Ground-penetrating Radar as Phenotyping Tool for Characterizing Intraspecific Variability in Root Traits of Pinus Halepensis

doi:10.21203/rs.3.rs-161358/v1

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Ground-penetrating Radar as Phenotyping Tool for Characterizing Intraspecific Variability in Root Traits of Pinus Halepensis

https://doi.org/10.21203/rs.3.rs-161358/v1

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Background and Aim

Drought is the main factor limiting Mediterranean forest ecosystem productivity. Root systems are responsible for water uptake but intraspecific variability in root morphology is poorly understood, mainly due to sampling complexity. The main aim of this study was to gain knowledge on the adaptive relevance of rooting traits for a widespread conifer using a non-invasive high-throughput technique.

Methods

Ground-Penetrating Radar (GPR) was used to characterize variability in coarse root features (frequency, depth, and diameter) among populations of the Mediterranean pine Pinus halepensis evaluated in a common garden. GPR records were analysed in relation to aboveground growth and also climate variables at origin of populations.

Results

Genotypic variability was detected for root traits among 56 range-wide populations categorized into 16 ecotypes. Root diameter of populations decreased eastward within the Mediterranean basin. Root frequency, but not depth and diameter, decreased following a northward gradient. Genotypic variation in root traits varied with climatic variables at origin such as summer to annual precipitation ratio, summer temperature and solar radiation. Particularly, root frequency increased with aridity, whereas root depth and diameter were maximum in ecotypes occupying the thermal midpoint of the species distribution range.

Conclusion

GPR is a high-throughput phenotyping tool that allows detection of intraspecific variation in root traits of Aleppo pine and its dependencies of eco-geographic characteristics at origin, thereby informing on the adaptive relevance of root systems for the species. It is also potentially suited for inferring population divergence in resource allocation above and belowground in forest genetic trials.

Plant Physiology and Morphology

Mediterranean trees

Common-garden experiment

Root morphology

Drought tolerance

Climate sensitivity

Intraspecific adaptation

Climate prediction models forecast an increase in temperature along with an intensification of extreme weather events that will likely lead to more prolonged and intense drought periods around the globe (IPCC 2007, 2014). This future climate may have serious impacts on ecosystem dynamics in the Mediterranean basin (Resco de Dios et el. 2007; Sardans and Peñuelas 2013). Seasonal drought is one of the main factors affecting Mediterranean forests, and forest tree species use different strategies to respond to changes in water availability. Among those, access to deep water pools is fundamental to survive dry periods (Dawson et al. 2020; Ripullone et al. 2020; Rossatto et al. 2012), because water uptake is dependent on root architecture (Hernández et al. 2010). A number of functional traits related to hydraulic conductivity such as root vessel diameter are determined by root xylem anatomy, which in turn is influenced by root diameter and rooting depth (Kirfel et al. 2017; Wang et al. 2015). Thus, root structure and morphology play a key role on the ability of plants to explore and access the deeper subsurface when shallower soil layers dry out (Andivia et al. 2019; Padilla and Pugnaire 2007). Species-specific and intraspecific differences in rooting patterns are still poorly characterized, however. This is mainly due to the inherent complexity of underground sampling and the fact that traditional approaches for root monitoring are destructive and non-repeatable.

The Ground-Penetrating Radar (GPR) is a non-destructive geophysical prospecting device that utilizes electromagnetic wave pulses for subsurface detection operating with dielectric properties (Lorenzo et al. 2010). GPR transmits electromagnetic signals into a surface and records the reflection of the signal transmitted by a receiving shielded antenna. It is widely used for many purposes, such as the detection of bedrock structure (Loudes et al. 2011; Valerio et al. 2012) or the quantification of soil water content (Klotzsche et al. 2018). Recently, it has been also used as non-invasive tool for the detection of root features and belowground biomass estimation (Barton et al. 2004; Butnor et al. 2001, 2003; Guo et al. 2013; Lorenzo et al. 2010). Roots have a different dielectric permittivity compared to soil and, as a result, GPR allows defining the number (frequency), the position (depth) and the size (diameter) of coarse roots through the timing and features of back-reflected signals induced by hyperbolic reflections on radargrams (Hirano et al. 2012, 2009; Wu et al. 2014). The reflected waves are detectable as hyperbolas, where the hyperbolic signatures’ peak (or the maximum amplitude of the hyperbola) corresponds to the center of every object detected, which is recorded on a portable control unit (Hirano et al. 2012). However, the successful identification of roots with GPR depends on different factors, with soil moisture, root water content and soil properties influencing wave frequency and thus root detection (Barton et al. 2004; Hirano et al. 2009).

