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As global change shifts the species composition of forests, we need to understand which species characteristics affect soil organic matter cycling to predict future soil carbon (C) storage. Recently, whether a tree species forms a symbiosis with arbuscular (AM) versus ectomycorrhizal (EcM) fungi has been suggested as a strong predictor of soil carbon storage, but there is wide variability within EcM systems. In this study, we investigated how mycorrhizal associations and the species composition of canopy trees and mycorrhizal fungi relate to the proportion of soil C and nitrogen (N) in mineral-associations and soil C:N across four sites representing distinct climates and tree communities in the Eastern U.S. broadleaf forest biome. In two of our sites, we found the expected relationship of declining mineral-associated C and N and increasing soil C:N ratios as the basal area of EcM-associating trees increased. However, across all sites these soil properties strongly correlated with canopy tree and fungal species composition. Sites where the expected pattern with EcM basal area was observed were 1) dominated by trees with lower quality litter in the Pinaceae and Fagaceae families and 2) dominated by EcM fungi with medium distance exploration type hyphae, melanized tissues, and the potential to produce peroxidases. This observational study demonstrates that differences in soil organic matter between AM andEcM systems are dependent on the taxa of trees and EcM fungi involved. Important information is lost when the rich mycorrhizal symbiosis is reduced to two categories. This article is protected by copyright. All rights reserved.

Long-term fire exclusion may weaken ecosystem resistance to the return of fire. We investigated how a surface wildfire that occurred after several decades of fire exclusion affected a southern Appalachian forest transitioning from a fire-adapted to a fire-intolerant state. Tree traits associated with fire adaptation often co-occur with traits for nutrient conservation, including the ectomycorrhizal (ECM) association. In the absence of fire, the ECM association may facilitate the accumulation of organic matter, which becomes colonized by fine roots that then become vulnerable to consumption or damage by fire. Therefore, a deeper organic horizon might make stands of fire-adapted, ECM trees less resistant to a surface wildfire than stands of arbuscular mycorrhizal (AM), fire-intolerant trees. To test this hypothesis, we established plots in stands that fall along a gradient of mycorrhizal tree relative abundance both inside and outside the perimeter of the 2016 Rock Mountain wildfire. With increasing relative abundance of ECM trees, we found increasing organic horizon depth and mass and slower rates of decay, even for litter of ECM tree species. We calculated a major (73–83%) reduction in fine root biomass and length in the organic horizon following the wildfire. Over three years post-fire, we observed a higher probability of crown decline, basal sprouting and aboveground biomass mortality with increasing abundance of ECM trees. We propose that the biogeochemistry of mycorrhizal associations can help explain why fire exclusion makes stands of fire-adapted trees less resistant to a surface wildfire than those with fire-intolerant trees.

Invasive plants have the potential to affect decomposition both directly, by introducing novel substrates that may differ from native species in key structural or chemical properties, and indirectly through changes to soil properties and microbial communities. The relative importance of these two mechanisms is unclear, especially with regard to wood decomposition. To explore these questions, we used a novel method to rapidly assess the wood decay rates of 11 native and 11 invasive non-native angiosperm species. The study was repeated at three pairs of sites, each consisting of an invaded and a relatively uninvaded forest. The invaded sites had either been colonized by a non-native grass (Microstegium vimineum (Trin.) A. Camus), a non-native woody shrub (Ligustrum sinense Lour.) or by multiple invasive species. After one year in the field, mass loss varied more than two-fold among the 22 wood species (24.2–52.3%). Wood origin (i.e., native or non-native) was only important at the Microstegium sites, with non-native species decomposing marginally faster than native species. Wood decomposed faster at both the Ligustrum-invaded and multiply-invaded sites than in their respective uninvaded sites but there were no differences between sites invaded or not by Microstegium. We detected positive relationships overall between mass loss and pH, K, P and NO3−, but invasion had no consistent effects on these soil properties. The results from this study show that the differences in wood decay rates between native and non-native species and the effects of invasion are highly idiosyncratic, with effects depending greatly on species and ecological context.

Symbiotic nitrogen (N) fixation has been shown to support carbon storage in young regenerating tropical forests, but N-fixing trees can also be strong competitors with non-fixing trees, making it unclear which mechanism drives long term patterns in biomass accretion. Many tropical forests have excess N, but factors such as rising atmospheric CO2 or selective cutting practices might induce additional N demand. Here we combine decades of stem inventory data, in-situ measures of symbiotic N fixation, and simulations of N demand to evaluate demographic and biogeochemical controls on biomass dynamics in legume-rich lowland forests of Trinidad. We document sustained net biomass accumulation and high rates of N fixation in these forests, regardless of the timing of selective timber harvests, including an old growth stand. The biomass accumulation was explained by growth of non-fixing trees, not N-fixing trees, but the total amount of symbiotic N fixation was sufficient to account for most of net above ground N demands, suggesting that N-fixers could contribute to the long-term C sink in these forests via fertilizing non-fixers.

The response of arctic ecosystems to global change will be critical for future climate, due to their vast stores of carbon. Climate warming appears to be linked to the expansion of shrubs across tundra, but it is unclear how shrubs affect the activity of soil microbes. We investigated three potential mechanisms by which shrubs may stimulate soil microbial activity, by 1) increasing the rate of litter inputs, 2) promoting soil microbial adaptation to litter and 3) reducing nutrient limitation. We created microcosms of root-free soils collected from shrub (Alnus fruticosa, Betula nana, and Salix pulchra) and non-shrub plots in arctic Alaska and conducted two experiments. We quantified heterotrophic soil respiration rates in response to litter inputs (experiment 1), or nutrient inputs (nitrogen, phosphorus, or both nutrients together, experiment 2). We found that shrub-conditioned soils maintained higher rates of soil respiration in both experiments. Shrub litter increased respiration in both soil types, but the relative response was greater in non-shrub soils. We found no evidence that shrubs reduce nutrient limitation to heterotrophic respiration, although we observed a short-term increase in respiration after phosphorus addition in both soil types. Collectively, our results suggest that higher rates of respiration in shrub-conditioned soils may be the result of higher litter input rates, but other factors such as organic matter quality or microbial community structure may also contribute to our observed differences in respiration.

