DOI: 10.5061/dryad.mgqnk990p
title: A genomic and morphometric analysis of alpine bumblebees: Ongoing reductions in tongue length but no clear genetic component

description: Over the last six decades, populations of the bumblebees Bombus sylvicola
 and Bombus balteatus in Colorado have experienced decreases in tongue
 length, a trait important for plant-pollinator mutualisms. It has been
 hypothesized that this observation reflects selection resulting from
 shifts in floral composition under climate change. Here we used
 morphometrics and population genomics to determine whether morphological
 change is ongoing, investigate the genetic basis of morphological
 variation, and analyse population structure in these populations. We
 analysed whole-genome sequencing data and morphometric measurements of 580
 samples of both species from seven high-altitude localities. Out of 281
 samples originally identified as B. sylvicola, 67 formed a
 separate genetic cluster comprising a newly-discovered cryptic species
 (“incognitus”). However, an absence of genetic structure within species
 suggests that gene flow is common between mountains. We did not discover
 any genetic associations with tongue length, but a SNP related to
 production of a proteolytic digestive enzyme was implicated in body size
 variation. We identified evidence of covariance between kinship and both
 tongue length and body size, which is suggestive of a genetic component of
 these traits, although it is possible that shared environmental effects
 between colonies are responsible. Our results provide evidence for ongoing
 modification of a morphological trait important for pollination and
 indicate that this trait probably has a complex genetic and environmental
 basis. This archive contains genetic variation data derived from genome
 sequencing of 580 bumblebee samples collected from high-elevation
 locations in Colorado. The species are Bombus sylvicola (n=214), Bombus
 balteatus (n=299) and "incognitus" (n=67).
description: We extracted DNA from the thoraces of bumblebees collected from
 across the seven sampling sites using the Qiagen Blood and Tissue kit. We
 prepared dual-indexed libraries using the Nextera Flex kit and performed
 sequencing on an Illumina HiSeq X to produce 2 × 150 bp reads, using an
 average of 36 samples per lane. The Bombus sylvicola and
 "incognitus" samples are mapped to the Bombus sylvicola
 reference assembly (GCA_019677175.1) and the Bombus balteatus samples are
 mapped to the Bombus balteatus assembly (GCA_019201815.1).We mapped reads
 to the two reference genome assemblies using the mem algorithm in BWA. We
 performed sorting and indexing of the resultant bam files using samtools
 and marked duplicate reads using Picard. We used the genome analysis
 toolkit (GATK) to call variants. We first ran HaplotypeCaller using
 default parameters on the bam file of each sample to generate a gVCF file
 for each sample. We then used GenomicsDBImport and GenotypeGVCFs with
 default parameters to call variants for all samples mapping to each
 reference assembly seperately. We applied a set of hard filters using the
 VariantFiltration tool to filter for reliable SNPs using the following
 thresholds: QD &lt;2, FS &gt;60, MQ &lt;40, MQRankSum
 &lt;−12.5, ReadPosRankSum &lt;−8 (see the GATK documentation for
 full descriptions of each filter). Only biallelic SNPs were considered for
 downstream analysis.
description: The files are VCF (variant call format) files generated by GATK
 (genome analysis toolkit). They are plain text files that can be read by a
 wide range of bioinformatic software including VCFtools. A description of
 the format is here: https://en.wikipedia.org/wiki/Variant_Call_Format