PERSPECTIVE
Ecosystem processes and interactions in a morass of diversity
James I. Prosser
Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, UK
Correspondence: James I. Prosser, Institute
of Biological and Environmental Sciences,
University of Aberdeen, Cruickshank Building,
St Machar Drive, Aberdeen AB24 3UU, UK.
Tel.: +441224 273254;
fax: +441224 272703;
e-mail: [email protected]
Received 5 March 2012; revised 14 June
2012; accepted 15 June 2012.
Final version published online 19 July 2012.
DOI: 10.1111/j.1574-6941.2012.01435.x
Editor: Max Häggblom
MICROBIOLOGY ECOLOGY
Keywords
diversity; biogeochemical cycles; interactions;
neutral theory; niche specialisation; signalling
compounds; scale; heterogeneity.
Abstract
High diversity in natural communities is indicated by both traditional, cultivation-based methods and molecular techniques, but the latter have significantly
increased richness estimates. The increased ease and reduced cost associated
with molecular analysis of microbial communities have fuelled interest in the
links between richness, community composition and ecosystem function, and
raise questions about our ability to understand mechanisms controlling interactions in highly complex communities. High-throughput sequencing is increasing the depth of sequencing but the relevance of such studies to important
ecological questions is often unclear. This article discusses, and challenges,
some of the often implicit assumptions made in community studies. It suggests
greater focus on ecological questions, more critical analysis of accepted concepts and consideration of the fundamental mechanisms controlling microbial
processes and interactions in situ. These considerations indicate that many
questions do not require deeper sequence analysis and increased phylogenetic
resolution but, rather, require analysis at smaller spatial scale, determination
of phenotypic diversity and temporal, rather than snapshot, studies. Increasing
realisation of the high richness of microbial communities, and potentially high
physiological diversity, also require new conceptual approaches.
Introduction
Axenic microbial cultures are rare in natural environments. The vast majority of microorganisms, outside laboratories, spend their lives interacting with other
microorganisms, often with plants and animals and
always with their environment. Biological interactions are
obviously essential for many of the ecosystem processes
performed by microorganisms. These range from biogeochemical cycling processes, in which the product of one
organism is the substrate for another, to more specific
interactions, traditionally exemplified by lichens, rumen
communities and symbiotic associations between plants
and nitrogen fixers or mycorrhizas.
Interactions are often described and explained in terms
of interactions between two species or functional groups.
For example, rhizobia in root nodules fix atmospheric
nitrogen, while the plant supplies rhizobia with organic
carbon. Even complex interactions that comprise biogeochemical cycles are often considered in terms of interactions between two functional groups. For example,
methanogens produce methane that is used by methanoFEMS Microbiol Ecol 81 (2012) 507–519
trophs. This view of microbial interactions in biogeochemical cycles is qualified by realisation that all cells
contain many elements, cycles of all elements are interlinked and each organism contributes to all biogeochemical cycles. It is also, traditionally, moderated by
realisation that diversity exists within microbial functional
groups and communities. Cultivated representatives of
functional groups exhibit physiological diversity, and this
may provide the basis for distributions of different representatives of functional groups and their contributions to
ecosystem function.
‘New, ridiculous diversity’
Traditional cultivation-based techniques demonstrated
high bacterial richness: large numbers of morphologically
distinct colonies grow on solid medium inoculated with
a soil suspension or seawater and further richness is
uncovered by the use of different growth media and
incubation conditions. Laboratory characterisation of
these organisms reveals their high taxonomic and physiological diversity. Estimates of richness have increased as
ª 2012 Federation of European Microbiological Societies
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508
molecular techniques for community analysis have developed. For example, cultivation-independent, 16S rRNA
gene-based techniques led to the estimates of 105–6 ‘distinct genomes’ (species?) g 1 soil or L 1 seawater (Curtis et al., 2002; Gans et al., 2005). The ability to
sequence genomes of individual cells extracted from
environmental samples (Rodrigue et al., 2009) enables
the application of techniques, such as multilocus
sequence typing (MLST) and analysis, that increase phylogenetic resolution. These will doubtless significantly
increase richness estimates. It is also possible to envisage
characterisation of communities by the analysis of complete genome sequences of individual cells rather than
the sequences of single or several genes. We may therefore soon have the technical ability, if not the need, to
distinguish 109–10 species or operational taxonomic units
(OTUs) or phylotypes g 1 soil. If this genome diversity
is linked to physiological diversity, we may find that,
like us, each individual microbial cell is different and
behaves differently.
The consequences of these developments can be exemplified by the ‘simple’ commensal interaction between an
autotrophic nitrite oxidiser growing on nitrite produced
by an autotrophic ammonia oxidiser. Laboratory studies
of a relatively small number of bacterial ammonia oxidisers reveal differences in potentially important ecophysiological characteristics, including maximum specific
growth rate, yield on, and affinity for ammonia and oxygen, carbon yield, pH and temperature growth optimum
and range, ammonia and nitrite inhibition, ureolytic
activity and salinity (Prosser, 1989; Koops & Pommerening-Röser, 2001). (Additional physiological diversity in
heterotrophs is reflected in the large number of organic
carbon compounds used in their phenotype-based taxonomy and identification.)
