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A long-standing question in the biology of woody plants has been to understand which portion of the root system dies relatively rapidly (within 1–2 yr) (Pregitzer, 2002; Trumbore & Gaudinski, 2003; Joslin et al., 2006). While the entire root system dies at the end of the growing season in annual plants, it has been difficult to identify the ephemeral roots in perennial woody plants (e.g. trees). For nearly half a century, ephemeral roots have been defined conveniently, but arbitrarily, as all roots less than a certain diameter, most commonly as those < 2 mm in diameter (Moir & Bachelard, 1969; Vogt et al., 1986; Jackson et al., 1997; Strand et al., 2008). Mounting evidence suggests, however, that fine roots defined in this way probably include both ephemeral roots and perennial roots in all woody species examined thus far (Gaudinski et al., 2001; Wells & Eissenstat, 2001; Matamala et al., 2003; Joslin et al., 2006; Guo et al., 2008a; Strand et al., 2008; Espeleta et al., 2009). The classic single-diameter-class approach fails to precisely define the ephemeral roots on a complex branching perennial root system (Pregitzer, 2002; Högberg & Read, 2006; Joslin et al., 2006; Guo et al., 2008a).
Identifying which roots are short-lived is crucial in quantifying terrestrial carbon (C) and nitrogen (N) cycles at the global scale (Jackson et al., 1997; Matamala et al., 2003; Strand et al., 2008). Assuming that the entire fine-root pool turns over once per year, it has been estimated that > 30% of global annual terrestrial net primary production (NPP) is devoted to fine-root production (Jackson et al., 1997). In forests, the estimate may be as high as 67% (Grier et al., 1981; Santantonio & Grace, 1987). However, if a substantial fraction of fine roots (<2mm) is long-lived, global estimates of root production and turnover will need to be revised. Recently, a few studies have reported evidence for conceiving woody plant root systems as composed of a two-tier system of root types (short-lived vs long-lived), and have modeled the consequences of such a conception on root dynamics (Riley et al., 2009; Gaudinski et al., 2010). Thus, identifying precisely which roots are ephemeral is a prerequisite for producing accurate estimates of belowground NPP at the global scale.
Previous studies have offered some clues about which roots might be ephemeral. For example, Lyford (1975) and Pregitzer et al. (2002) observed densely distributed root scars and ‘stumps’ on woody roots, implying frequent death of small lateral root branches. Guo et al. (2008c) and Valenzuela-Estrada et al. (2008) studied the relationship between root anatomy and branch order and found that across all 24 woody species examined, the distal two or three branch orders consistently had a nonwoody structure, an intact cortex and mycorrhizal colonization, whereas the higher-order mother roots showed secondary development, and lacked cortex and mycorrhizal colonization. Based on the anatomical structure of nonwoody roots, and the relationship of this structure to root function, it has been speculated that distal nonwoody branches, which have high metabolic intensity and are not as well-protected as woody roots, might be destined to die rapidly (Wells & Eissenstat, 2003; Guo et al., 2008c). This theory was supported by studies examining root survivorship for each of the distal nonwoody branch orders; these orders were all found to have similarly short life spans (Valenzuela-Estrada et al., 2008; Espeleta et al., 2009). Taken together, these studies suggest that the distal nonwoody branch orders are not only short-lived, but may grow and die simultaneously as intact lateral branches (or modules; sensu Harper, 1977).
If the distal nonwoody lateral branches constitute ephemeral modules in woody plants, they should exhibit a certain degree of physiological independence. Previous studies have found that they have higher N concentrations and respiration than higher-order woody roots in a wide range of tree species (Pregitzer et al., 1998, 2002; Valenzuela-Estrada et al., 2008; Li et al., 2010), suggesting that they have greater metabolic intensity. But are their metabolic activities decoupled from their woody mother roots? One way to test this is to see if nonwoody branches exhibit seasonal dynamics of N concentration and respiration that are distinctly different from those of higher-order woody roots.
To test whether nonwoody lateral root branches are ephemeral and physiologically semi-independent modules, it is necessary to track the life history and frequently sample both ephemeral and perennial roots on the same root system. One common method used to study root demography has been minirhizotrons; however, as a result of their small view (2.0 cm × 1.3 cm), minirhizotrons only observe the finest and most short-lived roots (Wells & Eissenstat, 2001; Withington et al., 2006; Valenzuela-Estrada et al., 2008), and infrequently observe thicker and longer-lived perennial fine roots (Guo et al., 2008a). Another technically sophisticated method that has gained prominence in recent years is the use of isotopic tracers, but this method is very expensive and uses a mass-balance approach that may focus more on long-lived coarser roots which dominate total fine-root mass (Gaudinski et al., 2001; Matamala et al., 2003; Joslin et al., 2006; Trumbore et al., 2006). It seems that only rhizotrons (Espeleta et al., 2009), combined with sequential sampling of a root cohort, can quantify the life span of individual roots on the branching root system and at the same time measure tissue N concentrations and respiration rates of all individual roots on the intact fine-root branching systems.
Here, we studied the fine-root system of a common forest tree in northeast China, Fraxinus mandshurica (Manchurian ash). The fine-root system of this species has been shown to contain the branch orders that have primary (nonwoody) structure and the branch orders that progress through secondary development and become woody roots (Guo et al., 2008c). Through an 820-d rhizotron observation of both nonwoody and woody branch orders, we tested the hypotheses that nonwoody branch orders, or the first three branch orders in this species, have much shorter life spans than higher-order woody roots, and that these roots grow and die as intact ephemeral units. We grew a root cohort and sampled this cohort by branch order at monthly intervals during two growing seasons to measure the temporal changes of anatomical traits, N concentration and respiration, to test whether ephemeral root modules also exhibit physiological changes over time that are distinct from their woody mother roots.