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Applied and Environmental Microbiology, October 2003, p. 6189-6200, Vol. 69, No. 10
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.10.6189-6200.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Statistical Approaches for Estimating Actinobacterial Diversity in Marine Sediments

James E. M. Stach,1* Luis A. Maldonado,2 Douglas G. Masson,3 Alan C. Ward,2 Michael Goodfellow,2 and Alan T. Bull1

Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ,1 School of Biology, University of Newcastle, Newcastle upon Tyne NE1 7RU,2 Southampton Oceanography Centre, Empress Dock, Southampton SO14 3ZH, United Kingdom3

Received 14 April 2003/ Accepted 5 August 2003

Bacterial diversity in a deep-sea sediment was investigated by constructing actinobacterium-specific 16S ribosomal DNA (rDNA) clone libraries from sediment sections taken 5 to 12, 15 to 18, and 43 to 46 cm below the sea floor at a depth of 3,814 m. Clones were placed into operational taxonomic unit (OTU) groups with >=99% 16S rDNA sequence similarity; the cutoff value for an OTU was derived by comparing 16S rRNA homology with DNA-DNA reassociation values for members of the class Actinobacteria. Diversity statistics were used to determine how the level of dominance, species richness, and genetic diversity varied with sediment depth. The reciprocal of Simpson's index (1/D) indicated that the pattern of diversity shifted toward dominance from uniformity with increasing sediment depth. Nonparametric estimation of the species richness in the 5- to 12-, 15- to 18-, and 43- to 46-cm sediment sections revealed a trend of decreasing species number with depth, 1,406, 308, and 212 OTUs, respectively. Application of the LIBSHUFF program indicated that the 5- to 12-cm clone library was composed of OTUs significantly (P = 0.001) different from those of the 15- to 18- and 43- to 46-cm libraries. FST and phylogenetic grouping of taxa (P tests) were both significant (P < 0.00001 and P < 0.001, respectively), indicating that genetic diversity decreased with sediment depth and that each sediment community harbored unique phylogenetic lineages. It was also shown that even nonconservative OTU definitions result in severe underestimation of species richness; unique phylogenetic clades detected in one OTU group suggest that OTUs do not correspond to real ecological groups sensu Palys (T. Palys, L. K. Nakamura, and F. M. Cohan, Int. J. Syst. Bacteriol. 47:1145-1156, 1997). Mechanisms responsible for diversity and their implications are discussed.

* Corresponding author. Mailing address: Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdom. Phone: 44 (0)1227 823336. Fax: 44 (0)1227 763912. E-mail: j.e.stach{at}kent.ac.uk.

Applied and Environmental Microbiology, October 2003, p. 6189-6200, Vol. 69, No. 10
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.10.6189-6200.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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