DOI: 10.5061/dryad.7d7wm37t4
title: Data from: Ultraconserved element phylogenomics and biogeography of the agriculturally important mason bee subgenus Osmia (Osmia)

description: One of the most important non‐Apis groups of bees for agriculture is the
 mason bee subgenus Osmia Panzer (Osmia), or Osmia s.s. (Hymenoptera:
 Megachilidae). Out of the 29 known species, four have been developed as
 managed pollinators of orchards. In addition, the group is important as a
 source of non‐native pollinators, given that several species have been
 introduced into new areas. Osmia s.s. occurs naturally throughout the
 northern temperate zone with greatest species richness in Europe and Asia.
 Here, we integrate phylogenomic data from ultraconserved elements (UCEs),
 near complete taxon sampling, and a diversity of analytical approaches to
 infer the phylogeny, divergence times, and biogeographic history of Osmia
 s.s. We also demonstrate how mitochondrial sequence data can be extracted
 from ultraconserved element data and combined with sequences from public
 repositories in order to test the phylogeny, examine species boundaries
 and identify specimen‐associated, non‐bee DNA. We resolve the phylogeny of
 Osmia s.s. and show strong support that Nearctic Osmia ribifloris is the
 sister group to the rest of the subgenus. Biogeographic analyses indicate
 that the group originated during the Late Miocene in the West Nearctic
 plus East Palearctic region following dispersal from the East Palearctic
 to the West Nearctic across the Bering land bridge prior to its closure
 5.5–4.8 Ma. The mitochondrial DNA results reveal potential taxonomic
 synonymies involving Osmia yanbianensis and Osmia opima, and Osmia rufina,
 Osmia rufinoides, and Osmia taurus.
description: See accompanying paper for more details. 
 <em>UCE Molecular Data Generation</em>
 To generate a genome-scale dataset of <em>Osmia
 </em>s.s<em> </em>we used ultra-conserved element (UCE)
 phylogenomics (Branstetter<em> et al.</em>, 2017;
 Faircloth<em> et al.</em>, 2012), an approach that combines
 the targeted enrichment of thousands of nuclear, UCE loci with multiplexed
 next-generation sequencing. Enrichment was performed using a recently
 published bait set that targets loci shared across all Hymenoptera
 (“hym-v2-bee-ant-specific”; Grab<em> et al.</em>, 2019). This
 bait set is a subset of the principal Hymenoptera bait set
 (Branstetter<em> et al.</em>, 2017) and includes probes
 synthesized from one ant and one bee genome only. It includes 9,068 unique
 baits targeting 2,545 UCE loci, plus an additional 264 baits targeting 12
 exons from 7 “legacy” markers (ArgK, CAD, EF1aF1 and F2, LwRh, NaK, PolD1,
 and TopI) that have been used extensively in Sanger sequencing-based
 studies. The bait set is currently available for purchase from Arbor
 Biosciences as a catalog kit. For all newly sequenced
 specimens, we extracted DNA using either the Qiagen DNeasy Blood and
 Tissue kit (48 specimens), or the Zymo Quick-DNA Miniprep Plus Kit (6
 specimens). All extractions were performed using mounted museum specimens
 that had been collected between 1971-2017. For most specimens we removed
 and destructively sampled one to two legs, leaving the rest of the
 specimen intact and able to serve as a “same specimen” DNA voucher. For a
 couple of smaller species, we performed whole body, non-destructive
 extractions, in which the specimens were removed from their pin and soaked
 in proteinase-K. Following the soak, the specimens were rinsed in 95%
 ethanol, dried, and re-mounted. For each extraction, we followed the
 manufacturer’s extraction protocol, except for the following modifications
 (applies to both kit types): (1) the proteinase-K digestion was performed
 overnight for 12-16 hours; (2) the elution buffer was warmed to 60º C
 prior to elution; (3) the elution buffer was allowed to incubate in the
 column for five minutes; and (4) DNA was eluted two times using 60-75 uL
 of fresh elution buffer each time. After extraction, the concentration of
 eluted DNA was measured using a Qubit 3.0 fluorometer (Thermo Fisher
 Scientific) and 1-50 ng of extracted DNA was used for library preparation
 and target enrichment. Each DNA sample was sheared
 using a Qsonica Q800R2 acoustic sonicator, with the target fragment size
 range being 400-600 bp. Having larger fragment sizes in the sequencing
 pool improves the amount of flanking DNA that is sequenced, which can
 improve UCE contig lengths following assembly. For recently collected,
 high quality samples, the sonicator was run for 90-120 seconds (shearing
 time) at 25% amplitude and with a 10-10 second on-off pulse. For older
 samples that likely had more degraded DNA, we adjusted the shearing times
 to between 30-60 seconds. Following sonication, fragmented DNA was
 purified at 3x volume using a homemade SPRI-bead substitute (Rohland
 & Reich, 2012). Illumina sequencing libraries were then generated
 using Kapa Hyper Prep kits and custom, 8 bp, dual-indexing adapters
