Robyn S. Cowan

A DNA barcode for land plants

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.

Towards writing the encyclopaedia of life: an introduction to DNA barcoding

An international consortium of major natural history museums, herbaria and other organizations has launched an ambitious project, the ‘Barcode of Life Initiative’, to promote a process enabling the rapid and inexpensive identification of the estimated 10 million species on Earth. DNA barcoding is a diagnostic technique in which short DNA sequence(s) can be used for species identification. The first international scientific conference on Barcoding of Life was held at the Natural History Museum in London in February 2005, and here we review the scientific challenges discussed during this conference and in previous publications. Although still controversial, the scientific benefits of DNA barcoding include: (i) enabling species identification, including any life stage or fragment, (ii) facilitating species discoveries based on cluster analyses of gene sequences (e.g. cox1=CO1, in animals), (iii) promoting development of handheld DNA sequencing technology that can be applied in the field for biodiversity inventories and (iv) providing insight into the diversity of life.

Selecting barcoding loci for plants: evaluation of seven candidate loci with species-level sampling in three divergent groups of land plants

There has been considerable debate, but little consensus regarding locus choice for DNA barcoding land plants. This is partly attributable to a shortage of comparable data from all proposed candidate loci on a common set of samples. In this study, we evaluated the seven main candidate plastid regions (rpoC1, rpoB, rbcL, matK, trnH‐psbA, atpF‐atpH, psbK‐psbI) in three divergent groups of land plants [Inga (angiosperm); Araucaria (gymnosperm); Asterella s.l. (liverwort)]. Across these groups, no single locus showed high levels of universality and resolvability. Interspecific sharing of sequences from individual loci was common. However, when multiple loci were combined, fewer barcodes were shared among species. Evaluation of the performance of previously published suggestions of particular multilocus barcode combinations showed broadly equivalent performance. Minor improvements on these were obtained by various new three‐locus combinations involving rpoC1, rbcL, matK and trnH‐psbA, but no single combination clearly outperformed all others. In terms of absolute discriminatory power, promising results occurred in liverworts (e.g. c. 90% species discrimination based on rbcL alone). However, Inga (rapid radiation) and Araucaria (slow rates of substitution) represent challenging groups for DNA barcoding, and their corresponding levels of species discrimination reflect this (upper estimate of species discrimination = 69% in Inga and only 32% in Araucaria; mean = 60% averaging all three groups).

300,000 species to identify: problems, progress, and prospects in DNA barcoding of land plants

DNA barcodes have been successfully applied to a limited number of animal groups with the application of the mitochondrial gene, cytochrome c oxidase subunit 1. Recently two DNA regions, the plastid trnH-psbA spacer and nuclear ribosomal ITS region, have been shown to have potential as an identification barcode for land plants, although with some significant drawbacks. The ideal barcode should be relatively short in length (∼700 bp), more variable between than within species, and easily amplifiable with universal primers. Building on current success, ongoing investigations are searching for the best barcode to apply to all land plants. Once established, a plant barcode may be effectively used in biodiversity inventories, conservation assessments, and applied forensic investigations. Advances in sequencing technology and the completion of the DNA barcode library have the potential to provide the public with increased access to information about the natural world.

How to Open the Treasure Chest? Optimising DNA Extraction from Herbarium Specimens

Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL (UAA) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10–143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (<200–300 bp) in order to maximise outcomes. Development of shorter barcoding regions, or mini-barcodes within existing ones should be of high importance as only a few options are currently available; this is particularly important if we hope to incorporate the millions of herbarium samples available into barcoding initiatives and other molecular studies.

AFLP fingerprinting in Capparis subgenus Capparis related to the commercial sources of capers

A genetic fingerprinting technique (AFLP) was used to determine the relationships among Capparis spp. Genetic distances, based on AFLP data were estimated for 45 accessions of Capparis species, from Spain, Morocco and Syria. The results of this analysis support the differentiation of four of the five taxa involved. The group of plants recognised as C. spinosa on the basis of morphological characters, includes several cultivars and appears in an intermediate position between C. orientalis and C. sicula and overlaps with C. orientalis. The other two species C. aegyptia and C. ovata are separate from the rest. Capparis spinosa had a low number of unique bands in comparison with the other species. Although these results cannot confirm the hybrid origin of C. spinosa, the distribution of the bands supports this hypothesis, the most likely parental species being C. orientalis and C. sicula.

Challenges in the DNA Barcoding of Plant Material

DNA barcoding, using a short gene sequence from a standardized region of the genome, is a species identification tool which would not only aid species discovery but would also have applications ranging from large-scale biodiversity surveys through to identification of a single fragment of material in forensic contexts. To fulfill this vision a universal, relatively cheap, scalable system needs to be in place. The mitochondrial locus being used for many animal groups and algae is not suitable for use in land plants, and an appropriate alternative is needed.Progress has been made in the selection of two alternative regions for plant DNA barcoding. There are however many challenges in finding a solution that fulfills all the requirements of a successful, universally applicable barcode, and in the short term a pragmatic solution that achieves as much as possible and has payoffs in most areas has been chosen. Research continues in areas ranging from the technicalities of sequencing the regions to data analysis and the potential improvements that may result from the developing technology and data analysis systems.The ultimate success of DNA barcoding as a plant identification tool for all occasions depends on the building of a reference database and it fulfilling the requirements of potential users such that they are able to achieve valid results through its use, that would be more time consuming and costly, and less reliable using other techniques.

