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Journal of Experimental Botany, Vol. 50, No. 340, pp. 1635–1645, November 1999
Assessing the ecological significance of molecular
diversity data in natural plant populations
N. McRoberts1,4, R.P. Finch1,5, W. Sinclair1, A. Meikle1, G. Marshall1, G. Squire2 and J. McNicol3
1 Plant Biology Department, SAC Auchincruive, Ayr KA6 5HW, UK
2 Scottish Crop Research Institute (SCRI), Invergowrie, Dundee DD2 5DA, UK
3 Biomathematics and Statistics Scotland (BioSS), SCRI, Invergowrie, Dundee DD2 5DA, UK
Received 25 January 1999; Accepted 6 August 1999
Abstract
Despite extensive research for several decades, there
remains a lack of understanding of the processes that
determine the dynamics of natural plant communities.
In this paper some current concepts in vegetation
dynamics are reviewed and an attempt is made to
provide a perspective of the way in which data for
molecular diversity might be used to help in developing
an understanding of population processes. It is proposed that data from assessments of general population diversity, and specific ecophysiological traits can
be used to assess the potential for individual species
to compete and substitute for each other in a
community.
Key words: Natural plant communities, dynamics, molecular diversity, population diversity, ecophysiological traits.
Introduction
Understanding the relationships between species richness,
species diversity and community stability remains a key
area of interest in vegetation ecology. At the theoretical
level a great deal of attention has been given to how the
processes of competition and reproduction determine
interactions amongst plant species in communities.
Maintaining stability in natural or managed vegetation is
important for practical reasons relating to habitat and
species preservation and rural sustainability. There is thus
a motivation to bring theoretical results into practical use
and, as a result, a considerable amount of research has
been conducted in both the theoretical and applied aspects
of vegetation ecology.
Recent developments in molecular methods for
assessing diversity, particularly the analysis of randomly
amplified polymorphic DNA fragments, have made it
possible to obtain estimates of intra- and inter-specific
diversity from nuclear genomes of natural plant populations with relative ease. However, methods for applying
the results of such analyses to improve our understanding
of the ecology of natural vegetation are still relatively
poorly developed. This is due, in part, to the difficulties
which arise in relating molecular marker data to important ecophysiological traits in natural plant populations.
The relationship between the traits displayed by individual species and their role in the ecological community
is a further poorly researched subject. Recent publications
(Aerts, 1999; Grime et al., 1997; Wardle et al., 1998) have
shown that insights into plant community structure can
be obtained from careful study of the ecophysiology of
individual species. However, it appears that a general,
theoretical framework for this aspect of vegetation ecology is only just being constructed.
In this paper the authors draw on their own findings
from a study of species-rich grassland in Scotland, as
well as the work of others, to illustrate connections
between community structure, ecophysiology and molecular diversity in plant communities. Some issues of methodology in the use of RAPD markers for the assessment
of molecular diversity and trait variation in natural plant
populations are also addressed.
Ecological background
Stability, complexity and diversity
Relationships between stability and diversity are a central
theme in standard, modern ecology texts (Begon et al.,
4 To whom correspondence should be addressed. Fax: +44 1292 525314. E-mail: [email protected]
5 Present address: Royal College of Obstetricians and Gynaecologists, 27 Sussex Place, Regent’s Park, London NW1 4RG, UK.
© Oxford University Press 1999
1636 McRoberts et al.
1996) and are an important issue in current ecological
theory (Southwood, 1996). It is now commonly accepted
that the stability of complex biological systems (such as
species-rich vegetation) does not derive per se from these
systems containing a large number of interacting species.
Computational and analytical results generated over 25
years ago (Gardner and Ashby, 1970; May, 1974) illustrate that, if interactions between species in model ecosystems occur in a purely random way, the stability of
the system declines with increasing species numbers for a
given level of interaction (or connectance). Similarly, in
systems containing a fixed number of species, stability
declines as the level of interaction (connectance) in the
system increases. Two additional implications of these
theoretical analyses have been noted (May, 1974): (1) if
a system contains a large number of interacting species,
the individual interactions will tend to be rather weak,
and; (2) a system containing a large number of species is
more likely to be stable if it is ‘organized’ into a set of
subsystems between which there is no interaction. Both
of these points are relevant to the discussion which
will follow.
