Prokaryotic diversity and its limits: microbial community structure in nature and implications for microbial ecology

Thomas P Curtis1 and William T Sloan2

Recent advances in the estimation of prokaryotic diversity have brought us insight into two questions: what is the extent of prokaryotic diversity, and perhaps more importantly, why bother nding out. In this review, we highlight the insights about the extent of diversity that may be gained by considering patterns that occur, or are likely to occur, in the relative abundance of prokaryotic taxa. We posit that global reservoirs of diversity are an important driving force behind patterns in localised diversity seen in leaves, intestines and wastewater treatment reactors. Thus, where the reservoir community is very large and relatively even, chance alone will prevent physically identical communities from having the same, or sometimes even stable, communities. By contrast, communities that tend to be similar (even when not physically identical) and stable are observed where the source diversity is low. Thus the relationship between structure and function in a community can only be understood, predicted and engineered through an understanding of the source of diversity from which the community is drawn.
Addresses 1 Department of Civil Engineering and Geosciences, Newcastle University, Newcastle, NE1 7RU, UK 2 Department of Civil Engineering, University of Glasgow, Glasgow, G12 8LT, UK e-mail: tom.curtis@ncl.ac.uk

Collectively these sages raise two questions, how can one measure microbial diversity and what does microbial diversity mean? Clearly if microbial diversity means nothing and cannot be measured the reader will be well advised to turn the page and seek enlightenment and advancement elsewhere. And yet one cannot help but wonder. The microbial world is immense more than 1030 individuals, which we know about [3]. That is, there are 109 times more bacteria on Earth than there are stars in the Universe. One needs no rationale to ponder on the extent of the microbial universe. We need merely to recognise it for what it is: an immense and unexplored frontier in science. A frontier of quite literally astronomical dimensions and of astonishing complexity the simplest microbe is arguably more complex than any star. From this perspective, we need to know about microbial diversity simply, because its there (The famous response of the mountaineer Mallory questioned on his motivation for climbing Mount Everest). Mallory and his colleagues at least knew the magnitude of their task. Yet for all our molecular sophistication, the diversity of the microbial world is not known with any certainty. In this review, we not only consider evidence about the magnitude of prokaryotic diversity, but also consider its importance as a driving force behind patterns in local taxon abundance in, and by implication the ecological characteristics of, leaves, intestines and wastewater treatment reactors.

Current Opinion in Microbiology 2004, 7:221226 This review comes from a themed issue on Ecology and industrial microbiology Edited by Elizabeth Wellington and Mike Larkin Available online 10th May 2004 1369-5274/$ see front matter 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2004.04.010 Abbreviations ARDRA amplied rDNA restriction analysis SSCP single strand conformation polymorphism

The practical calculation of diversity

The magnitude of diversity in the prokaryotic world is still a matter of debate. In essence there are two diametrically opposing views: there are many species of bacteria or, alternatively, there are very few. The rationale for low prokaryotic diversity is that microbial organisms are so abundant, that free-living representatives can be easily globally dispersed. Consequently allopatric speciation is discouraged in microbial communities by very high invasion rates [4]. This line of reasoning has its origins in the study of protozoa. The extension of these ideas to the prokaryotes and has been weakened by a lack of empirical evidence for low prokaryotic diversity. It was therefore very signicant that Hagstrom
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Introduction
Mumbo-Jumbo, that is how Thomas Brock [1] described the study of diversity in his pithy and prescient essay on the state of microbial ecology in 1987. Brock argued that measures of diversity were pointless because the dynamic nature of the microbial world meant that communities did not have a characteristic diversity but changed as the environment changed. An even grander old man, EO Wilson, reinforced the futility of the study of microbial diversity when he observed that microbial diversity is beyond practical calculation [2].
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222 Ecology and industrial microbiology

