Symbiotic bacteria and fungi proliferate in diapause and may enhance overwintering survival in a solitary bee.

Host-microbe interactions underlie the development and fitness of many macroorganisms, including bees. Whereas many social bees benefit from vertically transmitted gut bacteria, current data suggests that solitary bees, which comprise the vast majority of species diversity within bees, lack a highly specialized gut microbiome. Here we examine the composition and abundance of bacteria and fungi throughout the complete life cycle of the ground-nesting solitary bee Anthophora bomboides standfordiana. In contrast to expectations, immature bee stages maintain a distinct core microbiome consisting of Actinobacterial genera (Streptomyces, Nocardiodes) and the fungus Moniliella spathulata. Dormant (diapausing) larval bees hosted the most abundant and distinctive bacteria and fungi, attaining 33 and 52 times their initial copy number, respectively. We tested two adaptive hypotheses regarding microbial functions for diapausing bees. First, using isolated bacteria and fungi, we found that Streptomyces from brood cells inhibited the growth of multiple pathogenic filamentous fungi, suggesting a role in pathogen protection during overwintering, when bees face high pathogen pressure. Second, sugar alcohol composition changed in tandem with major changes in fungal abundance, suggesting links with bee cold tolerance or overwintering biology. We find that A. bomboides hosts a conserved core microbiome that may provide key fitness advantages through larval development and diapause, which raises the question of how this microbiome is maintained and faithfully transmitted between generations. Our results suggest that focus on microbiomes of mature or active insect developmental stages may overlook stage-specific symbionts and microbial fitness contributions during host dormancy.

Lessons from assembling UCEs: A comparison of common methods and the case of Clavinomia (Halictidae).

Sequence data assembly is a foundational step in high-throughput sequencing, with untold consequences for downstream analyses. Despite this, few studies have interrogated the many methods for assembling phylogenomic UCE data for their comparative efficacy, or for how outputs may be impacted. We study this by comparing the most commonly used assembly methods for UCEs in the under-studied bee lineage Nomiinae and a representative sampling of relatives. Data for 63 UCE-only and 75 mixed taxa were assembled with five methods, including ABySS, HybPiper, SPAdes, Trinity and Velvet, and then benchmarked for their relative performance in terms of locus capture parameters and phylogenetic reconstruction. Unexpectedly, Trinity and Velvet trailed the other methods in terms of locus capture and DNA matrix density, whereas SPAdes performed favourably in most assessed metrics. In comparison with SPAdes, the guided-assembly approach HybPiper generally recovered the highest quality loci but in lower numbers. Based on our results, we formally move Clavinomia to Dieunomiini and render Epinomia once more a subgenus of Dieunomia. We strongly advise that future studies more closely examine the influence of assembly approach on their results, or, minimally, use better-performing assembly methods such as SPAdes or HybPiper. In this way, we can move forward with phylogenomic studies in a more standardized, comparable manner.

Microbes, the 'silent third partners' of bee-angiosperm mutualisms.

While bee-angiosperm mutualisms are widely recognized as foundational partnerships that have shaped the diversity and structure of terrestrial ecosystems, these ancient mutualisms have been underpinned by 'silent third partners': microbes. Here, we propose reframing the canonical bee-angiosperm partnership as a three-way mutualism between bees, microbes, and angiosperms. This new conceptualization casts microbes as active symbionts, processing and protecting pollen-nectar provisions, consolidating nutrients for bee larvae, enhancing floral attractancy, facilitating plant fertilization, and defending bees and plants from pathogens. In exchange, bees and angiosperms provide their microbial associates with food, shelter, and transportation. Such microbial communities represent co-equal partners in tripartite mutualisms with bees and angiosperms, facilitating one of the most important ecological partnerships on land.

Evolutionary Origins and Patterns of Diversification in Animal Brood Parasitism.

