Phenotypic integration and developmental canalization have been hypothesized to constrain the degree of phenotypic plasticity, but little evidence exists, probably due to the lack in studies on the relationships among the three processes, especially for plants under different environments. We conducted a field experiment by subjecting plants of Abutilon theophrasti to three densities, under infertile and fertile soil conditions, and analyzing correlations among canalization, integration, and plasticity in a variety of measured morphological traits after 50 and 70 d, to investigate the relationships among the three variables in response to density and how these responses vary with soil conditions and growth stages. Results showed trait canalization decreased, phenotypic integration and the degree of plasticity (absolute plasticity) in traits increased with density. Phenotypic integration often positively correlated with absolute plasticity; whereas correlations between trait canalization and plasticity were insignificant in most cases, with a few positive ones between canalization and absolute plasticity at low and medium densities. As plants grew, these correlations intensified in infertile soil and attenuated in fertile soil. Our findings suggested the complexity of the relationship between canalization and plasticity: decreased canalization is more likely to facilitate active plastic responses under more favorable conditions; whereas increased level of integration should mainly be an outcome of plastic responses. Soil conditions and growth stage may affect responses of these correlations to density via modifying plant size, competition strength and plastic responses in traits. We also predicted that decreased canalization can be advantageous or disadvantageous, and the lack of response to stress may demonstrate a stronger ability of adaptation than passive response, thus should be adaptive plasticity as active response.

The experiment was conducted between June and August in 2007 at the Pasture Ecological Research Station of Northeast Normal University, Changling, Jilin province, China (44°45’N, 123°45’E). The environmental conditions for plant growth were very close to natural. We used a split plot design, with soil conditions as the main factor, density and block as a sub-factor. Two large plots were assigned as two (infertile and fertile) soil conditions, each was divided into nine 2 × 3 m sub-plots and randomly arranged with three treatments of densities and blocks. Seeds of A. theophrasti were sown on June 7, 2007, with three inter-planting distances of 30, 20 and 10 cm, to reach target plant densities of 13.4, 36 and 121 plants ▪ m^-2, assigned as relatively low, medium and high density treatments respectively.

Plants were harvested at 50 and 70 d of plant growth, representing developmental stages of early vegetative growth, late vegetative or early reproductive growth, and middle to late reproductive growth respectively. At each stage, six individual plants were randomly chosen from each plot, making a total of 6 replicates × 3 plots × 3 densities × 2 soils × 2 stages = 216 samplings. For each plant, the following traits were measured if applicable: the length of stem, diameter at the basal of stem, petiole length and angle, leaf number, lamina width (lamina size), branch length, angle and number, main root length, main root diameter, lateral root length and lateral root number (above or equal to 1 mm in diameter along the main root). Morphological traits of plants at 30 d of growth were not taken into account due to small plant sizes. Each individual plant was then separated into root, stem, petiole, leaf, reproductive and branch parts if any, oven-dried at 75^oC for two days and weighed.

All statistical analyses were conducted using SAS statistical software (SAS Institute 9.0 Inc. 2002). All traits were used in analyses (abbreviations see Table 1). All data were log-transformed except for petiole angles and branch angles (square root-transformed) to minimize variance heterogeneity before statistical analysis. Three-way ANOVA and ANCOVA were performed to evaluate the overall effects of growth stage, soil condition and population density and their interactions on all traits, with total mass nested in growth stage as a covariate in three-way ANCOVA. Within each soil condition at each stage, effects of density were analyzed by one-way ANOVA for total mass, and one-way ANCOVA for all the other traits with total mass as a covariate. Adjusted mean values of traits were produced from multiple comparisons by Least Significant Difference (LSD) method of the General Linear Model (GLM) program in ANCOVAs, and were used in calculation of plasticity.

The plasticity in traits was evaluated with the revised simplified Relative Distance Plasticity Index (RDPI_s), for its strong statistical power in tests of differences in plasticity (Valladares et al., 2006). We abbreviated RDPI_s to PI, and calculated it in a given trait in response to high and medium vs. low densities (H-L PI and M-L PI) as:

PI = (X – Y)/(X + Y)

where X was the adjusted mean trait value at high or medium density, and Y was the mean value at low density. Both the level and degree of plasticity (relative plasticity and absolute plasticity) in traits were calculated as PI_rel and PI_abs respectively.

Phenotypic canalization was evaluated by coefficient of variation (CV) for a given trait, calculated as the standard deviation divided by mean value of the trait. Phenotypic integration was estimated with the number of significant correlations of a trait with other traits (NC; p < 0.05) and coefficient of integration (CI; Cheverud et al., 1983). Correlations among traits were evaluated by Pearson Correlation Coefficients (PCC) produced by PROC CORR (Gianoli & Palacio-López, 2009). CI for traits was computed as:

I = [∑(λ-1)²/(n²-n)]^1/2

where n is the number of traits andλis an eigenvalue of the correlation matrix of the normalized data.

Both Correlation and regression analyses were applied to qualify and quantify the relationships between phenotypic plasticity (PI) and phenotypic canalization (CV) or integration (NC) at different densities for plants in each soil conditions at each stage. Results of correlations and regressions were also analyzed with three-way ANOVA to access effects of population density, soil conditions and growth stage and their interactions; and one-way ANOVA for effects of density on these relationships in each soil conditions at each stage.

Sheet 1 and 2 include all individual values of all trais used for calculating mean trait values for each density and soil conditions at each stage.

Sheet 3 and 4 are all values used for analyzing correlations and regressions between plasticity and canalization, integration.

Sheet 5 "note" explained all the abbreviations used in theses sheets.

abbrev.     abbrev.     trait                       unit

TM         total         total mass                   g

RMR         root/t     root mass/total mass          /

SMR         stem/t     stem mass/total mass          /

PMR         petiole/t     petiole mass/total mass        /

LMR         leaf/t     lamina (leaf) mass/total mass    /

REMR     repro/t     reproductive mass/total mass    /

H         height     height                      cm

SD         stemdia     stem diameter               mm

RL         rootl         main root length              cm

RD         rootdia     main root diameter           mm

LRL         lrootl     lateral root length             cm

LRN         lrootn     lateral root number            /

PL         plength     petiole length                cm

PA         pdegree     petiole angle                 ^o

LN         lnumber     leaf number                 /

LS         lsize         leaf size                    mm

NC-PI                 correlation between number of trait correlations and plasticity

CV-PI                 correlation between coefficient of variatin and plasticity

rel PI                 relative plasticity

abs PI                 absolute plasticity

M-L                     plasticity in response to medium vs. low density

H-L                     plasticity in response to high vs. low density

H-M                     plasticity in response to high vs. medium density