 (Glenn<em> et al.</em>, 2019). All library preparation steps
 were performed at quarter volume of the manufacturer’s recommendations,
 except for PCR, which was done at the full 50 uL volume. Limited cycle PCR
 was performed for 12 cycles on most samples, but for lower quality samples
 we increased the number of cycles to 14-16 cycles, usually as a re-PCR of
 the of the pre-PCR library. Each amplified library was cleaned using
 1.0-1.2x SPRI beads in order to remove contaminants and select out small
 fragments below 200 bp, including possible adapter dimer. The
 concentration of the final cleaned library was measured using
 Qubit. Enrichment was performed using the bee-ant UCE
 bait set described above and by following either a standard UCE protocol
 (enrichment protocol v1.5 available at ultraconserved.org), based on
 Blumenstiel<em> et al.</em> (2010), or by using a modified
 protocol in which we followed Arbor Biosciences v3.02 protocol for
 enrichment day 1 (more efficient than the standard protocol), and the
 standard UCE protocol for day 2. In either case, we combined up to 10
 samples into equimolar enrichment pools and used 500 ng of pooled DNA for
 enrichment. On the first day of enrichment, the pooled DNA samples were
 combined with biotinylated RNA-baits and a set of blocking agents that
 reduce non-specific binding (salmon sperm, adapter blockers, Cot-1). We
 used a 20k probe, custom order of the probe set (diluted 1 ul bait to 4 uL
 H2O) and our own blocking reagents, which included chicken Cot-1 DNA
 (Applied Genetics Laboratories, Inc.), rather than the human Cot-1 DNA
 that comes with Arbor Bioscience’s kits. We performed the enrichment
 incubation at 65ºC for 24 hours using strip tubes and a PCR thermal
 cycler. For the second day of enrichment we used 50 uL of streptavidin
 beads per sample and performed on-bead PCR following the three heated
 (65ºC) wash steps. The enriched pools were amplified for 18 cycles and the
 resulting products were cleaned with SPRI beads at 1x volume. Following
 enrichment, each enrichment pool was quantified using qPCR and pooled
 together into a final sequencing pool at equimolar concentrations.
 Sequencing pools were either sequenced locally on an in-lab Illumina
 MiniSeq (2x125) or sent to the University of Utah Genomics Core for
 sequencing on an Illumina HiSeq 2500 (2x125, v4 chemistry).
 <em>Sequence Processing and Matrix
 Generation</em> The raw sequence data were
 demultiplexed and converted to FASTQ format using BCL2FASTQ (Illumina).
 The resulting FASTQ reads were then cleaned and processed using the
 software package PHYLUCE v1.6 (Faircloth, 2016) and associated programs.
 Within the PHYLUCE environment, the raw reads were trimmed for adapter
 contamination using ILLUMIPROCESSOR v2.0 (Faircloth, 2013), which
 incorporates TRIMMOMATIC (Bolger<em> et al.</em>, 2014).
 Trimmed reads were assembled <em>de novo</em> into contigs
 using both TRINITY v2013-02-25 (Grabherr<em> et al.</em>,
 2011) and SPADES (Bankevich<em> et al.</em>, 2012) for
 comparative purposes. To identify and extract UCE contigs from the bulk
 set of contigs we used a PHYLUCE program (match_contigs_to_probes) that
 uses LASTZ v1.0 (Harris, 2007) to match probe sequences to contig
 sequences and create a relational database of hits. For this step, we
 adjusted the min-identity and min-coverage settings to 70 and 75,
 respectively, which we have found to recover the highest number of UCE
 loci in bees when using the hym-v2-bee-ant UCE probes and bait
 file. After extracting UCE contigs, we aligned each UCE
 locus using MAFFT v7.130b (Katoh & Standley, 2013), trying both
 the default algorithm setting in PHYLUCE (FFT-NS-i) and the slower but
 probably more accurate L-INS-i algorithm. To remove poorly aligned
 regions, we trimmed internal and external regions of alignments using
 GBLOCKS (Talavera & Castresana, 2007), with reduced stringency
 parameters (b1:0.5, b2:0.5, b3:12, b4:7). Using PHYLUCE, we then
 concatenated all loci into a supermatrix and inferred a preliminary tree
 using IQ-TREE v2.0-rc1 (Minh<em> et al.</em>, 2020). The
 resulting tree showed that several of the older, lower quality samples had
 exaggerated terminal branch lengths, likely resulting from difficulties in
 aligning shorter sequence fragments to more complete ones, an issue that
 is seemingly common in species-level target enrichment datasets containing
 old samples. To remove these poorly aligned sequences,
 we used the automated trimming program SPRUCEUP (Borowiec, 2019) on the
 complete set of UCE alignments. This program identifies outlier alignment
 sequences in individual samples, rather than alignment columns, and trims
 the sequences based on user defined cutoffs applied to all samples or to
 specific samples only. In the configuration file, we set SPRUCEUP to use
 the Jukes-Cantor-corrected distance calculation, a window size of 20 base
 pairs (bp), an overlap size of 15 bp, a lognormal distribution, and a
