Isotopic Signal Supports Physiological Integration in Root Suckers of Two Tree Species Differing in Shade Tolerance

Abstract

The physiological performance of clonal plants is largely linked with resource translocation among interconnected ramets. Whereas carbon (C) and nitrogen (N) transferences have been evidenced in several herbaceous clonal plants, empirical evidence in woody species is anecdotal. We evaluated physiological integration in two evergreen tree species, differing in the light requirements in a temperate rainforest of Southern Chile: Embothrium coccineum J.R. et. G. Forster (light-demanding) and Eucryphia cordifolia Cav. (shade-tolerant). We measured light availability for vegetative (root suckers) and sexual (seed-origin plants; hereafter, saplings) recruits of the two species. Then, we compared elemental and isotopic leaf traits between recruit types and species growing under similar light availability. A ¹³CO₂ field pulse labeling was performed on a set of Embothrium root suckers to quantify C transfer from moderately shaded suckers (donors) to highly shaded suckers (receivers). For the two species, leaf N concentration, δ¹³C, and δ¹⁵N were higher in suckers compared to saplings. In the labeling experiment, the δ¹³C and ¹²C equivalent excess did not differ between donor and receiver, indicating a weak C transfer between donors and receivers. Although the results from the pulse labeling were not conclusive, they suggest, together with the differences in natural isotope abundance, the existence of physiological integration in root suckers of both species. Our findings indicate that the formation of root suckers is more important for regeneration and persistence than for resource acquisition at an intermediate ecological succession of a temperate rainforest.

1. Introduction

Clonal growth in plants is the generation of genetically identical and potentially independent ramets by vegetative growth [1]. This strategy increases the persistence of an individual on a site, thus impacting both populations and forest succession dynamics [2,3,4,5,6]. In addition, clonal growth allows the colonization of environments where sexual reproduction is unsuccessful, extending the regeneration niche [7,8,9,10]. Physiological integration (i.e., resource translocation) among ramets explains clonal plants’ ecological advantages and high physiological performance while remaining interconnected [11,12]. This is particularly evident in highly heterogeneous environments, where resource transfer occurs towards ramets with a low availability of water and nutrients or in shade [13,14,15,16].

Embothrium coccineum J.R. et. G. Forster (Proteaceae) and Eucryphia cordifolia Cav. (Cunoniaceae) are two evergreen tree species able to recruit sexually and vegetatively through root suckering [17] that occur in the temperate rainforest of southern South America. Embothrium coccineum is a colonizer, light-demanding species that recruits mainly in gaps. The distribution of Embothrium makes it subject to severe disturbance events, such as fires and volcanic eruptions. After a severe disturbance, the species that can regenerate asexually are usually the first to colonize a space, allowing a faster reestablishment of the original flora, whereas Eucryphia cordifolia is a shade-tolerant species and typically recruits in the understory of mature forests [18,19,20]. Despite their contrasting shade tolerance, they coexist at intermediate stages of forest succession through root suckering under suboptimal conditions. Specifically, suckers of Embothrium coccineum establish in shady conditions (where saplings do not), while suckers of Eucryphia cordifolia can occupy open microsites (where sapling survival is very low) [10,17]. In both species, root suckers remain interconnected (at least) through the juvenile stage without having a root system of their own (rarely, a few rootlets). For both species, C translocation is a plausible explanation for the success of sucker establishment under suboptimal conditions: in the case of the light-demanding species (hereafter Embothrium), photosynthesis of shade-established suckers should be limited by the low light availability, whereas in the case of the shade-tolerant species (hereafter Eucryphia), photosynthesis should have diffusive limitations associated with the higher evaporative demand of air under open canopies [18,21]. In the case of N, the suckers of these species must necessarily obtain it through the parental root system since they have no root system of their own (at least) during the early growth stages.