Pinus halepensis Mill. (Aleppo pine) is the most widely distributed conifer species across the Mediterranean basin (Vennetier et al. 2018), occupying vastly different ecological niches ranging from mesic (annual precipitation exceeding 800 mm) to xeric environments (below 350 mm). The widespread distribution of Aleppo pine suggests the existence of intraspecific adaptive divergence. Indeed, there is strong evidence for genetic variability among populations originating from distinct environments to tolerate water stress, which involves traits such as water-use efficiency (Voltas et al. 2008), wood anatomy (Esteban et al. 2010), biomass allocation (Chambel et al. 2007), or use of water sources (Voltas et al. 2015). Deeper and more developed root systems might also be related to the ability of Aleppo pine to cope with water deficit (Andivia et al. 2019; Voltas et al. 2015). As a result, root morphology may be a key characteristic to understand water use strategies and, consequently, drought resistance mechanisms in this species. Although a large investment in roots is often related to enhanced survival under drought (Grossnickle et al. 2005; Matias et al. 2014), and Aleppo pine has a greater ability for colonizing the soil subsurface and produce a more efficient root system in dry environments than mountain pines (Andivia et al. 2019), the existence of intraspecific divergence in root traits for this Mediterranean pine has barely been investigated thus far (Voltas et al. 2015).

In this work, we applied GPR root detection to a common garden of adult Aleppo pine trees where populations representative of the circum-Mediterranean distribution of the species were tested. Our purpose was to evaluate if GPR is a valid tool to infer intraspecific variability for coarse root frequency, depth and diameter in a widespread tree species. We hypothesized that populations originating from xeric environments would present deeper, thicker and more numerous coarse roots than their mesic counterparts as a result of different adaptive responses to the use of soil water pools. In addition, we also hypothesized that variability in root traits among populations of the species is related, to some extent, to intraspecific divergence in aboveground growth, pointing to the existence of allocation trade-offs to aerial growth or belowground dry matter. Therefore, we aimed to: (1) test the use of GPR as a high-throughput root phenotypic technique to assess population differentiation in coarse root architecture for Aleppo pine; (2) relate intraspecific differences in coarse root traits (frequency, depth and diameter) among Aleppo pine populations to their climate at origin, which can be informative for understanding different adaptive strategies in the species; and (3) describe the associations between such root traits and aboveground growth and define potential allocation trade-offs at the intraspecific level.

Plant material and experimental site

Seeds from 56 populations of P. halepensis originating from mainland Spain, Balearic Islands, France, Greece, Italy and Tunisia were used in this study. These populations are representative of most of the current distribution range of the species (Fig. 1). The seeds were collected in 1995 from 20 to 30 adult individuals per population, spaced at least 100 m apart, and were sown in a forest nursery in Spain using standard practices the following year. In 1997, 896 one-year old seedlings (16 seedlings per population) were transplanted at the study site (provenance trial), which is located in Altura, Castellón province, Spain (39°49′29ʹʹN, 00°34′22ʹʹW, 640 m a.s.l.). The experimental design was a Latinised row-column design with four replicated blocks (John and Williams, 1998). The spacing between consecutive rows or columns was 2.5 m. Each row was ca. 70 m long, distributed in seven linear plots or experimental units consisting of four trees of the same provenance per plot. The total area of the provenance trial was about 0.8 ha. For this study, we used three of the four available blocks, as some individuals from the fourth block were affected by fire in 2012.

The soil at the study site is a calcic cambisol with loam texture (44.2% sand, 24.3% clay and 31.5% silt) and a maximum depth of ca. 40 cm followed by a petrocalcic horizon with vertical fractures. The general features of the soil are outlined in Table S1. The site has climatic characteristics similar to the average climate of the species across the Mediterranean basin (Santini et al. 2019a). The mean annual temperature is 13.8 °C and the mean annual precipitation is 468 mm, with 18% falling in summer.

