Molecular Phylogeny of Uncultivated Crenarchaeota in Great Basin Hot Springs of Moderately Elevated Temperature

Abstract

Uncultivated Crenarchaeota are distributed widely in low temperature (< 30°C) environments, and it has been hypothesized that they evolved from (hyper)thermophilic species thriving in marine hydrothermal vents or terrestrial hot springs. To further our understanding of the environmental distributions of Crenarchaeota, we studied mat samples collected from hot springs of moderately elevated temperature (∼ 49–67°C) in California and Nevada, USA. Clone libraries of archaeal 16S rRNA genes were constructed for selected samples using a PCR-based approach. Sequences from the Nevada hot springs (Rick's Hot Creek and Hard to Find) were closely related to uncultivated Crenarchaeota found near deep sea hydrothermal vents or from the subsurface geothermal system; sequences from the California hot spring (Surprise Valley), on the other hand, were closely related to sequences from freshwater sediments. Statistical analysis showed that the community structure of Archaea was significantly different between any two springs with greater differences occurring between the Nevada and California hot springs (P = 0.002). To determine whether these sequences represent indigenous microorganisms of geothermal springs, and not soil contaminants, archaeal 16S rRNA gene clone libraries were also constructed from soil samples taken from around Rick's Hot Creek and Surprise Valley hot springs. None of the hot spring sequences was closely related to those from the surrounding soil in Nevada or California or to the predominant soil Crenarchaeota in other locations, indicating that soil contamination to the hot spring environment was insignificant. Results of this study expand the distribution of Crenarchaeota into the moderately thermobiotic environment, which has been much less intensively studied than high temperature (> 70°C) or low temperature natural habitats, and demonstrates that thermophiles inhabiting moderate temperature portions of Great Basin hot springs are phylogenetically distinct from both cultivated hyperthermophilic Crenarchaeota and sympatric soil Crenarchaeota.

Keywords:

• Crenarchaeota
• diversity
• thermophiles
• hot springs
• soils
• Nevada
• California

Acknowledgments

We thank Christopher S. Romanek, Gary Mills, Rick Socki, Joy Hallmark, Kyle Costa, and Sue Lutz for help with field work in Nevada and California and Tristy Vick for editorial advice. David and Sandy Jamieson showed great hospitality and supported our sampling efforts. This research was supported by National Science Foundation grant MCB-0348180 to CLZ and JW and National Science Foundation grant MCB-0546865 to BPH. This publication was also made possible by support to the Nevada Genomics Center through Grant Number_P20RR016464_ from the National Center for Research Resources (NCRR), a component of the National Institutes of Health. The U.S. Department of Energy also supported this research through Financial Assistance Award DE- DE-FC09-07SR22506 to the University of Georgia Research Foundation (CLZ).

Notes

^a Operational taxonomic units (OTU) based on the similarities between sequences; sequences having or above 97% identity were defined as one OTU.

^b OTU that is only present in a particular sample.

^†Δ Cxy is the difference between a homologous coverage curve, CX(D), and a heterologous coverage curve, CXY(D), which is calculated using a Cramér-von Mises-type statistic. Each Δ Cxy is accompanied by a p value. Two libraries are considered significantly different when p is less than 0.05 and the bigger the Δ Cxy, the smaller the p value.