Biogeography of sulfur-oxidizing Acidithiobacillus populations in extremely acidic cave biofilms

Abstract

Extremely acidic (pH 0-1.5) Acidithiobacillus-dominated biofilms known as snottites are found in sulfide-rich caves around the world. Given the extreme geochemistry and subsurface location of the biofilms, we hypothesized that snottite Acidithiobacillus populations would be genetically isolated. We therefore investigated biogeographic relationships among snottite Acidithiobacillus spp. separated by geographic distances ranging from meters to 1000s of kilometers. We determined genetic relationships among the populations using techniques with three levels of resolution: (i) 16S rRNA gene sequencing, (ii) 16S-23S intergenic transcribed spacer (ITS) region sequencing and (iii) multi-locus sequencing typing (MLST). We also used metagenomics to compare functional gene characteristics of select populations. Based on 16S rRNA genes, snottites in Italy and Mexico are dominated by different sulfur-oxidizing Acidithiobacillus spp. Based on ITS sequences, Acidithiobacillus thiooxidans strains from different cave systems in Italy are genetically distinct. Based on MLST of isolates from Italy, genetic distance is positively correlated with geographic distance both among and within caves. However, metagenomics revealed that At. thiooxidans populations from different cave systems in Italy have different sulfur oxidation pathways and potentially other significant differences in metabolic capabilities. In light of those genomic differences, we argue that the observed correlation between genetic and geographic distance among snottite Acidithiobacillus populations is partially explained by an evolutionary model in which separate cave systems were stochastically colonized by different ancestral surface populations, which then continued to diverge and adapt in situ.

Figure 1

(a) Schematic depicting the snottite habitat in sulfidic caves. Snottites form in close proximity to H₂S-degassing cave streams, and typically occur where cave air H₂S(g) concentrations are between 0.2 and 25 ppm. (b, c) Representative photographs of snottites sampled in this study, from (b) Acquasanta, Italy and (c) Villa Luz, Mexico. Black scale bar in (b) is 1 cm.

Figure 2

Maximum likelihood phylogram of 16S rRNA gene sequences from the genus Acidithiobacillus. Representative sequences from this study are shown in bold. Numbers indicate bootstrap support by neighbor joining and maximum parsimony, in that order (only values >75% shown).

Figure 3

Phylogenetic analysis of ITS sequences from environmental and isolate At. thiooxidans strains. Sequence names are colored by cave location, names in bold indicate isolates and italicized names indicate environmental clones. The numbers of clones from the same sample represented by each sequence are given in parentheses. The base tree is a neighbor joining phylogram constructed after excluding alignment positions with >50% gaps, and numbers indicate maximum parsimony and neighbor joining bootstrap support for nodes connecting the four major clades. Stars indicate nodes compatible with both neighbor joining analysis and maximum parsimony consensus trees after excluding all gapped positions, and the solid circles indicate nodes compatible with maximum parsimony analysis of indels (Supplementary Figure S2). The dashed box in clade ITS_4 indicates ITS regions that were cloned from isolates RS30a and RS31a.

Figure 4

Maximum likelihood analyses of At. thiooxidans isolates based on MLST. Numbers at each node indicate posterior probabilities and bootstrap support from Bayesian, maximum parsimony and neighbor joining analyses, in that order. Sequence names are colored by cave location.

Figure 5

Genetic distance versus geographic distance for At. thiooxidans isolates from (a) the Frasassi and Acquasanta cave systems, and (b) the Frasassi cave system only. Isolates from RS30, RS31, RS2, GB30 and GB31 are excluded (see text for details). Genetic distance among strains was determined from MLST (Figure 4). Addition of a small random number was used to spread out overlapping points along the x-axis. Dashed lines are least squares regression lines, and Pearson's correlation for the depicted relationships are statistically significant (a: r=0.93, P<<0.001; b: r=0.75, P<<0.001).

Figure 6

Cartoon of enzymatic sulfur transformations involved in the oxidation of reduced inorganic sulfur compounds by snottite At. thiooxidans, inferred from metagenomic analysis. Abbreviations: HDR, heterodisulfide reductase; SDO, sulfur diooxygenase; SOR, sulfur oxygenase reductase; SOX, multicomponent sulfur oxidation pathway; SQR, sulfide:quinone oxidoreductase; TQO, thiosulfate:quinone oxidoreductase; TTH, tetrathionate hydrolase.

Figure 7

Proposed evolutionary histories of snottite populations in Italy. The caves could have been colonized from a single ancestral population (dashed arrow) that diverged by genetic drift or adaptation. Alternatively, the different cave populations could have originated from separate surface-dwelling populations with slight physiological differences (bold arrows), which then continued to diverge in situ.