Forest edges favor conservative resource-use and symbiotic belowground strategies in Eastern Amazonian forest fragments

Forest edges favor conservative resource-use and symbiotic belowground strategies in Eastern Amazonian forest fragments | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Forest edges favor conservative resource-use and symbiotic belowground strategies in Eastern Amazonian forest fragments RENAN DOMINGUES PACHECO, Tailane Silva Sousa, Paulo Bittencourt, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9406229/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Aims While edge effects on aboveground traits are relatively well-documented, little is known about how the edge–interior interface modulates root strategies and mycorrhizal associations under severe climatic conditions. This study evaluated how the environment (edge vs. interior) influences leaf water status and root functional traits of trees in forest fragments during the 2023 El Niño. Methods A total of 144 tree individuals were sampled across eight forest fragments in Pará, Brazil. We measured leaf water potential at predawn (Ψpd) and at midday (Ψmd), root morphological traits (specific root length - SRL, specific root area SRA, root tissue density RTD, and mean diameter) and mycorrhizal colonization (intensity of colonization in the root system M% and arbuscule abundance A%) in species occurring at the forest edge and in the interior of the fragments. We used GLMMs to test the effect of environment, including fragment and species as random effects, in addition to correlation analyses and PCA to explore functional coordination. Results Trees at the edge exhibited more negative Ψ md , with no difference in Ψ pd , indicating greater atmospheric evaporative demand. At the edge, we observed higher RTD, lower SRL, and greater mycorrhizal colonization and arbuscule abundance. In the interior, plants exhibited a more exploratory strategy, with higher SRL values. Root morphology and mycorrhizal symbiosis acted as independent strategies. Conclusion Fragmentation modulates belowground functional organization, promoting at the edge a conservative strategy coordinated with the reinforcement of the symbiotic pathway, which may be crucial for the resilience of Amazonian fragments under severe droughts. Forest fragmentation Root traits Arbuscular mycorrhizae Water stress Edge effect Drought Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION In recent decades, droughts caused by El Niño in the Amazon Forest have become longer and more intense (Espinoza et al. 2024 ; Feldpausch et al. 2016 ; Marengo et al. 2008 ). Global climate models predict that these droughts may become increasingly frequent (Nobre et al. 2007 ), leading to higher tree mortality and increased carbon (C) emissions to the atmosphere (Lapola et al. 2023 ). With increasing plant stress, the Amazon Forest is expected to cease being a carbon sink, as has already been observed in some regions (Gatti et al. 2021 ; Lapola et al. 2023 ; Tavares et al. 2023 ). In addition to droughts, another critical disturbance in the Amazon Forest is the exponential increase in forest edges, a direct consequence of deforestation and fragmentation (Aragão et al. 2018 ). In the Amazon, 45.5% of the biome already presents some level of fragmentation (Bauer et al. 2024 ). Although edge effects drastically alter microclimate, species composition, and biomass, most studies still focus on continuous forests (Nunes et al. 2023 ). Forest fragmentation alters the microclimate at edges by increasing solar radiation incidence, temperature, and atmospheric evaporative demand (Laurance et al. 2011 ; Magnago et al. 2015 ), subjecting vegetation to continuous water stress even when soil moisture has not yet reached critical levels (Barros et al. 2019 ; Didham and Lawton, 1999 ). Thus, in these already fragmented landscapes, there is still limited understanding of how the edge–interior gradient modulates the physiological and morphological responses of trees, especially during severe climatic events. At the forest edges, changes occur in species composition and functional traits compared to the interior (Cardoso et al. 2002; Figueiredo et al. 2018 ). These areas are often dominated by pioneer species with lower wood density and reduced height (Magnago et al. 2015 ; Michalski et al. 2007 ; Rabelo et al. 2015 ). In forest fragments in Eastern Amazon, it has been demonstrated that edge effects alter the ecological strategies of communities, leading to a decoupling between wood and leaf traits (Sousa et al. 2025 ). Comparisons of species traits between forest edges and interiors have mainly focused on aboveground traits, while much less is known about root traits and their strategies that may confer plant resistance and resilience to variations in water availability. The root system performs multiple functions, being responsible for water and nutrient uptake and soil carbon cycling due to the high turnover of fine roots, playing a central role in the functioning of terrestrial ecosystems (Brunner et al. 2015 ). Root traits involving morphology and mycorrhizal associations influence soil resource acquisition and are directly linked to plant growth, reproduction, and survival (Violle et al. 2007 ). Root morphological traits can be plastic, responding to nutrient and water availability (Freschet et al. 2015 ); characteristics such as roots length, diameter, and volume are related to water and nutrient acquisition, influencing plant productivity. Roots with higher specific root length (i.e. the ratio between root length and dry mass) exhibit more efficient hydraulic conductance (Comas et al. 2013 ), whereas higher root tissue density is associated with greater structural resistance, longevity, and resource conservation, at the expense of high rates of absorption and soil exploration (Eissenstat and Achor, 1999 ; Ryser, 1996 ). From a trait-based perspective, plant belowground strategies are structured along two main independent axes. The first is a conservation gradient, primarily reflected by root tissue density (RTD), which determines the balance between fast root turnover and structural persistence. The second is a collaboration gradient, which contrasts a 'do-it-yourself' resource acquisition strategy (characterized by high specific root length, SRL) with an 'outsourcing' strategy defined by a high reliance on mycorrhizal fungi (Bergmann et al. 2020 ). Together, these axes form the Root Economics Space, which provides a unifying framework to understand how plants adjust belowground strategies in response to environmental stress, such as severe droughts. Another trait influencing resource acquisition under water stress is the degree of root colonization by arbuscular mycorrhizal fungi (AMF) (Bergmann et al. 2020 ; Laliberté et al. 2017; Mccormack et al. 2015 ). This mutualistic relationship increases water uptake by expanding the volume of soil explored, positively influencing water potential and delaying stomatal closure (Augé, 2001 ); these responses are highly dependent on the environmental context (Freschet et al. 2015 ). This symbiosis may be particularly vital in edge environments, where water stress is elevated and evaporative demand is greater, favoring more acquisitive root strategies and high functional plasticity (Bergmann et al. 2020 ; Freschet et al. 2015 , 2021 ). However, it remains unexplored whether AMF colonization increases at edges as a compensatory plant strategy or whether the extreme microclimatic conditions of these Eastern Amazon fragments limit the efficiency of this association during severe drought events (Brunner et al. 2015 ), as well as understanding whether morphological variation changes between edges and interiors of forest fragments under conditions of extreme drought events (such as El Niño) in fragments as singular as those in this region. In this study, we investigated root functioning in eight forest fragments located in the state of Pará, Eastern Amazon, analyzing 144 tree individuals. The objective was to evaluate how the edge–interior environment influences water status, root functional traits, and species resource acquisition strategies. Thus, we hypothesized that root traits vary along the edge–interior gradient, with trees at the edge adjusting their root strategies in response to lower water availability. We expected (1) more negative water potential in species at the edge, indicating greater water limitation, compared to species in the fragment interior; (2) lower specific root length (SRL), lower specific root area (SRA), and higher root tissue density (RTD) compared to trees in the fragment interior, as a strategy for more efficient acquisition of water and nutrients; (3) higher mycorrhizal colonization and greater abundance of arbuscules at the edge, reflecting greater dependence on symbioses to mitigate water stress and sustain resource acquisition during severe drought events. MATERIAL AND METHODS STUDY AREA The study was conducted in eight forest fragments, distributed across three (3) municipalities: Barcarena (Lat: 01º30′21″ S; Long: 48º37′33″ W), Abaetetuba (Lat: 01°43′00″ S; Long: 48°52′00″ W), and Moju (Lat: 01°53′00″ S; Long: 48°46′00″ W) (Fig. 1 ), municipalities located in the Metropolitan Region of Belém, in the state of Pará, Brazil (IBGE, 2025; Mendonça et al. 2021) (Online Resource 1, Table S1 ). The region has an average annual precipitation of 2,500 mm and a mean temperature around 27°C (De Oliveira et al. 2020 ; Piratoba et al. 2017 ). The soils found in the Barcarena region are mainly characterized as Gleysols, Spodosols, and Oxisols (IDESP, 2022 ). Regarding vegetation, the region is composed of dense ombrophilous forest, with a predominance of evergreen species (Souza and Lisboa, 2005 ). DATA COLLETION The research was conducted in November 2023, during the dry season, characterized as a drought event due to El Niño (Espinoza et al. 2024 ). Eight forest fragments were selected for the study, and each fragment contained a watercourse (stream, igarapé ) within its interior. In each fragment, we evaluated three abundant species both at the edge (within the first 5 m) and in the interior (from 30 m from the edge). In each fragment, three individuals per species were selected for the collection of physiological and root traits, totalling 144 records corresponding to 24 distinct