AbstractBrood parasitism involves the exploitation of host parental care rather than the extraction of resources directly from hosts. We identify defining characteristics of this strategy and consider its position along continua with adjacent behaviors but focus on canonical brood parasites, where parasitism is obligate and hosts are noneusocial (thereby distinguishing from social parasitism). A systematic literature survey revealed 59 independently derived brood parasitic lineages with most origins (49) in insects, particularly among bees and wasps, and other origins in birds (seven) and fish (three). Insects account for more than 98% of brood parasitic species, with much of that diversity reflecting ancient (≥100-million-year-old) brood parasitic lineages. Brood parasites usually, but not always, evolve from forms that show parental care. In insects, brood parasitism often first evolves through exploitation of a closely related species, following Emery's rule, but this is less typical in birds, which we discuss. We conducted lineage-level comparisons between brood parasitic clades and their sister groups, finding mixed results but an overall neutral to negative effect of brood parasitism on species richness and diversification. Our review of brood parasites reveals many unanswered questions requiring new research, including further modeling of the coevolutionary dynamics of brood parasites and their hosts.

The evolutionary history of bees in time and space.

Bees are the most significant pollinators of flowering plants. This partnership began ca. 120 million years ago, but the uncertainty of how and when bees spread across the planet has greatly obscured investigations of this key mutualism. We present a novel analysis of bee biogeography using extensive new genomic and fossil data to demonstrate that bees originated in Western Gondwana (Africa and South America). Bees likely originated in the Early Cretaceous, shortly before the breakup of Western Gondwana, and the early evolution of any major bee lineage is associated with either the South American or African land masses. Subsequently, bees colonized northern continents via a complex history of vicariance and dispersal. The notable early absences from large landmasses, particularly in Australia and India, have important implications for understanding the assembly of local floras and diverse modes of pollination. How bees spread around the world from their hypothesized Southern Hemisphere origin parallels the histories of numerous flowering plant clades, providing an essential step to studying the evolution of angiosperm pollination syndromes in space and time.

Bee breweries: The unusually fermentative, lactobacilli-dominated brood cell microbiomes of cellophane bees.

Pathogens and parasites of solitary bees have been studied for decades, but the microbiome as a whole is poorly understood for most taxa. Comparative analyses of microbiome features such as composition, abundance, and specificity, can shed light on bee ecology and the evolution of host-microbe interactions. Here we study microbiomes of ground-nesting cellophane bees (Colletidae: Diphaglossinae). From a microbial point of view, the diphaglossine genus Ptiloglossa is particularly remarkable: their larval provisions are liquid and smell consistently of fermentation. We sampled larval provisions and various life stages from wild nests of Ptiloglossa arizonensis and two species of closely related genera: Caupolicana yarrowi and Crawfordapis luctuosa. We also sampled nectar collected by P. arizonensis. Using 16S rRNA gene sequencing, we find that larval provisions of all three bee species are near-monocultures of lactobacilli. Nectar communities are more diverse, suggesting ecological filtering. Shotgun metagenomic and phylogenetic data indicate that Ptiloglossa culture multiple species and strains of Apilactobacillus, which circulate among bees and flowers. Larval lactobacilli disappear before pupation, and hence are likely not vertically transmitted, but rather reacquired from flowers as adults. Thus, brood cell microbiomes are qualitatively similar between diphaglossine bees and other solitary bees: lactobacilli-dominated, environmentally acquired, and non-species-specific. However, shotgun metagenomes provide evidence of a shift in bacterial abundance. As compared with several other bee species, Ptiloglossa have much higher ratios of bacterial to plant biomass in larval provisions, matching the unusually fermentative smell of their brood cells. Overall, Ptiloglossa illustrate a path by which hosts can evolve quantitatively novel symbioses: not by acquiring or domesticating novel symbionts, but by altering the microenvironment to favor growth of already widespread and generalist microbes.

Genome of the bee Holcopasites calliopsidis-a species showing the common apid trait of brood parasitism.

Brood parasites represent a substantial but often poorly studied fraction of the wider diversity of bees. Brood parasitic bees complete their life cycles by infiltrating the nests of solitary host bees thereby enabling their offspring to exploit the food provisions intended for the host's offspring. Here, we present the draft assembly of the bee Holcopasites calliopsidis, the first brood parasitic species to be the subject of detailed genomic analysis. Consistent with previous findings on the genomic signatures of parasitism more broadly, we find that H. calliopsidis has the smallest genome currently known among bees (179 Mb). This small genome does not appear to be the result of purging of repetitive DNA, with some indications of novel repetitive elements which may show signs of recent expansion. Nor does H. calliopsidis demonstrate any apparent net loss of genic content in comparison with nonparasitic species, though many individual gene families do show significant contractions. Although the basis of the small genome size of this species remains unclear, the identification of over 12,000 putative genes-with functional annotation for nearly 10,000 of these-is an important step in investigating the genomic basis of brood parasitism and provides a valuable dataset to be compared against new genomes that remain to be sequenced.

