Airborne DNA reveals predictable spatial and seasonal dynamics of fungi.

Fungi are among the most diverse and ecologically important kingdoms in life. However, the distributional ranges of fungi remain largely unknown as do the ecological mechanisms that shape their distributions1,2. To provide an integrated view of the spatial and seasonal dynamics of fungi, we implemented a globally distributed standardized aerial sampling of fungal spores3. The vast majority of operational taxonomic units were detected within only one climatic zone, and the spatiotemporal patterns of species richness and community composition were mostly explained by annual mean air temperature. Tropical regions hosted the highest fungal diversity except for lichenized, ericoid mycorrhizal and ectomycorrhizal fungi, which reached their peak diversity in temperate regions. The sensitivity in climatic responses was associated with phylogenetic relatedness, suggesting that large-scale distributions of some fungal groups are partially constrained by their ancestral niche. There was a strong phylogenetic signal in seasonal sensitivity, suggesting that some groups of fungi have retained their ancestral trait of sporulating for only a short period. Overall, our results show that the hyperdiverse kingdom of fungi follows globally highly predictable spatial and temporal dynamics, with seasonality in both species richness and community composition increasing with latitude. Our study reports patterns resembling those described for other major groups of organisms, thus making a major contribution to the long-standing debate on whether organisms with a microbial lifestyle follow the global biodiversity paradigms known for macroorganisms4,5.

Variations of floral temperature in changing weather conditions.

Floral temperature is a flower characteristic that has the potential to impact the fitness of flowering plants and their pollinators. Likewise, the presence of floral temperature patterns, areas of contrasting temperature across the flower, can have similar impacts on the fitness of both mutualists. It is currently poorly understood how floral temperature changes under the influence of different weather conditions, and how floral traits may moderate these changes. The way that floral temperature changes with weather conditions will impact how stable floral temperatures are over time and their utility to plants and pollinators. The stability of floral temperature cues is likely to facilitate effective plant-pollinator interactions and play a role in the plant's reproductive success. We use thermal imaging to monitor how floral temperatures and temperature patterns of four plant species (Cistus 'snow fire' and 'snow white', Coreopsis verticillata and Geranium psilostemon) change with several weather variables (illumination, temperature; windspeed; cloud cover; humidity and pressure) during times that pollinators are active. All weather variables influenced floral temperature in one or more species. The directionality of these relationships was similar across species. In all species, light conditions (illumination) had the greatest influence on floral temperatures overall. Floral temperature and the extent to which flowers showed contrasting temperature patterns were influenced predominantly by light conditions. However, several weather variables had additional, lesser, influences. Furthermore, differences in floral traits, pigmentation and structure, likely resulted in differences in temperature responses to given conditions between species and different parts of the same flower. However, floral temperatures and contrasting temperature patterns that are sufficiently elevated for detection by pollinators were maintained across most conditions if flowers received moderate illumination. This suggests the presence of elevated floral temperature and contrasting temperature patterns are fairly constant and may have potential to influence plant-pollinator interactions across weather conditions.

Global Spore Sampling Project: A global, standardized dataset of airborne fungal DNA.

Novel methods for sampling and characterizing biodiversity hold great promise for re-evaluating patterns of life across the planet. The sampling of airborne spores with a cyclone sampler, and the sequencing of their DNA, have been suggested as an efficient and well-calibrated tool for surveying fungal diversity across various environments. Here we present data originating from the Global Spore Sampling Project, comprising 2,768 samples collected during two years at 47 outdoor locations across the world. Each sample represents fungal DNA extracted from 24 m3 of air. We applied a conservative bioinformatics pipeline that filtered out sequences that did not show strong evidence of representing a fungal species. The pipeline yielded 27,954 species-level operational taxonomic units (OTUs). Each OTU is accompanied by a probabilistic taxonomic classification, validated through comparison with expert evaluations. To examine the potential of the data for ecological analyses, we partitioned the variation in species distributions into spatial and seasonal components, showing a strong effect of the annual mean temperature on community composition.