species (Online Resource 1, Table S2). Species selection was based on their abundance and ecological relevance to the Amazonian flora. To characterize tree ecological strategies, we selected leaf physiological variables, root morphological traits, and mycorrhizal association (Online Resource 1, Table S3). PHYSIOLOGICAL TRAIT Water Potential We measured leaf water potential (ψ) in one leaf per individual for all 144 trees at the predawn (ψ predawn), between 4:00 and 5:00 h, and midday (ψ midday), between 12:00 and 13:00 h. Predawn ψ is an indicator of soil water status, since during this period the atmosphere presents low evaporative demand, leading leaf water status to equilibrate with soil water status (Bhaskar and Ackerly 2006 ; Taiz and Zeiger 2009 ). Midday ψ is an indicator of the driest condition experienced by the tree throughout a season, integrating atmospheric and soil water deficits (Bhaskar and Ackerly 2006 ; Taiz and Zeiger 2009 ). Water potential (ψ) was measured in situ using a pressure chamber (PMS 1505 D and PMS 1000, PMS Instruments), and immediately after sampling, the values for each individual were recorded (Pammenter and Van der Willigen 1998 ). ROOTS TRAITS Root Morphology We collected fine roots (diameter < 2 mm) from trees using a root-tracing method to ensure species-specific identification. From the base of the stem of each target tree, we carefully excavated the soil to a depth of 0–30 cm and followed the lateral coarse roots until they branched into the associated fine roots. This procedure followed the standardized sampling protocol of Freschet et al. ( 2021 ). After collection, fine roots were separated, cleaned, weighed, and kept under refrigeration until the determination of morphological parameters. We measured fresh mass and dry mass of fine roots using an analytical balance (Model AD430; Marte) with a resolution of 0.001 g. Dry mass was determined after drying the samples in a forced-air circulation oven at 70°C until constant weight was achieved (Freschet et al. 2021 ). To calculate dry mass content, we used the following equation: $$\:\text{M}\text{S}\:=\:\left(\frac{PS}{PF}\right)\times\:\:100$$ where MS represents dry mass content, PF is fresh weight, and PS is dry weight of the roots. Roots were scanned using a flatbed scanner in grayscale at 600 dpi. Image analysis was performed using the software RhizoVision Rxplorer v2.0.3, with algorithms described by Seethepalli et al. ( 2021 ), where root length and diameter were assessed. Specific root length (SRL) was obtained by dividing root length by dry mass (Gu et al. 2014 ; Zadworny et al. 2011). Specific root area (SRA) was calculated by dividing root surface area (obtained from scanned images) by dry mass (Wang et al. 2024 ). Root tissue density (RTD) was calculated by dividing dry mass by root volume (calculated from scanned images) (Gu et al. 2014 ; Kong et al. 2014 ). Mean root diameter was obtained using the software RhizoVision Rxplorer v2.0.3. Mycorrhizal Association To analyse the mutualistic relationship, we excavated and collected at least 30 cm of fine (absorptive) roots from each individual, in the superficial soil layer, between 0 and 30 cm depth. After collection, the roots were carefully washed in alternating baths to remove any particulate material and were stored in 50% ethanol for preservation. Subsequently, the roots were cleared with 10% potassium hydroxide (m/v), acidified in 2% HCl (v/v), and stained with 0.05% trypan blue (m/v) for the identification and quantification of colonization by mycorrhizal fungi (AMF). We used the MYCOCALC program, following the method described by TROUVELOT (1986), in which we evaluated the parameters: frequency of root system infection (F%), intensity of mycorrhizal colonization of the root system (M%), and abundance of arbuscules in the root system (A%). DATA ANALYSIS To assess the associations between root morphological traits and leaf physiological variables, we applied Spearman correlation analysis using the pairwise method. Statistical significance was determined based on p-values, considering a threshold of p < 0.05. All analyses were performed in the R statistical environment v.4.4.x (R Core Team, 2024 ), using the psych package to calculate correlation matrices and the corrplot package for graphical visualization of the results (Wei and Simko, 2021 ). To evaluate the effect of environment (edge vs. interior) on plant functional traits, we used Generalized Linear Mixed Models (GLMMs). In all models, the environment was treated as a fixed effect. To account for spatial variation and interspecific variability, we included sampling site and species nested within site (1 | Fragment/Species) as random effects. The significance of the environment was assessed using Likelihood Ratio Tests (LRT), comparing the full model with a null model using the anova function. All continuous variables were checked for distributional assumptions prior to model fitting. For Hypothesis 1, predawn water potential (Ψ pd ) was analyzed using a Gamma GLMM (log link), whereas midday water potential (Ψ md ) followed a Gaussian distribution and was analyzed using a Linear Mixed Model (LMM). For Hypothesis 2 (morphological traits: RTD, SRL, SRA, and DM), we fitted GLMMs with a Gamma distribution and a log link function. For Hypothesis 3 (mycorrhizal colonization: M% and A%), the models were also fitted using a Gamma distribution. To address heteroscedasticity detected through diagnostics from the DHARMa package, a dispersion formula for the environment term (dispformula = ~ Environment) was incorporated into these models. Model fit was verified by inspection of simulated residuals, and model quality was described using marginal R² and conditional R² values, calculated with the performance package. RESULTS Functional associations and root strategies Root attributes showed associations among themselves (Online Resource 1, Fig. S1 ). We observed negative correlations between root tissue density (RTD) and specific root area (SRA) (r = − 0.95, p < 0.001), as well as between RTD and specific root length (SRL) (r = − 0.88, p < 0.001). Mean diameter (DM) showed a negative correlation only with SRL (r = − 0.32, p = 0.001), indicating that roots with higher SRL tend to be thinner. On the other hand, mycorrhizal colonization intensity (M%) and arbuscule abundance (A%) showed a strong positive correlation with each other (r = 0.90, p < 0.001), but did not show significant associations with the root morphological attributes (RTD, SRA, SRL, and DM). The mean values and standard deviation for all morphological attributes evaluated are detailed in Online Resource 1, Table S5. Principal Component Analysis (PCA) showed that the first two components explained 65.8% of the total variance in the data. The first axis (PC1) explained 36.1% and was associated with specific root length (SRL) and specific root area (SRA), being negatively associated with root tissue density (RTD) and mean diameter (DM). The second axis was associated with mycorrhizal colonization (M%) and arbuscular colonization (A%). Thus, the PCA revealed an orthogonal axis separating mycorrhizal associations (A% and M%) from root morphological attributes (RTD, SRA, SRL, and DM) (Fig. 2 ). Regarding the environments, the biplot showed an overlap between the edge and interior groups, as indicated by the 95% confidence ellipses. However, samples from the interior environment (blue triangles) showed higher SRL and SRA values compared with those from the edge (orange circles), which appeared more centrally distributed in the multivariate space. Difference in water potential between trees in the fragment interior and edge Predawn water potential (Ψ pd ) did not vary between environments (χ² = 0.50; p = 0.480) (Fig. 3 a), with means of − 0.52 ± 0.29 MPa at the edge and − 0.55 ± 0.28 MPa in the interior (Online Resource 1, Table S5). Variance for this trait was explained by species (σ² = 0.105), with low influence of site (σ² = 0.011).Midday leaf water potential (Ψ md ) differed significantly between environments (χ² = 53.55; p < 0.001) (Fig. 3 b), with more negative values (− 2.08 ± 0.77 MPa) at the edge compared with the interior (− 1.79 ± 0.80 MPa). Variance partitioning revealed that species (σ² = 0.163) and site (σ² = 0.052) were the main drivers of the variation for this trait. Root morphological traits and strategies Specific root length (SRL) was significantly higher in the interior (117.44 ± 88.49 cm g⁻¹) than at the edge (59.15 ± 54.58 cm g⁻¹; χ² = 13.60; p = 0.003) (Fig. 4 a). SRL variability was predominantly explained by site (σ² = 0.097), with negligible contribution from species identity (σ² < 0.001) (Online Resource 1, Table S5). Root tissue density (RTD) was higher at the edge (0.021 ± 0.023 g cm⁻³) compared to the interior (0.008 ± 0.005 g cm⁻³; χ² = 34.92; p < 0.001) (Fig. 4 b). Variation in RTD was driven by both site (σ² = 0.114) and species (σ² = 0.068) (Online Resource 1, Table S5). Specific root area (SRA) showed no difference (χ² = 7.15; p = 0.067) (Fig. 4 c) between the forest interior (284.4 ± 177.9 cm² g⁻¹) and the edge (176.5 ± 152.1 cm² g⁻¹), and the main driver of SRA variation was site (σ² = 0.050) (Online Resource 1, Table S5). Mean fine-root diameter (DM) did not differ between the edge (0.66 ± 0.15 mm) and the interior (0.63 ± 0.16 mm; χ² = 3.11; p = 0.374) (Fig. 4 d). Most of the observed variability was attributed to species (σ² = 0.005), while site (σ² = 0.002) had a smaller contribution (Online Resource 1, Table S5). Mycorrhizal associations and symbiotic activity Arbuscule abundance in the root system (A%) was higher at the edge (30.6 ± 39.7%) than in the interior (2.6 ± 2.5%; χ² = 37.78; p < 0.001) (Fig. 5 a), with species being the dominant driver (σ² = 0.157) (Online Resource 1, Table S5). Mycorrhizal colonization intensity (M%) also was higher at the edge (3.45 ± 2.96%) compared to the interior (1.38 ± 0.67%; χ² = 31.62; p < 0.001) (Fig. 5 b). For colonization intensity, fragment (σ² = 0.048) explained more variance than species (σ² = 0.022) (Online Resource 1, Table S5). DISCUSSION Our study demonstrates that, under conditions of severe drought associated with El Niño, tree species exhibit contrasting belowground investments in root traits, reflecting different resource acquisition strategies between the edge and the interior of forest fragments