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Features including colonization routes (stream capture) and the existence of both epigean and cave-adapted hypogean populations make Astyanax mexicanus an attractive system for investigating the subterranean evolutionary time necessary for acquisition of the troglomorphic form. Using published sequences, we have estimated divergence times for A. mexicanus using: 1) two different population-level mitochondrial datasets (cytochrome b and NADH dehydrogenase 2) with both strict and relaxed molecular clock methods, and 2) broad phylogenetic approaches combining fossil calibrations and with four nuclear (recombination activating gene, seven in absentia, forkhead, and α-tropomyosin) and two mitochondrial (16S rDNA and cytochrome b) genes. Using these datasets, we have estimated divergence times for three events in the evolutionary history of troglomorphic A. mexicanus populations. First, divergence among cave haplotypes occurred in the Pleistocene, possibly correlating with fluctuating water levels allowing the colonization and subsequent isolation of new subterranean habitats. Second, in one lineage, A. mexicanus cave populations experienced introgressive hybridization events with recent surface populations (0.26-2.0 Ma), possibly also correlated with Pleistocene events. Finally, using divergence times from surface populations in the lineage without evidence of introgression as an estimate, the acquisition of the troglomorphic form in A. mexicanus is younger than 2.2 (fossil calibration estimates) – 5.2 (cytb estimate) Ma (Pliocene).
Sulfidic cave walls host abundant, rapidly-growing microbial communities that display a variety of morphologies previously described for vermiculations. Here we present molecular, microscopic, isotopic, and geochemical data describing the geomicrobiology of these biovermiculations from the Frasassi cave system, Italy. The biovermiculations are composed of densely packed prokaryotic and fungal cells in a mineral-organic matrix containing 5 to 25% organic carbon. The carbon and nitrogen isotope compositions of the biovermiculations (d13C 5 235 to 243%, and d15N 5 4 to 227%, respectively) indicate that within sulfidic zones, the organic matter originates from chemolithotrophic bacterial primary productivity. Based on 16S rRNA gene cloning (n567), the biovermiculation community is extremely diverse, including 48 representative phylotypes (.98% identity) from at least 15 major bacterial lineages. Important lineages include the Betaproteobacteria (19.5% of clones), Gammaproteobacteria (18%), Acidobacteria (10.5%), Nitrospirae (7.5%), and Planctomyces (7.5%). The most abundant phylotype, comprising over 10% of the 16S rRNA gene sequences, groups in an unnamed clade within the Gammaproteobacteria. Based on phylogenetic analysis, we have identified potential sulfur- and nitrite-oxidizing bacteria, as well as both auto- and heterotrophic members of the biovermiculation community. Additionally, many of the clones are representatives of deeply branching bacterial lineages with no cultivated representatives. The geochemistry and microbial composition of the biovermiculations suggest that they play a role in acid production and carbonate dissolution, thereby contributing to cave formation.
Although ecosystems thriving in the absence of photosynthetic processes are no longer considered unique phenomena, we have yet to understand how these ecosystems are energetically sustained via chemosynthesis. Ecosystem energetics were measured in microbial mats from active sulfidic caves (Movile Cave, Romania; Frasassi Caves, Italy; Lower Kane Cave, Wyoming, USA; and Cesspool Cave, Virginia, USA) using radiotracer techniques. We also estimated bacterial diversity using 16S rRNA sequences to relate the productivity measurements to the composition of the microbial communities. All of the microbial communities investigated were dominated by chemolithoautotrophic productivity, with the highest rates from Movile Cave at 281 g C/m2/yr. Heterotrophic productivities were at least one order of magnitude less than autotrophy from all of the caves. We generated 414 new 16S rRNA gene sequences that represented 173 operational taxonomic units (OTUs) with 99% sequence similarity. Although 13% of these OTUs were found in more than one cave, the compositions of each community were significantly different from each other (P≤0.001). Autotrophic productivity was positively correlated with overall species richness and with the number of bacterial OTUs affiliated with the Epsilonproteobacteria, a group known for sulfur cycling and chemolithoautotrophy. Higher rates of autotrophy were also strongly positively correlated to available metabolic energy sources, and specifically to dissolved sulfide concentrations. The relationship of autotrophic productivity and heterotrophic cycling rates to bacterial species richness can significantly impact the diversity of higher trophic levels in chemolithoautotrophically-based cave ecosystems, with the systems possessing the highest productivity supporting abundant and diverse macro-invertebrate communities.
We performed a microbial community analysis of biofilms inhabiting thermal (35 to 50°C) waters more than 60m below the ground surface near Acquasanta Terme, Italy. The groundwater hosting the biofilms has 400 to 830 mkM sulfide, <10 mkM O2, pH of 6.3 to 6.7, and specific conductivity of 8,500 to 10,500 mkS/cm. Based on the results of 16S rRNA gene cloning and fluorescent in situ hybridization (FISH), the biofilms have low species richness, and lithoautotrophic (or possibly mixotrophic) Gamma- and Epsilonproteobacteria are the principle biofilm architects. Deltaproteobacteria sequences retrieved from the biofilms have <90% 16S rRNA similarity to their closest relatives in public databases and may represent novel sulfate-reducing bacteria. The Acquasanta biofilms share few species in common with Frasassi cave biofilms (13°C, 80 km distant) but have a similar community structure, with representatives in the same major clades. The ecological success of Sulfurovumales-group Epsilonproteobacteria in the Acquasanta biofilms is consistent with previous observations of their dominance in sulfidic cave waters with turbulent water flow and high dissolved sulfide/oxygen ratios.
The main objective of this work was the comparative analysis of a large number of bacterial strains isolated from biodeteriorated surfaces in three different sites, namely the catacombs of St. Callistus in Rome, Italy, the catacombs dedicated to St. Agatha in Rabat, Malta and the Cave of Bats in Zuheros, Spain. Our results showed that even considering only culturable chemoorganotrophic bacteria the variability is very high, reflecting the great variety of microhabitats present. Hence any strategies to prevent, control or eliminate the biofilm-embedded microbiota from an archeological surface should take into account a number of considerations as stipulated in our study.
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