Prior to these analyses it had been proposed (for
example, Elton, 1958; Pimentel, 1961) that the presence
of many species within a habitat conferred stability directly through the buffering effects of the individual species
on each other against the effects of sources of instability.
This proposal is supported by observational evidence and
appears, superficially, to contradict the theoretical results
described above. However, as already noted (May, 1974),
natural systems are not randomly constructed. It is very
likely that the stability of complex natural systems results
from the particular subset of the possible interactions
between species that actually occurs, and the structure of
the environment in which the interactions take place.
Similar points have been made by several other authors
(Southwood and Way, 1970; Shmida and Ellner, 1984;
Tilman, 1994; Southwood, 1996). There is also empirical
evidence for a positive correlation between diversity and
stability in plant communities (Frank and McNaughton,
1991; Tilman and Downing, 1994).
Contrary to the results mentioned above, in a community of plants such as that studied at Cedar Creek by
Tilman, one might expect that interspecific competition
for resources would be greatest where numbers of species
were highest, and hence that diversity would decline over
time as stronger competitors excluded weaker ones. This
is the basic premise of the familiar competitive exclusion
principle (May, 1974).
If competition is to be accepted as an important driving
force in community ecology, one might ask whether
competitive exclusion (as conceived in theory) occurs in
the sort of grassland community which has been observed
by Tilman and others. A summary of results from the
Minnesota grassland experiments ( Tilman, 1994) illus-
trates the ability of Schizachyrium scoparium ( little
bluestem) rapidly and competitively to exclude other
species under experimental conditions, where competition
is predominantly for nitrogen only. However, in natural
conditions, where competition is also mainly for nitrogen,
little bluestem may co-exist with over 100 other species
for many years, before eventually becoming dominant.
Taking these observations together with the theoretical
results on connectance, it might be surmised that very
strong competitive effects are avoided or suppressed in
the communities studied.
The spatial patterns of competing species and interspecific trade-offs in relation to ecological traits have been
highlighted as mechanisms by which strong competition
might be avoided in multi-species communities (Crawley
and May, 1987; Shmida and Ellner, 1984; Tilman, 1994).
If different species have different and partially uncorrelated spatial patterns, the less competitive may be able to
persist by occupying patches that are not occupied by the
more competitive species. In order to maintain the
dynamic equilibrium which is suggested by this model, it
is necessary that there are interspecific trade-offs with
respect to traits determining competitive and reproductive
behaviour. A poor competitor will persist provided that
it allocates resources to producing large numbers of
propagules and that these disperse readily, allowing rapid
colonization of empty patches. Large-scale screening of
plant species in the British flora has revealed that such
trade-offs between competitive and reproductive traits do
indeed exist (Grime et al., 1997).
While much of the theoretical work on community
structure has been concerned with analytical models of
interactions amongst species, competition in the real
world occurs between individuals, or groups of individuals
of each species. In attempting to model interactions at
the level of the individual a simulation approach is often
more appropriate than an analytical approach. While
simulation models lack the generality that is provided by
analytical models, they do tend to focus on the mechanisms by which competition operates between individuals
(Benjamin, 1996) and thus to consider the traits involved
in the interspecific trade-offs discussed by other authors
( Tilman, 1994; Marshall and Squire, 1996; Grime et al.,
1997; Aerts, 1999).
Competition between ecophysiological traits
The general theory of community structure which has
developed in the last 25 years proposes that the number
of competing species which can co-exist in a habitat is a
function of the range of the available resources, the
differences between the preferred or optimum part of the
available resource that each species uses, and the stability
of the environment. The theory contends that an environment which is relatively stable over time (such as that
The ecological significance of molecular diversity data 1637
found in a tropical rainforest) allows a large number of
species to adapt to specialized niches (i.e. gain certain
sets of required traits), avoid direct competition, and thus
co-exist. The alternative, and less popular, energydiversity hypothesis proposes that species numbers are
determined not by the stability of the environment, but
by available energy. In simple terms, this theory suggest
that a large amount of available energy allows a large
number of species, which in turn forces each species into
having a narrow niche. Thus, ‘. . . tropical species have
extremely narrow niches because so many of them are
crammed into the forest!’ ( Turner, 1992).