in Eastern Amazonia. Edge trees experience greater water limitation throughout the day, with more negative midday leaf water potential (Ψ md ) values, while the stability of predawn water potential (Ψ pd ) between environments indicates that this water limitation is derived from greater atmospheric evaporative demand, rather than from differences in soil water availability. We observed a coordinated functional adjustment at the edge, where trees exhibit a more conservative root strategy, characterized by higher root tissue density (RTD) and lower specific root length (SRL), prioritizing structural resistance and greater root longevity (Bergmann et al. 2020 ; Ryser 1996 ). As part of this adjustment, these trees intensify the collaborative resource acquisition pathway, showing greater mycorrhizal colonization intensity and a higher abundance of arbuscules at the edge, suggesting a compensatory mechanism in response to the lower exploratory capacity of denser roots. In contrast, in the forest interior predominated a root strategy with greater foraging capacity, characterized by higher SRL values, favored by relatively more stable microclimatic conditions. Even under drought conditions associated with El Niño, trees at the edge exhibited more negative Ψmd values than species in the fragment interior. This suggests that the microclimate at forest edges, characterized by greater light incidence, higher temperatures, and higher vapor pressure deficit (Barros et al. 2019 ; Didham and Lawton 1999 ), intensifies the evaporative demand imposed on trees during the day. This result is consistent with previous studies documenting more extreme microclimatic conditions at forest edges, particularly in fragmented landscapes exposed to the anthropogenic matrix (Didham and Lawton, 1999 ; Haddad et al. 2015 ; Magnago et al. 2015 ). In contrast, we did not observe variation in Ψpd. This decoupling between daytime stress and nighttime recovery suggests that, under extreme drought conditions, water limitation manifests primarily through elevated atmospheric demand, rather than being determined exclusively by soil water status (Barros et al. 2019 ). The correlations observed between root tissue density (RTD) and traits associated with soil exploration (SRL), as well as the structuring of the data in the Principal Component Analysis (PCA), suggest strong functional coordination within the root system of the species. Trees at the edge showed higher RTD, indicating greater investment in structurally denser and more durable roots (Ryser, 1996 ; Roumet et al. 2016 ). These patterns are consistent with the concept of the Root Economics Space (Bergmann et al. 2020 ), in which investment in tissue density (RTD) represents an axis of functional conservation independent of the 'do-it-yourself' exploration strategy (Bergmann et al. 2020 ; Matthus et al. 2025 ). At the edge, the increase in RTD suggests a transition toward a conservative strategy, in which mechanical resistance and root longevity become priorities in response to the greater risk of hydraulic stress and functional instability in soils subject to higher water variability and intensified atmospheric demand (Eller et al. 2018 ; Vleminckx et al. 2021 ). The significant contribution of fragment identity (site) to RTD variation (σ² = 0.114) suggests that, although fragmentation acts as an environmental filter, the intensity of this functional adjustment is modulated by local characteristics of each remnant. This pattern indicates that edaphic variations or disturbance history among sites interact with the edge effect, favoring specific functional assemblages independently of species identity (Michalski et al. 2007 ; Violle et al. 2007 ). Thus, convergence toward more conservative strategies at the edge appears to be a response to atmospheric water stress, but whose magnitude is refined by the particular conditions of each forest fragment. In contrast, in the interior of the fragments, higher SRL values were observed, a trait associated with greater spatial soil exploration capacity (Eissenstat et al. 2000 ; Ostonen et al. 2007 ). Under non severe conditions, the relatively greater water availability in the fragment interior, often associated with proximity to watercourses and lower microclimatic variability, may act as a hydrological buffer, favoring more exploratory root strategies (Matthus et al. 2025 ). However, under the evaluated conditions, associated with extreme drought, this buffering effect appears to have been reduced, resulting in similar water conditions between edge and interior Even so, the functional organization of roots suggests that interior species maintain strategies associated with greater exploitation of the superficial soil, possibly reflecting environmental filtering between habitats (Santana et al. 2025 ) and belowground competition among coexisting species (Zangaro et al. 2016 ). In addition to morphological adjustments, our results indicate that mycorrhizal associations act as a complementary and independent mechanism in belowground strategies between the edge and the interior. The absence of significant correlations between morphological traits (RTD, SRA, SRL, DM) and mycorrhizal parameters (M% and A%), combined with the separation of these traits along distinct and orthogonal axes in the PCA (PC1 determining morphology and PC2 symbiosis), demonstrates that fungal colonization and root structure operate as autonomous strategies of environmental adjustment. This pattern suggests that trees at the edge reinforce the symbiotic pathway independently of changes in root density or extension, possibly to compensate for the greater evaporative demand of this environment. The expressive abundance of arbuscules at the edge (30.6%) compared to the interior (2.6%) shows that this association is functionally active, reflecting a directed investment in resource uptake, even in the face of low total mycorrhizal colonization (Lekberg and Koide, 2005 ). This coordination between increased root tissue density (RTD) and strengthening of the collaboration axis at the edge corroborates discussions on the bidimensionality of the Root Economics Space, in which investment in fungi acts as an autonomous compensatory strategy that does not depend on prior changes in root thickness or area (Bergmann et al. 2020 ; Matthus et al. 2025 ). The roots with higher RTD and mycorrhizal activity at the edge suggests an integrated strategy of tolerance to water stress and nutrientes uptake, in which morphological limitations associated with denser roots are partially compensated by symbiotic interactions. While roots with higher RTD tend to exhibit lower efficiency in the direct resources uptake (Comas and Eissenstat, 2004), mycorrhizal hyphae expand the volume of soil explored, functioning as a functional extension of the root system (Smith and Read, 2008 ). This functional arrangement highlights the importance of belowground biotic interactions as mediators of tree persistence in fragmented environments under high water stress, contributing to the stability and resilience of forest ecosystems (Bennett et al. 2017 ; Rillig et al. 2014 ). Despite the greater symbiotic association at the edge, the mycorrhizal colonization rates observed were low in both environments when compared to values frequently reported for tropical forests (Wang et al. 2024 ). This pattern suggests that additional factors, such as edaphic characteristics, land-use history, composition of fungal communities, and the combined effects of fragmentation and extreme drought, may be limiting the functional effectiveness of this association. Indeed, the intensity and benefits of mycorrhizal symbiosis are highly dependent on environmental context and resource limitation (Johnson et al. 2010 ). Macroecological-scale evidence indicates that colonization by arbuscular mycorrhizal fungi is strongly regulated by climatic variables, suggesting that the severe thermal and water variations associated with El Niño may have acted as an abiotic constraint on the responsiveness of the symbiosis (Soudzilovskaia et al. 2015 ). Thus, although symbiosis represents an important mechanism for mitigating water stress, its compensatory capacity may be reduced under extreme environmental conditions (Augé, 2001 ; Smith and Read, 2008 ). This functional arrangement highlights the importance of belowground biotic interactions as mediators of tree persistence in fragmented environments under high water stress. Evolutionary evidence suggests that mycorrhizae have played a role in facilitating plant adaptation to critical changes in water availability over time (Cosme, 2023 ), contributing to the stability and resilience of forest ecosystems (Bennett et al. 2017 ; Rillig et al. 2014 ). Our results demonstrate that forest fragmentation modulates the functional organization of roots and symbioses in Eastern Amazonia, establishing distinct responses between edge and interior under the influence of a severe drought event (El Niño). By revealing that trees at the edge operate in a more negative daytime water status and adopt a coordination between the root conservation axis (higher RTD) and the collaboration axis (greater abundance of arbuscules), this study fills a critical gap on how fragmented areas respond to the edge environment. Unlike continuous forests, forest fragments that comprise an increasing portion of the Amazonian landscape exhibit specific functional responses that suggest a structural adaptation to greater atmospheric evaporative demand. These findings reinforce the need to integrate edge heterogeneity into forest vulnerability models, since belowground persistence strategies differ drastically between the interior and the periphery of fragments. Therefore, conservation and restoration efforts must consider that the functional integrity of these forests depends on maintaining mycorrhizal connectivity and protecting root structure at the edges, essential components for the resilience of fragmented landscapes in the face of intensifying climatic anomalies in the region. Abbreviations A%, abundance of arbuscules in the root system AMF, arbuscular mycorrhizal fungi DM, mean root diameter F%, frequency of root system infection GLMMs, generalized linear mixed models LMM, linear mixed model LRT, likelihood ratio test M%, intensity of mycorrhizal colonization of the root system