Inverse relationship between species competitiveness and intraspecific trait variability may enable species coexistence in experimental seedling communities

Domain: earth_science
Published: 2024-04-03
Editorial outcome: minor_revision

Abstract
Theory suggests that intraspecific trait variability may promote species coexistence when competitively inferior species have higher intraspecific trait variability than their superior competitors. Here, we provide empirical evidence for this phenomenon in tree seedlings. We evaluated intraspecific variability and plastic response of ten traits in 6,750 seedlings of ten species in a three-year greenhouse experiment. While we observed no relationship between intraspecific trait variability and species competitiveness in competition-free homogeneous environments, an inverse relationship emerged under interspecific competition and in spatially heterogeneous environments. We showed that this relationship is driven by the plastic response of the competitively inferior species: Compared to their competitively superior counterparts, they exhibited a greater increase in trait variability, particularly in fine-root traits, in response to competition, environmental heterogeneity and their combination. Our findings contribute to understanding how interspecific competition and intraspecific trait variability together structure plant communities.

Introduction
There is substantial trait variation among individuals of the same plant species1. This intraspecific trait variability (ITV) has great potential to impact species coexistence2,3 because it can affect the competitiveness of species4–7 and their long-term population growth rates8,9. However, the evidence regarding whether ITV promotes or inhibits species coexistence is mixed10. For example, while Begon and Wall11 reported that ITV can promote coexistence by preventing competitively superior species from driving inferior ones to extinction, Hart et al.12 showed that ITV can accelerate competitive exclusion by increasing the dominance of superior competitors.

Although the relationship between ITV and species coexistence is intricate, the relative magnitude of ITV between competing species has been proposed to be a key determinant of this relationship13. For instance, ITV is expected to enhance coexistence when the competitively inferior species exhibit greater trait variation among its conspecifics than the superior species10,12. Such is the case of tree species whose seed quality strongly correlates with their competitive ability at the seedling stage, a high diversity of seed qualities can allow competitively inferior species to produce some highly competitive individuals13. Moreover, this variability enables some individuals of the inferior competitors to occupy niche space beyond the niche of the superior competitors. Consequently, when this negative relationship between ITV and species competitiveness occurs, some individuals of the inferior competitors have the same or greater trait value (i.e., seed quality) than do those of the superior competitors14, thus bolstering the persistence of their populations11,15. Although this negative relationship has been widely shown to promote species coexistence in mathematical models12–14,16–20, empirical evidence of this relationship remains insufficient.

There are conflicting predictions regarding the relative magnitude of ITV between competing species. First, if a competitively superior species occupies a small niche, the competing inferior species may exhibit greater ITV to utilize the remaining unoccupied niches in the environment21. For example, while superior competitors may benefit from deep roots to monopolize soil resources, inferior competitors might adapt either by extending their roots deeper22 or by spreading them laterally to access shallow soil resources23, resulting in a multipeaked root depth trait distribution with higher ITV (Fig. S1a). Conversely, if the superior competitors already occupies most of the niche space24,25, the inferior competitors may compress its trait values to maximize its survival in the remaining limited niche space. This may result in a smaller ITV in the inferior competitors. Alternatively, inferior competitors might inherently possess lower trait variability26,27 (Fig. S1b). Furthermore, if the above differences in resource use arise from inherent distinctions between species’ fundamental niches28, the magnitudes of ITV could diverge among species even in the absence of competition. Thus, any of these scenarios could occur even in competition-free environments.

The relative magnitude of ITV among competing species may change over a short time26,29. It is widely recognized that phenotypic plasticity can alter many functional traits30–32, and the direction and magnitude of these trait changes often vary among conspecifics28; thus, a trait’s plastic response to competition potentially increases ITV. Consequently, phenotypic plasticity resulting from competitive interactions can amplify ITV, resulting in a complex interplay between ITV and competition. For example, when the roots of neighboring plants come into contact, one species may respond by producing deeper roots, mitigating competition22. Alternatively, both species may simultaneously exhibit root elongation, intensifying competition33. Moreover, the sensitivity of ITV to competition may differ between competing species. For instance, one study showed that after six months of interspecific competition, the extent of the plastic response of the specific leaf area (SLA) of herbaceous plants in Central Europe increased in parallel to competitive suppression, which was quantified based on plant biomass6. This implies that the competitively inferior species experienced greater competition-induced trait changes, leading to a greater amount of competition-induced ITV. Similarly, in an experiment with Mediterranean annual plant species, the competitively inferior species experienced greater changes in trait values when competing with competitively superior species, resulting in reduced competitive hierarchies and favoring coexistence7. These findings imply that the impact of competition on ITV might be asymmetrical between competitively superior and inferior species34, triggering the inverse relationship between species competitiveness and ITV. Despite these examples, little is known about how much competition can change the relative magnitude of ITV among competing species or whether plastic responses to competition could alter the hypothesized negative relationship between competitive abilities and ITVs across different species.

