A starting guide to root ecology: strengthening ecological concepts and standardising root classification, sampling, processing and trait measurements.

In the context of a recent massive increase in research on plant root functions and their impact on the environment, root ecologists currently face many important challenges to keep on generating cutting-edge, meaningful and integrated knowledge. Consideration of the below-ground components in plant and ecosystem studies has been consistently called for in recent decades, but methodology is disparate and sometimes inappropriate. This handbook, based on the collective effort of a large team of experts, will improve trait comparisons across studies and integration of information across databases by providing standardised methods and controlled vocabularies. It is meant to be used not only as starting point by students and scientists who desire working on below-ground ecosystems, but also by experts for consolidating and broadening their views on multiple aspects of root ecology. Beyond the classical compilation of measurement protocols, we have synthesised recommendations from the literature to provide key background knowledge useful for: (1) defining below-ground plant entities and giving keys for their meaningful dissection, classification and naming beyond the classical fine-root vs coarse-root approach; (2) considering the specificity of root research to produce sound laboratory and field data; (3) describing typical, but overlooked steps for studying roots (e.g. root handling, cleaning and storage); and (4) gathering metadata necessary for the interpretation of results and their reuse. Most importantly, all root traits have been introduced with some degree of ecological context that will be a foundation for understanding their ecological meaning, their typical use and uncertainties, and some methodological and conceptual perspectives for future research. Considering all of this, we urge readers not to solely extract protocol recommendations for trait measurements from this work, but to take a moment to read and reflect on the extensive information contained in this broader guide to root ecology, including sections I-VII and the many introductions to each section and root trait description. Finally, it is critical to understand that a major aim of this guide is to help break down barriers between the many subdisciplines of root ecology and ecophysiology, broaden researchers' views on the multiple aspects of root study and create favourable conditions for the inception of comprehensive experiments on the role of roots in plant and ecosystem functioning.

Root morphology and mycorrhizal symbioses together shape nutrient foraging strategies of temperate trees.

Photosynthesis by leaves and acquisition of water and minerals by roots are required for plant growth, which is a key component of many ecosystem functions. Although the role of leaf functional traits in photosynthesis is generally well understood, the relationship of root functional traits to nutrient uptake is not. In particular, predictions of nutrient acquisition strategies from specific root traits are often vague. Roots of nearly all plants cooperate with mycorrhizal fungi in nutrient acquisition. Most tree species form symbioses with either arbuscular mycorrhizal (AM) or ectomycorrhizal (EM) fungi. Nutrients are distributed heterogeneously in the soil, and nutrient-rich "hotspots" can be a key source for plants. Thus, predicting the foraging strategies that enable mycorrhizal root systems to exploit these hotspots can be critical to the understanding of plant nutrition and ecosystem carbon and nutrient cycling. Here, we show that in 13 sympatric temperate tree species, when nutrient availability is patchy, thinner root species alter their foraging to exploit patches, whereas thicker root species do not. Moreover, there appear to be two distinct pathways by which thinner root tree species enhance foraging in nutrient-rich patches: AM trees produce more roots, whereas EM trees produce more mycorrhizal fungal hyphae. Our results indicate that strategies of nutrient foraging are complementary among tree species with contrasting mycorrhiza types and root morphologies, and that predictable relationships between below-ground traits and nutrient acquisition emerge only when both roots and mycorrhizal fungi are considered together.

Mycorrhizal fungi and roots are complementary in foraging within nutrient patches.

The roots of the majority of tree species are associated with either arbuscular mycorrhizal (AM) or ectomycorrhizal (EM) fungi. The absorptive roots of tree species also vary widely in their diameter. The linkages between root thickness, mycorrhiza type and nutrient foraging are poorly understood. We conducted a large root ingrowth experiment in the field to investigate how absorptive roots of varying thickness and their associated fungi (AM vs. EM) exploit different nutrient patches (inorganic and organic) in a common garden. In nutrient-rich patches, thin-root tree species more effectively proliferated absorptive roots than thick-root tree species, whereas thick-root tree species proliferated more mycorrhizal fungal biomass than thin-root tree species. Moreover, nutrient patches enriched with organic materials resulted in greater root and mycorrhizal fungal proliferation compared to those enriched with inorganic nutrients. Irrespective of root morphology, AM tree species had higher root foraging precision than mycorrhizal hyphae foraging precision within organic patches, whereas EM tree species exhibited the opposite. Our findings that roots and mycorrhizal fungi are complementary in foraging within nutrient patches provide new insights into species coexistence and element cycling in terrestrial ecosystems.

Linking root traits to nutrient foraging in arbuscular mycorrhizal trees in a temperate forest.

