Alpha and beta diversity
All statistical analyses were performed using R v.4.1.0. We examined
rarefaction curves to assess if sampling depth was adequate and all
samples plateaued (Supplementary Figure S2). Alpha diversity was
quantified using Chao1 as an estimate of species richness, and Shannon
diversity which also accounts for evenness of ASVs in a sample. To
determine if host species, tissue type, their interactions, or sex and
foraging type significantly affect diversity, a linear mixed effect
model was constructed using lmer (Bates et al. 2011), with
separate models for Chao1 and Shannon diversity. In both models,
individual bee was included as a random effect and p-values were
calculated using the lmerTest package (Kuznetsova et al. 2017).
To examine drivers of species composition among samples, we first
examined if predictor variables, including species, tissue, geographic
location, or sex contributed significantly to differences in species
composition, using permutational ANOVA (PERMANOVA) using adonis2and assessed variation in dispersion among groups usingbetadisper (Oksanen et al. 2012). We present results using
Bray-Curtis dissimilarities in the main text, but results are nearly
identical to those based on weighted Unifrac dissimilarities (both
presented in Supplementary Table S1). Differences between GI tract
tissues were confirmed using PERMANOVA on a subset of the data
containing only bees from which both the crop and gut sequenced
successfully.
Because not all species of bee were sampled at each site, we conducted
additional analyses to validate detected species and geographic effects
in our dataset using stratified PERMANOVA. First, we subset the dataset
to only samples collected in Davis, where both bee species were sampled
and examined effects of bee species. Second, we examined the effect of
sampling location separately for each bee species. In both cases, we
used PERMANOVA implemented in adonis2 .
Additionally, to examine if bees caught foraging or nesting differed in
bacterial diversity or composition, we repeated all alpha and beta
diversity analyses on only X. sonorina samples, the only bee
species for which we had nesting and foraging individuals. Models
included sampling location, tissue, behavior and sex.