DOI: 10.5061/dryad.crjdfn3h9
title: Data from: Pollen diet, more than geographic distance, shapes provision microbiome composition in two species of cavity-nesting bees

description: The microbial composition of stored food can influence its stability and
 determine the microbial species consumed by the organism feeding on it.
 Many bee species store nectar and pollen in provisions constructed to feed
 developing offspring. Yet whether microbial composition is determined by
 the pollen types within provisions, variation between bee species at the
 same nesting sites, or geographic distance was unclear. Here, we sampled
 two species of co-occurring cavity nesting bees in the genus Osmia at 13
 sites in California and examined the composition of pollen, fungi, and
 bacteria in provisions. Pollen composition explained 15% of variation in
 bacterial composition and ~30% of variation in fungal composition, whereas
 spatial distance among sites explained minimal additional variation.
 Symbiotic microbe genera Ascosphaera, Sodalis, and Wolbachia showed
 contrasting patterns of association with pollen composition, suggesting
 distinct acquisition and transmission routes for each. Comparing
 provisions from both bee species comprised of the same pollens points to
 environmental acquisition rather than bee species as a key factor shaping
 the early stages of the bee microbiome in Osmia. The patterns we observed
 also contrast with the Apilactobacillus-dominated provision
 microbiome in other solitary bee species, suggesting variable mechanisms
 of microbial assembly in stored food among bee species.
description: <strong>Study system</strong> We
 sampled the stored nectar and pollen within brood cells of two species of
 cavity-nesting <em>Osmia</em> (mason bees, Megachilidae) that
 nest in early spring with partially overlapping phenology, and that often
 co-occur. It is common to see nests of the two species within the same
 piece of wood nesting substrate within a microsite. <em>Osmia
 lignaria</em> is managed as a pollinator for early-flowering orchard
 crops and is a diet generalist (Williams & Tepedino 2003).
 <em>Osmia ribifloris</em> is a diet specialist that forages
 mainly on host plants in the Ericaceae (Sampson <em>et
 al.</em> 2013). Both <em>Osmia</em> species are common
 across the western United States and restricted to North America as of
 2025 (discoverlife.org). Nests of each species are distinguished by
 nesting material: <em>O. lignaria</em> constructs nest
 partitions using mud, while <em>O. ribifloris</em> uses
 masticated leaves (Fig. 1). <strong>Sampling
 design</strong> We sampled
 <em>Osmia</em> populations at 13 geographic locations,
 hereafter ‘sites’, in the foothills of the western slope of the Sierra
 Nevada in California (Fig 2) where populations of each species were
 established or bees were newly released. At each site, nesting was
 encouraged by the placement of clean (washed and heat-treated) nesting
 blocks in early spring. In 2020, populations were sampled at 10 sites,
 where sites hosted natural populations of either or both species (Table
 S1). In 2022, populations of <em>O. ribifloris</em> were
 released at 3 agricultural sites and a subset of the natural source
 populations were also re-sampled (Table S1). To collect
 provision material, we transferred whole provisions to sterile 1.7 mL
 centrifuge tubes using single use plastic pipets or sterile toothpicks.
 Provisions were sampled when brood cells contained either an egg (most
 common), 1<sup>st</sup> or rarely 2<sup>nd</sup>
 instar larvae, and no frass was present in brood cells. Larvae of these
 species have a blind gut and do not defecate until the
 5<sup>th</sup> instar (Torchio 1989). Collected provisions
 were stored at -80°C until DNA extraction. At each site, we sampled
 provisions from 3 nests per species and 2 brood cells within each nest, or
 as many as were available. <strong>Sample
 Processing</strong> DNA was extracted from
 approximately a third of each provision using the Qiagen PowerSoil kit
 (Germantown, MD, USA) following manufacturer’s instructions. Extracted DNA
 was quantified using nanodrop and submitted for amplicon sequencing at
 Dalhousie Integrated Microbiome Resource (IMR, Dalhousie University, Nova
 Scotia CA) Sequencing facility. Plant sequences were targeted using rbcL
 primers rbcLaF (5’-ATGTCACCACAAACAGAGACTAAAGC-3’) and rbcL506R
 (5’-AGGGGACGACCATACTTGTTCA-3’), bacterial 16S rRNA sequences were
 amplified using 799F (799F=5'-AACMGGATTAGATACCCKG-3) and 1115R
 (1115R=5'-AGGGTTGCGCTCGTTG-3') to reduce contamination with
 mitochondrial and chloroplast sequences and fungal ITS sequences were
 amplified using ITS86 (5’-GTGAATCATCGAATCTTTGAA) and ITS4
 (5’-TCCTCCGCTTATTGATATGC-3’). Kit extraction blanks were also included in
 sequencing runs for all sample types (N=2/region). PCR was performed in
 duplicate using high-fidelity Phusion Plus polymerase, followed by
 amplicon cleanup and normalization using Charm Just-a-Plate Normalization
