Introduction 

Quercus pyrenaica Willd. is a vigorous root-resprouting species that has been intensively coppiced for firewood, charcoal and woody pastures for centuries ([20]). Due to the appearance of new energy sources and rural exodus that occurred in the 1970s, coppicing has mostly ceased, and symptoms of decline - slow stem growth, branch dieback, and scarce acorn production - are widely observed in abandoned stands ([26]). Coppice stagnation entails ecologic, economic and social problems, namely increased fire risk, stand over-aging, low productivity, absence of sexual regeneration, and consequently, hindered application of alternative management plans. Due to the wide distribution and significant ecological value of coppiced stands of Mediterranean oak species, silviculture faces the crucial challenge of finding new alternative uses for these abandoned coppices ([6], [4]). Attempts to conversion into high forests via thinning have not been successful to date, partly due to the lack of a comprehensive understanding of the physiological mechanisms underpinning tree stagnation ([25]).

Disproportionate respiratory costs of large root systems grown after centennial coppicing have been suggested as a potential driver of Q. pyrenaica decay ([4]), but assessments on carbon expenditures have not been essayed to date. Quantification of the relative weight of respiratory carbon sinks for the plant is crucial for a better understanding of tree carbon budgets ([32], [1], [17]). Notwithstanding, our comprehension of respiratory processes, particularly of woody organs, is limited compared to our knowledge of photosynthesis ([10], [17], [13]). In resprouting deciduous species, nonstructural carbohydrates are stored in large amounts in woody organs ([3]) that can contain a large portion of living parenchyma ([19]). The penalty in terms of respiratory carbon loss associated to the maintenance of these storage tissues ([14], [9]) could be of particular relevance in carbon budgets of root-resprouting Q. pyrenaica. Long lasting coppicing might lead to massive systems of living roots ([21], [31]) that store but also consume a large portion of carbohydrates assimilated aboveground.

To better understand the role of respiratory carbon loss in Q. pyrenaica decay, we gathered and extended previous published work on Q. pyrenaica biomass and respiration of woody tissues ([22], [21], [23], [24]) to scale up stem and root respiration (R_S and R_R, respectively) to the tree and stand levels. We aimed at comparing respiratory expenditures (i) between above- and below-ground woody organs across one growing season, and (ii) in relation to data gathered from various forests to provide an insight of the magnitude of carbon invested for woody tissue respiration in Q. pyrenaica coppices. We expect R_S and particularly R_R to be important carbon sinks for the plant (i.e., from the plant perspective), and therefore high R_R/R_S ratios as well as woody tissue respiration rates relative to other forest stands.

  Materials and methods 

To estimate respiratory carbon loss of woody organs in a coppice system of Q. pyrenaica, we reviewed our previous work on Q. pyrenaica root development and biomass ([21]), and internal and external stem CO₂ fluxes ([22], [23], [24]), together with unpublished data of soil CO₂ efflux. These studies were performed in a one-hectare experimental plot located in the Monte Matas de Valsaín (Segovia, Spain) at an altitude of 1140 m a.s.l. Climate is sub-Mediterranean with an average annual rainfall and temperature of 885 mm and 10 °C, respectively. It consists on a monospecific one-storied regular coppice of Q. pyrenaica with a stand density of 781 stems ha^-1. The forest has been subjected to coppicing since at least the XII century, and traditional management was abandoned in the 1970s. Stems within the plot were geo-referenced and leaves collected for genetic analyses to delineate the commonly inconspicuous clonal structure of Q. pyrenaica coppiced stands. Note that Q. pyrenaica is a root resprouting species, hence one clone can be constituted by several stems located far away (dozens of meters) from each other ([30]). The clonal assignment of stems was based on nuclear microsatellite molecular markers ([29] - see Fig. 1 in [22]). Data on woody tissue respiration was collected from the eight stems belonging to a single clonal genotype (clone). Four 24-h measurement campaigns were conducted across the growing season of 2013, at the end of which stems and roots were harvested for biomass quantification and scaling up of R_S and R_R to the clone and stand levels. The root system was hydraulically excavated with a high-pressure water pump down to 1 m depth over an area of 81 m² (Fig. 1). Biomass was partitioned into leaves, branches, stems, taproots, coarse roots and fine roots. Woody biomass was further partitioned into bark, sapwood and heartwood tissues from allometric equations adjusted by means of exhaustive sampling of branches, stems, taproots and coarse roots. Leaf area index (LAI) was estimated from measurements of specific leaf area of sampled leaves and total leaf biomass. Further details on stand characteristics, excavation methodology, and above- and below- ground biomass measurements can be seen in Salomón et al. ([21]).

