Study area
The study was conducted in a rocky Amazonian savanna community in the municipality of Nova Canaã do Norte in the state of Mato Grosso, Brazil (Figure 1), 10º53’98,7” S, 55º46’68,7” W). The soil of the study area is classified as Litholic Neosols that are poorly drained, dystrophic, alic, extremely acidic and sandy, and with low nutrient concentrations (Pessoa et al., 2021). The climate of the area is equatorial (Am) hot and humid, according to the Köppen classification, with temperatures varying from 20° C to 36° C with an annual average above 28° C (Alvares et al., 2013). Total annual rainfall can reach 2,180 mm, with two well-defined seasons — a rainy season encompassing November, December, January, February, and March (1,180 mm) and a dry season encompassing June, July, August, and September (108 mm) — with the other months being considered transition periods (Alvares et al., 2013).
Data collection
The seven most abundant woody plant species in the study area were selected from vegetation representing 75% of the community plant biomass (Table 1). The following morphological, anatomical, and hydraulic measurements were made for each species: leaf area, leaf succulence, leaf specific mass, stomatal density, adaxial cuticle, maximum theoretical stomatal conductance and carbon isotopic composition (water use efficiency - WUE); theoretical hydraulic conductivity, vessel lumen area, vessel frequency, vessel element length (including the tips, Figure S1), lumen fraction, ray frequency and ray width; fiber lumen, fiber length, wood specific gravity, vessel wall thickness, intervessel pits (often horizontal in tangential sections, Figure S2) and pit membrane thickness (resistance mechanism); and xylem embolism resistance and hydraulic safety margin (embolism vulnerability proxy). Trait sampling and measurements for the species are detailed below.
Leaf
Morphological measurements were made of samples from five fully expanded leaves from three individuals of each species. Discs of 0.5 cm in diameter were removed from the median third of the leaves. The discs were hydrated for 24 h and dried with paper for subsequent determination of saturated mass (Msaturated) using a digital balance (AY220, Shimadzu), and thickness with a digital caliper (Stainless, Hardened). The hydrated discs were then placed in an oven set at 55 ºC for 72 h to obtain dry mass (Mdry). These parameters were used to calculate leaf succulence (Msaturated - Mdry/Areadisc) (Kluge and Ting, 1978) and leaf specific mass (Mdry/Areadisc) (Kluge and Ting, 1978). Leaf area was determined by digitally scanning the leaves used for the other morphological analyses and then measuring their area using ImageJ digital image processing system.
Leaf cross-sections were made in the middle of the leaf by freehand for measurements of leaf anatomical attributes and to observe the adaxial cuticle. Stomatal density (mm2) and stomatal pore length (µm) were determined from the analysis of images of epidermis dissociated by the Franklin method (Franklin, 1945). Theoretical maximum stomatal conductance was calculated from the relationship between stomatal pore density and size (de Boer et al., 2016), according to the following equation:
Gmax = D x L (Eq 1.)
where Gmax is maximum stomatal conductance (mm.s-1), D is stomatal density (mm²) and L is stomatal length (µm).
Scanning electron microscopy
Two leaves were selected for each species for investigation of stomatal morphology on the abaxial leaf face. Fragments of the median third of the leaf blade were fixed in an aqueous solution of 2.5% glutaraldehyde, 4% formaldehyde, and 0.05M sodium cacodylate buffer at pH 7.2 (Karnovsky, 1965 modified by Da Cunha et al., 2000) and then post-fixed in 1% osmium tetroxide and 0.05M sodium cacodylate buffer for 2 h at room temperature. After fixation, the samples were submitted to acetone dehydration, followed by CO2 critical point drying (CPD 030, Baltec). The samples were then adhered to stubs with carbon tape and covered with a layer of approximately 20 nm of gold (SCD 050, Baltec, Switzerland). Images were obtained using a ZEISS EVO 40 (Germany) scanning electron microscope at a voltage of 15 kV.
Carbon isotopic composition (δ13C)
For determining δ13C, five leaves were selected from three individuals of each species. The leaves were dried in an oven at 60 °C for 72 h and then macerated. After maceration, the five leaves for each individual were homogenized. The homogenized material was subsequently weighed (1.5 mg) with a precision analytical balance. Data were obtained using a Thermo Finnigan Delta V Advantage mass spectrometer coupled to a Flash 2000 (Thermo 26 Fisher Scientific in Bremen, Germany) elemental analyzer at the Laboratório de Ciências Ambientais from Universidade Estadual do Norte Fluminense Darcy Ribeiro. Pee Dee Belemnite (PDB) was used as the standard value for C. The analytical precision was ± 0.1‰, while the precision of the elemental and isotopic compositions was determined by certified standard (Protein OAS/IsotopeCert 114859; Elemental Microanalysis).
