Allocation plasticity
Our results showed plasticity of modular mass allocation in response to density varied with different stages of plant growth, and true plasticity mainly occurred at the earlier two stages. At the first stage, medium density increased root mass, root:stem and root:leaf ratios, consistent with other studies (Gersani et al. 2001; O'Brien et al. 2005), indicating low to intermediate interactions is more likely to facilitate root growth, and root interactions occurred before aboveground interactions. In spite of no response in leaf mass, the significant increases of root:leaf and stem:leaf ratios by high relative to low density indicated a relatively lower investment of resource into leaves, than into stems and roots under competition. As plants grew larger at 50 d, they began to interfere with each other and competing for aboveground resources, when greater mass allocated to stems was required for an extra elongation (Bell & Galloway 2007; Weiner & Fishman 1994). Greater stem mass and smaller mass allocated to other modules led to increased ratios of stem mass to other modular mass by high versus low density. Meanwhile, because of the greater importance of laminas to reproductive organs (Cheplick 2006), lamina:petiole and lamina:reproductive ratios also increased with density. The decreases in leaf mass and reproductive mass might be a cost of extensive stem elongation. However, the decrease of leaf mass may also be a direct effect of density, as it is reported that root mass and leaf mass were still decreased by high vs. low density despite the induced stem elongation has been suppressed (Maliakal et al. 1999). No response was found in root mass at this stage, consistent with some studies (Cahill Jr 2003; Casper et al. 1998), but not with others (Huang et al. 2010). Under both competition and limited belowground resources, root allocation usually decreased, as resource deficiency aggravated belowground competition. However, the sufficient soil nutrients in this study may ameliorated root competition, leading to no response in root mass.
There has been less studies on responses of reproductive modules to density, which showed inconsistent results. Reduction of productive mass can be due to competition (Gersani et al. 2001; Japhet et al. 2009; Murphy & Dudley 2007), or just an effect of plant size (Arenas et al. 2002; Wang et al. 2006). We found both decrease and canalization in reproductive mass and its ratios to other modular mass, indicating reproductive plasticity depends on specific stages. At the initial stage of reproduction, a trade-off occurs during resource distribution at the physiological level, either towards vegetative growth or towards reproduction (Fox 1995), as plant resource allocation or development is limited (Karlsson & Méndez 2005; Levins 1968). Increased vegetative mass was at the cost of reproductive mass, consistent with other studies (Álvarez-Cansino et al. 2010; Matsuyama & Sakimoto 2008).
At 70 d, we did not find responses in any modular mass or allocation traits. This may be due to attenuated competition and its effects over time. As plants grew, competition increases and then decreases over time in a dense population (Hutchings & Budd 1981), resulting in shifts in resource availability and plant-plant interactions (Bouvet et al. 2005; Zhou et al. 2005). As plants continued to grow in the dense populations, reaching its carrying capacity, smaller individuals may be obsoleted (Hutchings & Budd 1981), and competition attenuated again.
Allometric plasticity
Our results showed plasticity in mass allocation and allometric relationships did not coincide. We found true plasticity in allocation after removal of size effects in covariance analyses, as well as the growth-dependence in allometric relationships. Growth period or duration can significantly affect the pattern of allometry (Thompson 2019; Tobler & Nijhout 2010), results from allometry analyses based on single stages differs from those across difference stages (Li et al. 2013; Wang 2006). The discrepancy in the two sets of results and stage-dependence of allometric relationships (Table 3) implied that allometric plasticity might be apparent plasticity rather than true plasticity. If the one-point allometric plasticity is apparent plasticity, plasticity of developmental trajectory might also be apparent plasticity per se. Because differences in developmental trajectories should be caused by shifts in allometric relationships at one or multiple points. Therefore allometric plasticity might be only a function of plant size or developmental rate.
An organism is capable to buffer developmental pathways against genetic or environmental perturbations (Kitano 2004; Masel & Siegal 2009; Mestek Boukhibar & Barkoulas 2016; Wilkins 1997), to maintain developmental stability and make sure the precision of developmental progression, in order to produce an “ideal” form regardless of different circumstances (Auffray et al. 1999; Palmer 1994; Van Dongen & Lens 2000). Once the equilibrium is broken, deviation from developmental trajectory due to mis-regulation of allometry might be lethal (Vea & Shingleton 2020) and prevented as best as possible. The ability to maintain developmental stability is thereby regarded as a premise for plants surviving different stressful environments (Elgart et al. 2015). Consequently, the occurrence of real significant deviations from a programmed development should be very difficult and rare. Evidences come from the little effects of phenology on leaf-shoot and other scaling relationships in woody species (Smith 2020) and the highly stabilized allometric pattern between size and shape of Drosophila wings over 40 million years (Houle et al. 2019). Voje et al. (2013) analyzed over 300 empirical estimates of allometry, and also found limited evidence for microevolutionary changes in allometric slopes for allometric relationships among morphological traits of animals (Voje et al. 2013). This is probably because there is a lack of genetic variation of allomerty or genetic constraints to allometry (Lines et al. 2012), due to a potentially large number of pleiotropic effects(Houle et al. 2019).
Plant strategy revealed by stage-dependent allocation and allometry plasticity
Comprehensively, the two sets of results demonstrated the transition of the strategy of plants dealing with increased density. If plasticity in allocation traits and allometric relationships were true plasticity and apparent plasticity respectively, then: 1) at the first and second stages when allocation traits responded to density but allometry remained stable at the earlier stages, plants altered strategy of resource partitioning without changing growth rate or ontogenetic trajectory; 2) at the third stage when allometry responded to density but allocation traits did not, plants began to alter growth rate or developmental trajectory, leading to divergent developmental stages at different densities; and 3) stage-dependent plasticity in allocation traits and allometry suggested density effects became increasingly severe over time, as delayed developmental processes should be more detrimental than altered allocation strategy.
Two components in trait plasticity
The definition of plasticity did not define but explicitly implies that the difference in phenotype is a complex result of environmental effects (positive or negative) and active responses of organisms (Nicotra et al. 2010; Pigliucci 2005; van Kleunen & Fischer 2005). In other words, phenotypic plasticity intrinsically comprises two components of plant responses and environmental effects. In spite of its great significance to relevant investigations, no studies have attempted to disentangle the two components in plasticity of a given trait. In this study, we are trying to address this by virtue of covariance analysis. In covariance analyses, the variation in a given trait includes effects of both plant size and density. If the response of plant size can reflect whether an environmental effect is benign or malignant and its extent, then the effect of plant size in the variation of a given trait can be considered as an indirect environmental effect, and the left proportion of variation after removal of the size effect indicates the direct response of the trait. For example, when plant size decreased in response to high vs. low density, it suggested high density was less beneficial than low density for plants. In the responses of modular traits to density, effects of plant size can be considered as an indirect effect of density (which was mostly adverse), while the variation in adjusted mean trait values reflected their actual responses. By discerning the two components in plasticity, we may be able to evaluate relative extents of environmental effects versus plant responses, which should be essential for understanding the nature and consequences of plasticity.