Latitudinal patterns in stabilizing density dependence of forest communities.

Numerous studies have shown reduced performance in plants that are surrounded by neighbours of the same species1,2, a phenomenon known as conspecific negative density dependence (CNDD)3. A long-held ecological hypothesis posits that CNDD is more pronounced in tropical than in temperate forests4,5, which increases community stabilization, species coexistence and the diversity of local tree species6,7. Previous analyses supporting such a latitudinal gradient in CNDD8,9 have suffered from methodological limitations related to the use of static data10-12. Here we present a comprehensive assessment of latitudinal CNDD patterns using dynamic mortality data to estimate species-site-specific CNDD across 23 sites. Averaged across species, we found that stabilizing CNDD was present at all except one site, but that average stabilizing CNDD was not stronger toward the tropics. However, in tropical tree communities, rare and intermediate abundant species experienced stronger stabilizing CNDD than did common species. This pattern was absent in temperate forests, which suggests that CNDD influences species abundances more strongly in tropical forests than it does in temperate ones13. We also found that interspecific variation in CNDD, which might attenuate its stabilizing effect on species diversity14,15, was high but not significantly different across latitudes. Although the consequences of these patterns for latitudinal diversity gradients are difficult to evaluate, we speculate that a more effective regulation of population abundances could translate into greater stabilization of tropical tree communities and thus contribute to the high local diversity of tropical forests.

Linking fine root lifespan to root chemical and morphological traits-A global analysis.

Fine root lifespan is a critical trait associated with contrasting root strategies of resource acquisition and protection. Yet, its position within the multidimensional "root economics space" synthesizing global root economics strategies is largely uncertain, and it is rarely represented in frameworks integrating plant trait variations. Here, we compiled the most comprehensive dataset of absorptive median root lifespan (MRL) data including 98 observations from 79 woody species using (mini-)rhizotrons across 40 sites and linked MRL to other plant traits to address questions of the regulators of MRL at large spatial scales. We demonstrate that MRL not only decreases with plant investment in root nitrogen (associated with more metabolically active tissues) but also increases with construction of larger diameter roots which is often associated with greater plant reliance on mycorrhizal symbionts. Although theories linking organ structure and function suggest that root traits should play a role in modulating MRL, we found no correlation between root traits associated with structural defense (root tissue density and specific root length) and MRL. Moreover, fine root and leaf lifespan were globally unrelated, except among evergreen species, suggesting contrasting evolutionary selection between leaves and roots facing contrasting environmental influences above vs. belowground. At large geographic scales, MRL was typically longer at sites with lower mean annual temperature and higher mean annual precipitation. Overall, this synthesis uncovered several key ecophysiological covariates and environmental drivers of MRL, highlighting broad avenues for accurate parametrization of global biogeochemical models and the understanding of ecosystem response to global climate change.

Extreme drought impacts have been underestimated in grasslands and shrublands globally.

Climate change is increasing the frequency and severity of short-term (~1 y) drought events-the most common duration of drought-globally. Yet the impact of this intensification of drought on ecosystem functioning remains poorly resolved. This is due in part to the widely disparate approaches ecologists have employed to study drought, variation in the severity and duration of drought studied, and differences among ecosystems in vegetation, edaphic and climatic attributes that can mediate drought impacts. To overcome these problems and better identify the factors that modulate drought responses, we used a coordinated distributed experiment to quantify the impact of short-term drought on grassland and shrubland ecosystems. With a standardized approach, we imposed ~a single year of drought at 100 sites on six continents. Here we show that loss of a foundational ecosystem function-aboveground net primary production (ANPP)-was 60% greater at sites that experienced statistically extreme drought (1-in-100-y event) vs. those sites where drought was nominal (historically more common) in magnitude (35% vs. 21%, respectively). This reduction in a key carbon cycle process with a single year of extreme drought greatly exceeds previously reported losses for grasslands and shrublands. Our global experiment also revealed high variability in drought response but that relative reductions in ANPP were greater in drier ecosystems and those with fewer plant species. Overall, our results demonstrate with unprecedented rigor that the global impacts of projected increases in drought severity have been significantly underestimated and that drier and less diverse sites are likely to be most vulnerable to extreme drought.

The forest, the cicadas and the holey fluxes: Periodical cicada impacts on soil respiration depends on tree mycorrhizal type.

