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Abstract Understanding the myriad avenues through which spatial and environmental factors shape evolution is a major focus in biological research. From a molecular perspective, much work has been focused on genomic sequence variation; however, recently there has been increased interest in how epigenetic variation may be shaped by different variables across the landscape. DNA methylation has been of particular interest given that it is dynamic and can alter gene expression, potentially offering a path for a rapid response to environmental change. We utilized whole genome enzymatic methyl sequencing to evaluate the distribution of CpG methylation across the genome and to analyze patterns of spatial and environmental association in the methylomes of two broadly distributed montane bumble bees (Bombus vancouverensis Cresson and Bombus vosnesenskii Radoszkowski) across elevational gradients in the western US. Methylation patterns in both species are similar at the genomic scale with ∼1% of CpGs being methylated and most methylation being found in exons. At the landscape scale, neither species exhibited strong spatial or population structuring in patterns of methylation, although some weak relationships between methylation and distance or environmental variables were detected. Differential methylation analysis suggests a stronger environment association in B. vancouverensis given the larger number of differentially methylated CpG's compared to B. vosnesenskii. We also observed only a handful of genes with both differentially methylated CpGs and previously detected environmentally associated outlier SNPs. Overall results reveal a weak but present pattern in variation in methylation over the landscape in both species. 

ABSTRACT Bumble bees are common in cooler climates and many species likely experience periodic exposure to very cold temperatures, but little is known about the temporal dynamics of cold response mechanisms following chill exposure, especially how persistent effects of cold exposure may facilitate tolerance of future events. To investigate molecular processes involved in the temporal response by bumble bees to acute cold exposure, we compared mRNA transcript abundance in Bombus impatiens workers exposed to 0°C for 75 min (inducing chill coma) and control bees maintained at a constant ambient temperature (28°C). We sequenced the 3′ end of mRNA transcripts (TagSeq) to quantify gene expression in thoracic tissue of bees at several time points (0, 10, 30, 120 and 720 min) following cold exposure. Significant differences from control bees were only detectable within 30 min after the treatment, with most occurring at the 10 min recovery time point. Genes associated with gluconeogenesis and glycolysis were most notably upregulated, while genes related to lipid and purine metabolism were downregulated. The observed patterns of expression indicate a rapid recovery after chill coma, suggesting an acute differential transcriptional response during recovery from chill coma and return to baseline expression levels within an hour, with no long-term gene expression markers of this cold exposure. Our work highlights the functions and pathways important for acute cold recovery, provides an estimated time frame for recovery from cold exposure in bumble bees, and suggests that cold hardening may be less important for these heterothermic insects. 

Abstract Bombus vosnesenskii Radowszkowski, 1862 is one of three bumble bee species commercially available for pollination services in North America; however, little is documented about B. vosnesenskii colony life cycle or the establishment of ex situ rearing, mating, and overwintering practices. In this study, we documented nest success, colony size, and gyne production; recorded the duration of mating events; assessed overwintering survival of mated gynes; and evaluated second-generation nest success for colonies established from low- and high-elevation wild-caught B. vosnesenskii gynes. Of the 125 gynes installed, 62.4% produced brood cells (nest initiation) and 43.2% had at least 1 worker eclose (nest establishment). High-elevation B. vosnesenskii gynes had significantly higher nest initiation and establishment success than low-elevation gynes. However, low-elevation colonies were significantly larger with queens producing more gynes on average. Mating was recorded for 200 low-elevation and 37 high-elevation gynes, resulting in a mean duration of 62 and 51 min, respectively. Mated gynes were then placed into cold storage for 54 days to simulate overwintering, which resulted in 59.1% of low-elevation gynes surviving and 91.9% of high-elevation gynes surviving. For second-generation low-elevation gynes, 26.4% initiated nesting and 14.3% established nesting. Second-generation high-elevation gynes did not initiate nesting despite CO2 narcosis treatments. Overall, these results increase our understanding of B. vosnesenskii nesting, mating, and overwintering biology from 2 elevations. Furthermore, this study provides information on successful husbandry practices that can be used by researchers and conservationists to address knowledge gaps and enhance the captive rearing of bumble bees.