Drawing on the ‘stability-diversity’ and ‘energydiversity’ theories outlined above, one could consider that
the traits which a species expresses determine its function
in the community, or that the available roles in the
community determine the traits that a species is allowed
to express. In either case, by examining ecophysiological
traits in species that differ in behaviour it might be
possible to understand how competition operates in complex communities. In turn, this might allow an understanding of how the communities themselves are
constructed and the factors which are important in their
long-term stability ( Tilman, 1994; Wardle et al., 1998;
Aerts, 1999).
The relationship between plant communities and their
environments has been shown to be more subtle than is
implied by the simple interpretations of the stabilitydiversity and energy-diversity theories given above
( Tilman, 1994; Aerts, 1999). In particular it has been
shown that feedback between plants and their environment through soil processes can have a significant role
in changing the environment to suit species expressing
particular sets of traits (Aerts, 1999).
When considering competition among species in terms
of traits it is useful to consider the interactions as taking
place in a trait-space in which each species occupies a
certain region determined by the values of each trait that
its members express. A simple bivariate example of a
trait-space is presented in Fig. 1 (based on data presented
by Wardle et al., 1998). Examination of Fig. 1a makes
apparent the fact that some areas of the trait-space
defined by specific leaf area and leaf nitrogen content are,
for the 20 species examined, more densely occupied than
others. If a large number of traits is examined, data
reduction methods can be used to project the multidimensional trait-space onto a small number of composite
axes (dimensions). The technical details of these multivariate methods have been described ( Krzanowski, 1990)
and a clear account of their application to ecological data
has been given (Digby and Kempton, 1987). Figure 1b
shows the results of applying principal coordinates analysis (PCO) to data for 12 traits from Tables 1, 4 and 6
in Wardle et al. ( Wardle et al., 1998) and gives an
impression of the way in which the 20 species occupy the
Fig. 1. (a) The distribution of 20 herbaceous plant species in a bivariate
trait-space: Leaf nitrogen content is plotted against specific leaf area
from the data of Wardle et al. ( Wardle et al., 1998). The error bars are
the standard errors reported by the original authors. (b) An ordination
of 20 herbaceous plant species in two principal co-ordinates derived
from data taken from Wardle et al. ( Wardle et al., 1998). Numbers
next to the data points identify individual species: 1, Achillea millefolium;
2, Anthemis cotula; 3, Brassica rapa; 4, Carduus tenuifolius; 5, Cerastium
glomeratum; 6, Chrysanthemum leucanthemum; 7, Cirsium arvense; 8,
Crepis capillaris; 9, Daucus carota; 10, Hypochaeris radicata; 11,
Leontodon taraxicoides; 12, Plantago lanceolata; 13, Ranunculus sardous;
14, Rumex obtusifolius; 15, Rumex pulcher; 16, Silene gallica; 17,
Sisymbrium officinale; 18, Spergula arvensis; 19, Stellaria medea; 20,
Taraxacum officinale. Some of the numbers have been omitted from (a)
for clarity.
1638 McRoberts et al.
trait-space defined by those 12 traits. As in the simpler
two-trait example in Fig. 1a, certain regions of the traitspace in Fig. 1b are more densely occupied than others,
suggesting that certain combinations of traits are more
common among the species examined by Wardle et al.
than others ( Wardle et al., 1998).
Considering the long-term stability of communities and
their ability to recover from periods of extreme environmental conditions, species which share similar traits
should be able to substitute for one another in the
community as a whole. Thus, the degree of potential
competition between two species might also be viewed as
their potential for mutual substitution. The most direct
and long-term competition is likely to occur between
species which behave in similar ways (i.e. display similar
sets of traits and are closest in trait-space).
Although experiments of the type conducted by Wardle
et al. ( Wardle et al., 1998) provide useful insights into
the mechanisms through which competition occurs, and
the interspecific trade-offs which exist, some caution is
required in extrapolating from the experimental situation
to the natural multi-species community. It remains unclear
exactly the extent to which the expression of ecophysiological traits might differ under natural and experimental
conditions.
Molecular versus phenotypic variation
So far, diversity has been considered from a predominantly phenotypic point of view. The problems which can
be encountered in trying to relate phenotypic variation
to underlying genotypic variation in natural plant populations have already been discussed (Bachmann, 1994).