Climate variables

For each population, climate data at origin for the period 1970-2000 were obtained from the WorldClim database (Fick and Hijmans, 2017) at 1 km² spatial resolution. The following climate

variables were retrieved, based on previous studies on the climatic drivers of ecotypic variation in Mediterranean pines (Climent et al. 2008; Tapias et al. 2004): mean annual temperature (MAT), mean summer temperature (MST), temperature annual range (TAR; T_max-T_min), mean annual precipitation (MAP) and summer to annual precipitation ratio (PsP). Moreover, solar radiation accounting for cloud cover (SR) was retrieved from WorldClim and monthly means of day-time vapour pressure deficit for the warmest (summer) quarter (VPD_s) were calculated for each population by subtracting the water vapour pressure (VP) from the monthly average of saturation water vapour (VP_sat) obtained at daily level. VP was estimated as in Ferrio and Voltas (2005):

where T_m is mean monthly temperature, P_m is monthly precipitation and Z is altitude (in m).

In turn, VP_sat was estimated from day-time temperature (T_day) as (Jones 1992):

The populations were further categorised into 16 ecotypes (Fig. 1, Table S2) following previously published approaches based on climate attributes (Climent et al. 2008).

Vertical growth data

Tree height (H) and diameter at breast height (DBH), measured in 2013 (at age 17 years), were used as aboveground growth traits. Additionally, tree crown area was estimated from aerial RGB imagery taken in summer 2016 (at age 20 years) through a canopy height model using the package ForestTools (Plowright 2018), as reported in Santini et al. (2020). We assumed constancy of genotypic ranking in tree growth from age 15 onwards, as previously reported for P. halepensis (Sbay and Zas, 2018).

GPR data collection

The Ground-Penetrating Radar (GPR) is a geophysical detection device that uses electromagnetic waves to identify subterranean anomalies. Partial signals are reflected by a change in dielectric properties through the subsurface medium, and data collected in real time can be manipulated to provide an estimation of the number (frequency), depth and diameter of coarse roots in the subsurface (Barton et al. 2004). GPR records were obtained in a single day in mid-June 2015, when trees were 19 years old, and the number, depth and diameter of coarse roots were estimated using a MALÅ RAMAC X3M GPR (MALÅ Geoscience AB, Sweden) equipped with an 800 MHz shielded antenna coupled to an inspection cartwheel. The 800 MHz antenna was used because it has been shown to provide the best possible resolution in calcic soils (Rodríguez-Robles et al. 2017), which are distinctive of the study site. In particular, roots reaching a depth of up to ca. 0.9 m could be identified within the petrocalcic fractures underneath the lower soil layer of 40 cm, and roots with a diameter of ca. 2 cm or higher were also detectable.

GPR measurements were taken continuously along three linear profiles (P1, P2, P3) following the trial’s column direction (Fig. 2a, b). The profiles were positioned between the two central trees of each experimental unit at a row distance of ca. 0.30 m from the main trunk of one (P1) or another tree (P3), and also halfway (1.25 m) between both trees (P2). In order to assign each detected root to a particular experimental unit, the position of trees along each linear profile was manually recorded in the RAMAC XV Monitor implemented in the GPR along with GPR measurements. With this information, relevant data could be retrieved at sub-plot level in the profile (see “GPR data processing” subsection).

GPR data processing

Coarse roots were identified through hyperbolic signatures on radargrams. GPR radargrams were examined, through RadExplorer v1.42 software (MALÅ Geoscience AB, Sweden), following visual inspection of the hyperbolae (“manual dataset”), which allowed to determine trends in the linear distribution of roots along the linear profiles.

Based on this preliminary information, a second dataset was created in RadExplorer using a built-in detection procedure along predefined sub-plots (“automatic dataset”), which was subjected to a noise removal process in order to correct for both object position (stolt F-K migration function) and parallel or low frequency bands (background removal filter function) (see Rodríguez-Robles et al. 2017, for details). This procedure did not allow for a case-by-case assessment of hyperbolae; however, it was able to detect a larger number of roots, being free of the subjectivity that flaws the visual inspection of radargrams and, thus, allowing a more robust analysis.

The automatic dataset was generated for three different sub-plot sizes. In all cases, their cross-sections extended from P1 through P3 and, therefore, were centred at each experimental unit’s mid-point, but had different lengths of 50, 100 and 250 cm (hereafter S₅₀, S₁₀₀ and S₂₅₀ sub-plots, respectively; Fig. 2c). Note that the largest sub-plot length (S₂₅₀) was equivalent to the mean inter-plot distance (Fig. 2c). This strategy allowed to identify a particular sub-plot area maximising population differences in root traits, as described in section 2.5.

