REACTION NORMS OF Arabidopsis. III. RESPONSE TO NUTRIENTS IN 26
POPULATIONS FROM A WORLD-WIDE COLLECTION
Massimo Pigliucci* and Carl D. Schlichting
Department of Ecology and Evolutionary Biology
University of Connecticut
* Author for correspondence. Currently at: Ecology and
Evolutionary Biology, Box G-W, Brown University, Providence, RI
American Journal of Botany, in press
Key-words: Arabidopsis, phenotypic plasticity, nutrients,
The study of phenotypic plasticity, the ability of a given
genotype to express different phenotypes as environments change,
is becoming a central focus of ecological genetics and
evolutionary theory. Studying 26 populations, we found
significant among population genetic variation for eight of the
nine traits measured, as well as plasticity in four traits. The
multivariate association of the nine traits defines four major
groups of covarying characters, each of which may be plastic or
not, depending on the particular population.
key-words: Arabidopsis thaliana, phenotypic plasticity,
The primary ecological and evolutionary role of the interactions
between genotypes and environments in shaping phenotypes has been
increasingly appreciated by students of phenotypic evolution
(Schlichting 1986; Sultan 1987; Scheiner 1993). In this paper we
address two basic questions concerning reaction norms of A.
thaliana as a model system: (i) is there natural variation for
reaction norms within the species? In the past, some authors
have used only a restricted number of lines from this species
(Jones 1971) or, if more were tested (Westerman and Lawrence
1970) most of them are no longer available for further
studies. It is important to characterize the populations that are
currently available from standardized sources such as the
Arabidopsis Biological Research Center. (ii) Which groups
of phenotypic characters tend to covary in A. thaliana, and how
are these sets of covariation affected by environmental changes?
The existence of groups of character correlations has long
been pointed out, and its importance for the ecology of the
species (Clausen and Hiesey 1960; Schlichting 1986) or as
indicators of developmental constraints (Gould 1984; Stearns,
de Jong, and Newman 1991) widely debated. We demonstrate the
existence of distinct and ecologically meaningful trait
covariation in A. thaliana, as well as of genetic variability for
the response to the environment of the principal components
defined by trait covariation.
Materials and Methods
Seed stocks from 26 populations of Arabidopsis thaliana were
obtained from the Arabidopsis Biological Resource Center at Ohio
State University. Seeds were soaked in water on filter
paper in petri dishes for 24 hours to induce germination. They
were then individually transferred to 5.7 cm pots filled with
Fafard Superfine Germinating Mix, and put in a growth
chamber at a constant temperature of 25xC and an average relative
humidity of 65%. The light cycle was set at a daylength of 14h.
Two levels of nutrients were supplied, adding either 1 or 5
pellets of 14-14-14 N-P-K Slater Osmocote at potting time. Nine
characters were measured during or at the end of the ontogeny of
each plant, in order to summarize important aspects of the
phenotype of A. thaliana: bolting time, flowering time,
senescence time, height at flowering, final height, number of
basal branches, number of lateral branches, and fruit production.
A two-way analysis of variance was performed on the nine traits,
investigating the effects of the following sources of variation:
population (genetic variation for character means among
provenances); treatment (amount of phenotypic plasticity); and
population x treatment interaction (PxT, pattern of phenotypic
plasticity, or genetic variation for plastic responses
among provenances). Reaction norms (graphs of environment vs.
phenotype) were plotted for each population and each character.
To investigate the multivariate relationships among the nine
characters, and how they change with the environment, a principal
component analysis was carried out on the total data set. Sets of
covarying characters were identified by the relative loadings of
the traits on the major eigenvectors.
Results and Discussion
The univariate analyses of variance and the scatterplots of the
norms of reaction strongly indicate the presence of genetic
variation, plasticity, and genetic variation for plasticity for
several traits in Arabidopsis thaliana. In this study most of the
significant Population x Treatment interaction terms are due to a
relatively small number of populations characterized by highly
distinctive reaction norms (see Figure 1 for an example concerning
Figure 1. Population reaction norms for bolting time. Notice the
markedly different behavior of a few populations (e.g.,
Netherlands and Italy-2).
The contrasts among these populations should
therefore be a primary focus of further research looking into
both the genetic basis and the ecological significance of
differences in plasticity. Zhang and Lechowicz (1994) found
heritable phenotypic variation for flowering time in 13
populations coming from an extensive latitudinal range, together
with a positive correlation between flowering time and
reproductive output. Later flowering genotypes also showed an
increased plasticity to changes in nutrient conditions.
Therefore, they concluded that selection for later flowering
would also produce higher plasticity by correlated response.
The variation for the nine traits measured in this study was
distributed predominantly along four major axes in multivariate
space (see Table 1, below).
