High Small-Scale Variation of Leaf Traits And Their Plasticity Within And Among Genotypes of A Clonal Species

Genetic variation of plant traits and their phenotypic plasticity are two supplementary ways of plant adaptation to temporarily uctuating and spatially heterogeneous environmental conditions. Genetic variability and plasticity of leaf traits have been studied extensively as important indicators of the plant survival. In the case of clonal species with a patchy local distribution of clonal individuals, it would be important to investigate leaf traits at a small spatial scale. Here, small-scale variability of leaf traits and their plasticity within and among clonally spread genotypes in small 2 x 2 m plots was examined on the example of the clonal legume Trifolium alpestre. Seven leaf traits, leaet length, area, width, fresh and dry weights, dry matter content (LDMC), and specic leaf area (SLA), were measured for ramets of various clonal genotypes sampled from ve natural populations of T. alpestre. High variation of leaf traits and their plasticity was detected among the individual ramets of genotypes in 2 x 2 m plots of within the same population, as well as differential variation among the genotypes from different populations. The extent of variation in leaf traits and plasticity was found to be specic for the particular trait, genotype and site. The observed high variation of leaf traits and their plasticity within and among the clonally spread genotypes in local sites of populations is attributed to their differential combined response on the small-scale heterogeneity in the habitat conditions and genetic factors. High variation of leaf traits and their plasticity allows plants effectively respond to spatiotemporally uctuating environmental conditions.


Introduction
Persistence and stability of plant species in natural communities has been shown regulated by plant functional traits (Polley et al., 2020). Among the plant functional traits, leaf traits are of special importance because leaves have a key role in carbon assimilation by photosynthesis and transpiration, thus ensuring the use of light and nutrients for the plant growth (Wilson et al., 1999;Evans and Poorter, 2001;Hodgson et al., 2011). Numerous studies have analysed variability and plasticity of leaf traits among various plant species under diverge habitat conditions (review: Gratani, 2014). These studies have shown that variability and plasticity of leaf traits are important indicators of the persistence of plant species and their populations. Among the leaf traits, SLA and LDMC are the two most important leaf functional traits that are related to plant performance in natural communities but respond differently to particular environmental factors related to light conditions and soil fertility (Wilson et al., 1999;Hodgson et al., 2011). SLA has been shown positively associated with relative growth rate, carbon acquisition through photosynthesis and competitive ability of plant species across biomes at the global level (Reich et al., 1999). Poorter and Bongers, (2006) found that tree species with higher photosynthesis and respiration rates also higher SLA values. Majeková et al., (2014) showed that LDMC is the best predictor of population stability: herbaceous species with high LDMC were be more stable over 13 years in a grassland community studied. The above studies showed that LDMC and SLA vary largely among and within the species of different functional groups, indicating the need to study the extent of variability within individual species.
Among the plant functional groups, clonal plants are of special interest because the ability for the clonal growth favours the plant ability to survive in adverse habitat conditions. Clonal plants are widely represented in environments with increased stress conditions, dominating frequently in cold and wet habitats, in light-and nutrient-limited habitats, along forest edges and in species-rich semi-natural grasslands (reviews: Oborny and Bartha, 1995;Klimeš et al., 1997). They are characterized by the continuous horizontal spatial spread of individual plants (ramets of the same clone) during the growth period. Clonal plants form local patches of individuals that are interconnected by underground rhizomes or aboveground spacers and thus form spatial structure of genotypes (clones) at a local scale. Therefore, in the case of clonal species with a patchy local distribution of clonal individuals, it would be important to investigate leaf traits at a small spatial scale. Variation of leaf traits at the level of local populations has been studied for several clonal grasses of different growth form (Bittebiere et al., 2013;Bittebiere and Mony, 2015). It has been reported that higher plasticity and variation of functionally important leaf traits in clonal plants support the performance and survival of species, particularly in spatially and timely uctuating conditions of light intensity, soil fertility and water availability (Navas and Garnier, 2002).
To my knowledge, the extent small-scale variation in leaf traits and their plasticity has not been studied in clonal species. Here, Trifolium alpestre L., growing in rare fragmented populations at its northern distribution range in Estonia, was used as a model clonal species for simultaneous assessing of variability in morphological and functional leaf traits and their plasticity among and within genets in small 2 x 2 m plots from different sites of natural populations. Given that both biotic and abiotic conditions are found to be variable at a local small spatial scales in various natural communities and that plants respond to micro-environmental variations by changes in plant phenotypes and physiology (review: Denney et al., 2020), I expect to nd variation in leaf traits of plants at local sites. The major aim of the study is to measure the extent of small-scale variability in the leaf trait values and their plasticity within and between clones from different fragmented populations.

