Leading dimensions of root functional trait variation
Absorptive roots are not simple, discrete structures such as leaves or needles; rather, they have a complex branching architecture, and form intimate associations with mycorrhizal fungi. This hierarchical structural complexity has hindered our understanding of absorptive root trait variation because it is very difficult to identify which roots are truly absorptive and what unit is functionally identical for cross-species trait comparisons (Guo et al., 2008; Xia et al., 2010). Here we focused on the first-order (on a stream-based ordering system) roots, which are the most distal and metabolically active component of the woody root system (Pregitzer et al., 2002; Xia et al., 2010). The choice of this branch order allows us to better understand cross-species root trait variation. Our results clearly suggest that the 14 root traits we measured showed different degrees of variation and were segregated in two major trait dimensions.
The first dimension was dominated by root diameter-related traits, including root diameter, cortex thickness, stele diameter, vessel diameter, and MC (Fig. 4a), all of which were highly correlated with each other (with all Pearson's correlation coefficients > 0.62; Table 3). The second dimension, largely independent of the first dimension (i.e. forming an orthogonal axis to the first axis; Fig. 4a), was dominated by the root branching ratio and branching intensity, the two parameters that were either weakly correlated (in the case of branching intensity, all correlation coefficients < 0.40 with the exception of the correlation with root length) or not correlated (in the case of branching ratio) to all parameters on the first axis (Table 3). The identification of these two dimensions supported our first two hypotheses. These two largely independent dimensions represent two important aspects of the adaptation of roots to their environment and offer new, fundamental understanding of root functioning in resource foraging and acquisition, and root trait evolution.
The first dimension, or diameter-related dimension, describes the coordinated variation among root diameter, cortex thickness, stele diameter, and MC rate. The degree of coordination among these traits is very high, such that thick roots always have thicker cortex and stele, particularly in angiosperms, which show large variation in diameter (Fig. 2), and higher MC rates (Fig. 3). The tight linkage among root diameter, cortex thickness and stele diameter (Fig. 2) should be particularly useful for predicting how the root cortex and stele would vary with root diameter across other angiosperms species. Moreover, these size-related root traits are among the most variable traits we measured here (all with a CV > 50%; Table 1); they are also phylogenetically conservative (Table 2), showing limited convergence (Fig. S1). Clearly, variation in diameter, the associated anatomical structure, and the degree of mycotrophy is the most significant way in which the first-order roots of different subtropical tree species in China are differentiated.
Variation of root diameter-related traits across species may represent distinct strategies of root construction, maintenance, and persistence. First, species with thicker first-order roots devote more C and nutrients per unit area (or length) to root construction. This type of species (termed ‘magnoid type’ by Baylis (1975)) may seem less efficient in producing root surface area. However, the loss of surface area resulting from this thick root strategy can be compensated by increased MC (Fig. 3), and consequently increased density of extramatrical hyphae (although this has yet to be verified). Thus, species with thick first-order roots may have no less total surface area per unit root mycorrhizal biomass in comparison with species with thin roots. Also, given the large cortex space conferred by thick roots (Fig. 2), the association between mycorrhizal fungi and thick roots may be particularly strong (Brundrett, 2002), contributing to the relative evolutionary conservatism of both root diameter and MC rate (Table 2), particularly in ancestral families (e.g. close clustering of Magnoliaceae species in Figs 4b, S3).
Secondly, species with magnoid-type roots should have higher [N] as a consequence of their larger proportion of cortex because the majority (> 70%) of root N is in the cortex (D. L. Guo, unpublished; Fig. 2). Indeed, we found a positive yet weak correlation between root diameter and root [N] (Table 3), a finding similar to that of Holdaway et al. (2011). Thus, it would be interesting to determine in the future if root respiration also relates positively to root diameter for first-order roots across a large number of species as a result of the relatively close link between [N] and root maintenance respiration (Reich et al., 2008; but see Bouma et al., 2001, in which citrus (Citrus paradisi Macf.) had a root diameter twice as great as that of apple (Malus domestica Borkh.), yet the two species had similar root respiration rates). It should be noted that MC is high in most species studied here, so that maintenance respiration would have to be quantified for roots and mycorrhizal fungi at the same time. If AM species generally tend to have low hyphal biomass within the root, as suggested by Ouimette et al. (2013), then mycorrhizal respiration may mainly originate from extrametrical hyphae. Therefore, we would need a better understanding of both the standing biomass and turnover of extramatrical hyphae or, better, a direct measurement of hyphal respiration. Hyphae can turn over on weekly time-scales (Hernandez & Allen, 2013) but roots usually turn over on annual time-scales (Withington et al., 2006; McCormack et al., 2012). The maintenance costs incurred by hyphae may be reduced if plants produce and maintain hyphae only when nutrient and water uptake rates are high. Hyphal turnover of different species should be a profitable avenue of future research in understanding root–fungal trait economics.