Table 1 Principal component analysis of the multivariate
response of nine characters measured in 26 populations of
Arabidopsis thaliana exposed to two levels of nutrients.
PC-1 PC-2 PC-3 PC-4
eigenvalue 2.7 1.8 1.4 1.2
% of expl. var. 29.5 20.0 15.0 13.1
Bolting time -0.48 +0.25 +0.37 +0.11
Flowering time +0.25 -0.04 +0.66 +0.08
Senescence time -0.20 +0.35 -0.39 +0.22
N. leaves -0.44 +0.35 +0.36 +0.11
Hgt. at flowering +0.46 0.00 +0.33 +0.02
Final height +0.15 +0.43 +0.07 -0.59
Basal branches +0.26 +0.27 +0.01 +0.67
Lateral branches +0.13 +0.53 -0.03 -0.29
N. fruits +0.38 +0.39 -0.17 +0.19
The first principal component, mostly
influenced by developmentally early characters and fruit
production, shows interesting connections among key characters in
A. thaliana. In particular, there is a non surprising direct
covariation between bolting and number of leaves: the later a
plant bolts, the more leaves it has in its rosette. At the same
time, these two traits are negatively related to height at
flowering and fruit production: this means that if the plant has
a rapid life cycle and produces a small rosette, it eventually is
able to grow taller and have a higher reproductive fitness. The
second principal component represents a compound measure of
fitness, in that final plant size, number of lateral branches on
the main stem, and total fruit output all weigh positively on it.
Therefore, larger plants have a highly branched architecture and
a higher reproductive fitness. The third principal component
points to an interesting trade-off between flowering time and
senescence time. It is useful to recall that these two traits are
defined as segments of the life cycle (from bolting to flowering
and from flowering to senescence), and therefore there is no
a priori statistical expectation for them to be correlated (and
certainly not negatively correlated). We suggest that these two
phases of the life cycle might be influenced by largely
overlapping genes with pleiotropic effects, which together might
set constraints to the total life span of the plant (if an
individual flowers later it senesces faster, and vice versa). A
fascinating possibility that remains to be explored is that such
a constraint may in fact be the result of selection for an
overall short life cycle under disturbed natural settings. The
fourth principal component also highlights an interesting
possible trade-off: plants that have a taller central stem tend
to have fewer basal branches, i.e., fewer secondary stems.
Secondary stems (otherwise known as coflorescences) represent a
developmentally open fate, in the sense that they can originate
more coflorescences, or differentiate into flowers (Schultz and
Haughn 1993). If there is a genetic switch responsible for the
two strategies, the genes involved might be controlling the
whole-plant architecture, and might be doing so by modulating
their response to environmental conditions. Further
characterization of these populations might yield very
interesting insights into the possible role of regulatory genes
that respond directly to environmental variation ("plasticity
genes", sensu Schlichting and Pigliucci 1995).
Most populations show little plasticity for any of the principal
components, and do not show much genetic variation either: their
centroids are concentrated toward the central portion of all four
components. This means that the "typical" A. thaliana (including
the laboratory lines Landsberg and Columbia) displays a not too
rapid phenology, a good reproductive output, and tend to be short
with few lateral branches or tall with few basal branches. In
contrast, values for some populations indicate genetic variation,
and very distinctive patterns of plasticity, at the multivariate
level. We have provenances that are distinct from the average
population from a genetic point of view, but do not show any
plasticity for any principal component (e.g., France-2). All
aspects of their multivariate phenotype are highly canalized
against environmental variation (at least of the type induced by
changes in nutrients). Some populations show strong environmental
effects on some principal components, while being canalized for
others (e.g., Russia-1 and Italy-1). There is no consistency
among populations for which aspects of the multivariate phenotype
are canalized or plastic, which suggests natural variability for
the degree of multivariate plasticity, and the possibility of
investigating its genetic bases by controlled crosses of these
ecotypes or by selection experiments. Finally, some populations
appear to have all their covariance sets environmentally
affected, although to different degrees (e.g., Italy-2). Again,
the existence of such a range is a promising feature for studies
aimed at addressing the very complex problem of plasticity vs.
canalization at the whole phenotype level. The debate between the
adaptive advantage of integrated responses (Schlichting 1986) and
their nonadaptive role as indicators of constraints (Gould 1984)
is fundamental to our understanding of phenotypic evolution, and
it is easy to concede points to both schools of thought, given
the complexity of the observable patterns. Clearly more efforts
in this direction are likely to yield fascinating insights into
multivariate phenotypic evolution.
We are in debt to Richard Abbott for comments
on a previous draft of this manuscript. This work was partially
supported by NSF dissertation grant DEB-9122762 and a Sigma-Xi
Research-in-Aid Grant to MP, and by NSF grant DEB-9220593 to CDS.
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