Phylogenomics and biogeography of the small carpenter bees (Apidae: Xylocopinae: Ceratina).

Small carpenter bees in the genus Ceratina are behaviourally diverse, species-rich, and cosmopolitan, with over 370 species and a range including all continents except Antarctica. Here, we present the first comprehensive phylogeny of the genus based on ultraconserved element (UCE) phylogenomic data, covering a total of 185 ingroup specimens representing 22 of the 25 current subgenera. Our results support most recognized subgenera as natural groups, but we also highlight several groups in need of taxonomic revision - particularly the nominate subgenus Ceratina sensu stricto - and several clades that likely need to be described as new subgenera. In addition to phylogeny, we explore the evolutionary history of Ceratina through divergence time estimation and biogeographic reconstruction. Our findings suggest that Ceratinini split from its sister tribe Allodapini about 72 million years ago. The common ancestor of Ceratina emerged in the Afrotropical realm approximately 42 million years ago, near the Middle Eocene Climatic Optimum. Multiple subsequent dispersal events led to the present cosmopolitan distribution of Ceratina, with the majority of transitions occurring between the Afrotropics, Indomalaya, and the Palearctic. Additional movements also led to the arrival of Ceratina in Madagascar, Australasia, and a single colonization of the Americas. Dispersal events were asymmetrical overall, with temperate regions primarily acting as destinations for migrations from tropical source regions.

Microbiome and floral associations of a wild bee using biodiversity survey collections.

The health of bees can be assessed through their microbiome, which serves as a biomarker indicating the presence of both beneficial and harmful microorganisms within a bee community. This study presents the characterisation of the bacterial, fungal, and plant composition on the cuticle of adult bicoloured sweat bees (Agapostemon virescens). These bees were collected using various methods such as pan traps, blue vane traps and sweep netting across the northern extent of their habitat range. Non-destructive methods were employed to extract DNA from the whole pinned specimens of these wild bees. Metabarcoding of the 16S rRNA, ITS and rbcL regions was then performed. The study found that the method of collection influenced the detection of certain microbial and plant taxa. Among the collection methods, sweep net samples showed the lowest fungal alpha diversity. However, minor differences in bacterial or fungal beta diversity suggest that no single method is significantly superior to others. Therefore, a combination of techniques can cater to a broader spectrum of microbial detection. The study also revealed regional variations in bacterial, fungal and plant diversity. The core microbiome of A. virescens comprises two bacteria, three fungi and a plant association, all of which are commonly detected in other wild bees. These core microbes remained consistent across different collection methods and locations. Further extensive studies of wild bee microbiomes across various species and landscapes will help uncover crucial relationships between pollinator health and their environment.

Gene regulation supporting sociality shared across lineages and variation in complexity.

Across evolutionary lineages, insects vary in social complexity, from those that exhibit extended parental care to those with elaborate divisions of labor. Here, we synthesize the sociogenomic resources from hundreds of species to describe common gene regulatory mechanisms in insects that regulate social organization across phylogeny and levels of social complexity. Different social phenotypes expressed by insects can be linked to the organization of co-expressing gene networks and features of the epigenetic landscape. Insect sociality also stems from processes like the emergence of parental care and the decoupling of ancestral genetic programs. One underexplored avenue is how variation in a group's social environment affects the gene expression of individuals. Additionally, an experimental reduction of gene expression would demonstrate how the activity of specific genes contributes to insect social phenotypes. While tissue specificity provides greater localization of the gene expression underlying social complexity, emerging transcriptomic analysis of insect brains at the cellular level provides even greater resolution to understand the molecular basis of social insect evolution.

Wild bee and pollen microbiomes across an urban-rural divide.

