A genomic and morphometric analysis of alpine bumblebees: Ongoing reductions in tongue length but no clear genetic component

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).

Methods
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 <2, FS >60, MQ <40, MQRankSum <−12.5, ReadPosRankSum <−8 (see the GATK documentation for full descriptions of each filter). Only biallelic SNPs were considered for downstream analysis.

过去六十年来，科罗拉多州的森林熊蜂（Bombus sylvicola）与环带熊蜂（Bombus balteatus）种群的喙长这一影响植物-传粉者互利共生的关键性状出现了缩短现象。已有假说提出，该现象反映了气候变化下植物群落组成改变所带来的选择压力。本研究借助形态测量学与群体基因组学手段，旨在探明形态性状变化是否仍在持续，解析形态变异的遗传基础，并分析这些种群的群体结构。

我们对采自7个高海拔采样点的580份两类熊蜂样本的全基因组测序数据与形态测量数据展开分析。在最初鉴定为森林熊蜂的281份样本中，有67份构成了一个独立的遗传聚类群，对应一个新发现的隐存种（"incognitus"）。不过，物种内部缺乏遗传结构这一结果表明，不同山地种群间存在频繁的基因流。本研究未发现与喙长相关的遗传关联，但鉴定到一个与蛋白水解消化酶合成相关的单核苷酸多态性（SNP，Single Nucleotide Polymorphism）位点与体型变异相关。我们检测到亲缘关系与喙长、体型均存在协变关系，这暗示这些性状存在遗传基础，不过也不排除蜂群间共享的环境效应是其成因。本研究结果证实，影响传粉功能的重要形态性状仍在持续演变，并表明该性状的遗传与环境基础均较为复杂。

本数据集存档包含采自科罗拉多州高海拔区域的580份熊蜂样本的基因组测序遗传变异数据。所涉物种包括森林熊蜂（n=214）、环带熊蜂（n=299）以及隐存种"incognitus"（n=67）。

方法

本研究使用Qiagen血液与组织提取试剂盒，从7个采样点采集的熊蜂胸部组织中提取基因组DNA。采用Nextera Flex试剂盒构建双索引文库，并在Illumina HiSeq X测序平台上进行测序，生成2×150 bp的测序读段，每泳道平均分配36个样本。将森林熊蜂与隐存种"incognitus"的测序读段比对至森林熊蜂参考基因组组装版本（GCA_019677175.1），环带熊蜂的测序读段则比对至环带熊蜂参考基因组组装版本（GCA_019201815.1）。

本研究使用BWA软件的mem算法将测序读段比对至两套参考基因组组装版本。使用samtools对生成的BAM文件进行排序与索引，并通过Picard工具标记重复读段。采用基因组分析工具包（GATK，Genome Analysis Toolkit）进行变异检测：首先对每个样本的BAM文件以默认参数运行HaplotypeCaller，为每个样本生成gVCF文件；随后分别对比对至两套参考基因组的所有样本，以默认参数运行GenomicsDBImport与GenotypeGVCFs进行联合变异检测。通过VariantFiltration工具应用一系列硬过滤参数以筛选可靠的单核苷酸多态性（SNP），过滤阈值如下：QD <2、FS >60、MQ <40、MQRankSum <−12.5、ReadPosRankSum <−8（各过滤参数的详细说明可参见GATK官方文档）。后续分析仅考虑双等位基因SNP。