Thirdly, compared with the thin-root strategy, the apparent disadvantage of building thick roots with less root surface area per unit biomass may be further compensated by living longer, and by having better chemical defense and thus less tissue loss as a result of herbivory. It is possible that the total return of nutrient and water uptake may be the same relative to root lifespan between thin- and thick-root species. In fact, there may be two distinct strategies of nutrient uptake for species of different root morphologies: the fast strategy, or high uptake rate over a short lifespan (which corresponds to small root diameter), and the slow strategy, or low uptake rate over a long lifespan (which corresponds to a thick root diameter; Eissenstat et al., 2000; Bouma et al., 2001), although other strategies may also exist. If the positive correlation between root diameter and root lifespan found in McCormack et al. (2012) is broadly true, then we may finally be able to link root morphology and chemistry with root lifespan in a trait economics framework analogous to the leaf mass per area lifespan relationship reported for leaf economics traits (Wright et al., 2004).
The second dimension, represented by branching intensity and branching ratio, may be critical for nutrient foraging in the soil. Root branching is a key trait determining root plastic responses to nutrient patches. Many studies have shown that roots branch extensively into nutrient-rich patches (so-called morphological plasticity; Drew & Saker, 1975, 1978; Pregitzer et al., 1993; and reviewed by Hodge, 2004). This local proliferation and enhanced nutrient uptake in diverse natural ecosystems may be critical for species competition (Jackson & Caldwell, 1989; Jackson et al., 1990; Robinson et al., 1999). In our study, branching intensity (the number of first-order roots per cm of second-order roots) ranged from 0.44 to 7.37 first-order roots per centimeter of second-order roots, with an overall CV of 67.1% (Table 1). The wide variation in branching intensity across species may be an indicator of large inter-specific and inter-individual differences (although the inter-individual differences were not as strong as the inter-specific differences; Table S2) in the plasticity of the absorptive root system, sensitivity to patchy and pulsed nutrient supply, and competitive capacity. For example, species with a high branching intensity may be capable of rapid proliferation into nutrient and water patches, conferring on them a competitive advantage in relatively nutrient-poor environments.
In addition to branching intensity, we also used branching ratio as a measure of root architecture. Branching ratio is the number of first-order roots per second-order root without considering the length of the second-order roots. This parameter ranged from 1.2 to 10, with an overall variation of 47%, also suggesting quite large variability across species (Table 1). Moreover, in contrast to some degree of correlation between branching intensity and root diameter (which was driven by the correlation of both parameters to root length), branching ratio was unrelated to root diameter in Pearson's correlation with both original data and PIC data (Table 3), suggesting that this trait can vary independently of root diameter-related indices (Fig. 4a).
Both branching intensity and branching ratio had small Blomberg's K values and showed weak phylogenetic conservatism (Table 2), suggesting the strong influence of environmental factors (possibly soil nutrient and water conditions) on root branching. Holdaway et al. (2011) found that branching intensity, defined as the number of tips divided by the total length of the first two to three branch orders, was negatively correlated with soil available phosphorus (P) and N across species and sites, suggesting that higher branching intensity may be required at low-fertility sites.
Mechanisms underlying major trait dimensions
The two major dimensions of root traits reported may be explained by several underlying mechanisms. From a biophysical perspective, absorptive roots composed of the first two to three branch orders can only vary in two major ways: the thickness of an individual root segment, and the branching intensity, because any root branch can do only one thing: occupy and divide a soil volume of limited size. As root diameter is strongly related to root length (Table 3 in this study; Chen et al., 2013), thick first-order roots are also longer, and thick-root species should occupy the same area with much less dense roots than thin-root species. Also, the space between individual roots on a root branch may be thoroughly exploited by extramatrical hyphae (Fig. S4).
From an ecological perspective, species differ in growth rates, competitive ability and dominance in natural ecosystems, and variation in root form may contribute to the competitive abilities of different species. In the cold-desert plant community of the Great Basin in the USA, the invader species Agropyron desertorum, also a superior competitor in the system, had greater rooting densities, which was mainly attributable to having thinner roots rather than having higher root biomass (Eissenstat & Caldwell, 1988a,b). In addition, the ability to branch out in nutrient patches can be important for competition (Robinson et al., 1999), and there are only a few ways in which root proliferation can be achieved: producing many lateral roots (probably the most likely strategy for species with thin and densely branched roots; Johnson et al., 2008), many root hairs (which also seems to occur mostly in thin-root and less mycotrophic species (Baylis, 1975)), or abundant mycorrhizal hyphae (the most likely strategy for species with thick and sparsely branched roots).
From an evolutionary perspective, a trend of decreasing root diameter and increasing root branching from more primitive species to more modern species has been observed (Baylis, 1975; Fitter, 1991; Comas et al., 2012; Chen et al., 2013). Baylis (1975) reported that modern plants with ‘magnolioid’ roots, that is, thick, sparsely branched root systems, are associated with the primitive family Magnoliaceae, and this was supported by our data (Fig. 1). Also, more modern families are associated with thin first-order roots (Comas & Eissenstat, 2009; Comas et al., 2012; Chen et al., 2013). This thinning trend in first-order root form appears to coincide with an increasingly drier global environment and local habitats since the mid-Cretaceous (Comas et al., 2012; Chen et al., 2013). Thus, selection pressures such as water supply patterns may be instrumental in creating the large differences in root form among species of different lineages and for maintaining these differences in the present environment. Additionally, between the two main clades in the phylogenetic tree, we observed contrasting values for PC1 based on Abouheif's metric, and for traits such as root diameter, stele diameter and cortex thickness (Fig. S1). These contrasting patterns across different phylogenetic clades may provide a basis for inferring how different angiosperm groups altered their functional traits during species divergence and evolution.