Wild pollinators and their microbiota are sensitive to land use changes from anthropogenic activities that disrupt landscape and environmental features. As urbanization and agriculture affect bee habitats, human-led disturbances are driving changes in bee microbiomes, potentially leading to dysbiosis detrimental to bee fitness. This study examines the bacterial, fungal, and plant compositions of the small carpenter bee, Ceratina calcarata, and its pollen provisions across an urban-rural divide. We performed metabarcoding of C. calcarata and provisions in Toronto by targeting the 16S rRNA, ITS, and rbcL regions. Despite similar plant composition and diversity across bees and their provisions, there was a greater microbial diversity in pollen provisions than in bees. By characterizing the differences in land use, climate, and pesticide residues that differentiate urban and rural landscapes, we find that urban areas support elevated levels of microbial diversity and more complex networks between microbes and plants than rural areas. However, urban areas may lead to lower relative abundances of known beneficial symbionts and increased levels of pathogens, such as Ascosphaera and Alternaria fungi. Further, rural pollen provisions indicate elevated pesticide residues that may dysregulate symbiosis. As anthropogenic activities continue to alter land use, ever changing environments threaten microbiota crucial in maintaining bee health.

The effects of maternal care on the developmental transcriptome and metatranscriptome of a wild bee.

Maternal care acts as a strong environmental stimulus that can induce phenotypic plasticity in animals and may also alter their microbial communities through development. Here, we characterize the developmental metatranscriptome of the small carpenter bee, Ceratina calcarata, across developmental stages and in the presence or absence of mothers. Maternal care had the most influence during early development, with the greatest number and magnitude of differentially expressed genes between maternal care treatments, and enrichment for transcription factors regulating immune response in motherless early larvae. Metatranscriptomic data revealed fungi to be the most abundant group in the microbiome, with Aspergillus the most abundant in early larvae raised without mothers. Finally, integrative analysis between host transcriptome and metatranscriptome highlights several fungi correlating with developmental and immunity genes. Our results provide characterizations of the influence of maternal care on gene expression and the microbiome through development in a wild bee.

Brain Gene Expression of Foraging Behavior and Social Environment in Ceratina calcarata.

Rudimentary social systems have the potential to both advance our understanding of how complex sociality may have evolved and our understanding of how changes in social environment may influence gene expression and cooperation. Recently, studies of primitively social Hymenoptera have greatly expanded empirical evidence for the role of social environment in shaping behavior and gene expression. Here, we compare brain gene expression profiles of foragers across social contexts in the small carpenter bee, Ceratina calcarata. We conducted experimental manipulations of field colonies to examine gene expression profiles among social contexts including foraging mothers, regular daughters, and worker-like dwarf eldest daughters in the presence and absence of mother. Our analysis found significant differences in gene expression associated with female age, reproductive status, and social environment, including circadian clock gene dyw, hexamerin, and genes involved in the regulation of juvenile hormone and chemical communication. We also found that candidate genes differentially expressed in our study were also associated with division of labor, including foraging, in other primitively and advanced eusocial insects. Our results offer evidence for the role of the regulation of key developmental hormones and circadian rhythms in producing cooperative behavior in rudimentary insect societies.

The impact of geography and climate on the population structure and local adaptation in a wild bee.

Deciphering processes that contribute to genetic differentiation and divergent selection of natural populations is useful for evaluating the adaptive potential and resilience of organisms faced with various anthropogenic stressors. Insect pollinator species, including wild bees, provide critical ecosystem services but are highly susceptible to biodiversity declines. Here, we use population genomics to infer the genetic structure and test for evidence of local adaptation in an economically important native pollinator, the small carpenter bee (Ceratina calcarata). Using genome-wide SNP data (n = 8302), collected from specimens across the species' entire distribution, we evaluated population differentiation and genetic diversity and identified putative signatures of selection in the context of geographic and environmental variation. Results of the analyses of principal component and Bayesian clustering were concordant with the presence of two to three genetic clusters, associated with landscape features and inferred phylogeography of the species. All populations examined in our study demonstrated a heterozygote deficit, along with significant levels of inbreeding. We identified 250 robust outlier SNPs, corresponding to 85 annotated genes with known functional relevance to thermoregulation, photoperiod, and responses to various abiotic and biotic stressors. Taken together, these data provide evidence for local adaptation in a wild bee and highlight genetic responses of native pollinators to landscape and climate features.