and colleagues [5] published an analysis of the rate of accumulation of new sequences based on the rate of accession in GenBank. The rate of discovery appeared to be reducing and the inventory appeared to be nearing completion at about 1117 unique sequences (see Update). The implication was that the prokaryotic diversity, in the marine environment at least, is small. This stands in stark contrast to other recent estimates of diversity in a wide variety of environments made by two groups in the UK and the USA. Dunbar [6] and our group [7] (independently) had the same essential idea: that prokaryotic communities might have characteristic species abundance curves and that parameterising the curve will give insights into the extent of diversity. A species abundance curve is a graphical representation of the relative abundance of the taxa in a given community, and the area under such a curve is the total diversity. Unfortunately, no complete, or even nearly complete, species abundance curve has ever been described for a prokaryotic community and so the description of the curve is not a straightforward matter. The two groups tackled this issue in contrasting manners. Curtis et al. [7] developed a rough and ready universal prokaryotic diversity estimator. They used a substantial body of theory to suggest that prokaryotic communities would have a characteristic species curve and that that curve was likely to be lognormal. They then derived an equation to calculate the area under the species abundance curve. The equation exploited the observation that the area under a lognormal curve would be a function of the ratio between the number of individuals (NT) and the abundance of the most abundant taxon (Nmax). The equation also assumed either a minimum abundance (typically 1 species at an abundance of 1) or the well known canonical form of the lognormal. The former was felt to be simpler and therefore preferable. Because NT/ Nmax can be approximately estimated from clone libraries and NT is easy to determine (it is the direct microscopic count), the equation can be used to roughly estimate the maximum possible diversity of any microbial community of any size. This approach does not assume a particular species denition, simply a credible method for distinguishing between taxa. More discriminating taxonomic methods will give higher NT/Nmax values and thus higher estimates of diversity. Curtis et al. [7] used published data on ribosomal RNA gene based clone libraries. They found that there could be 7000 species in a gram of soil, while a ton of soil could theoretically hold three million different taxa. By contrast, there could be no more than 160 species in a millilitre of seawater and the entire sea could hold no more than two million different taxa. These rough estimates are surprisingly similar to estimates made by
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DNA:DNA hybridisation of 300011,000 and 160 different genomes for small samples of soil and seawater respectively [8]. The two datasets are not completely comparable and at least one commentator has suggested that Torsviks estimates are too conservative [9]. Nevertheless, the paper offers substantial theoretical support for the early work of the Bergen group suggesting incredibly high soil diversity. More generally, the paper also made estimates of the variation of the extent of diversity between environments, community sizes and phylogenetic groups. It appeared that some environments and phylogenetic groups are much much more diverse than others. So what? To begin to answer this question we must look to John Dunbar. Dunbar and colleagues [6] took the lognormal as the best null distribution of species abundance. Within this distribution they assumed that there was only one species that attained the maximum abundance and only one at the minimum abundance. They proceeded to generate a suite of potential lognormal distributions to describe a community. These were constrained to give diversities within the range suggested by hybridisation kinetics experiments and by the observed total number of individuals. The potential curves were sampled 200 times and the diversity compared with the diversity in 200 clones from four Arizona soils. They found that there had to be between 4000 and 8000 species in the hypothetical communities if the 200 samples were to give the same diversity as 200 clones. Dunbars sample size calculations are sobering. In a sample with 4000 species it would take 25,000 samples (i.e. clones) to reveal 2000 different species and 285,000 samples to be reasonably sure of sampling the 2000 most abundant species. The inference is clear; in soil at least, we are still in the foothills of microbial diversity assessed using rRNA genes. However, the issue of sampling raises many subtler and probably more important issues, which could easily go unnoticed. We can see straight away, that the decline in the rate of new accessions discussed earlier [5] is inevitable; the small number of very abundant taxa will obscure the larger number of moderately rare or very rare organisms (see Update). Thus we cannot draw conclusions about the number of species in the sea from the rate of acquisition of new sequences unless we have some measure of sampling effort and the rate of acquisition of previously observed sequences. It is important at this point to remember that the lognormal species abundance curve is really just a useful starting point and that other distributions are plausible. Patterns in clone libraries suggest that other patterns exist in nature including the at or nearly at that will permit hyperdiverse communities [10], the apparently lognormal
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Prokaryotic diversity and its limits: Curtis and Sloan 223

[11] and the geometric series (seen in the Archaea in [12]). Moreover, Narang and Dunbar have illustrated that the diversity we observe from random sampling will be a function of the underlying distribution from which we are sampling [13]. In the next section we will attempt to demonstrate why these sampling issues could be of profound practical importance in the microbial world. We will show how the size of a microbial meta-community, and the manner it is drawn upon to make local microbial communities, could explain certain patterns in microbial ecology.

