Preface to the Princeton Landmarks in Biology Edition vii Preface xi Symbols Used xiii 1. The Importance of Islands 3 2. Area and Number of Speicies 8 3. Further Explanations of the Area-Diversity Pattern 19 4. The Strategy of Colonization 68 5. Invasibility and the Variable Niche 94 6. Stepping Stones and Biotic Exchange 123 7. Evolutionary Changes Following Colonization 145 8. Prospect 181 Glossary 185 References 193 Index 201

The Theory of Island Biogeography

Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

Because a natural entity “species” cannot be recognized as a group of strains that is genetically well separated from its phylogenetic neighbors, a pragmatic approach was taken to define a species by a polyphasic approach (L. G. Wayne, D. J. Brenner, R. R. Colwell, P. A. D. Grimont, O. Kandler, M. I. Krichevsky, L. H. Moore, W. E. C. Moore, R. G. E. Murray, E. Stackebrandt, M. P. Starr, and H. G. Truper, Int. J. Syst. Bacteriol. 37:463-464, 1987), in which a DNA reassociation value of about 70% plays a dominant role. With the establishment of rapid sequence analysis of 16S rRNA and the recognition of its potential to determine the phylogenetic position of any prokaryotic organism, the role of 16S rRNA similarities in the present species definition in bacteriology needs to be clarified. Comparative studies clearly reveal the limitations of the sequence analysis of this conserved gene and gene product in the determination of relationships at the strain level for which DNA-DNA reassociation experiments still constitute the superior method. Since today the primary structure of 16S rRNA is easier to determine than hybridization between DNA strands, the strength of the sequence analysis is to recognize the level at which DNA pairing studies need to be performed, which certainly applies to similarities of 97% and higher.

Taxonomic Note: A Place for DNA-DNA Reassociation and 16S rRNA Sequence Analysis in the Present Species Definition in Bacteriology

For centuries, biologists have studied patterns of plant and animal diversity at continental scales. Until recently, similar studies were impossible for microorganisms, arguably the most diverse and abundant group of organisms on Earth. Here, we present a continental-scale description of soil bacterial communities and the environmental factors influencing their biodiversity. We collected 98 soil samples from across North and South America and used a ribosomal DNA-fingerprinting method to compare bacterial community composition and diversity quantitatively across sites. Bacterial diversity was unrelated to site temperature, latitude, and other variables that typically predict plant and animal diversity, and community composition was largely independent of geographic distance. The diversity and richness of soil bacterial communities differed by ecosystem type, and these differences could largely be explained by soil pH (r(2) = 0.70 and r(2) = 0.58, respectively; P < 0.0001 in both cases). Bacterial diversity was highest in neutral soils and lower in acidic soils, with soils from the Peruvian Amazon the most acidic and least diverse in our study. Our results suggest that microbial biogeography is controlled primarily by edaphic variables and differs fundamentally from the biogeography of "macro" organisms.

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The diversity and biogeography of soil bacterial communities

Evolution of Protein Molecules

Identification of major subgroups of ammonia-oxidizing bacteria in environmental samples by T-RFLP analysis of amoA PCR products.

A novel species, Methylocella tundrae, is proposed for three methanotrophic strains (T4T, TCh1 and TY1) isolated from acidic Sphagnum tundra peatlands. These strains are aerobic, Gram-negative, non-motile, dinitrogen-fixing rods that possess a soluble methane monooxygenase and utilize the serine pathway for carbon assimilation. Strains T4T, TCh1 and TY1 are moderately acidophilic organisms capable of growth between pH 4·2 and 7·5 (optimum 5·5–6·0) and between 5 and 30 °C (optimum 15 °C). The major phospholipid fatty acid is 18 : 1ω7c. The DNA G+C content of strain T4T is 63·3 mol%. The three strains possess almost identical 16S rRNA gene sequences and are most closely related to two previously identified species of Methylocella, Methylocella palustris (97 % similarity) and Methylocella silvestris (97·5 % similarity). DNA–DNA hybridization values of strain T4T with Methylocella palustris KT and Methylocella silvestris BL2T were respectively 27 and 36 %. Thus, the tundra strains represent a novel species, for which the name Methylocella tundrae sp. nov. is proposed. Strain T4T (=DSM 15673T=NCIMB 13949T) is the type strain.

Methylocella tundrae sp. nov., a novel methanotrophic bacterium from acidic tundra peatlands.