Exosymbiotic microbes within fermented pollen provisions are as important for the development of solitary bees as the pollen itself.

Developing bees derive significant benefits from the microbes present within their guts and fermenting pollen provisions. External microbial symbionts (exosymbionts) associated with larval diets may be particularly important for solitary bees that suffer reduced fitness when denied microbe-colonized pollen.To investigate whether this phenomenon is generalizable across foraging strategy, we examined the effects of exosymbiont presence/absence across two solitary bee species, a pollen specialist and generalist. Larvae from each species were reared on either microbe-rich natural or microbe-deficient sterilized pollen provisions allocated by a female forager belonging to their own species (conspecific-sourced pollen) or that of another species (heterospecific-sourced pollen). Our results reveal that the presence of pollen-associated microbes was critical for the survival of both the generalist and specialist larvae, regardless of whether the pollen was sourced from a conspecific or heterospecific forager.Given the positive effects of exosymbiotic microbes for larval fitness, we then examined if the magnitude of this benefit varied based on whether the microbes were provisioned by a conspecific forager (the mother bee) or a heterospecific forager. In this second study, generalist larvae were reared only on microbe-rich pollen provisions, but importantly, the sources (conspecific versus heterospecific) of the microbes and pollen were experimentally manipulated.Bee fitness metrics indicated that microbial and pollen sourcing both had significant impacts on larval performance, and the effect sizes of each were similar. Moreover, the effects of conspecific-sourced microbes and conspecific-sourced pollen were strongly positive, while that of heterospecific-sourced microbes and heterospecific-sourced pollen, strongly negative.Our findings imply that not only is the presence of exosymbionts critical for both specialist and generalist solitary bees, but more notably, that the composition of the specific microbial community within larval pollen provisions may be as critical for bee development as the composition of the pollen itself.

Phylogenetic relationships and the evolution of host preferences in the largest clade of brood parasitic bees (Apidae: Nomadinae).

Brood parasites (also known as cleptoparasites) represent a substantial fraction of global bee diversity. Rather than constructing their own nests, these species instead invade those of host bees to lay their eggs. Larvae then hatch and consume the food provisions intended for the host's offspring. While this life history strategy has evolved numerous times across the phylogeny of bees, the oldest and most speciose parasitic clade is the subfamily Nomadinae (Apidae). However, the phylogenetic relationships among brood parasitic apids both within and outside the Nomadinae have not been fully resolved. Here, we present new findings on the phylogeny of this diverse group of brood parasites based on ultraconserved element (UCE) sequence data and extensive taxon sampling with 114 nomadine species representing all tribes. We suggest a broader definition of the subfamily Nomadinae to describe a clade that includes almost all parasitic members of the family Apidae. The tribe Melectini forms the sister group to all other Nomadinae, while the remainder of the subfamily is composed of two sister clades: a "nomadine line" representing the former Nomadinae sensu stricto, and an "ericrocidine line" that unites several mostly Neotropical lineages. We find the tribe Osirini Handlirsch to be polyphyletic, and divide it into three lineages, including the newly described Parepeolini trib. nov. In addition to our taxonomic findings, we use our phylogeny to explore the evolution of different modes of parasitism, detecting two independent transitions from closed-cell to open-cell parasitism. Finally, we examine how nomadine host-parasite associations have evolved over time. In support of Emery's rule, which suggests close relationships between hosts and parasites, we confirm that the earliest nomadines were parasites of their close free-living relatives within the family Apidae, but that over time their host range broadened to include more distantly related hosts spanning the diversity of bees. This expanded breadth of host taxa may also be associated with the transition to open-cell parasitism.

Solitary bee larvae modify bacterial diversity of pollen provisions in the stem-nesting bee, Osmia cornifrons (Megachilidae).

