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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) (Fig. 1). For each station we sampled three different sites with three-fold replication during the wet and dry seasons. We have successfully completed 3 sampling campaigns in October 2002, September 2003 and January of 2004. We have begun applying a new molecular diversity survey method called SARST-V6 (Serial Analysis of Ribosomal Sequence Tags) 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. From a pilot study of 4545 tags, we have identified 126 bacterial phylotypes. Figure 2 shows a preliminary analysis examining linkages between this diversity and geochemistry in the river. Figure 3 shows a partial list of tags with 100% identity to known bacterial sequences in GenBank. We have also obtained many additional tags with less than 100% similarity to a reported sequence in GenBank for which we are designing primers to extract full-length sequence data. These sequences await further phylogenetic analyses. An analysis of a total of 11,951 tags is underway and this may significantly increase the total number of phylotypes known to date.

Alongside SARST analyses, we are also conducting phylogenetic analyses of the unique phylotypes using small subunit (SSU) rRNA gene clone libraries. Initial surveys of this diversity based on full-length SSU rRNA genes for the three domains of life are summarized in Figures 4-6. We have also begun to further study the newly discovered phylotypes in the Río Tinto by constructing fluorescently-labeled oligonucleotide rRNA probes and conducting fluorescence in situ hybridization (FISH) experiments on environmental samples. Figure 7 shows a CY3-labeled probe specific for the endosymbiont from the Iron Mountain Candidatus captivus acidiprotistae, also present in the Rio Tinto

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