 cutoff value of 0.98. Additionally, we used manual cutoffs for the samples
 with the most exaggerated terminal branch lengths
 (Osmia_maxillaris_BLX43:0.039, Osmia_melanocephala_BLX45:0.035,
 Osmia_emarginata_infuscata_BLX38:0.04,
 Osmia_mustelina_umbrosa_BLX47:0.047, Osmia_cornuta_BLX35:0.037,
 Osmia_cerinthidis_cerinthidis_BLX32:0.04,
 Osmia_cerinthidis_cerinthidis_BLX176:0.05, Osmia_rufina_BLX368:0.04,
 Osmia_bicornis_globosa_BLX174:0.037). We arrived at these settings over
 several rounds of testing, with the objective being to do minimal trimming
 for most species and more aggressive trimming for the problematic samples
 (trimming results show in Fig. 1). Following trimming
 with SPRUCEUP, we used PHYLUCE to re-trim all alignments with GBLOCKS
 (same settings as above) and then to filter the alignments for taxon
 occupancy, requiring loci to have 0%, 50%, 75%, 90%, 95%, 98%, and 100%
 taxon completeness. For each resulting set of filtered loci, we used
 PHYLUCE to generate descriptive statistics for the data and to concatenate
 the loci into a supermatrix for analysis. For each supermatrix we
 performed an unpartitioned phylogenetic analysis with IQ-TREE v2
 (Minh<em> et al.</em>, 2020), selecting GTR+F+G4 as the model
 of sequence evolution and performing 1,000 Ultrafast Bootstrap replicates
 (UFB; Hoang<em> et al.</em>, 2018) and 1,000 replicates of the
 SH-like approximate likelihood ratio test (SH-aLRT; Guindon<em> et
 al.</em>, 2010). For most analyses we used the GTR+F+G4 model only
 and did not perform model testing. This was done because using more
 complex models does not negatively impact phylogenetic results (Abadi
 <em>et al</em>., 2019), even when a less complex model might
 be favored. We also avoided using the proportion of invariant sites (+I)
 model with gamma distributed rates (+G) for among-site rate heterogeneity
 because the two parameters cannot be optimized together (Yang, 2006).
 Based on a review of the matrix statistics and the results of the
 unpartitioned analyses, we selected the 75% complete locus set as the
 focal set for additional analyses.
description: # Data from: Ultraconserved element phylogenomics and biogeography of the
 agriculturally important mason bee subgenus Osmia (Osmia)
 [https://doi.org/10.5061/dryad.7d7wm37t4](https://doi.org/10.5061/dryad.7d7wm37t4) UCE phylogenomics and biogeography of the agriculturally important mason bee subgenus Osmia (Osmia) Michael G. Branstetter1, Andreas Müller2, Terry L. Griswold1, Michael C. Orr3, Chao-Dong Zhu3 1U.S. Department of Agriculture, Agricultural Research Service, Pollinating Insects Research Unit, 5310 Old Main Hill, Utah State University, Logan, UT 84322, USA; ORCID-iD: [https://orcid.org/0000-0002-3734-6166](https://orcid.org/0000-0002-3734-6166) 2ETH Zurich, Institute of Agricultural Sciences, Biocommunication and Entomology, Schmelzbergstrasse 9/LFO, 8092 Zurich, Switzerland 3Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing, 100101, P. R. China. ## Description of the data and file structure Files in Dryad Package: * Bulk-Assembly-Contigs: Contains bulk assemblies for ALL samples included in the publication. Data were assembled using Trinity and SPAdes. Taxon names have been updated to the final published names. * uce-contigs-unaligned.fasta: Monolithic fasta file output by phyluce after running the get_fastas_from_match_counts script. Contains all UCE contigs, untrimmed and unaligned. Taxon names have been updated to the final published names. * UCE-Alignments-Untrimmed: All UCE alignments before trimming (Gblocks or Spruceup). Loci aligned using MAFFT-LINSi algorithm. Taxon names have been updated to the final published names. * DNA-Matrices-and-Partition-Files: Concatenated DNA matrices and associated partition files. Taxon names have been updated to the final published names. * Tree-and-Log-Files: Tree files and analysis log files. Taxon names have been updated to the final published names in the tree files but not the log files. File names start with the figure number used in the main publication. * BioGeoBEARS: Input files and script files for BioGeoBEARS. Taxon names represent final published names. * Divergence-Dating: Input files for BEAST2 and MCMCTree divergence dating analyses. Taxon names have not been updated to the final published names. * uce-baits-hymV2-bee-ant-uniques.fasta: UCE bait file used to enrich UCE loci and to extract out UCE contigs from the bulk set of contigs. --- Additional Notes * All sample names include an extraction number (e.g. BLX#). This can be used to match up samples with additional information in the published tables. * The matrix and tree file names include information about the dataset and analysis. The file name usually includes the number of samples (e.g. 59t), the percent taxon completeness (e.g. 75p), information about locus filtering (e.g. clock500r), the analysis program (e.g. iqtree), and partitioning scheme used (e.g. swsc). Additional information about the analysis can be found in the paper and associated supplemental files.