Fig. 1 - Root systems of two hydraulically excavated clones of Quercus pyrenaica located in the Monte Matas de Valsaín (Segovia, Spain). The root system of the large clone surveyed in this study (lower part of the photograph) covered at least 81 m². Before harvesting, the clone was composed by eight stems connected through root grafts and parental roots.

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Stem CO₂ efflux to the atmosphere (E_A) was measured in every stem with a portable infrared gas analyzer (LI-6400^®, Li-Cor Inc., Lincoln, NE, USA) and a soil chamber (LI-6400-09^®) using PVC collars attached to the stems. Stem E_A measured on a surface area basis (E_A(S)) was expressed on a volume basis (E_A(V) - eqn. 1):

where S and V are the axial surface area and the volume of living tissues (bark and sapwood) of the stem segment, respectively; r_h and r_h+l denote the radius of heartwood and heartwood plus living tissues, respectively, and L is the vertical length of the stem segment ([24]). Stem respiration (R_S) was estimated as the sum of E_A(V) and the internal CO₂ flux through xylem (F_T) as R_S = E_A + F_T (adapted from [16]). F_T was calculated as a function of the sap flux and the vertical gradient of CO₂ dissolved in sap solution (sap [CO₂^*]). Sap flux density was measured using Granier-type thermal dissipation probes, and sap [CO₂^*] was estimated from measurements of xylem [CO₂] in the gas phase, sap temperature, and sap pH in each stem applying Henry’s law. Briefly, xylem [CO₂] was measured with solid non-dispersive infrared (NDIR) CO₂ sensors (model GMM221^®, Vaisala, Helsinki, Finland) inserted into the stem above and below the stem collar. Stem temperature was measured with type-T thermocouples inserted 5 cm away from the NDIR probe. Sap pH was measured with a micro-pH electrode and a portable pH meter (Crison, Barcelona, Spain) on sap samples expressed from detached twigs using a pressure chamber (see [23] for further details). Overall E_A, F_T and R_S at the clone level were estimated by aggregating E_A, F_T and R_S scaled up for each stem (and their branches) according to their corresponding volume of living woody biomass. Aboveground clonal respiratory fluxes were eventually expressed on a soil surface area basis taking into account the clonal surface extension.

Soil CO₂ efflux (E_S) was measured with a portable infrared gas analyzer and a soil chamber using soil PVC collars (see [22] for detailed methodology). Unpublished data from four soil collars located below the canopy of the eight monitored stems were averaged to obtain clonal E_S on a soil area basis (m²). Since roots barely extended beyond the excavated area (81 m²), a buffer of 0.63 m was added to estimate the clone extension (102 m²). This buffer distance was chosen to meet actual stand density (8 stems in 102 m² = 784 stems ha^-1) and scale up results to the stand level. Root-respired CO₂ diffusing to the atmosphere through soil (E_S-ROOT) was estimated from E_S measurements and the relative contribution of autotrophic respiration to E_S. Seasonality of root autotrophic contribution to E_S was obtained from two studies in a Mediterranean Quercus cerris coppice cut one ([18]) and 17 ([27]) years before E_S measurements. Spring contributions reported in these studies were attributed to the first measurement campaign (DOY 143-144), summer contributions to the second (DOY 183-184) and third (DOY 218-219) campaigns, and autumn contributions to the fourth campaign (DOY 266-267). To account for F_T on R_R estimates (R_R = E_S-ROOT + F_T - [2]), internal and external CO₂ fluxes were measured at the base of the stem (0.1 m).

