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Project Progress

Our population studies in the Rio Tinto focus on three stations located in the more extreme headwaters of the river: the Origin (OR), Anabel’s Garden (AG) and Berrocal (BE)

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We have begun analyzing data from a new molecular diversity survey method called SARST-V6 (Serial Analysis of Ribosomal Sequence Tags) applied to the October 2002 samples. With SARST, the PCR products from orthologous hypervariable regions (~100 bp long for the bacterial V6 region) in rRNA genes are ligated together to form large concatemers. Our SARST data are currently only available for bacteria. A total of 10,575 RSTs (ribosomal sequence tags) were BLASTED against the GenBank and RDP Databases. The most abundant tags matched known typical species from this river including Acidithiobacillus and Leptospirillum spp.. Other tags matched taxa that were previously unknown from the Tinto, including uncultured bacteria reported from Iron Mountain that represented more than 17% of the total diversity (Uncultured TRA3-2) and Sulfobacillus disulfidooxidans. We recovered numerous rare taxa.

We have used the program Clusterer (http://web.mit.edu/polz/clusterer/clusterer.html) to determine Operational Taxonomic Units (OTUs) in our study and are currently determining alpha and beta diversity indices based on these results.
Figure 2 demonstrates the application of the Clusterer Program. The number of clusters decreased by 51.33% at 2bp differences or 98.65% similarity cut-off level. This result indicates that the cut-off similarity value for neutral microdiversity in the V6 region for our data is situated at 99% diversity.

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To capture the relative diversity of Bacteria, Archaea and Eukarya, we have devised a three-domain approach that employs universal primers targeting one of the most hypervariable regions of rRNA — the V4-V8 region. We have completed sequencing 384 clones from all three replicate samples from the three sites of the Anabel’s Garden and Berrocal stations and are currently processing samples from the Origin Site.
Figure 3 shows preliminary placement of archaeal clones from Anabel’s Garden including some phylotypes never reported from the Rio Tinto.

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