Pulse-chase labeling experiments, a technique used to trace the fate of resources from source to sink organs (typically autotrophs and heterotrophs, respectively) [22,23,24], have been applied to assess physiological integration between interconnected ramets. By doing this, carbon (C) and nitrogen (N) transference has been evidenced in several herbaceous clonal plants (including forbs, sedges, grasses, and bamboos), in both natural populations or greenhouse conditions (e.g., [13,15,25,26,27,28,29,30,31,32]). Although many works use pulse labeling to trace resource allocation, to our knowledge, only a few field experiments have been conducted on massive woody plants of natural populations [33,34]. While in one of these studies, there was evidence of N transfer towards the ramets with the highest demand [33], in the other study, authors concluded that no clonal integration of photosynthates occurred among ramets, likely because they were not C-limited [34]. However, resource transfer can be stimulated by enhancing the sink effect of the potential receiver’s ramets, for instance, by increasing their metabolic demands or decreasing their resource availability [11,13,35].

As an alternative to pulse labeling, the natural abundance of δ¹³C can also be used as a proxy for C translocation between ramets. Carbon is translocated from the source to sink organs/tissues as sucrose. Sucrose synthesis is based on the ¹³C-depleted triose-phosphates exported from chloroplast. The aldolase reaction within the Calvin cycle favors ¹³C during the production of fructose 1,6-biphosphate, which leads to a relative enrichment of starch [36,37]. Additionally, invertases cause progressive ¹³C enrichment in the sucrose [38,39,40]. This chain of metabolic reactions causes an enrichment of sucrose in source organs and the result is that the heterotrophic-sink organs of woody plants (basically, stems and roots) tend to be ¹³C enriched after C translocation [39,41]. Similarly, the physiological integration of ramets must be reflected in a natural enrichment of ¹³C in the leaves of the sink ramets, compared with saplings [42].

Analogously, the natural abundance of δ¹⁵N could be used to track N sharing among ramets. The variation in the natural abundance of δ¹⁵N in chemical fractions of higher plants depends on the amount of N available in the soil, the form that the plant absorbs it (i.e., nitrate >> ammonium > amino acids), and the enzymatic isotope fractionation within the cell [43,44]. After plant uptake, nitrate and ammonium are assimilated by (respectively) nitrate reductase and glutamine synthetase enzymes, both of which fractionate against ¹⁵N [44]. When nitrate reduction preferentially consumes most of the ¹⁴NO₃ available, no ¹⁴N/¹⁵N net discrimination occurs during N reduction [45], resulting in a net increment of δ¹⁵N. Nitrate is reduced to ammonium and incorporated into the amino acid glutamine by the glutamine synthetase activity [46,47]. Ammonium ¹⁵N isotope fractionation by glutamine synthetase occurs similarly to nitrate reductase [43,48]. As clonal plants tend to be bigger than stand-alone plants and need to support a more substantial initial growth, higher N demand from N sources occurs; therefore, less ¹⁵N discrimination is expected in clonal plants. Furthermore, if N is translocated from the parent plant, a less depleted δ¹⁵N is expected in juvenile ramets compared to stand-alone saplings, as large plants feed deeper in the soil, where the δ¹⁵N becomes enriched due to the microbial activity [49,50].

Here, firstly, we hypothesized that suckers will show higher δ¹³C and δ¹⁵N natural abundances than their counterpart saplings due to the metabolic enrichment in ¹³C and the lower isotopic discrimination for ¹⁵N during translocation through interconnected ramets. To test this, we first conducted a comparative elemental and isotopic leaf analysis. In addition, the greater access of suckers to N through the parental root system should be reflected in a lower foliar C/N ratio of suckers compared to saplings, explaining their higher competitiveness [51]. We predicted that, if suckers act as net sinks for elemental and isotopic C and N, regardless of the species’ light requirements, then their enhanced recruitment [17] is explained by physiological integration. Secondly, we hypothesized that if most C is transferred from a donor to a receiver sucker, then the C isotopic signal should be significantly higher in the non-labeled (receivers) than in labeled suckers (donors). Alternatively, if the C transfer is negligible, labeled suckers should show a significantly higher C isotopic signal than non-labeled ones, indicating that most assimilated C remains in the donor sucker. We conducted a field pulse-labeling experiment in interconnected suckers to further test C translocation in the light-demanding, colonizer species (i.e., Embothrium). We selected Embothrium rather than Eucryphia because it is easier to control light availability (i.e., the environmental limiting factor of photosynthesis in Embothrium) than air humidity (i.e., the environmental limiting factor of photosynthesis in Eucryphia). Finally, Embothrium was the species that had more chances to rely on resource translocation due to the advanced successional stage of the forest.