Experimental warming of forest ecosystems typically stimulates soil respiration (CO2 efflux), but most warming experiments have been conducted in northern latitudes (> 40°N) with relatively young soils. We quantified the influence of experimental warming on soil respiration (RT) in two adjacent forest habitats—a mature, closed canopy forest and a gap where trees were manually removed— on highly-weathered Ultisols of the southeastern U.S. (33°N). Using temperature variation, both natural and induced by experimental warming, we also quantified the temperature sensitivity of RT, defined as the activation energy, EA in the Arrhenius equation. Experimental warming (either + 3 °C or + 5 °C above ambient) did not significantly increase soil respiration rate or cumulative CO2 loss over the 3 years of the experiment, and did not influence the temperature sensitivity of soil respiration, once the influence of natural temperature variation was taken into consideration. Despite the absence of an experimental warming effect, we observed that EA varied on monthly time scales, and varied differently in each habitat. Soil moisture and habitat also influenced RT, but the effects were not consistent, and varied by month. Our results suggest that although RT does depend on temperature, the sensitivity of RT to temperature variation is influenced primarily by factors like microclimate and plant phenology that can change on relatively short (

The reintroduction of disease‐resistant hybrids is a commonly proposed solution to the introduction of pathogens and pests that weaken or eliminate native plant species. Plant interactions with soil biota result in plant–soil feedbacks (PSFs), which have consequences for individual plant growth and survival as well as broader community‐level processes, such as species diversity and coexistence. Because of their importance, species reintroduction should consider these interactions, yet little work has integrated this perspective. Here, we investigate the effects of hybrid Castanea (chestnut) reintroduction on PSFs and how these mechanisms may influence the recruitment of other species in contemporary forests. We also examine how blight‐resistant Castanea hybrids perform in the soil conditions of contemporary forests and we compare their belowground interactions with those of Castanea dentata. We conducted a reciprocal greenhouse experiment testing the effect of species‐specific soil inoculum on the growth and survival of C. dentata, Castanea hybrids, and other forest dominants. Our results suggest that C. dentata and hybrids had similar belowground interactions and were regulated by negative PSFs, meaning soil microbial communities reduced conspecific growth and survival. These negative PSFs may involve the presence of the non‐native pathogen Phytophthera cinnamomi. Soil inoculum of C. dentata and Castanea hybrids had similar effects on heterospecific growth, suggesting Castanea restoration will have neutral effects on natural regeneration in restoration plantings. We conclude that Castanea hybrids may fill a similar belowground niche to their parent species, and that site selection, screening for soil pathogens, and site planting density will be important to restoration.

International audience; The effects of plants on the biosphere, atmosphere and geosphere are key determinants of terrestrial ecosystem functioning. However, despite substantial progress made regarding plant belowground components, we are still only beginning to explore the complex relationships between root traits and functions. Drawing on the literature in plant physiology, ecophysiology, ecology, agronomy and soil science, we reviewed 24 aspects of plant and ecosystem functioning and their relationships with a number of root system traits, including aspects of architecture, physiology, morphology, anatomy, chemistry, biomechanics and biotic interactions. Based on this assessment, we critically evaluated the current strengths and gaps in our knowledge, and identify future research challenges in the field of root ecology. Most importantly, we found that belowground traits with the broadest importance in plant and ecosystem functioning are not those most commonly measured. Also, the estimation of trait relative importance for functioning requires us to consider a more comprehensive range of functionally relevant traits from a diverse range of species, across environments and over time series. We also advocate that establishing causal hierarchical links among root traits will provide a hypothesis-based framework to identify the most parsimonious sets of traits with the strongest links on functions, and to link genotypes to plant and ecosystem functioning.

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. ISSN:0028-646X ISSN:1469-8137

Root systems show a tremendous diversity both between and within species, suggesting a large variability in plant functioning and effects on ecosystem properties and processes. In recent decades, developments in many areas of root research have brought considerable advances in our understanding of root traits and their contribution to plant and ecosystem functioning. However, despite major progress, a comprehensive overview—bridging research fields—is lacking. Furthermore, considerable uncertainties exist in the identification of root entities, and the selection and standardized measurement of traits. Here, we provide a comprehensive overview on root entities, exemplify recent advances in our understanding of both theoretical and demonstrated relationships between root traits and plant or ecosystem functioning, discuss trait-trait relationships and hierarchies among traits, and critically assess current strengths and gaps in our knowledge.