The identification of plant functional traits that can be linked to ecosystem processes is of wide interest, especially for predicting vegetational responses to climate change. Root diameter of the finest absorptive roots may be one plant trait that has wide significance. Do species with relatively thick absorptive roots forage in nutrient-rich patches differently from species with relatively fine absorptive roots? We measured traits related to nutrient foraging (root morphology and architecture, root proliferation, and mycorrhizal colonization) across six coexisting arbuscular mycorrhizal (AM) temperate tree species with and without nutrient addition. Root traits such as root diameter and specific root length were highly correlated with root branching intensity, with thin-root species having higher branching intensity than thick-root species. In both fertilized and unfertilized soil, species with thin absorptive roots and high branching intensity showed much greater root length and mass proliferation but lower mycorrhizal colonization than species with thick absorptive roots. Across all species, fertilization led to increased root proliferation and reduced mycorrhizal colonization. These results suggest that thin-root species forage more by root proliferation, whereas thick-root species forage more by mycorrhizal fungi. In mineral nutrient-rich patches, AM trees seem to forage more by proliferating roots than by mycorrhizal fungi.

Variability in root production, phenology, and turnover rate among 12 temperate tree species.

The timing of fine root production and turnover strongly influences both the seasonal potential for soil resource acquisition among competing root systems and the plant fluxes of root carbon into soil pools. However, basic patterns and variability in the rates and timing or fine root production and turnover are generally unknown among perennial plants species. We address this shortfall using a heuristic model relating root phenology to turnover together with three years of minirhizotron observations of root dynamics in 12 temperate tree species grown in a common garden. We specifically investigated how the amount and the timing of root production differ among species and how they impact estimates of fine root turnover. Across the 12 species, there was wide variation in the timing of root production with some species producing a single root flush in early summer and others producing roots either more uniformly over the growing season or in multiple pulses. Additionally, the pattern and timing of root production appeared to be consistent across years for some species but varied in others. Root turnover rate was related to total root production (P < 0.001) as species with greater root production typically had faster root turnover rates. We also found that, within species, annual root production varied up to a threefold increase between years, which led to large interannual differences in turnover rate. Results from the heuristic model indicated that shifting the pattern or timing of root production can impact estimates of root turnover rates for root populations with life spans less than one year while estimates of root turnover rate for longer lived roots were unaffected by changes in root phenology. Overall, we suggest that more detailed observations of root phenology and production will improve fidelity of root turnover estimates. Future efforts should link patterns of root phenology and production with whole-plant life history traits and variation in annual and seasonal climate.

Predicting fine root lifespan from plant functional traits in temperate trees.

Although linkages of leaf and whole-plant traits to leaf lifespan have been rigorously investigated, there is a limited understanding of similar linkages of whole-plant and fine root traits to root lifespan. In comparisons across species, do suites of traits found in leaves also exist for roots, and can these traits be used to predict root lifespan? We observed the fine root lifespan of 12 temperate tree species using minirhizotrons in a common garden and compared their median lifespans with fine-root and whole-plant traits. We then determined which set of combined traits would be most useful in predicting patterns of root lifespan. Median root lifespan ranged widely among species (95-336 d). Root diameter, calcium content, and tree wood density were positively related to root lifespan, whereas specific root length, nitrogen (N) : carbon (C) ratio, and plant growth rate were negatively related to root lifespan. Root diameter and plant growth rate, together (R² = 0.62) or in combination with root N : C ratio (R² = 0.76), were useful predictors of root lifespan across the 12 species. Our results highlight linkages between fine root lifespan in temperate trees and plant functional traits that may reduce uncertainty in predictions of root lifespan or turnover across species at broader spatial scales.

Topographical shifts in fine root lifespan in a mixed, mesic temperate forest.

Root lifespan, often is estimated in landscape- and ecosystem-level carbon models using linear approximations. In water manipulation experiments, fine root lifespan can vary with soil water content. Soil water content is generally structured by complex topography, which is largely unaccounted for in landscape- and ecosystem-scale carbon models. Topography governs the range of soil water content experienced by roots which may impact their lifespan. We hypothesized that root lifespan varied nonlinearly across a temperate, mesic, forested catchment due to differences in soil water content associated with topographic position. We expected regions of the landscape that were too wet or too dry would have soils that were not optimal for roots and thus result in shorter root lifespans. Specifically, we hypothesized that root lifespan would be longest in areas that consistently had soil water content in the middle of the soil water content spectrum, while in soils at either very low or very high soil water content, root lifespan would be relatively short. We tested this hypothesis by collecting and analyzing two years of minirhizotron and soil moisture data in plots widely distributed in the Shale Hills catchment of the Susquehanna-Shale Hills Critical Zone Observatory in Pennsylvania. We found that fine root lifespans were longer in traditionally wetter topographic regions, but detected no short term (biweekly) effect of soil moisture on root lifespan. Additionally, depth in soil, soil series, slope face orientation, and season of birth strongly affected root lifespans across the catchment. In contrast, lifespan was unaffected by root diameter or mycorrhizal association. Failure to account for these variables could result in erroneous estimates of fine root lifespan and, consequentially, carbon flux in temperate forested regions.