The emergence of billions of periodical cicadas affects plant and animal communities profoundly, yet little is known about cicada impacts on soil carbon fluxes. We investigated the effects of Brood X cicadas (Magicicada septendecim, M. cassinii and M. septendeculain) on soil CO2 fluxes (RS ) in three Indiana forests. We hypothesized RS would be sensitive to emergence hole density, with the greatest effects occurring in soils with the lowest ambient fluxes. In support of our hypothesis, RS increased with increasing hole density and greater effects were observed near AM-associating trees (which expressed lower ambient fluxes) than near EcM-associating trees. Additionally, RS from emergence holes increased the temperature sensitivity (Q10 ) of RS by 13%, elevating the Q10 of ecosystem respiration. Brood X cicadas increased annual RS by ca. 2.5%, translating to an additional 717 Gg of CO2 across forested areas. As such, periodical cicadas can have substantial effects on soil processes and biogeochemistry.

Understanding the ecological versatility of Tetracladium species in temperate forest soils.

Although Tetracladium species have traditionally been studied as aquatic saprotrophs, the growing number of metagenomic and metabarcoding reports detecting them in soil environments raises important questions about their ecological adaptability and versatility. We investigated the factors associated with the relative abundance, diversity and ecological dynamics of Tetracladium in temperate forest soils. Through amplicon sequencing of soil samples collected from 54 stands in six forest sites across the eastern United States, we identified 29 distinct Amplicon Sequence Variants (ASVs) representing Tetracladium, with large differences in relative abundance and small changes in ASV community composition among sites. Tetracladium richness was positively related to soil pH, soil temperature, total sulphur and silt content, and negatively related to plant litter quality, such as the lignin-to-nitrogen ratio and the lignocellulose index. Co-occurrence network analysis indicated negative relationships between Tetracladium and other abundant fungal groups, including ectomycorrhizal and arbuscular mycorrhizal fungi. Collectively, our findings highlight the ecological significance of Tetracladium in temperate forest soils and emphasize the importance of site-specific factors and microbial interactions in shaping their distribution patterns and ecological dynamics.

Contrasting drought tolerance traits of woody plants is associated with mycorrhizal types at the global scale.

It is well-known that the mycorrhizal type of plants correlates with different modes of nutrient cycling and availability. However, the differences in drought tolerance between arbuscular mycorrhizal (AM) and ectomycorrhizal (EcM) plants remains poorly characterized. We synthesized a global dataset of four hydraulic traits associated with drought tolerance of 1457 woody species (1139 AM and 318 EcM species) at 308 field sites. We compared these traits between AM and EcM species, with evolutionary history (i.e. angiosperms vs gymnosperms), water availability (i.e. aridity index) and biomes considered as additional factors. Overall, we found that evolutionary history and biogeography influenced differences in hydraulic traits between mycorrhizal types. Specifically, we found that (1) AM angiosperms are less drought-tolerant than EcM angiosperms in wet regions or biomes, but AM gymnosperms are more drought-tolerant than EcM gymnosperms in dry regions or biomes, and (2) in both angiosperms and gymnosperms, variation in hydraulic traits as well as their sensitivity to water availability were higher in AM species than in EcM species. Our results suggest that global shifts in water availability (especially drought) may alter the biogeographic distribution and abundance of AM and EcM plants, with consequences for ecosystem element cycling and ultimately, the land carbon sink.

Asymmetric effects of hydroclimate extremes on eastern US tree growth: Implications on current demographic shifts and climate variability.

Forests around the world are experiencing changes due to climate variability and human land use. How these changes interact and influence the vulnerability of forests are not well understood. In the eastern United States, well-documented anthropogenic disturbances and land-use decisions, such as logging and fire suppression, have influenced forest species assemblages, leading to a demographic shift from forests dominated by xeric species to those dominated by mesic species. Contemporarily, the climate has changed and is expected to continue to warm and produce higher evaporative demand, imposing stronger drought stress on forest communities. Here, we use an extensive network of tree-ring records from common hardwood species across ~100 sites and ~1300 trees in the eastern United States to examine the magnitude of growth response to both wet and dry climate extremes. We find that growth reductions during drought exceed the positive growth response to pluvials. Mesic species such as Liriodendron tulipifera and Acer saccharum, which are becoming more dominant, are more sensitive to drought than more xeric species, such as oaks (Quercus) and hickory (Carya), especially at moderate and extreme drought intensities. Although more extreme droughts produce a larger annual growth reduction, mild droughts resulted in the largest cumulative growth decreases due to their higher frequency. When using global climate model projections, all scenarios show drought frequency increasing substantially (3-9 times more likely) by 2100. Thus, the ongoing demographic shift toward more mesic species in the eastern United States combined with drier conditions results in larger drought-induced growth declines, suggesting that drought will have an even larger impact on aboveground carbon uptake in the future in the eastern United States.

