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.

Abstract. 1. Sown wildflower strips can support insects that collect pollen for their larvae. How these strips affect flower visitors with carnivorous larvae, however, is almost unknown. We studied the impact of wildflower strips and their surroundings on two common solitary wasps: the caterpillar-hunting Ancistrocerus nigricornis Curtis and the spider-hunting Trypoxylon figulus Linnaeus.2. Trap-nest locations at 22 semi-natural habitats in central Germany formed independent gradients in landscape complexity and distance to either one or several wildflower strips in their surroundings. For each brood cell, we recorded the number of prey items, total caterpillar weight and spider species richness.3. Ancistrocerus nigricornis built more cells in proximity to wildflower strips and with increasing amount of surrounding grassland. Fewer prey items provided in landscapes with large shares of semi-natural habitats suggest that in these landscapes high-quality prey is available. In contrast, T. figulus built more cells with increasing distance of nests to wildflower strips. If there were few strips, T. figulus built more cells in grassland-rich landscapes, whereas low shares of grassland were compensated when several wildflower strips were present. Benefits of flowering strips for T. figulus seem related to flower resources for adults, rather than through prey provisioning.4. In conclusion, wildflower strips promote prey-hunting wasps through species-specific effects on adult and larval food provisioning. Considering the differential effects of wasps on crop pests (caterpillars) and natural enemies (spiders), the functional role of wildflower strips in agroecosystems may be much greater than assumed when solely focusing on pollination.