2.1 Methods for evaluating ARQ

We report tree stem ARQ results based on measurement methods described in Hilman and Angert (2016). These methods overcome the difficulty of measuring small changes in O₂ against the high atmospheric background by using a static stem chamber, in which the O₂ changes are considerably larger than in an open flow chamber.

We used three different approaches to measuring ARQ: two are based on discrete gas samples of headspace air, and one is based on direct measurement of the headspace air using gas sensors (“continuous” sampling). Discrete gas samples are either taken within 30 min to several hours after chamber sealing (“instantaneous” sampling) or after the chamber has been sealed to the stem for more than 24 h, once steady-state conditions have been achieved (“steady state”). These methods and the time required for achieving steady state were confirmed by comparing with continuous measurements (Hilman and Angert, 2016). For each site and experiment described in Sect. 2.2, we identify the method used to estimate ARQ as instantaneous, steady state (for flask samples), or continuous (Table 1).

Table 1Study sites, tree species sampled at each site, stem chambers, stems diameters, and experiments done in the site (A–G list in Sect. 2.2).

^a All chambers were installed at ∼1.3 m above the ground, except for the Q. calliprinos on Carmel Ridge that were placed near to the ground due to the shrubby canopy, the low branching of the trunk, and the constraint of the size of the chamber. ^b Chambers were made of a 10 cm × 12 cm Perspex^® plate with four connectors to allow attachment of sampling flasks. The chamber on the M. domestica was slightly larger, 12 cm × 19 cm, with six flasks connectors. Chambers were placed on top of a closed cell foam frame that allowed an air-tight seal between the rigid chamber and the uneven surface of the tree stem. We used nylon straps to compress the foam, while the sealant was applied between the foam and stem for ensuring the seal (Hilman and Angert, 2016). Sealants were silicone-based vacuum grease (Silicaid^® 1010 manufactured by Aidchim Ltd., Raanana, Israel) or hot glue applied by a hot-glue gun. ^c The chambers are described in Muhr et al. (2013) and Carbone et al. (2013). Briefly, the chambers were made from an opaque plastic polypropylene pipe T fitting with fittings for sampling flasks. Sealants were caulking (Nautiflex; OASE GmbH, Oerel-Barchel, Germany) or hot glue applied by a hot-glue gun. ^d Chambers were built from a 15 cm long piece of polypropylene (PP) tubing (6.5 cm OD) that was welded shut on both sides with a PP disc (6.7 cm diameter). By cutting off a segment (height 2 cm) the tube was turned into an incubation chamber. Opposite the chamber opening, three fittings (Sprint ESKV 20, Wiska, Germany) were installed and sealed around the edges with liquid rubber (Dichtfix, Bindulin, Fürth, Germany). For sampling, chambers were attached to the trees with four lashing straps. To achieve a gas-tight seal, a frame (25 mm thick) made from closed-porous cellular rubber (EPDM quality, REIFF Technische Produkte GmbH, Reutlingen, Germany) was placed between the chamber and the stem.

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2.1.1 ARQ measurement from discrete samples

The evaluation of ARQ from discrete gas measurements is based on a one-box model that describes gas dynamics in the headspace of a static chamber sealed to the surface of a tree stem (Angert and Sherer, 2011; Angert et al., 2012; Hilman and Angert, 2016). In the model, the gas in the chamber headspace has initial mean atmospheric values (20.95 % O₂, 0.04 % CO₂), ensured by flushing the chamber with ambient air before measurement. Once the chamber is closed and the headspace above the stem surface is isolated, metabolic reactions in the stem control the chamber's air composition. For the first few hours, headspace concentrations of CO₂ increase and O₂ decrease at rates that are roughly linear with time (instantaneous incubation, Figs. 1 and S1 in the Supplement). During this linear stage, ARQ is calculated by

$$\begin{array}{}\text{(1)}& \mathrm{ARQ}={\displaystyle \frac{{\mathrm{CO}}_{\mathrm{2}}\mathrm{efflux}}{{\mathrm{O}}_{\mathrm{2}}\mathrm{influx}}}={\displaystyle \frac{\mathrm{\Delta }{\mathrm{CO}}_{\mathrm{2}}}{\mathrm{\Delta }{\mathrm{O}}_{\mathrm{2}}}},\end{array}$$

where ΔCO₂ and ΔO₂ are the changes in [CO₂] and [O₂] during the initial period after the chamber was sealed, and for discrete samples can also be determined from the difference in concentrations between the chamber air sampled at a specific time and the initial atmosphere. Instantaneous fluxes of CO₂ and O₂ reported here are obtained either by monitoring concentration change during the first hour following chamber closure with sensors directly in the field or by sampling headspace air with glass flasks within 30 min to a few hours of closing the chamber. The flasks were transported to the laboratory for measurement of CO₂ and O₂.