Molecular analysis of single genes (not MLST or genome sequences) suggests that we must consider many
hundreds of different bacterial ammonia oxidiser phylotypes and their physiological diversity. We also have to
consider the role of both archaeal and bacterial ammonia
oxidisers (Prosser & Nicol, 2008), and nitrite oxidisers
presumably exhibit similar diversity (Daims et al., 2001;
Freitag et al., 2005). This diversity may (or may not)
relate to combinations of environmental characteristics to
which each is perfectly suited, determining their environmental distribution and activity.
We can never be certain that we have measured all of
the phylogenetic or physiological diversity in a functional
group, such as ammonia or nitrite oxidisers, or in ‘total’
bacterial or archaeal communities. This unavoidable inadequacy, coupled with the dramatic decrease in DNA
sequencing costs, can lead to deeper and deeper
sequence-based characterisation of communities, merely
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
J.I. Prosser
because it is possible. (Unfortunately, there is no such
drive to achieve more in-depth analysis of phenotypic
diversity, which is equally important but technically more
difficult.) In some senses, the diversity now being measured is ridiculously high, in terms of its relevance to ecological questions and our ability to deal with it
technically and, more importantly, conceptually. Are there
important ecological questions that require characterisation of every 16S rRNA gene sequence in a sample? Will
the major deficiencies in our understanding of microbial
interactions be remedied by considering interactions
between thousands of phylogenetically and phenotypically
distinct organisms? The remainder of this article will consider some of the questions we ask, the approaches
adopted and the relevance of high levels of richness to
these questions.
The ‘big’ questions
There are many important ecological questions, and views
of their relative importance will vary, but two fundamental questions are highlighted by the ability to characterise
previously uncultivated organisms and molecular identification and phylogenetic analysis of natural microbial
communities.
(1) What determines diversity? What are the mechanisms
determining the number and relative abundances of
different phylotypes?
(2) So what? Does it matter which and how many phylotypes are present. To what extent do they (all?), and
their interactions, influence ecosystem functions, such
as nutrient cycling?
These two questions are linked, as activities and interactions will depend on environmental characteristics,
which may determine richness, but richness will also
depend on immigration, generation of genetic and phenotypic diversity, rates of ‘speciation’ and removal
through emigration and death. Ultimately, we are interested in prediction and control. Do we have sufficient
understanding to predict ecosystem function quantitatively on the basis of which and how many phylotypes
are present and can we manage ecosystems by changing
these?
Terminology
To avoid the vexed and unresolvable questions of whether
and how we can or need to define bacterial and archaeal
species, and eukaryotic microbial species, I will use the
term phylotype. This is generally what we attempt to
measure when characterising communities using molecular techniques and classifying cultivated organisms. It has
greater relevance than OTU, which by definition has
FEMS Microbiol Ecol 81 (2012) 507–519
509
Processes and interactions in a morass of diversity
operational value, whereas phylotype describes a group
that may have evolutionary significance.
It is also necessary to consider the term ‘diversity’
and to distinguish between richness, evenness, community composition and community structure. Richness
(the total number of phylotypes) and evenness (the distribution of individuals between the different phylotypes)
are actually combined in the most commonly used measure of diversity, the Shannon–Weaver diversity index,
and opposing changes in richness and evenness can
result in the same value for this, and other indices.
Thus, for example, a reduction in evenness, through
growth of, and dominance by a particular component
within the community, results in reduced values for
diversity indices, even though species richness is
unchanged. The term ‘diversity’ is therefore ambiguous,
but is often used to mean richness, without explicitly
stating this. It is feasible to assess dominance and evenness of microbial communities, even using molecular
fingerprinting techniques, although this is rarely
attempted. It is not feasible to measure richness with
these techniques (Bent et al., 2007), as numbers of
T-RFLP peaks or DGGE bands will not detect or distinguish the total number of phylotypes present in most
environmental samples at the resolution at which they
are normally studied. Rarefaction analysis can provide
estimates of richness but depend strongly on assumptions of the nature of phylotype abundance distribution
curves (Gans et al., 2005). Their use with high-throughput sequencing data is now beginning to provide more
meaningful estimates of richness, as depth of coverage
increases, but is still limited by ignorance of these distributions.
Coverage of richness increases as phylogenetic resolution decreases; for example, it requires much less
sequencing effort to obtain 50% coverage of phyla than
of genera. At the resolution usually employed (97% or
99% 16S rRNA gene (fragment) sequence diversity), we
have only a vague idea of richness, but in reality this matters little. Most environmental studies characterise communities in samples (e.g. 1 g soil, 1 L seawater) that
contain hundreds of thousands of phylotypes. The number of phylotypes present is usually not important, either
a priori (when devising the study) or a posteriori (when
analysing the data), and attention usually focuses on
community composition.
Community composition and community
structure
Community composition and community structure are
commonly used when describing microbial communities.
Community composition comprises the phylotypes
FEMS Microbiol Ecol 81 (2012) 507–519
present and their relative abundances and is what is actually estimated. The term ‘community structure’, additionally, implies that the different phylotypes are ordered,
through interactions, into a network specifically selected
in a particular environment, potentially providing optimal
‘efficiency’. That is, the term suggests that the whole is
different to the sum of the individual parts and also
implies that if one member of this network is removed,
or ceases to function, then the ecosystem will work less
‘efficiently’, be less robust or cease to function. This will
obviously be the case if at least one representative of a
particular group performing an essential ecosystem function is absent or becomes extinct. The nitrogen cycle will
obviously be affected if denitrifiers or ammonia oxidisers
are completely absent. It is less obvious when considering
extinction of one of the myriad of phylotypes that can
perform these functions.