Microbes, including diverse bacteria and fungi, play an important role in the health of both solitary and social bees. Among solitary bee species, in which larvae remain in a closed brood cell throughout development, experiments that modified or eliminated the brood cell microbiome through sterilization indicated that microbes contribute substantially to larval nutrition and are in some cases essential for larval development. To better understand how feeding larvae impact the microbial community of their pollen/nectar provisions, we examine the temporal shift in the bacterial community in the presence and absence of actively feeding larvae of the solitary, stem-nesting bee, Osmia cornifrons (Megachilidae). Our results indicate that the O. cornifrons brood cell bacterial community is initially diverse. However, larval solitary bees modify the microbial community of their pollen/nectar provisions over time by suppressing or eliminating rare taxa while favoring bacterial endosymbionts of insects and diverse plant pathogens, perhaps through improved conditions or competitive release. We suspect that the proliferation of opportunistic plant pathogens may improve nutrient availability of developing larvae through degradation of pollen. Thus, the health and development of solitary bees may be interconnected with pollen bacterial diversity and perhaps with the propagation of plant pathogens.

Wild insect diversity increases inter-annual stability in global crop pollinator communities.

While an increasing number of studies indicate that the range, diversity and abundance of many wild pollinators has declined, the global area of pollinator-dependent crops has significantly increased over the last few decades. Crop pollination studies to date have mainly focused on either identifying different guilds pollinating various crops, or on factors driving spatial changes and turnover observed in these communities. The mechanisms driving temporal stability for ecosystem functioning and services, however, remain poorly understood. Our study quantifies temporal variability observed in crop pollinators in 21 different crops across multiple years at a global scale. Using data from 43 studies from six continents, we show that (i) higher pollinator diversity confers greater inter-annual stability in pollinator communities, (ii) temporal variation observed in pollinator abundance is primarily driven by the three-most dominant species, and (iii) crops in tropical regions demonstrate higher inter-annual variability in pollinator species richness than crops in temperate regions. We highlight the importance of recognizing wild pollinator diversity in agricultural landscapes to stabilize pollinator persistence across years to protect both biodiversity and crop pollination services. Short-term agricultural management practices aimed at dominant species for stabilizing pollination services need to be considered alongside longer term conservation goals focussed on maintaining and facilitating biodiversity to confer ecological stability.

(More than) Hitchhikers through the network: the shared microbiome of bees and flowers.

Growing evidence reveals strong overlap between microbiomes of flowers and bees, suggesting that flowers are hubs of microbial transmission. Whether floral transmission is the main driver of bee microbiome assembly, and whether functional importance of florally sourced microbes shapes bee foraging decisions are intriguing questions that remain unanswered. We suggest that interaction network properties, such as nestedness, connectedness, and modularity, as well as specialization patterns can predict potential transmission routes of microbes between hosts. Yet microbial filtering by plant and bee hosts determines realized microbial niches. Functionally, shared floral microbes can provide benefits for bees by enhancing nutritional quality, detoxification, and disintegration of pollen. Flower microbes can also alter the attractiveness of floral resources. Together, these mechanisms may affect the structure of the flower-bee interaction network.

Landscape Composition and Fungicide Exposure Influence Host-Pathogen Dynamics in a Solitary Bee.

Both ecosystem function and agricultural productivity depend on services provided by bees; these services are at risk from bee declines which have been linked to land use change, pesticide exposure, and pathogens. Although these stressors often co-occur in agroecosystems, a majority of pollinator health studies have focused on these factors in isolation, therefore limiting our ability to make informed policy and management decisions. Here, we investigate the combined impact of altered landscape composition and fungicide exposure on the prevalence of chalkbrood disease, caused by fungi in the genus Ascosphaera Olive and Spiltoir 1955 (Ascosphaeraceae: Onygenales), in the introduced solitary bee, Osmia cornifrons (Radoszkowski 1887) (Megachilidae: Hymenoptera). We used both field studies and laboratory assays to evaluate the potential for interactions between altered landscape composition, fungicide exposure, and Ascosphaera on O. cornifrons mortality. Chalkbrood incidence in larval O. cornifrons decreased with high open natural habitat cover, whereas Ascosphaera prevalence in adults decreased with high urban habitat cover. Conversely, high fungicide concentration and high forest cover increased chalkbrood incidence in larval O. cornifrons and decreased Ascosphaera incidence in adults. Our laboratory assay revealed an additive effect of fungicides and fungal pathogen exposure on the mortality of a common solitary bee. Additionally, we utilized phylogenetic methods and identified four species of Ascosphaera with O. cornifrons, both confirming previous reports and shedding light on new associates. Our findings highlight the impact of fungicides on bee health and underscore the importance of studying interactions among factors associated with bee decline.