For calibration, we measured in situ (i.e. through soil digging) the depth and diameter of 12 coarse roots from different plots. Root depth and diameter were then GPR-estimated through the time interval between zero crossing. This time interval is the time elapsed between signal emission and registration by the shielded antenna. Time zero is an adjustment made before processing the data in relation to soil surface (depth= 0), in order to set the instant in which the radar signal leaves the shielded antenna. The calibration of root diameter was done by linear regression of measured diameters on time interval values. Although root depth does not generally require a site-specific calibration (Hirano et al. 2009), we removed the initial direct current signal component from the GPR control unit (RAMAC XV Monitor and ProEx system – adjust signal position) to guarantee that depth estimates started from 0. For full methodological details, see Rodríguez-Robles et al. (2017).

Statistical analyses

For the definition of a proper sub-plot size for GPR-based root detection, we considered that a sub-plot too large could potentially capture coarse roots not just from the targeted experimental unit, but also from neighbouring units. Conversely, a sub-plot too small could underestimate the actual number of coarse roots of an experimental unit. To try circumvent this issue, we examined the total root number of the abovementioned sub-plot sizes obtained from the manual dataset (S₅₀, S₁₀₀, S₂₅₀). We counted the number of roots as a function of linear distance from the trunk (sub-plot length) using evenly spaced bins of 10 cm. The general tendency was a high number of root at small and medium distances from the trunk, followed by a progressive decrease in root number and a sudden increase after some point (see Results subsection 3.1).

The ‘optimal’ sub-plot area was additionally explored through linear mixed-effects analysis of variance (ANOVA) fitted to the automatic dataset independently for each sub-plot size. The ANOVA included replicate, row, column and population as fixed effects, while row within block and column crossed to replicate were defined as random effects. We retained as optimal sub-plot size the one that maximised the F value for population differences in the ANOVAs for coarse root traits (diameter, depth, and frequency), which approximately matched the optimal sub-plot size previously identified in the manual dataset (Table 1, S3). The mixed-effects ANOVAs were further extended to partition the variability among populations into fixed ecotype and between-population within-ecotype effects, hence testing for ecotypic structure in root traits.

As indirect validation of our phenotyping approach, we compared the information obtained through GPR measurements with estimates of the relative contribution of two consecutive soil layers (0-15 cm, upper soil; 15-40 cm, lower soil) to the total amount of water taken up by the different populations, as inferred through the analysis of the isotopic composition (δ¹⁸O and δ²H) of soil and xylem water in 2010 (data retrieved from Voltas et al. 2015). Additionally, simple correlations were calculated between GPR-based root traits and xylem water δ¹⁸O at plot level.

Population (or ecotype) least squares means of root depth, diameter and frequency were subjected to linear and quadratic regressions as a function of long-term climate conditions of each population (or ecotype) at origin. The three populations of ecotype 16MC were not used in the regression analysis because they have an uncertain geographic origin. Linear and quadratic regressions of root traits were also performed on Euclidean geographic distances and climatic distances (Gower’s distances) of each population (or ecotype) from the trial site. Gower’s distances indicate dissimilarities among entities, in this case climate dissimilarities between populations (ecotypes) at origin and the trial site, and were calculated following Rutter and Fenster (2007):

Where p is the number of climate variables, A_iand B_i are the values of each climate variable (MAT, TAR, MAP, PsP) at site A (trial) and B (population or ecotype origin), and r_i is the range of each climate variable in the dataset.

Finally, linear and quadratic regressions were calculated for root traits as a function of aboveground growth variables (H, DBH and crown area), both at population and ecotype level. The associations of aboveground growth variables with those climate variables at origin found to be related to population or ecotypic variability in root traits were also evaluated. For model selection, the best fitting model for each explanatory variable (linear, quadratic) was chosen considering its overall significance and fit (higher F-value and adjusted R²).

