Associations between geography, host genetics, and the skin
microbiome
There were 845 microeukaryotic OTUs and 303 bacterial OTUs recovered
across all samples after filtering and rarefaction. Microeukaryotic
diversity was positively correlated with bacterial diversity across all
samples (number of OTUs; Spearman’s ρ = 0.25, p < 0.001).
Mantel tests revealed significant positive associations between
geographic distance and beta diversity in both eukaryotic and bacterial
microbes: populations that were geographically closer showed
significantly more similar microbiome community structure (eukaryotic
taxa: r = 0.18, p < 0.05; bacterial taxa: r = 0.40, p
< 0.01, Fig. 2). Neither MHC IIB genetic distance nor neutral
genetic distance (from microsatellite data published in Duryea et al.
2015) were associated with microbiome community structure (p
> 0.1 for all Mantel tests between 18S or 16S beta
diversity and FST matrices for MHC IIB or
microsatellites).
In the 16S bacterial dataset, Proteobacteria were dominant across all
samples, both by number of OTUs and sequence reads (Fig. 3A&B).
Proteobacteria also formed the core bacterial microbiome across samples
(Fig. 3C). Among the eukaryotic microbiota, fungi were dominant by both
number of OTUs and sequence reads (Fig. 3D&E). No core group of
eukaryotic taxa was recovered, though some fungal OTUs were found in
approximately 50% of samples (Fig. 3F). These common fungal OTUs
included Ascomycota, Basidiomycota, and unidentified fungi.
Bacteria diversity was similar across site types (Mann-Whitney tests, p
> 0.05), but island frogs had fewer eukaryotic OTUs than
mainland frogs (85.5 OTUs on average on islands vs. 110.5 on average on
the mainland; Mann-Whitney test, U = 2,604, p = 0.001). However, site
type was associated with variation in composition of both bacteria and
microeukaryotes (Fig. 4A&B). Eight bacterial groups were significantly
associated with site type: Cyanobacteria and Proteobacteria were
statistically associated with coastal mainland sites, while six
bacterial groups were statistically associated with island sites. Among
the microeukaryotes, Rhizaria, Nucleariids, Ichthyosporeans, and
Apusozoans were statistically associated with coastal sites while Fungi
and Apicomplexans were statistically associated with island sites.
While the number of bacterial OTUs was not significantly different
between MHC IIB homozygotes and heterozygotes (t-test, p >
0.05), MHC IIB homozygotes possessed fewer microeukaryote OTUs than MHC
IIB heterozygotes (16.6 on average on homozygotes vs. 29.7 on
heterozygotes; Mann-Whitney U, U = 3,729.5, p < 0.01).
However, this result could be confounded by differences across island
and mainland sites: both microbial community composition (Fig. 4A&B)
and the number of MHC IIB heterozygotes and homozygotes (Table 1) vary
across site types. Therefore, the analysis of microeukaryote diversity
against MHC genotypes was repeated on a subset of the data only
including individuals from mainland sites. MHC IIB homozygotes possessed
significantly fewer microeukaryote OTUs than heterozygotes when only
mainland frogs were included in the analysis (19.2 on average on
homozygotes vs. 39.1 on heterozygotes; Mann-Whitney U, U = 681.5, p
< 0.01).
Microbiome community composition varied between MHC IIB heterozygotes
and homozygotes for both bacteria and microeukaryotes when compared with
null expectations based on genotype randomizations. MHC IIB
heterozygotes hosted significantly more unidentified Bacteria,
Bacteroidetes, and Firmicutes, but fewer Actinobacteria and
Proteobacteria OTUs than homozygotes (Fig. 4C). In terms of
microeukaryotes, MHC IIB heterozygotes hosted significantly more OTUs
belonging to the Ciliates, Rhizaria, and Stramenopiles, but
significantly fewer Fungi and Algae OTUs than homozygotes (Fig. 4D).