Gene Tree Estimation Error with Ultraconserved Elements: An Empirical Study on Pseudapis Bees.

Summarizing individual gene trees to species phylogenies using two-step coalescent methods is now a standard strategy in the field of phylogenomics. However, practical implementations of summary methods suffer from gene tree estimation error, which is caused by various biological and analytical factors. Greatly understudied is the choice of gene tree inference method and downstream effects on species tree estimation for empirical data sets. To better understand the impact of this method choice on gene and species tree accuracy, we compare gene trees estimated through four widely used programs under different model-selection criteria: PhyloBayes, MrBayes, IQ-Tree, and RAxML. We study their performance in the phylogenomic framework of $>$800 ultraconserved elements from the bee subfamily Nomiinae (Halictidae). Our taxon sampling focuses on the genus Pseudapis, a distinct lineage with diverse morphological features, but contentious morphology-based taxonomic classifications and no molecular phylogenetic guidance. We approximate topological accuracy of gene trees by assessing their ability to recover two uncontroversial, monophyletic groups, and compare branch lengths of individual trees using the stemminess metric (the relative length of internal branches). We further examine different strategies of removing uninformative loci and the collapsing of weakly supported nodes into polytomies. We then summarize gene trees with ASTRAL and compare resulting species phylogenies, including comparisons to concatenation-based estimates. Gene trees obtained with the reversible jump model search in MrBayes were most concordant on average and all Bayesian methods yielded gene trees with better stemminess values. The only gene tree estimation approach whose ASTRAL summary trees consistently produced the most likely correct topology, however, was IQ-Tree with automated model designation (ModelFinder program). We discuss these findings and provide practical advice on gene tree estimation for summary methods. Lastly, we establish the first phylogeny-informed classification for Pseudapis s. l. and map the distribution of distinct morphological features of the group. [ASTRAL; Bees; concordance; gene tree estimation error; IQ-Tree; MrBayes, Nomiinae; PhyloBayes; RAxML; phylogenomics; stemminess].

Macro-conformality of coatings deposited using high-speed spatial plasma-enhanced atomic layer deposition.

Rotational spatial plasma-enhanced atomic layer deposition has been used to deposit thin films on half-sphere lenses. Non-uniformity of less than ±1% is demonstrated for Nb2O5 deposited at 1.4 Å/s and for SiO2 deposited at 6 Å/s.

Pollen defenses negatively impact foraging and fitness in a generalist bee (Bombus impatiens: Apidae).

Plants may benefit from limiting the community of generalist floral visitors if the species that remain are more effective pollinators and less effective pollenivores. Plants can reduce access to pollen through altered floral cues or morphological structures, but can also reduce consumption through direct pollen defenses. We observed that Eucera (Peponapis) pruinosa, a specialist bee on Cucurbita plants, collected pure loads of pollen while generalist honey bees and bumble bees collected negligible amounts of cucurbit pollen, even though all groups of bees visited these flowers. Cucurbit flowers have no morphological adaptations to limit pollen collection by bees, thus we assessed their potential for physical, nutritional, and chemical pollen traits that might act as defenses to limit pollen loss to generalist pollinators. Bumble bee (Bombus impatiens) microcolonies experienced reduced pollen consumption, mortality, and reproduction as well as increased stress responses when exposed to nutritional and mechanical pollen defenses. These bees also experienced physiological effects of these defenses in the form of hindgut expansion and gut melanization. Chemical defenses alone increased the area of gut melanization in larger bees and induced possible compensatory feeding. Together, these results suggest that generalist bumble bees avoid collecting cucurbit pollen due to the physiological costs of physical and chemical pollen defenses.

Omnivory in Bees: Elevated Trophic Positions among All Major Bee Families.

