This study describes for the first time small-scale variation of leaf traits concomitantly with their plasticity on the example of the clonal legume Trifolium alpestre. The most impressive result of the study is finding remarkably high fine-scale variation of leaf traits and their plasticity among individual ramets within the same clones (genets) in small 2 x 2 m plots. Thus, the CV values for LDMC and SLA individual genotypes (AGs) of T. alpestre (table 1) are largely overlapping those estimated for various clonal specie (grasses and forbs) at the population level by Bittebiere et al. (2013). Such high small-scale intraclonal variation of leaf traits most likely reflects plastic responses of ramets to fine-scale heterogeneous habitat conditions existing even within such small plots. Apparently, leaf traits of the same genet respond differentially to the fine-scale variation in surrounding environmental conditions. This raises a question about the kind of habitat variables that might cause so notable fine-scale differences in the leaf traits. Trifolium alpestre populations in Saaremaa are adapted to similar thin calcareous soils that are characteristic for all study sites. Resource sharing among the plants of the same clone through the clonal integration is shown to diminish the effect of soil heterogeneity on the leaf traits in clonal species (review: Liu et al., 2016a). Therefore, the high variability of leaf traits within the allozyme-based clones (AGs) is somewhat surprising. Several studies have shown large effects of biotic neighbourhood, the local vegetation density and the species identity on various plant functional traits at small spatial scales (Bittebiere and Mony, 2015; Abakumova et al., 2016). The density and height of neighbour plants will cause differential shading of individual plants, and numerous studies have shown that light intensity and quality greatly affect the plant performance and morphology (Liu et al., 2016b). In addition, variable density and species composition of neighbour plants will affect the traits measures between clonal ramets through the competition for various abiotic resources, e.g. light, water and nutrients (Bittebiere and Mony, 2015; Wang et al., 2016). Based on the above literature data, we assume that variation in the density and species identity may be the major factor for the observed high variability of leaf traits within clones.
In addition to the effects of environmental factors, the genetic differences among the genets should also be considered. The allozyme genotypes analysed for the leaf traits (putative genets) may consist of several sub-clones differing by changes in the DNA structure of genes affecting the expression of leaf traits by inclusion of mutations that were detected in plant species with the use of various hypervariable DNA markers (review: Nybom, 2004). In addition, the accumulation of epigenetic changes has been shown to be a characteristic feature of clonal plants that has contributed to their ability for rapid plastic response to environmental variables (review: Douhovnikoff and Dodd, 2015). Epigenetic mutations caused by the methylation of histones and gene promoters induce heritable changes in the expression of respective genes and in the plant characters that they control. It is appropriate to assume that the observed fine-scale variability of leaf traits among MGs is partially caused by the differential accumulation of various DNA-based mutations in the same AG.
The results of the study show differential variability of plasticity of traits (CV) among clonal genotypes from different local populations. Principal component analysis showed that the five AGs derived from different populations are well distinguished from each other by their PC 1 and PC 2 values, illustrating significant differentiation between all clones. Inter-clonal competition among the initial sexual recruits and selection of plastic genotypes adapted to the particular local site has presumably contributed to the formation of differently adapted local AGs 1–5. The formation clonal clumps in natural populations in result of clonal competition and selection of superior genotypes that are adapted for a set of local environmental conditions has been shown for several clonal species (Arens et al., 2005; Vandepitte et al., 2009). It is thus reasonable to assume that clonal competition has contributed the spread of genotypes with more plastic leaf traits that are better fit to the local microenvironments in natural populations of T. alpestre with different sets of biotic conditions. The genotypes with differently variable leaf traits likely have superior competitive ability that favours their subsequent spread due to a better fit to local microsite habitats. Inter-clonal competition among the initial sexual recruits and selection of plastic genotypes adapted to the particular local site has presumably also contributed to the formation of differently adapted local clones.