Certainly one should expect to find considerable phenotypic variation in some species, even for populations with
an apparently narrow genetic base. Moreover, there is
good evidence that mean values of ecologically significant
traits change over time in competing plant species (Gray,
1987) and, given this, one might expect to find that the
level of underlying genetic variation will also change over
time. In the context of habitat management and maintenance, the phenotypic plasticity of plants introduces the
question of how much underlying genetic diversity it is
necessary to conserve in order to safeguard the survival
of individual species or communities. Given the potential
practical and fundamental value of reliable genetic diversity data, it is not surprising that methods for the analysis
of molecular diversity have been used increasingly to
study natural plant populations (Bachmann, 1994). In
the following section the authors draw on their own
analyses of molecular variation in natural plant populations to illustrate how correlations between diversity and
competition can be made with reference to the ecological
theory outlined above.
Molecular analysis of diversity
Reproducibility and reliability of RAPDs
A number of methods are available for analysing molecular diversity in plant populations. Recent reviews
(Bachmann, 1994; Karp et al., 1996) have discussed the
relative merits of these methods. This study will be
concerned with issues arising from the use of RAPD
(randomly amplified polymorphic DNA) markers ( Welsh
and McClelland, 1990; Williams et al. 1990). One frequently cited limitation of RAPDs, in a variety of applications in molecular genetics, is a lack of reproducibility in
the fingerprints which they produce (see, for example, He
et al., 1994; Brown and Kresovich, 1996; Karp et al.,
1996).
Experience with Agrostis capillaris, Festuca rubra, and
Rumex acetosa has shown that it is possible to generate
reproducible RAPD fingerprints (Fig. 2), but only by
placing great importance on careful repetition of robust,
experimental protocols. Even with reproducible fingerprints, however, it must be remembered that the presence
of co-migrating non-homologous markers is a possibility
( Williams et al., 1993). Developments of the basic RAPD
approach such as SCARs (sequence-characterized amplified regions) increase the reproducibility of PCR-based
marker systems and avoid the occurrence of nonhomologous markers of equal molecular weight (Paran
and Michelmore, 1993). However, increases in the complexity of the basic RAPD method reduce its ease of use
and its practicality in handling large numbers of samples.
Despite the potential problems mentioned above, the use
of RAPDs in assessing diversity and population structure
in natural plant populations is now commonplace
(Gabrielsen et al., 1997; Le Corre et al., 1997; Martin
et al., 1997). In some cases where direct comparisons
have been made between RAPDs and other types of
Fig. 2. Examples of RAPD markers from Festuca rubra plants. Lanes
1 and 14 are molecular weight markers. Lanes 2–5, 6–9 and 10–13 are,
respectively, four-replicate samples of three different F. rubra plants;
i.e. the template DNA in each lane was derived from one of four
different vegetative shoots for each plant. The markers were generated
by primer AB2–13 (5∞-CCCGATTCGG-3∞) using 5 ng DNA and 5 pmol
of primer. Amplification conditions were: 5 min at 94 °C, then 45 cycles
of (i) 94 °C for 1 min, (ii) 41 °C for 1 min, and (iii) 72 °C for 2 min.
Samples were then held at 72 °C for 5 min, then cooled to, and held at,
4 °C prior to electrophoresis.
The ecological significance of molecular diversity data 1639
marker, comparable estimates of genetic diversity have
been obtained (Finch et al., 1997; Le Corre et al., 1997;
Sydes and Peakall, 1998; Buso et al., 1998).
General analyses of intraspecific diversity
Ignoring for the moment the more difficult question of
how to use molecular diversity data as indicators of
potential phenotypic diversity, the relatively simple question of how to gather the molecular data can be addressed.
The examples used are drawn mainly from a five-year
study of the dynamics of mixed species plant communities
on unimproved upland grazing at two sites in Scotland;
Kirkton near Crianlarich in Perthshire, and Cleish, in
Fife. At each site samples were collected from a plot
50×40 m by sampling 11 randomly selected loci along 5
transects 10 m apart. The nearest plant of each species to
each sampling point was selected for analysis.