Arbuscular Mycorrhizal Tree Communities Have Greater Soil Fungal Diversity and Relative Abundances of Saprotrophs and Pathogens than Ectomycorrhizal Tree Communities.

Trees associating with different mycorrhizas often differ in their effects on litter decomposition, nutrient cycling, soil organic matter (SOM) dynamics, and plant-soil interactions. For example, due to differences between arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) tree leaf and root traits, ECM-associated soil has lower rates of C and N cycling and lower N availability than AM-associated soil. These observations suggest that many groups of nonmycorrhizal fungi should be affected by the mycorrhizal associations of dominant trees through controls on nutrient availability. To test this overarching hypothesis, we explored the influence of predominant forest mycorrhizal type and mineral N availability on soil fungal communities using next-generation amplicon sequencing. Soils from four temperate hardwood forests in southern Indiana, United States, were studied; three forests formed a natural gradient of mycorrhizal dominance (100% AM tree basal area to 100% ECM basal area), while the fourth forest contained a factorial experiment testing long-term N addition in both dominant mycorrhizal types. We found that overall fungal diversity, as well as the diversity and relative abundance of plant pathogenic and saprotrophic fungi, increased with greater AM tree dominance. Additionally, tree community mycorrhizal associations explained more variation in fungal community composition than abiotic variables, including soil depth, SOM content, nitrification rate, and mineral N availability. Our findings suggest that tree mycorrhizal associations may be good predictors of the diversity, composition, and functional potential of soil fungal communities in temperate hardwood forests. These observations help explain differing biogeochemistry and community dynamics found in forest stands dominated by differing mycorrhizal types. IMPORTANCE Our work explores how differing mycorrhizal associations of temperate hardwood trees (i.e., arbuscular [AM] versus ectomycorrhizal [ECM] associations) affect soil fungal communities by altering the diversity and relative abundance of saprotrophic and plant-pathogenic fungi along natural gradients of mycorrhizal dominance. Because temperate hardwood forests are predicted to become more AM dominant with climate change, studies examining soil communities along mycorrhizal gradients are necessary to understand how these global changes may alter future soil fungal communities and their functional potential. Ours, along with other recent studies, identify possible global trends in the frequency of specific fungal functional groups responsible for nutrient cycling and plant-soil interactions as they relate to mycorrhizal associations.

Coupling of plant and mycorrhizal fungal diversity: its occurrence, relevance, and possible implications under global change.

First principles predict that diversity at one trophic level often begets diversity at other levels, suggesting plant and mycorrhizal fungal diversity should be coupled. Local-scale studies have shown positive coupling between the two, but the association is less consistent when extended to larger spatial and temporal scales. These inconsistencies are likely due to divergent relationships of different mycorrhizal fungal guilds to plant diversity, scale dependency, and a lack of coordinated sampling efforts. Given that mycorrhizal fungi play a central role in plant productivity and nutrient cycling, as well as ecosystem responses to global change, an improved understanding of the coupling between plant and mycorrhizal fungal diversity across scales will reduce uncertainties in predicting the ecosystem consequences of species gains and losses.

The xylem of anisohydric Quercus alba L. is more vulnerable to embolism than isohydric codominants.

The coordination of plant leaf water potential (ΨL ) regulation and xylem vulnerability to embolism is fundamental for understanding the tradeoffs between carbon uptake and risk of hydraulic damage. There is a general consensus that trees with vulnerable xylem more conservatively regulate ΨL than plants with resistant xylem. We evaluated if this paradigm applied to three important eastern US temperate tree species, Quercus alba L., Acer saccharum Marsh. and Liriodendron tulipifera L., by synthesizing 1600 ΨL observations, 122 xylem embolism curves and xylem anatomical measurements across 10 forests spanning pronounced hydroclimatological gradients and ages. We found that, unexpectedly, the species with the most vulnerable xylem (Q. alba) regulated ΨL less strictly than the other species. This relationship was found across all sites, such that coordination among traits was largely unaffected by climate and stand age. Quercus species are perceived to be among the most drought tolerant temperate US forest species; however, our results suggest their relatively loose ΨL regulation in response to hydrologic stress occurs with a substantial hydraulic cost that may expose them to novel risks in a more drought-prone future.