PCA, principal component analysis PC1, first principal component PC2, second principal component RTD, root tissue density SRA, specific root area SRL, specific root length Ψ md , midday leaf water potential Ψ pd , predawn leaf water potential Declarations Competing interests The authors have no relevant financial or non-financial interests to disclose. Funding This study was financially supported by Hydro Alunorte through the project “Assessment of Aquatic Biota and Riparian Vegetation of the Hydrography that Influences the Murucupi Basin and Surrounding Areas of Hydro Alunorte”, including scholarships granted to Renan Domingues Pacheco and Tailane da Silva Sousa. This study was also supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES), Finance Code 001. Author contributions All authors contributed to the conception and design of the study. Material preparation, data collection, and data analysis were performed by Renan Domingues Pacheco, Tailane da Silva Sousa, Paulo Bittencourt, Patrícia de Britto Costa, Keven Adrian Furtado Alves, Thaisa Michelan, and Grazielle Sales Teodoro. The first draft of the manuscript was written by Renan Domingues Pacheco, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. ACKNOWLEDGEMENTS We would like to thank Hydro Alunorte for the financial support provided to the project “Assessment of Aquatic Biota and Riparian Vegetation of the Hydrography that Influences the Murucupi Basin and Surrounding Areas of Hydro Alunorte”, as well as for the scholarships granted to Renan Domingues Pacheco and Tailane da Silva Sousa. We also thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES), Finance Code 001. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Aragão LEOC, Anderson LO, Fonseca MG et al (2018) 21st Century drought-related fires counteract the decline of Amazon deforestation carbon emissions. Nat Commun 9:536. https://doi.org/10.1038/s41467-017-02771-y Augé RM (2001) Water relations, drought and vesicular-arbuscular mycorrhizal symbiosis. 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Plant Phenomics Smith SE, Read DJ (2008) Mycorrhizal symbiosis. Academic Soudzilovskaia NA, Van Der Heijden MGA, Cornelissen JHC et al (2015) Quantitative assessment of the differential impacts of arbuscular and ectomycorrhiza on soil carbon cycling. New Phytol 208:280–293. https://doi.org/10.1111/nph.13447 Sousa TS, Pacheco RD, Pereira L et al (2025) Edge effect influences the ecological strategies of plant communities in tropical forest fragments. Plant Biol J 28:509–519. https://doi.org/10.1111/plb.70137 Souza APDS, Lisboa RCL (2005) Musgos (Bryophyta) na Ilha Trambioca, Barcarena, PA, Brasil. Acta Bot Bras 19:487–492. https://doi.org/10.1590/S0102-33062005000300008 Taiz L, Zeiger E (2009) Plant physiology, 4th edn. Sinauer Associates, Porto Alegre, p 744 Tavares JV, Oliveira RS, Mencuccini M et al (2023) Basin-wide variation in tree hydraulic safety margins predicts the carbon balance of Amazon forests. 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J Trop Ecol 32:300–313. https://doi.org/10.1017/S0266467416000274 Supplementary Files ESM1.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 02 May, 2026 Reviewers invited by journal 29 Apr, 2026 Editor invited by journal 16 Apr, 2026 Editor assigned by journal 16 Apr, 2026 First submitted to journal 15 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9406229","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":631907782,"identity":"e2ebe924-9bdc-4374-91eb-9f3456f65d56","order_by":0,"name":"RENAN DOMINGUES PACHECO","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYNACNjACAQkeefYGIG1gQbQWGxnDngMgLRKEtUBBmg3DjQSwdTgVm7f3HvxcUGYTzSfdfk3iw5/DPIwzn1/d8KNAgoG/vTsBmxaZM+eSpWecS8ttkzlTJjmz7TAPu3RO2c0eoMMkzpzdgE2LhESOgTRv2+HcNomcNGneBqAts3PSbvAAtRhI5GLXIv/G+Ddcyx+gwxhunkm7+QefFgkeM6gt6cekGdjSeBhusB+7jdcWnhwzax6QXyRymC1722x4DHty2G7LGEjw4PQL+xnj2zxlNrnzZ6Q/vPHjj4S9PPvxZzff/LGR42/vxaoFCfAYoDB4CCgHAfYH6IxRMApGwSgYBWAAALGTXhMxKPICAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0009-5809-9692","institution":"Universidade Federal Rural da Amazonia","correspondingAuthor":true,"prefix":"","firstName":"RENAN","middleName":"DOMINGUES","lastName":"PACHECO","suffix":""},{"id":631907783,"identity":"6791dcbb-2692-4dfa-903f-84dc22cde86e","order_by":1,"name":"Tailane Silva Sousa","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tailane","middleName":"Silva","lastName":"Sousa","suffix":""},{"id":631907784,"identity":"5d5c2e16-f302-492c-9df4-30eb2696ab31","order_by":2,"name":"Paulo Bittencourt","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Paulo","middleName":"","lastName":"Bittencourt","suffix":""},{"id":631907785,"identity":"60d4597c-5905-4ba4-97ae-6c089eaa9ab1","order_by":3,"name":"Patrícia De Britto Costa","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Patrícia","middleName":"De Britto","lastName":"Costa","suffix":""},{"id":631907786,"identity":"7fb2d3c4-4d39-47ce-b319-32d0fe17aa4a","order_by":4,"name":"Keven Alves","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Keven","middleName":"","lastName":"Alves","suffix":""},{"id":631907787,"identity":"0b519384-f8bd-49ef-9957-89615ec5541a","order_by":5,"name":"Thaisa Sala Michelan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Thaisa","middleName":"Sala","lastName":"Michelan","suffix":""},{"id":631907788,"identity":"e6f190de-8eb1-4440-aa5c-58ef54165793","order_by":6,"name":"Grazielle Sales Teodoro","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Grazielle","middleName":"Sales","lastName":"Teodoro","suffix":""}],"badges":[],"createdAt":"2026-04-13 15:41:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9406229/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9406229/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108788999,"identity":"ecffbaef-79e2-4eeb-b22f-f756a1b6f4ca","added_by":"auto","created_at":"2026-05-08 11:58:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":147638,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of the forest fragments studied in the municipalities of Barcarena, Abaetetuba, and Moju, Metropolitan Mesoregion of Belém, Pará.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9406229/v1/0b4ef2c770734f7108199f6d.png"},{"id":108789000,"identity":"03fd1ea2-0d8b-4ece-b45e-b6d59ea34892","added_by":"auto","created_at":"2026-05-08 11:58:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2085963,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Component Analysis (PCA) for the functional traits and environments. Orange circles represent individuals from the “Edge” and blue triangles represent those from the “Interior”. The larger symbols represent the group centroids (multivariate mean), while the 95% confidence ellipses indicate the dispersion of each group. The PC1 (36.1%) and PC2 (29.7%) axes explain 65.8% of the total data variation.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9406229/v1/c34edd8b0a03346bcf371e3c.png"},{"id":108807610,"identity":"a472d2f7-9829-4f55-b80d-9abe0b7c0df9","added_by":"auto","created_at":"2026-05-08 15:30:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120868,"visible":true,"origin":"","legend":"\u003cp\u003eLeaf water dynamics in different forest environments. (a) Predawn leaf water potential (Ψ\u003cem\u003epd\u003c/em\u003e) and (b) midday leaf water potential (Ψ\u003cem\u003emd\u003c/em\u003e). The circles represent mean values per site at the Edge (orange) and interior (green), with gray lines connecting the same pair of sites between environments.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9406229/v1/a4c37e04a2257cc6616e2e68.png"},{"id":108789002,"identity":"3835ab14-4998-4f52-a709-fae2dc3a7066","added_by":"auto","created_at":"2026-05-08 11:58:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":839923,"visible":true,"origin":"","legend":"\u003cp\u003eRoot morphological traits in trees in the Eastern Amazon. The graph presents (a) specific root length (SRL), (b) root tissue density (RTD), (c) specific root area (SRA), and (d) mean root diameter (DM). The points represent species means in each of the eight studied forest fragments, separated by environment (orange circles = edge; green circles = interior), and the lines connect paired measurements between edge and interior within the same fragment.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9406229/v1/af1c6e880440bfdd690009e4.png"},{"id":108977208,"identity":"c3cdb212-4853-4bd3-a7a7-dff844e40258","added_by":"auto","created_at":"2026-05-11 11:30:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":473180,"visible":true,"origin":"","legend":"\u003cp\u003eFor mycorrhizal symbiotic relationships, arbuscule abundance (A%) (a) and mycorrhizal colonization intensity (M%) (b). The points represent species means in each of the eight studied forest fragments, separated by environment (orange circles = edge; green circles = interior), and the lines connect paired measurements between edge and interior within the same fragment.