Plant diversity has long been assumed to predict soil microbial diversity. However, contradictory results have been found when examining their relationships, particularly at broad spatial scales. To address this issue, we conducted a meta‐analysis to evaluate the patterns in the correlation between plant diversity and soil microbial diversity and the underlying factors driving the relationship. We collected correlation data from 84 studies covering more than 3900 natural terrestrial samples globally. Using the hierarchical mixed‐effects model, we investigated factors including targeted taxonomic group, microbial examination method, sampling extent, biome type, soil type, and environmental factors to assess the patterns of the plant–microbial correlation and the determinants of their variations. We found that microbial richness displayed a modest but positive correlation with plant diversity (r = 0.333, CI = 0.220–0.437). In spite of variability among taxonomic groups and their relationship with plant diversity, positive correlations were more pronounced in the intermediate sampling extent of latitude and elevation coverage, and tropical forests. Among examined environmental factors, soil pH was negatively associated with the plant and soil microbial relationships at large spatial scales. The plant–microbial correlation appears more sensitive to edaphic factor variation in the poor nutrients and soil less compact systems. Collectively, our results point to key differences across taxonomic groups, spatial scales and biomes, and the modulating effects of climate and soil. The findings shed light on our deep understanding in plant–microbial diversity relationships at broad spatial scales and ecosystem sensitivity to biodiversity loss and environmental change.

Longleaf pine savannas house a diverse community of herbaceous N2-fixing legume species that have the potential to replenish nitrogen (N) losses from fire. Whether legumes fill this role depends on the factors that regulate symbiotic fixation, including soil nutrients such as phosphorus (P) and molybdenum (Mo) and the growth and fixation strategies of different species. In greenhouse experiments, we determined how these factors influence fixation for seven species of legumes grown in pure field soil from two different regions of the southeastern US longleaf pine ecosystem. We first added P and Mo individually and in combination, and found that P alone constrained fixation. Phosphorus primarily influenced fixation by regulating legume growth. Second, we added N to plants and found that species either downregulated fixation (facultative strategy) or maintained fixation at a constant rate (obligate strategy). Species varied nearly fourfold in fixation rate, reflecting differences in growth rate, taxonomy and fixation strategy. However, fixation responded strongly to P addition across all species in our study, suggesting that the P cycle regulates N inputs by herbaceous legumes.

Most land plants acquire nitrogen (N) through associations with arbuscular (AM) and ectomycorrhizal (ECM) fungi, but these symbionts employ contrasting strategies for N acquisition, which may lead to different stocks of soil carbon (C). We experimentally test this hypothesis with a mesocosm system where AM and ECM tree seedling roots, or their hyphae only, could access mineral soils with 13 C- and 15 N-enriched organic matter. We quantified loss of soil C and N, plant uptake of N and new inputs of plant C to soil. We found that AM, but not ECM, seedlings reduced soil C relative to controls. Soil C loss was greater in the presence of roots relative to hyphae only for both AM and ECM seedlings, but was correlated with plant N uptake for AM seedlings only. While new plant C inputs stimulated soil C loss in both symbioses, we detected plant C inputs more frequently and measured higher rates of decomposer activity in soils colonized by AM relative to ECM seedlings. Our study experimentally demonstrates how mycorrhizal strategies for N can affect soil C and C:N, even at the scale of an individual plant. Such effects may contribute to broad patterns in soil C across terrestrial ecosystems.

Summary Climate change is increasing drought frequency, which may affect symbiotic N2 fixation (SNF), a process that facilitates ecosystem recovery from disturbance. Here, we assessed the effect of drought frequency on the ecophysiology and SNF rate of a common N2-fixing tree in eastern US forests. We grew Robinia pseudoacacia seedlings under the same mean soil moisture, but with different drought frequency caused by wet–dry cycles of varying periodicity. We found no effect of drought frequency on final biomass or mean SNF rate. However, seedlings responded differently to wet and dry phases depending on drought frequency. Under low-frequency droughts, plants fixed carbon (C) and nitrogen (N) at similar rates during wet and dry phases. Conversely, under high-frequency droughts, plants fixed C and N at low rates during dry phases and at high rates during wet phases. Our findings suggest that R. pseudoacacia growth is resistant to increased drought frequency because it employs two strategies – drought tolerance or drought avoidance, followed by compensation. SNF may play a role in both by supplying N to leaf tissues for acclimation and by facilitating compensatory growth following drought. Our findings point to SNF as a mechanism for plants and ecosystems to cope with drought.

Climate change may affect the microbial production and temperature sensitivity of extracellular enzymes that release carbon (C) and nutrients from soil organic matter. We measured the response of six hydrolytic enzymes involved in C, nitrogen (N), and phosphorus (P) degradation to experimental warming in a mixed-deciduous forest persisting on highly-weathered Ultisols (Whitehall Forest, Georgia, US). We found that warming produced no consistent enzyme response. However, we observed significant seasonal variation in enzyme activities, temperature sensitivities and elemental enzyme ratios (C:N and C:P). Seasonal differences in enzyme activity and temperature sensitivity were best explained by soil moisture and temperature. Our results suggest that seasonal dynamics in soil microclimate, organic matter supply, and microbial demand exert more control on enzyme dynamics than does a uniform increase in soil temperature.

Increased drought intensity and frequency due to climate change may reduce the abundance and activity of nitrogen (N2 )-fixing plants, which supply new N to terrestrial ecosystems. As a result, drought may indirectly reduce ecosystem productivity through its effect on the N cycle. Here, we manipulated growing season net rainfall across a series of plots in an early successional mesic deciduous forest to understand how drought affects the aboveground productivity of the N2 -fixing tree Robinia pseudoacacia and three co-occurring nonfixing tree species. We found that lower soil moisture was associated with reduced productivity of R. pseudoacacia but not of nonfixing trees. As a result, the relative biomass and density of R. pseudoacacia declined in drier soils over time. Greater aboveground biomass of R. pseudoacacia was also associated with greater total soil N, extractable inorganic N, N mineralization rates, and productivity of nonfixing trees. These soil N effects may reflect current N2 fixation by R. pseudoacacia saplings, or the legacy effect of former trees in the same location. Our results suggest that R. pseudoacacia promotes the growth of nonfixing trees in early succession through its effect on the N cycle. However, the sensitivity of R. pseudoacacia to dry soils may reduce N2 fixation under scenarios of increasing drought intensity and frequency, demonstrating a mechanism by which drought may indirectly diminish potential forest productivity and recovery rate from disturbance.

