DOI: 10.25829/bexis.27229-0
title: Plant species diversity based on rDNA gene sequences (ITS2) of trap nesting solitary bee species 2017-2018

description: Biodiversity loss, often found in intensively managed agricultural landscapes, correlates with reduced ecosystem functioning, e.g. insect pollination and altered floral resource diversity, abundance and composition. How this affects bee foraging and larval diets in solitary wild bees is largely unclear. To better understand impacts of land-use intensification on wild bee-plant interactions, we investigated communities of trap nesting solitary bees, their pollen foraging and the nutritional quality of larval food composed in grasslands along a land-use intensity gradient across three bioregions (Swabian Alb, Hainich- Dün, Schorfheide-Chorin) in Germany.
description: Theory: We investigate how land-use and subsequent changes in plant species diversity and composition affected the taxonomic composition of larval pollen provisions. Trap nests were located on each of the 27 plots. Each trap nest were checked for occupied reed sticks every 8-10 weeks from March to October to cover the whole flight period of trap nesting solitary bees. We collected samples (i.e. occupied reed sticks) five times in 2017 and three times in 2018. We collected pollen provisions from nests of bee species which were found at least three times across bioregions. |Measurements Type: High-throughput sequencing (Next-generation sequencing (NGS))|instruments: Trap nests: - Each trap nest contained a plastic tube (sewer pipes (length 110-120 cm); cut smaller pieces with 20 cm length) with 60-80 hollow reed sticks (length ~ 20 cm, width 4-12 mm). - Trap nest protection (wire mesh; mesh width (min 1.3-1.5 mm)). - Reed sticks with closed entrances were removed from plastic tubes, replaced with new reed sticks, and then directly and gently (temperature protection!) transported to the lab |calibration: - Sterile forceps - DNA/RNA Shield (Zymo) - ZymoBIOMICS™ DNA extraction kit - PCR cycler - Qubit II Fluorometer - Bioanalyzer 2200 - Illumina MiSeq sequencer|procedures: We followed the dual-indexing strategy based on (Sickel et al., 2015) in order to generate a pooled amplicon library based on the ITS2 rDNA region used for pollen metabarcoding for the Illumina platform. We used USEARCH v11.0.667 (Edgar, 2013; 2016) to merge forward with corresponding reverse reads, accepted if longer than 200bp and passing quality filtering. For determination of amplicon sequence variants (ASVs), we deduplicated, sorted by length, denoised and chimera-filtered reads over all samples and removed singletons with the same software. ASVs were taxonomically classified using first global alignment searches using USEARCH against a reference database containing all plant species known for the Biodiversity Exploratories created with the BCdatabaser software (Keller et al., 2014; Ankenbrand et al., 2015; Keller et al., 2020). Sickel W., Ankenbrand M., Grimmer G., Holzschuh A., Härtel S., Lanzen J. 2015 Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach. BMC Ecol 15. (doi:10.1186/s12898-015-0051-y) Edgar R.C. 2013 UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nature Methods 10(10), 996-998. (doi:10.1038/nmeth.2604) Edgar R.C. 2016 SINTAX: a simple non-Bayesian taxonomy classifier for 16S and ITS sequences. bioRxiv, 074161. (doi:10.1101/074161) Keller A., Hohlfeld S., Kolter A., Schultz J., Gemeinholzer B., Ankenbrand M. 2020 BCdatabaser: on-the-fly reference database creation for (meta-)barcoding. Bioinformatics (Oxford, England).(doi:10.1093/bioinformatics/btz960) Ankenbrand M., Keller A., Wolf M., Schultz J., Förster F. 2015 ITS2 database V: Twice as much. Molecular Biology and Evolution 32, 3030-3032. (doi:10.1093/molbev/msv174) |