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9406229/v1/935b4b14940b0f07b9805a7b.png"},{"id":108979608,"identity":"299bd195-993a-4506-9c7a-c5b7e959bc51","added_by":"auto","created_at":"2026-05-11 12:00:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3499050,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9406229/v1/05ef10ea-909d-4bc7-8877-434ff95956a7.pdf"},{"id":108789004,"identity":"464c089f-d00b-45ea-86e7-5782bf9bac9d","added_by":"auto","created_at":"2026-05-08 11:58:55","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":416051,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9406229/v1/588fec6b11857e74d5e48b31.pdf"}],"financialInterests":"","formattedTitle":"Forest edges favor conservative resource-use and symbiotic belowground strategies in Eastern Amazonian forest fragments","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIn recent decades, droughts caused by El Ni\u0026ntilde;o in the Amazon Forest have become longer and more intense (Espinoza et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Feldpausch et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Marengo et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Global climate models predict that these droughts may become increasingly frequent (Nobre et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), leading to higher tree mortality and increased carbon (C) emissions to the atmosphere (Lapola et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). With increasing plant stress, the Amazon Forest is expected to cease being a carbon sink, as has already been observed in some regions (Gatti et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lapola et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Tavares et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition to droughts, another critical disturbance in the Amazon Forest is the exponential increase in forest edges, a direct consequence of deforestation and fragmentation (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the Amazon, 45.5% of the biome already presents some level of fragmentation (Bauer et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Although edge effects drastically alter microclimate, species composition, and biomass, most studies still focus on continuous forests (Nunes et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Forest fragmentation alters the microclimate at edges by increasing solar radiation incidence, temperature, and atmospheric evaporative demand (Laurance et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Magnago et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), subjecting vegetation to continuous water stress even when soil moisture has not yet reached critical levels (Barros et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Didham and Lawton, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Thus, in these already fragmented landscapes, there is still limited understanding of how the edge\u0026ndash;interior gradient modulates the physiological and morphological responses of trees, especially during severe climatic events.\u003c/p\u003e \u003cp\u003eAt the forest edges, changes occur in species composition and functional traits compared to the interior (Cardoso et al. 2002; Figueiredo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These areas are often dominated by pioneer species with lower wood density and reduced height (Magnago et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Michalski et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Rabelo et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In forest fragments in Eastern Amazon, it has been demonstrated that edge effects alter the ecological strategies of communities, leading to a decoupling between wood and leaf traits (Sousa et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Comparisons of species traits between forest edges and interiors have mainly focused on aboveground traits, while much less is known about root traits and their strategies that may confer plant resistance and resilience to variations in water availability.\u003c/p\u003e \u003cp\u003eThe root system performs multiple functions, being responsible for water and nutrient uptake and soil carbon cycling due to the high turnover of fine roots, playing a central role in the functioning of terrestrial ecosystems (Brunner et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Root traits involving morphology and mycorrhizal associations influence soil resource acquisition and are directly linked to plant growth, reproduction, and survival (Violle et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Root morphological traits can be plastic, responding to nutrient and water availability (Freschet et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); characteristics such as roots length, diameter, and volume are related to water and nutrient acquisition, influencing plant productivity. Roots with higher specific root length (i.e. the ratio between root length and dry mass) exhibit more efficient hydraulic conductance (Comas et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), whereas higher root tissue density is associated with greater structural resistance, longevity, and resource conservation, at the expense of high rates of absorption and soil exploration (Eissenstat and Achor, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Ryser, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). From a trait-based perspective, plant belowground strategies are structured along two main independent axes. The first is a conservation gradient, primarily reflected by root tissue density (RTD), which determines the balance between fast root turnover and structural persistence. The second is a collaboration gradient, which contrasts a 'do-it-yourself' resource acquisition strategy (characterized by high specific root length, SRL) with an 'outsourcing' strategy defined by a high reliance on mycorrhizal fungi (Bergmann et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Together, these axes form the Root Economics Space, which provides a unifying framework to understand how plants adjust belowground strategies in response to environmental stress, such as severe droughts.\u003c/p\u003e \u003cp\u003eAnother trait influencing resource acquisition under water stress is the degree of root colonization by arbuscular mycorrhizal fungi (AMF) (Bergmann et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lalibert\u0026eacute; et al. 2017; Mccormack et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This mutualistic relationship increases water uptake by expanding the volume of soil explored, positively influencing water potential and delaying stomatal closure (Aug\u0026eacute;, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2001\u003c/span\u003e); these responses are highly dependent on the environmental context (Freschet et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This symbiosis may be particularly vital in edge environments, where water stress is elevated and evaporative demand is greater, favoring more acquisitive root strategies and high functional plasticity (Bergmann et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Freschet et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, it remains unexplored whether AMF colonization increases at edges as a compensatory plant strategy or whether the extreme microclimatic conditions of these Eastern Amazon fragments limit the efficiency of this association during severe drought events (Brunner et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), as well as understanding whether morphological variation changes between edges and interiors of forest fragments under conditions of extreme drought events (such as El Ni\u0026ntilde;o) in fragments as singular as those in this region.\u003c/p\u003e \u003cp\u003eIn this study, we investigated root functioning in eight forest fragments located in the state of Par\u0026aacute;, Eastern Amazon, analyzing 144 tree individuals. The objective was to evaluate how the edge\u0026ndash;interior environment influences water status, root functional traits, and species resource acquisition strategies. Thus, we hypothesized that root traits vary along the edge\u0026ndash;interior gradient, with trees at the edge adjusting their root strategies in response to lower water availability. We expected (1) more negative water potential in species at the edge, indicating greater water limitation, compared to species in the fragment interior; (2) lower specific root length (SRL), lower specific root area (SRA), and higher root tissue density (RTD) compared to trees in the fragment interior, as a strategy for more efficient acquisition of water and nutrients; (3) higher mycorrhizal colonization and greater abundance of arbuscules at the edge, reflecting greater dependence on symbioses to mitigate water stress and sustain resource acquisition during severe drought events.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSTUDY AREA\u003c/h2\u003e \u003cp\u003eThe study was conducted in eight forest fragments, distributed across three (3) municipalities: Barcarena (Lat: 01\u0026ordm;30\u0026prime;21\u0026Prime; S; Long: 48\u0026ordm;37\u0026prime;33\u0026Prime; W), Abaetetuba (Lat: 01\u0026deg;43\u0026prime;00\u0026Prime; S; Long: 48\u0026deg;52\u0026prime;00\u0026Prime; W), and Moju (Lat: 01\u0026deg;53\u0026prime;00\u0026Prime; S; Long: 48\u0026deg;46\u0026prime;00\u0026Prime; W) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), municipalities located in the Metropolitan Region of Bel\u0026eacute;m, in the state of Par\u0026aacute;, Brazil (IBGE, 2025; Mendon\u0026ccedil;a et al. 2021) (Online Resource 1, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The region has an average annual precipitation of 2,500 mm and a mean temperature around 27\u0026deg;C (De Oliveira et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Piratoba et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The soils found in the Barcarena region are mainly characterized as Gleysols, Spodosols, and Oxisols (IDESP, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Regarding vegetation, the region is composed of dense ombrophilous forest, with a predominance of evergreen species (Souza and Lisboa, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDATA COLLETION\u003c/h3\u003e\n\u003cp\u003eThe research was conducted in November 2023, during the dry season, characterized as a drought event due to El Ni\u0026ntilde;o (Espinoza et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Eight forest fragments were selected for the study, and each fragment contained a watercourse (stream, \u003cem\u003eigarap\u0026eacute;\u003c/em\u003e) within its interior. In each fragment, we evaluated three abundant species both at the edge (within the first 5 m) and in the interior (from 30 m from the edge). In each fragment, three individuals per species were selected for the collection of physiological and root traits, totalling 144 records corresponding to 24 distinct species (Online Resource 1, Table S2). Species selection was based on their abundance and ecological relevance to the Amazonian flora. To characterize tree ecological strategies, we selected leaf physiological variables, root morphological traits, and mycorrhizal association (Online Resource 1, Table S3).