Fire is a critical force in structuring ecosystems, but it also removes substantial amounts of nitrogen (N), which can limit plant growth. Biological N fixation (BNF) may alleviate fire-induced N deficiencies that inhibit ecosystem recovery, yet if and how BNF achieves this under frequent fire is unclear. This problem is further complicated in the context of modern human influences (such as land-use history and atmospheric N deposition), which may confound the relationship between fire and fixation. Here, we investigate whether BNF supplies the N necessary to replace fire-induced N losses in restored longleaf pine savannas, and, if so, what factors control fixation. We established 54 1-ha plots of longleaf pine capturing 227 yr of forest recovery and a broad gradient of fire return interval (1.5-20 yr) at two sites in the southeastern United States. We quantified N fixation from three functional groups (herbaceous legumes, soil crusts, and asymbiotic N fixing bacteria), N losses from individual fire events and ecosystem dynamics of N supply and demand. We found that BNF rates were low but sustained over stand age but were substantially below estimated rates of atmospheric N deposition. While fire temporarily stimulated BNF from herbaceous legumes, neither BNF nor atmospheric N deposition were sufficient to balance N losses from fire and soil N stocks declined over stand age. However, rates of N mineralization were surprisingly high and tree productivity was unrelated to N availability, questioning the importance of N limitation in these temperate savannas. While it is possible that progressive N losses signal a decline in ecosystem resiliency, N enrichment from multiple land-use transitions and anthropogenic N deposition may suppress rates of BNF or diminish its importance as a long-term N balancing source in these pyrogenic ecosystems. In this case, fire may be acting as relief mechanism, critical for returning the modern longleaf pine landscape to its historical oligotrophic condition.

Natural forest disturbance events can influence soil biogeochemical processes in two ways - by creating downed woody debris (DWD; fallen tree boles or branches) and by creating canopy gaps that alter forest microclimate. DWD represents a substrate for microbial growth and a persistent store of carbon and nutrients, but microbial activity is also sensitive to temperature and moisture. We studied the potential interaction of DWD and canopy gaps on soil microbial processes, and wondered if microclimatic conditions resulting from the manipulation of forest structure would be enough to inhibit production, thereby altering a critical ecosystem process. Gaps and DWD (>10 cm diameter) were added to a maturing, even-aged, second-growth northern hardwood forest (the Flambeau Experiment; N Wisconsin, USA) to enhance structural complexity and promote key ecosystem processes typically associated with late-successional forests. We investigated the influence of DWD and gaps on soil microbial community composition, extracellular enzyme activity and soil characteristics. Soils were sampled near intermediately and highly decayed DWD and 2 m away from DWD (control) in gaps and closed canopy a decade after manipulation. DWD decomposition influenced the surrounding soil differentially depending on decay class and canopy condition. Mean C- and P-potential extracellular enzyme activities (BG, BX and AP) were enhanced near highly decayed DWD in gaps. The relative abundance of bacteria (actinomycete, anaerobic, gram-negative and gram-positive) remained constant in gaps but decreased from May to August in closed canopy. In gaps, soil total exchangeable cations increased by 34.6%, available phosphorus by 152% and fungal to bacterial ratios by 23.3% but temperatures decreased by 3.42% suggesting that canopy condition continues to affect soil properties and microbial processes a decade after gap creation. These results highlight the contribution of DWD to the forest floor and the influence of decaying wood characteristics on belowground ecosystems critical to future forest productivity. Retaining or adding heterogeneously distributed DWD of varying decay status may be essential to maintain ecosystem functions associated with nutrient cycling and microbial community dynamics in managed forests.

Increased drought intensity and frequency due to climate change may reduce the abundance and activity of nitrogen (N

In Neotropical forests, lianas are increasing in abundance relative to trees. This increased species richness may reflect a positive response to global change factors including increased temperature, atmospheric CO2, habitat fragmentation, and drought severity; however, questions remain as to the specific mechanisms facilitating the response. Previous work suggests that lianas may gain an ecological advantage over trees through leaf functional traits that offer a quick return on investment of resources, although it is unknown whether this pattern extends to root traits and relationships with fungal or bacterial symbionts belowground. We sampled confamilial pairs of liana and tree species and quantified morphological and chemical traits of leaves and fine roots, as well as root symbiont abundance, to determine whether functional traits associated with resource acquisition differed between the two. Compared to trees, lianas possessed higher specific leaf area, specific root length, root branching intensity, and root nitrogen (N) and phosphorus (P) concentrations, and lower leaf and root tissue density, leaf and root carbon (C), root diameter, root C:P and N:P, and mycorrhizal colonization. Our study provides new evidence that liana leaf and root traits are characterized by a rapid resource acquisition strategy relative to trees. These liana functional traits may facilitate their response to global change, raising questions about how increased liana dominance might affect ecosystem processes of Neotropical forests.

