Panamanian frog species host unique skin bacterial communities

Abstract

Vertebrates, including amphibians, host diverse symbiotic microbes that contribute to host disease resistance. Globally, and especially in montane tropical systems, many amphibian species are threatened by a chytrid fungus, Batrachochytrium dendrobatidis (Bd), that causes a lethal skin disease. Bd therefore may be a strong selective agent on the diversity and function of the microbial communities inhabiting amphibian skin. In Panamá, amphibian population declines and the spread of Bd have been tracked. In 2012, we completed a field survey in Panamá to examine frog skin microbiota in the context of Bd infection. We focused on three frog species and collected two skin swabs per frog from a total of 136 frogs across four sites that varied from west to east in the time since Bd arrival. One swab was used to assess bacterial community structure using 16S rRNA amplicon sequencing and to determine Bd infection status, and one was used to assess metabolite diversity, as the bacterial production of anti-fungal metabolites is an important disease resistance function. The skin microbiota of the three Panamanian frog species differed in OTU (operational taxonomic unit, ~bacterial species) community composition and metabolite profiles, although the pattern was less strong for the metabolites. Comparisons between frog skin bacterial communities from Panamá and the US suggest broad similarities at the phylum level, but key differences at lower taxonomic levels. In our field survey in Panamá, across all four sites, only 35 individuals (~26%) were Bd infected. There was no clustering of OTUs or metabolite profiles based on Bd infection status and no clear pattern of west-east changes in OTUs or metabolite profiles across the four sites. Overall, our field survey data suggest that different bacterial communities might be producing broadly similar sets of metabolites across frog hosts and sites. Community structure and function may not be as tightly coupled in these skin symbiont microbial systems as it is in many macro-systems.

Keywords: Batrachochytrium dendrobatidis; Kolmogorov-Smirnov measure; amphibian; chytrid fungus; microbiome; microbiota; structure-function relationship.

Figure 1

Map of Panamá showing sampled field sites (numbered circles) and the frog species sampled at each location (colored circles: red, Agalychnis callidryas; gold, Dendropsophus ebraccatus; blue, Craugastor fitzingeri) during a field survey assessing the diversity of bacterial communities on amphibian skin. The three species of amphibians we sampled have persisted with the fungal pathogen, Batrachochytrium dendrobatidis, for different lengths of time across the four sampled sites, numbered one to four from west to east (i.e., longest at Cerro Campana and shortest at Nuevo Vigia; Woodhams et al., ; Rebollar et al., 2014).

Figure 2

Example of how the K-S test statistic is determined and K-S measures plotted in descending order for species and zone analyses. (A) The relative abundance distributions of a single bacterial OTU across two groups: temperate amphibians (solid line) vs. tropical amphibians (long dash). (B) The K-S test statistic assesses the difference between the two distributions based on the largest absolute distance between empirical distribution functions (short dash). The empirical distribution function is an observed cumulative distribution function, where, in this case, the y-axis represents the proportion of observations less than or equal to a given relative abundance on the x-axis. (C) K-S measures for each OTU plotted in descending order for species analysis. (D) K-S measures for each OTU plotted in descending order for zone analysis.

Figure 3

Mean relative abundance of bacterial phyla present on the skin of (A) three frog species surveyed in Panamá in 2012 (Agalychnis callidryas, Dendropsophus ebraccatus, Craugastor fitzingeri; this study) and (B) a subset of individuals of each of the three frog species surveyed in Panamá compared to three frog species surveyed in Virginia, USA (Anaxyrus americanus, Lithobates catesbeianus, Pseudacris crucifer; Walke et al., ; Belden et al. unpublished data).

Figure 4

Beta diversity of bacterial communities (OTUs) (left column) and bacterially-produced metabolite profiles (right column) sampled from the skin of three frog species from Panamá, grouped by frog species (A,B, red, Agalychnis callidryas; gold, Dendropsophus ebraccatus; blue, Craugastor fitzingeri) and the frog's infection status (C,D, white, infected; black, not infected) by the fungal pathogen, Batrachochytrium dendrobatidis. NMDS ordinations are based on Bray–Curtis and Jaccard dissimilarities for OTUs and metabolites, respectively. Each point represents a single individual.