The relative magnitude of ITV among competing species can become highly complex when considering multispecies interactions in heterogeneous abiotic environments35,36. Unlike simple pairwise competition, multispecies interactions involve more than two species, thus possibly reducing unoccupied niche space as the total number of species increases37. However, heterogeneous abiotic environments can expand the overall available niche space, potentially increasing unoccupied niches36,38. The uncertainty surrounding vacant niches in such ecosystems makes it difficult to predict how competitively inferior species might adapt their ITV to access untapped resources and respond to competition. Furthermore, abiotic environments may also affect the magnitude of ITV by either directly filtering out individuals with unfit trait values20,39 or indirectly adjusting interspecific competition, which in turn alters the extent and direction of plastic responses of traits40. Therefore, inferior competitors face more complex abiotic and biotic filters (e.g., different environmental filters and various interspecific competitions) in spatially heterogeneous environments than in homogeneous environments, making it uncertain whether the negative relationship between ITV and species competitiveness remains across spatially heterogeneous environments.

Here, we investigated whether the negative relationship exists between species under competition-free, pairwise competition and multispecies competition conditions in both homogeneous and heterogeneous environments. We conducted a two-phase seedling competition experiment (Fig. 1) and monitored ten key traits in 6,750 seedlings of ten coexisting native tree species (Table 1) over a three-year period. Three different competition scenarios were considered: two-species competition in a spatially homogeneous environment (Phase I), multispecies competition in a homogeneous environment, and multispecies competition in a spatially heterogeneous environment (Phase II). Each scenario included two treatments: competition-free and competition. We quantified the mean competitive ability of the species by comparing total seedling biomass between the competition and competition-free treatments in each scenario. We correspondingly estimated the ITVs for each species in both treatments in each scenario using two methods: multidimensional trait space41–43 and individual trait variability (Bao’s coefficient of variation)6,7,44,45. Based on the competitiveness and ITVs of these species, we aimed to address the following questions: (1) Do competitively inferior species have larger ITV than competitively superior species—i.e., negative relationships between ITV and species competitiveness – in environments with homogeneous abiotic conditions and no competition? (2) Does this relationship between ITV and species competitiveness under homogeneous abiotic conditions persist in the presence of pairwise competition? (3) Does this relationship under homogeneous abiotic conditions persist in the presence of multispecies competition? (4) Do the patterns change under heterogeneous abiotic conditions? Our results showed that a negative relationship between ITV and species competitiveness is absent from competition-free homogeneous environments but occurs under pairwise or multispecies competition, as well as in heterogeneous abiotic environments. We found that the emergence of the relationship is driven by a greater increase in ITV in the inferior competitors than in the superior competitors in response to pairwise or multispecies competition and environmental heterogeneity.

Results
Absence of the negative relationship in a competition-free environment
Under a competition-free and homogeneous abiotic environment, no significant difference was found in the ITV between the competitively inferior and superior species in the eight pairs of species examined in the Phase I experiment (pairwise Wilcoxon test, V=23, P=0.273; Fig. 2a). At the species level, five out of the eight inferior competitors had significantly greater ITVs than did their superior counterparts (black asterisks, Fig. S2a), while the other three inferior competitors (Cyclobalanopsis myrsinifolia, Quercus chenii and Schima superba) had significantly lower ITVs (red asterisks, Fig. S2a). Similarly, there was no significant linear relationship between the ITV and the competitiveness of species under the competition-free treatment in the homogeneous environment of the Phase II experiment (P=0.099; Fig. 2b and Table S1). Furthermore, visualizations of the reduced-dimensional hypervolume of the traits revealed a more detailed but similar pattern (Figs. S3a–c and S4a–c).