Mega-Barcoding Projects: Delivering National DNA Barcoding Initiatives for Plants.

Plant DNA barcoding has a multitude of applications ranging from species detection and biomonitoring to investigating ecological networks and checking food quality. The ability to accurately identify species, using DNA barcoding, depends on the quality and comprehensiveness of the reference library that is used. This chapter describes how to create plant reference libraries using the rbcL, matK, and ITS2 DNA barcode regions. It covers the creation of species lists, the collection of specimens from the field and herbarium, DNA extraction, PCR amplification, and DNA sequencing. This methodology gives special attention to using samples from herbaria, as they represent important collections of easily accessible, taxonomically verified plant material.

Genetic factors predict hybrid formation in the British flora.

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.

Temporal change in floral availability leads to periods of resource limitation and affects diet specificity in a generalist pollinator.

Generalist species are core components of ecological networks and crucial for the maintenance of biodiversity. Generalist species and networks are expected to be more resilient, and therefore understanding the dynamics of specialization and generalization in ecological networks is a key focus in a time of rapid global change. Whilst diet generalization is frequently studied, our understanding of how it changes over time is limited. Here we explore temporal variation in diet specificity in the honeybee (Apis mellifera), using pollen DNA metabarcoding of honey samples, through the foraging season, over two years. We find that, overall, honeybees are generalists that visit a wide range of plants, but there is temporal variation in the degree of specialization. Temporal specialization of honeybee colonies corresponds to periods of resource limitation, identified as a lack of honey stores. Honeybees experience a lack of preferred resources in June when switching from flowering trees in spring to shrubs and herbs in summer. Investigating temporal patterns in specialization can identify periods of resource limitation that may lead to species and network vulnerability. Diet specificity must therefore be explored at different temporal scales in order to fully understand species and network stability in the face of ecological change.

Plants, pollinators and their interactions under global ecological change: The role of pollen DNA metabarcoding.

Anthropogenic activities are triggering global changes in the environment, causing entire communities of plants, pollinators and their interactions to restructure, and ultimately leading to species declines. To understand the mechanisms behind community shifts and declines, as well as monitoring and managing impacts, a global effort must be made to characterize plant-pollinator communities in detail, across different habitat types, latitudes, elevations, and levels and types of disturbances. Generating data of this scale will only be feasible with rapid, high-throughput methods. Pollen DNA metabarcoding provides advantages in throughput, efficiency and taxonomic resolution over traditional methods, such as microscopic pollen identification and visual observation of plant-pollinator interactions. This makes it ideal for understanding complex ecological networks and their responses to change. Pollen DNA metabarcoding is currently being applied to assess plant-pollinator interactions, survey ecosystem change and model the spatiotemporal distribution of allergenic pollen. Where samples are available from past collections, pollen DNA metabarcoding has been used to compare contemporary and past ecosystems. New avenues of research are possible with the expansion of pollen DNA metabarcoding to intraspecific identification, analysis of DNA in ancient pollen samples, and increased use of museum and herbarium specimens. Ongoing developments in sequencing technologies can accelerate progress towards these goals. Global ecological change is happening rapidly, and we anticipate that high-throughput methods such as pollen DNA metabarcoding are critical for understanding the evolutionary and ecological processes that support biodiversity, and predicting and responding to the impacts of change.

Environmental DNA analysis as an emerging non-destructive method for plant biodiversity monitoring: a review.