Approaches to develop a road map for the long-term conservation of an island endemic genus Cylindrocline

Habitat fragmentation and invasive alien species contribute to genetic bottlenecks and the threat of extinction of many endemic species of Mauritius, part of the Madagascan and Indian Ocean biodiversity hotspot. The genus Cylindrocline has two species, C. commersonii (critically endangered) and C. lorencei (extinct in the wild). The last living specimens of C. lorencei disappeared in the wild after the recorded collecting of seeds in 1982 by Conservatoire Botanique National de Brest (CBN Brest). Embryo rescue was used as a method to germinate these seeds and the seedlings raised this way were shared by CBN Brest with Royal Botanic Gardens Kew (RBG Kew) as part of an exchange programme in 2001. Mature plants both at CBN Brest and RBG Kew stopped producing viable seeds and this has made the long-term conservation of C. lorencei even more difficult. Seeds of C. commersonii collected from the wild in 2010 have a very low viability while ex situ grown C. commersonii produce non-viable seeds. Molecular studies conducted in C. lorencei using amplified fragment length polymorphisms (AFLP) showed no genetic variability among remaining individuals. Two samples of C. commersonii showed a very small amount of genetic variability. The variability between the two species was well within the limits commonly found within species or between closely related species and the long-term conservation of the genus requires a radical (to a degree) approach to avoid its extinction. The importance of novel approaches for restoration and long-term conservation are discussed.

A Universal Probe Set for Targeted Sequencing of 353 Nuclear Genes from Any Flowering Plant Designed Using k-medoids Clustering

Sequencing of target-enriched libraries is an efficient and cost-effective method for obtaining DNA sequence data from hundreds of nuclear loci for phylogeny reconstruction. Much of the cost associated with developing targeted sequencing approaches is preliminary data needed for identifying orthologous loci for probe design. In plants, identifying orthologous loci has proven difficult due to a large number of whole-genome duplication events, especially in the angiosperms (flowering plants). We used multiple sequence alignments from over 600 angiosperms for 353 putatively single-copy protein-coding genes to design a set of targeted sequencing probes for phylogenetic studies of any angiosperm lineage. To maximize the phylogenetic potential of the probes while minimizing the cost of production, we introduce a k-medoids clustering approach to identify the minimum number of sequences necessary to represent each coding sequence in the final probe set. Using this method, five to 15 representative sequences were selected per orthologous locus, representing the sequence diversity of angiosperms more efficiently than if probes were designed using available sequenced genomes alone. To test our approximately 80,000 probes, we hybridized libraries from 42 species spanning all higher-order lineages of angiosperms, with a focus on taxa not present in the sequence alignments used to design the probes. Out of a possible 353 coding sequences, we recovered an average of 283 per species and at least 100 in all species. Differences among taxa in sequence recovery could not be explained by relatedness to the representative taxa selected for probe design, suggesting that there is no phylogenetic bias in the probe set. Our probe set, which targeted 260 kbp of coding sequence, achieved a median recovery of 137 kbp per taxon in coding regions, a maximum recovery of 250 kbp, and an additional median of 212 kbp per taxon in flanking non-coding regions across all species. These results suggest that the Angiosperms353 probe set described here is effective for any group of flowering plants and would be useful for phylogenetic studies from the species level to higher-order lineages, including all angiosperms.

Flower preferences and pollen transport networks for cavity-nesting solitary bees: Implications for the design of agri-environment schemes

Abstract Floral foraging resources are valuable for pollinator conservation on farmland, and their provision is encouraged by agri‐environment schemes in many countries. Across Europe, wildflower seed mixtures are widely sown on farmland to encourage pollinators, but the extent to which key pollinator groups such as solitary bees exploit and benefit from these resources is unclear. We used high‐throughput sequencing of 164 pollen samples extracted from the brood cells of six common cavity‐nesting solitary bee species (Osmia bicornis, Osmia caerulescens, Megachile versicolor, Megachile ligniseca, Megachile centuncularis and Hylaeus confusus) which are widely distributed across the UK and Europe. We documented their pollen use across 19 farms in southern England, UK, revealing their forage plants and examining the structure of their pollen transport networks. Of the 32 plant species included currently in sown wildflower mixes, 15 were recorded as present within close foraging range of the bees on the study farms, but only Ranunculus acris L. was identified within the pollen samples. Rosa canina L. was the most commonly found of the 23 plant species identified in the pollen samples, suggesting that, in addition to providing a nesting resource for Megachile leafcutter bees, it may be an important forage plant for these species. Higher levels of connectance and nestedness were characteristic of pollen transport networks on farms with abundant floral resources, which may increase resilience to species loss. Our data suggest that plant species promoted currently by agri‐environment schemes are not optimal for solitary bee foraging. If a diverse community of pollinators is to be supported on UK and European farmland, additional species such as R. canina should be encouraged to meet the foraging requirements of solitary bees.