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 generated a genome assembly of B. balteatus. We then 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 found a significant decrease in tongue length between bees collected between 2012-2014 and in 2017, indicating that morphological shifts are ongoing. 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.

Genetic Barriers to Historical Gene Flow between Cryptic Species of Alpine Bumblebees Revealed by Comparative Population Genomics.

Evidence is accumulating that gene flow commonly occurs between recently diverged species, despite the existence of barriers to gene flow in their genomes. However, we still know little about what regions of the genome become barriers to gene flow and how such barriers form. Here, we compare genetic differentiation across the genomes of bumblebee species living in sympatry and allopatry to reveal the potential impact of gene flow during species divergence and uncover genetic barrier loci. We first compared the genomes of the alpine bumblebee Bombus sylvicola and a previously unidentified sister species living in sympatry in the Rocky Mountains, revealing prominent islands of elevated genetic divergence in the genome that colocalize with centromeres and regions of low recombination. This same pattern is observed between the genomes of another pair of closely related species living in allopatry (B. bifarius and B. vancouverensis). Strikingly however, the genomic islands exhibit significantly elevated absolute divergence (dXY) in the sympatric, but not the allopatric, comparison indicating that they contain loci that have acted as barriers to historical gene flow in sympatry. Our results suggest that intrinsic barriers to gene flow between species may often accumulate in regions of low recombination and near centromeres through processes such as genetic hitchhiking, and that divergence in these regions is accentuated in the presence of gene flow.

Flight of the bumble bee: Buzzes predict pollination services.

Multiple interacting factors drive recent declines in wild and managed bees, threatening their pollination services. Widespread and intensive monitoring could lead to more effective management of wild and managed bees. However, tracking their dynamic populations is costly. We tested the effectiveness of an inexpensive, noninvasive and passive acoustic survey technique for monitoring bumble bee behavior and pollination services. First, we assessed the relationship between the first harmonic of the flight buzz (characteristic frequency) and pollinator functional traits that influence pollination success using flight cage experiments and a literature search. We analyzed passive acoustic survey data from three locations on Pennsylvania Mountain, Colorado to estimate bumble bee activity. We developed an algorithm based on Computational Auditory Scene Analysis that identified and quantified the number of buzzes recorded in each location. We then compared visual and acoustic estimates of bumble bee activity. Using pollinator exclusion experiments, we tested the power of buzz density to predict pollination services at the landscape scale for two bumble bee pollinated alpine forbs (Trifolium dasyphyllum and T. parryi). We found that the characteristic frequency was correlated with traits known to affect pollination efficacy, explaining 30-52% of variation in body size and tongue length. Buzz density was highly correlated with visual estimates of bumble bee density (r = 0.97), indicating that acoustic signals are predictive of bumble bee activity. Buzz density predicted seed set in two alpine forbs when bumble bees were permitted access to the flowers, but not when they were excluded from visiting. Our results indicate that acoustic signatures of flight can be deciphered to monitor bee activity and pollination services to bumble bee pollinated plants. We propose that applications of this technique could assist scientists and farmers in rapidly detecting and responding to bee population declines.

Functional mismatch in a bumble bee pollination mutualism under climate change.

Ecological partnerships, or mutualisms, are globally widespread, sustaining agriculture and biodiversity. Mutualisms evolve through the matching of functional traits between partners, such as tongue length of pollinators and flower tube depth of plants. Long-tongued pollinators specialize on flowers with deep corolla tubes, whereas shorter-tongued pollinators generalize across tube lengths. Losses of functional guilds because of shifts in global climate may disrupt mutualisms and threaten partner species. We found that in two alpine bumble bee species, decreases in tongue length have evolved over 40 years. Co-occurring flowers have not become shallower, nor are small-flowered plants more prolific. We argue that declining floral resources because of warmer summers have favored generalist foraging, leading to a mismatch between shorter-tongued bees and the longer-tubed plants they once pollinated.

Bumble bee nest abundance, foraging distance, and host-plant reproduction: implications for management and conservation.