Microbial mechanisms and ecosystem flux estimation for aerobic NOy emissions from deciduous forest soils.

Reactive nitrogen oxides (NOy; NOy = NO + NO2 + HONO) decrease air quality and impact radiative forcing, yet the factors responsible for their emission from nonpoint sources (i.e., soils) remain poorly understood. We investigated the factors that control the production of aerobic NOy in forest soils using molecular techniques, process-based assays, and inhibitor experiments. We subsequently used these data to identify hotspots for gas emissions across forests of the eastern United States. Here, we show that nitrogen oxide soil emissions are mediated by microbial community structure (e.g., ammonium oxidizer abundances), soil chemical characteristics (pH and C:N), and nitrogen (N) transformation rates (net nitrification). We find that, while nitrification rates are controlled primarily by chemoautotrophic ammonia-oxidizing archaea (AOA), the production of NOy is mediated in large part by chemoautotrophic ammonia-oxidizing bacteria (AOB). Variation in nitrification rates and nitrogen oxide emissions tracked variation in forest communities, as stands dominated by arbuscular mycorrhizal (AM) trees had greater N transformation rates and NOy fluxes than stands dominated by ectomycorrhizal (ECM) trees. Given mapped distributions of AM and ECM trees from 78,000 forest inventory plots, we estimate that broadleaf forests of the Midwest and the eastern United States as well as the Mississippi River corridor may be considered hotspots of biogenic NOy emissions. Together, our results greatly improve our understanding of NOy fluxes from forests, which should lead to improved predictions about the atmospheric consequences of tree species shifts owing to land management and climate change.

Root-derived inputs are major contributors to soil carbon in temperate forests, but vary by mycorrhizal type.

Roots promote the formation of slow-cycling soil carbon (C), yet we have a limited understanding of the magnitude and controls on this flux. We hypothesised arbuscular mycorrhizal (AM)- and ectomycorrhizal (ECM)-associated trees would exhibit differences in root-derived C accumulation in the soil, and that much of this C would be transferred into mineral-associated pools. We installed δ13 C-enriched ingrowth cores across mycorrhizal gradients in six Eastern U.S. forests (n = 54 plots). Overall, root-derived C was 54% greater in AM versus ECM-dominated plots. This resulted in nearly twice as much root-derived C in putatively slow-cycling mineral-associated pools in AM compared to ECM plots. Given that our estimates of root-derived inputs were often equal to or greater than leaf litter inputs, our results suggest that variation in root-derived soil C accumulation due to tree mycorrhizal dominance may be a key control of soil C dynamics in forests.

Ectomycorrhizal fungi are associated with reduced nitrogen cycling rates in temperate forest soils without corresponding trends in bacterial functional groups.

Microbial processes play a central role in controlling the availability of N in temperate forests. While bacteria, archaea, and fungi account for major inputs, transformations, and exports of N in soil, relationships between microbial community structure and N cycle fluxes have been difficult to detect and characterize. Several studies have reported differences in N cycling based on mycorrhizal type in temperate forests, but associated differences in N cycling genes underlying these fluxes are not well-understood. We explored how rates of soil N cycle fluxes vary across gradients of mycorrhizal abundance (hereafter "mycorrhizal gradients") at four temperate forest sites in Massachusetts and Indiana, USA. We paired measurements of N-fixation, net nitrification, and denitrification rates with gene abundance data for specific bacterial functional groups associated with each process. We find that the availability of NO3 and rates of N-fixation, net nitrification, and denitrification are reduced in stands dominated by trees associated with ECM fungi. On average, rates of N-fixation and denitrification in stands dominated by trees associated with arbuscular mycorrhizal fungi were more than double the corresponding rates in stands dominated by trees associated with ectomycorrhizal fungi. Despite the structuring of flux rates across the mycorrhizal gradients, we did not find concomitant shifts in the abundances of N-cycling bacterial genes, and gene abundances were not correlated with process rates. Given that AM-associating trees are replacing ECM-associating trees throughout much of the eastern US, our results suggest that shifts in mycorrhizal dominance may accelerate N cycling independent of changes in the relative abundance of N cycling bacteria, with consequences for forest productivity and N retention.