To compare E_S, R_R and R_S averaged over the growing season within the monitored clone, ANOVA and pairwise comparisons were performed in R software (version 3.4.0). Respiratory fluxes at the stand level from 15 sites were gathered (Tab. S1 in Supplementary material) to evaluate the relative magnitude of respiratory costs of the surveyed Q. pyrenaica coppice. Inter-site statistical comparisons were not performed because only one site was surveyed in this study.

  Results and discussion 

Seasonal and diel variation in E_A, F_T and E_S on a soil area basis are shown in Fig. 2. The contribution of F_T to aboveground R_S was less than 10% ([24]), whereas F_T belowground was less than 2% of E_S-ROOT ([22]). The modest contribution of axial CO₂ transport to total respiration rates is explained by the low xylem [CO₂] observed in Q. pyrenaica, generally lower than 1%. This concentration is about one order of magnitude lower than that reported for other tree species using this methodology ([28]). The limited build-up of CO₂ in the xylem was partly ascribed to the low resistance to radial CO₂ diffusion, likely related to the poor plant water status of species distributed across drought-prone regions ([23]). Averaged over the growing season, E_S (38.9 mol CO₂ clone^-1 day^-1) was greater than R_S (11.7 mol CO₂ clone^-1 day^-1) and R_R (8.5 mol CO₂ clone^-1 day^-1 - P < 0.001; Tab. 1, Fig. 2), being R_S and R_R not significantly different (P > 0.10). Due to the magnitude of E_S, the contribution of autotrophic respiration to E_S substantially determined the root-to-shoot ratio of respiration (R_R/R_S). To illustrate this, a contribution of R_R to E_S ranging between 14 and 27% ([18], [27] - data reported for a Mediterranean oak coppice at different states of maturity) yielded R_R/R_S ratios ranging between 0.42 and 1.18 across the growing season (Tab. 1). Alternatively, if heterotrophic and autotrophic contributions to E_S were considered equal, as generally assumed for different forest biomes ([11], [2]), R_R/R_S would reach values ranging from 1.36 to 2.18.

Fig. 2 - Diel variations in stem CO₂ efflux to the atmosphere (E_A), stem internal CO₂ transport through xylem (F_T) and soil CO₂ efflux (E_S) on four dates over the growing season in an abandoned coppice of Quercus pyrenaica. Fluxes registered from one stem segment and one soil collar intensively monitored (18 times day^-1) are shown. Additional stem segments and collars used to average E_A, F_T and E_S are not displayed in this figure as they were monitored less intensively (4 times day^-1). E_A and F_T on a volume basis was obtained from previous work ([24]) and expressed on a soil area basis for comparison with E_S. Shaded areas indicate night-time.

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Above- and below-ground functional woody biomass (bark and sapwood) was similar in the surveyed clone: 1026 Kg aboveground and 972 Kg belowground ([21]). Consequently, seasonal deviations of R_R/R_S from unity reflected differences in the metabolic activity between below- and above-ground organs over time. R_R/R_S ratio above one was uniquely observed during spring (Tab. 1), likely explained by an earlier growth of roots relative to stems ([15]), particularly by intense fine root growth and belowground cambial activity at this time of the year ([8]). The decrease in R_R/R_S observed onward (ratios below one) resulted from the moderate root activity relative to the intensification of aboveground metabolism, namely stem growth, leaf development and phloem transport. Predominant R_R/R_S ratios below one along the growing season evidenced an unexpected large weight of aboveground woody tissue respiration as a carbon sink for the plant. The accumulation of woody biomass in stems and branches in this over-aged coppice (cut for the last time around 1967), together with the remarkably high portion of living parenchyma observed in Q. pyrenaica stems ([19]) may contribute to the high respiratory costs of woody organs aboveground.