Branch
Branch samples were sectioned (15–20 μm thickness) in transversal and longitudinal tangential planes using a sliding microtome (SM2010 R, LEICA, Germany). The sections were then clarified in sodium hypochlorite (50%) and acidulated water (0.1%), dehydrated in an ascending ethanol series (50% to 100%) (Johansen, 1940), stained with Astra blue and hydro-alcoholic Safranin, and immersed in xylene P.A. Permanent slides were made using Entellan® (Merck) synthetic resin.
We used a maceration method for the measurements of vessel element length (including tips) and fiber length. Maceration of branch material followed Franklin (1945). Small branch fragments were removed from each sample and placed in bottles containing a macerating solution of glacial acetic acid and hydrogen peroxide (1:1). The bottles were then sealed and placed in an oven at 60 °C for 24 h or until the complete dissociation of cells. The material was then washed in distilled water, stained with 1% aqueous Safranin, and mounted on semi-permanent slides with glycerin. Imperforate tracheary elements were not observed in macerations for any of our samples, as it is common among Angiosperm species.
Quantitative analysis was performed using 12 slides per individual. All descriptions, counts and branch cellular measurements followed IAWA Committee standards (1989). Permanent and semi-permanent slides were analyzed using a light-field light microscope (Axioplan, ZEISS, USA), with image capture via a coupled camera (Power shot A640, CANON, USA).
Transmission electron microscopy
Two branches of each species were selected for analysis of the intervascular pit membrane. Branch fragments were fixed in modified Karnovsky solution (Karnovsky, 1965 modified by Da Cunha et al., 2000). Post-fixed in 1% osmium tetroxide and 0.05M sodium cacodylate buffer 2 h at room temperature, and then dehydrated in an increasing acetone series and infiltrated and embedded with epoxy resin (Epon®). Ultrathin sections (80 nm) were made using an ultramicrotome (Reichert Ultracuts Leica Instruments) with a diamond knife (Diatome®), which were collected in copper grids (300 mesh) and contrasted with 1.0% alcoholic uranyl acetate, followed by 5.0% aqueous lead citrate (Reynolds, 1963). Ultrastructure analysis of pit membranes was performed using a JEM 1400 Plus JEOL transmission electron microscope at a voltage of 80 kV, with 20 observations per individual. Measurements of anatomical attributes (Table 2) were performed using Image Pro-Plus 4.0 digital image processing system. Pit membrane thickness was measured cuts made in the sapwood at three different points, later averaged to represent the sample pit membrane thickness (Figure S4). All measurements for each species were made from three individuals with similar height and two branches between 1.5 and 2 cm diameter per individual.
Wood specific gravity
Wood specific gravity was calculated by first measuring the fresh volume of wood samples by displacement of a water column (Williamson and Wiemann, 2010). Samples were immersed in a beaker containing water on top of a digital balance and sample volume was converted from the weight of displaced water (e.g., 1g = 1 cm3). Dry mass was obtained by drying the samples in an oven at 105 °C for 72 h. Wood specific gravity was then calculated as:
WSG = Dm/Dv (Eq 2)
where WSG = wood specific gravity (g.cm-3), Dm = dry mass, and Dv = displaced volume.
Theoretical hydraulic conductivity
Theoretical hydraulic conductivity (Kth) was calculated for each sampled individual from vessel lumen area using the Hagen-Poiseuille equation:
Kth = πD4/128η (Eq 3)
where Kh = theoretical hydraulic conductivity in kg.s-1.m-1.MPa-1, η = water viscosity at 20 °C (1.002 x 10-3 Pa.s) and D = hydraulically weighted vessel diameter in mm.
Because cross-sections of vessels are not perfect circles, vessel lumen area was used to calculate equivalent vessel diameter (d) (Scholz et al., 2013) as:
d = √4A.π (Eq 4)
where A = vessel lumen area.
Hydraulically weighted vessel diameter (D) was calculated as:
D = (Σd4/N)0.25 (Eq 5)
where d = equivalent vessel diameter in mm, and N = number of measured vessels.
