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Applied and Environmental Microbiology, February 2009, p. 668-675, Vol. 75, No. 3
0099-2240/09/$08.00+0     doi:10.1128/AEM.01757-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Extensive Phylogenetic Analysis of a Soil Bacterial Community Illustrates Extreme Taxon Evenness and the Effects of Amplicon Length, Degree of Coverage, and DNA Fractionation on Classification and Ecological Parameters ^,

Sergio E. Morales,¹ Theodore F. Cosart,^2,3 Jesse V. Johnson,^2,3 and William E. Holben^1,3*

Microbial Ecology Program, Division of Biological Sciences,¹ Department of Computer Science,² Montana—Ecology of Infectious Diseases Program, The University of Montana, Missoula, Montana³

Received 30 July 2008/ Accepted 2 November 2008

To thoroughly investigate the bacterial community diversity present in a single composite sample from an agricultural soil and to examine potential biases resulting from data acquisition and analytical approaches, we examined the effects of percent G+C DNA fractionation, sequence length, and degree of coverage of bacterial diversity on several commonly used ecological parameters (species estimation, diversity indices, and evenness). We also examined variation in phylogenetic placement based on multiple commonly used approaches (ARB alignments and multiple RDP tools). The results demonstrate that this soil bacterial community is highly diverse, with 1,714 operational taxonomic units demonstrated and 3,555 estimated (based on the Chao1 richness estimation) at 97% sequence similarity using the 16S rRNA gene. The results also demonstrate a fundamental lack of dominance (i.e., a high degree of evenness), with 82% of phylotypes being encountered three times or less. The data also indicate that generally accepted cutoff values for phylum-level taxonomic classification might not be as applicable or as general as previously assumed and that such values likely vary between prokaryotic phyla or groups.

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* Corresponding author. Mailing address: Microbial Ecology Program, Division of Biological Sciences, University of Montana, Missoula, MT 59812-1006. Phone: (406) 243-6163. Fax: (406) 243-4184. E-mail: bill.holben{at}mso.umt.edu

Published ahead of print on 14 November 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, February 2009, p. 668-675, Vol. 75, No. 3
0099-2240/09/$08.00+0     doi:10.1128/AEM.01757-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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