Emergence of the negative relationship under competition
At the end of our experiment, interspecific competition caused significant reductions in biomass in all the species (Table 2, S2). Visualization of hypervolume showed that competition altered the shape, range, and center of the multidimensional trait hypervolumes of most species (Fig. S4). The three inferior competitors with lower ITVs in the competition-free environment of Phase I had significantly greater trait variability under the competition treatment (Fig. S2b). Consequently, inferior competitors exhibited significantly greater ITV than their superior counterparts in pairwise competition in a homogeneous environment (V=36, P=0.004; Fig. 2d). There was also a significant negative relationship between ITV and the competitiveness of species under multispecies competition in the same abiotic environment (that is, environmental block 1) (R2=0.60, slope=−8.84, P=0.025; Fig. 2e and Table S1). Furthermore, similar negative patterns were also found in homogeneous environments by mixed-effects models based on 7-dimensional (pseudo R2=0.061, slope=−3.68, P=0.033; Fig. S5b) and reduced-dimensional hypervolumes (pseudo R2=0.074, slope=−1.55, P=0.017; Fig. S5d). Similar trends were also found when the two deciduous species were excluded (Fig. S6). All of these results support that an inverse relationship between species competitiveness and ITV occurs under interspecific competition.

We further discovered that the emergence of the negative relationship when transitioning from competition-free to competition treatment was caused by the different responses of competitively inferior and superior species to competition. Inferior competitors exhibited greater increases in ITV than did their superior counterparts in pairwise competition (V=35, P=0.007; Tables S3). Under multispecies competition, the change in ITV when transitioning from the competition-free to the competitive treatment was significantly and negatively correlated with species competitiveness (Table S4). Thus, species that suffered more competitive suppression responded to competition with a greater increase in ITV (R2=0.61, slope=−13.25, P<0.001; Fig. 3a).

The negative relationship is also influenced by heterogeneous environmental conditions, including light, soil moisture and phosphorus content. In the competition-free treatment, we observed the emergence of the negative relationship with transitioning from homogeneous to heterogeneous environments (Fig. 2b, c). Under competition, the negative relationships between ITV and species competitiveness became even stronger, with changes in slope and R2 from −8.84 and 0.60 in homogeneous environments to −14.96 and 0.85 in heterogeneous environments, respectively (Fig. 2e, f and Table S1). Most of the change in slope was attributed to the greater increase in ITV for competitively inferior species in heterogeneous environments than for superior species (Fig. 3b).

Similar patterns at the individual trait level
The patterns of intraspecific variability in individual traits were similar to those observed when considering all traits using multidimensional hypervolume, although there were some differences (Fig. S7). Overall, competitively inferior species had significantly greater intraspecific variability in individual traits than competitively superior species in the competition treatment (P<0.001, black solid line in Fig. S7b), while no significant difference was found in the competition-free treatment (P=0.062, black dashed line Fig. S7a). Specifically, inferior competitors had significantly greater conspecific variability in terms of root tissue density and specific root length in the competitive treatment (Fig. S7b) but lower variability in leaf toughness in both the competition-free and competitive treatments (Fig. S7a, b). In addition, intraspecific variability increased for most of the competing species for all the root and leaf traits that were measured except for leaf toughness, whose variability decreased (Fig. 4 and Table S5). Additionally, three fine-root traits exhibited the greatest relative increase in intraspecific variability (124.80±19.51%, Fig. 4).

Methods
Study site and species selection
The experiment was performed in a greenhouse located at the southern foot of Taibai Mountain in Tiantong National Forest Park, Ningbo, Zhejiang Province, China (29°48.817´N, 121°47.116´E), which has a subtropical monsoon climate with hot and humid summers, dry and cold winters, an annual mean temperature of 16.2°C and precipitation of 1374.7 mm. The soil in the study area is a montane yellow-red soil characterized by a predominantly loamy and clay texture with a slightly acidic soil pH ranging from 4.4 to 5.1. The forests in this area are subtropical evergreen broad-leaved forests with deciduous tree species69.

We initially selected 16 dominant and codominant native tree species based on previous knowledge about the forest community composition in the area69, life forms (11 evergreen and 5 deciduous species, as listed in Table S7), phylogenetic relationships (Fig. S11), and seed availability. In 2016 and 2018, more than 2000 healthy and intact seeds per species were collected. Specifically, we exclusively sourced seeds from a single mature, healthy adult tree located within our study area. This approach was used to minimize the genetic variation among conspecific seeds and maternal effects among species.

These collected seeds were then sterilized with insecticides (imidacloprid and carbendazim) before being stored over winter for sowing in seedling trays in April of the respective experimental years. Throughout the germination period, the seedlings were exposed to full sunlight, and the seedlings were adequately watered. The soil used for seedling rearing was collected from the topsoil (15–30 cm, excluding nitrogen-rich soil at 0–15 cm depth) of the forest where the mother trees grew. The soils were sieved through a 1-cm mesh to remove large particles and organic debris and homogenized before the experiment. The prepared soil had 6.10 g/kg total carbon, 0.4 g/kg total nitrogen, 0.45 g/kg total phosphorus and a pH of 5.21. Due to the low germination rates of some species (Table S7), we ultimately reduced the number of experimental species to 10, with 8 species in Phase I and 7 species in Phase II (Table 1). The seedling height at transplantation was approximately 6–25 cm, with deciduous species averaging 12–25 cm and evergreen species averaging 6–13 cm (Table S7). This difference in height between deciduous and evergreen species may cause interspecific asymmetric competition, which is observed at the seedling stage in tree species70.

