Introduction
Gut microbial communities can be important mediators of host health and fitness (McFall-Ngai et al. 2013). Many bee species (Hymenoptera: Apoidea: Anthophila) host distinct and functionally important bacterial communities in the GI tract (Martinson et al. 2011, Lee et al. 2015, Moran 2015, Engel et al. 2016). As a low-diversity and tractable experimental system, the bee gut represents an excellent model to examine metabolic specialization, function and coexistence within microbial communities (Engel et al. 2016). However, bee species vary in microbiome composition, including the presence of specialized taxa and the relative abundance of environmental bacteria (Kwong et al. 2017, McFrederick et al. 2017). The factors that predict this variation among species and their functions remain poorly understood (Engel et al. 2016), but sociality has been proposed as an important driver of gut microbiome evolution for bees, like other macroorganisms (Moeller et al. 2016, Kwong et al. 2017, Moran et al. 2019).
Social corbiculate bees in the subfamily Apinae (‘pollen basket’ bees), including honey bees and bumble bees, are characterized by distinctive gut microbial communities that are relatively consistent among individuals within a species (Kwong et al. 2017). In honey bees and bumble bees, gut bacterial communities are consistent among individuals and transmitted by social interactions (Koch and Schmid-Hempel 2011, Powell et al. 2014, Billiet et al. 2017). By contrast, in non-social bee species, including those closely related to social corbiculates (McFrederick and Rehan 2019), individuals host more variable and less distinctive microbiomes, likely driven by environmental rather than social acquisition of microbes (McFrederick et al. 2012, McFrederick and Rehan 2019, Cohen et al. 2020). However, key tests of the sociality hypothesis using bee species in the genus Halictus (which contains solitary and social species) found limited influence of sociality on bacterial composition (McFrederick et al. 2014, Rubin et al. 2018). These results raise the possibility that other traits instead of or in addition to sociality may be more important in shaping microbiome composition and specialization among bees.
Carpenter bee species in the genus Xylocopa (Apidae: Xylocopini) offer a unique system to study the relative role of sociality in structuring microbiome composition. Xylocopa are large-bodied bees and close phylogenetic relatives of social corbiculate apids (Bossert et al. 2019). Xylocopa are locally common and economically important pollinators in some systems (Keasar 2010, Giannini et al. 2015), and nest in timber or dead stalks of plants (Barrows 1980). Of particular note, several species of carpenter bees have been characterized as facultatively or incipiently social (Gerling et al. 1989, Michener 1990). In characterized species, the oldest female in a nest maintains reproductive dominance and feeds younger nestmates via trophallaxis (Lucia et al. 2015), but cooperative brood care is rarely documented. One species in which sociality has been well-studied is X. sonorina , which lives in small, fluid, dominance-based societies with reproductive division of labor, where the proportion of individuals nesting socially is temporally dynamic (Ostwald et al. 2020). Moreover, in all of the best-studied Xylocopa species, both social and solitary nests are present within the same population (Gerling et al. 1989). Investigation of the microbiome of carpenter bees—close relatives of corbiculates with contrasting social structure– may offer insights into the role of sociality in the evolution of the bee microbiome.
Here, we examine the composition of bacterial full-length 16S rRNA genes in Xylocopa sonorina Smith [previously X. varipunctaPatton] and Xylocopa tabaniformis orpifex Smith (Bezark 2013), two carpenter bee species common in the western North America. We leverage PacBio Sequencing and a sample inference method with single-nucleotide resolution (Callahan et al. 2019) to examine strain-level resolution and phylogenetic relationships to previously characterized bee microbial taxa. These Xylocopa species often co-occur locally, collect nectar and pollen from the same plant hosts, and show a similar seasonal phenology in activity and reproduction. We examined microbiome composition in two tissues, the crop (foregut) and gut (combined midgut and hindgut), which are disparate in function and separated by a proventricular valve affecting movement between these regions. We hypothesized that the crop would be variable in bacterial composition among individuals, due to frequent intake of food including pollen, low microbial biomass and predominance of environmentally-sourced microbes, (Anderson et al. 2013). We hypothesized that if Xylocopa is similar to social apids, the gut would host a core microbiome distinctive from the crop that was consistent among individuals (Moran 2015), or if Xylocopamicrobiomes resemble solitary bees sampled to date, the gut would host a variable microbiome with high similarity to the crop (Voulgari-Kokota et al. 2019).
To address these hypotheses, we sampled bees from three geographic locations and first compared how bacterial alpha and beta-diversity differ among tissues, species and geographic locations. We also examined if sex or foraging status was associated with bacterial composition. Next, we defined the core bacterial taxa for these species and examined phylogenetic patterns among strains within these core clades. Lastly, to examine if shorter regions of the 16S rRNA gene could also detect these patterns, we repeated analyses using the full-length data that had been trimmed to the V4 region only. Our results suggest that carpenter bees host distinctive gut bacterial communities including bacterial clades previously detected in corbiculates. In addition, Xylocopaspecies host phylogenetically and geographically distinct lineages within core clades which are revealed by the full-length 16S but not by the V4 region alone.