Ecosystem function
Microbial ecosystem function typically relates to biogeochemical cycling, but many others exist, for example,
maintenance of soil structure and quality and pathogen
control (Ritz et al., 2009). These are important functions,
but are frequently ignored in microbial diversityecosystem function studies because they are difficult to
associate with a particular phylogenetic or functional
gene. Similarly, physiological characteristics targeted in
molecular studies are usually restricted to those associated
with specific functional genes, rather than potentially
more important characteristics, for example, survival,
dormancy (Lennon & Jones, 2011), growth rate, substrate
affinity or susceptibility to predation.
Evolution and ecology
Niche specialisation and differentiation
The majority of microbial community composition studies are based implicitly, but rarely explicitly, on links
between evolution and ecology, and between phylogenetic
and phenotypic diversity, embraced in the concept of
niche differentiation. They commonly involve analysis of
community composition in environments with different
characteristics or under different conditions. For example,
bacterial community composition might be measured in
soils following different fertiliser treatments, in different
regions or before and after pollution.
The rationale for such studies is that:
(1) Recombination and mutation generate new phylotypes with different phenotypes.
(2) Phylotypes whose phenotypes are better adapted to
the environment will be selected.
ª 2012 Federation of European Microbiological Societies
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510
(3) Selection increases the relative abundance of betteradapted phylotypes and, potentially, leads to the development of distinctive phylogenetic groups possessing the
new phenotype, that is, we have niche specialisation and
differentiation.
(4) Dispersal of members of the new phylotype leads to
its colonisation of, and selection in other, similar environments.
(5) Sites with similar environmental characteristics will
be dominated by phylotypes whose phenotypes are
adapted to those characteristics.
(6) Analysis of 16S rRNA genes provides information on
phylogeny, enabling both detection and identification of
adapted phylotypes and giving information on their evolution.
(7) 16S rRNA gene analysis of communities will identify
links between phylogeny, phenotype and environmental
characteristics.
(8) Analysis of functional genes provides similar tracking
of evolution and adaptation, but within particular functional groups.
For various reasons, this experimental approach has
limited value.
(1) A tenet of 16S rRNA gene analysis is that phylogeny
provides only limited information on function. Philippot
et al. (2010) discuss this with respect to ecological coherence and taxonomic rank. Sometimes there are patterns,
often there are not and, even more often, we do not
know.
(2) There are many examples of phenotypes (functions)
of environmental significance that are distributed among
many phylogenetic groups. Denitrification is represented
within approximately 50% of phylogenetic groups,
ammonia oxidisers are found within the Beta- and Gammaproteobacteria and the thaumarchaea and nitrite
oxidisers occur within the Alpha-, Beta- and Deltaproteobacteria, Nitrospirae and Nitrospina.
(3) Links between phylogeny and function are seriously
disrupted by lateral gene transfer, over both evolutionary
and ecological time scales.
(4) Plasmids often determine important, selective environmental characteristics, notably antibiotic resistance
and heavy metal resistance, but many more.
(5) 16S rRNA gene-based phylogeny discriminates at the
level of traditionally defined genera, but is much less useful
for the discrimination of species or at higher taxonomic
resolution. Significant phenotypic variation occurs within
genera; this is how species are traditionally distinguished.
(6) Finer scale resolution is possible using other molecular approaches and indicates fine scale physiological
diversity. For example, MLST analysis of cultivated organisms demonstrates that phylotypes differing in very few,
or just a single gene, can have critically different characª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
J.I. Prosser
teristics, for example, the presence or absence of virulence. There are good reasons to expect that similar
differences will exist in organisms preforming important
ecosystem functions.
(7) Functional gene analysis restricts phylogenetic analysis to particular functional groups, but gives no more
information than 16S rRNA genes on phenotypic diversity, unless adaptation results from changes in the gene
product itself.
(8) Analysis of nucleic acid-based phylogeny will not
detect epigenetic effects, in which gene expression or phenotype is determined by mechanisms that are not based
on DNA sequence. Epigenetics is rarely considered by
microbial ecologists, but is increasingly considered to be
important in plant and in animal ecology (Bossdorf et al.,
2008).
(9) It is difficult to detect effects of biological interactions
(e.g. predation) by analysing sequences.
This list is not exhaustive but demonstrates that we
know very little of the extent to which 16S rRNA gene or
functional gene diversity tell us about phenotypic diversity. Thus, even if community composition is driven by
adaptation, phenotypic diversity, differences in environmental characteristics and selection, it will be very difficult to detect links between phylotype diversity,
phenotypic diversity and ecosystem function by the analysis of 16S rRNA or functional genes.