Questions remain as to which soil nutrients limit primary production in tropical forests. Phosphorus (P) has long been considered the primary limiting element in lowland forests, but recent evidence demonstrates substantial heterogeneity in response to nutrient addition, highlighting a need to understand and diagnose nutrient limitation across diverse forests. Fine-root characteristics including their abundance, functional traits, and mycorrhizal symbionts can be highly responsive to changes in soil nutrients and may help to diagnose nutrient limitation. Here, we document the response of fine roots to long-term nitrogen (N), P, and potassium (K) fertilization in a lowland forest in Panama. Because this experiment has demonstrated that N and K together limit tree growth and P limits fine litter production, we hypothesized that fine roots would also respond to nutrient addition. Specifically we hypothesized that N, P, and K addition would reduce the biomass, diameter, tissue density, and mycorrhizal colonization of fine roots, and increase nutrient concentration in root tissue. Most morphological root traits responded to the single addition of K and the paired addition of N and P, with the greatest response to all three nutrients combined. The addition of N, P, and K together reduced fine-root biomass, length, and tissue density, and increased specific root length, whereas root diameter remained unchanged. Nitrogen addition did not alter root N concentration, but P and K addition increased root P and K concentration, respectively. Mycorrhizal colonization of fine roots declined with N, increased with P, and was unresponsive to K addition. Although plant species composition remains unchanged after 14 years of fertilization, fine-root characteristics responded to N, P, and K addition, providing some of the strongest stand-level responses in this experiment. Multiple soil nutrients regulate fine-root abundance, morphological and chemical traits, and their association with mycorrhizal fungi in a species-rich lowland tropical forest.

Climate change is increasing drought frequency, which may affect symbiotic N

Summary Symbiotic di-nitrogen (N2) fixation is a critical biogeochemical process in tropical forests, yet it remains unresolved how fixation is controlled by the availability of soil nitrogen and phosphorus, two nutrients often considered limiting in terrestrial ecosystems. We examine whether individual N2-fixing trees can overcome nitrogen and phosphorus constraints by employing different strategies of nutrient acquisition and use: N2 fixation, phosphatase exudation, mycorrhizal symbiosis and changes in root–shoot ratio or tissue stoichiometry. We grew a common and widespread N2 fixer, Inga punctata, in a full factorial nitrogen and phosphorus addition experiment (each nutrient at three levels) and evaluated whether trees adjusted their strategies of nutrient acquisition to overcome limitation. N2 fixation was controlled by nitrogen availability in phosphorus-sufficient soils, but both fixation and plant growth were constrained by phosphorus in the unamended native phosphorus-poor soils. Despite the investment in both extracellular phosphatases and mycorrhizal symbionts, plants were unable to overcome phosphorus limitation. Our findings support the hypotheses that: (i) N2 fixation is proximately controlled by nitrogen availability, consistent with a facultative fixation strategy, and (ii) N2 fixation and N2 fixer biomass growth are ultimately constrained by soil phosphorus. We found no support for the hypothesis that fixers can overcome phosphorus limitation by trading fixed N2 for soil phosphorus. Synthesis. This study provides new knowledge about how nitrogen and phosphorus interact to regulate tropical N2 fixation by examining a suite of strategies that plants may employ to overcome nutrient limitation. These findings, focused at the organismal level, have broader implications for biogeochemical controls at the ecosystem level in tropical forests.

Ericoid mycorrhizal fungi (ERM) may specialize in capturing nutrients from their host's litter as a strategy for regulating nutrient cycles in terrestrial ecosystems. In spite of their potential significance, we know little about the structure of ERM fungal communities and the genetic basis of their saprotrophic traits (e.g., genes encoding extracellular enzymes). Rhododendron maximum is a model ERM understory shrub that influences the nutrient cycles of montane hardwood forests in the southern Appalachians (North Carolina, USA). We sampled ERM roots of R. maximum from organic and mineral soil horizons and identified root fungi by amplifying and sequencing internal transcribed spacer (ITS) ribosomal DNA (rDNA) collected from cultures and clones. We observed 71 fungal taxa on ERM roots, including known symbionts Rhizoscyphus ericae and Oidiodendron maius, putative symbionts from the Helotiales, Chaetothyriales, and Sebacinales, ectomycorrhizal symbionts, and saprotrophs. Supporting the idea that ERM fungi are adept saprotrophs, richness of root-fungi was greater in organic than in mineral soil horizons. To study the genetic diversity of oxidative enzymes that contribute to decomposition, we amplified and sequenced a portion of genes encoding multicopper oxidases (MCOs) from ERM ascomycetes. Most fungi possessed multiple copies of MCO sequences with strong similarities to known ferroxidases and laccases. Our findings indicate that R. maximum associates with a taxonomically and ecologically diverse fungal community. The study of MCO gene diversity and expression may be useful for understanding how ERM root fungi regulate the cycling of nutrients between the host plant and the soil environment.

Summary 1. Relationships between mycorrhizal plants and soil nitrogen (N) have led to the speculation that the chemistry of plant litter and the saprotrophy of mycorrhizal symbionts can function together to closely couple the N cycle between plants and soils. We hypothesized that a tannin-rich, ericoid mycorrhizal (ERM) plant promotes the retention of protein‐tannin N in soil, and that this N source is accessible to saprotrophic ERM symbionts and their hosts, but remains less available to co-occurring ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) symbionts and their hosts. 2. We tested this feedback hypothesis in a southern Appalachian forest community composed of two microsites: a hardwood microsite with ECM and AM trees in the overstorey and understorey, and an AM herb layer; and a rhododendron microsite where the understorey and herb layer are replaced by ERM rhododendron. We synthesized 15 N-enriched protein‐tannin complexes from leaf litter extracts representing each forest microsite and examined the fate of 15 N in soil volumes 3 months and 1 year after the complexes were placed in the field. 3. Protein‐tannin complexes derived from the rhododendron microsite led to a higher retention of 15 N in soil organic matter and a lower recovery in dissolved N pools than those from the hardwood microsite, supporting the hypothesis that rhododendron tannins create stable complexes that increase organic N retention in soils. 4. Rhododendron complexes led to greater 15 N-enrichment in ERM roots than in AM and ECM roots, supporting the hypothesis that rhododendron can better access the N complexed by its own litter tannins than can co-occurring forest trees and plants. Our results suggest that both fungal saprotrophy and a high specific root length contribute to the ability of ERM roots to acquire N from complex organic sources. 5. Synthesis. This study provides evidence of an intricate N feedback where plant litter chemistry influences the cycle of N to maximize N acquisition by the host’s mycorrhizal roots, while hindering N acquisition by mycorrhizal roots of co-occurring plants. Feedback processes such as these have the potential to drive patterns in nitrogen cycling and productivity in many terrestrial ecosystems.