Mycorrhizal roots slow the decay of belowground litters in a temperate hardwood forest.

There is increasing evidence that plant roots and mycorrhizal fungi, whether living or dead, play a central role in soil carbon (C) cycling. Root-mycorrhizal-microbial interactions can both suppress and enhance litter decay, with the net result dependent upon belowground nutrient acquisition strategies and soil nutrient availability. We measured the net effect of living roots and mycorrhizal fungi on the decay of dead roots and fungal hyphae in a hardwood forest dominated by either sugar maple (Acer saccharum) or white oak (Quercus alba) trees. Root and fungal litter were allowed to decompose within root-ingrowth bags and root-exclusion cores. In conjunction with root effects on decay, we assessed foraging responses and root induced changes in soil moisture, nitrogen (N) availability and enzyme activity. After 1 year, maple root production increased, and mycorrhizal fungal colonization decreased in the presence of decaying litter. In addition, we found that actively foraging roots suppressed the decay of root litter (- 14%) more than fungal litter (- 3%), and suppression of root decay was stronger for oak (- 20%) than maple roots (- 8%). Suppressive effects of oak roots on decay were greatest when roots also reduced soil N availability, which corresponded with reductions in hydrolytic enzyme activity and enhanced oxidative enzyme activities. These findings further our understanding of context-dependent drivers of root-mycorrhizal-microbial interactions and demonstrate that such interactions can play an underappreciated role in soil organic matter accumulation and turnover in temperate forests.

Vapor pressure deficit helps explain biogenic volatile organic compound fluxes from the forest floor and canopy of a temperate deciduous forest.

Biogenic volatile organic compounds (BVOCs) play critical roles in ecological and earth-system processes. Ecosystem BVOC models rarely include soil and litter fluxes and their accuracy is often challenged by BVOC dynamics during periods of rapid ecosystem change like spring leaf out. We measured BVOC concentrations within the air space of a mixed deciduous forest and used a hybrid Lagrangian/Eulerian canopy transport model to estimate BVOC flux from the forest floor, canopy, and whole ecosystem during spring. Canopy flux measurements were dominated by a large methanol source and small isoprene source during the leaf-out period, consistent with past measurements of leaf ontogeny and theory, and indicative of a BVOC flux situation rarely used in emissions model testing. The contribution of the forest floor to whole-ecosystem BVOC flux is conditional on the compound of interest and is often non-trivial. We created linear models of forest floor, canopy, and whole-ecosystem flux for each study compound and used information criteria-based model selection to find the simplest model with the best fit. Most published BVOC flux models do not include vapor pressure deficit (VPD), but it entered the best canopy, forest floor, and whole-ecosystem BVOC flux model more than any other study variable in the present study. Since VPD is predicted to increase in the future, future studies should investigate how it contributes to BVOC flux through biophysical mechanisms like evaporative demand, leaf temperature and stomatal function.

Nitrogen cycling microbiomes are structured by plant mycorrhizal associations with consequences for nitrogen oxide fluxes in forests.

Volatile nitrogen oxides (N2 O, NO, NO2 , HONO, …) can negatively impact climate, air quality, and human health. Using soils collected from temperate forests across the eastern United States, we show microbial communities involved in nitrogen (N) cycling are structured, in large part, by the composition of overstory trees, leading to predictable N-cycling syndromes, with consequences for emissions of volatile nitrogen oxides to air. Trees associating with arbuscular mycorrhizal (AM) fungi promote soil microbial communities with higher N-cycle potential and activity, relative to microbial communities in soils dominated by trees associating with ectomycorrhizal (ECM) fungi. Metagenomic analysis and gene expression studies reveal a 5 and 3.5 times greater estimated N-cycle gene and transcript copy numbers, respectively, in AM relative to ECM soil. Furthermore, we observe a 60% linear decrease in volatile reactive nitrogen gas flux (NOy  ≡ NO, NO2 , HONO) as ECM tree abundance increases. Compared to oxic conditions, gas flux potential of N2 O and NO increase significantly under anoxic conditions for AM soil (30- and 120-fold increase), but not ECM soil-likely owing to small concentrations of available substrate ( NO 3 - ) in ECM soil. Linear mixed effects modeling shows that ECM tree abundance, microbial process rates, and geographic location are primarily responsible for variation in peak potential NOy flux. Given that nearly all tree species associate with either AM or ECM fungi, our results indicate that the consequences of tree species shifts associated with global change may have predictable consequences for soil N cycling.

