DOI: 10.5061/dryad.vdncjsxqh
title: Molecular sequencing and morphological identification reveal similar patterns in native bee communities across public and private grasslands of eastern North Dakota

description: Bees play a key role in the functioning of human-modified and natural
 ecosystems by pollinating agricultural crops and wild plant communities.
 Global pollinator conservation efforts need large-scale and long-term
 monitoring to detect changes in species’ demographic patterns and shifts
 in bee community structure. The objective of this project was to test a
 molecular sequencing pipeline that would utilize a commonly used locus,
 produce accurate and precise identifications consistent with morphological
 identifications, and generate data that are both qualitative and
 quantitative. We applied this amplicon sequencing pipeline to native bee
 communities sampled across Conservation Reserve Program (CRP) lands and
 native grasslands in eastern North Dakota. We found the 28S LSU locus to
 be more capable of discriminating between species than the 18S SSU rRNA
 locus, and in some cases even resolved instances of cryptic species or
 morphologically ambiguous species complexes. Overall, we found the
 amplicon sequencing method to be a qualitatively accurate representation
 of the sampled bee community richness and species identity, especially
 when a well-curated database of known 28S LSU sequences is available. Both
 morphological identification and molecular sequencing revealed similar
 patterns in native bee community structure across CRP lands and native
 prairie. Additionally, a genetic algorithm approach to compute
 taxon-specific correction factors using a small subset of the most
 concordant samples demonstrated that a high level of quantitative accuracy
 could be possible if the specimens are fresh and processed soon after
 collection. Here we provide a first step to a molecular pipeline for
 identifying insect pollinator communities. This tool should prove useful
 for future national monitoring efforts as use of molecular tools becomes
 more affordable and as numbers of 28S LSU sequences for pollinator species
 increase in publicly-available databases.
description: Pollinator communities were sampled from four paired grassland
 “locations” within the Prairie Pothole Region of eastern North Dakota in
 2012 and 2013. At each NPAM and CRP location, 20 transects were
 established at least 65 meters from each other, and 5 meters away from
 fences, wetlands, and shelterbelts. Transects were sampled approximately
 once every other week from May to September in 2012 and 2013, with a
 sample consisting of a single unscented Springstar blue vane trap placed
 at one fixed end of the transect for 24 hours. Pollinator specimens were
 identified morphologically to the lowest possible taxonomic level at the
 NPWRC invertebrate lab in Jamestown, North Dakota. For molecular
 identification, one mesothoracic leg from each specimen was placed into a
 2.0 ml microcentrifuge tube (one tube per sample), and genomic DNA was
 extracted and purified with a bead bashing protocol. PCR Amplicons for the
 18S and 28S loci were amplified and sequenced on the Illumina MiSeq using
 2x300 bp paired-end reads. These paired reads were merged into one read,
 filtered to remove any reads with at least one predicted error, and
 dereplicated using USEARCH -fastq_mergepairs -fastq_filter -fastx_uniques
 commands.
description: Files associated with Darby et al. "Molecular sequencing and
 morphological identification reveal similar patterns in native bee
 communities across public and private grasslands of eastern North
 Dakota": ~~~~~~~~~~~~~~~~~
 NDbeesequences.fasta -FASTA-formated file
 containing all new and unique bee sequences obtained from 18S and 28S
 amplicon sequencing ~~~~~~~~~~~~~~~~~~~
 sequencedatalong.csv -comma-delimited file
 containing the sequencing read counts for all community samples:
 --Sample: unique internal identifier for each community sample
 (1-224)<br> --Location: one of four sampling locations (Arrowwood,
 Kulm, SullysHill, Tewaukon)<br> --Management: one of two grassland
 management regimes (CRP - Conservation Reserve Program, or NPAM - Northern
 Prairie Adaptive Management)<br> --Date: Sampling Date
 (MM/DD/YYY)<br> --Year: Sampling Year (2012 or 2013)<br>
 --Month: Sampling Month (integer 5 through 9)<br> --Species: Species
 name, code, and locus (corresponding to fasta headers in
 "NDbeesequences.fasta" file)<br> --Locus: one of two
 genetic loci (18S [small subunit rRNA] or 28S [large subunit
 rRNA])<br> --Readcount: number of high-throughput sequencing reads
 matching to the species. ~~~~~~~~~~~~~~~~~~~~
 sequencedatalong.csv -comma-delimited file
 containing the specimen counts for all community samples:
 --Location: one of four sampling locations (Arrowwood, Kulm,
 SullysHill, Tewaukon)<br> --Management: one of two grassland
 management regimes (CRP - Conservation Reserve Program, or NPAM - Northern
 Prairie Adaptive Management)<br> --Date: Sampling Date
 (MM/DD/YYY)<br> --Species: Species name, code, and locus
 (corresponding to fasta headers in "NDbeesequences.fasta"
 file)<br> --Specimencount: number of specimens identified
 morphological to the species.<br>