Integrative population genetics and metagenomics reveals urbanization increases pathogen loads and decreases connectivity in a wild bee.

As urbanization continues to increase, it is expected that two-thirds of the human population will reside in cities by 2050. Urbanization fragments and degrades natural landscapes, threatening wildlife including economically important species such as bees. In this study, we employ whole genome sequencing to characterize the population genetics, metagenome and microbiome, and environmental stressors of a common wild bee, Ceratina calcarata. Population genomic analyses revealed the presence of low genetic diversity and elevated levels of inbreeding. Through analyses of isolation by distance, resistance, and environment across urban landscapes, we found that green spaces including shrubs and scrub were the most optimal pathways for bee dispersal, and conservation efforts should focus on preserving these land traits to maintain high connectivity across sites for wild bees. Metagenomic analyses revealed landscape sites exhibiting urban heat island effects, such as high temperatures and development but low precipitation and green space, had the highest taxa alpha diversity across all domains even when isolating for potential pathogens. Notably, the integration of population and metagenomic data showed that reduced connectivity in urban areas is not only correlated with lower relatedness among individuals but is also associated with increased pathogen diversity, exposing vulnerable urban bees to more pathogens. Overall, our combined population and metagenomic approach found significant environmental variation in bee microbiomes and nutritional resources even in the absence of genetic differentiation, as well as enabled the potential early detection of stressors to bee health.

Environmental Effects on Bee Microbiota.

Anthropogenic activities and increased land use, which include industrialization, agriculture and urbanization, directly affect pollinators by changing habitats and floral availability, and indirectly by influencing their microbial composition and diversity. Bees form vital symbioses with their microbiota, relying on microorganisms to perform physiological functions and aid in immunity. As altered environments and climate threaten bees and their microbiota, characterizing the microbiome and its complex relationships with its host offers insights into understanding bee health. This review summarizes the role of sociality in microbiota establishment, as well as examines if such factors result in increased susceptibility to altered microbiota due to environmental changes. We characterize the role of geographic distribution, temperature, precipitation, floral resources, agriculture, and urbanization on bee microbiota. Bee microbiota are affected by altered surroundings regardless of sociality. Solitary bees that predominantly acquire their microbiota through the environment are particularly sensitive to such effects. However, the microbiota of obligately eusocial bees are also impacted by environmental changes despite typically well conserved and socially inherited microbiota. We provide an overview of the role of microbiota in plant-pollinator relationships and how bee microbiota play a larger role in urban ecology, offering microbial connections between animals, humans, and the environment. Understanding bee microbiota presents opportunities for sustainable land use restoration and aiding in wildlife conservation.

Molecular patterns and processes in evolving sociality: lessons from insects.

Social insects have provided some of the clearest insights into the origins and evolution of collective behaviour. Over 20 years ago, Maynard Smith and Szathmáry defined the most complex form of insect social behaviour-superorganismality-among the eight major transitions in evolution that explain the emergence of biological complexity. However, the mechanistic processes underlying the transition from solitary life to superorganismal living in insects remain rather elusive. An overlooked question is whether this major transition arose via incremental or step-wise modes of evolution. We suggest that examination of the molecular processes underpinning different levels of social complexity represented across the major transition from solitary to complex sociality can help address this question. We present a framework for using molecular data to assess to what extent the mechanistic processes that take place in the major transition to complex sociality and superorganismality involve nonlinear (implying step-wise evolution) or linear (implying incremental evolution) changes in the underlying molecular mechanisms. We assess the evidence for these two modes using data from social insects and discuss how this framework can be used to test the generality of molecular patterns and processes across other major transitions. This article is part of a discussion meeting issue 'Collective behaviour through time'.