Diversity and community assembly

Consider a microbial community, any such community, the gut of a child newly born, or a waste reactor newly built, a new leaf or a new root. When the community forms it must be initiated by drawing, at random, from the environment around it. Yet we can see from the work of Dunbar, that two random samples from a lognormal community can have quite different compositions and many thousands of sampling events are required before we can be assured that all the representatives of the metacommunity can be seen in the new community. If you place these ndings in the context of the large source diversities envisaged by Curtis et al. [7] then differences between samples of large metacommunities seem inevitable. In practical terms this means that physically identical environments will have differing compositions if they are formed at random from very large metacommunities. As a consequence, the same physical environments could react in subtly (or not so subtly) different ways to variations in environmental conditions. Conversely, two microbial communities can only be identical if they are drawn from a metacommunity whose distribution and diversity is such that virtually the same initial species will arrive by chance. There is evidence to support this conception. Since Fernandez and her colleagues noted that the bacterial communities in anaerobic reactors were not reproducible [14] the same observations have been made in aerobic biological treatment communities [15], oil contaminated microcosms [16] and other anaerobic communities [17,18]. Similarly, since Zoetendal et al. [19] observed differences between the faecal ora of humans it has become apparent that these ndings can be extended to any part of the intestine of a wide range of organisms including pigs [11], mice [20] social wasps [21] and species of termite [22]. The leaves of plants are also different [23]. At present, these differences are typically attributed to host selection or feeding differences (for commensal communities) or chaotic dynamics (for biological treatment communities). The latter explanation is offered because the bacterial diversity of small biological treatment communities are often very unstable [15,16].
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Interestingly, this very dynamic behaviour is not seen in large biological treatment systems [24] or in samples taken from a river [25] (a similarly large and homogenous system) or in the Archaea in biological treatment systems (discussed below). Thus, if these systems are chaotic, only the Bacteria are chaotic and only in small biological treatment systems (intestines it appears are stable). Though, an elegant, indeed beautiful, mechanism for chaotic dynamics in microbial communities has been proposed. The theory rests on the assumption that competition between taxa is nely poised; this has not yet been demonstrated. Indeed the original theorists would have needed to choose their kinetic coefcients using a special searching algorithm to get just the right balance required to obtain chaotic behaviour [26,27]. Environmental and host factors do not appear to account for all the differences in commensal communities. In a heroic survey of over 4000 clones from 24 pigs Leser et al. [11] were unable to attribute the presence or absence of a phylotype to any environmental factor. They did however observe that the differing clones were log-normally distributed. Similarly, in a DGGE (denaturing gradient gel electrophoresis) survey of the intestines of wasps from 21 nests, no explanatory environmental factor was discerned [21]. There is some evidence that host factors play a role (including a very nice comparison of twins) [28]. However, even in the most carefully controlled study of inbred mice in identical conditions and receiving autoclaved food and only using fatty acid proles to compare diversity, some differences between mice persisted [20]. Similarly in comparisons between hosts species of plants Yang et al. [23] found greater similarity between plants of the same species, but there were still substantial dissimilarities between the leaves of plants of the same species in the same eld. The implication of the forgoing is clear. No two, naturally assembled bacterial communities appear to be the same. If random invasion from outside has a role the characteristics of any given community will be inuenced by i) the size of the reservoir or metacommunity of diversity from which it is drawn, ii) the distribution of taxa within the metacommunity, iii) the rate of carriage of bacteria from the reservoir to the source community, iv) the size of the source community and (v) the spatial structure of the community. These concepts are not new, not even for the microbial world. MacArthur and Wilson [29] put many of these ideas forward in the early sixties and supported them with data from experiments on microbes. Now that the notion that bacteria can have biogeography has been so elegantly restated in a recent study of Sulfolobus in hot spring communities [30], perhaps this theory and its more explicitly neutral descendants [31,32] should be revisited. Neutral theories rather controversially assume
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no member of a particular functional group is, on average, any more competitive than any other member of that functional group. The advantage of such an approach is that it is simple and therefore there is some chance of its application in the complex and vast microbial world. If these ideas have at least some predictive power then where one had a smaller reservoir of diversity in the metacommunity one might expect not only a smaller amount of diversity in each such community, but also a greater reproducibility between similar communities. Moreover, although such communities might have greater stability of diversity (there is less metacommunity diversity to drive change) they could, paradoxically, have less stability of function because they would have less functional redundancy [33,34]. A suite of papers on the diversity of Euryarchaeota in anaerobic reactors would appear to be a case in point. When Fernandez et al. [14] compared the community structure of the methanogens (members of the Euryarchaeota) in two pairs of replicated anaerobic reactors the diversity appeared very similar. Pender et al. [17] were also able to demonstrate stable replicated Euryarchaeota diversity using amplied rDNA restriction analysis (ARDRA) in two laboratory reactors. This nding is supported by a T-RFLP (terminal-restriction fragment length polymorphism) analysis by the same group [18]. Godons group found the Archaea diversity (comprising almost exclusively Euryarchaeota), assessed using single strand conformation polymorphism (SSCP), to be stable during start up [35] and operation [36]. The bacterial diversity was unstable in the French [36], Irish [18] and American studies [14]. The implication of this reproducibility and stability is that the metacommunity of Euryarchaeota in anaerobic reactors has low diversity. To test this inference, we would need to examine the diversity of the metacommunity for this group of organisms. How does one measure a metacommunity? One way would be to sample many different representatives of the same type of community, since each local community is implicitly a sample of the metacommunity. Le Clerc et al. [37] have done just this, by examining the Archaea in 44 anaerobic reactors from all over the world, at a variety of scales and treating a variety of wastes. They found 23 different sequences in the rst 24 reactors and no new diversity in the next 20. Any undetected diversity must be extremely rare indeed. All the sequences detected were from the Euryarchaeota, virtually all were very closely related to known cultured strains or environmental clones. Two sequences (a sequence close to Methanosaeta concilli and a sequence close to a clone from an uncultured organism, vadinDC06, from the Methanobacterium clade) were found in 84% and 73% of reactors respectively. Excitingly, the same two sequences were found to occur in 100% and 83% of
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reactors in a smaller, and entirely unconnected ARDRA based survey of six full and lab scale reactors in the Republic of Ireland [38]. This study also found only a handful of differing sequences [38]. Together, these two studies strongly suggest that the source or metacommunity for Euryarchaeota in anaerobic digesters is indeed low at least when assessed using the rDNA sequences. This would undoubtedly explain why the methanogenic diversity of such reactors is both easily replicated and stable. Interestingly, some of these ndings are consistent with (but not proof of) neutral community models [31,32]. For example, neutral theories of biogeography (and incidentally the Curtis et al. paper [7]) suggest that larger reactors have more diversity: LeClerc [37] appears to observe this, the modal observed number of Euryarchaeota being greater in the latter. In addition, to nd the same organisms in a wide variety of apparently very different reactors at relatively high abundance would suggest that these organisms appear frequently, by chance because they are highly represented in the meta-community. The deterministic alternative is that these organisms are superb generalists that can compete in any design of reactor receiving any waste. The truth is probably somewhere between the two extremes. The implicit lack of functional redundancy in the methanogens may also explain why engineers consider methanogenesis to be a delicate process that is easy to inhibit [34]. Similarly, we posit that autotrophic ammonia oxidation in wastewater treatment is also governed by a small global meta-community. It will therefore be easy to replicate the diversity and the diversity will be stable, but low and therefore correspondingly easy to disrupt. Space precludes an extensive consideration of spatial structure and random colonisation. But our intuition is that spatial structure (for example biolms) will preserve diversity from random colonisation while homogeneity will tend to obscure it.