There is also considerable, apparent, functional redundancy within microbial communities. Certainly, there are
many hundreds of phylotypes at the traditional species
level that can perform basic ecosystem processes, for
example, metabolism of simple organic carbon compounds, nitrification and denitrification. However, even if
we find two strains that appear to have identical characteristics and, by implication, identical ecosystem function,
we cannot eliminate the possibility that they will respond
differently to a new, previously untried set of environmental characteristics. It is therefore never possible to
rule out or disprove functional redundancy within phylotypes. This ‘inability to disprove’ makes meaningless the
frequently cited concept that ‘everything is everywhere
but the environment selects’ (Bass Becking, 1934). This
may be useful as a way of thinking of about niche differentiation but, as a hypothesis, it cannot be refuted and
therefore lacks value. It is not possible to measure everything (undiscovered phylotypes may be present) and it is
not possible to say with confidence that any phylotype is
absent, only that is below the detection limit. In addition,
it is usually quoted with no attempt to define ‘everything’
or ‘everywhere’. If ‘everywhere’ refers to 1 mL of lake
water, it is not possible for such a volume to contain
everything, if ‘everything’ refers to every known 16S
rRNA gene-defined phylotype.
FEMS Microbiol Ecol 81 (2012) 507–519
511
Processes and interactions in a morass of diversity
Fig. 1. Ranked phylotype abundance distributions for bacterial
communities in seven of 29 tree holes. Lines represent phylotype
abundance distributions predicted by the neutral model using
parameters derived from the smallest (50 mL) tree hole. From
Woodcock et al. (2007), with permission.
FEMS Microbiol Ecol 81 (2012) 507–519
Log10 (number of taxa, S)
Log10 (number of taxa, S)
Niche differentiation combined with interactions, notably
competition, is intuitively attractive as a mechanism
determining community composition, even if it is difficult to assess and demonstrate. Neutral theory (Bell,
2000; Hubbell, 2001) presents an alternative mechanism
in which all phylotypes in a community are assumed to
follow the same rules, regardless of phenotype, and their
relative abundances are determined by random immigration, birth, death and speciation. If speciation is considered to be negligible, relationships between numbers of
phylotypes and area (or volume) can be predicted from
knowledge of the size of the community and two parameters. The first, m, is the probability of replacing a dying
organism with one entering randomly from the source
community in the surrounding environment, rather than
reproduction within the local community. The second, h,
describes the log-series distribution that is assumed to
exist for phylotype abundance; h increases with total richness in the source community. Neutral theory therefore
combines both phylotype abundance distributions and
phylotype area distributions.
Woodcock et al. (2007) tested neutral theory predictions of the relationship between phylotype abundance in
bacterial communities (characterised by 16S rRNA-DGGE
analysis) and the volume (rather than area) of tree holes
(the water-filled holes remaining after tree death). The
two parameters, m and h, were estimated from data for
the smallest tree hole and then used to predict phylotype
abundance in other tree holes. Figure 1 shows the quality
of fit of predicted and experimental phylotype abundance
distributions over a wide volume range. Relationships
between phylotype abundance and tree-hole volume
determined empirically were almost identical to those calculated by neutral theory using the estimated values of m
and h (Fig. 2a and b) and there was a good fit between
observed and predicted phylotype richness (Fig. 2c). Most
of the variability in richness could therefore be explained
solely on the basis of two parameters, without consideration of environmental differences (similar ecosystems in
close proximity were characterised), links between phylogenetic and phenotypic diversity, niche differentiation or
interactions. Predictions were based on the assumption
Predicted number of taxa, S
Neutral theory
1.8
(a)
1.4
1
S = 2.11V 0.26
0.6
1.5
1.8
2.5
3
3.5
Log10 (treehole volume, V)
2
4
4.5
(b)
1.4
1
S = 2.19V 0.25
0.6
1.5
2
2.5
3
3.5
4
Log10 (treehole volume, V )
4.5
30
(c)
25
20
15
10
5
5
10
15
20
25
Observed number of taxa
30
Fig. 2. Bacterial taxon richness in 29 tree holes. (a) Observed
bacterial richness in all tree holes and a solid line representing the
power–law relationship, S = 2.11 V0.26, fitted empirically using linear
regression. (b) Bacterial richness predicted by the neutral model (see
legend to Fig. 1) and a solid line representing the power-law
relationship, S = 2.19 V0.25, fitted using linear regression. (c)
Observed and predicted richness in each tree hole. Perfect fit is
represented by the line, and the two squares indicate where the
neutral model was rejected. From Woodcock et al. (2007), with
permission.
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512
that phylotype abundance is described by a log-series distribution, which is not possible to determine using DGGE
(as discussed earlier), but neutral theory offers the most
parsimonious explanation for phylotype abundance relationships with tree-hole volume.
Ofiţeru et al. (2010) extended this approach using
T-RFLP time series data on ‘total’ bacterial communities
and bacterial ammonia oxidiser communities in a wastewater treatment system. This gave more information on
phylotype abundance distributions, and the basic neutral
model was extended to allow variation in growth rates of
different phylotypes, potentially through differences in
phenotype associated with particular environmental factors. Variability arising from a purely neutral model and
additional variability because of environmental factors
could therefore be estimated. The proportion of variability indicated by the purely neutral model was 0.2 and
0.23 for the bacteria and ammonia oxidisers, respectively,
and increased to respective values of 0.28 and 0.37 with
inclusion of major environmental factors. Substantial variation remained unexplained, suggested reasons being
effects of unmeasured environmental factors, more complex effects of environmental factors, measurement error
and lack of relationship between T-RFLP-defined phylotypes and ecotypes (i.e. between phylogeny and function).