Experimental design
To test the assumption that a negative relationship exists between species competitiveness and ITV across various competition scenarios, we conducted a two-phase tree seedling competition experiment over three years (Fig. 1). In Phase I (2017–2018), we examined the relationship in a two-species pairwise competition in a homogeneous environment (Fig. 1a). We expanded this examination to Phase II (2019–2021) in which multispecies competition in both homogeneous (Fig. 1b) and heterogeneous abiotic environments (Fig. 1c) were considered.

Phase I: pairwise competition in a homogeneous environment. From June 2017 to August 2018, we carried out a pairwise competition test with eight tree species in a spatially homogeneous environment within a greenhouse. Given the constraints of seedling availability, we chose eight species pairs in this phase (Table 2). The species pairs were selected to have an even distribution of phylogenetic distances among species (Fig. S11) to maximize the representation of species with different competitive abilities based on the competition-relatedness hypothesis71. In June 2017, we transplanted seedlings of the eight selected species into pots with a diameter of 15 cm and a height of 15 cm. The seedlings were divided into two planting treatments (Fig. 1a): competition-free and competition. In the competition-free treatment, only one seedling of each selected species was planted in a single pot. In the competition treatment, two seedlings, one from each competing species, were planted in a pot. To ensure a fair comparison between treatments, the initial height and base diameter of the seedlings were kept similar for each species among the pots. If a seedling died within the first month after transplanting, it was replaced with a similar-sized conspecific seedling. We carefully kept the distance between two seedlings within a pot within 3–5 cm to facilitate competition among seedlings within the pots while keeping the inter-pot distance fixed at 15–20 cm to minimize potential interference between pots. For each species in the competition-free treatment and for each species pair in the competition treatment, 45 replicate pots were established, resulting in a total of 720 pots with 1080 seedlings. A total of 78 individuals with at least one missing trait value were excluded from the following analyses in Phase I.

Since we focused primarily on the effects of competition on ITV, we installed insect screens and regularly sprayed pesticides to prevent potential herbivory. We also maintained similar abiotic conditions (particularly light intensity, soil moisture, and soil nutrient content) within and between all pots in Phase I. To ensure this homogeneity, all pots contained the same quantity (2±0.01 kg) of well-mixed soil as that used for germinating seedlings. Additionally, the light intensity was kept as similar as possible in the greenhouse, and the pots were randomly rearranged every two weeks during the experiment to minimize the effects of ambient conditions. The soil moisture was maintained at approximately 33.04±0.28% by adding 200 ml of water to each pot every 3–5 days. In this study, we refer to the experiment in Phase I as a two-species competition experiment in a homogeneous environment.

Phase II: Multispecies competition in homogeneous and heterogeneous environments. In June 2019, we conducted a multispecies competition experiment with seven tree species (Table 1) in nine abiotic environmental blocks (Fig. 1c) in the same greenhouse. Seedlings within each environmental block shared the same abiotic conditions, so that each environmental block represented a spatially homogeneous environment. However, seedlings in different environmental blocks experienced different abiotic conditions; thus, the nine environmental blocks comprised a spatially heterogeneous environment (Fig. S12), similar to the findings of the studies of Allesina & Levine72. Both the homogeneous and heterogeneous environments included competition-free and multispecies treatments.

The nine abiotic environmental blocks (Fig. 1c) were selected according to a three-factor and three-level orthogonal design (Table S8). These abiotic environments effectively represented the abiotic conditions in the study area while considering practical constraints and experimental feasibility. The three factors considered were light (L), soil moisture (M), and soil phosphorus content (P), all of which are critical for seedling growth. Different layers of shade netting around and on top of the greenhouse, along with varying watering frequencies and fertilizer concentrations (see next paragraph for details), were used to control the three abiotic environmental factors at low, medium and high levels (represented by 1-3) based on real environmental data from previous surveys69. The abiotic conditions for the nine blocks (named blocks 1-9) were as follows: L3-M3-P1, L3-M1-P3, L3-M2-P2, L2-M2-P1, L1-M1-P2, L1-M3-P3, L2-M2-P3, L2-M1-P2 and L2-M3-P1 (Fig. S12). In Block 1 (L3-M3-P1), the abiotic environment was identical to that in Phase I, which facilitated a direct ITV comparison between pairwise and multispecies competition.