Biological nitrogen fixation limits plant growth and carbon exchange at local to global scales. Long-term nutrient manipulation experiments in forests and short-term manipulation experiments in microcosms suggest that the micronutrient molybdenum, a component of the nitrogen-fixing enzyme nitrogenase, limits nitrogen fixation by asymbiotic bacteria in tropical soils in Panama. Nitrogen fixation, the biological conversion of di-nitrogen to plant-available ammonium, is the primary natural input of nitrogen to ecosystems1, and influences plant growth and carbon exchange at local to global scales2,3,4,5,6. The role of this process in tropical forests is of particular concern, as these ecosystems harbour abundant nitrogen-fixing organisms1,4 and represent one third of terrestrial primary production4,7,8. Here we show that the micronutrient molybdenum, a cofactor in the nitrogen-fixing enzyme nitrogenase, limits nitrogen fixation by free-living heterotrophic bacteria in soils of lowland Panamanian forests. We measured the fixation response to long-term nutrient manipulations in intact forests, and to short-term manipulations in soil microcosms. Nitrogen fixation increased sharply in treatments of molybdenum alone, in micronutrient treatments that included molybdenum by design and in treatments with commercial phosphorus fertilizer, in which molybdenum was a ‘hidden’ contaminant. Fixation did not respond to additions of phosphorus that were not contaminated by molybdenum. Our findings show that molybdenum alone can limit asymbiotic nitrogen fixation in tropical forests and raise new questions about the role of molybdenum and phosphorus in the tropical nitrogen cycle. We suggest that molybdenum limitation may be common in highly weathered acidic soils, and may constrain the ability of some forests to acquire new nitrogen in response to CO2 fertilization9.

The recent infestation of southern Appalachian eastern hemlock stands by hemlock woolly adelgid (HWA) is expected to have dramatic and lasting effects on forest structure and function. We studied the short-term changes to the carbon cycle in a mixed stand of hemlock and hardwoods, where hemlock was declining due to either girdling or HWA infestation. We expected that hemlock would decline more rapidly from girdling than from HWA infestation. Unexpectedly, in response to both girdling and HWA infestation, hemlock basal area increment (BAI) reduced substantially compared to reference hardwoods in 3 years. This decline was concurrent with moderate increases in the BAI of co-occurring hardwoods. Although the girdling treatment resulted in an initial pulse of hemlock needle inputs, cumulative litter inputs and O horizon mass did not differ between treatments over the study period. Following girdling and HWA infestation, very fine root biomass declined by 20–40% in 2 years, which suggests hemlock root mortality in the girdling treatment, and a reduction in hemlock root production in the HWA treatment. Soil CO2 efflux (Esoil) declined by approximately 20% in 1 year after both girdling and HWA infestation, even after accounting for the intra-annual variability of soil temperature and moisture. The reduction in Esoil and the concurrent declines in BAI and standing very fine root biomass suggest rapid declines in hemlock productivity from HWA infestation. The accelerated inputs of detritus resulting from hemlock mortality are likely to influence carbon and nutrient fluxes, and dictate future patterns of species regeneration in these forest ecosystems.

Summary Rhododendron maximum L., a spreading understory shrub, inhibits overstory regeneration and alters forest community structure in southern Appalachian hardwood forests. Using paired plots and reciprocal litter transplants in forests with and without R. maximum cover, we examined the influence of R. maximum on litter mass and quality, N cycling and soil extracellular enzymes. Standing stocks of soil organic matter, soil N, leaf litter mass and fine root biomass were greater in forests with R. maximum than those without. Tannin extracts from R. maximum foliage, and leaf litter and fine roots collected under R. maximum had a relatively high capacity to precipitate protein compared to extracts from trees. Across the growing season, soil inorganic N availability was generally lower under R. maximum , mostly due to reduced NO 3 − availability. Our data suggest that R. maximum litter alters N cycling through the formation of recalcitrant polyphenol–organic N complexes. Soil extracellular enzymes indicate the potential processing rates of organic substrates. Between forest types, polyphenol oxidase activity was greatest in R. maximum O horizons, regardless of litter type, suggesting that the local microbial community can better degrade and access protein–tannin-complexed N. Protease activity did not differ between forest types, but was greater on R. maximum leaf litter than hardwood leaf litter. The alteration of the N cycle via the formation of polyphenol–organic N complexes may contribute to hardwood seedling suppression, while the enzymatic release of these complexes by ericoid mycorrhizal fungi may increase N acquisition for R. maximum and contribute to its expansion in southern Appalachian forests.