Figure 5

Relative abundance of 41 OTUs selected based on K-S measures that best defined the differences in skin bacterial community structure of three frog species from Panamá (left, Agalychnis callidryas; right, Dendropsophus ebraccatus; center, Craugastor fitzingeri). OTU relative abundances ranged from 0 to 0.32. Lighter shades indicate lower relative abundances (white relative abundance = 0) and darker shades indicate higher relative abundances (darkest relative abundance = 0.32). OTUs are ordered top to bottom based on K-S measures (see Table 2 for exact values and additional taxonomic information for each OTU).

Figure 6

Beta diversity of skin bacterial communities of tropical (circles) and temperate (diamonds) amphibians. Three species from each zone were included in the analysis [red, Agalychnis callidryas; gold, Dendropsophus ebraccatus; blue, Craugastor fitzingeri from Panamá (tropical zone); brown, Anaxyrus americanus; green, Lithobates catesbeianus; cream, Pseudacris crucifer from Virginia, USA (temperate zone)]. NMDS ordination based on Bray–Curtis dissimilarities. Each point represents a single individual.

Figure 7

Relative abundance of 31 OTUs selected based on K-S measures that best defined the differences in skin bacterial community structure of tropical (left) and temperate (right) amphibians. Three species from each zone were included in the analysis: Agalychnis callidryas, Dendropsophus ebraccatus, Craugastor fitzingeri from Panamá (tropical zone) and Anaxyrus americanus, Lithobates catesbeianus, Pseudacris crucifer from Virginia, USA (temperate zone). OTU relative abundances ranged from 0 to 0.37. Lighter shades indicate lower relative abundances (white, relative abundance = 0) and darker shades indicate higher relative abundances (darkest relative abundance = 0.37). OTUs are ordered top to bottom based on K-S measures (see Table 3 for exact values and additional taxonomic information for each OTU).

Figure 8

Alpha diversity of bacterial OTUs (A,B, richness; C,D, phylogenetic diversity; E,F, evenness) and bacterially-produced metabolites (G,H, richness) sampled from the skin of Agalychnis callidryas (left column) and Dendropsophus ebraccatus (right column). Both species were sampled at each of four locations during a field survey conducted in Panamá in 2012. Sites are ordered left to right along the x-axis based on the relative length of time these species have persisted with the fungal pathogen, Batrachochytrium dendrobatidis, at each site (i.e., longest at Cerro Campana and shortest at Nuevo Vigia; Woodhams et al., ; Rebollar et al., 2014). The letters above the bars indicate statistically significant differences among sites.

Figure 9

Beta diversity of bacterial communities (OTUs) (A,B) and bacterially-produced metabolite profiles (C,D) sampled from the skin Agalychnis callidryas (left column), Dendropsophus ebraccatus (right column), grouped by sampling location. Both species were sampled at each of four locations during a field survey conducted in Panamá in 2012. The color of the circles indicates the four sites and relative length of time these species have persisted with the fungal pathogen, Batrachochytrium dendrobatidis, at each site (i.e., aquamarine = longest at Cerro Campana and purple = shortest at Nuevo Vigia; Woodhams et al., ; Rebollar et al., 2014). NMDS ordinations are based on Bray–Curtis and Jaccard dissimilarities for OTUs and metabolites, respectively. Each point represents a single individual.

Figure 10

Dispersion of the bacterial communities (OTUs) sampled from the skin of (A) Agalychnis callidryas and (B) Dendropsophus ebraccatus. Both species were sampled at each of four locations during a field survey conducted in Panamá in 2012. Sites are ordered left to right along the x-axis based on the relative length of time these species have persisted with the fungal pathogen, Batrachochytrium dendrobatidis, at each site (i.e., longest at Cerro Campana and shortest at Nuevo Vigia; Woodhams et al., ; Rebollar et al., 2014). Analyses are based on Bray–Curtis and Jaccard dissimilarities for OTUs and metabolites, respectively. The letters above the bars indicate statistically significant differences among sites.