\u003c/p\u003e\n\u003ch3\u003ePHYSIOLOGICAL TRAIT\u003c/h3\u003e\n\u003cp\u003eWater Potential\u003c/p\u003e \u003cp\u003eWe measured leaf water potential (ψ) in one leaf per individual for all 144 trees at the predawn (ψ predawn), between 4:00 and 5:00 h, and midday (ψ midday), between 12:00 and 13:00 h. Predawn ψ is an indicator of soil water status, since during this period the atmosphere presents low evaporative demand, leading leaf water status to equilibrate with soil water status (Bhaskar and Ackerly \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Taiz and Zeiger \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Midday ψ is an indicator of the driest condition experienced by the tree throughout a season, integrating atmospheric and soil water deficits (Bhaskar and Ackerly \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Taiz and Zeiger \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Water potential (ψ) was measured in situ using a pressure chamber (PMS 1505 D and PMS 1000, PMS Instruments), and immediately after sampling, the values for each individual were recorded (Pammenter and Van der Willigen \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eROOTS TRAITS\u003c/h3\u003e\n\u003cp\u003eRoot Morphology\u003c/p\u003e \u003cp\u003eWe collected fine roots (diameter\u0026thinsp;\u0026lt;\u0026thinsp;2 mm) from trees using a root-tracing method to ensure species-specific identification. From the base of the stem of each target tree, we carefully excavated the soil to a depth of 0\u0026ndash;30 cm and followed the lateral coarse roots until they branched into the associated fine roots. This procedure followed the standardized sampling protocol of Freschet et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). After collection, fine roots were separated, cleaned, weighed, and kept under refrigeration until the determination of morphological parameters. We measured fresh mass and dry mass of fine roots using an analytical balance (Model AD430; Marte) with a resolution of 0.001 g. Dry mass was determined after drying the samples in a forced-air circulation oven at 70\u0026deg;C until constant weight was achieved (Freschet et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To calculate dry mass content, we used the following equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{M}\\text{S}\\:=\\:\\left(\\frac{PS}{PF}\\right)\\times\\:\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere MS represents dry mass content, PF is fresh weight, and PS is dry weight of the roots.\u003c/p\u003e \u003cp\u003eRoots were scanned using a flatbed scanner in grayscale at 600 dpi. Image analysis was performed using the software RhizoVision Rxplorer v2.0.3, with algorithms described by Seethepalli et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), where root length and diameter were assessed. Specific root length (SRL) was obtained by dividing root length by dry mass (Gu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zadworny et al. 2011). Specific root area (SRA) was calculated by dividing root surface area (obtained from scanned images) by dry mass (Wang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Root tissue density (RTD) was calculated by dividing dry mass by root volume (calculated from scanned images) (Gu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kong et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Mean root diameter was obtained using the software RhizoVision Rxplorer v2.0.3.\u003c/p\u003e \u003cp\u003eMycorrhizal Association\u003c/p\u003e \u003cp\u003eTo analyse the mutualistic relationship, we excavated and collected at least 30 cm of fine (absorptive) roots from each individual, in the superficial soil layer, between 0 and 30 cm depth. After collection, the roots were carefully washed in alternating baths to remove any particulate material and were stored in 50% ethanol for preservation.\u003c/p\u003e \u003cp\u003eSubsequently, the roots were cleared with 10% potassium hydroxide (m/v), acidified in 2% HCl (v/v), and stained with 0.05% trypan blue (m/v) for the identification and quantification of colonization by mycorrhizal fungi (AMF). We used the MYCOCALC program, following the method described by TROUVELOT (1986), in which we evaluated the parameters: frequency of root system infection (F%), intensity of mycorrhizal colonization of the root system (M%), and abundance of arbuscules in the root system (A%).\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDATA ANALYSIS\u003c/h2\u003e \u003cp\u003eTo assess the associations between root morphological traits and leaf physiological variables, we applied Spearman correlation analysis using the pairwise method. Statistical significance was determined based on p-values, considering a threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All analyses were performed in the R statistical environment v.4.4.x (R Core Team, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), using the psych package to calculate correlation matrices and the corrplot package for graphical visualization of the results (Wei and Simko, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To evaluate the effect of environment (edge vs. interior) on plant functional traits, we used Generalized Linear Mixed Models (GLMMs). In all models, the environment was treated as a fixed effect. To account for spatial variation and interspecific variability, we included sampling site and species nested within site (1 | Fragment/Species) as random effects. The significance of the environment was assessed using Likelihood Ratio Tests (LRT), comparing the full model with a null model using the anova function. All continuous variables were checked for distributional assumptions prior to model fitting.\u003c/p\u003e \u003cp\u003eFor Hypothesis 1, predawn water potential (Ψ\u003cem\u003epd\u003c/em\u003e) was analyzed using a Gamma GLMM (log link), whereas midday water potential (Ψ\u003cem\u003emd\u003c/em\u003e) followed a Gaussian distribution and was analyzed using a Linear Mixed Model (LMM). For Hypothesis 2 (morphological traits: RTD, SRL, SRA, and DM), we fitted GLMMs with a Gamma distribution and a log link function. For Hypothesis 3 (mycorrhizal colonization: M% and A%), the models were also fitted using a Gamma distribution. To address heteroscedasticity detected through diagnostics from the DHARMa package, a dispersion formula for the environment term (dispformula\u0026thinsp;=\u0026thinsp;~\u0026thinsp;Environment) was incorporated into these models. Model fit was verified by inspection of simulated residuals, and model quality was described using marginal R\u0026sup2; and conditional R\u0026sup2; values, calculated with the performance package.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eFunctional associations and root strategies\u003c/p\u003e \u003cp\u003eRoot attributes showed associations among themselves (Online Resource 1, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We observed negative correlations between root tissue density (RTD) and specific root area (SRA) (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.95, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as well as between RTD and specific root length (SRL) (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.88, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Mean diameter (DM) showed a negative correlation only with SRL (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.32, p\u0026thinsp;=\u0026thinsp;0.001), indicating that roots with higher SRL tend to be thinner. On the other hand, mycorrhizal colonization intensity (M%) and arbuscule abundance (A%) showed a strong positive correlation with each other (r\u0026thinsp;=\u0026thinsp;0.90, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but did not show significant associations with the root morphological attributes (RTD, SRA, SRL, and DM). The mean values and standard deviation for all morphological attributes evaluated are detailed in Online Resource 1, Table S5. Principal Component Analysis (PCA) showed that the first two components explained 65.8% of the total variance in the data. The first axis (PC1) explained 36.1% and was associated with specific root length (SRL) and specific root area (SRA), being negatively associated with root tissue density (RTD) and mean diameter (DM). The second axis was associated with mycorrhizal colonization (M%) and arbuscular colonization (A%). Thus, the PCA revealed an orthogonal axis separating mycorrhizal associations (A% and M%) from root morphological attributes (RTD, SRA, SRL, and DM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Regarding the environments, the biplot showed an overlap between the edge and interior groups, as indicated by the 95% confidence ellipses. However, samples from the interior environment (blue triangles) showed higher SRL and SRA values compared with those from the edge (orange circles), which appeared more centrally distributed in the multivariate space.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDifference in water potential between trees in the fragment interior and edge\u003c/p\u003e \u003cp\u003ePredawn water potential (Ψ\u003cem\u003epd\u003c/em\u003e) did not vary between environments (χ\u0026sup2; = 0.50; p\u0026thinsp;=\u0026thinsp;0.480) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), with means of \u0026minus;\u0026thinsp;0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 MPa at the edge and \u0026minus;\u0026thinsp;0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 MPa in the interior (Online Resource 1, Table S5). Variance for this trait was explained by species (σ\u0026sup2; = 0.105), with low influence of site (σ\u0026sup2; = 0.011).Midday leaf water potential (Ψ\u003cem\u003emd\u003c/em\u003e) differed significantly between environments (χ\u0026sup2; = 53.55; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), with more negative values (\u0026minus;\u0026thinsp;2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77 MPa) at the edge compared with the interior (\u0026minus;\u0026thinsp;1.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80 MPa). Variance partitioning revealed that species (σ\u0026sup2; = 0.163) and site (σ\u0026sup2; = 0.052) were the main drivers of the variation for this trait.