2. Materials and Methods

2.1. Study Site

This study was carried out in the Puyehue National Park (40°39′ S, 72°11′ W, 350 m a.s.l.), located in the western foothill of the Andes in south-central Chile. Most of the park is covered by old-growth forest; however, this study was conducted in a ca. 50-years-old second-growth forest fragments dominated by Nothofagus dombeyi and Caldcluvia paniculata, in addition to Eucryphia cordifolia and Embothrium coccineum (Figure 1). The occurrence of suckers and saplings of Embothrium, Eucryphia, and other species was very scarce in old-growth forests. A second-growth forest was therefore selected over the old-growth forest because, in these forest patches, it was possible to find adult trees of Embothrium and Eucryphia together, allowing us to sample co-occurring saplings and root suckers from both species along a wide gradient of light availability, crucial to address our hypotheses [17]. The study plots were established in the Anticura sector of the park, close to the weather station managed by the Forestry National Corporation (CONAF). Records from this station (for the period 1980 to 2016) show that the area experiences a temperate maritime climate, averaging 2725 mm of annual rainfall. Monthly rainfall sharply decreases during summer but is greater than 100 mm on average for most months. The warmer month is January, with an average temperature of 14.4 °C and the colder is July, with 5.4 °C on average, being the mean annual temperature of 9.8 °C.

2.2. Recruitment Type and Light Environment

The study was conducted in two permanent plots (25 × 60 m each, Figure 1) established in previous research in which we evaluated the regeneration niche and functional traits of suckers and saplings of the two species studied here (see details in [17]). Each plot was established in a different secondary forest patch (separated by ca. 400 m), both embedded in the same old-growth forest matrix. Each plot included >5 adult individuals of the studied species (Embothrium and Eucryphia) and the two plots together covered the light availability gradient described previously for the same study site (Global Site Factor (GSF) range [0.006–0.325]) [52,53]. In a central subplot of 5 × 50 m, all the recruits of the studied species between 2 and 150 cm in length were identified as either root suckers (i.e., from vegetative reproduction) or saplings (i.e., from seed origin). To determine whether a recruit was a sapling or a sucker, the surface soil around the recruit was temporarily removed, thus carefully revealing the root collar. Recruits were identified as root suckers when their root collars were still connected to their parental roots and as saplings when they did not show any subsidiary root connections or root scars indicating past connections [21]. The light availability for each recruit was determined according to the Global Site Factor (GSF), which is the proportion of global radiation (direct plus diffuse) under a plant canopy relative to that in the open site [54], calculated from hemispherical photographs recorded above each recruit apex under homogeneous overcast conditions. For this purpose, we used a Coolpix 4500 digital camera equipped with an FC-E8 fisheye lens (Nikon, Tokyo, Japan). The GSF was obtained from each photograph using the canopy analysis software HemiView version 2.1 (1999, Delta-T Devices Ltd., Burwell, Cambridge, UK).

2.3. Sampling for Natural C and N Stable Isotope Abundances

In September 2017, we randomly selected 40 individuals per combination of species and recruit type from the total pool of recruits within the two plots to quantify their natural isotope abundances. Recruit selection was conducted using the “randbetween” function of Microsoft Office Excel software (Microsoft Office Enterprise 2007; Microsoft Corporation, Redmond, WA, USA). Once in the field, damaged recruits were discarded, resulting in sample sizes as follows: 36 suckers and 28 saplings of Embothrium, and 25 suckers and 39 saplings of Eucryphia. Selected plant recruits were carefully harvested in the afternoon, kept under humid conditions, and processed in the field laboratory within one hour after they were harvested; this procedure was performed to avoid changes in the isotopic signal [55]. None of the sampled suckers showed their own root system. Leaves were separated from the stems, stored in paper bags, and dried in a forced-air oven for 72 h at 60 °C. Total C and N content and their natural stable isotope signatures were determined for all harvested individuals (see Section 2.5).