Neutral theory and niche theory are not necessarily
mutually exclusive but the former is usually ignored. It is
sometimes criticised for its simplicity, but simplicity is
arguably a major aim of models, hypotheses and theories.
At the other extreme, consideration of the many possible
phenotypic characteristics that might be influenced by
many environmental factors in different ways in many
thousands of phylotypes is unlikely to be technically or
conceptually tractable, and unlikely to generate insight,
understanding or predictive power.
J.I. Prosser
logeny and function involve ‘snapshot’ sampling and correlation of community composition and environmental
characteristics. Temporal investigations are of greater
value, but we know little of the rates of invasion, adaptation and selection in natural environments and, importantly, even less about the rates of decrease in abundance
of phylotypes that are ‘de-selected’ following environmental change. In this respect, survival, dormancy and resuscitation rate may be more important in determining
community composition than growth parameters such as
maximum specific growth rate and substrate affinity or
more easily measured physiological characteristics associated with functional genes.
The impact of individual phylotypes
It is therefore dangerous to assume that phylogeny is
linked to function and that links, even if they exist, can
be determined using experimental approaches currently
or imminently available. Nevertheless, let us consider a
hypothetical stable community containing phylotypes
whose phenotypic characteristics are perfectly adapted to
their immediate, stable environment. How might we
quantify the impact of a particular phylotype on ecosystem function, in an attempt to assess the importance of
richness in complex natural ecosystems?
One approach is to apply the concept of metabolic
control theory (MCT; Kacser et al., 1995). This can be
illustrated (Fig. 3) by imagining conversion of a substrate,
Xo, through several intermediate substrates, S1–S3, to a
product S4, catalysed by enzymes E1–E4. MCT quantifies
the influence of each enzyme on flux through the pathway as a flux control coefficient, defined as the proportional increase in flux resulting from a proportional
increase in enzyme concentration. Enzymes at low
Temporal heterogeneity and scale
The degree to which communities reflect environmental
characteristics is also influenced by differences in temporal scales of community change and environmental
change. Some factors, for example, ocean salinity or subsurface temperature, are stable over relatively long time
scales. Others change regularly over short time scales, for
example, diurnal cycles of light and temperature in surface waters or upper soil layers. Other events are much
less predictable and more variable, for example, changes
in concentrations of many nutrients, invasion, predation
and rainfall. Niche differentiation and selection through
competitive interactions assume relatively constant conditions, or at least environmental conditions that prevail
for sufficiently long to enable adaptation, selection and
detectable community change. Most attempts to link phyª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Fig. 3. Calculation of flux control coefficient for enzyme E2 in a
linear metabolic pathway converting substrate Xo to product S4, via
three intermediates.
FEMS Microbiol Ecol 81 (2012) 507–519
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Processes and interactions in a morass of diversity
concentration might be expected to have greater control
than those at high concentration. All flux control coefficients will sum to 1, and the average flux control coefficient for the pathway in Fig. 3, which contains four
enzymes, will be 0.25. If E2 has a flux control coefficient
of 0.97, and therefore significantly greater control (equivalent to the traditional, qualitative concept of a rate-limiting step), the remaining enzymes will have an average
control coefficient of 0.01, and negligible control. Doubling the amount of E2 will significantly affect flux, while
changing amounts of E1, E3 or E4 will have little effect.
If we imagine a typical metabolic map of a cell, and
assume 100 enzymes, the average flux control coefficient
for each enzyme will be 0.01. If we apply this concept to
an equivalent map of all biogeochemical cycling processes
occurring within an ecosystem, the average control of a
particular functional group will equal the inverse of the
number of functional groups contributing to ecosystem
function. So, for example, production of N2 will depend
on denitrifiers, but also on other groups active in the
nitrogen and carbon cycles that provide them with their
major substrates. All will therefore have flux control coefficients for N2 production. The impact of denitrifier biomass, per se, on N2 flux will therefore depend on flux
control coefficients for functional groups directly involved
in nitrate reduction, but also those involved in supply of
carbon and other essential nutrients. The average impact
of an individual process or functional group on an ecosystem function will therefore be very low.
The situation is much more complex in microbially
mediated ecosystems than metabolic pathways because
each step in a nutrient cycle is mediated by many 10s or
100s or even 1000s of phylotypes. The influence and
importance of a particular phylotype involved in a function will therefore be, on average, very, very low. One
phylotype may be important, that is, may have a high
flux control coefficient, but, if so, the average contribution of other phylotypes will be negligible. This approach
has been tested for simple microbial interactions in a ‘stable’ system (Allison et al., 1993) but is more difficult to
apply to non-equilibrium, growing systems (Röling,
2007), and flux control coefficients will also be subject to
environmental change. Nevertheless, it predicts that most
phylotypes will have negligible impact on ecosystem function and if we double the biomass of the average phylotype, it will have no measurable impact. Analysis of the
effects of richness or community composition on ecosystem function may therefore not require increasing expenditure on the analysis of phylotypes that are present at
low relative abundance and greater effort should be
expended on finding ways to determine which phylotypes
control function. This has particular implications for our
ability to manipulate and manage microbial communities.
FEMS Microbiol Ecol 81 (2012) 507–519
Does richness matter for ecosystem
function and interactions?