Recent reports of global declines in pollinator species imply an urgent need to assess the abundance of native pollinators and density-dependent benefits for linked plants. In this study, we investigated (1) pollinator nest distributions and estimated colony abundances, (2) the relationship between abundances of foraging workers and the number of nests they represent, (3) pollinator foraging ranges, and (4) the relationship between pollinator abundance and plant reproduction. We examined these questions in an alpine ecosystem in the Colorado Rocky Mountains, focusing on four alpine bumble bee species (Bombus balteatus, B. flavifrons, B. bifarius, and B. sylvicola), and two host plants that differ in their degrees of pollinator specialization (Trifolium dasyphyllum and T. parryi). Using microsatellites, we found that estimated colony abundances among Bombus species ranged from ~18 to 78 colonies/0.01 km2. The long-tongued species B. balteatus was most common, especially high above treeline, but the subalpine species B. bifarius was unexpectedly abundant for this elevation range. Nests detected among sampled foragers of each species were correlated with the number of foragers caught. Foraging ranges were smaller than expected for all Bombus species, ranging from 25 to 110 m. Fruit set for the specialized plant, Trifolium parryi, was positively related to the abundance of its Bombus pollinator. In contrast, fruit set for the generalized plant, T. dasyphyllum, was related to abundance of all Bombus species. Because forager abundance was related to nest abundance of each Bombus species and was an equally effective predictor of plant fecundity, forager inventories are probably suitable for assessing the health of outcrossing plant populations. However, nest abundance, rather than forager abundance, better reflects demographic and genetic health in populations of eusocial pollinators such as bumble bees. Development of models incorporating the parameters we have measured here (nest abundance, forager abundance, and foraging distance) could increase the usefulness of foraging worker inventories in nionitoring, managing, and conserving pollinator populations.

Tracing impacts of partner abundance in facultative pollination mutualisms: from individuals to populations.

Partner abundance affects costs and benefits in obligate mutualisms, but its role in facultative partnerships is less clear. We address this gap in a pollination web consisting of two clovers (Trifolium) that differ in specialization on a bumble bee pollinator Bombus balteatus. We examine how pollination niche breadth affects plant responses to pollinator abundance, comparing early-flowering (specialized) and late-flowering (generalized) cohorts of T. parryi and early T. parryi to T. dasyphyllum, a pollination generalist. Co-pollinators disrupt the link between B. halteatus visitation and pollination rate for both clovers. Only for early-flowering T. parryi do visitation, pollination, and seed set increase with density of B. balteatus. Bumble bee density also alters timing of seed germination in T. parryi, with seeds from plants receiving augmented B. balteatus germinating sooner than seeds of open-pollinated counterparts. Benefits saturate at intermediate bumble bee densities. Despite strong effects of B. balteatus density on individual plant fitness components, population models suggest little impact of B. balteatus density on lamda in T. parryi or T. dasyphyllum. Findings show that functional redundancy in a pollinator guild mediates host-plant responses to partner density. Unexpected effects of pollinator density on life history schedule have implications for recruitment under pollinator decline.

Density-dependent effects of ants on selection for bumble bee pollination in Polemonium viscosum.

Mutualisms are commonly exploited by cheater species that usurp rewards without providing reciprocal benefits. Yet most studies of selection between mutualist partners ignore interactions with third species and consequently overlook the impact of cheaters on evolution in the mutualism. Here, we explicitly investigate how the abundance of nectar-thieving ants (cheaters) influences selection in a pollination mutualism between bumble bees and the alpine skypilot, Polemonium viscosum. As suggested in past work with this species, bumble bees accounted for most of the seed production (78% +/- 6% [mean +/- SE]) in our high tundra study population and, in the absence of ants, exerted strong selection for large flowers. We tested for indirect effects of ant abundance on seed set through bumble bee pollination services (pollen delivery and pollen export) and a direct effect through flower damage. Ants reduced seed set per flower by 20% via flower damage. As ant density increased within experimental patches, the rate of flower damage rose, but pollen delivery and export did not vary significantly, showing that indirect effects of increased cheater abundance on pollinator service are negligible in this system. To address how ants affect selection for plant participation in the pollination mutualism we tested the impact of ant abundance on selection for bumble bee-mediated pollination. Results show that the impact of ants on fitness (seed set) accruing under bumble bee pollination is density dependent in P. viscosum. Selection for bumble bee pollination declined with increasing ant abundance in experimental patches, as predicted if cheaters constrain fitness returns of mutualist partner services. We also examined how ant abundance influences selection on flower size, a key component of plant investment in bumble bee pollination. We predicted that direct effects of ants would constrain bumble bee selection for large flowers. However, selection on flower size was significantly positive over a wide range of ant abundance (20-80% of plants visited by ants daily). Although high cheater abundance reduces the fitness returns of bumble bee pollination, it does not completely eliminate selection for bumble bee attraction in P. viscosum.