2.5 Statistical analysis
Statistical analysis was conducted on the ASV level of theclr -transformed 16S rRNA dataset. Prokaryotic community profiles
were constructed at the phylum level, and the relative abundance of
major phyla (> 0.1 % of the total microbial community) in
environmental samples was compared between sites by Analysis of
Compositions of Microbiomes with Bias Correction (ANCOM-BC) (Lin &
Peddada, 2020). Alpha diversity metrics including the abundance-based
coverage estimator (ACE), diversity (Shannon diversity index), and
evenness (Inverse Simpson’s diversity index) were computed. A comparison
between each of the alpha diversity indices by sites and source was
performed by two-way ANOVA followed by a post-hoc Tukey honestly
significant difference (Tukey HSD) test after ensuring data normality
and homogeneity with tests as above.
To address the community variation between samples collected from
different sites and sources, we adopted a permutational multivariate
analysis of variance (PERMANOVA) analysis and Analysis of Similarity
(ANOSIM) with 999 permutations to compare their compositions. A distance
matrix based on the ASVs was first constructed with the distancefunction in the “vegan” R package (Oksanen et al., 2022) with the
Euclidean distance (Gloor et al., 2017). Then, the Adonis method via theadonis function with 999 permutations was used for the comparison
of communities. Pairwise PERMANOVA was carried out withpairwise.adonis with the “pairwiseAdonis” package (Arbizu,
2023). Permutation tests for homogeneity of multivariate dispersions
(PERMDISP) were conducted using betadisper and permutestto verify significant PERMANOVA outcomes. Principal Component Analysis
(PCA) was used to visualize the beta diversity matrix in the
“phyloseq” package (McMurdie & Holmes, 2013).