Discussion
Our results demonstrate that the Xylocopa species examined here, although incipiently social, host a microbiome that exhibits similarities to previously characterized social corbiculates in at least two ways. First, Xylocopa individuals host a specific and distinctive set of bacterial taxa found consistently in bees that span geographic sampling locations, sexes, and individuals displaying different behaviors (nest-caught vs. foraging), unlike most previously characterized solitary bees (Voulgari-Kokota et al. 2019). Second, many of the bacterial lineages detected at high abundance in the gut have been previously described in social corbiculates (Kwong et al. 2017). Below, we discuss the specific taxa found and the implications for the effect of sociality on the evolution of the bee microbiome.
The bacterial taxa that we describe here are comprised of primarily bee-associated taxa or those that associate with diverse insects, similar to the previously characterized Xylocopa tenuiscapa(Subta et al. 2020). Xylocopa gut samples containedBombilactobacillus (previously Lactobacillus Firm4),Bombiscardovia ( Bifidobacteriaceae), and Lactobacillus bxid5692 (similar to Lactobacillus Firm5) in nearly every individual, often with multiple co-occurring ASVs, and typically at high abundance, similar to previously characterized Apis andBombus species (Kwong et al. 2017). Bombilactobacillus andBombiscardovia are considered core symbionts of adult bumble bees (Killer et al. 2010, Kwong et al. 2017, Parmentier et al. 2018) and primarily transmitted via social contact (Billiet et al. 2017). In addition, the full-length Bombiscardovia sequences inXylocopa are diverse and cluster with B. coagulansisolated from Bombus , or closely related to the newly describedBifidobacterium xylocopae and B. actinocoloniiforme from European Xylocopa sp; suggesting that this bacterial group has a larger host range than previously recognized (Kwong et al. 2017, Alberoni et al. 2019, Hammer et al. 2021). In Apis mellifera ,Bombilactobacillus and Bifidobacterium sp. colonize the hindgut and are implicated in saccharide breakdown and fermentation (Lee et al. 2015). Despite similarities to the A. mellifera andBombus microbiome, the Xylocopa microbiomes characterized here are also distinct in the consistent presence of Apibacter , which is found consistently in Asian honey bees (Kwong et al. 2018) but only sporadically in Bombus. In contrast to Bombus ,Xylocopa appears to mostly lack Snodgrassella— we detected a single ASV in low abundance. Gilliamella was frequently detected, but differed between species: X. sonorinahosted Gilliamella more consistently and at higher abundance in than did X. tabaniformis (Figure 2). In addition, the gut of bothXylocopa species consistently contained Entomomonas at relatively high frequencies (Supplementary Figure 6), a bacterium with a highly reduced genome and limited metabolic capabilities which was previously described in a diverse group of insects including Diptera, Coleoptera, and other Hymenoptera, including ants andApis (Wang et al. 2020). The ecological role of this genus is not understood. In X. sonorina , the crop was highly dominated byApilactobacillus (Supplementary Figures S5a; S6a),previously documented in solitary bees and in the crop of social bees (McFrederick et al. 2018). Apilactobacillus may dominate in the provisions of some bee species where they have been hypothesized to inhibit pathogen growth or prevent spoilage of stored pollen (Vásquez and Olofsson 2009, McFrederick et al. 2018, Kapheim et al. 2021).
Since Xylocopa are incipiently social rather than classically eusocial (Gerling et al. 1981), their distinctive microbiome raises questions about the mechanisms required for effective social transmission of the microbiome. Dominant Xylocopa females will feed newly emerged nestmates via trophallaxis, and allow consumption of the stored provision (Gerling et al. 1989, Ostwald et al. 2021, Vickruck and Richards 2021). In this way, Xylocopa exhibit behavioral similarities to Apis , which engage in trophallaxis, andBombus , which do not engage in trophallaxis yet feed from shared food resources and engage in coprophagy (Näpflin and Schmid-Hempel 2016). In addition, Xylocopa individuals migrate among nests, including those of non-kin (Ostwald et al. 2021, Vickruck and Richards 2021). We hypothesize that these behaviors, as well as the relatively long lifespan (1-2 years), and large body size of Xylocopa could help explain the maintenance of specialized microbial taxa. Differences between Xylocopa species in microbiome composition and richness also suggest areas for future study. Although we lack details onX. tabaniformis sociality, this species is likely incipiently social like X. sonorina (Breed 1976) yet differs in breeding systems: males patrol flowers for mating, while X. sonorina males host non-resource based territories (Marshall and Alcock 1981). This could explain the differences in sex-specific microbiomes between species. Additional information on social structure or nesting biology may inform reduced sequence variant richness and more variable crop community in X. tabaniformis compared to X. sonorina .
In previous work, the microbiome of halictid bees that nest socially, including those that exhibit trophallaxis and eusociality (Kapheim et al. 2016), did not differ substantially from those that nest non-socially (McFrederick et al. 2014, Rubin et al. 2018), and resembled solitary bee microbiomes (Voulgari-Kokota et al. 2019), which are primarily environmentally acquired (McFrederick et al. 2012, Kapheim et al. 2021). The primary difference between solitary and social halictids was the abundance of Sodalis , an insect endosymbiont (Rubin et al. 2018). Combined, these studies suggest that not only social behaviors but perhaps additional other biological differences may be required for the maintenance of a distinctive bee microbiome.
Both Xylocopa species displayed microbiome differences among collection locations, suggesting population-level differentiation in microbiome composition. Indeed, many ASVs of core taxa were location-specific (Supplementary Figures 7, 8, 9), while only a few dominant ASVs (e.g. Lactobacillus bxid5692 ASV 9) was detected at all locations in both bee species. Sampling location explained between 9-16% of variation in bacterial composition, more than previous studies examining geographic signatures in honey bees (Ge et al. 2021) stingless bees (Liu et al. 2021), and even some solitary Osmia (Rothman et al. 2020). Although long-read sequences likely enable us to detect such patterns (Supplementary Table 1), distinctive Xylocopa sociality and patterns of microbial transmission may also contribute to geographic structuring.
As expected, we found that restricting our analysis to the V4 region resulted in fewer taxa detected, a loss of phylogenetic resolution (Supplementary Figure S7), and the loss of genetic information that could distinguish bacterial taxa between host species and among geographic locations (Supplementary Table S4). Due to reduced sequence length, ASVs that previously distinguished species and locations were collapsed into a single ASV (Supplementary Figure S 9), yet species and geographic location could still largely be distinguished using V4 sequences only (Supplementary Table S1). This comparison suggests that first-generation sequencing may be able to detect drivers of microbial community composition, but fail to show the extent of strain-level differentiation that exists among populations and species. However, we caution that our comparative approach does not account for realistic primer bias or sequencing bias (Quail et al. 2012, Tedersoo et al. 2018) and as a result, may overestimate the similarity of these regions and their ecological inference. Nevertheless our data support the conclusion that long reads enable enhanced ecological insights into the strain-level composition and evolution of the microbiome, suggesting that despite its greater cost, this approach may be warranted when strain-level information may differentiate populations or closely related species (Tedersoo et al. 2021).
Overall, our results provide evidence that the microbiomes of species with simple social groups can have characteristics typically associated with the more complex eusociality of the corbiculate bees. Further work will be necessary to determine the role that specific features of sociality, such as trophallaxis, play in shaping the microbiome in the earliest stages of social evolution, and uncover the functional consequences of a specialized microbiome.