2.4. ¹³CO₂ Isotope Labeling

A pulse-labeling experiment was performed in December 2017 to quantify resource translocation among interconnected root suckers of Embothrium. The experiment was replicated in four pairs of closely and directly interconnected root suckers, i.e., a pair of suckers that were less than 30 cm apart and without any sucker in between. From the two suckers, the smallest and most illuminated was considered as the donor (i.e., the one to be isotopically labeled), whereas the potential receiver was the largest size (i.e., the longest) and shadiest sucker (Supplementary Materials, Table S2). This experimental design was intended to strengthen the C sink in receivers and thus increase the resolution of the ¹³C signal. During the morning, the donors were covered with an air-tight plexiglass chamber of 45 ×10 × 10 cm equipped with a high fluidity box fan (VN-2350, DC 12V, 130 mA; Techman Electronics, La Verne, CA, USA) and with three independent silicon tubes, which include three-way stopcocks for the inner control of the CO₂ concentration (hereafter, [CO₂]) (Supplementary Materials, Figure S1). The forest floor and the donors at their root collars were covered with four layers of plastic film to isolate them from the atmospheric CO₂. The plastic film was joined to the chamber using neutral liquid silicon to avoid leaks. This design was previously tested in the laboratory to assess the chamber gas-tight. No leaks were detected after four simulations.

For the field pulse labeling, the [CO₂] inside the chambers was monitored using the infra-red gas analyzer incorporated into the handheld photosynthesis system CI-340 (CID-Bio-Sciences, Inc., Camas, WA, USA) with airflow set at 0.2 L min⁻¹ (step 1, Supplementary Materials, Figure S2). The [CO₂] inside the chambers was scrubbed down by passing the chamber air through soda-lime columns. The chamber emptying started after a diminishing of 30 µmol of [CO₂] relative to the initial measurements of [CO₂] inside the chamber, or after a maximum of eight minutes. In all cases, the pulse labeling started when the [CO₂] inside the chamber descended to ca. 250 ppm (step 2, Supplementary Materials, Figure S2). Chambers were filled up with CO₂ enriched with the heavy stable C isotope (99.9% ¹³C; Cambridge Isotope Laboratories, Andover MA, USA) until they reached lectures of ca. 700 ppm [CO₂] (step 3, Supplementary Materials, Figure S2) and monitored during the subsequent three minutes (step 4, Supplementary Materials, Figure S2). The selected thresholds of [CO₂] used for emptying and filling up the chamber did not produce alterations in the RuBisCO activity and activation, according to the global patterns of RuBisCO responses to [CO₂] gradients [56]. To stimulate gas exchange, the donor was illuminated from the top outside of the chamber with the IRGA’s red/blue light source delivering between 320 and 800 µmol of photons m⁻² s⁻¹ at the top of the plant inside the chamber. That amount of light is around the light saturation point for Embothrium suckers inhabiting the understory forest to avoid light limitation of photosynthesis. During the pulse labeling, neighbor plants were carefully covered with thick plastic bags to prevent accidental free-air enrichment. The temperature and the relative humidity inside the chamber during the pulse labeling were measured with an iButton Hygrochron temperature/humidity logger (DS1923; Maxim Integrated Products, Inc., San José, CA, USA) with (respectively) 0.0625 °C and 0.04% resolution. The temperature and the relative humidity were 12.5 °C ± 1.3 and 90.9% ± 4.6 on average (±SD), respectively. The chamber was removed three hours after the beginning of the pulse labeling. Donor and receiver shoots were harvested after seven days, following the C transport velocity from leaves to below-ground organs and soil, previously reported for 1.5-year-old beech trees in a temperate climate [22]. Harvested plants were quickly carried out to the field laboratory, where fully mature leaves of the current year were placed in small paper bags and heated for 7.5 to 10 min to stop any enzymatic activity in a Thomas TH-34DGM microwave at the highest power [57]; a beaker with water was placed inside the microwave to maintain air humidity [58]. This procedure preserved plant material for determining organic compounds without quantifiable effects on N levels [57,58].

