes; and the depth of sequence coverage can be increased as sequencing costs fall. Its non-targeted nature does, however, present a major informatics challenge for upscaling to large sample sets.This article is part of the themed issue 'From DNA barcodes to biomes'., (© 2016 The Authors.)

Prologue 'As the study of natural science advances, the language of scientific description may be greatly simplified and abridged. This has already been done by Linneaus and may be carried still further by other invention. The descriptions of natural orders and genera may be reduced to short definitions, and employment of signs, somewhat in the manner of algebra, instead of long descriptions. It is more easy to conceive this, than it is to conceive with what facility, and in how short a time, a knowledge of all the objects of natural history may ultimately be acquired; and that which is now considered learning and science, and confined to a few specially devoted to it, may at length be universally possessed in every civilized country and in every rank of life'. J. C. Louden 1829. Magazine of natural history, vol. 1: This article is part of the themed issue 'From DNA barcodes to biomes'., (© 2016 The Author(s).)

Premise of the Study: Cryptic species represent a conservation challenge, because distributions and threats cannot be accurately assessed until species are recognized and defined. Cryptic species are common in diminutive and morphologically simple organisms, but are rare in charismatic and/or highly visible groups such as conifers. New Caledonia, a small island in the southern Pacific is a hotspot of diversity for the emblematic conifer genus Araucaria (Araucariaceae, Monkey Puzzle trees) where 13 of the 19 recognized species are endemic., Methods: We sampled across the entire geographical distribution of two closely related species (Araucaria rulei and A. muelleri) and screened them for genetic variation at 12 nuclear and 14 plastid microsatellites and one plastid minisatellite; a subset of the samples was also examined using leaf morphometrics., Key Results: The genetic data show that populations of the endangered A. muelleri fall into two clearly distinct genetic groups: one corresponding to montane populations, the other corresponding to trees from lower elevation populations from around the Goro plateau. These Goro plateau populations are more closely related to A. rulei, but are sufficiently genetically and morphological distinct to warrant recognition as a new species., Conclusions: Our study shows the presence of a previously unrecognized species in this flagship group, and that A. muelleri has 30% fewer individuals than previously thought. Combined, this clarification of species diversity and distributions provides important information to aid conservation planning for New Caledonian Araucaria., (© 2016 Botanical Society of America.)

DNA barcoding has had a major impact on biodiversity science. The elegant simplicity of establishing massive scale databases for a few barcode loci is continuing to change our understanding of species diversity patterns, and continues to enhance human abilities to distinguish among species. Capitalizing on the developments of next generation sequencing technologies and decreasing costs of genome sequencing, there is now the opportunity for the DNA barcoding concept to be extended to new kinds of genomic data. We illustrate the benefits and capacity to do this, and also note the constraints and barriers to overcome before it is truly scalable. We advocate a twin track approach: (i) continuation and acceleration of global efforts to build the DNA barcode reference library of life on earth using standard DNA barcodes and (ii) active development and application of extended DNA barcodes using genome skimming to augment the standard barcoding approach., (© 2016 John Wiley & Sons Ltd.)

Obtaining accurate phylogenies and effective species discrimination using a small standardized set of plastid genes is challenging in evolutionarily young lineages. Complete plastid genome sequencing offers an increasingly easy-to-access source of characters that helps address this. The usefulness of this approach, however, depends on the extent to which plastid haplotypes track morphological species boundaries. We have tested the power of complete plastid genomes to discriminate among multiple accessions of 11 of 13 New Caledonian Araucaria species, an evolutionarily young lineage where the standard DNA barcoding approach has so far failed and phylogenetic relationships have remained elusive. Additionally, 11 nuclear gene regions were Sanger sequenced for all accessions to ascertain the success of species discrimination using a moderate number of nuclear genes. Overall, fewer than half of the New Caledonian Araucaria species with multiple accessions were monophyletic in the plastid or nuclear trees. However, the plastid data retrieved a phylogeny with a higher resolution compared to any previously published tree of this clade and supported the monophyly of about twice as many species and nodes compared to the nuclear data set. Modest gains in discrimination thus are possible, but using complete plastid genomes or a small number of nuclear genes in DNA barcoding may not substantially raise species discriminatory power in many evolutionarily young lineages. The big challenge therefore remains to develop techniques that allow routine access to large numbers of nuclear markers scaleable to thousands of individuals from phylogenetically disparate sample sets., (© 2015 John Wiley & Sons Ltd.)

