Temporal Patterns of Honeybee Foraging in a Diverse Floral Landscape Revealed Using Pollen DNA Metabarcoding of Honey.

Understanding the plants pollinators use through the year is vital to support pollinator populations and mitigate for declines in floral resources due to habitat loss. DNA metabarcoding allows the temporal picture of nectar and pollen foraging to be examined in detail. Here, we use DNA metabarcoding to examine the forage use of honeybees (Apis mellifera L.) within a florally diverse landscape within the UK, documenting the key forage plants used and seasonal progression over two years. The total number of plant taxa detected in the honey was 120, but only 16 of these were found with a high relative read abundance of DNA, across the main foraging months (April-September). Only a small proportion of the available flowering genera in the landscape were used by the honeybees. The greatest relative read abundance came from native or near-native plants, including Rubus spp., Trifolium repens, the Maleae tribe including Crataegus, Malus, and Cotoneaster, and Hedera helix. Tree species were important forage in the spring months, followed by increased use of herbs and shrubs later in the foraging season. Garden habitat increased the taxon richness of native, near-native and horticultural plants found in the honey. Although horticultural plants were rarely found abundantly within the honey samples, they may be important for increasing nutritional diversity of the pollen forage.

Barcode UK: A complete DNA barcoding resource for the flowering plants and conifers of the United Kingdom.

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.

Shifts in honeybee foraging reveal historical changes in floral resources.

Decreasing floral resources as a result of habitat loss is one of the key factors in the decline of pollinating insects worldwide. Understanding which plants pollinators use is vital to inform the provision of appropriate floral resources to help prevent pollinator loss. Using a globally important pollinator, the honeybee, we show how changes in agricultural intensification, crop use and the spread of invasive species, have altered the nectar and pollen sources available in the UK. Using DNA metabarcoding, we analysed 441 honey samples from 2017 and compared these to a nationwide survey of honey samples from 1952. We reveal that shifts in major plants foraged by honeybees are driven by changes in the availability of these plants within the landscape. Improved grasslands are the most widespread habitat type in the UK, and management changes within this habitat have the greatest potential to increase floral resource availability.

Temperate airborne grass pollen defined by spatio-temporal shifts in community composition.

Grass pollen is the world's most harmful outdoor aeroallergen. However, it is unknown how airborne pollen assemblages change across time and space. Human sensitivity varies between different species of grass that flower at different times, but it is not known whether temporal turnover in species composition match terrestrial flowering or whether species richness steadily accumulates over the grass pollen season. Here, using targeted, high-throughput sequencing, we demonstrate that all grass genera displayed discrete, temporally restricted peaks of incidence, which varied with latitude and longitude throughout Great Britain, revealing that the taxonomic composition of grass pollen exposure changes substantially across the grass pollen season.

Pollen metabarcoding reveals broad and species-specific resource use by urban bees.

Bee populations are currently undergoing severe global declines driven by the interactive effects of a number of factors. Ongoing urbanisation has the potential to exacerbate bee declines, unless steps are taken to ensure appropriate floral resources are available. Sown wildflower strips are one way in which floral resources can be provided to urban bees. However, the use of these strips by pollinators in urban environments remains little studied. Here, we employ pollen metabarcoding of the rbcL gene to compare the foraging patterns of different bee species observed using urban sown wildflower strips in July 2016, with a goal of identifying which plant species are most important for bees. We also demonstrate the use of a non-destructive method of pollen collection. Bees were found to forage on a wide variety of plant genera and families, including a diverse range of plants from outside the wildflower plots, suggesting that foragers visiting sown wildflower strips also utilize other urban habitats. Particular plants within the wildflower strips dominated metabarcoding data, particularly Papaver rhoeas and Phacelia tanacetifolia. Overall, we demonstrate that pollinators observed in sown wildflower strips use certain sown foodplants as part of a larger urban matrix.

Generalisation and specialisation in hoverfly (Syrphidae) grassland pollen transport networks revealed by DNA metabarcoding.

Pollination by insects is a key ecosystem service and important to wider ecosystem function. Most species-level pollination networks studied have a generalised structure, with plants having several potential pollinators, and pollinators in turn visiting a number of different plant species. This is in apparent contrast to a plant's need for efficient conspecific pollen transfer. The aim of this study was to investigate the structure of pollen transport networks at three levels of biological hierarchy: community, species and individual. We did this using hoverflies in the genus Eristalis, a key group of non-Hymenopteran pollinators. We constructed pollen transport networks using DNA metabarcoding to identify pollen. We captured hoverflies in conservation grasslands in west Wales, UK, removed external pollen loads, sequenced the pollen DNA on the Illumina MiSeq platform using the standard plant barcode rbcL, and matched sequences using a pre-existing plant DNA barcode reference library. We found that Eristalis hoverflies transport pollen from 65 plant taxa, more than previously appreciated. Networks were generalised at the site and species level, suggesting some degree of functional redundancy, and were more generalised in late summer compared to early summer. In contrast, pollen transport at the individual level showed some degree of specialisation. Hoverflies defined as "single-plant visitors" varied from 40% of those captured in early summer to 24% in late summer. Individual hoverflies became more generalised in late summer, possibly in response to an increase in floral resources. Rubus fruticosus agg. and Succisa pratensis were key plant species for hoverflies at our sites Our results contribute to resolving the apparent paradox of how generalised pollinator networks can provide efficient pollination to plant species. Generalised hoverfly pollen transport networks may result from a varied range of short-term specialised feeding bouts by individual insects. The generalisation and functional redundancy of Eristalis pollen transport networks may increase the stability of the pollination service they deliver.

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.

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.

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.

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.