2.5. Elemental and Isotopic Analyses

Foliar samples of both natural abundance and pulse labeling were ground in an agate mortar until passing a 1 mm mesh and ground in a ball mill to pass to 0.425 mm mesh (Spex Sample Prep 8000M Mixer/Mill, Metuchen, NJ, USA). To avoid cross-contamination, labeled leaf material was ground with different equipment. The leaf C and N concentrations (respectively LCC and LNC) and isotopic analyses were performed using approximately 2 µg of fine powder encapsulated in a tin capsule. Materials were combusted, and the gas evolved was analyzed to determine LCC and LNC, as well ¹³C/¹²C and ¹⁵N/¹⁴N ratios with an Isotope Ratio Mass Spectrometer CHNS-IRMS autoanalyzer (20-22 IRMS, SERCON, Crewe, UK) at the Soil, Water and Forest Research Lab (LISAB) at the University of Concepción. The element isotope composition (δ^xxE) was calculated as follows:

$${\mathsf{\delta }}^{\mathrm{x}\mathrm{x}}\mathrm{E}=\left({\displaystyle \frac{{\mathrm{R}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}{{\mathrm{R}}_{\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{d}}}}-1\right)\times 1000,$$

where R is the ratio between the heaviest and lightest isotope of the element. C and N isotopes ratios were relativized to the wheat flour SC0464 (SERCON, Crewe, UK) standard, previously calibrated using a series of primary standards (IAEA-600, IAEA-CH-3, IAEA V9, IAEA-C3, USGS-40, and USGS-41).

For the pulse-labeling samples, the δ¹³C of donor and receiver suckers was converted into mg of ¹²C equivalent excess to measure the enrichment level of the labeled sample relative to the ¹³C background level before tracer administration [59]. We used a modified version of the procedure and calculations simplified in [60]. Background samples consisted of one-year-old leaves harvested one day before the start of labeling from six suckers growing in the same permanent plots and under a GSF similar to that of the donors (donors background; ANOVA test: F_1,5 = 0.005; p = 0.95) and receivers (receivers background; ANOVA test: F_1,5 = 0.009; p = 0.93), thus avoiding the effect of short-term temporal variations in the isotopic signal [61,62,63].

2.6. Data Analysis

The leaf chemical traits were compared between recruit types (i.e., root suckers and saplings) and species (Embothrium and Eucryphia) by means of two-way ANOVAs, using a Type-I sum of squares (SSs). The evaluated traits were LCC and LNC, the C/N ratio, as well as the isotopic composition of ¹³C (δ¹³C) and ¹⁵N (δ¹⁵N). The post-hoc differences were evaluated by estimating the marginal means (least-squares means) through the functions “emmeans” and “cld” of the R packages emmeans [64] and multcompView [65], adjusting p-values with the Tukey method at α = 0.05. Finally, the significance level (α) was established by means of the step-up false discovery rate (FDR) procedure to control for the probability of Type-I error under repeated testing [66].

To evaluate the diffusion of photoassimilates between the Embothrium root suckers used in the ¹³CO₂ isotope pulse-labeling experiment, we compared LCC, LNC, δ¹³C, δ¹⁵N and the ¹²C equivalent excess between the donor and receiver by means of the ANOVA of linear mixed models, including the donor/receiver pair of root suckers as a random effect in order to consider the interdependence of certain data pairs [67]. The random effect was included to reduce the probability of Type-I (false positives) and Type-II (false negatives) error rates [67]. The models were subjected to Type-III sum of squares (SSs) ANOVA (“anova” function). The post-hoc differences and the significance level (αFDR) were managed as mentioned above.

For all the analyses, the Shapiro–Wilk test and the Non-constant Variance Score (NCV) test were used to verify, respectively, the normality and homoscedasticity of the models’ residuals. The NCV test was applied through the “ncvTest” function available at the car library of the RStudio software (R version 4.1.3) [68]. In the case of linear mixed models, the homoscedasticity assumption was tested for a model considering the fixed effects only. The Box-Cox transformation was applied when necessary to meet the assumptions of normality and homoscedasticity of the model residuals.

3. Results

3.1. Light Availability

The GSF of the sampled plants did not differ between recruit types (p = 0.18), between species (p = 0.21), or for the interaction of these factors (p = 0.73) (Supplementary Materials, Table S1). Therefore, differences in leaf chemical traits among recruit types and/or species could not be attributed to differences in the light environment.

3.2. Differences Between Recruit Types and Species in Leaf Elemental Concentration

All the evaluated elemental analyses of leaves (except for leaf C concentration) differed between recruit types, which were equivalent to the two studied species (i.e., non-significant interaction between recruit types and species;

Table 1. Summary results of ANOVAs comparing the leaf chemical composition between recruit types and species sampled in the study of isotope natural abundance. Significant differences are indicated in bold (based on the step-up false discovery rate (αFDR). * Box-Cox transformed variable. p probability value. See full results of ANOVA and residual tests in Table S1.