Network analysis
Interactions within an ecosystem have been represented
as networks linking pairs of phylotypes based on the
strength of associations between each pair, quantified by
the correlation of phylotype relative abundances in time
series data (Fuhrman & Steele, 2008; Steele et al., 2011)
and following environmental change (He et al., 2012).
Fuhrman & Steele (2008) used network analysis to investigate interactions within marine bacterial communities.
A threshold value (equivalent to a P-value associated
with correlation analysis) was chosen to distinguish
phylotypes that were interacting ‘significantly’ with each
other and also with environmental variables that had
greatest influence. Analysis is illustrated by networks of
SAR11 OTUs, and biotic and abiotic environmental variables (Fig. 4). An interesting feature is that a single
phylotype interacts with only a limited number of others, a maximum of 11 in this example. One interacted
with only one other phylotype and two showed no significant interactions, although they presumably interact
with phylotypes below the detection limit or not targeted. The analysis highlights the importance of quantifying the strength of interactions to assess which are
significant and shows that the majority of interactions
will be insignificant and immeasurable. He et al. (2012)
describe a similar approach, Random-Matrix Theory,
using microarray data on soil communities subjected to
elevated carbon dioxide.
Basic network analysis is empirical and suffers the disadvantages of any association- or correlation-based
method. The complexity of the network will depend on
the threshold value used to assess significance of associations and phylotypes and their links with abiotic factors
will be restricted to those that are measured, which may
not include the major drivers. It does have the potential,
however, to uncover unsuspected interactions between
phylotypes that might then be the subject of more rigorous and critical investigation. More importantly, exploration of network characteristics may reveal mechanistic
control of system properties. For example, complexity
(average numbers of significant associations per phylotype) may be linked to stability; connectance (the proportion of potential associations observed) may be linked to
behaviour; focal phylotypes (those associated directly with
a high proportion of other phylotypes) may be critical for
ecosystem functioning (analogous to keystone species);
and other aspects of networks (nestedness, compartmentalisation) may reflect different behaviours and interactions. Network analysis therefore provides the potential
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514
J.I. Prosser
Fig. 4. A network representing significant (local similarity analysis correlation, P < 0.05) interactions between SAR11 (asterisked circles) and
other bacterial phylotypes (circles) and abiotic (hexagons) and biotic (squares) environmental factors in samples taken from the San Pedro Ocean
Time Series site. Solid and dashed lines represent positive and negative interactions, respectively, arrows represent 1-month time lag correlations
and numbers on lines are correlation values. See original publication for details of phylotypes and environmental factors. From Fuhrman & Steele
(2008) with permission.
for investigation of fundamental ecological concepts using
microbial communities.
Richness reduction
While there is evidence that many microbial communities
are sensitive to environmental change and perturbation,
the importance of community composition, and particularly richness, for ecosystem function is less clear (Allison
& Martiny, 2008). This is partly because of technical difficulties in distinguishing effects of environmental conditions and community composition on function. Richness
of complex natural communities can be reduced by
removal of a portion of the community, through partial
killing or dilution, followed by incubation to enable reestablishment of the original biomass concentration. In one
example (Griffiths et al., 2000) progressive reduction in
richness by chloroform fumigation did not affect broadscale function (e.g. decomposition) but reduced rates of
more specific functions (nitrification, denitrification,
methane oxidation). Resilience of all processes, however,
decreased with decreasing richness. Girvan et al. (2005)
reduced diversity by addition of benzene or copper sulphate to soil. Benzene had a greater effect on microbial
community composition than copper sulphate, and there
was evidence for a link between diversity and resistance
to perturbation. Richness reduction again did not affect
broad-scale function (wheat shoot mineralisation)
but reduced narrow-niche function (mineralisation of
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2,4-dichlorophenol), with greater resilience in the more
diverse soil.
Fumigation methods are potentially selective and require
uniform penetration of killing agents throughout soil.
These limitations may be reduced by inoculation of sterilised soil with dilutions of a soil suspension obtained from
the same soil. Using this approach, Griffiths et al. (2001)
and (Wertz et al., 2006) found that richness did not influence a wide range of broad and specific soil functions,
unless dilution led to complete extinction or biomass
recovery was incomplete (Wertz et al., 2006). Richness also
had no effect on resistance and resilience of soil denitrifier
and nitrite oxidiser communities (Wertz et al., 2007).
Both of these approaches have disadvantages and reestablishment of communities may select for faster growing
organisms. Nevertheless, the results, particularly those
from dilution-based experiments, suggest that the species
present at high relative abundance are capable of performing major ecosystem functions, even under transient
conditions, as long as they are present and at sufficient
biomass concentration. It suggests, again, that greater evidence for the importance of less abundant species on ecosystem function is required before significant resources
are expended on their characterisation.
Community construction
Richness–ecosystem function relationships can also be
investigated by the construction of communities with
FEMS Microbiol Ecol 81 (2012) 507–519
515
Processes and interactions in a morass of diversity
defined composition. For example, Langenheder et al.