Specifically, two-layer and one-layer shade nets around and above the plants created two light gradients: L1 and L2, respectively. The third light level, L3, was one with no shade net. The mean (±SE) light intensity, measured by an LI-1500 (LI-COR, USA) on a sunny summer day from 8 am–5 pm, was 14.54±1.99 μmol⋅m−2⋅s−1 in L1, 502.91±103.09 μmol⋅m−2⋅s−1 in L2, and 1237.20±195.45 μmol⋅m−2⋅s−1 in L3. For soil moisture, watering intervals of six days (M1), four days (M2), and two days (M3) were utilized in summer, reducing these intervals by one day for every 3°C temperature decrease. The mean (±SE) volumetric soil moistures throughout the year were 21.36±0.28% in M1, 28.76±0.22% in M2 and 33.04±0.28% in M3. To create three levels of soil phosphate availability, each pot was fertilized monthly with 200 ml of 0 g⋅ml−1 (P1), 0.2 g⋅ml−1 (P2) or 0.4 g⋅ml−1 (P3) liquid phosphate (NaHPO4). After two years, the mean (±SE) soil phosphate concentration, measured by the Mo-Sb colorimetric method, was 0.49±0.04 g/kg in the P1, 0.89±0.02 g/kg in the P2, and 1.07±0.05 g/kg in the P3.

As in the Phase I experiment, we imposed two treatments in each environmental block: competition-free and competition (Fig. 1b). In the competition-free treatment, only one seedling of each species was grown in a separate pot. In the competition treatment, seven seedlings, each from a different species, were grown together in a pot. Healthy seedlings of similar initial heights were selected for each species (Table S7) and transplanted into the pots. The distance between seedlings within a pot was 3–5 cm. The pot size, spacing between pots, soil used, replacement of dead seedlings and insecticidal activity were all consistent with those used in the Phase I experiment. For each species in the competition-free and competition treatments in each abiotic environmental block, 45 replicate pots were established (360 pots per environmental block), resulting in a total of 3240 pots with 5670 seedlings. A total of 599 individuals with at least one missing trait value were excluded from the subsequent analyses in Phase II.

Estimation of competitive rankings
The competitive ranks among species were determined using the relative interaction intensity index (RII), which is a commonly used, unbiased estimation of competition intensity73. The smaller the RII is, the greater the level of competitive suppression experienced by the species. Specifically, for a group of species i and j in Phase I, the RII of species i was calculated as (Bi,paired - Bi,alone)/(Bi,paired + Bi,alone), where Bi,paired and Bi,alone are the average individual dry biomasses of species i in the competition and competition-free treatments, respectively. If RIIij ≥ RIIji, species i is less affected by species j and is thus considered to have a greater competitive ability than species j. This means that in this group, species i is competitively superior and species j is inferior. Species i and j will retain the labels of superior and inferior species, respectively, for that species group in the competition-free treatment. We use the term ‘competitiveness’ to denote the competitive ability of species as determined by the above RII. Importantly, there were eight groups of species in Phase I, and a species could be either competitively superior or inferior in different groups, as competitive ability is not a constant attribute of a species and can be altered by different competitors and by resource availability.

For the Phase II experiment, the RII for species i in environmental block n was calculated as (Bi,n,mixed - Bi,n,alone)/(Bi,n,mixed + Bi,n,alone), where Bi,n,mixed and Bi,n,alone are the average individual dry biomasses of species i in the competition and competition-free treatments of the block, respectively. We calculated the RII of each species in each environmental block (Fig. S13). As RII values for the same species varied across different blocks, species competitiveness was determined independently in each homogeneous environment. In heterogeneous environments, we determined species competitiveness by calculating the mean RII for each species in all nine blocks. To estimate the total biomass of individual seedlings (B), we harvested each seedling, including both aboveground and belowground parts, at the end of each experimental phase and dried them at 75°C for 72 h to determine the total dry biomass (sum of aboveground and belowground dry weights).

Notably, the biomass-based RII was more appropriate than the methods based on relative abundance or frequency. This approach aligned with Begon’s definition of competition, which he characterizes as an interaction among individuals competing for limited resources, leading to reduced growth, survival, and reproduction rates11. The RII estimates competition by comparing biomass, mortality or reproduction for a given species between the presence and absence of competition73. Experimental investigations often prioritize the seedling stage of trees, opting for biomass as a proxy for competition due to the challenges of obtaining mortality and reproduction data over short durations, such as a year6,74,75. On the other hand, the intensity of competition varies with the life history and abiotic environment of the plant, which causes it to be impossible to determine the competitive rank of two species in advance by pre-experiments or the distribution range of species in natural communities76. And the latter is affected by both competitive ability, soil seed bank, spatial and temporal heterogeneity, successional stage and resource situation24,77,78. This shows that species with high abundance in the community do not necessarily have high growth rates at the seedling stage. Therefore, in our seedling experiments, the competitive superior and inferior were defined after the experiment based on the final biomass, which represent the total competitive benefit of the plant.