Figure 1Modeled changes in a tree stem chamber of the concentrations of CO₂, O₂, and the ratio between ΔCO₂ and ΔO₂, which are the changes in the gas concentrations from their initial values and are also the difference in concentrations between the chamber and the atmosphere. The gas dynamics are based on a one-box model with arbitrary fluxes and ARQ (ratio of CO₂ efflux∕O₂ influx for tree stems) equal to 0.5. The two time frames in which ARQ can be measured from the ratio ΔCO₂∕ΔO₂ are indicated in the figure.

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After the first hours, the initially linear rates of change in headspace gas concentration with time decline and concentrations eventually remain constant (Figs. 1, S1). In this phase the gases in the chamber and the outer part of the stem, where most of the metabolism takes place, are assumed to be in equilibrium. This steady state occurs when the rates of addition of CO₂ and loss of O₂ from the stem to the chamber headspace are balanced by diffusive (assuming no strong wind) exchange of headspace air with outside air through porous portions of the outer stem. For steady-state samples, the chamber is sealed to the surface of the stem and left for a period longer than 24 h, after which the headspace air is sampled using glass flasks. The CO₂ and O₂ concentrations must be corrected for differences in diffusivity between CO₂ and O₂, as detailed in Angert and Sherer (2011), Angert et al. (2012), and Hilman and Angert (2016) in order to estimate the ratio of the gas fluxes from the concentrations in the static chamber:

$$\begin{array}{}\text{(2)}& \mathrm{ARQ}={\displaystyle \frac{{\mathrm{gCO}}_{\mathrm{2}}\times \mathrm{\Delta }{\mathrm{CO}}_{\mathrm{2}}}{{\mathrm{gO}}_{\mathrm{2}}\times \mathrm{\Delta }{\mathrm{O}}_{\mathrm{2}}}},\end{array}$$

where gCO₂ and gO₂ are the CO₂ and O₂ conductance values in the outer layer of the stem between the chamber and the atmosphere. The structure of the path along which diffusion occurs is the same for CO₂ and O₂ and hence the conductance ratio gCO₂∕gO₂ depends solely on the ratio of diffusivities of the gases in air, which is 0.76 (Massman, 1998). As a result, at steady state

$$\begin{array}{}\text{(3)}& \mathrm{ARQ}=\mathrm{0.76}\times {\displaystyle \frac{\mathrm{\Delta }{\mathrm{CO}}_{\mathrm{2}}}{\mathrm{\Delta }{\mathrm{O}}_{\mathrm{2}}}}.\end{array}$$

Assuming constant CO₂∕O₂ fluxes over time, samples taken either by instantaneous or steady-state methods will yield the same ARQ values. Indeed, Hilman and Angert (2016) demonstrated excellent agreement for direct comparisons of the instantaneous and steady-state measurement methods, and the results are further compared here.

2.2 Study sites and experimental design

For addressing the variation in stem ARQ values across a range of tree species and environments, our study included trees located in tropical forests (Panama and Brazil), in temperate forests (Bartlett and Harvard, USA), and in a Mediterranean savanna (Spain) and a Mediterranean shrubland (Carmel Ridge, Israel). We also included five trees located on the Hebrew University campus in Jerusalem (Israel) and in the adjacent Jerusalem Botanical Gardens. The trees in Panama were part of a fertilization and litter manipulation project (Wright et al., 2011; Sayer and Tanner, 2010). No treatment effects were found (Fig. S2, this topic is not in the scope of this paper). Details about the sites, tree species, stem chambers, stem dimensions, and experiments conducted in each of the sites are presented in Table 1 and Fig. 3. The list below summarizes what data were available from the different sites and what questions in particular we addressed with these data (the numbering of the experiments matches Table 1):

• A.

  Seasonal and/or phenological measurements of stem ARQ were performed in Jerusalem, US, and Brazil sites. In Jerusalem, five individual trees from five different species (first five species in Table 1) were measured. The phenological state of deciduous trees (all except Quercus calliprinos) was separated into four classes (Fig. 4). In the US sites, trees measured at the northern site (Bartlett Experimental Forest) had fall color development, while leaves at Harvard Forest (southern site) were still green. After analysis of flasks, we excluded results from three trees because of suspected air leakage from the chamber (O₂>20 % after 6 days of stem incubation). In Brazil, six trees were measured. After analysis we excluded results from 4 out of 12 instantaneous measurements because of a weak signal (O₂>20.7 % and SD>0.1 after 3 h of incubation).