	LCC (%)		LNC (%) *		Leaf C/N Ratio *
Factors	p	αFDR	p	αFDR	p	αFDR
Recruit type (RT)	0.690	0.050	0.000	0.019	0.000	0.011
Species (SP)	0.000	0.036	0.000	0.042	0.000	0.044
RT × SP	0.540	0.014	0.049	0.008	0.058	0.022

, Figure 2A). Root suckers showed significantly higher LNC and lower C/N ratio than saplings (Figure 2B,C).

Leaf elemental traits also differed significantly among the two studied species (Table 1). LCC was 2% higher in Eucryphia compared to Embothrium (Figure 2A). LNC was 0.43% higher in Embothrium than in Eucryphia (Figure 2B). Accordingly, the leaf C/N ratio was significantly higher in Eucryphia compared to Embothrium (Figure 2C). Even though the residuals of the model developed for C/N ratio were not normal after Box-Cox transformation (Table S1), the results have statistical support considering that (1) the p-values for the corresponding ANOVA had values far from the margin of significance, (2) the residuals were homoscedastic, and (3) the absence of normality only had significant effects when it implied heterocedasticity [69].

3.3. Differences Between Recruit Types and Species in Natural Abundance Isotope Composition

Both leaf δ¹³C and δ¹⁵N differed significantly between recruit types and species, with no significant interaction (

Table 2. Summary results of ANOVAs comparing the natural stable isotope composition of C (δ¹³C) and N (δ¹⁵N) between recruit types and species sampled in the study of isotope natural abundance. Significant differences are indicated in bold (based on the step-up false discovery rate (αFDR). p probability value. See full results of ANOVA and residual tests in Table S1.

	Leaf δ13C (‰)		Leaf δ15N (‰)
Factors	p	αFDR	p	αFDR
Recruit type (RT)	0.001	0.031	0.000	0.039
Species (SP)	0.000	0.006	0.000	0.033
RT × SP	0.949	0.047	0.938	0.003

). Both leaf δ¹³C and δ¹⁵N were significantly higher in suckers than in saplings (Figure 3). On the other hand, leaf δ¹³C and δ¹⁵N were significantly higher in Embothrium than in Eucryphia (Figure 3).

3.4. Carbon Transfer Between Interconnected Root Suckers of Embothrium

In the labeling experiment, no significant differences were found between donor and receiver root suckers of Embothrium for elemental LCC and LNC, nor for isotopic compositions of ¹³C and ¹⁵N (

Table 3. Summary results of the linear mixed models (LMMs) comparing leaf chemical traits for the Embothrium root suckers used in the ¹³CO₂ labeling experiment. Mean values (±SD) for each variable are also shown. The significance level was established by means of the step-up false discovery rate (αFDR) procedure. * Box-Cox transformed variable. p probability value. The full results of LMM and assumptions tests of the models are available in Table S3.

Variable	p	αFDR	Root Suckers
			Receiver	Donor
LCC (%)	0.288	0.02	45.87 ± 0.55	44.71 ± 2.18
LNC (%)	0.443	0.04	2.16 ± 0.47	2.34 ± 0.64
Leaf δ13C (‰)	0.327	0.03	−33.12 ± 1.67	−31.42 ± 2.72
Leaf δ15N (‰)	0.752	0.05	0.81 ± 3.07	0.7 ± 2.64
12Ceq (mg) *	0.020	0.01	0.49 ± 0.01	0.54 ± 0.033

; Figure 4A). Similarly, no statistical differences were found between donors and receivers in ¹²C equivalent excess (i.e., the parameter used to quantify carbon translocation). However, the ¹²C equivalent excess was consistently positive in both donor and receiver suckers, based on the natural abundance background values (Table 3; Figure 4B).