To validate the competitive rank estimations derived from the RII, we also employed additional methods, such as the log response ratio (lnRR)79 and the neighbor-effect intensity index with commutative symmetry (NIntC)80. Given their consistent results with the RII, we exclusively report the findings from the RII for simplicity and clarity.

Trait measurement
To ensure accurate estimation of the ITV45, 45 replicates were performed for each species in the competition treatment in each environmental block. This process resulted in a total of 1080 and 5670 seedlings in the Phase I and Phase II experiments, respectively. After one year (two years for Phase II) of the experiment, we measured ten key functional traits for each seedling (Table S9) following a standardized protocol81. These traits included relative leaf chlorophyll content, leaf dry matter content, leaf mass per area, leaf toughness, leaf thickness, stem-specific density, stem moisture content, specific root length, specific root area and root tissue density. A detailed description of the trait measurements is given in Part 2 of the Supplementary Methods.

Specifically, relative leaf chlorophyll content (Chl) was measured on fully expanded leaves in situ using the Chl meter SPAD-502Plus (Konica-Minolta, Japan). The meter probe was placed at the adaxial leaf surface while avoiding pinching a mid-vein, measured three times, and averaged per leaf in each seedling. Chl of all seedlings was measured on the same day from 9:00 to 12:00 AM82 (Marenco et al.82). Three leaves were randomly selected for leaf toughness (LTO) measurements using the “punch test” method83 with a digital force gauge (precision 0.001 N, HADPI, Leqing, China). The induction needle (diameter is 1 mm) of the digital force gauge moved down at a constant speed of 10 mm⋅s−1, piercing the middle position of each blade, and avoided primary and secondary veins. This was repeated three times at different positions, and then averaged the maximum force of three times as leaf toughness (GN) measurements were averaged. For measuring other leaf traits, twenty complete and healthy mature leaves were randomly collected for each seedling at 5 a.m. in the following days and taken to the laboratory in a refrigerated incubator. The leaf thickness (LTh, mm) was first measured with a micrometer at five locations on the selected leaf. Then we immediately weighed all leaves to quantify leaf fresh weight (g) and measured leaf area (cm2) with a leaf area meter (LI-3000C, America). Finally, all leaves were dried to a constant weight in a well-ventilated oven at 75°C as leaf dry biomass (g). Leaf dry matter content (LDMC) was the leaf dry biomass of an individual divided by its leaf fresh weight. Leaf matter area (LMA) was the ratio of leaf dry biomass to leaf area.

After measuring the leaf traits, all seedlings were harvested to measure the traits of stem and root traits. To estimate stem-specific density (SSD) and stem moisture content (SMC), one main stem was taken on 5 cm above the base of each seedling. Fresh volume was measured using the water displacement method within 24 h. Then the same section was dried for 72 h in a well-ventilated oven at 75°C until a constant mass was achieved as stem dry weight. SSD was calculated as the ratio of stem dry weight and the fresh volume. SMC was defined as the difference between the fresh and dry weight of the stem divided by the dry weight of the same section. To measure root traits, we carefully cleaned the root surface from soil and attached organic particles with a 2 mm sieve under the shower head faucet until it was clean. Then, about 2 g of fine root samples less than 2 mm in diameter were used to scan fine root images (400 dpi) using a WinRhizo (Epson Expression 10000XL Scanner) flatbed scanner. Before scanning, the fine roots were scattered in trays without overlapping each other. Each fine root image was manually removed from background impurities (e.g., shadows and stains) using Adobe Photoshop (Adobe Systems), and was analyzed using the root analysis software WinRHIzO (Arabidopsis version 2012b, Regents Instruments Inc., Quebec Canada) to obtain total root length (cm), root area (cm2) and root volume (cm3) of the fine roots. Thereafter, each scanned root sample was placed in an envelope and dried to obtain its dry biomass (mg). Specific root length (SRL) and specific root area (SRA) were calculated as the ratios of total root length and root area to root dry biomass, respectively. Root tissue density (RTD) was the ratio of fine root dry weight to fine root volume.