Root detection using GPR

Manual measurements of root diameter during calibration showed a positive correlation (r = 0.95; p < 0.001) with time interval between zero crossing (Fig. 3a). Measured and GPR-estimated root depth also showed a positive correlation (r = 0.98; p < 0.001) (Fig. 3b). During visual inspection of radargrams, we detected the largest number of roots at distances between 10 cm and 50 cm from each tree trunk along the trial’s column direction, with a progressive decrease at larger distances (Fig. 3c). However, the number of detected roots increased again starting at 1 m apart from each tree. This observation suggested that GPR was detecting roots corresponding to trees of adjacent experimental units for column distances higher than 90 cm from each targeted tree trunk (Fig. 3c). This was confirmed by analyses of variance on the automatic dataset carried out for three different sub-plot sizes (S₅₀, S₁₀₀, S₂₅₀). Particularly, the results showed that the sub-plot size that maximised population differences in root traits was S₁₀₀ (Tables 1 and S3). Further analyses were therefore performed for S₁₀₀. At sub-plot level (for S₁₀₀), GPR estimates of coarse root traits varied between 3.0 cm and 5.0 cm (diameter), between 14.7 cm and 44.3 cm (depth) and between 3 and 19 roots (frequency).

Genotypic variation in root traits

We detected significant variation in coarse root diameter among populations (p = 0.030), but the effect was only marginally significant (p = 0.091) among ecotypes (Table 1). Mean values of ecotypes varied between 3.5 cm (15TU, Tunisia; Table S2) and 4.0 cm (3ALC, Spanish Southern Plateau). Differences among populations in coarse root depth (Table 1) were marginally significant (p = 0.086), while significant differences among ecotypes were detected (p = 0.011). Mean values of ecotypes varied between 21 cm (15TU) and 30 cm (16MC, reforestations of Northern Spain). These results suggested that rooting depth had a stronger ecotypic pattern than root diameter. We did not observe significant differences in root frequency among populations (p = 0.460), whereas a marginally significant effect was detected among ecotypes (p = 0.096). Mean root number of ecotypes varied between 8.9 (2MO, Spanish Ebro depression) and 13.1 (7BM, Spanish Baetic system). Estimates of coarse root depth and diameter were significantly and positively correlated both at population (r = 0.416, p = 0.001) (Fig. S1a) and ecotype level (r = 0.628, p = 0.009), but root frequency did not correlate significantly with root depth or diameter (Fig. S1b, c).

The variability of root traits was in good agreement with xylem water isotopes records obtained in 2010 for the same trial, with the exception of root frequency. At population level, we observed significant negative correlations between population means of coarse root diameter or depth and δ¹⁸O of xylem water in early autumn (r = 0.622, p < 0.001, and r = 0.497, p < 0.001, respectively; Fig. S2), but not in peak summer (mid-July) (p > 0.1, Fig. S2). At ecotype level, a negative association between root diameter and the relative contribution of the upper soil layer to xylem water in early autumn was also observed (Fig. 4a), although this relationship was non-significant for root depth (Fig. 4b) and frequency (Fig. 4c). There was also a negative association between the relative contribution of bottom soil layer in early autumn and root diameter at ecotype level (Fig. 4d), but not involving root depth (Fig. 4e) or frequency (Fig. 4f).

Genotypic associations with aboveground growth

The ecotypic variation in coarse root diameter and depth showed marginally significant quadratic relationships (p = 0.064 and p = 0.102, respectively) with tree height (Fig. 5, Table S4), while root frequency did not show any relation with vertical growth. These results suggested that shallower and smaller roots were indistinctly found in ecotypes having lower or higher than average height growth. Other aboveground growth variables (DBH, crown area) were unrelated to root depth, diameter and frequency (Table S4).

Associations with geographic variables and climate at origin

Coarse root traits were related to some geographic and climatic variables at origin of populations, either linearly (Table S5a) or quadratically (Table S6a). Root traits decreased linearly (albeit weakly) with geographic distance to the trial and also with longitude (Table S5a). These results suggested geographically-structured differences between populations located near the trial site (i.e. the westernmost distribution of Aleppo pine) and those from the eastern Mediterranean basin. A significant quadratic dependence of root depth on Gower’s distance (Table S6a) was also observed, indicating that populations with climate at origin either most similar or most dissimilar to the trial site presented shallower roots. In particular, root diameter showed a quadratic dependence on solar radiation (R² = 0.157; p = 0.014), with thicker roots occurring in populations having intermediate solar radiation at origin. In turn, root depth showed a quadratic dependence on PsP (R² = 0.126; p = 0.035) and, marginally, on MST (R² = 0.103; p = 0.067), with shallower roots observed under low and high values of both climate variables (Table S6a). Root frequency was linearly dependent on solar radiation (R² = 0.222; p ˂ 0.001) and marginally on PsP (R² = 0.060; p = 0.060), with more roots observed under high solar radiation and low PsP at origin. In addition, we also detected significant and marginally significant positive linear dependencies of root frequency on VPD_s (R² = 0.161; p = 0.003) and TAR (R² = 0.061; p =0.073) respectively.