 kit, and sequencing using MiSeq PE300 was performed by Dalhousie
 IMR.  <strong>Sequence data
 processing</strong> Sequence data were quality
 filtered, trimmed, error-corrected, merged, and had chimeras removed using
 the DADA2 pipeline (Callahan <em>et al.</em> 2016), see full
 code in accompanying Dryad and Zenodo repository. This generated a table
 of actual sequence variants (ASVs) present across each sample. Sequence
 variants detected in the negative controls were removed from the dataset
 using Decontam (Davis <em>et al.</em> 2018), resulting in the
 removal of 8 fungal ASVs, 3 pollen ASVs and 14 bacterial ASVs. Taxonomic
 annotation was performed using the UNITE database (v7.2) for ITS sequences
 (Nilsson <em>et al.</em> 2019), BEExact v2023.01.30 and SILVA
 v138.1 for 16S sequences, and plant database for rbcL sequences (Bell
 & Brosi 2016). Manual BLAST searches were performed for the most
 abundant 100 ASVs in each dataset when unannotated at the family level, or
 when 16S databases did not match, and updated when BLAST searches
 indicated high confidence annotations. Data wrangling was performed using
 Phyloseq (McMurdie & Holmes 2013). All ASVs that were not assigned
 to an appropriate kingdom based on the sequencing region (e.g. fungi for
 ITS) and a phylum within that kingdom were filtered out; reads assigned as
 mitochondria or chloroplast were also filtered out from ITS and 16S
 datasets. Following sequence curation, species accumulation curves were
 examined to ensure saturation (Figure S1), with samples dropped below a
 minimum of 500 reads for pollen (mean = 25,110, 2 samples dropped), 450
 reads/sample for fungi (mean=11,564, none dropped), and 500 for bacteria
 (mean=6,570; 20 samples dropped).
description: # Data from: Pollen diet, more than geographic distance, shapes provision
 microbiome composition in two species of cavity-nesting bees Dataset DOI:
 [10.5061/dryad.crjdfn3h9](10.5061/dryad.crjdfn3h9) ## Description of the
 data and file structure ### Files and variables #### File:
 bacterialtaxonomy.csv **Description:**  ##### Variables * ASV: Actual
 sequence variant * X: Placeholder * Kingdom: Assigned Kingdom * Phylum:
 Assigned Phylum * Class: Assigned Class * Order: Assigned Order * Family:
 Assigned Family * Genus: Assigned Genus * Species: Assigned Species ####
 File: sample_data_export-mod.csv **Description:**  ##### Variables *
 SampleLabel: Sample ID * Site: Sampling site label * Species: Bee species,
 L = Osmia lignaria, R= Osmia ribifloris * Nest: Nest ID * CellPos: Cell
 position within the nest, with 1 indicating the first cell provisioned in
 a given nest * Type: Indicates if samples were taken in an agricultural or
 natural site  * Year: Year sampled * FungiCode: Code for matching with the
 fungal dataset * Kit: Which kit was used for DNA extraction * SiteMatch:
 Site label used for matching * CollectionDate: Date that the cell was
 collected * Lat: Latitude of collection site * Lon: Longitude of
 collection site * County: County of collection site * RibNests: Estimated
 total number of O. ribifloris nests at that site * LigNests: Estimated
 total number of O. lignaria nests at that site * Sample_or_Control:
 Indicates if this is a sample or control (blanks removed for this dataset)
 * is.neg: Indicates if this is a negative control #### File: pollenasv.csv
 **Description:**  ##### Variables * SampleLabel: Sample label for matching
 with sample data * The remaining columns indicate pollen ASVs that match
 to Pollen taxonomy worksheet, and values indicate the raw sequence counts
 within a given sample  #### File: pollentaxonomy.csv **Description** #####
 Variables * Sequence: Sequence (matches to pollen ASV) * X: Placeholder *
 Kingdom: Assigned Kingdom * Phylum: Assigned Phylum * Class: Assigned
 Class * Order: Assigned Order * Family: Assigned Family * Genus: Assigned
 Genus * Species: Assigned Species #### File: fungalasv.csv
 **Description:**  ##### Variables * SampleLabel: Sample label to match to
 sample metadata. The  * remaining columns are counts of each ASV present
 within each sample #### File: bacterialasv.csv **Description:**  #####
 Variables * SampleLabel: Sample label to match to sample metadata. The  *
 remaining columns are counts of each ASV present within each sample ####
 File: fungaltaxonomy.csv **Description:**  ##### Variables * Sequence:
 Sequence (matches to fungal ASV) * X: Placeholder * Kingdom: Assigned
 Kingdom * Phylum: Assigned Phylum * Class: Assigned Class * Order:
 Assigned Order * Family: Assigned Family * Genus: Assigned Genus *
 Species: Assigned Species ## Code/software No specific software is needed
 to view these data, but we used R and RStudio, along with the R package
 "phyloseq," to combine these files into a single phyloseq object
 and perform data manipulations and statistical analyses.  ## Access
 information Other publicly accessible locations of the data: * Sequence
 data have been deposited to NCBI SRA under BioProject ID PRJNA1246220.  
 Data was derived from the following sources: * NA