Drought legacies are dependent on water table depth, wood anatomy and drought timing across the eastern US.

Severe droughts can impart long-lasting legacies on forest ecosystems through lagged effects that hinder tree recovery and suppress whole-forest carbon uptake. However, the local climatic and edaphic factors that interact to affect drought legacies in temperate forests remain unknown. Here, we pair a dataset of 143 tree ring chronologies across the mesic forests of the eastern US with historical climate and local soil properties. We found legacy effects to be widespread, the magnitude of which increased markedly in diffuse porous species, sites with deep water tables, and in response to late-season droughts (August-September). Using an ensemble of downscaled climate projections, we additionally show that our sites are projected to drastically increase in water deficit and drought frequency by the end of the century, potentially increasing the size of legacy effects by up to 65% and acting as a significant process shaping forest composition, carbon uptake and mortality.

Leaf litter decay rates differ between mycorrhizal groups in temperate, but not tropical, forests.

Whereas the primary controls on litter decomposition are well established, we lack a framework for predicting interspecific differences in litter decay within and across ecosystems. Given previous research linking tree mycorrhizal association with carbon and nutrient dynamics, we hypothesized that the two dominant mycorrhizal groups in forests - arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi - differ in litter decomposition rates. We compiled leaf litter chemistry and decay data for AM- and ECM-associating angiosperms and gymnosperms (> 200 species) from temperate and tropical/subtropical, and investigated relationships among decay rates, mycorrhizal association, phylogeny and climate. In temperate forests, AM litters decayed faster than ECM litters, with litter nitrogen and phylogeny best explaining variation in litter decay. In sub/tropical forests, we found no significant difference in litter decay rate between mycorrhizal groups, and variation in decay rates was best explained by litter phosphorus. Our results suggest that knowledge of tree mycorrhizal association may improve predictions of species effects on ecosystem processes, particularly in temperate forests where AM and ECM species commonly co-occur, providing a predictive framework for linking litter quality, organic matter dynamics and nutrient acquisition in forests.

Anisohydric behavior linked to persistent hydraulic damage and delayed drought recovery across seven North American tree species.

The isohydry-anisohydry spectrum has become a popular way to characterize plant drought responses and recovery processes. Despite the proven utility of this framework for understanding the interconnected physiological changes plants undergo in response to water stress, new challenges have arisen pertaining to the traits and tradeoffs that underlie this concept. To test the utility of this framework for understanding hydraulic traits, drought physiology and recovery, we applied a 6 wk experimental soil moisture reduction to seven tree species followed by a 6 wk recovery period. Throughout, we measured hydraulic traits and monitored changes in gas exchange, leaf water potential, and hydraulic conductivity. Species' hydraulic traits were not coordinated, as some anisohydric species had surprisingly low resistance to embolism (P50 ) and negative safety margins. In addition to widespread hydraulic damage, these species also experienced reductions in photosynthesis and stem water potential during water stress, and delayed recovery time. Given that we observed no benefit of being anisohydric either during or after drought, our results indicate the need to reconsider the traits and tradeoffs that underlie anisohydric behavior, and to consider the environmental, biological and edaphic processes that could allow this strategy to flourish in forests.

Linking variation in intrinsic water-use efficiency to isohydricity: a comparison at multiple spatiotemporal scales.

Species-specific responses of plant intrinsic water-use efficiency (iWUE) to multiple environmental drivers associated with climate change, including soil moisture (θ), vapor pressure deficit (D), and atmospheric CO2 concentration (ca ), are poorly understood. We assessed how the iWUE and growth of several species of deciduous trees that span a gradient of isohydric to anisohydric water-use strategies respond to key environmental drivers (θ, D and ca ). iWUE was calculated for individual tree species using leaf-level gas exchange and tree-ring δ13 C in wood measurements, and for the whole forest using the eddy covariance method. The iWUE of the isohydric species was generally more sensitive to environmental change than the anisohydric species was, and increased significantly with rising D during the periods of water stress. At longer timescales, the influence of ca was pronounced for isohydric tulip poplar but not for others. Trees' physiological responses to changing environmental drivers can be interpreted differently depending on the observational scale. Care should be also taken in interpreting observed or modeled trends in iWUE that do not explicitly account for the influence of D.