For our second objective, to explore the potential for low ARQ values to reflect dissolution and transport of CO₂ in the xylem sap, we measured instantaneous ARQ at varying sap flow velocities and at different times of a day. Transport of CO₂ was previously reported to be correlated with sap flow (McGuire and Teskey, 2004; Bowman et al., 2005; McGuire et al., 2007). Thus, anti-correlation of ARQ with sap flux, expressed via maximal ARQ values during the night when transport is at a minimum, would provide evidence that low ARQ can be explained by export of locally respired CO₂ (as DIC) out of the stem region being measured (experiments B, C, and E, below). If transport of dissolved CO₂ is the main driver of low ARQ values, we would also expect that (D) higher ARQ values will be observed at higher temperatures (due to differential temperature dependences of CO₂∕O₂ solubility coefficients), (F) ARQ values will increase with stem height due to DIC accumulation and stem tapering that induce stronger CO₂ diffusive loss, (F) ARQ values will decrease with depth in the stem (due to the greater proximity to the water conducting vessel elements), and (G) ARQ values in incubated stem cores will be higher than measured values at the stem surface (due to the detachment from the transport system).We performed a number of experiments to test each of these predictions (additional details in Table 1):

• B.

  ARQ (instantaneous) was measured simultaneously with sap flux density measurements in nine Quercus ilex trees with similar diameter (0.35 to 0.49 m at breast height) at the site in Spain.

• C.

  ARQ (instantaneous) was measured during daytime, at predawn when the transpiration stream should reach its minimum, and again during the next day. We conducted two day–night campaigns on the trees at the site in Jerusalem. Additionally, during 4 days, ARQ (continuous) values were measured every 4 h from the M. domestica tree in Jerusalem.

• D.

  Nighttime results of the continuous ARQ measurements on the M. domestica enabled us to examine the relationship between temperature and ARQ. During the night, when sap flux is minimal, the temperature effect on the gases' solubility should have its maximum effect on ARQ values.

• E.

  ARQ (steady state) was measured over spring, summer, and winter for Quercus calliprinos trees on the Carmel Ridge site, simultaneously with predawn shoot water potential (Ψ_pd). Ψ_pd is a measure for available soil water and therefore is also a rough proxy for seasonal differences in transpiration rates (Aranda et al., 2005; Bucci et al., 2005).

• F.

  ARQ was measured at different heights on the same tree stems, while simultaneously ARQ was determined from air sampled inside the stem. During the seasonal measurements in Jerusalem, ARQ (steady state) was measured at the stem base of the Q. calliprinos and the Platanus occidentalis trees as well as at breast height. In Brazil, we measured ARQ (instantaneous) from stem chambers and in-stem probes to sample in-stem gases from the tree base up to 11 m above the ground on a single Scleronema micranthum tree on two separate days.

• G.

  ARQ (steady state) measured from stem chambers was compared with ARQ measurements through incubation of stem cores. Measurement of stem tissues should provide better estimation for the stem outer layers' RQ by excluding dissolution and advection in the xylem stream. Incubations were performed on cores taken from four species in four different sites (Table 1). In Jerusalem, we compared repeated stem incubation ARQ with that of leaf incubation.

2.3 Sap flux density

Sap flux density was monitored in nine trees at the site in Spain using heat ratio method (HRM) sensors (SFM1 Sap Flow Meter, ICT International). A description of the installation and measurement is presented in Methods S1. The detailed procedures for sap flux corrections and calculations are described in Perez-Priego et al. (2017). We tested whether the daily maximum sap flux density (i.e., average of measurements between 10:00 and 17:00 local time during the day of the ARQ measurement), which correlated with CO₂ dissolution fluxes (Bowman et al., 2005), could explain variability in ARQ (instantaneous).

2.4 Shoot water potential

Predawn shoot water potential (Ψ_pd) on Carmel Ridge was measured using a pressure chamber (PMS Instrument Company, Corvallis, Oregon, USA). At each sampling time, we sampled 2–3 terminal twigs containing 5–10 leaves from each tree. The samples were wrapped in plastic, placed on ice, and measured within an hour of sampling using the pressure chamber technique (Scholander et al., 1965).