Legumes capable of fixing atmospheric N2 are abundant and diverse in many tropical forests, but the factors determining ecological patterns in fixation are unresolved. A long-standing idea is that fixation depends on soil nutrients (N, P or Mo), but recent evidence shows that fixation may also differ among N2-fixing species. We sampled canopy-height trees across five species and one species group of N2-fixers along a landscape P gradient, and manipulated P and Mo to seedlings in a shadehouse. Our results identify taxonomy as the major determinant of fixation, with P (and possibly Mo) only influencing fixation following tree-fall disturbances. While 44% of trees did not fix N2, other trees fixed at high rates, with two species functioning as superfixers across the landscape. Our results raise the possibility that fixation is determined by biodiversity, evolutionary history and species-specific traits (tree growth rate, canopy stature and response to disturbance) in the tropical biome.

We used morphotyping and molecular methods to characterize ectomycorrhizas of bishop pine (Pinus muricata D. Don) and Bolander pine (Pinus contorta ssp. bolanderi (Parl.) Critchf.) from mixed conifer and hydric pygmy forests on the northern California coast. Sixteen ectomycorrhizal morphotypes were described, producing 15 internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) types, and 12 were identified via ITS sequencing. From a given site, all root tips of a specific morphotype produced identical ITS-RFLP patterns. However, sometimes two morphotypes produced the same ITS-RFLP type, and sometimes samples of the same morphotype from two different sites produced two different ITS-RFLP types. These results indicate that surveys of ectomycorrhizal fungi based on morphology alone are not sufficient, and that grouping morphotypes prior to molecular analysis can expedite the process. Ectomycorrhizas from mixed conifer included Russuloid sp., Tomentella sublilacina (Ellis & Holw.) Wakef., Tuber sp., and two Thelephoroid species. Ectomycorrhizas from hydric pygmy included two Dermocybe spp., a Cortinarius sp., two Thelephoroid spp., and Suillus tomentosus (Kauffman) Singer. Both plant communities contained Cenococcum geophilum Fr.:Fr. The hydric pygmy sites were more similar to each other than to the mixed conifer site (Jaccard similarity). The presence of ectomycorrhizal taxa in one plant community type may reflect biotic (host specificity) or abiotic (soil fertility or hydrology) adaptation.Key words: ectomycorrhiza, bishop pine, Pinus muricata, Bolander pine, Pinus contorta ssp. bolanderi, morphotyping, ITS-RFLP.

On the northern California coast, mixed conifer forests occur on younger, relatively fertile terraces, whereas mesic and hydric pygmy forests occur on older, infertile, poorly drained terraces. We characterized mycorrhizal associations, ectomycorrhizal morphotypes, and colonization levels for nine plant species from three plant communities (mixed conifer, mesic pygmy, and hydric pygmy). Pinus contorta ssp. bolanderi (Parl.) Critchf. and Pinus muricata D. Don were ectomycorrhizal; all ericaceous plants formed ericoid mycorrhizas except Arctostaphylos nummularia A. Gray, which formed only arbutoid mycorrhizas. Arbuscular mycorrhizas were not observed, even on Cupressus goveniana ssp. pigmaea (Lemmon) J. Bartel. Ectomycorrhizal colonization was significantly lower in pygmy forests, while ericoid mycorrhizal colonization was significantly higher, as compared with the mixed conifer forest. Ectomycorrhizal and ericoid mycorrhizal colonization was greater on hummocks than in swales at hydric pygmy sites. Thirteen distinct ectomycorrhizal and arbutoid mycorrhizal morphotypes were observed: eight only on Pinus spp. and two only on A. nummularia. Two morphotypes were found only in mixed conifer, and eight were found only in pygmy communities. Distribution of morphotypes may reveal habitat and (or) host adaptation by ectomycorrhizal fungi.Key words: ectomycorrhizal morphotypes, ericoid mycorrhizas, pygmy forest, Ericaceae, mycorrhizal colonization, low-fertility soils.

We used morphotyping and molecular methods to characterize ectomycorrhizas of bishop pine (Pinus muricata D. Don) and Bolander pine (Pinus contorta ssp. bolanderi (Parl.) Critchf.) from mixed conifer and hydric pygmy forests on the northern California coast. Sixteen ectomycorrhizal morphotypes were described, producing 15 in- ternal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) types, and 12 were identified via ITS sequencing. From a given site, all root tips of a specific morphotype produced identical ITS-RFLP patterns. However, sometimes two morphotypes produced the same ITS-RFLP type, and sometimes samples of the same morphotype from two different sites produced two different ITS-RFLP types. These results indicate that surveys of ectomycorrhizal fungi based on morphology alone are not sufficient, and that grouping morphotypes prior to molecular analysis can expedite the process. Ectomycorrhizas from mixed conifer included Russuloid sp., Tomentella sublilacina (Ellis & Holw.) Wakef., Tuber sp., and two Thelephoroid species. Ectomycorrhizas from hydric pygmy included two Dermocybe spp., a Cortinarius sp., two Thelephoroid spp., and Suillus tomentosus (Kauffman) Singer. Both plant communities contained Cenococcum geophilumFr.:Fr. The hydric pygmy sites were more similar to each other than to the mixed conifer site (Jaccard similarity). The presence of ectomycorrhizal taxa in one plant community type may reflect biotic (host speci- ficity) or abiotic (soil fertility or hydrology) adaptation.

