The identification of pollen plays an important role in ecology, palaeo-climatology, honey quality control and other areas. Currently, expert knowledge and reference collections are essential to identify pollen origin through light microscopy. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach, but the assessment of mixed pollen samples originating from multiple plant species is still a tedious and error-prone task. Next-generation sequencing has been proposed to avoid this hindrance. In this study we assessed mixed pollen probes through next-generation sequencing of amplicons from the highly variable, species-specific internal transcribed spacer 2 region of nuclear ribosomal DNA. Further, we developed a bioinformatic workflow to analyse these high-throughput data with a newly created reference database. To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. We assessed in total 16 mixed pollen samples, 14 originated from honeybee colonies and two from solitary bee nests. The sequencing technique resulted in higher taxon richness (deeper assignments and more identified taxa) compared to light microscopy. Abundance estimations from sequencing data were significantly correlated with counted abundances through light microscopy. Simulation analyses of taxon specificity and sensitivity indicate that 96% of taxa present in the database are correctly identifiable at the genus level and 70% at the species level. Next-generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialised palynological expert knowledge.

BackgroundMeta-barcoding of mixed pollen samples constitutes a suitable alternative to conventional pollen identification via light microscopy. Current approaches however have limitations in practicability due to low sample throughput and/or inefficient processing methods, e.g. separate steps for amplification and sample indexing.ResultsWe thus developed a new primer-adapter design for high throughput sequencing with the Illumina technology that remedies these issues. It uses a dual-indexing strategy, where sample-specific combinations of forward and reverse identifiers attached to the barcode marker allow high sample throughput with a single sequencing run. It does not require further adapter ligation steps after amplification. We applied this protocol to 384 pollen samples collected by solitary bees and sequenced all samples together on a single Illumina MiSeq v2 flow cell. According to rarefaction curves, 2,000–3,000 high quality reads per sample were sufficient to assess the complete diversity of 95% of the samples. We were able to detect 650 different plant taxa in total, of which 95% were classified at the species level. Together with the laboratory protocol, we also present an update of the reference database used by the classifier software, which increases the total number of covered global plant species included in the database from 37,403 to 72,325 (93% increase).ConclusionsThis study thus offers improvements for the laboratory and bioinformatical workflow to existing approaches regarding data quantity and quality as well as processing effort and cost-effectiveness. Although only tested for pollen samples, it is furthermore applicable to other research questions requiring plant identification in mixed and challenging samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12898-015-0051-y) contains supplementary material, which is available to authorized users.

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The availability of pollen in agricultural landscapes is essential for the successful growth and reproduction of honey bee colonies (Apis mellifera L.). The quantity and diversity of collected pollen can influence the growth and health of honey bee colonies, but little is known about the influence of landscape structure on pollen diet. In a field experiment, we rotated 16 honey bee colonies across 16 agricultural landscapes, used traps to collect samples of collected pollen and observed intra-colonial dance communication to gain information about foraging distances. DNA metabarcoding was applied to analyze mixed pollen samples. Neither the amount of collected pollen nor pollen diversity was related to landscape diversity. However, we found a strong seasonal variation in the amount and diversity of collected pollen in all sites independent of landscape diversity. The observed increase in foraging distances with decreasing landscape diversity suggests that honey bees compensated for lower landscape diversity by increasing their pollen foraging range in order to maintain pollen amount and diversity. Our results underscore the importance of a diverse pollen diet for honey bee colonies. Agri-environmental schemes aiming to support pollinators should focus on possible spatial and temporal gaps in pollen availability and diversity in agricultural landscapes.

Honey bees (Apis mellifera L.) show a large variation in foraging distances and use a broad range of plant species as pollen resources, even in regions with intensive agriculture. However, it is unknown how increasing areas of mass-flowering crops like oilseed rape (Brassica napus; OSR) or a decrease of seminatural habitats (SNH) change the temporal and spatial availability of pollen resources for honey bee colonies, and thus foraging distances and frequency in different habitat types. We studied pollen foraging of honey bee colonies in 16 agricultural landscapes with independent gradients of OSR and SNH area within 2 km and used waggle dances and digital geographic maps with major land cover types to reveal the distance and visited habitat type on a landscape level. Mean pollen foraging distance of 1347 decoded bee dances was 1015 m (± 26 m; SEM). In spring, increasing area of flowering OSR within 2 km reduced mean pollen foraging distances from 1324 m to only 435 m. In summer, increasing cover of SNH areas close to the colonies (within 200 m radius) reduced mean pollen foraging distances from 846 to 469 m. Frequency of pollen foragers per habitat type, measured as the number of dances per hour and hectare, was equally high for SNH, grassland, and OSR fields, but lower for other crops and forests. In landscapes with a small proportion of SNH a significantly higher density of pollen foragers on SNH was observed, indicating that pollen resources in such simple agricultural landscapes are more limited. Overall, we conclude that SNH and mass-flowering crops can reduce foraging distances of honey bee colonies at different scales and seasons with possible benefits for the performance of honey bee colonies. Further, mixed agricultural landscapes with a high proportion of SNH reduce foraging densities of honey bees in SNH and thus possible competition for pollen resources.