Similar dependencies of coarse root traits on geographic and climatic factors occurred at ecotype level, with relationships being either linear (Table S5b) or quadratic (Table S6b). In particular, we observed a negative linear regression of root diameter on longitude and a positive linear regression of root frequency on latitude. There were also marginally significant quadratic relationships between root depth and latitude and between both root depth or diameter and altitude (Fig. S3). The quadratic relationships were such that northern and southern ecotypes tended to present shallower roots, while deeper and thicker roots were distinctive of ecotypes from either low or high altitudes. Ecotypes from the Iberian Peninsula were characterized by a large variability in root traits (Fig. S4).

Regarding climate variables, root frequency showed a negative linear dependence on PsP (Fig. S4), while quadratic dependencies of coarse root traits on MST were detected for root diameter and depth, but not for frequency (Fig. 6a,b,c). Both root depth and diameter showed also a quadratic relationship with solar radiation, while root frequency showed a positive relation with this latter variable (Fig. 6d,e,f). These results suggested that shallower and finer roots were present in ecotypes having either relatively low and high MST or solar radiation values at origin. Also, root depth showed a marginally significant (negative) linear dependence on VPD_s(Fig. 6e,f), denoting that ecotypes subjected to higher summer transpirative demand tended to show shallower roots. As for root depth, tree height showed a negative dependence on VPD_s (R² = 0.315; p = 0.029), but (contrary to the pattern observed for root traits) it was negatively related to SR (R² = 0.299; p = 0.035) and unrelated to MST (results not shown).

GPR applicability to root phenotyping of forest trees

This study showcases the use of the Ground Penetrating Radar device as high-throughput phenotyping tool to assess intraspecific differentiation in coarse roots potentially related to water use and drought tolerance strategies in a widespread conifer. It broadens the customary use of GPR for tree root detection in ecological studies carried out since the turn of this century (Guo et al. 2013; Hruska et al. 1999). By using an 800 MHz antenna we were able to detect 1,835 roots with diameters equal to or above 2.2 cm at a maximum depth of 88 cm for about 330 trees monitored in a common garden of ca. 0.8 ha.

In situ calibration provided direct evidence of the adequacy of GPR measurements for root detection. An additional (indirect) indication of the potential of GPR for root detection was the observation that the variability detected in coarse root traits for Aleppo pine agreed (at least partly) with the information derived from xylem water isotopes on the use of water sources by the same trees (Voltas et al., 2015). However, we should note that only associations involving δ¹⁸O of xylem water for early autumn were significant, whereas no relevant relationships with GPR records were detected in peak summer. This suggests that the existing electromagnetic gradient between roots and soil at the time of GPR measurements (mid-June) could have identified more roots than those roots effectively taking up water during the acute summer drought period (end of July). Hence, a fraction of coarse roots might have remained in a quiescent condition during soil dry-down in summer as avoidance mechanism against hydraulic failure for an isohydric species such as Aleppo pine. Changes in root turnover and turgor are some of the prevailing consequences of high soil temperature and water deficit stress in plants (Brunner et al. 2015, Gill and Jackson 2000), and both have been widely described for the case of fine roots (Gill and Jackson 2000; Kitajima et al. 2010; Montagnoli et al. 2019). However, there is no evidence of inactivation of coarse roots in forest tree species thus far. This possibility would deserve detailed investigation through e.g. the temporal assessment of GPR signals following changes in soil water status during the growing season.

Evidence for intraspecific variability in root traits of Aleppo pine

It is known that root system architecture varies among Iberian pines, with mountain species (e.g. Pinus sylvestris) usually having shallower roots than typically Mediterranean species (e.g. Pinus pinaster, P. halepensis) (Andivia et al. 2019). This variability correlates with the species’ ecological niches and the intensity of drought stress present in their environments (Andivia et al. 2019). However, there is still a lack of information on the variability of functional root traits at the intraspecific level, which determines the efficiency of water acquisition by trees (Kirfel et al. 2017). A main goal of this work was to fill this knowledge gap for a Mediterranean pine species and, particularly, to characterize intraspecific patterns of coarse root traits potentially related to different strategies of water uptake and use in Aleppo pine.