Future directions: testing hypotheses related to root trait dimensions
Our studies clearly point to a number of testable hypotheses for the future. First, root lifespan, a critical but difficult-to-measure root trait, may be hypothesized to be positively related to root diameter. As already discussed, compared with building thin roots, building thick roots of the same length would carry higher construction costs, leading to a lower nutrient uptake rate per unit time per unit biomass, and thus a longer root lifespan may be necessary for a net gain of nutrients equal to that of thin roots. One caveat in testing this hypothesis is that the lifespan–root diameter relationship may be mediated by other plant traits such as plant growth rate and wood (tissue) density (McCormack et al., 2012).
Secondly, hypotheses linking root diameter with strategies of nutrient uptake can be tested so that the functional significance of root trait variation can be better understood. For example, slow and fast strategies were found to be associated with a long and a short root lifespan, respectively, and root lifespan was negatively associated with root diameter (Eissenstat et al., 2000). Determining whether these patterns are general is a high-priority goal for future research. Moreover, how mycorrhizal fungi are involved in the lifespan–root diameter relationship deserves attention in light of our observation that the MC rate increased with root diameter and then leveled off at a root diameter of c. 470 μm (Fig. 3). Does this suggest that we may assume a constant level of colonization for species with coarser roots (e.g. first-order root diameter > 470 μm) and that the root lifespan of these species is less plastic because of the lack of substantial changes in mycorrhizal colonization rate, thus a lack of mycorrhizal influence on root lifespan.
Last but not least, a better understanding of the root–mycorrhizal association needs to be achieved. The present finding that thick roots had higher colonization rates confirms earlier work on British flora (Peat & Fitter, 1993), suggesting that this is a common pattern. Yet we still lack mechanistic understanding and elucidation of the functional significance of this pattern. For example, does a high MC rate in thick roots serve mainly nutrient uptake functions, or alternatively other key functions such as defense? It has been shown that the root MC rate may be negatively correlated to extramatrical hyphae production (fig. 2a,b in Maherali & Klironomos, 2007). In addition, Resendes et al. (2008) found that mycorrhizal colonization and nonmycorrhizal fungal colonization were mutually exclusive in the first 25 d of root life, suggesting that mycorrhizal fungi may be an important factor preventing nonmycorrhizal fungi from colonizing roots. The relationship between root form and fungal identity/abundance may be an area of great importance for both theoretical and practical endeavors, as recognized by Newsham et al. (1995).
As a consequence of the lack of knowledge of the broad-scale patterns of root trait variation, we still lack consensus on which root traits to choose in a root trait study. Our results and those of previous root trait studies (e.g. Pregitzer et al., 2002; Tjoelker et al., 2005; Roumet et al., 2006; Withington et al., 2006; Holdaway et al., 2011) suggest that root morphology, anatomy, and chemistry are the basic parameters in any root trait study. These traits have several features: they are relatively easy to measure, having clear functional significance at the individual root and whole-plant levels, and have been linked to ecosystem-scale belowground processes, such as root production, mortality and decomposition, and to aboveground traits. Our study also suggests that consideration of anatomy may be essential for a better understanding of the linkage between root form and function. In future studies, root lifespan and nutrient uptake rates are urgently needed for broad comparisons and more comprehensive understanding of root functional traits.
By measuring 14 root traits on 96 species of diverse phylogeny, we found two leading dimensions of trait variation: a diameter-related dimension that may integrate root construction, and possibly maintenance and persistence, with MC, and a branching density dimension that may express differences in root plastic responses to environment. Knowledge of these two readily measured dimensions offers a promising path for understanding root trait economics and root ecological strategies.
The patterns and arguments presented here are only a peek into the tremendous diversity of root traits and strategies. Progress in recent decades supports a view that roots are complex structures and play a multifaceted role in plant functioning and ecosystem processes. Roots are at the same time structures of nutrient acquisition (Pregitzer et al., 2002) and active resource foraging (Kembel & Cahill, 2005), hosts and organizers of mutualistic and nonmutualistic microbial communities (Brundrett, 2002), circuit breakers of the plant hydraulic system (Hacke & Sauter, 1996; Johnson et al., 2012), stations of signaling and below–aboveground communication (Bais et al., 2006; Parniske, 2008), and ‘weapons’ against competitors (Dybzinski et al., 2011), to name just a few. Extraordinary structural diversity and plasticity in root form and function are needed to achieve the complex role that absorptive roots play, and our study represents a small but promising step toward a full understanding of this highly intriguing and critical plant organ.