New Caledonia is a global biodiversity hotspot. Hypotheses for its biotic richness suggest either that the island is a 'museum' for an old Gondwana biota or alternatively it has developed following relatively recent long distance dispersal and in situ radiation. The conifer genus Araucaria (Araucariaceae) comprises 19 species globally with 13 endemic to this island. With a typically Gondwanan distribution, Araucaria is particularly well suited to testing alternative biogeographic hypotheses concerning the origins of New Caledonian biota. We derived phylogenetic estimates using 11 plastid and rDNA ITS2 sequence data for a complete sampling of Araucaria (including multiple accessions of each of the 13 New Caledonian Araucaria species). In addition, we developed a dataset comprising 4 plastid regions for a wider taxon sample to facilitate fossil based molecular dating. Following statistical analyses to identify a credible and internally consistent set of fossil constraints, divergence times estimated using a Bayesian relaxed clock approach were contrasted with geological scenarios to explore the biogeographic history of Araucaria. The phylogenetic data resolve relationships within Araucariaceae and among the main lineages in Araucaria, but provide limited resolution within the monophyletic New Caledonian species group. Divergence time estimates suggest a Late Cretaceous-Cenozoic radiation of extant Araucaria and a Neogene radiation of the New Caledonian lineage. A molecular timescale for the evolution of Araucariaceae supports a relatively recent radiation, and suggests that earlier (pre-Cenozoic) fossil types assigned to Araucaria may have affinities elsewhere in Araucariaceae. While additional data will be required to adequately resolve relationships among the New Caledonian species, their recent origin is consistent with overwater dispersal following Eocene emersion of New Caledonia but is too old to support a single dispersal from Australia to Norfolk Island for the radiation of the Pacific Araucaria sect. Eutacta clade.

Background: New Caledonia harbours a highly diverse and endemic flora, and 13 (out of the 19 worldwide) species of Araucaria are endemic to this territory. Their phylogenetic relationships remain largely unresolved. Using nuclear microsatellites and chloroplast DNA sequencing, we focused on five closely related Araucaria species to investigate among-species relationships and the distribution of within-species genetic diversity across New Caledonia., Results: The species could be clearly distinguished here, except A. montana and A. laubenfelsii that were not differentiated and, at most, form a genetic cline. Given their apparent morphological and ecological similarity, we suggested that these two species may be considered as a single evolutionary unit. We observed cases of nuclear admixture and incongruence between nuclear and chloroplast data, probably explained by introgression and shared ancestral polymorphism. Ancient hybridization was evidenced between A. biramulata and A. laubenfelsii in Mt Do, and is strongly suspected between A. biramulata and A. rulei in Mt Tonta. In both cases, extensive asymmetrical backcrossing eliminated the influence of one parent in the nuclear DNA composition. Shared ancestral polymorphism was also observed for cpDNA, suggesting that species diverged recently, have large effective sizes and/or that cpDNA experienced slow rates of molecular evolution. Within-species genetic structure was pronounced, probably because of low gene flow and significant inbreeding, and appeared clearly influenced by geography. This may be due to survival in distinct refugia during Quaternary climatic oscillations., Conclusions: The study species probably diverged recently and/or are characterized by a slow rate of cpDNA sequence evolution, and introgression is strongly suspected. Within-species genetic structure is tightly linked with geography. We underline the conservation implications of our results, and highlight several perspectives.

Aim: To examine the phylogeography of Ficus insipida subsp. insipida in order to investigate patterns of spatial genetic structure across the Neotropics and within Amazonia., Location: Neotropics., Methods: Plastid DNA ( trn H- psb A; 410 individuals from 54 populations) and nuclear ribosomal internal transcribed spacer (ITS; 85 individuals from 27 populations) sequences were sampled from Mexico to Bolivia, representing the full extent of the taxon's distribution. Divergence of plastid lineages was dated using a Bayesian coalescent approach. Genetic diversity was assessed with indices of haplotype and nucleotide diversities, and genetic structure was examined using spatial analysis of molecular variance (SAMOVA) and haplotype networks. Population expansion within Amazonia was tested using neutrality and mismatch distribution tests., Results: trn H- psb A sequences yielded 19 haplotypes restricted to either Mesoamerica or Amazonia; six haplotypes were found among ITS sequences. Diversification of the plastid DNA haplotypes began c . 14.6 Ma. Haplotype diversity for trn H- psb A was higher in Amazonia. Seven genetically differentiated SAMOVA groups were described for trn H- psb A, of which two were also supported by the presence of unique ITS sequences. Population expansion was suggested for both markers for the SAMOVA group that contains most Amazonian populations., Main Conclusions: Our results show marked population genetic structure in F. insipida between Mesoamerica and Amazonia, implying that the Andes and seasonally dry areas of northern South America are eco-climatic barriers to its migration. This pattern is shared with other widespread pioneer species affiliated to wet habitats, indicating that the ecological characteristics of species may impact upon large-scale phylogeography. Ficus insipida also shows genetic structure in north-western Amazonia potentially related to pre-Pleistocene historical events. In contrast, evident population expansion elsewhere in Amazonia, in particular the presence of genetically uniform populations across the south-west, indicate recent colonization. Our findings are consistent with palaeoecological data that suggest recent post-glacial expansion of Amazonian forests in the south.