(2010) isolated six bacteria from a soil sample that could
grow on glucose, xylose or galactose as the sole carbon
and energy source. These were used to construct communities with all levels of richness (1–6) in all possible combinations. Each isolate was present at the same biomass
concentration, and the system was designed to maintain
evenness during incubation on all possible combinations
of the three carbon sources. This enabled investigation of
the effect of richness, species composition and environmental (substrate) complexity on broad ecosystem function (respiratory activity). Richness–function relationships
were initially linear but then became saturating at species
and substrate richness values of 4 (Fig. 5) and 2, respectively. Species richness saturation was presumably due to
functional redundancy or negative interactions among
competitors. Effects of species and resource richness were
independent, suggesting complex, species–substrate-specific interactions that, unfortunately, prevent widely applicable prediction of behaviour.
These strains were isolated from the same soil and
potentially competed for the same organic carbon substrates, but results indicate that high natural richness will
not influence the rate of organic C degradation. Changes
in relative abundance or extinction of phylotypes may
therefore be less important than physiological flexibility
within existing communities, which may maintain ecosystem function without the requirement for community
change. Using a similar approach, Wittebolle et al. (2009)
constructed microcosms with communities containing 18
denitrifier strains, but with differences in initial evenness.
Denitrification activity during incubation and growth of
the communities increased with evenness, particularly
under stress conditions. This study highlights the different
aspects of diversity that must be considered and potential
differences between growing communities, in which relative abundances will change, and ‘stable’ communities.
Spatial heterogeneity and
microenvironments
Microbial cells in natural environments are rarely distributed uniformly. This is most evident in soil and sediments, but is also true for planktonic marine
communities, where particulate material (marine snow)
provides foci for microbial growth and activity. Spatial
distribution in soil is illustrated in Fig. 6 (Nunan et al.,
2003). In topsoil (Fig. 6b), cells appear to form communities of different size containing (presumably) interacting
cells. In subsoil, cells are rare (Fig. 6c) and ‘uniform’ colonisation is only approached when soil is supplemented
with high levels of organic carbon (Fig. 6a). Only a fraction of the soil surface is colonised (Grundmann, 2004),
and the number of cells in an area of 0.282 mm2 ranged
from approximately one in subsoil to six to eight in
cropped soil (Nunan et al., 2003). If cells are homogeneously distributed, therefore, distances between cells will
be great in relation to cell size. Heterogeneous distribution means that interacting cells and microcolonies will
be separated even further and these calculations apply to
all cells that can be detected microscopically. Intercellular
distances between members of particular subgroups or
Fig. 5. The influence of species and resource richness on respiratory activity of defined bacterial communities. The boundaries of boxes indicate
the interquartile range and median, whiskers indicate 1.5 times the interquartile range and open circles indicate outliers. From Langenheder et al.
(2010), with permission.
FEMS Microbiol Ecol 81 (2012) 507–519
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516
(a)
(b)
(c)
Fig. 6. Bacterial colonisation of soil after the addition of glucose (a),
in topsoil sampled after harvest (b) and in subsoil (c). The scale bar
represents 20 lm. From Nunan et al. (2003), with permission.
functional groups will be even greater. For example, distances between 2,4-D degraders and nitrite oxidisers were
estimated to be in the sub-millimetre range (Grundmann,
2004). These large intercellular distances exist despite high
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J.I. Prosser
cell concentrations (107–9 cells g 1 soil) and high numbers of phylotypes. There is very little information on
community composition of soil microcolonies, but presumably some will contain monocultures and others
mixed cultures of interacting phylotypes.
Colonisation of 1 g soil by unicellular microbes may be
seen as analogous to our colonisation of the earth. Each
system contains several billion individuals and each contains communities of varying size. Interactions within
both communities can be envisaged, but interactions
between spatially separated organisms will obviously be
restricted and dependent on a wide range of factors. The
probability of two cells within a gram of soil meeting
may be similar to that of two humans meeting, but it will
certainly be very low and the scope for significant interaction will be similarly low.
If microbial interactions are mediated by soluble compounds (substrates, products, antibiotics, signalling compounds), the probability of these compounds reaching
target organisms at effective concentrations must be considered. In soil, this will depend on intercellular distance,
diffusion, production rate, path length, tortuosity, pore
structure, moisture content, bulk flow and compound
degradation. As a consequence, the majority of such
interactions may be restricted to the 10–100 lm scale,
with organisms interacting within soil pores or microcolonies. If so, interactions are likely to involve only a limited number of phylotypes and the observations described
earlier (‘saturation’ of effects on ecosystem function at
low richness) may apply at the scale over which interactions are taking place.
These considerations have enormous implications, conceptually and experimentally. The latter are obvious, but
usually ignored. If interactions operate at the 10–100 lm
scale, then it will be difficult to gain understanding using
techniques that destroy spatial structure (i.e. lysing all
cells and solubilising nucleic acids) and analysing 1 g soil
samples. (It would be equally difficult to understand
human interactions, or interactions between nations, by
homogenising the total human population and analysing
a small subsample.) Network analysis may give hints of
interactions but more rapid progress requires study at the
spatial scale at which interactions operate. This may be
more difficult technically, but much more relevant and
potentially much more rewarding.