Co-expression Gene Networks and Machine-learning Algorithms Unveil a Core Genetic Toolkit for Reproductive Division of Labour in Rudimentary Insect Societies.

The evolution of eusociality requires that individuals forgo some or all their own reproduction to assist the reproduction of others in their group, such as a primary egg-laying queen. A major open question is how genes and genetic pathways sculpt the evolution of eusociality, especially in rudimentary forms of sociality-those with smaller cooperative nests when compared with species such as honeybees that possess large societies. We lack comprehensive comparative studies examining shared patterns and processes across multiple social lineages. Here we examine the mechanisms of molecular convergence across two lineages of bees and wasps exhibiting such rudimentary societies. These societies consist of few individuals and their life histories range from facultative to obligately social. Using six species across four independent origins of sociality, we conduct a comparative meta-analysis of publicly available transcriptomes. Standard methods detected little similarity in patterns of differential gene expression in brain transcriptomes among reproductive and non-reproductive individuals across species. By contrast, both supervised machine learning and consensus co-expression network approaches uncovered sets of genes with conserved expression patterns among reproductive and non-reproductive phenotypes across species. These sets overlap substantially, and may comprise a shared genetic "toolkit" for sociality across the distantly related taxa of bees and wasps and independently evolved lineages of sociality. We also found many lineage-specific genes and co-expression modules associated with social phenotypes and possible signatures of shared life-history traits. These results reveal how taxon-specific molecular mechanisms complement a core toolkit of molecular processes in sculpting traits related to the evolution of eusociality.

The effects of urban land use gradients on wild bee microbiomes.

Bees and their microbes interact in complex networks in which bees form symbiotic relationships with their bacteria and fungi. Microbial composition and abundance affect bee health through nutrition, immunity, and fitness. In ever-expanding urban landscapes, land use development changes bee habitats and floral resource availability, thus altering the sources of microbes that wild bees need to establish their microbiome. Here, we implement metabarcoding of the bacterial 16S and fungal ITS regions to characterize the diversity and composition of the microbiome in 58 small carpenter bees, Ceratina calcarata, across urban land use gradients (study area 6,425 km2). By categorizing land use development, green space, precipitation, and temperature variables as indicators of habitat across the city, we found that land use variables can predict microbial diversity. Microbial composition was also found to vary across urban land use gradients, with certain microbes such as Acinetobacter and Apilactobacillus overrepresented in less urban locations and Penicillium more abundant in developed areas. Environmental features may also lead to differences in microbe interactions, as co-occurrences between bacteria and fungi varied across percent land use development, exemplified by the correlation between Methylobacterium and Sphingomonas being more prevalent in areas of higher urban development. Surrounding landscapes change the microbial landscape in wild bees and alter the relationships they have with their microbiome. As such, urban centres should consider the impact of growing cities on their pollinators' health and protect wild bees from the effects of anthropogenic activities.

Comparative metagenomics reveals expanded insights into intra- and interspecific variation among wild bee microbiomes.

The holobiont approach proposes that species are most fully understood within the context of their associated microbiomes, and that both host and microbial community are locked in a mutual circuit of co-evolutionary selection. Bees are an ideal group for this approach, as they comprise a critical group of pollinators that contribute to both ecological and agricultural health worldwide. Metagenomic analyses offer comprehensive insights into an organism's microbiome, diet, and viral load, but remain largely unapplied to wild bees. Here, we present metagenomic data from three species of carpenter bees sampled from around the globe, representative of the first ever carpenter bee core microbiome. Machine learning, co-occurrence, and network analyses reveal that wild bee metagenomes are unique to host species. Further, we find that microbiomes are likely strongly affected by features of their local environment, and feature evidence of plant pathogens previously known only in honey bees. Performing the most comprehensive comparative analysis of bee microbiomes to date we discover that microbiome diversity is inversely proportional to host species social complexity. Our study helps to establish some of the first wild bee hologenomic data while offering powerful empirical insights into the biology and health of vital pollinators.