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRoot morphological traits and strategies\u003c/p\u003e \u003cp\u003eSpecific root length (SRL) was significantly higher in the interior (117.44\u0026thinsp;\u0026plusmn;\u0026thinsp;88.49 cm g⁻\u0026sup1;) than at the edge (59.15\u0026thinsp;\u0026plusmn;\u0026thinsp;54.58 cm g⁻\u0026sup1;; χ\u0026sup2; = 13.60; p\u0026thinsp;=\u0026thinsp;0.003) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). SRL variability was predominantly explained by site (σ\u0026sup2; = 0.097), with negligible contribution from species identity (σ\u0026sup2; \u0026lt; 0.001) (Online Resource 1, Table S5). Root tissue density (RTD) was higher at the edge (0.021\u0026thinsp;\u0026plusmn;\u0026thinsp;0.023 g cm⁻\u0026sup3;) compared to the interior (0.008\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005 g cm⁻\u0026sup3;; χ\u0026sup2; = 34.92; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Variation in RTD was driven by both site (σ\u0026sup2; = 0.114) and species (σ\u0026sup2; = 0.068) (Online Resource 1, Table S5). Specific root area (SRA) showed no difference (χ\u0026sup2; = 7.15; p\u0026thinsp;=\u0026thinsp;0.067) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) between the forest interior (284.4\u0026thinsp;\u0026plusmn;\u0026thinsp;177.9 cm\u0026sup2; g⁻\u0026sup1;) and the edge (176.5\u0026thinsp;\u0026plusmn;\u0026thinsp;152.1 cm\u0026sup2; g⁻\u0026sup1;), and the main driver of SRA variation was site (σ\u0026sup2; = 0.050) (Online Resource 1, Table S5). Mean fine-root diameter (DM) did not differ between the edge (0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 mm) and the interior (0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 mm; χ\u0026sup2; = 3.11; p\u0026thinsp;=\u0026thinsp;0.374) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Most of the observed variability was attributed to species (σ\u0026sup2; = 0.005), while site (σ\u0026sup2; = 0.002) had a smaller contribution (Online Resource 1, Table S5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMycorrhizal associations and symbiotic activity\u003c/p\u003e \u003cp\u003eArbuscule abundance in the root system (A%) was higher at the edge (30.6\u0026thinsp;\u0026plusmn;\u0026thinsp;39.7%) than in the interior (2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5%; χ\u0026sup2; = 37.78; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), with species being the dominant driver (σ\u0026sup2; = 0.157) (Online Resource 1, Table S5). Mycorrhizal colonization intensity (M%) also was higher at the edge (3.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96%) compared to the interior (1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67%; χ\u0026sup2; = 31.62; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). For colonization intensity, fragment (σ\u0026sup2; = 0.048) explained more variance than species (σ\u0026sup2; = 0.022) (Online Resource 1, Table S5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur study demonstrates that, under conditions of severe drought associated with El Ni\u0026ntilde;o, tree species exhibit contrasting belowground investments in root traits, reflecting different resource acquisition strategies between the edge and the interior of forest fragments in Eastern Amazonia. Edge trees experience greater water limitation throughout the day, with more negative midday leaf water potential (Ψ\u003cem\u003emd\u003c/em\u003e) values, while the stability of predawn water potential (Ψ\u003cem\u003epd\u003c/em\u003e) between environments indicates that this water limitation is derived from greater atmospheric evaporative demand, rather than from differences in soil water availability. We observed a coordinated functional adjustment at the edge, where trees exhibit a more conservative root strategy, characterized by higher root tissue density (RTD) and lower specific root length (SRL), prioritizing structural resistance and greater root longevity (Bergmann et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ryser \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). As part of this adjustment, these trees intensify the collaborative resource acquisition pathway, showing greater mycorrhizal colonization intensity and a higher abundance of arbuscules at the edge, suggesting a compensatory mechanism in response to the lower exploratory capacity of denser roots. In contrast, in the forest interior predominated a root strategy with greater foraging capacity, characterized by higher SRL values, favored by relatively more stable microclimatic conditions.\u003c/p\u003e \u003cp\u003eEven under drought conditions associated with El Ni\u0026ntilde;o, trees at the edge exhibited more negative Ψmd values than species in the fragment interior. This suggests that the microclimate at forest edges, characterized by greater light incidence, higher temperatures, and higher vapor pressure deficit (Barros et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Didham and Lawton \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), intensifies the evaporative demand imposed on trees during the day. This result is consistent with previous studies documenting more extreme microclimatic conditions at forest edges, particularly in fragmented landscapes exposed to the anthropogenic matrix (Didham and Lawton, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Haddad et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Magnago et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In contrast, we did not observe variation in Ψpd. This decoupling between daytime stress and nighttime recovery suggests that, under extreme drought conditions, water limitation manifests primarily through elevated atmospheric demand, rather than being determined exclusively by soil water status (Barros et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe correlations observed between root tissue density (RTD) and traits associated with soil exploration (SRL), as well as the structuring of the data in the Principal Component Analysis (PCA), suggest strong functional coordination within the root system of the species. Trees at the edge showed higher RTD, indicating greater investment in structurally denser and more durable roots (Ryser, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Roumet et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These patterns are consistent with the concept of the Root Economics Space (Bergmann et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), in which investment in tissue density (RTD) represents an axis of functional conservation independent of the 'do-it-yourself' exploration strategy (Bergmann et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Matthus et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). At the edge, the increase in RTD suggests a transition toward a conservative strategy, in which mechanical resistance and root longevity become priorities in response to the greater risk of hydraulic stress and functional instability in soils subject to higher water variability and intensified atmospheric demand (Eller et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Vleminckx et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The significant contribution of fragment identity (site) to RTD variation (σ\u0026sup2; = 0.114) suggests that, although fragmentation acts as an environmental filter, the intensity of this functional adjustment is modulated by local characteristics of each remnant. This pattern indicates that edaphic variations or disturbance history among sites interact with the edge effect, favoring specific functional assemblages independently of species identity (Michalski et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Violle et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Thus, convergence toward more conservative strategies at the edge appears to be a response to atmospheric water stress, but whose magnitude is refined by the particular conditions of each forest fragment.\u003c/p\u003e \u003cp\u003eIn contrast, in the interior of the fragments, higher SRL values were observed, a trait associated with greater spatial soil exploration capacity (Eissenstat et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Ostonen et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Under non severe conditions, the relatively greater water availability in the fragment interior, often associated with proximity to watercourses and lower microclimatic variability, may act as a hydrological buffer, favoring more exploratory root strategies (Matthus et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, under the evaluated conditions, associated with extreme drought, this buffering effect appears to have been reduced, resulting in similar water conditions between edge and interior Even so, the functional organization of roots suggests that interior species maintain strategies associated with greater exploitation of the superficial soil, possibly reflecting environmental filtering between habitats (Santana et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and belowground competition among coexisting species (Zangaro et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to morphological adjustments, our results indicate that mycorrhizal associations act as a complementary and independent mechanism in belowground strategies between the edge and the interior. The absence of significant correlations between morphological traits (RTD, SRA, SRL, DM) and mycorrhizal parameters (M% and A%), combined with the separation of these traits along distinct and orthogonal axes in the PCA (PC1 determining morphology and PC2 symbiosis), demonstrates that fungal colonization and root structure operate as autonomous strategies of environmental adjustment. This pattern suggests that trees at the edge reinforce the symbiotic pathway independently of changes in root density or extension, possibly to compensate for the greater evaporative demand of this environment. The expressive abundance of arbuscules at the edge (30.6%) compared to the interior (2.6%) shows that this association is functionally active, reflecting a directed investment in resource uptake, even in the face of low total mycorrhizal colonization (Lekberg and Koide, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This coordination between increased root tissue density (RTD) and strengthening of the collaboration axis at the edge corroborates discussions on the bidimensionality of the Root Economics Space, in which investment in fungi acts as an autonomous compensatory strategy that does not depend on prior changes in root thickness or area (Bergmann et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Matthus et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe roots with higher RTD and mycorrhizal activity at the edge suggests an integrated strategy of tolerance to water stress and nutrientes uptake, in which