4. Discussion

Our results suggest the existence of resource translocation through the network of interconnected woody ramets, regardless of the shade tolerance of the species. The strongest evidence is the natural ¹³C and ¹⁵N enrichment of sucker leaves relative to saplings. Resource translocation between ramets is necessary in the case of N, as suckers do not have their own root system during the early stages of development, so N must be supplied by the parental plant. In fact, the ¹⁵N enrichment of the suckers can be explained by the lower discrimination of ¹⁵N during nitrate and ammonium reduction due to the high N demand of the whole genet (i.e., ramets and parental genotype altogether) [43,45,48]. In addition, the higher δ¹⁵N values of suckers compared to saplings can be explained by the access of the parental root system to the deeper soil layers, which are richer in ¹⁵N [49,50,70]. Although we did not analyze nitrogen isotope composition in the soil, the relatively small range of variation in saplings (fully dependent on the soil) suggests that source variations in δ¹⁵N alone cannot explain the variability observed in suckers.

Physiological integration among ramets explains why sucker leaves are enriched in N, even though they do not have their own root system. The lower C/N ratio of the suckers, as predicted, indicates a higher allocation of resources to the N-rich metabolic machinery, which favors rapid growth and increased competitiveness under high N availability [51]. In the case of Embothrium, the higher LNC in leaves of young suckers is likely related to their higher chlorophyll content compared to saplings of the same species [17], since, typically, the nitrogen and chlorophyll contents in leaves are positively correlated [71]. This is not the case with Eucryphia, whose suckers have the same or lower chlorophyll concentration, depending on the study site [17,21]. However, in contrast to the higher LNC of suckers studied here, Farahat et al. [72] have reported similar foliar N and chlorophyll content between Fagus grandifolia (deciduous tree species) root suckers and saplings. Therefore, the fate of the extra N concentration in the suckers of this later species remains an open question.

Translocation of photoassimilates through the interconnected ramets is the most plausible explanation for the natural ¹³C enrichment of suckers, relative to saplings of the same species. A priori, the higher δ¹³C of sucker leaves can be explained by different mechanisms: (1) higher carboxylation rates; (2) lower transpiration rates; and (3) translocation of sucrose between suckers [38,39,40,73,74] (see details in the Introduction). However, an eco-physiological study with Eucryphia, indicated that suckers have both lower carboxylation rates in saturating light and higher stomatal conductance than saplings [21], which discard that the differences in δ¹³C between vegetative and sexual recruits of this species are due to differences in the gas exchange strategy. Both the low carbon gain and the poor stomatal control of Eucryphia suckers have been explained because of parental subsidy in terms of photoassimilates and water [17]. The same explanation is plausible in the case of Embothrium, considering that suckers’ leaves of this species are also enriched in ¹³C and that no differences were found for species × recruit type.

Our pulse-labeling experiment showed no significant differences between donor and receiver suckers for elemental and isotopic composition or ¹²C equivalent excess. Therefore, although we partially accepted our hypothesis (based on the fact that translocation of ¹³C to the recipient was not observed, and there was also no evidence of higher ¹³C accumulation by the donor), we did not find conclusive evidence of a strong transfer of C between Embothrium ramets. However, since a weak labeling signal was found in both the donor and receiver, we cannot rule out a certain transfer of C. On the one hand, ¹²C_eq values were consistently positive for both donor and receiver suckers, indicating that both were enriched in ¹³C with respect to non-labeled background suckers developed under similar light conditions. Furthermore, no significant differences were detected between donors and receivers in either δ¹³C or ¹²C_eq, which also supports the transfer of ¹³C from labeled donors to receivers. In this sense, the lack of statistical differences in both measured parameters suggests that the acquired ¹³C was proportionally transported out of the donor leaves, but evenly distributed among donors and receivers (i.e., the latter did not act as a stronger sink). In this sense, Magda et al. [13] found that carbon transfer among ramets of the herb Lathyrus sylvestris was partially consistent, suggesting facultative carbon transfer. However, our interpretation should be taken cautiously, and we suggest further experiments to provide unequivocal evidence of C translocation. Likely, in our case, the ¹³C pulse labeling was diluted, as a consequence of the multiple suckers in the genet, which could be driven by other ramets larger and/or developed in very low light acting as stronger C sinks than the ones selected for the experiment as potential receivers. In humid and rainy sites, ramets of Eucryphia showed to be water spenders when compared to saplings [21], increasing the probability of uptake of depleted¹³CO₂ from the leaf surrounding air due to greater stomatal opening [16,75,76]. This may have been exacerbated by CO₂ recycling from the forest soil, typically depleted in ¹³C [75]. Concomitantly, the higher affinity of RuBisCO for ¹²C [75,76] may dilute the ¹³C labeling because of the preferential uptake of ¹²CO₂. It is noteworthy that, having mentioned that, suckers still have a higher δ¹³C than saplings, evidencing parental/ramets subsidy. Dilution of a tracer also depends on the labeling amount and duration, as well as the post-label sampling time and the size of the pool to be labeled [62]. Regarding the size, Pinno and Wilson [33] evaluated nutrient flow between interconnected Populus tremuloides parent and ramets trees inhabiting nutrient-rich and poor sites, respectively. They found that bigger parents inhabiting N-richer soils were N subsidized from ramets inhabiting N-poor soils, contrary to the patterns commonly observed in herbs [15,77,78]. Additionally, as parent trees can supply resources to their connected ramets, keeping them alive in shaded understory microsites [2,79], our results suggest that root suckers were subsidized, thus masking the slight C transfer between the studied ramets.

