DOI: 10.5061/dryad.ncjsxksx1
title: Morphological integration, canalization, and plasticity in response to emergence time in Abutilon theophrasti

description: The relationships between trait plasticity and canalization, and between
 phenotypic integration and plasticity, have been under debate, largely
 because direct evidence is still scarce for their associations, especially
 in response to environments. To investigate the relationships between
 canalization, integration, and phenotypic plasticity in response to
 emergence time, we conducted a field experiment with an annual herbaceous
 species of Abutilon theophrasti, by subjecting plants to four treatments
 of emergence time (spring, late spring, summer, and late summer), to
 measure several morphological traits and analyze correlations of
 plasticity with canalization and integration in these traits, at two
 stages of plant growth. Results showed plants with delayed emergence had
 higher phenotypic integration and more positive correlations between
 integration and plasticity, but less negative correlations between
 decreased canalization and plasticity, compared to those that emerged in
 spring. Results suggested significant environmental changes that induce
 plastic responses, rather than environmental stress, can result in greater
 phenotypic integration in plants. Negative correlations between decreased
 canalization and plasticity occurred more frequently in plants emerging in
 spring and the least frequently in those emerging in summer, suggesting
 their relationship depends on specific environmental conditions and the
 degree of plasticity. Both increased phenotypic integration and decreased
 canalization might merely be the outcome of plastic responses, rather than
 mechanisms constraining or facilitating plasticity.
description: <strong>Experimental design</strong>
 We conducted the field experiment in 2007 at the Pasture
 Ecological Research Station of Northeast Normal University, Changling,
 Jilin Province, China (44°45’ N, 123°45’ E). The original soil of the
 experimental field (aeolian sandy soil, pH = 8.3) at the station had been
 used annually for many years, with nutrients availability of organic C 3.1
 mg kg<sup>–1</sup>, available N 21.0 mg
 kg<sup>–1</sup>, and available P 1.1 mg
 kg<sup>–1</sup> during the growth season of 2007. Seeds of
 <em>A. theophrasti </em>were collected from local wild
 populations near the research station in late August 2006 and dry stored
 at -4°C. We applied a randomized block design, with emergence timing (ET)
 as the main factor, and block as the sub-factor. The whole plot was
 divided into twelve 2 × 3 m sub-plots, which were randomly assigned with
 four ET treatments and three blocks. Plants of <em>A. theophrasti
 </em>were grown on June 7, June 27, July 17, and August 7, to make
 them emerge in different periods of the season, as four ET treatments of
 spring (ET1), late spring (ET2), summer (ET3) and late summer (ET4). The
 treatments of emergence timing accorded with the time range of emergence
 of <em>A. theophrasti</em> in its natural habitats in
 northeast China. Most of the seeds emerged four days after sowing. Seeds
 were sown at an inter-planting distance of 10 cm, and seedlings were
 thinned at the four-leaf stage. Plots were hand-weeded when necessary and
 watered regularly to prevent drought.
 <strong>Data collection</strong>
 For each ET treatment, we arranged two times sampling (at days 50
 and 70 of growth; Appendix S1; see Supplemental Data with this article),
 according to the lengths of their life cycles, generally at the stages of
 vegetative growth, late vegetative or early reproductive growth, and
 middle to late reproductive growth respectively. For each sampling, five
 to six individual plants were randomly chosen from each plot, making a
 maximum total of 6 replicates × 3 blocks × 4 treatments × 2 samplings =
 144 samplings. For each plant, the following traits were measured (if
 applicable): main root length, diameter at the basal of the main root,
 length and number of lateral roots (above or equal to 1 mm in diameter
 along the main root), the length of stem, diameter at the base of stem,
 petiole length and angle, leaf number, leaf size (lamina width), branch
 length and angle, and branch number (Appendix S1; see Supplemental Data
 with this article). Each plant individual was then separated into roots,
 stems, petioles, laminas, reproductive modules, and branches (if there
 were any), oven-dried at 75°C for two days, and weighed. Reproductive
 modules consisted of flowers and fruits produced along the main stem and
 branches, and branches included the stems and leaves on branches. Total
 mass and mass allocation traits were calculated.
 <strong>Statistical analysis</strong>
 Statistical analyses were conducted using IBM SPSS statistics 19.
 All measured and calculated traits were used for analysis (Appendix S2;
 see Supplemental Data with this article). To minimize variance
 heterogeneity, all data were log-transformed, except for petiole angles
 and branch angles (square root-transformed), before statistical analysis.
 For plant total mass, we applied two-way ANOVA to analyze the effects of
 emergence time, growth stage, and their interactions, and one-way ANOVA to
 analyze the effects of emergence time or growth stage within each or
 across all of the other treatments. Plant size (e. g. total mass) can have
 very significant effects on other traits, which may bias the effects of
 emergence time. Therefore, for all the other traits, we applied two-way
 ANCOVA to evaluate the overall effects of emergence time, growth stage,
 and their interactions, and one-way ANCOVAs for effects of emergence time
 or growth stage within each or across all of the other treatments, with
 total mass as a covariate. Multiple comparisons used the Least Significant
 Difference (LSD) method in the General Linear Model (GLM)
 program. For each trait, the percent number of
 significant correlations of it with other traits (NC;
 <em>p</em> &lt; 0.05) was used as the index of phenotypic
 integration, and NC values were arcsine- and square-root-transformed
 before analyses; coefficient of variation (CV) was used to evaluate the
 level of canalization, which was calculated as the standard deviation
 divided by the mean value of the trait. Both the level and degree of
 plasticity (relative plasticity and absolute plasticity,
 PI<sub>rel</sub> and PI<sub>abs</sub>) for each
 trait were calculated with the revised simplified Relative Distance
 Plasticity Index (RDPI<sub>s</sub>, abbreviated as PI), for
 its strong statistical power in tests of differences in plasticity
 (Valladares et al., 2006), with the formula as:
 PI<sub>rel</sub> = (<em>X</em> –
 <em>Y</em>) / (<em>X</em> +
 <em>Y</em>) PI<sub>abs</sub> =
 |<em>X</em> – <em>Y</em> | /
 (<em>X</em> + <em>Y</em>) where
 <em>X</em> was the adjusted mean trait value in the treatment
 of earlier emergence (ET1, ET2, and ET3), and <em>Y</em> was
 the adjusted mean value in the treatment of delayed emergence (ET2, ET3,
 and ET4). The adjusted mean values and standard errors were produced in
 one-way ANCOVA, with emergence time as effect within each stage, and total
 mass as a covariate. Since there were four treatments of emergence timing,
 we calculated the PI in response to each treatment versus another one.
 Consequently, there were six kinds of plasticity in total, including the
 responses to ET4 (vs. ET1, ET2, ET3), ET3 (vs. ET1, ET2) and ET2 (vs.
 ET1). Correlations between PI and NC, and between PI
 and CV, were evaluated by Pearson Correlation Coefficients (PCC) produced
 by PROC CORR (Gianoli and Palacio-López 2009). We then applied regression
 analyses to quantify the relationships between PI and CV and between PI
 and NC for plants in different emergence treatments at both stages.
 Results of correlations and regressions were analyzed with two-way ANOVA
 for the effects of emergence time and growth stage and their interactions;
 and one-way ANOVA for the effects of emergence time on these relationships
 at each stage.
description: # Morphological integration, canalization, and plasticity in response to
 emergence time in *Abutilon theophrasti*
 [https://doi.org/10.5061/dryad.ncjsxksx1](https://doi.org/10.5061/dryad.ncjsxksx1) This dataset contains one Excel file with three sheets.