Finally, the remaining aboveground and root (excluding the parts used to measure leaf, stem, and fine root traits) were dried in an oven for 72 h at 75°C, and their corresponding dry biomass for each seedling was obtained. Total shoot biomass was the sum of the dry weights of the leaves, stem, and remaining aboveground parts, and total root biomass was the sum of the fine roots and remaining root.

Estimation of intraspecific trait variability
The ITV of each species was quantified using two different approaches: a multidimensional trait approach and an individual trait approach. The first emphasizes the importance of trait covariance in determining species fitness41–43,84, while the latter highlights the unequal roles traits play in fitness6,7,44. Both approaches have unique strengths and can offer different insights into the relationship between ITV and species competitiveness. Specifically, the ITV of multidimensional traits was quantified using the size of the multidimensional trait space, also known as hypervolume85,86. To estimate the hypervolume for each species, we used the Silverman bandwidth estimator and applied a 5% quantile threshold to create a multidimensional Gaussian kernel density. To ensure robust estimation of hypervolumes and fair comparisons between hypervolumes, we employed two methods. First, we used seven functional traits (Table S9) with correlation coefficients less than 0.8 (Fig. S14) to estimate hypervolume in both the Phase I and II experiments. Additionally, we estimated a reduced-dimensional hypervolume based on the scores of the first three principal component axes according to the aforementioned traits (Table S10 and Fig. S15)87. The number of principal components required was determined using Horn’s parallel analysis88.

To robustly compare hypervolumes between species across treatments, we first calculated 999 hypervolumes for each species based on random samples of 20 conspecifics for Phase I and 15 conspecifics from either a single homogeneous environment (Fig. 2b, e) or nine different environments with varying conditions (Fig. 2c, f) for Phase II. This process was performed separately under both competition-free and competition treatments using the same method as that used by Bittebiere et al.47. We then took the mean and standard error of the 999 hypervolume values to obtain the mean and variability in the ITV for each species under each scenario (e.g., competition-free homogeneous, competition-free heterogeneous, etc.) (Fig. 2). All trait values were centered and scaled for all individuals across all pots in both Phase I and Phase II, as recommended by Blonder86. The sizes of all hypervolumes in this study are reported in units of standard deviation raised to the power of the number of trait dimensions used, i.e., SD7 and SD3 for the seven-dimensional and the reduced three-dimensional PCA hypervolumes, respectively.

In addition, an individual trait approach was also used to quantify the effect of competition on intraspecific variability for each trait. We first calculated the intraspecific variability for each trait in each species using the improved coefficient of variation (Bao’s CV)45. We then calculated both the absolute and relative changes in ITV from competition-free to competition treatments across the three competitive scenarios (pairwise competition in a homogeneous environment and multispecies competition in homogeneous and heterogeneous environments). A positive change in ITV indicates that the relative variability of the target trait increased under competition; a negative change indicates a decrease in the relative variability of the target trait. In Phase II, we deliberately selected seven (with correlation coefficients less than 0.8) out of ten traits to calculate the ITV, while in Phase I, we used all ten traits. This selection aimed to make the multidimensional trait space comparable between phases. To test whether trait selection introduced bias in Phase II, similar analyses were performed using all the functional traits.

Comparison of intraspecific trait variability between competing species
For Phase I, we used the Paired Wilcoxon rank-sum test to examine whether competitively inferior species had higher ITVs than superior species did, separately, in the competition-free and competition treatments. In Phase II, we tested the relationship between ITV and competitiveness (RII) and whether this relationship differed between competition treatments, separately, in spatially heterogeneous environments and in environmental block 1, which had the same homogeneous environment as in Phase I, allowing comparison of results between the two phases. This was done by fitting mixed-effects models with competition treatments (competition-free and competition), competitiveness (RII), and their interaction as fixed effects and species as a random effect. To confirm the robustness of the results from block 1, we expanded our models to include all nine homogeneous environmental blocks shown in Fig. 1c, with species and environmental blocks as two crossover random factors. We quantified the ITV and RII in spatially heterogeneous environments using aggregated populations from the nine environmental blocks (Fig. S12). To evaluate the potential bias of life forms, we performed similar analyses after excluding the two deciduous species.