Reliance on shallow soil water in a mixed-hardwood forest in central Pennsylvania.

We investigated depth of water uptake of trees on shale-derived soils in order to assess the importance of roots over a meter deep as a driver of water use in a central Pennsylvania catchment. This information is not only needed to improve basic understanding of water use in these forests but also to improve descriptions of root function at depth in hydrologic process models. The study took place at the Susquehanna Shale Hills Critical Zone Observatory in central Pennsylvania. We asked two main questions: (i) Do trees in a mixed-hardwood, humid temperate forest in a central Pennsylvania catchment rely on deep roots for water during dry portions of the growing season? (ii) What is the role of tree genus, size, soil depth and hillslope position on the depth of water extraction by trees? Based on multiple lines of evidence, including stable isotope natural abundance, sap flux and soil moisture depletion patterns with depth, the majority of water uptake during the dry part of the growing season occurred, on average, at less than ∼60 cm soil depth throughout the catchment. While there were some trends in depth of water uptake related to genus, tree size and soil depth, water uptake was more uniformly shallow than we expected. Our results suggest that these types of forests may rely considerably on water sources that are quite shallow, even in the drier parts of the growing season.

The continuous incorporation of carbon into existing Sassafras albidum fine roots and its implications for estimating root turnover.

Although understanding the timing of the deposition of recent photosynthate into fine roots is critical for determining root lifespan and turnover using isotopic techniques, few studies have directly examined the deposition and subsequent age of root carbon. To gain a better understanding of the timing of the deposition of root carbon, we labeled four individual Sassafras albidum trees with 99% 13C CO2. We then tracked whether the label appeared in roots that were at least two weeks old and no longer elongating, at the time of labeling. We found that not only were the non-structural carbon pools (soluble sugars and starch) of existing first-order tree roots incorporating carbon from current photosynthate, but so were the structural components of the roots, even in roots that were more than one year old at the time of labeling.Our findings imply that carbon used in root structural and nonstructural pools is not derived solely from photosynthate at root initiation and have implications regarding the determination of root age and turnover using isotopic techniques.

Foraging strategies in trees of different root morphology: the role of root lifespan.

Resource exploitation of patches is influenced not simply by the rate of root production in the patches but also by the lifespan of the roots inhabiting the patches. We examined the effect of sustained localized nitrogen (N) fertilization on root lifespan in four tree species that varied widely in root morphology and presumed foraging strategy. The study was conducted in a 12-year-old common garden in central Pennsylvania using a combination of data from minirhizotron and root in-growth cores. The two fine-root tree species, Acer negundo L. and Populus tremuloides Michx., exhibited significant increases in root lifespan with local N fertilization; no significant responses were observed in the two coarse-root tree species, Sassafras albidum Nutt. and Liriodendron tulipifera L. Across species, coarse-root tree species had longer median root lifespan than fine-root tree species. Localized N fertilization did not significantly increase the N concentration or the respiration of the roots growing in the N-rich patch. Our results suggest that some plant species appear to regulate the lifespan of different portions of their root system to improve resource acquisition while other species do not. Our results are discussed in the context of different strategies of foraging of nutrient patches in species of different root morphology.

Above- and belowground controls on water use by trees of different wood types in an eastern US deciduous forest.

Stomata control tree transpiration by sensing and integrating environmental signals originating in the atmosphere and soil, and co-occurring species may differ in inherent stomatal sensitivity to these above- and belowground signals and in the types of signals to which they respond. Stomatal responsiveness to environmental signals is likely to differ across species having different types of wood (e.g., ring-porous, diffuse-porous and coniferous) because each wood type differs in the structure, size and spatial distribution of its xylem conduits as well as in the scaling of hydraulic properties with stem diameter. The objective of this study was to evaluate the impact of variation in soil water availability and atmospheric evaporative demand on stomatal regulation of transpiration in seven co-occurring temperate deciduous forest species representing three wood types. We measured whole-tree sap flux and soil and atmospheric variables in a mixed deciduous forest in central Pennsylvania over the course of a growing season characterized by severe drought and large fluctuations in atmospheric vapor pressure deficit (D). The relative sensitivity of sap flux to soil drying was ∼2.2-2.3 times greater in the diffuse-porous and coniferous species than in the ring-porous species. Stomata of the ring-porous oaks were only about half as responsive to increased D as those of trees of the other two wood types. These differences in responsiveness to changes in the below- and aboveground environment implied that regulation of leaf water potential in the ring-porous oaks was less stringent than that in the diffuse-porous angiosperms or the conifers. The results suggest that increases in the frequency or intensity of summer droughts in the study region could have multiple consequences for forest function, including altered successional time courses or climax species composition and cumulative effects on whole-tree architecture, resulting in a structural and physiological legacy that would restrict the ability of trees to respond rapidly to more favorable growth conditions.