We found population differentiation for root traits in P. halepensis, which were geographically structured following an ecotypic pattern. The presence of ecotypic patterns in this species has been previously reported for traits related to the tree’s water budget such as water-use efficiency (Voltas et al. 2008), transpiration (Santini et al. 2019b) and the use of water sources (Voltas et al. 2015). Despite the loss of genetic diversity that Aleppo pine suffered after the post-glacial recolonization from the eastern Mediterranean (Grivet et al. 2009), we observed greater variability in coarse root traits among populations originating from the Iberian Peninsula than among their eastern Mediterranean counterparts. This result requires further corroboration, however, since comparatively less populations were available from this latter region (and distributed within a narrower latitudinal gradient) compared to the western Mediterranean basin.

Relationships between root traits and aboveground growth

We detected quadratic associations of root depth with tree height and diameter at the intraspecific level. Although these relationships were only marginally significant, they suggest the presence of predictable intraspecific allometric patterns linking below and aboveground growth, which are partially consistent with the general allometric scaling theory (West et al. 1999). In particular, they point to proportionality of carbon investment to above and belowground growth for ecotypes reaching up to ca. 6 m height at age 19 years in the study site, which is representative of the average climate conditions of the species range-wide. Above this height threshold, a negative association between aerial growth and investment in roots could be observed at the ecotype level. This suggests a preferential mass allocation to aerial carbon stocks for mesic ecotypes having high growth potential (Climent et al. 2008; Voltas et al. 2018), as exemplified by ecotypes 12GR and 14IT originating from the center-eastern Mediterranean basin. These ecotypes experience mild climate conditions at origin (either having MAP> 500 mm or MSP> 60 mm) and are known to exhibit a larger height plasticity compared with xeric ecotypes across water availability gradients (Patsiou et al. 2020). This superior plasticity might be associated with a progressively large carbon allocation to roots under harsher (i.e. drier or nutrient-poor) conditions. In any case, our results suggest that these mesic ecotypes allocate relatively more resources to aerial growth than to root development under the relatively favorable conditions encountered in the trial compared with xeric ecotypes.

Variability in root traits of Aleppo pine follows geographical and climatic gradients

The assessment of geographic dependencies of roots traits suggests that root diameter decreases across an eastward longitudinal cline in Aleppo pine populations. This geographical pattern agrees with other P. halepensis studies, that could be indirectly linked to allocation to roots, reporting decreases in reproductive investment (Climent et al. 2008) and in water-use efficiency (Voltas et al. 2008) in eastern populations. On the other hand, the larger proportion of shallow roots in populations originating from either extreme of the species’ latitudinal range disagrees with our initial hypothesis. However, root frequency decreased as latitude increased, suggesting that southern populations, which generally are exposed to a drier climate, have more abundance of coarse roots than northern populations, regardless of their root depth. This discrepancy could be related to the higher costs of construction and maintenance of deep roots (Schenk 2008a) in ecotypes with low growth potential and high reproductive allotment, leading to carbon allocation shifts towards shallower and more abundant roots in dry and resource-poor habitats. This could be an advantageous strategy in habitats where the uppermost soil layers may present higher nutrients and moisture as a result of discrete precipitation pulses (Schenk 2008a, b).

Previous studies assessed that rooting depth increases with water shortage in pine species (Andivia et al. 2019), a general interspecific pattern that only partially agrees with our results. That is, root frequency increased with aridity at origin. However, genotypic relationships between root depth or diameter and temperature were hump-shaped, and peaked towards the thermal midpoint of the species distribution range (Albert et al. 2010). This finding may be related to the existence of different adaptive strategies imprinted in a number of life-history traits and their potential trade-offs – in addition to rooting traits – to cope with the conditions encountered by the species along its distribution range as modulated by stresses (drought, cold temperatures, pests) and disturbances (fire). For example, the different responses of aerial growth and rooting traits to incoming radiation might indicate allocation trade-offs related to higher selective pressures for increased competition for light in the case of Greek and Italian mesic ecotypes. This result also suggests that the existence of trade-offs between reproduction, defense and vegetative growth, as described for Aleppo pine (Climent et al. 2008; Santini et al. 2019a; Voltas et al. 2008), may also affect carbon allocation to coarse roots in fire-prone, dry and warm habitats. In this regard, the negative dependency of rooting depth on VPD_s has to be carefully interpreted, since this relation is mostly driven by the Tunisian ecotype (15TU), which has shallow roots despite being exposed to high summer evapotranspiration. This could be explained as the result of conflicting functional strategies typical of stressed and disturbed environments (Santini et al. 2019a), implying a relatively low allocation to growth compared with reproduction (Climent et al. 2008; Santos-del-Blanco et al. 2013). This possibility is indeed supported by the negative association found between tree height and VPD_s.