Conclusions
Perhaps diversity is not all mumbo-jumbo. Perhaps patterns in global microbial diversity affect community composition, stability and functionality at a local level. If, as we argue, diversity matters, then patterns in global diversity could have a substantial effect on studies that seek to link community function and structure, strategies for seeking new drugs, for probiotics, bioaugmentation or studies to determine the persistence of chemicals. To understand microbial systems at a local level we will have to understand something of the metacommunity from which it is drawn. Moreover, we will have to correctly understand the relationship between random factors and deterministic factors. Microbiologists have typically ignored the former. It may be fruitful to rectify this situation and to develop a deeper appreciation of the
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role of chance in microbial ecology, simple neutral models, might be a good place to start.

8.

Torsvik V, Ovreas L, Thingstad TF: Prokaryotic diversity Magnitude, dynamics, and controlling factors. Science 2002, 296:1064-1066. Dykhuizen DE: Santa Rosalia revisited: Why are there so many species of bacteria? Antonie Van Leeuwenhoek Int J Gen Mol Microbiol 1998, 73:25-33.

Update
The recent publication of an environmental genome shotgun survey of the Sargasso Sea [39] resonates with some of the ndings of this review and underscores the importance of sampling considerations in the study of diversity in general and environmental genomics in particular. The survey found 148 new phylotypes in the already well-characterised Sargasso Sea, suggesting that increased sampling effort will reveal many rare sequences. The authors estimated that the true diversity of the samples ranged from about 1800 to 45 000 species and that far greater depth of sampling was required just to permit assembly of the genomes of the 50 most abundant species. This strongly echoes the earlier insights of Dunbar [6] and calls for a sober and strategic assessment of the resources required to truly characterise a metacommunity. If we are to command these resources then we must have a clear idea of what microbial diversity is for. Finally Mary Lunn has developed a method for estimating bacterial diversity from clone libraries with at rank abundance distributions that employs no contestable assumptions. Applied to a well-known soil dataset this method shows that it is quite unlikely that that there are less than 104 taxa in the soil from which this dataset was collected [40].

