Quantification and statistical analysis
Alpha diversity was estimated by different indices including Shannon,
Inverse Simpson, Observed Species, and Phylogenetic Index on raw count
ASV table after filtering out contaminants using Phyloseq R package .
The significance of diversity differences was tested with one-way ANOVA
with Tukey’s post hoc test including morphotype and habitat as variables
(Indices ~ morphotype * habitat). To estimate beta
diversity across samples, we computed Bray-Curtis, Unweighted Unifrac ,
and Jaccard distance using the vegan v2.6-2 and Phyloseq R package. We
visualized differences across samples using Principal Coordinate
Analysis (PCoA) ordination. Variation in microbial diversity was
assessed with permutational multivariate analyses of variance (adonis in
R) with population and location as factors using 9999 permutations for
significance testing. To generate the microbiota dendrogram we collapsed
raw ASV table counts from laboratory samples by host morphotype identity
to get representative microbiota profiles using microbiome R package .
We calculated unweighted unifrac distances for each table and the
matrices were Unweighted Pair group Method with Arithmetic mean (UPGMA)
clustered to generate dendrograms of interspecific relatedness using
phangorn v 2.9.0 and ggtree packages. All analyses were conducted in the
R environment . Data were plotted using ggplot2 R package.