Methodological limitations

Despite its high detection capability and non-destructive assessment of root features, GPR is a method not devoid of technical limitations for the assessment of root morphology and distribution in tree phenotyping studies. These are related to the existing variability in root orientation, since overlapping roots and roots underneath the tree trunk are underestimated by the device (Butnor et al. 2016; Li et al. 2016). Also, the existing electromagnetic gradient between roots and soil makes a previous calibration strictly necessary to reduce the background noise produced by the particular physicochemical characteristics of the soil. In particular, it should be noted that GPR detection capacity is seriously reduced in both nearly saturated (Hirano et al. 2009; Rodríguez-Robles et al 2017) and very dry soils (Hirano et al. 2009). In this last case, GPR detection is impaired if roots have low water content (e.g. under 20% volumetric water content in Cryptomeria japonica; Hirano et al. 2009). In this regard, our field campaign targeted a period of full vegetative activity of trees while avoiding such extremes in soil water status (abundant precipitation in April-early May and acute drought in July-August).

This study provides new avenues for the examination of intraspecific variability in root traits of forest tree species using GPR as a high-throughput phenotyping method. We observed population differentiation in P. halepensis following east-west (root diameter) and north-south (root frequency) geographical patterns. However, the interspecific pattern described for the genus Pinus, by which species originating from xeric conditions exhibit large allocation to roots, was not strictly found at the intraspecific level for Aleppo pine. This observation may be attributable to the existence of different adaptive strategies under varying environmental conditions for the species, which likely lead to trade-offs involving the use of resources. In this regard, our work contributes to unravel the evolutionary complexity of a widespread conifer of high ecological significance in the Mediterranean basin.

Funding

This work was partly funded by the Spanish Government, grant numbers AGL2015-68274-C3-3-R (MINECO/FEDER) and RTI2018-094691-B-C31 (MCIU/AEI/FEDER, EU). E. Lombardi was supported by a AGAUR FI-2020 pre-doctoral fellowship (Generalitat de Catalunya). J. P. Ferrio was supported by Reference Group H09_20R (Gobierno de Aragón). U. Rodríguez-Robles acknowledges the National Council for Science and Technology of Mexico (CONACyT), grant number 332356.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Availability of data

Data will be made publicly through Mendeley repository, upon acceptance of the article.

Authors’ contribution

Erica Lombardi: Formal analysis, Writing - Original Draft, Visualization. Juan Pedro Ferrio: Conceptualization, Formal analysis, Writing - Review & Editing. Ulises Rodríguez-Robles: Methodology, Data curation, Formal analysis, Writing - Review & Editing. Víctor Resco de Dios: Formal analysis, Writing - Review & Editing. Jordi Voltas: Conceptualization, Formal analysis, Writing - Review & Editing, Funding acquisition.

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Table 1 Mixed-effects model analysis of variance for root diameter (cm), root depth (cm) and frequency of 56 populations of Aleppo pine categorized into 16 ecotypes grown in a common garden in Altura (Spain). Data refer to sub-plot S₁₀₀. Only fixed effects are reported

		Root diameter				Root depth			Root frequency
Source of variation	Num. df	Den. df		F-value	P > F	Den. df	F-value	P > F	Den. df	F-value	P > F

Block	2	13.38	1.49		0.2260	9.78	0.26	0.7726	7.57	1.92	0.1459
Column	6	92.39	4.05		0.0005	12.19	1.30	0.2516	11.63	2.62	0.0153
Population (P)	55	84.56	1.39		0.0300	73.19	1.27	0.0860	89.76	1.01	0.4600

Block	2	13.38	1.49		0.2260	9.78	0.36	0.7726	7.57	1.92	0.1459
Column	6	92.39	4.05		0.0005	12.19	1.3	0.2516	11.63	2.62	0.0153
Region (Reg)	15	97.31	1.51		0.0913	87.85	2.00	0.0118	97.58	1.50	0.0957
P(Reg)	40	90.55	1.16		0.2267	81.11	1.03	0.4098	93.87	0.84	0.7555

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Ground-penetrating Radar as Phenotyping Tool for Characterizing Intraspecific Variability in Root Traits of Pinus Halepensis

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