DOI: 10.5061/dryad.612jm643m
title: Associations between leaf developmental stability, variability, canalization and phenotypic plasticity in Abutilon theophrasti

description: Developmental stability, canalization, and phenotypic plasticity are the
 most common sources of phenotypic variation, yet comparative studies
 investigating the relationships between these sources, specifically in
 plants, are lacking. To investigate the relationships among developmental
 stability or instability, developmental variability, canalization and
 plasticity in plants, we conducted a field experiment with Abutilon
 theophrasti, by subjecting plants to three densities under infertile vs.
 fertile soil conditions. We measured the leaf width (leaf
 size) and calculated fluctuating asymmetry (FA), coefficient of
 variation within and among individuals (CVintra and CVinter), and
 plasticity (PIrel) in leaf size at day 30, 50 and 70 of plant growth, to
 analyze the correlations among these variables in response to density and
 soil conditions, at each of or across all growth stages. Results showed
 increased density led to lower leaf FA, CVintra and
 PIrel and higher CVinter in fertile soil. A positive
 correlation between FA and PIrel occurred in infertile soil,
 while correlations between CVinter and PIrel and between
 CVinter and CVintra were negative at high density and/or
 in fertile soil, with non-significant correlations among
 them in other cases. Results suggested the complexity of responses of
 developmental instability, variability and canalization in leaf size as
 well as their relationships, which depend on the strength of stresses.
 Intense aboveground competition that accelerates the decrease in leaf size
 (leading to lower plasticity) will be more likely to reduce developmental
 instability, variability and canalization in leaf size. Increased
 developmental instability and intra- and inter-individual variability
 should be advantageous and facilitate adaptive plasticity in less
 stressful conditions, thus they are more likely to positively correlate
 with plasticity; whereas developmental stability and canalization with
 lower developmental variability should be beneficial for stabilizing plant
 performance in more stressful conditions, where they tend to have more
 negative correlations with plasticity. 
description: The experiment used a split plot design, with infertile and
 fertile soil conditions assigned as two whole plots, each of which was
 divided into nine 2 × 3 m sub-plots, with three plant densities and three
 blocks randomly distributed. Low, medium and high densities were set up by
 sowing seeds at three inter-planting distances of 30, 20 and 10 cm, to
 reach the target plant densities of 12.8, 27.5 and 108.5
 plants·m-2 respectively. Most seeds emerged four to five days later.
 The initial densities were a little higher than the target ones, thus
 seedlings were thinned to the target densities when almost all of them
 reached four-leaf stage. Plots were hand weeded when necessary and
 regularly irrigated to prevent drought. Contrasting
 soil conditions were set up by using the soil of experimental plots as
 infertile soil, in comparison with covering a layer of more-fertile soil
 on the plots as fertile soil 14. The soil of the experimental
 field at the station (aeolian sandy soil, pH = 8.3) had been utilized
 repeatedly every year, thus in low nutrient availability: organic C 3.1
 mg·kg-1, available N 21.0 mg·kg-1, available P 1.1 mg·kg-1 during the
 growth season of 2007 47. Fertile soil conditions were
 created by covering 5-10 cm virgin soil (meadow soil, pH = 8.2)
 transported from the nearby meadow in the north of the research station,
 which had never been cultivated before, with main nutrients of organic C
 18.7 mg·kg-1, available N 47.5 mg·kg-1, available P 4.0 mg·kg-1 during the
 growth season of 2007 48. Data
 collection and analysis Plants were harvested at 30, 50
 and 70 days of growth from emergence. At each of three stages, five to six
 individuals were randomly chosen from each plot, making the maximum total
 of 6 replicates × 3 blocks × 3 densities × 2 soils × 3 stages = 324
 sampling. For each individual in each treatment, the widths of right and
 left sides per leaf were measured twice with a digital caliper for all
 leaves on the main stem, to calculate leaf size (LS, the average width of
 right and left sides) and leaf fluctuating asymmetry (FA). We attempted to
 apply almost all kinds of indexes (FA1-FA8 and FA10) to calculate leaf FA
 as an estimate of developmental stability 20,49. Results from ANOVA analyses
 indicated similar conclusions from most of the indexes. We adopted three
 of FA1, FA2 and FA10 from all indexes, as FA1 and FA2 are ones with and
 without effects of leaf size respectively, and FA10 is the only index with
 measurement error variance partitioned out of the total between-sides
 variance, with formulas as follows 20,49: FA1 =
 ∑│R - L│/n
                                                  (1-1)
 FA2 = ∑[(R - L)/LS]/n
                                                (1-2)
 FA10 = 0.798 × √ (MSsj - MSm) /
 M                                      (1-3) where
 R and L were the widths of right and left sides of a leaf, n was the total
 number of leaves, and LS (leaf size) was calculated by (R+L)/2, MSsj was
 the mean squares of side × individual interaction, MSm was the mean
 squares of measurement error, M was the number of replicate measurements
 per side, from a side × individual ANOVA on untransformed replicate
 measurements of R and L. Developmental canalization in
 leaf size was evaluated by coefficient of variation (CV, the standard
 deviation divided by mean value of the trait), including among-leaf CV
 (CVleaf) and among-individual CV (CVin). Plasticity in
 leaf size was calculated with the formula of simplified Relative Distance
 Plasticity Index (RDPIs)50. We abbreviated RDPIs to PI
 for convenience, and we also calculated the degree of plasticity in leaf
 size as absolute plasticity (PIabs), corresponding to its relative
 plasticity (PIrel): PIrel = (X – Y)/(X + Y)
                                                  (2-1)
    PIabs = | (X – Y)/(X + Y) |
                                                (2-2)
 where X was the adjusted mean leaf size at high or medium
 density, and Y was the adjusted mean leaf size at low density. Therefore,
 plasticity indexes included those in response to high vs. low density
 (PIrel-HL and PIabs-HL) and in response to medium vs. low density
 (PIrel-ML and PIabs-ML). All variables for traits were
 used in statistics, and the original data was log-transformed, petiole
 angles were square root-transformed, before any analysis to minimize
 variance heterogeneity. All analyses were conducted using SAS statistical
 software (SAS Institute 9.0 Inc. 2002). Three-way ANOVA was performed for
 overall effects of growth stage, soil conditions, population density and
 their interactions on all variables. Then we used one-way ANOVA for
 effects of density on all variables in each soil conditions at each stage
 and across all soils and stages. Multiple comparisons used LSD method in
 General Linear Model (GLM) program. For each of and across all treatments,
 correlations among all variables were analyzed with PROC CORR, producing
 Pearson Correlation Coefficients (PCC) for all correlations and Partial
 Pearson Correlation Coefficients (PPCC) for correlations among leaf FAs,
 CVs and PIs, with leaf size as covariate in partial correlation
 analyses.