In common with analyses of genetic variation in other
natural plant populations (Bachmann, 1994, Martin et al.,
1997), these analyses have revealed high levels of diversity
in RAPD markers in the three species which were examined. Figure 3 illustrates typical results from PCO of
similarity matrices generated from small data sets for
A. capillaris, F. rubra and R. acetosa. In all three cases,
similarities among the individuals, estimated using
Jaccard’s coefficient, are relatively low (typically between
20% and 80%). When average similarities within and
between sites are considered ( Table 1) it can be seen that
although mean between-site similarities are lower than
within-site similarities, the differences are small, suggesting that there is as much variation within sites as
between sites. Note that in the plots presented in Fig. 3
the PCO axes have been chosen to illustrate the presence
of inter-site variability in the data.
The high level of variability detected among individuals
of the three species that were examined generally results
in the dominant axes of the PCO capturing a low proportion of the variation in the similarity matrix. Comparable
studies on apomictic species (Palacios and GonzàlezCandelas, 1998) and inbreeding species (Buso et al., 1998)
have shown that interpopulation (i.e. intersite) differences
can readily be identified by this method when they are
sufficiently large, in comparison with within-population
differences. Returning to the data sets illustrated in Fig. 3,
the first five principal coordinates in total in each case
accounted for: 61% (A. capillaris), 53% (F. rubra) and
49% (R. acetosa) of the variation. Multivariate analysis
of variance (MANOVA) conducted on the PCO scores
for the plants along the sets of five axes suggested that a
significant difference existed between sites only in the case
of F. rubra ( Table 1). These results were corroborated by
examining the sources of variance within the original
similarity matrices. The between-sites to within-sites variance ratios ( Table 2) were small in all three cases, but
Fig. 3. Ordinations of approximately 20 individuals from two sites of
(a) Agrostis capillaris, (b) Festuca rubra and (c) Rumex acetosa
following principal coordinates analysis of similarity matrices constructed from RAPD marker data using Jaccard’s coefficient. (&)
Plants from Kirkton, Perthshire; (#) plants from Cleish, Fife.
Table 1. Mean intra- and inter-site similarities for three plant
species sampled at two different sites
Samples are of approximately 20 individuals in each case and similarities
were calculated using Jaccard’s coefficient on 50–100 RAPD markers.
Kirkton
Cleish
A. capillaris
F. rubra
45.5
43.5
Kirkton
40.8
36.9
Kirkton
47.9
Cleish
R. acetosa
37.1
Cleish
42.9
42.4
Kirkton
44.8
Cleish
1640 McRoberts et al.
Table 2. Within and between site variances based on similarity matrices generated from RAPD profiles and the results from MANOVA
of the first five principal coordinates of each similarity matrix
Per cent variance within sitesa
Per cent variance between sitesa
Per cent variancea accounted for in first five PCO axes
F statisticb
A. capillaris
F. rubra
R. acetosa
49
51
61
2.92 (5, 13 d.f.)
46
54
53
6.99 (5,12 d.f.)
56
44
49
1.66 (5,12 d.f.)
aVariances of inter-individual similarity matrix.
bApproximate F statistic from MANOVA of differences between sites for first five PCO axes.
slightly larger in the case of F. rubra than with the
other species.
Prior to analysis of the marker data it had been
expected that greater levels of variation might be observed
in R. acetosa (an obligate outbreeder) than in A. capillaris
and F. rubra. In the A. capillaris and F. rubra, it was
expected that genetic diversity might be relatively low as
a result of either inbreeding, in the case of F. rubra, or
suppression of sexual reproduction in both species by
grazing. However, in all three species, no evidence of
clonal development was observed in the samples of plants
collected in the first year of the study. It is possible that
the relatively large inter-sample distance used in the first
year of sampling reduced the probability of detecting
clones in any of the species. Indeed, data collected for
R. obtusifolius suggested that clonal development over
periods of up to 5 years would be unlikely to extend
beyond 1 m from the initial seedling (Pino et al., 1995).
In subsequent years of this study a range of sampling
distances have been employed in order to determine
whether clonal development is present in any of the
species.
Sampling among and within genomes
Although levels of molecular variation within sites are as
high as between sites, between-site differences can still be
detected as a source of variation in the RAPD data. It is
possible to determine which markers, and therefore which
primers, are most closely associated with inter-site variation. Reanalysis of data for those primers alone can
increase discrimination between sites (results not shown).
Such judicious use of particular RAPD primers for
the analysis of diversity and population structure in
buffalograss was reported (Huff et al., 1995).