As pollen and nectar foragers, bees have long been considered strictly herbivorous. Their pollen provisions, however, are host to abundant microbial communities, which feed on the pollen before and/or while it is consumed by bee larvae. In the process, microbes convert pollen into a complex of plant and microbial components. Since microbes are analogous to metazoan consumers within trophic hierarchies, the pollen-eating microbes are, functionally, herbivores. When bee larvae consume a microbe-rich pollen complex, they ingest proteins from plant and microbial sources and thus should register as omnivores on the trophic "ladder." We tested this hypothesis by examining the isotopic compositions of amino acids extracted from native bees collected in North America over multiple years. We measured bee trophic position across the six major bee families. Our findings indicate that bee trophic identity was consistently and significantly higher than that of strict herbivores, providing the first evidence that omnivory is ubiquitous among bee fauna. Such omnivory suggests that pollen-borne microbes represent an important protein source for larval bees, which introduces new questions as to the link between floral fungicide residues and bee development.

Agriculturally dominated landscapes reduce bee phylogenetic diversity and pollination services.

Land-use change threatens global biodiversity and may reshape the tree of life by favoring some lineages over others. Whether phylogenetic diversity loss compromises ecosystem service delivery remains unknown. We address this knowledge gap using extensive genomic, community, and crop datasets to examine relationships among land use, pollinator phylogenetic structure, and crop production. Pollinator communities in highly agricultural landscapes contain 230 million fewer years of evolutionary history; this loss was strongly associated with reduced crop yield and quality. Our study links landscape-mediated changes in the phylogenetic structure of natural communities to the disruption of ecosystem services. Measuring conservation success by species counts alone may fail to protect ecosystem functions and the full diversity of life from which they are derived.

Combining transcriptomes and ultraconserved elements to illuminate the phylogeny of Apidae.

Two increasingly popular approaches to reconstruct the Tree of Life involve whole transcriptome sequencing and the target capture of ultraconserved elements (UCEs). Both methods can be used to generate large, multigene datasets for analysis of phylogenetic relationships in non-model organisms. While targeted exon sequencing across divergent lineages is now a standard method, it is still not clear if UCE data can be readily combined with published transcriptomes. In this study, we evaluate the combination of UCEs and transcriptomes in a single analysis using genome-, transcriptome-, and UCE data for 79 bees in the largest and most biologically diverse bee family, Apidae. Using existing tools, we first developed a workflow to assemble phylogenomic data from different sources and produced two large nucleotide matrices of combined data. We then reconstructed the phylogeny of the Apidae using concatenation- and coalescent-based methods, and critically evaluated the resulting phylogenies in the context of previously published genetic, genomic, and morphological data sets. Our estimated phylogenetic trees are robustly supported and largely congruent with previous molecular hypotheses, from deep nodes to shallow species-level phylogenies. Moreover, the combined approach allows us to resolve controversial nodes of the apid Tree of Life, by clarifying the relationships among the genera of orchid bees (Euglossini) and the monophyly of the Centridini. Additionally, we present novel phylogenetic evidence supporting the monophyly of the diverse clade of cleptoparasitic Apidae and the placement of two enigmatic, oil-collecting genera (Ctenoplectra and Tetrapedia). Lastly, we propose a revised classification of the family Apidae that reflects our improved understanding of apid higher-level relationships.

Viral transmission in honey bees and native bees, supported by a global black queen cell virus phylogeny.

In recent decades, we have realized that honey bee viruses are not, in fact, exclusive to honey bees. The potential impact of Apis-affiliated viruses on native pollinators is prompting concern. Our research addresses the issue of virus crossover between honey bees and native bees foraging in the same localities. We measured the presence of black queen cell virus (BQCV), deformed wing virus (DWV) and sacbrood virus (SBV) in managed Apis mellifera (honey bees) and native Andrena spp. (subgenus Melandrena) bee populations in five commercial orchards. We identified viral presence across sites and bees and related these data to measures of bee community diversity. All viruses were found in both managed and native bees, and BQCV was the most common virus in each. To establish evidence for viral crossover between taxa, we undertook an additional examination of BQCV where 74 samples were sequenced and placed in a global phylogenic framework of hundreds of BQCV strains. We demonstrate pathogen sharing across managed honey bees and distantly related wild bees. This phylogenetic analysis contributes to growing evidence for host switching and places local incidence patterns in a worldwide context, revealing multispecies viral transmission.