Conservation policies usually focus on in situ protection of native populations, a priority that requires accurate assessment of population status. Distinction between native and introduced status can be particularly difficult (and at the same time, is most important) for species whose natural habitat has become both rare and highly fragmented. Here, we address the status of the white elm (Ulmus laevis Pallas), a European riparian tree species whose populations have been fragmented by human activity and is protected wherever it is considered native. Small populations of this species are located in Iberia, where they are unprotected because they are considered introductions due to their rarity. However, Iberia and neighbouring regions in southwestern France have been shown to support discrete glacial refuge populations of many European trees, and the possibility remains that Iberian white elms are native relicts. We used chloroplast RFLPs and nuclear microsatellites to establish the relationship between populations in Iberia and the Central European core distribution. Bayesian approaches revealed significant spatial structure across populations. Those in Iberia and southwestern France shared alleles absent from Central Europe, and showed spatial population structure within Iberia common in recognized native taxa. Iberian populations show a demographic signature of ancient population bottlenecks, while those in Central European show a signature of recent population bottlenecks. These patterns are not consistent with historical introduction of white elm to Iberia, and instead strongly support native status, arguing for immediate implementation of conservation measures for white elm populations in Spain and contiguous areas of southern France.

New Caledonia is a global biodiversity hotspot facing extreme environmental degradation. Given the urgent need for conservation prioritisation, we have made a first-pass quantitative assessment of the distribution of Narrow Endemic Species (NES) in the flora to identify species and sites that are potentially important for conservation action. We assessed the distributional status of all angiosperm and gymnosperm species using data from taxonomic descriptions and herbarium samples. We characterised species as being NES if they occurred in 3 or fewer locations. In total, 635 of the 2930 assessed species were classed as NES, of which only 150 have been subjected to the IUCN conservation assessment. As the distributional patterns of un-assessed species from one or two locations correspond well with assessed species which have been classified as Critically Endangered or Endangered respectively, we suggest that our distributional data can be used to prioritise species for IUCN assessment. We also used the distributional data to produce a map of "Hotspots of Plant Narrow Endemism" (HPNE). Combined, we used these data to evaluate the coincidence of NES with mining activities (a major source of threat on New Caledonia) and also areas of conservation protection. This is to identify species and locations in most urgent need of further conservation assessment and subsequent action. Finally, we grouped the NES based on the environments they occurred in and modelled the habitat distribution of these groups with a Maximum Entropy Species Distribution Model (MaxEnt). The NES were separable into three different groups based primarily on geological differences. The distribution of the habitat types for each group coincide partially with the HPNE described above and also indicates some areas which have high habitat suitability but few recorded NES. Some of these areas may represent under-sampled hotspots of narrow endemism and are priorities for further field work.

To understand the evolutionary consequences of hybridization between the outcrossing plant Geum rivale (Rosaceae) and the selfer Geum urbanum, we tested the predictions of two simple models that assume either (A) low or (B) high pollen fitness in hybrids. Model A predicts only four genotypic classes (G. rivale, G. rivale backcross [BC(R)], F1, and Geum urbanum) and asymmetric introgression from inbreeding to outbreeding species. Model B predicts additional genotypic classes and potential generation of novel inbreeding lines in the hybrid swarm. Amplified fragment length polymorphism (AFLP) analysis of adults revealed only the four genotypes predicted by model A. However, microsatellite analysis of parent-progeny arrays demonstrated production of selfed offspring by F1 and BC(R) maternal parents and contribution of these genotypes to outcross pollen pools, as predicted by model B. Moreover, AFLP and morphological analysis showed that the offspring generation comprised genotypes and phenotypes covering the entire spectrum of variation between the two parental species, in line with model B. A common garden experiment indicated no systematic reduction in fitness of offspring derived from hybrid parents. The genetic structure of the adults in the Geum hybrid swarm cannot be explained by restricted mating patterns but may result from ecological selection acting on a diverse offspring population., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

Premise of the Study: Microsatellite markers were developed for the hemiparasitic plant Melampyrum sylvaticum to investigate the breeding system, genetic diversity, and structure of populations in the United Kingdom, Sweden, and Norway., Methods and Results: Microsatellites were isolated from genomic DNA using an enrichment protocol. Twenty-nine loci were characterized in two individuals from each of 15 geographically disparate populations ("global"). Seven polymorphic loci were further characterized in one population ("local"). The number of alleles per locus ranged from two to 12 in the global sample and one to seven in the local sample. The expected heterozygosity ranged from 0-0.75, the observed heterozygosity from 0-0.1, and the inbreeding coefficient from 0.84-1 in the local sample., Conclusions: The results show the utility of these novel polymorphic microsatellite markers for further conservation genetic analyses. The strong deficit of heterozygosity across all loci in the local sample suggests the species may be inbreeding.