The differences between the two studied species in the leaf chemical attributes reflect their contrasting ecological strategies. The lower LCC of Embothrium (compared to that of Eucryphia) is consistent with the reported association between leaf mass per area (LMA) and shade tolerance in evergreens [17,80], whereas its higher LNC agrees with the generalized negative relationship between shade tolerance and leaf N [81]. The higher δ¹³C and δ¹⁵N of Embothrium compared to Eucryphia reflect the higher carboxylation rates of the light-demanding species [82], and support differences in soil foraging strategies [83] of the species due to cluster roots activity [84,85] vs. arbuscular mycorrhizal association [86].

For these particular forests, it has been reported that the vegetative recruitment through root suckering is very profuse and it is more relevant than sexual reproduction when effective recruitment was considered (i.e., grown over 50 cm length) [17]. The high success of suckers, at least during the early ontogeny of the species, increases the residence time of a plant on a site [87], impacting both population and forest succession dynamics [2,3,4,5,6,88]. Although the importance of persistence has been largely neglected in favor of recruitment [87], our study shows that recruitment of root suckers favors the persistence of a species on a site through physiological integration. In particular, we evidenced that the reported success of root suckers of Embothrium and Eucryphia [17] could be explained by C and N exchange across the clonal network of interconnected ramets. Consequently, the effect of parental subsidy can be an explanatory variable to comprehend the regeneration of light niche extensions, as well as the persistence of the tree species ([17,21], this work). In addition, our results suggest that suckers and saplings do not compete for edaphic resources, which could favor the coexistence of both types of recruitment along the forest succession.

5. Conclusions

By studying the natural abundance of stable isotopes in dominant species of the temperate rainforest of southern South America, we provide evidence supporting physiological integration within young ramets in two tree species with contrasting light requirements. This study is novel in assessing the subsidy of suckers based on their different isotopic signatures (¹³C and ¹⁵N), as compared with seed-originated individuals. In particular, given the differences in the isotopic composition of suckers and saplings, we infer that young suckers are subsidized either by the parent trees and/or other ramets. Therefore, during the early ontogenetic development of suckers, physiological integration does occur regardless of the level of shade tolerance of the studied species. This helps to explain that the formation of root suckers is more important for regeneration and persistence than for resource acquisition at an intermediate ecological succession of a temperate rainforest. Due to the lack of clear evidence of C translocation from pulse labeling, both the magnitude and direction of photoassimilates translocation through the network of interconnected ramets are modulated by the strength of C demand of sink suckers. Certainly, it would be stronger to assess the second hypothesis using the two-tree species, but it was possible just to evaluate C translocation on Embothrium due to its higher chances of relying on resource sharing. Another limitation was the lack of strong evidence for C translocation, given that donors and receivers were similar in the evaluated parameters (Table S3). In order to strengthen evidence and to achieve a better understanding of the implications of shade tolerance on resource translocation, future works need to include as factors: (1) the level of tolerance; (2) the microenvironmental parameters in the experimental design; and (3) the ramets size and their interconnections, and the parent tree (this means to consider the entire genet) as explanatory variables for resources translocation. Applying all these considerations to a study will help us to elucidate the functional role of clonal growth in trees by means of root suckering, increasing our knowledge of the ecophysiology of clonal propagation through roots in woody species and improving our understanding of its effects and advantages on the dynamics of temperate rainforests.