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.
Bibliographical noteFunding Information:
This work was supported by a generous graduate research fellowship to DSJ from the Cave Conservancy Foundation, and grants to JLM from the Biogeosciences Program of the National Science Foundation (EAR 0311854 and EAR 0527046) and NASA NAI (NNA04CC06A). We thank A Montanari for logistical support and the use of facilities and laboratory space at the Osservatorio Geologico di Coldigioco (Italy), and C Alberto Cordero Martinez for lodging and logistical support in Villa Luz Park. Permits for access to Villa Luz were granted by C Rogers Morales Mendez and L Felino Arevalo Gallegos. We thank S Mariani, S Galdenzi, S Cerioni and M Mainiero for expert advice and field assistance with research in Italy. F Baldoni, S Recanatini, S Dattagupta and the members of the Gruppo Speleologico CAI di Fabriano and the Gruppo Speleologico Marchigiano CAI di Ancona provided additional assistance. We thank L Rosales-Lagarde, L Hose and S Dattagupta for field assistance in Mexico, T Jones and E Lyon for laboratory assistance at Penn State, and C House and C Grettenberger for insightful discussion.
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