Using a newly developed 16S rRNA gene (rDNA)-targeted PCR assay with proposed group specificity for planctomycetes, we examined anoxic bulk soil of flooded rice microcosms for the presence of novel planctomycete-like diversity. For comparison, oxic rice roots were included as an additional sample in this investigation. The bacterial diversity detectable by this PCR assay was assessed by using a combined approach that included terminal restriction fragment length polymorphism (T-RFLP) analysis and comparative sequence analysis of cloned 16S rDNA. T-RFLP fingerprint patterns generated from rice roots contained 12 distinct terminal restriction fragments (T-RFs). In contrast, the T-RFLP fingerprint patterns obtained from the anoxic bulk soil contained 33 distinct T-RFs, a clearly higher level of complexity. A survey of 176 bulk soil 16S rDNA clone sequences permitted correlation of 20 T-RFs with phylogenetic information. The other 13 T-RFs remained unidentified. The predominant T-RFs obtained from rice roots could be assigned to members of the genus Pirellula within the Planctomycetales, while most of the T-RFs obtained from the bulk soil corresponded to novel lines of bacterial descent. Using a level of 16S rDNA sequence dissimilarity to cultured microorganisms of approximately 20% as a threshold value, we detected 11 distinct bacterial lineages for which pure-culture representatives are not known. Four of these lineages could be assigned to the order Planctomycetales, while one lineage was affiliated with the division Verrucomicrobia and one lineage was affiliated with the spirochetes. The other five lineages either could not be assigned to any of the main lines of bacterial descent or clearly expanded the known diversity of division level lineages WS3 and OP3. Our results indicate the presence of bacterial diversity at a subdivision and/or division level that has not been detected previously by the so-called universal 16S rDNA PCR assays.

Novel bacterial lineages at the (sub)division level as detected by signature nucleotide-targeted recovery of 16S rRNA genes from bulk soil and rice roots of flooded rice microcosms.

Rice paddy soils are one of the major sources of atmospheric methane (15). These flooded soils contain largely uncharacterized microbial populations which degrade polymeric organic matter to low-molecular-weight compounds, such as acetate, H2, and CO2. These compounds are finally converted to methane by methanogenic archaea (16). Several cultivation studies to characterize autochthonous bacterial inhabitants of rice paddy soils have been performed (18, 19, 34, 51). It is not clear how successful such studies have been in characterizing bacterial communities. It has been estimated, for example, that the portion of microbial diversity which has been obtained in pure culture by conventional cultivation techniques amounts to only 0.1 to 1% of the total diversity (1, 12). Similarly, attempts to cultivate the bacteria in soils typically yield culturable cell numbers that are less than 5% (usually less than 1%) of the total microscopically countable cell numbers (3, 29). These observations indicate the inadequacy of culture methods to characterize complex microbial communities. One reason for this inadequacy is that the growth conditions used for direct enrichment cultures (i.e., cultures grown with undiluted inoculum) often do not reflect the natural conditions in the environment examined and thus select for fast-growing bacteria which are best adapted to the growth medium (16, 29, 49, 50). Consequently, the microorganisms obtained in pure culture may not have numerical or ecological significance for the biogeochemical processes in the environment, and abundant or even dominant populations may not be detected. Underestimation of microbial diversity by culture methods has favored the use of cultivation-independent, molecular approaches to investigate the microbial diversity in natural environments. The studies which have been performed have focused mainly on retrieval and comparative analysis of 16S rRNA-encoding gene (rDNA) sequences. The results not only have dramatically changed our view of the evolution and phylogenetic diversity of prokaryotes but also have confirmed the immense underrepresentation of naturally occurring bacteria and archaea obtained so far in pure culture (4, 11, 17, 23, 24, 29, 32).

It has, however, become clear that the cultivation-independent PCR-based 16S rDNA approach is also characterized by limitations and pitfalls (29). The steps used can lead to biases in the proportions of the 16S rDNA sequence types recovered. Consequently, this approach is more qualitative than quantitative. In addition, inferring an ecological role or even a defined phenotype on the basis of the phylogenetic position of an environmental 16S rDNA sequence is often not possible.

In an attempt to address the problems mentioned above, we applied to the same sample of rice paddy soil a culture method in which we used serial liquid dilutions of inoculum and a technique to recover environmental 16S rDNA. Cultivation was carried out by using the anaerobic most-probable-number (MPN) counting format. Using this dual approach, a previous study was able to gain insight into the structural and functional composition of the methanogenic community in anoxic rice paddy soil (16). In the present study we focused on the bacterial community, which was probably more diverse. Isolation of 19 saccharolytic and cellulolytic bacteria obtained from terminal and subterminal positive dilution steps of the MPN counting procedure and phenotypic characterization of these bacteria are described in the accompanying paper (8). Below, these bacteria are referred to as MPN isolates. Here we describe the results of a comparative phylogenetic analysis of two different 16S rDNA data sets generated during this investigation; i.e., sequence data for the 19 MPN isolates were compared to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets revealed that the isolates were representatives of predominant bacterial groups, and thus our findings resulted in a more objective view of the structural and functional composition of the bacterial community inhabiting the anoxic rice paddy soil which we studied.

Comparative Phylogenetic Assignment of Environmental Sequences of Genes Encoding 16S rRNA and Numerically Abundant Culturable Bacteria from an Anoxic Rice Paddy Soil

Werner Liesack

Papers

Identification of major subgroups of ammonia-oxidizing bacteria in environmental samples by T-RFLP analysis of amoA PCR products.

Methylocella tundrae sp. nov., a novel methanotrophic bacterium from acidic tundra peatlands.

Novel bacterial lineages at the (sub)division level as detected by signature nucleotide-targeted recovery of 16S rRNA genes from bulk soil and rice roots of flooded rice microcosms.

Comparative Phylogenetic Assignment of Environmental Sequences of Genes Encoding 16S rRNA and Numerically Abundant Culturable Bacteria from an Anoxic Rice Paddy Soil

Microbial diversity in soil: the need for a combined approach using molecular and cultivation techniques.