Reduced neural investment in post-reproductive females of the bee Ceratina calcarta.

Many insects show plasticity in the area of the brain called the mushroom bodies (MB) with foraging and social experience. MBs are paired neuropils associated with learning and memory. MB volume is typically greater in mature foragers relative to young and/or inexperienced individuals. Long-term studies show that extended experience may further increase MB volume, but long-term studies have only been performed on non-reproductive social insect workers. Here we use the subsocial bee Ceratina calcarata to test the effect of extended foraging experience on MB volume among reproductive females. Ceratina calcarata females forage to provision their immature offspring in the spring, and then again to provision their adult daughters in the late summer. We measured the volume of the MB calyces and peduncle, antennal lobes (AL), optic lobes (OL), central complex (CX), and whole brains of three groups of bees: newly emerged females, reproductive females in spring (foundresses), and post-reproductive mothers feeding their adult daughters in late summer. Post-reproductive late summer mothers had smaller MB calyces and ALs than foundresses. Moreover, among late mothers (but not other bees), wing wear, which is a measure of foraging experience, negatively correlated with both MB and OL volume. This is contrary to previously studied non-reproductive social insect workers in which foraging experience correlates postiviely with MB volume, and suggests that post-reproductive bees may reduce neural investment near the end of their lives.

Phylogenomics and historical biogeography of the cleptoparasitic bee genus Nomada (Hymenoptera: Apidae) using ultraconserved elements.

The genus Nomada Scopoli (Hymenoptera: Apidae) is the largest genus of brood parasitic bees with nearly 800 species found across the globe and in nearly all biogeographic realms except Antarctica. There is no previous molecular phylogeny focused on Nomada despite their high species abundance nor is there an existing comprehensive biogeography for the genus. Using ultraconserved element (UCE) phylogenomic data, we constructed the first molecular phylogeny for the genus Nomada and tested the monophyly of 16 morphologically established species groups. We also estimated divergence dates using fossil calibration points and inferred the geographic origin of this genus. Our phylogeny recovered 14 of the 16 previously established species groups as monophyletic. The superba and ruficornis groups, however, were recovered as non-monophyletic and need to be re-evaluated using morphology. Divergence dating and historic biogeographic analyses performed on the phylogenetic reconstruction indicates that Nomada most likely originated in the Holarctic âˆ¼ 65 Mya. Geodispersal into the southern hemisphere occurred three times: once during the Eocene into the Afrotropics, once during the Oligocene into the Neotropics, and once during the Miocene into Australasia. Geodispersal across the Holarctic was most frequent and occurred repeatedly throughout the Cenozoic era, using the De Geer, Thulean, and the Bering Land Bridges. This is the first instance of a bee using both the Thulean and De Geer land bridges and has implications of how early bee species dispersed throughout the Palearctic in the late Cretaceous and early Paleogene.

Social divergence: molecular pathways underlying castes and longevity in a facultatively eusocial small carpenter bee.

Unravelling the evolutionary origins of eusocial life is a longstanding endeavour in the field of evolutionary-developmental biology. Descended from solitary ancestors, eusocial insects such as honeybees have evolved ontogenetic division of labour in which short-lived workers perform age-associated tasks, while a long-lived queen produces brood. It is hypothesized that (i) eusocial caste systems evolved through the co-option of deeply conserved genes and (ii) longevity may be tied to oxidative damage mitigation capacity. To date, however, these hypotheses have been examined primarily among only obligately eusocial corbiculate bees. We present brain transcriptomic data from a Japanese small carpenter bee, Ceratina japonica (Apidae: Xylocopinae), which demonstrates both solitary and eusocial nesting in sympatry and lives 2 or more years in the wild. Our dataset captures gene expression patterns underlying first- and second-year solitary females, queens and workers, providing an unprecedented opportunity to explore the molecular mechanisms underlying caste-antecedent phenotypes in a long-lived and facultatively eusocial bee. We find that C. japonica's queens and workers are underpinned by divergent gene regulatory pathways, involving many differentially expressed genes well-conserved among other primitively eusocial bee lineages. We also find support for oxidative damage reduction as a proximate mechanism of longevity in C. japonica.