2.5 In-stem measurements

For sampling gas from inside the stem, stainless-steel tubes (1.3 cm diameter) were installed 4, 8, and 12 cm deep into the stem, in various stem heights on the same tree in Brazil where the vertical ARQ transects were measured. Installation procedure was according to Muhr et al. (2013) and tubes were sealed between sampling dates. Using rubber tubing we connected the sampling flasks to the tubes for incubation of 4 days. The flasks were then analyzed for CO₂ and O₂ in the Hampadah. Assuming steady state, ARQ was calculated using Eq. (3) (Angert et al., 2012).

2.6 Measuring ARQ of incubated tissues

Stem cores were extracted immediately after the chamber incubation experiment in Panama, Spain, and Jerusalem using a 1.2 cm diameter cork borer. The outer bark and green tissues, as well as sapwood sieves (with paler color than the phloem tissues), were removed from the cores. The cores were cut into longitudinal halves, wrapped with moist gauze cloth to avoid desiccation, and placed in an air-tight incubation system to which glass flasks (two or three) were connected by Swagelok Ultra-Torr fittings (Swagelok, Solon, OH, USA, Fig. S4). At the end of the incubation period, the flasks were closed and analyzed in the Hampadah. Since the incubations took place in a closed system (no diffusive exchange with outside air), the changes with time in [CO₂] and [O₂] are assumed to be linear, and ARQ can be calculated using Eq. (1).

In Panama and Spain the incubations were started immediately upon core extraction, at ambient temperature, and lasted 8 and 3 h, respectively. In Jerusalem the cores were kept on moist gauze cloth for 2 h before being sealed in the incubation system and kept at 25 ^∘C in an environmental chamber. Repeated incubations were performed in series, with the incubation systems flushed in between with ambient air. Simultaneously, from each tree, four leaves from an understory branch were cut and inserted into the same incubation systems, for the same incubation durations. The O₂ uptake rate (nmol O₂ g FW⁻¹ s⁻¹) was calculated as follows (adopted from Pruyn et al., 2002):

where ΔO₂ is the decrease in [O₂] during the incubation, V_H is the volume of headspace (mL), T is the incubation period (s), M_FW is the fresh weight (g), V_m is the molar volume, and 10⁹ converts units to nmol. We dried the samples in an oven at 60 ^∘C for 2 days for the dry weight.

In Brazil, stem cores were extracted by using a 5.15 mm diameter increment corer. After bark was removed the cores were cut to a length of 6 cm each and then allowed to equilibrate with the atmosphere for 6–8 h, while continually being kept moist. After equilibration, each core was transferred to an incubation chamber equipped with flasks. Prior to starting the incubation, a few milliliters of water were added to keep the core tissue moist. In this case, incubations were left at room temperature (∼25 ^∘C) for 24 h before flasks were closed and removed.

2.7 Statistical analysis

All statistical analysis was done using JMP (JMP^®, JMP Pro 13, SAS Institute Inc., Cary, NC, USA). Repeated-measures analysis of variance (ANOVA) was used to evaluate how the interaction of tissue (stem core and leaves) with ARQ and O₂ uptake varies with time in the repeated incubations of the tissues from the trees in Jerusalem. Mauchly's test indicated violation of sphericity in the ARQ response in the repeated incubations experiment (χ²=18.132, P=0.021); therefore, the Greenhouse–Geisser adjusted F test was chosen. One-way ANOVA followed by Tukey–Kramer HSD (honestly significant difference) was used to perform comparisons among time points in every tissue. A Student's t test was used for comparisons between stem cores and leaves at each time point.

3.1 ARQ values under varying xylem stream flow and temperature

ARQ (instantaneous) values of nine Q. ilex trees in Spain were invariable (mean ± SD of 0.42±0.04) in comparison with the larger variation in maximum daily sap flux density among these trees (0.15±0.05 m³ H₂O m⁻² h⁻¹), and no correlation was found between the ARQ and sap flux density (r²=0, P=0.9891).

Mean ARQ ± SD values (steady state) of the trees at the Carmel Ridge site were 0.62±0.06, 0.68±0.07, and 0.69±0.08 for spring, summer, and winter, respectively. Repeated-measures analysis of variance found no significant difference between seasons ($F_{\mathrm{2},\mathrm{2}}=\mathrm{2.52}$, P=0.28), while Ψ_pd varied significantly with seasons ($F_{\mathrm{2},\mathrm{2}}=\mathrm{207.85}$, P=0.0048). During summer, Ψ_pd was −2.65 MPa, much lower than the spring and winter values (−0.64 and −0.86 MPa, respectively).

