Natasha de Vere

DNA Barcoding the Native Flowering Plants and Conifers of Wales

We present the first national DNA barcode resource that covers the native flowering plants and conifers for the nation of Wales (1143 species). Using the plant DNA barcode markers rbcL and matK, we have assembled 97.7% coverage for rbcL, 90.2% for matK, and a dual-locus barcode for 89.7% of the native Welsh flora. We have sampled multiple individuals for each species, resulting in 3304 rbcL and 2419 matK sequences. The majority of our samples (85%) are from DNA extracted from herbarium specimens. Recoverability of DNA barcodes is lower using herbarium specimens, compared to freshly collected material, mostly due to lower amplification success, but this is balanced by the increased efficiency of sampling species that have already been collected, identified, and verified by taxonomic experts. The effectiveness of the DNA barcodes for identification (level of discrimination) is assessed using four approaches: the presence of a barcode gap (using pairwise and multiple alignments), formation of monophyletic groups using Neighbour-Joining trees, and sequence similarity in BLASTn searches. These approaches yield similar results, providing relative discrimination levels of 69.4 to 74.9% of all species and 98.6 to 99.8% of genera using both markers. Species discrimination can be further improved using spatially explicit sampling. Mean species discrimination using barcode gap analysis (with a multiple alignment) is 81.6% within 10×10 km squares and 93.3% for 2×2 km squares. Our database of DNA barcodes for Welsh native flowering plants and conifers represents the most complete coverage of any national flora, and offers a valuable platform for a wide range of applications that require accurate species identification.

Using DNA Metabarcoding to Identify the Floral Composition of Honey: A New Tool for Investigating Honey Bee Foraging Preferences

Identifying the floral composition of honey provides a method for investigating the plants that honey bees visit. We compared melissopalynology, where pollen grains retrieved from honey are identified morphologically, with a DNA metabarcoding approach using the rbcL DNA barcode marker and 454-pyrosequencing. We compared nine honeys supplied by beekeepers in the UK. DNA metabarcoding and melissopalynology were able to detect the most abundant floral components of honey. There was 92% correspondence for the plant taxa that had an abundance of over 20%. However, the level of similarity when all taxa were compared was lower, ranging from 22–45%, and there was little correspondence between the relative abundance of taxa found using the two techniques. DNA metabarcoding provided much greater repeatability, with a 64% taxa match compared to 28% with melissopalynology. DNA metabarcoding has the advantage over melissopalynology in that it does not require a high level of taxonomic expertise, a greater sample size can be screened and it provides greater resolution for some plant families. However, it does not provide a quantitative approach and pollen present in low levels are less likely to be detected. We investigated the plants that were frequently used by honey bees by examining the results obtained from both techniques. Plants with a broad taxonomic range were detected, covering 46 families and 25 orders, but a relatively small number of plants were consistently seen across multiple honey samples. Frequently found herbaceous species were Rubus fruticosus, Filipendula ulmaria, Taraxacum officinale, Trifolium spp., Brassica spp. and the non-native, invasive, Impatiens glandulifera. Tree pollen was frequently seen belonging to Castanea sativa, Crataegus monogyna and species of Malus, Salix and Quercus. We conclude that although honey bees are considered to be supergeneralists in their foraging choices, there are certain key species or plant groups that are particularly important in the honey bees environment. The reasons for this require further investigation in order to better understand honey bee nutritional requirements. DNA metabarcoding can be easily and widely used to investigate floral visitation in honey bees and can be adapted for use with other insects. It provides a starting point for investigating how we can better provide for the insects that we rely upon for pollination.

Environmental DNA metabarcoding: transforming how we survey animal and plant communities

The genomic revolution has fundamentally changed how we survey biodiversity on earth. High‐throughput sequencing (“HTS”) platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed “environmental DNA” or “eDNA”). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called “eDNA metabarcoding” and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education.

Pollen DNA barcoding : Current applications and future prospects.

Identification of the species origin of pollen has many applications, including assessment of plant-pollinator networks, reconstruction of ancient plant communities, product authentication, allergen monitoring, and forensics. Such applications, however, have previously been limited by microscopy-based identification of pollen, which is slow, has low taxonomic resolution, and has few expert practitioners. One alternative is pollen DNA barcoding, which could overcome these issues. Recent studies demonstrate that both chloroplast and nuclear barcoding markers can be amplified from pollen. These recent validations of pollen metabarcoding indicate that now is the time for researchers in various fields to consider applying these methods to their research programs. In this paper, we review the nascent field of pollen DNA barcoding and discuss potential new applications of this technology, highlighting existing limitations and future research developments that will improve its utility in a wide range of applications.

Using DNA metabarcoding to investigate honey bee foraging reveals limited flower use despite high floral availability.

Understanding which flowers honey bees (Apis mellifera) use for forage can help us to provide suitable plants for healthy honey bee colonies. Accordingly, honey DNA metabarcoding provides a valuable tool for investigating pollen and nectar collection. We investigated early season (April and May) floral choice by honey bees provided with a very high diversity of flowering plants within the National Botanic Garden of Wales. There was a close correspondence between the phenology of flowering and the detection of plants within the honey. Within the study area there were 437 genera of plants in flower during April and May, but only 11% of these were used. Thirty-nine plant taxa were recorded from three hives but only ten at greater than 1%. All three colonies used the same core set of native or near-native plants, typically found in hedgerows and woodlands. The major plants were supplemented with a range of horticultural species, with more variation in plant choice between the honey bee colonies. We conclude that during the spring, honey bees need access to native hedgerows and woodlands to provide major plants for foraging. Gardens provide supplementary flowers that may increase the nutritional diversity of the honey bee diet.