Embolism resistance and hydraulic safety (P50 and HSM)
Branches of 1.5 to 2.0 m in length were collected at dawn for assessing embolism resistance and hydraulic safety (P50 and HSM). Ten to 15 cm long segments were cut from the base of each branch under water and were allowed to rehydrate for 12 h, keeping them covered and sealed by black plastic bags. Hydraulic measurements were then made on the distal end of each branch to ensure there were none artificially embolized vessels in the measured sample. All samples used for hydraulic measurements were from first or second-order branches that were 30–55 cm in length and 2–4 cm in diameter and were cut under water with a sharp blade before connecting to the apparatus to ensure that all vessels were open. We measure the water potential of the leaves after 12 hours of hydration, taken to represent the timepoint when transpiration is at its minimum and the water potential of the plant is closest to equilibrium with that of the soil. We also determined midday water potential (Ψmd), to capture the minimum Ψ of the plant in the dry season (Figure S5), leaf water potentials were measured using a pressure chamber (Model 1505, PMS). This measure is affected by any cuticular or stomatal transpiration and, thus, broadly captures the integrated effects of plant traits and the environment water demand on the minimum water potential a plant reaches in natural conditions.
P50 was used as an embolism resistance index, which is water potential corresponding to a 50% loss of xylem conductivity. P50 was used to calculate the hydraulic safety margin (HSM; i.e., the difference between P50 and Ψmd), which is a good predictor of drought resistance (Barros et al., 2019). Xylem embolism resistance of each branch was measured using the pneumatic method in the manual measurements set-up (Pereira et al., 2016; Zhang et al., 2018). With this method, loss of hydraulic conductance is estimated from the increase in air volume inside the wood caused by the formation of an embolism, as the branch dehydrates (for details of the methods see Bittencourt et al., 2018). Branches were dehydrated using the bench dehydration method (Sperry et al., 1988). The branches were bagged for an hour to balance the water potential of the xylem with that of the leaves before each air removal measurement. The volume of air reservoir was as adjusted when a rapid drop in air discharge or values close to atmospheric pressure were detected to preserve the accuracy of the method. The reservoir volume varied between 1.305 to 2.610 depending on the species and/or individuals the volume. Water potential of one or two leaves was measured immediately after air removal. Embolism resistance is then given by increasing air removal (PAD = the percentage of air removed) with each tree xylem water potential. To calculate P50, we gathered data for the repetitions of two branches of the same tree and adjusted a sigmoid curve to the data where P50 and slope are the adjusted parameters (Pammenter and Willigen, 1998).
𝑃𝐴𝐷 = 100 / (1 + exp (𝑎 (Ψ – 𝑃50). (Eq 5)
Characterization of P50 was done for the seven dominant species of the community. The pneumatic method was applied to the branches to construct vulnerability curves for the xylem. We were not able to produce reliable vulnerability curves to Macareia radula and Alchornea discolor using the pneumatic method most likely due to an undetected leakage during the measurements in the field. After inspecting the curves, we observed large amount of variation in the initial and final measurements, and consequently, poor fit of the curves. Both species have very tick bark and fast shrinkage requiring fitting adjustments during the measurements. We believe that this may have caused air leakage and for this reason we were not confident to use the data collected for both species for the analysis. The P50 and HSM are presented for the remaining five dominant species: N. guianensis, S. versicolor, P. cachimboensis, K. rubriflora and M. guianensis (Figure S6).
Statistical analysis
To determine morphological, anatomical, and ecophysiological variables related to transpiration, water transport, and xylem vulnerability, we made pairwise tests of the relationships between attributes due to the high degree of correlation. Only attributes directly related to our hypotheses were considered for pairwise relationships.
The Shapiro-Wilk normality test was used to test the assumptions of regression and choose methods for data analysis. A correlation matrix was constructed to observe associations among the variables of this study. Correlations were performed using the non-parametric statistics to calculate Spearman's Coefficient (rho) as data and variables did not follow a normal distribution even after transformation. A graph was constructed from the correlation matrix to show the observed relationships within an intuitive model of expected relationships. The model was proposed based on leaf and wood anatomical and hydraulic attributes to explain correlated mechanisms between leaf transpiration, water transport mechanism, and resistance to embolism.
Pairwise regression models were adjusted for water use efficiency, xylem embolism vulnerability, hydraulic safety margin, and anatomical variables to evaluate the anatomical basis for observed variation in ecophysiological attributes. Adjusted coefficients of determination (R2), 95% confidence intervals and p-values are reported. The adjusted regression models were corrected through a multiple comparison test (Bonferroni test). The data were transformed when necessary to meet the assumptions of regression (linearity, homoscedasticity, normality, and low leverage).
The data were scaled to standard deviation units to perform a principal component analysis (PCA), to ordinate possible plant strategies and select the variables that contribute most to species clustering. Descriptive statistical analysis, correlation matrix, regression models, and principal component analysis were performed using R© software (R Core Team, 2019).