Rare and threatened subarctic willow scrub communities in the UK are the subject of ongoing conservation programmes, yet little is known about the diversity of fungal taxa that they support. Isolates of the rust genus Melampsora were sampled from 112 leaves of eight subarctic willow (Salix) taxa and their hybrids from twelve sites in the UK. In order to determine the number of Melampsora taxa present in the samples, isolates were sequenced for the Internal Transcribed Spacer (ITS) region of rDNA and data were subject to phylogenetic analysis. Maximum likelihood and Bayesian analysis indicated that the isolates fell into three strongly supported host-associated clades. Clade I contained only isolates from Salix herbacea and was distinguished morphologically by dense urediniospore echinulation and thin cell walls. Clade II contained isolates from Salix arbuscula and Salix reticulata only. These could not be distinguished morphologically from isolates in Clade III which were found on Salix lapponum, Salix myrsinites, Salix myrsinifolia, Salix aurita, Salix lanata, and their hybrids. Clade II was most distinct in ITS sequence, differing by 50 bases from Clades I and III, while the latter clades differed in sequence by only 24 bases on average. Clades I and III are likely to represent the previously recognised taxa Melampsora alpina Juel 1894 and Melampsora epitea Thüm. 1879 respectively, but Clade II has not apparently been described before. Significant differences in the intensity of infection by isolates of Clade III were found among different Salix species at a single site, suggesting either differences in resistance among Salix taxa, or the presence of further cryptic taxa within Clade III. The study illustrates the power of molecular phylogenetic analysis to reveal cryptic biodiversity within Melampsora, and suggests that conserving Salix host diversity within subarctic willow communities will ensure that a diversity of associated Melampsora taxa is maintained., (Copyright © 2012 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Premise of the Study: Despite its small size, New Caledonia is characterized by a very diverse flora and striking environmental gradients, which make it an ideal setting to study species diversification. Thirteen of the 19 Araucaria species are endemic to the territory and form a monophyletic group, but patterns and processes that lead to such a high species richness are largely unexplored., Methods: We used 142 polymorphic AFLP markers and performed analyses based on Bayesian clustering algorithms, genetic distances, and cladistics on 71 samples representing all New Caledonian Araucaria species. We examined correlations between the inferred evolutionary relationships and shared morphological, ecological, or geographic parameters among species, to investigate evolutionary processes that may have driven speciation., Key Results: We showed that genetic divergence among the present New Caledonian Araucaria species is low, suggesting recent diversification rather than pre-existence on Gondwana. We identified three genetic groups that included small-leaved, large-leaved, and coastal species, but detected no association with soil preference, ecological habitat, or rainfall. The observed patterns suggested that speciation events resulted from both differential adaptation and vicariance. Last, we hypothesize that speciation is ongoing and/or there are cryptic species in some genetically (sometimes also morphologically) divergent populations., Conclusions: Further data are required to provide better resolution and understanding of the diversification of New Caledonian Araucaria species. Nevertheless, our study allowed insights into their evolutionary relationships and provides a framework for future investigations on the evolution of this emblematic group of plants in one of the world's biodiversity hotspots.

DNA barcoding in the land plants presents a number of challenges compared to DNA barcoding in many animal clades. The CO1 animal DNA barcode is not effective for plants. Plant species hybridize frequently, and there are many cases of recent speciation via mechanisms, such as polyploidy and breeding system transitions. Additionally, there are many life-history trait combinations, which combine to reduce the likelihood of a small number of markers effectively tracking plant species boundaries. Recent results, however, from the two chosen core plant DNA barcode regions rbcL and matK plus two supplementary regions trnH-psbA and internal transcribed spacer (ITS) (or ITS2) have demonstrated reasonable levels of species discrimination in both floristic and taxonomically focused studies. We describe sampling techniques, extraction protocols, and PCR methods for each of these two core and two supplementary plant DNA barcode regions, with extensive notes supporting their implementation for both low- and high-throughput facilities.

DNA barcoding of a group of European liverwort species from the genus Herbertus was undertaken using three plastid (matK, rbcL and trnH-psbA) and one nuclear (ITS) marker. The DNA barcode data were effective in discriminating among the sampled species of Herbertus and contributed towards the detection of a previously overlooked European Herbertus species, described here as H. norenus sp. nov. This species shows clear-cut differences in DNA sequence for multiple barcode regions and is also morphologically distinct. The DNA barcode data were also useful in clarifying taxonomic relationships of the European species with some species from Asia and North America. In terms of the discriminatory power of the different barcode markers, ITS was the most informative region, followed closely by matK. All species were distinguishable by ITS alone, rbcL + matK and various other multimarker combinations., (© 2011 Blackwell Publishing Ltd.)