Note that the process of selecting primers that are
particularly associated with inter-site differences makes
explicit the fact that some regions of the genome appear
to differ among sites while others do not. Parallel observations can be made at a lower spatial scale. For example,
it is possible to identify primers which generate similarity
matrices that show significant correlations with matrices
of physical distance between individuals within a site.
Table 3 gives a set of results for 13 primers which were
tested on F. rubra plants from Kirkton. Of the primers
Table 3. Mantel test statistics and associated probabilities for the
significance of correlations between genetic distance matrices and
the physical distance matrix for a set of Festuca rubra plants
sampled at Kirkton, Perthshire
Correlations significant at P≤0.05 are shown in bold.
Primer
Mantel statistic
(m)
Probability (M≥m)
AB207
AB208
AB209
AB210
AB211
AB212
AB213
AB214
AB215
AB216
AB217
AB218
AB219
0.3533
−0.2831
−0.1951
0.2164
0.7035
−0.4195
0.6853
−0.1273
0.6281
−0.2970
0.6364
−0.2744
−0.4390
0.4000
0.7853
0.6750
0.2750
0.0333
0.8583
0.0417
0.7000
0.0333
0.6833
0.0500
0.7853
0.9000
examined, four (AB211, AB213, AB125, and AB217)
produced matrices of genetic distance (distance=1–similarity) which showed significant positive correlations with
the matrix of physical distances among the plants.
It follows from the observations above that, by chance,
it is possible to select different sets of primers which give
very different estimates of molecular diversity both within
and between sites. Similarly, for a given set of primers,
the variation which is detected will depend on the particular sample of plants that is used for analysis. In part,
both of these potential problems can be overcome by
maximizing the number of individuals sampled and the
number of primers used. However, it should be noted
that in making inferences about diversity in natural plant
populations at least two sources of sampling variation
occur. The genotypes which are assessed are a sample of
the available population and the molecular markers examined are a sample of all potential markers in the genome.
These sources of variation may individually, or in conjunction, introduce as much uncertainty into the use of
RAPDs for the analysis of molecular diversity as the
reported lack of reproducibility in marker profiles; however, they are issues which are not often discussed in
detail in published analyses.
In cases where a primary objective is to examine
The ecological significance of molecular diversity data 1641
differences in diversity, genotype incidence or evolutionary history among a set of distinct populations (Dawson
et al., 1993; Huff et al., 1995; Peakall et al., 1995; Gillies
et al., 1997; Buso et al., 1998), or to examine the variation
of RAPD markers with physical separation (Graham
et al., 1997), at least one level of stratification (that of
populations) is imposed on the sampling of individuals.
Within each population the aim should be to collect
a representative selection of genotypes. Irrespective of
whether the sampling within populations is conducted
according to a predetermined sampling protocol, or is
random, or arbitrary, the assumption most commonly
made, for the purposes of statistical inference, is that the
resulting sample is random. These issues were discussed
in relation to sampling from small populations of
Limomium dufourii species on the Iberian Peninsula., and
also provided a method for assessing the reproducibility
of RAPD markers (Palacios and Gonzàlez-Candelas,
1998).
In selecting the primers that will be used to generate
the molecular marker data, care is required if estimates
of diversity within populations are not to be biased. Some
preliminary screening of primers may be needed to
identify those which give reproducible marker profiles.
However, this step introduces the possibility of unintentional selection either of primers which produce a relatively high, or a relatively low, proportion of polymorphic
bands. In a recent study of diversity in wild rice, Buso
et al. (Buso et al., 1998) explicitly addressed ways in
which pre-selecting biased primers can be avoided (as did
Palacios and Gonzàlez-Candelas, 1998), but the issue is
not commonly dealt with in such a manner in similar
studies.
The difficulty in extrapolating from estimates of diversity based on DNA marker data to estimates of diversity
in adaptive characters was highlighted (Bachmann, 1994).
As he pointed out, in order to use RAPD data to make
inferences about diversity in functional genes, it is necessary to assume that levels of variation revealed by RAPDs
are representative of the genome as a whole. The fact
that RAPD markers may be distributed preferentially in
certain regions of the genome (Noli et al., 1997), and
may therefor provide estimates of diversity which vary
in their accuracy across the genome, should make one
cautious in drawing inferences about levels of diversity in
functional genes on the basis of RAPD data. The issues
of obtaining representative samples from the plant population and unbiased estimates of marker variation from
those samples are also of key importance in any effort to
relate markers to particular traits.