9.

10. Zhou JZ, Xia BC, Treves DS, Wu LY, Marsh TL, ONeill RV, Palumbo AV, Tiedje JM: Spatial and resource factors inuencing high microbial diversity in soil. Appl Environ Microbiol 2002, 68:326-334. 11. Leser TD, Amenuvor JZ, Jensen TK, Lindecrona RH, Boye M, Moller K: Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited. Appl Environ Microbiol 2002, 68:673-690. 12. Godon JJ, Zumstein E, Dabert P, Habouzit F, Moletta R: Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl Environ Microbiol 1997, 63:2802-2813. 13. Narang R, Dunbar J: Modeling bacterial species abundance from small community surveys. Microb Ecol 2003, Epub ahead of print. 14. Fernandez AS, Hashsham SA, Dollhopf SL, Raskin L, Glagoleva O, Dazzo FB, Hickey RF, Criddle CS, Tiedje JM: Flexible community structure correlates with stable community function in methanogenic bioreactor communities perturbed by glucose. Appl Environ Microbiol 2000, 66:4058-4067. 15. Kaewpipat K, Grady CPL: Microbial population dynamics in laboratory-scale activated sludge reactors. Water Sci 2002, 46:19-27. 16. Roling WFM, Milner MG, Jones DM, Lee K, Daniel F, Swannell RJP, Head IM: Robust hydrocarbon degradation and dynamics of bacterial communities during nutrient-enhanced oil spill bioremediation. Appl Environ Microbiol 2002, 68:5537-5548. 17. Pender S, Toomey M, Carton M, Eardly D, Patching JW, Colleran E, OFlaherty V: Long-term effects of operating temperature and sulphate addition on the methanogenic community structure of anaerobic hybrid reactors. Water Res 2004, 38:619-630. 18. Collins G, Woods A, McHugh S, Carton MW, OFlaherty V: Microbial community structure and methanogenic activity during start-up of psychrophilic anaerobic digesters treating synthetic industrial wastewaters. FEMS Microbiol Ecol 2003, 46:159-170. 19. Zoetendal EG, Akkermans ADL, De Vos WM: Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specic communities of active bacteria. Appl Environ Microbiol 1998, 64:3854-3859. 20. Vaahtovuo J, Toivanen P, Eerola E: Bacterial composition of murine fecal microora is indigenous and genetically guided. FEMS Microbiol Ecol 2003, 44:131-136. 21. Reeson AF, Jankovic T, Kasper ML, Rogers S, Austin AD: Application of 16S rDNA-DGGE to examine the microbial ecology associated with a social wasp Vespula germanica. Insect Mol Biol 2003, 12:85-91. 22. Schmitt-Wagner D, Friedrich MW, Wagner B, Brune A: Axial dynamics, stability, and interspecies similarity of bacterial community structure in the highly compartmentalized gut of soil-feeding termites (Cubitermes spp.). Appl Environ Microbiol 2003, 69:6018-6024. 23. Yang CH, Crowley DE, Borneman J, Keen NT: Microbial phyllosphere populations are more complex than previously realized. Proc Natl Acad Sci USA 2001, 98:3889-3894. 24. Smith NR, Yu ZT, Mohn WW: Stability of the bacterial community in a pulp mill efuent treatment system during normal operation and a system shutdown. Water Res 2003, 37:4873-4884. 25. Brummer IHM, Felske A, Wagner-Dobler I: Diversity and seasonal variability of beta-proteobacteria in biolms of polluted rivers: Analysis by temperature gradient gel electrophoresis and cloning. Appl Environ Microbiol 2003, 69:4463-4473. Current Opinion in Microbiology 2004, 7:221226

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. Brock T: The study of microorganisms in situ: progress and problems. In Ecology of Microbial Communities. Edited by Fletcher M, Gray TRG and Jones JG. Cambridge University Press; 1987. Wilson EO: The Diversity of Life: Penguin; 1994. Whitman WB, Coleman DC, Wiebe WJ: Prokaryotes: The unseen majority. Proc Natl Acad Sci USA 1998, 95:6578-6583. Finlay BJ: Global dispersal of free-living microbial eukaryote species. Science 2002, 296:1061-1063. Hagstrom A, Pommier T, Rohwer F, Simu K, Stolte W, Svensson D, Zweifel UL: Use of 16S ribosomal DNA for delineation of marine bacterioplankton species. Appl Environ Microbiol 2002, 68:3628-3633.