In the Jerusalem day–night campaigns, ARQ (instantaneous) values ranged between 0.52 and 1.05, across all trees, seasons, and sampling times (Fig. 5). Predawn ARQ values exceeding than daylight values (by amounts larger than the differences between duplicates) were observed during the summer in M. domestica and in the upper chamber on Q. calliprinos. No significant diurnal effect was found in repeated-measures analysis of variance of the breast height chambers, neither when results of all the trees were grouped by season nor when results were grouped by stem chamber. In continuous measurements of M. domestica, with ARQ values obtained every 4 h, ARQ during the night (0.70; n=12) was not significantly (P>0.76 in a Student's t test) greater than in the day (0.71; n=11; Fig. 6). The variations among the nighttime values were best explained using temperatures measured 235 min before the ARQ measurement (r²=0.84, P=0.0001, $\mathrm{ARQ}=\mathrm{0.01}\times \mathrm{temperature}\left({}^{\circ }\mathrm{C}\right)+\mathrm{0.54}$). With the same time lag, the coefficient of determination for the daytime values is r²=0.44 (P=0.0266).

Figure 5Instantaneous ARQ (ratio CO₂ efflux∕O₂ influx for tree stems ± SD of duplicates) values measured over a day–night–day transition during July 2012 (a) and April 2013 (b) from different trees growing on the Hebrew University campus in Jerusalem, Israel. Quercus calliprinos was measured at two different heights on the stem. The first sampling was taken during daylight (day 1), next sampling before dawn (predawn), and last sampling during daylight of the successive day (day 2).

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3.2 Stem surface and in-stem ARQ vertical transects

In Q. calliprinos, measured over 3 years in Jerusalem, ARQ did not differ significantly (P>0.33 in a Student's t test) between breast height and stem base (ARQ of 0.56 vs. 0.59, respectively, n=14, Fig. 4). For P. occidentalis measured for the same period the ARQ measured at breast height was significantly higher than ARQ measured at the stem base (0.74 vs. 0.64, respectively, n=12, P=0.003 in a Student's t test, Fig. 4). For a single S. micranthum tree in Brazil, ARQ values measured at heights of 6.5 and 11 m above the ground were similar to ARQ measured at breast height (Fig. 7), but also show differences with the stem base. In this tree, ARQ measured in March (0.46±0.11; wet season) was lower than in October (0.89±0.16; dry season). The in-stem ARQ values ranged between 0.25 and 0.56, with average ± SD of 0.46±0.07 in both seasons and at all stem positions and depths. The in-stem ARQ, as well as [CO₂] values, had no clear vertical trend (Figs. 7; S5).

Figure 6Diurnal patterns of (a) O₂ influx to the stem and CO₂ efflux from the stem, (b) chamber temperature, and (c) instantaneous ARQ (ratio CO₂ efflux∕O₂ influx for tree stems). Shaded areas indicate night periods. Error bars are 95 % confidence bounds. All data were obtained from a single M. domestica tree during 24–28 April 2013 on the Hebrew University campus in Jerusalem, Israel.

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Figure 7Instantaneous ARQ (ratio of CO₂ efflux∕O₂ influx for tree stems) measured from stem chambers installed at different heights above the ground on a S. micranthum tree in Brazil. At the same heights ARQ was measured from 4 cm in-stem probes. The measurements were conducted during 30 March and 18 October 2012. Error bars represents SD of duplicate samples from the same stem chamber.

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3.3 Tissue incubations

The average ARQ values of the stem core incubations were similar to the stem chamber ARQ for the four sites and trees where these comparisons were made (Fig. 8). When incubations were repeated over time for Q. ilex stem cores and leaves, significant effects of time, tissue (leaves, stem cores), and their interactions (time × tissue) on ARQ and O₂ uptake rates were observed. ARQ of the stem cores increased from 0.44±0.08 (mean ± SD, n=4) after 3 h to 0.94±0.03 at the end of the experiment (32 h; Fig. 9). The ARQ of incubated leaves of the same trees showed higher initial ARQ of 0.80±0.02, with an increase over time to 0.92±0.02.

Figure 8Comparisons of stem chamber steady-state ARQ (ratio CO₂ efflux∕O₂ influx for tree stems) to ARQ measured from incubations of stem cores (ratio CO₂ increase∕O₂ decrease), by species (n individuals) in different sites. Values are means ± SD.

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