Respiratory fluxes at the stand level, and extrapolated to the whole year, were compared with those reported for several forest sites (see Tab. s1 in Supplementary material for details on the extrapolation). Average E_S and E_A across 15 stands were 776 and 162 g C m^-2 year^-1, respectively, whereas these values increased up to 1164.2 and 297.0 g C m^-2 year^-1 in the surveyed Q. pyrenaica coppice (Tab. S1). That is, E_S and E_A were 1.5 and 1.8 times higher in this study, suggesting greater carbon losses from soil and stem respiration. These ratios could be substantially reduced, however, if xylem transport of respired CO₂ was accounted for ([2]) in the 15 forest stands. Leaf area index, a key ecophysiological determinant of carbon gas exchange at the canopy ([5]), was additionally considered for comparison. LAI in our site was 3.8, a value that falls within the reported range for the species ([12]) and the genus Quercus ([5]). The slightly low LAI relative to that averaged across different forest types (4.2 - Tab. S1 in Supplementary material) does not suggest a greater potential for carbon assimilation. Taken together, these rough comparisons suggest a strong carbon sink for the plant associated to respiratory processes that do not scale with the carbon input within the surveyed Q. pyrenaica coppice.

To summarize, the relative importance of R_R and R_S as carbon sinks for the plant shifted along the growing season in accordance to the root and stem phenology and metabolic activity. R_R/R_S ratios lower than one point to an unexpected importance of aboveground woody tissue respiration in carbon budgets of Q. pyrenaica coppices. Large carbon losses via soil and stem respiration relative to those observed in several forest types supports the hypothesis of an imbalance between carbon sources and sinks, contributing to the decline of abandoned coppices ([7], [22], [21]). Nonetheless, these conclusions should be handled with caution due to the lack of data on carbon assimilation, and the limited sample size of the study, largely constrained by the laborious task of root excavation. Firstly, temporal patterns of leaf carbon exchange should be analyzed in detail to gain a comprehensive perspective of tree carbon budgets and to better address the role of a potential root-to-shoot physiological imbalance in Q. pyrenaica coppice decline. Secondly, a greater number of monitored individuals as well as soil collars within the plot and across longer periods would improve spatial and temporal upscaling of respiratory fluxes. In this line, comparisons between coppiced and non-coppiced sites would provide stronger empirical support to the hypothesis that historical coppicing leads to massive root development and constrained aboveground performance.

  List of abbreviations 

E_A, stem CO₂ efflux to the atmosphere; E_A-LEAF, foliage CO₂ efflux to the atmosphere; E_S, soil CO₂ efflux; E_S-ROOT, root-respired CO₂ that diffused to the atmosphere through soil; F_T, internal CO₂ flux through xylem; R_R, root respiration; R_S, stem respiration

  Acknowledgements 

We are grateful to Javier Donés (Centro de Montes y Aserradero de Valsaín) for economic and logistical support. We also thank Elena Zafra, Guillermo González, César Otero, Manuel Iglesias, Paula Guzmán, Aida Rodríguez, Jose Carlos Miranda, Rosa Ana López, Eva Miranda and Matías Millerón for their enthusiastic help with field work. This work was funded by the Comunidad de Madrid through CAM P2009/AMB-1668 and S2013/MAE-2760 projects and by the Organismo Autónomo de Parques Nacionales through the PPNN 1148/2014 project. Roberto L. Salomón was supported by a Ph.D. scholarship from the Universidad Politécnica de Madrid.

  References

  Supplementary Material

Tab. S1 - Stand density, leaf area index (LAI) and partitioning of annual ecosystem respiration into soil (E_S), stem (E_A) and leaf (E_A-LEAF) CO₂ efflux to the atmosphere across different forest stands.