Study species and sites
Trifolium alpestre L. is a perennial diploid (2n = 16) clover species which is distributed throughout central, southern and east Europe, but has sparse distribution mainly in dry meadows, forest fringes, steppes and light woodlands (Coombe, 1968). The species is able to reproduce both sexually from seeds and further by the local clonal spread of sexually derived individuals (Kaljund et al., 1998). In Estonia, its distribution of this species is limited only to the Island Saaremaa in the westernmost part of Estonia where the species is at its northern range edge and grows in small and isolated forest-edge populations on thin calcareous soils. My common garden observations showed that the species has epigeogenous, phalanx type of rhizomes with short spacer length that favours clumped clonal spread. In our common garden experiment, we observed that each mother plant formed new shoots from the two outer buds that grew up to 12-15 cm in a sandy soil during the summer growth period. Field excavations showed that genets formed nets of interconnected rhizomes with short spacers of about 5-10 cm length, indicating phalanx type clonal growth form for this species. Therefore, 2 x 2 m plots with sixteen 25 x 25 cm subplots were chosen as suitable for the study.
Ramets from ve 2 x 2 m local plots from ve different sites of high plant density in the Viidumäe Nature Reserve on the island Saaremaa (58º17'54'' N 22º 2'55'' E) were collected for analyses. Sites 1 and 2 were growing near a road verge, about 200 m apart from each other. Sites 3 and 4 were from roadside forest gaps, separated by a 40 m coppice, while site 5 was under a sparse pine forest about 50 km kilometres apart from other populations. In total, 80 individuals were sampled among the ve plots studied. All plots are derived from similar thin calcareous soils that are characteristic for the whole study area. However, the plots differed in the density and species identity of neighbour plants.
Leaf sampling and trait measurements MLGs was described by the coe cient of variation (CV, %).The CV was calculated by dividing standard deviation by trait mean and multiplied by 100.

Results
The studied ve plots were found to have each a different dominating AG that were clustered in neighbouring subplots, indicating their patchy small-scale clonal spread as expected for a species with short epigeogenous, phalanx type rhizomes of limited annual spread ( Table 1). The same dominant AG was recorded in 9-16 subplots of each plot and from one to three different AGs in boarder subplots (data not shown, but see Kaljund et al. 2018). The spatial aggregation of dominating AGs indicates that they are derived through the clonal spread and represent clones. The data show remarkable differences in the variability by trait means and in their plasticity (CV) of lea et traits among individual plants of AGs spread in 2 x 2 m plots ( Table 1). The data also show that intra-clonal variability and plasticity of the seven leaf traits in local microsites are speci c for particular traits and clones. The two functional leaf traits, LDMC and SLA, showed moderate, but differential variation among the ve clones in their mean values.   Leaf fresh and dry weights are generally the most variable and plastic traits within clones, showing the CV values ranging from 20.6 to 33.7 % among the clonemates of the ve clones, followed by the lea et area (CV range 17.3-24.9 %). Statistical analysis of the data in Table 1 for Pearson's linear r coe cients shows that all differences between trait means within clones are statistically signi cant at P ≤ 0.01, except AG3 at P < 0.02. Principal component analysis shows that the ve AGs (C1-C5) are well distinguished from each other by their PC 1 and PC 2 values, illustrating statistically signi cant genetic differentiation between all ve clones (Fig. 1). Remarkably, the clone pair AG1 and AG2 as well as AG3 and AG4 originating from closely spaced populations are clearly differentiated by PC1 and PC2. In contrast, spatially far populations AG4 and AG5 from spatially far populations appear in the same PC1 and PC2 sector, presumably indicating that rather differences in habits than geographic distances cause larger differentiation between clones.  Table 1 shows that character means and CVs of clones are not correlated: higher trait means in clones often have lower CV values and reversely. For example, lea et length of AG5 had the lowest mean 42.4 among AGs, but the highest CV. Reversely, LDMC of AG4 had the highest value among AGs, but the lowest CV. Similarly, lea et fresh weight of AG3 hat the highest value among AGs, but a low value of CV. Statistical correlation analysis of the data showed that the mean linear Pearson's coe cient r values between the trait mean and plasticity values varied largely between clones from negative (AG1 -0.049; AG2 -0.029; and AG4 -0.27) to positive (AG3 0.11 and AG5 0.062). This result implies that the trait values and their plasticity vary differentially and independently among the clones.

Discussion
This study describes for the rst 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 nding remarkably high ne-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 re ects plastic responses of ramets to ne-scale heterogeneous habitat conditions existing even within such small plots. Apparently, leaf traits of the same genet respond differentially to the ne-scale variation in surrounding environmental conditions. This raises a question about the kind of habitat variables that might cause so notable ne-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 ne-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 ve AGs derived from different populations are well distinguished from each other by their PC 1 and PC 2 values, illustrating signi cant 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 t 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 t 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, exempli ed 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 ne-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 exibility 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 mm 2 /mg (Table 1), being within 15.7 and 39.2 mm 2 /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 ve 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 ve 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 signi cantly depending on the light, nutrient and water availability, indicating plastic responses to these abiotic factors. The ve 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. Our study revealed a signi cantly 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