2. 3. 4. 5.

Dunbar J, Barns SM, Ticknor LO, Kuske CR: Empirical and theoretical bacterial diversity in four Arizona soils. Appl Environ Microbiol 2002, 68:3035-3045. An insightful and important paper that points the way forward because it seeks to deal with the consequences of the size of the microbial world in a quantitative way. 6.  7. Curtis TP, Sloan W, Scannell J: Estimating prokaryotic diversity  and its limits. Proc Natl Acad Sci USA 2002, 99:10494-10499. This paper represents a rst attempt at estimating prokaryotic diversity. Substantial improvements are likely to be possible. Nevertheless we suspect that many of the initial estimates will prove to be roughly right. Not because all the assumptions are perfect but because the estimates are insensitive to modest departures from the assumptions. There is a small error in equation 1 which is not carried over to the subsequent equations used in calculating diversity. A Matlab programme to solve the equations may be found at: http://people.civil.gla.ac.uk/$sloan/ www.sciencedirect.com

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26. Huisman J, Weissing FJ: Fundamental unpredictability in multispecies competition. Am Nat 2001, 157:488-494. 27. Huisman J, Weissing FJ: Biodiversity of plankton by species oscillations and chaos. Nature 1999, 402:407-410. 28. Zoetendal EG, Akkermans ADL, Akkermans-van Vliet WM, De Visser AGM, De Vos WM: The host genotype affects the bacterial community in the human gastrointestinal tract. Microb Ecol Health 2001, 13:129-134. 29. MacArthur R, Wilson E: The Theory of Island Biogeography. Princeton: Princeton Univerity Press; 1967. 30. Whitaker RJ, Grogan DW, Taylor JW: Geographic barriers  isolate endemic populations of hyperthermophilic archaea. Science 2003, 301:976-978. A delightful study and a clear demonstration that the biogeography we see depends on the level of resolution we use to demonstrate differences between taxa. 31. Hubbell SP: The Unied Neutral Theory of Biodiversity and Biogeography. Princeton: Princeton University Press; 2001. 32. Bell G: Ecology - Neutral macroecology. Science 2001, 293:2413-2418. 33. Norberg J, Swaney DP, Dushoff J, Lin J, Casagrandi R, Levin SA: Phenotypic diversity and ecosystem functioning in changing environments: A theoretical framework. Proc Natl Acad Sci USA 2001, 98:11376-11381. 34. Briones A, Raskin L: Diversity and dynamics of microbial communities in engineered environments and their implications for process stability. Curr Opin Biotechnol 2003, 14:270-276.

35. Leclerc M, Delbes C, Moletta R, Godon JJ: Single strand conformation polymorphism monitoring of 16S rDNA Archaea during start-up of an anaerobic digester. FEMS Microbiol Ecol 2001, 34:213-220. 36. Zumstein E, Moletta R, Godon JJ: Examination of two years of community dynamics in an anaerobic bioreactor using uorescence polymerase chain reaction (PCR) single-strand conformation polymorphism analysis. Environ Microbiol 2000, 2:69-78. 37. Leclerc M, Delgenes J-P, Godon JJ: Diversity of the archael  community in 44 anaerobic digesters as determined by single stranded conformation polymorphism analysis and 16S rDNA sequencing. Environmental Microbiology 2004: in press. This survey of Euryarchaeota in anaerobic reactors is remarkable because it appears to be almost complete and thus the meta-community appears to be dened. Obviously more discriminatory methods than single stranded conformation polymorphism analysis would nd more diversity. 38. McHugh S, Carton M, Mahony T, OFlaherty V: Methanogenic population structure in a variety of anaerobic bioreactors. FEMS Microbiol Lett 2003, 219:297-304. 39. Venter J, Remmington K, Heidelberg J, Halpern A, Ruscch D,  Eisen J, Wu D, Paulsen I, Nelson K, Nelson W et al.: Environmental genome shotgun sequencing of the Sargasso sea. Science 2004, 304:66-74. A technical tour de force that never the less attempts to grapple with thorny sampling issues in a manner that reveals the magnitude of our ambition. 40. Lunn M, Sloan WT, Curtis TP: Estimating bacterial diversity from clone libraries with at rank abundance distributions. Environ Microbiol: in press.

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