Tropical bee species abundance differs within a narrow elevational gradient.

Insect pollination is among the most essential ecosystem services for humanity. Globally, bees are the most effective pollinators, and tropical bees are also important for maintaining tropical biodiversity. Despite their invaluable pollination service, basic distributional patterns of tropical bees along elevation gradients are globally scarce. Here, we surveyed bees at 100 m elevation intervals from 800 to 1100 m elevation in Costa Rica to test if bee abundance, community composition and crop visitor assemblages differed by elevation. We found that 18 of 24 bee species spanning three tribes that represented the most abundantly collected bee species showed abundance differences by elevation, even within this narrow elevational gradient. Bee assemblages at the two crop species tested, avocado and squash, showed community dissimilarity between high and low elevations, and elevation was a significant factor in explaining bee community composition along the gradient. Stingless bees (Tribe Meliponini) were important visitors to both crop species, but there was a more diverse assemblage of bees visiting avocado compared to squash. Our findings suggest that successful conservation of tropical montane bee communities and pollination services will require knowledge of which elevations support the highest numbers of each species, rather than species full altitudinal ranges.

Conservation insights from wild bee genetic studies: Geographic differences, susceptibility to inbreeding, and signs of local adaptation.

Conserving bees are critical both ecologically and economically. Genetic tools are valuable for monitoring these vital pollinators since tracking these small, fast-flying insects by traditional means is difficult. By surveying the current state of the literature, this review discusses how recent advances in landscape genetic and genomic research are elucidating how wild bees respond to anthropogenic threats. Current literature suggests that there may be geographic differences in the vulnerability of bee species to landscape changes. Populations of temperate bee species are becoming more isolated and more genetically depauperate as their landscape becomes more fragmented, but tropical bee species appear unaffected. These differences may be an artifact of historical differences in land-use, or it suggests that different management plans are needed for temperate and tropical bee species. Encouragingly, genetic studies on invasive bee species indicate that low levels of genetic diversity may not lead to rapid extinction in bees as once predicted. Additionally, next-generation sequencing has given researchers the power to identify potential genes under selection, which are likely critical to species' survival in their rapidly changing environment. While genetic studies provide insights into wild bee biology, more studies focusing on a greater phylogenetic and life-history breadth of species are needed. Therefore, caution should be taken when making broad conservation decisions based on the currently few species examined.

Supporting Bees in Cities: How Bees Are Influenced by Local and Landscape Features.

Urbanization is a major anthropogenic driver of decline for ecologically and economically important taxa including bees. Despite their generally negative impact on pollinators, cities can display a surprising degree of biodiversity compared to other landscapes. The pollinating communities found within these environments, however, tend to be filtered by interacting local and landscape features that comprise the urban matrix. Landscape and local features exert variable influence on pollinators within and across taxa, which ultimately affects community composition in such a way that contributes to functional trait homogenization and reduced phylogenetic diversity. Although previous results are not easily generalizable, bees and pollinators displaying functional trait characteristics such as polylectic diet, cavity-nesting behavior, and later emergence appear most abundant across different examined cities. To preserve particularly vulnerable species, most notably specialists that have become underrepresented within city communities, green spaces like parks and urban gardens have been examined as potential refuges. Such spaces are scattered across the urban matrix and vary in pollinator resource availability. Therefore, ensuring such spaces are optimized for pollinators is imperative. This review examines how urban features affect pollinators in addition to ways these green spaces can be manipulated to promote greater pollinator abundance and diversity.