The Principal Component Analysis data (Fig. 1) also show that leaf traits are largely differentiated among the AGs (C 1–5) that have spread and occupied local sites, exemplified by 2 x 2 m plots. Trifolium alpestre natural populations in Saaremaa are adapted to similar thin calcareous soils that are characteristic for all study sites. Resource sharing among the plants of the same clonal AG through the clonal integration should further minimize the effect of soil heterogeneity on the plant traits (Liu et al., 2016). Therefore, the fine-scale biotic heterogeneity in the density and species composition of neighbour plants is presumably the main cause of the high intra-clonal variation of leaf traits among plants in small 2 x 2 m plots, as described in several studies (Bittebiere and Mony, 2015; Abakumova et al., 2016). The density and height of neighbour plants will cause differential shading of individual plants, and numerous studies have shown that light intensity and quality greatly affect the plant performance and morphology (Liu et al., 2016). In addition, variable density and species composition of neighbour plants will affect the traits measures between clonal ramets through the competition for various abiotic resources, e.g. light, water and nutrients (Bittebiere and Mony, 2015; Wang et al., 2016). High flexibility and plasticity of leaf traits observed in a single genotype will provide greater opportunity for its persistence in spatially and temporarily variable conditions.
The SLA values among the Estonian clones of T. alpestre varied between 16.9 and 23.8 mm2/mg (Table 1), being within 15.7 and 39.2 mm2/mg reported for this species in the TRY database for the genotypes of the Central European populations (Kattge et al., 2020). This comparison shows that clones from an Estonian northern population that has persisted since the post-glacial colonisation up to our days are characterised by the same range of SLA values as the Central European populations. However, the mean SLA plasticity CV value 32.7 computed from the TRY data for the eleven Central European plants of T. alpestre, is much higher than the mean CV 15.3 among the five Estonian clones. The higher SLA plasticity range among the Central European plants may be explained because they were sampled from more variable habitat conditions than the Estonian plants that grow in similar thin calcareous soils. The main difference among the plots studied was in the density and species identity of plants that affect the neighbouring light conditions for each ramet and may be the main reason for the observed variation in the SLA values and plasticity. This assumption is supported by the study of Poorter and Evans (1998) who showed that the SLA values among six herb species were 1.8–2.1 times higher under five times lower irradiance than at high irradiance. Meziane and Shipley (1999) found that SLA of herbaceous species varies depending on the interacting light intensity and nutrient availability combinations. Similarly, Navas and Garnier (2002) showed that SLA of a small clonal shrub Rubia pergrina varies significantly depending on the light, nutrient and water availability, indicating plastic responses to these abiotic factors. The five T. alpestre clones studied are collected from forest roadside sites with similar calcareous soils, further indicating that rather variable light conditions caused by different neighbour plants and competition among the neighbouring plants may be the prime factors that caused the observed high intra-clonal variation in the leaf traits, but not differences in soil conditions.
Leaf dry matter content (LDMC) is another important leaf functional trait related to the plant performance in variable environments, being associated with multiple environmental conditions (Wilson et al., 1999). LDMC is considered a better indicator of soil fertility than SLA (Hodgson et al., 2011), biomass production and population stability (Majeková et al., 2014; Smart et al., 2017) than SLA. The LDMC among the five Estonian clones varied from 31.0 to 39.8. This range is similar to 30.6 to 34.8 retrieved for the Central European individuals from the TRY database. However, the mean LDMC plasticity value 10.2 among the five Estonian clones is much higher than the mean 5.8 among nine Central European individuals. SLA is a more plastic than LDMC among the clones from the Estonian populations, 15.3 and 10.2 respectively. This result is consistent with the study of Harze et al. (2016) who reported higher SLA plasticity range in comparison with the LDMC plasticity for four calcareous grassland herbs from three populations.
Our study revealed a significantly negative correlation between LDMC and SLA. This is consistent with the literature data showing overall negative association between LDMC and SLA in perennial herbs (Li et al., 2005). The negative association between LDMC and SLA has evidently contributed to their role as alternative indicators of plant responses to the use of habitat resources (Wilson et al., 1999; Hodgson et al., 2011). Significant positive association between leaf length, area, fresh and dry weights evidently indicates their similar inherent response to the existing ecological conditions.
Overall, our data show that the two leaf functional traits generally are more variable within clones than between them. Several studies have shown that SLA and LDMC vary notably depending on the specific environmental factors and multiple interactions between them (Price et al,. 2017; Stark et al., 2017). The results obtained illustrate astonishingly large fine-scale variation in leaf traits and their plasticity both among and within clones of T. alpestre that has presumably contributed to the persistence of this locally rare clonal species at its northern range limit.