morphological limitations associated with denser roots are partially compensated by symbiotic interactions. While roots with higher RTD tend to exhibit lower efficiency in the direct resources uptake (Comas and Eissenstat, 2004), mycorrhizal hyphae expand the volume of soil explored, functioning as a functional extension of the root system (Smith and Read, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This functional arrangement highlights the importance of belowground biotic interactions as mediators of tree persistence in fragmented environments under high water stress, contributing to the stability and resilience of forest ecosystems (Bennett et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rillig et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the greater symbiotic association at the edge, the mycorrhizal colonization rates observed were low in both environments when compared to values frequently reported for tropical forests (Wang et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This pattern suggests that additional factors, such as edaphic characteristics, land-use history, composition of fungal communities, and the combined effects of fragmentation and extreme drought, may be limiting the functional effectiveness of this association. Indeed, the intensity and benefits of mycorrhizal symbiosis are highly dependent on environmental context and resource limitation (Johnson et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Macroecological-scale evidence indicates that colonization by arbuscular mycorrhizal fungi is strongly regulated by climatic variables, suggesting that the severe thermal and water variations associated with El Ni\u0026ntilde;o may have acted as an abiotic constraint on the responsiveness of the symbiosis (Soudzilovskaia et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Thus, although symbiosis represents an important mechanism for mitigating water stress, its compensatory capacity may be reduced under extreme environmental conditions (Aug\u0026eacute;, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Smith and Read, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This functional arrangement highlights the importance of belowground biotic interactions as mediators of tree persistence in fragmented environments under high water stress. Evolutionary evidence suggests that mycorrhizae have played a role in facilitating plant adaptation to critical changes in water availability over time (Cosme, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), contributing to the stability and resilience of forest ecosystems (Bennett et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rillig et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur results demonstrate that forest fragmentation modulates the functional organization of roots and symbioses in Eastern Amazonia, establishing distinct responses between edge and interior under the influence of a severe drought event (El Ni\u0026ntilde;o). By revealing that trees at the edge operate in a more negative daytime water status and adopt a coordination between the root conservation axis (higher RTD) and the collaboration axis (greater abundance of arbuscules), this study fills a critical gap on how fragmented areas respond to the edge environment. Unlike continuous forests, forest fragments that comprise an increasing portion of the Amazonian landscape exhibit specific functional responses that suggest a structural adaptation to greater atmospheric evaporative demand. These findings reinforce the need to integrate edge heterogeneity into forest vulnerability models, since belowground persistence strategies differ drastically between the interior and the periphery of fragments. Therefore, conservation and restoration efforts must consider that the functional integrity of these forests depends on maintaining mycorrhizal connectivity and protecting root structure at the edges, essential components for the resilience of fragmented landscapes in the face of intensifying climatic anomalies in the region.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003e\u003cp\u003eA%, abundance of arbuscules in the root system\u003c/p\u003e\u003cp\u003eAMF, arbuscular mycorrhizal fungi\u003c/p\u003e\u003cp\u003eDM, mean root diameter\u003c/p\u003e\u003cp\u003eF%, frequency of root system infection\u003c/p\u003e\u003cp\u003eGLMMs, generalized linear mixed models\u003c/p\u003e\u003cp\u003eLMM, linear mixed model\u003c/p\u003e\u003cp\u003eLRT, likelihood ratio test\u003c/p\u003e\u003cp\u003eM%, intensity of mycorrhizal colonization of the root system\u003c/p\u003e\u003cp\u003ePCA, principal component analysis\u003c/p\u003e\u003cp\u003ePC1, first principal component\u003c/p\u003e\u003cp\u003ePC2, second principal component\u003c/p\u003e\u003cp\u003eRTD, root tissue density\u003c/p\u003e\u003cp\u003eSRA, specific root area\u003c/p\u003e\u003cp\u003eSRL, specific root length\u003c/p\u003e\u003cp\u003eΨ\u003cem\u003emd\u003c/em\u003e, midday leaf water potential\u003c/p\u003e\u003cp\u003eΨ\u003cem\u003epd\u003c/em\u003e, predawn leaf water potential\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was financially supported by Hydro Alunorte through the project \u0026ldquo;Assessment of Aquatic Biota and Riparian Vegetation of the Hydrography that Influences the Murucupi Basin and Surrounding Areas of Hydro Alunorte\u0026rdquo;, including scholarships granted to Renan Domingues Pacheco and Tailane da Silva Sousa. This study was also supported by Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; Brasil (CAPES), Finance Code 001.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eAll authors contributed to the conception and design of the study. Material preparation, data collection, and data analysis were performed by Renan Domingues Pacheco, Tailane da Silva Sousa, Paulo Bittencourt, Patr\u0026iacute;cia de Britto Costa, Keven Adrian Furtado Alves, Thaisa Michelan, and Grazielle Sales Teodoro. The first draft of the manuscript was written by Renan Domingues Pacheco, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eWe would like to thank Hydro Alunorte for the financial support provided to the project \u0026ldquo;Assessment of Aquatic Biota and Riparian Vegetation of the Hydrography that Influences the Murucupi Basin and Surrounding Areas of Hydro Alunorte\u0026rdquo;, as well as for the scholarships granted to Renan Domingues Pacheco and Tailane da Silva Sousa. We also thank the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; Brasil (CAPES), Finance Code 001.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArag\u0026atilde;o LEOC, Anderson LO, Fonseca MG et al (2018) 21st Century drought-related fires counteract the decline of Amazon deforestation carbon emissions. 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J Trop Ecol 32:300\u0026ndash;313. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S0266467416000274\u003c/span\u003e\u003cspan address=\"10.1017/S0266467416000274\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Forest fragmentation, Root traits, Arbuscular mycorrhizae, Water stress, Edge effect, Drought","lastPublishedDoi":"10.21203/rs.3.rs-9406229/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9406229/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003eWhile edge effects on aboveground traits are relatively well-documented, little is known about how the edge\u0026ndash;interior interface modulates root strategies and mycorrhizal associations under severe climatic conditions. This study evaluated how the environment (edge vs. interior) influences leaf water status and root functional traits of trees in forest fragments during the 2023 El Ni\u0026ntilde;o.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 144 tree individuals were sampled across eight forest fragments in Par\u0026aacute;, Brazil. We measured leaf water potential at predawn (Ψpd) and at midday (Ψmd), root morphological traits (specific root length - SRL, specific root area SRA, root tissue density RTD, and mean diameter) and mycorrhizal colonization (intensity of colonization in the root system M% and arbuscule abundance A%) in species occurring at the forest edge and in the interior of the fragments. We used GLMMs to test the effect of environment, including fragment and species as random effects, in addition to correlation analyses and PCA to explore functional coordination.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTrees at the edge exhibited more negative Ψ\u003cem\u003emd\u003c/em\u003e, with no difference in Ψ\u003cem\u003epd\u003c/em\u003e, indicating greater atmospheric evaporative demand. At the edge, we observed higher RTD, lower SRL, and greater mycorrhizal colonization and arbuscule abundance. In the interior, plants exhibited a more exploratory strategy, with higher SRL values. Root morphology and mycorrhizal symbiosis acted as independent strategies.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eFragmentation modulates belowground functional organization, promoting at the edge a conservative strategy coordinated with the reinforcement of the symbiotic pathway, which may be crucial for the resilience of Amazonian fragments under severe droughts.\u003c/p\u003e","manuscriptTitle":"Forest edges favor conservative resource-use and symbiotic belowground strategies in Eastern Amazonian forest fragments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-08 11:58:50","doi":"10.21203/rs.3.rs-9406229/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-05-02T09:54:06+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-29T16:05:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2026-04-16T07:16:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-16T06:23:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2026-04-15T11:19:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dd8b66db-cf5a-40a4-b99a-9fc5eb354fad","owner":[],"postedDate":"May 8th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"","date":"2026-05-02T09:54:06+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-29T16:05:33+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T11:58:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-08 11:58:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9406229","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9406229","identity":"rs-9406229","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}