A phylogenetic analysis of the British flora sheds light on the evolutionary and ecological factors driving plant invasions

Darwins naturalization hypothesis predicts that invasive species should perform better in their novel range in the absence of close relatives in the native flora due to reduced competition. Evidence from recent taxonomic and phylogenetic-based studies, however, is equivocal. We test Darwins naturalization hypothesis at two different spatial scales using a fossil-dated molecular phylogenetic tree of the British native and alien flora (ca. 1600 species) and extensive, fine-scale survey data from the 1998 Countryside Survey. At both landscape and local scales, invasive species were neither significantly more nor less related to the native flora than their non-invasive alien counterparts. Species invasiveness was instead correlated with higher nitrogen and moisture preference, but not other life history traits such as life-form and height. We argue that invasive species spread in Britain is hence more likely determined by changes in land use and other anthropogenic factors, rather than evolutionary history. Synthesis. The transition from non-invasive to invasive is not related to phylogenetic distinctiveness to the native community, but instead to their environmental preferences. Therefore, combating biological invasions in the Britain and other industrialized countries need entirely different strategies than in more natural environments.

Research-based residential fieldwork learning: Double bonus?

Abstract In the current Higher Education climate, there is a pressing need to integrate research and teaching in the student learning experience. In order to create synergy between research and teaching activities, to the benefit of students and instructors, we provided students with the opportunity to participate in a high profile collaboration between two scientific institutions. We planned and developed an integrated module leading the student through all the steps necessary in a large-scale, collaborative research project. Student feedback resulting from the intervention showed impressive levels of improvement in general appreciation of the course. Students also suggested that they would have liked a longer module, despite the intensive workload they experienced. All areas explored (general knowledge, research-based evaluation criteria, group and individual work) showed improvements in student evaluation after the module. We conclude that a residential, integrated experience of scientific research, from initial data collection to presentation at a scientific conference, can produce significant positive, active learning experiences to the students. A double bonus comes from the benefits towards research, both by compilation of data and long-term collaboration opportunities.

DNA Barcoding for Plants

DNA barcoding uses specific regions of DNA in order to identify species. Initiatives are taking place around the world to generate DNA barcodes for all groups of living organisms and to make these data publically available in order to help understand, conserve, and utilize the worlds biodiversity. For land plants the core DNA barcode markers are two sections of coding regions within the chloroplast, part of the genes, rbcL and matK. In order to create high quality databases, each plant that is DNA barcoded needs to have a herbarium voucher that accompanies the rbcL and matK DNA sequences. The quality of the DNA sequences, the primers used, and trace files should also be accessible to users of the data. Multiple individuals should be DNA barcoded for each species in order to check for errors and allow for intraspecific variation. The worlds herbaria provide a rich resource of already preserved and identified material and these can be used for DNA barcoding as well as by collecting fresh samples from the wild. These protocols describe the whole DNA barcoding process, from the collection of plant material from the wild or from the herbarium, how to extract and amplify the DNA, and how to check the quality of the data after sequencing.

New universal ITS2 primers for high-resolution herbivory analyses using DNA metabarcoding in both tropical and temperate zones

DNA metabarcoding is a rapidly growing technique for obtaining detailed dietary information. Current metabarcoding methods for herbivory, using a single locus, can lack taxonomic resolution for some applications. We present novel primers for the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) designed for dietary studies in Mauritius and the UK, which have the potential to give unrivalled taxonomic coverage and resolution from a short-amplicon barcode. In silico testing used three databases of plant ITS2 sequences from UK and Mauritian floras (native and introduced) totalling 6561 sequences from 1790 species across 174 families. Our primers were well-matched in silico to 88% of species, providing taxonomic resolution of 86.1%, 99.4% and 99.9% at the species, genus and family levels, respectively. In vitro, the primers amplified 99% of Mauritian (n = 169) and 100% of UK (n = 33) species, and co-amplified multiple plant species from degraded faecal DNA from reptiles and birds in two case studies. For the ITS2 region, we advocate taxonomic assignment based on best sequence match instead of a clustering approach. With short amplicons of 187–387 bp, these primers are suitable for metabarcoding plant DNA from faecal samples, across a broad geographic range, whilst delivering unparalleled taxonomic resolution.

Floral resource partitioning by individuals within generalised hoverfly pollination networks revealed by DNA metabarcoding

Pollination is a key ecosystem service for agriculture and wider ecosystem function. However, most pollination studies focus on Hymenoptera, with hoverflies (Syrphidae) frequently treated as a single functional group. We tested this assumption by investigating pollen carried by eleven species of hoverfly in five genera, Cheilosia, Eristalis, Rhingia, Sericomyia and Volucella, using DNA metabarcoding. Hoverflies carried pollen from 59 plant taxa, suggesting they visit a wider number of plant species than previously appreciated. Most pollen recorded came from plant taxa frequently found at our study sites, predominantly Apiaceae, Cardueae, Calluna vulgaris, Rubus fruticosus agg., and Succisa pratensis, with hoverflies transporting pollen from 40% of entomophilous plant species present. Overall pollen transport network structures were generalised, similar to other pollination networks elsewhere. All hoverfly species were also generalised with few exclusive plant/hoverfly interactions. However, using the Jaccard Index, we found significant differences in the relative composition of pollen loads between hoverfly genera, except for Volucella, demonstrating some degree of functional complementarity. Eristalis and Sericomyia species had significant differences in relative pollen load composition compared to congeners. Our results demonstrate the range of pollens transported by hoverflies and the potential pollination function undertaken within this ecologically and morphologically diverse guild.