Associating markers with ecophysiological traits
In cultivated species, associations between RAPD markers
and traits can be established by QTL (Quantitative
Trait Locus) analysis. Essentially, the co-segregation of
markers, or groups of markers and quantitative traits in
controlled crosses is studied through the use of variance
analysis or regression techniques (Lebowitz et al., 1987;
Lander and Botstein, 1989; Paterson, 1996). In trying to
study natural plant populations in situ, there are considerable difficulties in following a standard QTL approach.
First, it is difficult to establish parent–offspring relationships among groups of plants sampled in such populations. Secondly, many traits are difficult to assess in a
non-destructive way. One partial solution to these problems is to assess plants which have been removed from
their natural habitats and grown under controlled conditions. This approach, of course, suffers from the weakness
that one cannot be certain of functional significance of
the traits in the wild, when studying their variation in
controlled experimental conditions. This problem notwithstanding, it has been possible to demonstrate correlations (not formal linkages) between RAPD markers and
relative root growth rate in F. rubra and R. acetosa plants.
In fact, given a sample of plants for which both RAPD
marker data and quantitative trait data are available, it
is straightforward to obtain initial tests for associations
between the presence of each marker and expression of
the trait.
The first step in identifying associations between
markers and traits is to select an appropriate method for
screening the large number of RAPD markers which will
be examined. The approach outlined below was found to
be useful, because it employs a graphical method and
allows explicit examination of the distribution of the trait
value in the sample of plants under study; several other
methods based around commonly available statistical
tests (e.g. t-tests, or Mann-Whitney tests) would produce
similar results.
To give a description of the method, consider a single
polymorphic RAPD marker, the incidence of which has
been determined in a sample of plants for which quantitative trait data are also available. The incidence of the
RAPD marker can be used to divide the sample of plants
into two groups, those which have the marker present
and those which do not. For each of these groups it is
possible to plot a frequency distribution of the number
of individuals against values of the trait. If the presence
of the marker is correlated with expression of the trait,
then the frequency distributions of the two groups of
individuals will tend to overlap to a smaller degree than
they would if there was no correlation between marker
and trait. A hypothetical example is shown in Fig. 4.
Taking any value of the trait from the region in which
the two groups of individuals overlap (indicated by the
vertical line in Fig. 4), two further groups can be defined.
Those that have the marker and that have values of the
trait greater than the chosen threshold value, are referred
to as the true positive proportion ( TPP). Those that do
1642 McRoberts et al.
Fig. 4. A hypothetical example of trait frequency distributions for
individuals which either have (above the x-axis) or do not have (below
the x-axis) a particular RAPD marker. In both cases the trait is
assumed to be Normally distributed in the sample. Presence of the
marker is partly, and positively, correlated with higher values of the trait.
not have the marker and which have values of the trait
less than or equal to the threshold are referred to as the
true negative proportion ( TNP). In medical diagnostic
scales testing, from where the technique and terminology
outlined here are borrowed, the TPP is known as the
specificity (of the diagnostic) and the value (1–TNP) is
known as the sensitivity. If the threshold value of the
trait is varied across many points in its measured range,
and at each point the specificity and sensitivity is recorded,
by plotting sensitivity against (1–specificity) a graphical
display of the degree of association of the marker with
the trait is generated (for example, Fig. 5c). This type of
plot is known as the Receiver Operating Characteristic
(ROC ) curve (Metz, 1978; Zweig and Campbell, 1993).
A detailed account of the method in relation the assessment of ecological indicators has been given (Murtaugh,
1996).
If marker incidence is particularly well correlated with
expression of the trait, the marker will discriminate well
between two groups of individuals in the population and
the ROC curve associated with it will tend to be bowed
toward the top left-hand corner of the plot. A marker
which has no particular association with the trait will
tend to generate a ROC curve which follows the straight
diagonal from the bottom left to top right hand corner
of the plot (as in Fig. 5f ).
As an example, consider a preliminary study of RAPD
markers associated with root growth in R. acetosa. Root
length (mm) was assessed in pot-grown R. acetosa plants
over a period of 9 weeks from March to May 1997.