General circulation models project more intense and frequent droughts over the next century, but many questions remain about how terrestrial ecosystems will respond. Of particular importance, is to understand how drought will alter the species composition of regenerating temperate forests wherein symbiotic dinitrogen (N2)-fixing plants play a critical role. In experimental mesocosms we manipulated soil moisture to study the effect of drought on the physiology, growth and competitive interactions of four co-occurring North American tree species, one of which (Robinia pseudoacacia) is a symbiotic N2-fixer. We hypothesized that drought would reduce growth by decreasing stomatal conductance, hydraulic conductance and increasing the water use efficiency of species with larger diameter xylem vessel elements (Quercus rubra, R. pseudoacacia) relative to those with smaller elements (Acer rubrum and Liriodendron tulipifera). We further hypothesized that N2 fixation by R. pseudoacacia would decline with drought, reducing its competitive ability. Under drought, growth declined across all species; but, growth and physiological responses did not correspond to species' hydraulic architecture. Drought triggered an 80% increase in nodule biomass and N accrual for R. pseudoacacia, improving its growth relative to other species. These results suggest that drought intensified soil N deficiency and that R. pseudoacacia's ability to fix N2 facilitated competition with non-fixing species when both water and N were limiting. Under scenarios of moderate drought, N2 fixation may alleviate the N constraints resulting from low soil moisture and improve competitive ability of N2-fixing species, and as a result, supply more new N to the ecosystem.

Ericoid mycorrhizal fungi (ERM) may specialize in capturing nutrients from their host's litter as a strategy for regulating nutrient cycles in terrestrial ecosystems. In spite of their potential significance, we know little about the structure of ERM fungal communities and the genetic basis of their saprotrophic traits (e.g., genes encoding extracellular enzymes). Rhododendron maximum is a model ERM understory shrub that influences the nutrient cycles of montane hardwood forests in the southern Appalachians (North Carolina, USA). We sampled ERM roots of R. maximum from organic and mineral soil horizons and identified root fungi by amplifying and sequencing internal transcribed spacer (ITS) ribosomal DNA (rDNA) collected from cultures and clones. We observed 71 fungal taxa on ERM roots, including known symbionts Rhizoscyphus ericae and Oidiodendron maius, putative symbionts from the Helotiales, Chaetothyriales, and Sebacinales, ectomycorrhizal symbionts, and saprotrophs. Supporting the idea that ERM fungi are adept saprotrophs, richness of root-fungi was greater in organic than in mineral soil horizons. To study the genetic diversity of oxidative enzymes that contribute to decomposition, we amplified and sequenced a portion of genes encoding multicopper oxidases (MCOs) from ERM ascomycetes. Most fungi possessed multiple copies of MCO sequences with strong similarities to known ferroxidases and laccases. Our findings indicate that R. maximum associates with a taxonomically and ecologically diverse fungal community. The study of MCO gene diversity and expression may be useful for understanding how ERM root fungi regulate the cycling of nutrients between the host plant and the soil environment.

We maintained a factorial nitrogen (N), phosphorus (P), and potassium (K) addition experiment for 11 years in a humid lowland forest growing on a relatively fertile soil in Panama to evaluate potential nutrient limitation of tree growth rates, fine-litter production, and fine-root biomass. We replicated the eight factorial treatments four times using 32 plots of 40×40 m each. The addition of K was associated with significant decreases in stand-level fineroot biomass and, in a companion study of seedlings, decreases in allocation to roots and increases in height growth rates. The addition of K and N together was associated with significant increases in growth rates of saplings and poles (1-10 cm in diameter at breast height) and a further marginally significant decrease in stand-level fine-root biomass. The addition of P was associated with a marginally significant (P = 0.058) increase in fine-litter production that was consistent across all litter fractions. Our experiment provides evidence that N, P, and K all limit forest plants growing on a relatively fertile soil in the lowland tropics, with the strongest evidence for limitation by K among seedlings, saplings, and poles. © 2011 by the Ecological Society of America.

Plants can acquire carbon from sources other than atmospheric carbon dioxide (CO(2)), including soil-dissolved inorganic carbon (DIC). Although the net flux of CO(2) is out of the root, soil DIC can be taken up by the root, transported within the plant, and fixed either photosynthetically or anaplerotically by plant tissues. We tested the ability of Pinus taeda L. seedlings exposed to (13)C-labeled soil DIC and two NH(4)(+) availability regimes to take up and fix soil DIC. We also measured the concentration and distribution of the fixed soil DIC within the plant and mycorrhizal tissues, and quantified the contribution of soil DIC to whole-plant carbon (C) gain. Seedlings exposed to labeled DIC were significantly enriched in (13)C compared with seedlings exposed to unlabeled DIC (6.7 versus -31.7 per thousand). Fixed soil DIC was almost evenly distributed between above- and belowground biomass (55 and 45%, respectively), but was unevenly distributed among tissues. Aboveground, stem tissue contained 65% of the fixed soil DIC but represented only 27% of the aboveground biomass, suggesting either corticular photosynthesis or preferential stem allocation. Belowground, soil DIC had the greatest effect (measured as (13)C enrichment) on the C pool of rapidly growing nonmycorrhizal roots. Soil DIC contributed approximately 0.8% to whole-plant C gain, and approximately 1.6% to belowground C gain. We observed a slight but nonsignificant increase in both relative C gain and the contribution of soil DIC to C gain in NH(4)(+)-fertilized seedlings. Increased NH(4)(+) availability significantly altered the distribution of fixed soil DIC among tissue types and increased the amount of fixed soil DIC in ectomycorrhizal roots by 130% compared with unfertilized seedlings. Increased NH(4)(+) availability did not increase fixation of soil DIC in nonmycorrhizal roots, suggesting that NH(4)(+) assimilation may be concentrated in ectomycorrhizal fungal tissues, reflecting greater anaplerotic demands. Soil DIC is likely to contribute only a small amount of C to forest trees, but it may be important in C fixation processes of specific tissues, such as newly formed stems and fine roots, and ectomycorrhizal roots assimilating NH(4)(+).