Diversity of uncultured Epsilonproteobacteria from terrestrial sulfidic caves and springs

Abstract

This study expands the phylogenetic diversity of Epsilonproteobacteria using the 16S rRNA gene framework. Of the 73 lineages defined by sequence similarities at or greater than 99%, most were found at only one site. In contrast, eight lineages were retrieved from sites spanning geographic distances from 1,000 to >10,000 km.

FIG. 1.

Maximum likelihood phylogeny of the newly characterized Epsilonproteobacteria terrestrial sequences, rooted to basal epsilonproteobacterial sequences (e.g., Caminibacter, AY691429 and AY691430; Hydrogenimonas, AB105048; Lebetimonas, AB167820; Nautilia, AF357197 and AJ575809; Nitratiruptor, AB175499; Thioreductor, AB175498), other proteobacterial sequences (e.g., Alphaproteobacteria, AY180103 and D84529; Deltaproteobacteria, Y11560 and X95181; Gammaproteobacteria, AJ543327 and AY776275), and Aquifex aeolicus (AJ309733) and Thermotoga maritima (AJ401021) sequences (outgroups not shown). Accession numbers for previously acquired sequences are included in brackets. Nodal supports from 100 bootstrap replicates are indicated on branches; bootstrap proportions less than 70% are not shown. Species level OTUs are based on monophyletic clades identified by DOTUR clustering at the 99% similarity level and are indicated as follows: circles represent OTUs consisting of sequences from a single location, squares represent OTUs consisting of sequences from two locations found within the same region, and stars represent OTUs consisting of sequences from two or more sites located on different continents. For clarity, monophyletic clades (i.e., a group of sequences containing an ancestor and all of its descendants) consisting of sequences from a single location are collapsed to a single branch; some of these sequence groups consist of more than one OTU (as defined by sequences with ≥99% similarity), which is indicated by more than one symbol per branch. For collapsed clades, the number of sequences is indicated in parentheses behind each location name. Abbreviations correspond to sampling sites listed in Table 1. The four main “Thiovulgaceae” clades from Campbell et al. (3) are indicated.

FIG. 2.

Average pairwise sequence distances for each of the eight OTUs containing representatives from more than one site plotted against the average geographic distance among all of the sites within the OTU. The phylogenetic distances were calculated using the PHYLIP v3.6 DNADIST module (10), which was used as DOTUR input to determine OTUs. Error bars represent ± 1 standard deviation.