Relative root growth was calculated as the difference in
root length between the start date and end date divided
by root length at the start date. Figure 6 shows ROCs
from six RAPD markers which were generated in the
analysis of diversity in R. acetosa described above. The
first five ROC curves ( Fig. 5a–e) are for markers which
were identified as positively correlated with expression of
the trait. The sixth ROC curve, included for comparison,
is from a marker not associated with the trait.
Of 114 markers screened, 11 generated ROC curves
that suggested they were correlated with the trait. The
association between this set of 11 markers and the trait
was examined further by multiple regression analysis.
Two multiple regression models were identified that
included the five markers identified in Fig. 5a–e. The
more complex model, which accounted for 81% of the
variation in relative root growth, included markers m106,
m60, m46, and m14. The simpler model which included
only m2 and m14 accounted for 64% of the variation in
relative root growth. The sources of the markers and
their approximate molecular weights are given in Table 4.
Note that two of the markers were generated by the same
primer (AB4–08). Before proceeding with the isolation
and sequencing of the markers identified in R. acetosa,
their association with root growth will be tested on a
larger sample of plants in order to eliminate the possibility
that the results reported here are a sampling effect.
Discussion
It has been shown that, in a particular class of competition
model, that specifically accounts for allele frequencies in
both intra- and interspecific competition, the effect of
increasing competition between genotypes is to reduce
fluctuations in gene frequencies (Clarke and Beaumont,
1992). Thus, at least theoretically, competition can buffer
species from the loss of genes; i.e. it can help to maintain
diversity. Clarke and Beaumont note that their findings
are due to the structure of the model which they investi-
Table 4. Summaries of RAPD markers correlated with root growth in pot-grown Rumex acetosa plants
Primer
Primer sequence
Marker identity
Marker molecular
weight (base pairs)
AB4-08
5∞-CCTCCAGTGT-3∞
AB2-19
AB4-09
AB5-18
5∞-ACGGCGTATG-3∞
5∞-TCCCACGCAA-3∞
5∞-GATGCCAGAC-3∞
m2
m14
m46
m60
m106
2400
1000
250
1500
930
The ecological significance of molecular diversity data 1643
Fig. 5. ROC curves for six RAPD markers and relative root growth in Rumex acetosa. (a–e) The markers are positively correlated with the trait.
(f ) The ROC curve is included for comparison and is for a marker not correlated with the trait.
gated, and that there are obvious cases where competition
will not act to preserve diversity (Clarke and Beaumont,
1992). Nonetheless, their work does suggest that there
need not be a contradiction between the existence of
competition and diversity within plant communities.
Continued investigation of the links between molecular
diversity and the expression of ecologically significant
traits in plant communities should allow us a better
understanding of the processes that stabilize communities
over time.
1644 McRoberts et al.
However, the stochastic nature of both the trait data
and the molecular data that are collected for such purposes should be kept in mind. There is a contradiction in
the way in which molecular data appear to be viewed in
molecular ecology. In order to establish indices of diversity or to relate marker incidence to traits, it is necessary
that there should be variation in marker incidence among
individuals in a population. However, the very existence
of this variation means that estimates of diversity, or
associations between markers and traits derived from
samples of populations and samples of markers of those
individuals, are subject to uncertainty. In molecular ecology, as in other areas of population biology (Turner,
1992), it might be wise not to ignore the impact that
this stochasticity might have on our models of population processes, particularly when dealing with small
populations.
The above reservations notwithstanding, careful manipulative experiments as advocated previously (Crawley,
1990), in conjunction with observational studies, will
provide detailed information on the competitive interactions between natural plants species, and on the expression of ecologically significant traits relating to physiology
and reproduction. The identification of markers associated with expression of such traits (Luo, 1998) offers the
possibility of extending our analysis of natural populations in situ even further, provided care is taken in
extrapolating from the results of controlled experiments
to natural conditions.
Acknowledgements
We wish to thank Ursula Bausenwein and Peter Millard of the
MacCaulay Land Use Research Institute (MLURI ) for the
root growth data. Thanks to Gareth Hughes, Bruce Marshall
and two anonymous referees for helpful comments. BioSS,
SAC, SCRI, and MLURI receive financial support from the
Scottish Executive (SERAD) which funded the work on
vegetation dynamics reported here.
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