Environmental DNA (eDNA) analysis has recently transformed and modernized biodiversity monitoring. The accurate detection, and to some extent quantification, of organisms (individuals/populations/communities) in environmental samples is galvanizing eDNA as a successful cost and time-efficient biomonitoring technique. Currently, eDNA's application to plants remains more limited in implementation and scope compared to animals and microorganisms. This review evaluates the development of eDNA-based methods for (vascular) plants, comparing its performance and power of detection with that of traditional methods, to critically evaluate and advise best-practices needed to innovate plant biomonitoring. Recent advancements, standardization and field applications of eDNA-based methods have provided enough scope to utilize it in conservation biology for numerous organisms. Despite our review demonstrating only 13% of all eDNA studies focus on plant taxa to date, eDNA has considerable environmental DNA has considerable potential for plants, where successful detection of invasive, endangered and rare species, and community-level interpretations have provided proof-of-concept. Monitoring methods using eDNA were found to be equal or more effective than traditional methods; however, species detection increased when both methods were coupled. Additionally, eDNA methods were found to be effective in studying species interactions, community dynamics and even effects of anthropogenic pressure. Currently, elimination of potential obstacles (e.g. lack of relevant DNA reference libraries for plants) and the development of user-friendly protocols would greatly contribute to comprehensive eDNA-based plant monitoring programs. This is particularly needed in the data-depauperate tropics and for some plant groups (e.g., Bryophytes and Pteridophytes). We further advocate to coupling traditional methods with eDNA approaches, as the former is often cheaper and methodologically more straightforward, while the latter offers non-destructive approaches with increased discrimination ability. Furthermore, to make a global platform for eDNA, governmental and academic-industrial collaborations are essential to make eDNA surveys a broadly adopted and implemented, rapid, cost-effective and non-invasive plant monitoring approach.

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.

A taxonomic, genetic and ecological data resource for the vascular plants of Britain and Ireland.

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.

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.

Predicting the severity of the grass pollen season and the effect of climate change in Northwest Europe.

Allergic rhinitis is an inflammation in the nose caused by overreaction of the immune system to allergens in the air. Managing allergic rhinitis symptoms is challenging and requires timely intervention. The following are major questions often posed by those with allergic rhinitis: How should I prepare for the forthcoming season? How will the season's severity develop over the years? No country yet provides clear guidance addressing these questions. We propose two previously unexplored approaches for forecasting the severity of the grass pollen season on the basis of statistical and mechanistic models. The results suggest annual severity is largely governed by preseasonal meteorological conditions. The mechanistic model suggests climate change will increase the season severity by up to 60%, in line with experimental chamber studies. These models can be used as forecasting tools for advising individuals with hay fever and health care professionals how to prepare for the grass pollen season.

Environmental DNA reveals links between abundance and composition of airborne grass pollen and respiratory health.

Grass (Poaceae) pollen is the most important outdoor aeroallergen,1 exacerbating a range of respiratory conditions, including allergic asthma and rhinitis ("hay fever").2-5 Understanding the relationships between respiratory diseases and airborne grass pollen with a view to improving forecasting has broad public health and socioeconomic relevance. It is estimated that there are over 400 million people with allergic rhinitis6 and over 300 million with asthma, globally,7 often comorbidly.8 In the UK, allergic asthma has an annual cost of around US$ 2.8 billion (2017).9 The relative contributions of the >11,000 (worldwide) grass species (C. Osborne et al., 2011, Botany Conference, abstract) to respiratory health have been unresolved,10 as grass pollen cannot be readily discriminated using standard microscopy.11 Instead, here we used novel environmental DNA (eDNA) sampling and qPCR12-15 to measure the relative abundances of airborne pollen from common grass species during two grass pollen seasons (2016 and 2017) across the UK. We quantitatively demonstrate discrete spatiotemporal patterns in airborne grass pollen assemblages. Using a series of generalized additive models (GAMs), we explore the relationship between the incidences of airborne pollen and severe asthma exacerbations (sub-weekly) and prescribing rates of drugs for respiratory allergies (monthly). Our results indicate that a subset of grass species may have disproportionate influence on these population-scale respiratory health responses during peak grass pollen concentrations. The work demonstrates the need for sensitive and detailed biomonitoring of harmful aeroallergens in order to investigate and mitigate their impacts on human health.

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.

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.

DNA barcoding a taxonomically complex hemiparasitic genus reveals deep divergence between ploidy levels but lack of species-level resolution.

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.

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.