Signalling compounds are thought to mediate many
microbial social interactions (see reviews by Keller &
Surette, 2006; West et al., 2007). Such compounds control production of exoenzymes, biosurfactants, antibiotics
and exopolysaccharides that are presumed to be of ecological significance. Discovery of their widespread occurrence (e.g. acyl-homoserine lactone and oligopeptide
quorum sensing molecules in Gram-negative and
FEMS Microbiol Ecol 81 (2012) 507–519
517
Processes and interactions in a morass of diversity
Gram-positive bacteria, respectively) promised insight
into interactions within natural communities, but few
studies have assessed their in situ environmental relevance. In heterogeneous natural environments, the impact
of signalling compounds will depend on their transport
and degradation, as indicated above, but also on specificity of action in complex communities and potential alternative functions (Redfield, 2002; Hense et al., 2007). They
therefore illustrate the more important conceptual implications of spatial heterogeneity. The scale at which interactions operate will depend on the mechanisms involved
and those mediated by signalling compounds are likely to
occur at small spatial scale. Dispersal of soil organisms
through motility, bulk flow, belowground transport (soil
animals) and above-ground dispersal (birds, wind, aeroplanes) will operate at widely different spatial scales.
The importance of spatial scale is illustrated in a single
study of signalling in Bacillus subtilis in which competence and production of surfactins, extracellular enzymes
and exopolymers are controlled by quorum sensing
through an extracellular pheromone. Bacillus subtilis
strains are grouped into pherotypes; members of a particular pherotype can induce competence in members of the
same pherotype, but not in members of other pherotypes
(Ansaldi et al., 2002; Mandic-Mulec et al., 2003; Stefanic
& Mandic-Mulec, 2009), providing a potential mechanism for diversification. It could therefore be predicted
that competition between different pherotypes would lead
to competitive exclusion and a reduction in richness, but
only at the (small) scale at which interactions are taking
place. To test this, Stefanic & Mandic-Mulec (2009)
found four pherotypes represented in B. subtilis isolates
obtained from two closely located 1-cm3 soil samples.
Pherotype richness decreased in subsamples of decreasing
size, as predicted, but even 1/16-sections often contained
more than one pherotype. This gives an indication of the
spatial scale at which interactions involving signalling
compounds, and other soluble compounds, might operate
but community analyses are normally performed at scales
(e.g. 1 g, 1 L) that are much greater than those at which
communication will take place. Interestingly, the four
pherotypes found by Stefanic & Mandic-Mulec (2009) in
a Slovenian riverbank soil are the same as those found in
North American desert soil samples. This indicates additional large-scale dispersal mechanisms, high migration
rates and lack of geographical isolation, as observed previously (Roberts & Cohan, 1995). Further genetic and
physiological analysis suggests a strong, but not complete
link between pherotypes and ecotypes (Stefanic et al.,
2012).
Mechanisms controlling interactions will therefore
determine the scale at which they influence community
composition and processes. Biogeographical studies and
FEMS Microbiol Ecol 81 (2012) 507–519
network analysis approaches must consider this, not just
when interpreting results, but also when planning experiments. Similarly, biogeographical effects seen at scales of
metres or larger are likely to result from larger scale environmental effects, rather than small-scale interactions.
Measuring biogeographical patterns of phylotypes without
regard to the mechanisms that might be driving interactions and other driving forces associated with those phylotypes is likely to generate descriptive findings, rather
than understanding, insight and generic theories. A further important factor is the need for approaches for scaling up small-scale interactive effects in heterogeneous
environments to the scales at which we are interested in
measuring processes.
Conclusion
The aim of this essay is not to provide an in-depth,
comprehensive analysis of the various topics discussed.
This is outside its scope and would require major
reviews or textbooks. Many of the citations provide evidence that could be countered by evidence from other
studies and many of the questions raised do not have
‘correct’ answers. Rather, the essay is intended to
encourage reflection on the basis for studies on microbial communities, interactions and ecosystem function.
It challenges the rationale for some studies of microbial
communities, suggests the need for greater clarity in
what we mean by diversity and why we are studying it
and suggests the importance of hypothesis-driven
approaches, before deciding whether resource-intensive,
if ‘cutting-edge’, techniques are relevant. It may be
increasingly easy to determine the in-depth community
composition through deeper and deeper sequencing, but
this may not be required to address many important
ecological questions. Certainly, it is unlikely that 1 g soil
or 1 L seawater requires 100 000 different 16S rRNA
gene-defined phylotypes to function efficiently and
understanding of microbial interactions requires much
greater consideration of environmental heterogeneity and
the spatial scale at which interactions are mediated. Similarly, it is unlikely that a community within 1 g soil or
1 L seawater is exquisitely adapted to and selected for
the unique combination of physicochemical and biological characteristics unique to that environment. If niche
specialisation and differentiation drive community composition in natural environments, sequence diversity data
are redundant without knowledge of the degree to which
sequence diversity translates into true phenotypic diversity and without knowledge of temporal changes in communities and links to processes. In the rush to obtain
and analyse data, it is easy to sweep difficult issues
under the carpet. This applies to both methodological
ª 2012 Federation of European Microbiological Societies
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518
issues, for example, biases and experimental design, and
conceptual issues, for example, neutral theory and relevance of 16S rRNA gene phylogeny to function. Techniques are available to characterise vast diversity within
microbial communities. In assessing the links between
microbial community composition, interactions and their
relevance to ecosystem function, the challenge is to identify key questions and to address them through sound
conceptual approaches and with appropriate techniques
and experimental design.
Acknowledgements
I am very grateful for helpful reviewer comments on this
manuscript.
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