To quantify the effect of competition on ITV, also known as competition-induced intraspecific variability, we calculated the absolute and relative changes in multidimensional ITV for each species from the competition-free to competition treatments in both Phases I and II. These changes were compared to the RIIs of competing species using a simple linear regression model. Similar analyses were used to test the relationship between changes in individual trait variability and species competitiveness. The statistical analyses in this study were performed using R software (v.4.0.5; R Core Team, 2019), and the R package hypervolume (v.3.1.1) was used to calculate the multidimensional hypervolumes from the trait data89.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study uses a series of experiments to assess whether the “intraspecific mean-variance trade off” that has been explored with theoretical studies emerges in empirical work. The size of the experiment is impressive and the theoretical framework for this investigation is solid. The results are noteworthy because the authors do in fact demonstrate this trade-off with their experimental work.\nHowever, after reviewing the manuscript several times, I am still confused about why and how specific approaches were taken and wary of the conclusions made in relation to coexistence. Considerable restructuring and clearer justification for specific approaches and experimental decisions is needed to make the manuscript understandable and appealing to a broad ecological audience. My main concerns apply throughout all sections of the manuscript and include: (1) Confusion about what exactly the “interspecific mean-variance trade-off” is. This trade-off needs to be more clearly defined. In providing this information in the introduction, it would be helpful to provide information about how this trade-off can be explored and tested empirically prior to introducing specific hypotheses associated with the study. (2) The experimental design is confusing to me. Specifically, how and why are the heterogenous environments included? Why would someone expect the trade-off to emerge in heterogenous environments but not in a homogenous one? Are the specific treatments (i.e, light, soil moisture, phosphorous) in the heterogenous environment comparable and is grouping these results together reasonable? More information for this experimental decision and associated analyses is warranted. (3) I am skeptical that the experimental design and results of this work actually inform our understanding of coexistence even though implications for coexistence are implied throughout the manuscript. Justification for why competition studies with the specific performance metrics used (i.e., biomass) are reasonable to infer coexistence is needed. Finally, (4) I find parts of the manuscript tangential to the core ideas of this work. For example, IV resulting from adaptations vs. plasticity are discussed in the introduction but not mentioned again in the manuscript. I recommend the authors narrow in on the core questions, core results, and eliminate text related to ideas that cannot be tested and do not add to the overarching message of the work. Specific line comments are below.\nLines 37 – 38: What is the competitive response of a trait? Because this term is frequently used in competition literature in regard to performance outcomes of competition, its use here is confusing. To me, if a trait is responding to competition, this may be assessed as IV resulting from plastic responses to a stressor (i.e., competition in this case), but it is not a “competitive response” in performance outcomes.\nLines 53 – 56: Given the IV is a population or species-level metric, I am unclear about how it affects competitiveness of *individual* members of a species? Here and throughout the manuscript, I think it is important to note that outcomes of pairwise or group competition trails may not directly translate coexistence outcomes. Long-term population growth rates would be needed to make these claims.\nLines 63 – 66: This is a bit unclear. Is the IV for a specific trait being measured, is it multi-dimensional, or does it apply to a performance outcome as suggested here (i.e., seed production)? This ought to be defined early in the manuscript.\nLine 68: As far as I can see, Bolnick et al. 2011 does not refer to differences in competing species IV as the “interspecific mean-variance trade-off”. Because of this omission, I am having a tough time conceptualizing this trade-off. Please explicitly define the trade-off and provide citations for the use of this term that readers can refer to.\nLines 70 – 71: The second part of this sentence seems tangential. If stressful conditions are to be explicitly tested this ought to discussed to a greater extent in the introduction and related to the expected patterns.\nLines 75 – 76: Is the effect or impact of this trade-off for coexistence also absent? If yes, it is worth stating that here.\nLines 77 - 110: I appreciate the discussion of adaptations vs. plastic responses resulting in IV. But, within the present study the authors do not determine whether IV results from adaptations (i.e., genetic differences among individuals within a population), maternal effects (i.e., differences in traits resulting from the maternal environment), or plasticity. I also do not this it is possible within the context of the study. If this text is to be included, it ought to be more directly related to the current study and the implication of these difference origins of IV relation to competition and coexistence ought to be discussed.\nLines 129 – 132: This is actually two questions and I think they ought to be listed individually. Also, please clarify when the environment is homogenous vs. heterogenous for each of these questions.\nLines 132 (and above): Please discuss how IV resulting from heterogenous environments vs. competition are related.\nLine 137: Trade-off is misspelled.\nLines 148 – 155: I am concerned about the potential for maternal effects with this design. Plants could express traits related to the maternal environments in which they are grown, so assessing whether IV results from adaptations vs. plasticity is difficult if seeds from a common environment are not used in competition studies. If the origin of trait differences is not relevant or does not affect results, this ought to be mentioned.\nLine 171: Given that this section also describes the heterogenous environment component of the study, I think this ought to be reflected in the section heading.\nLines 194 – 195: This is confusing given the various abiotic treatments described above. Please clarify.\nLines 239 – 241: Were both aboveground and belowground biomass collected? Could results differ is belowground biomass was also assessed. Please discuss limitations here and throughout.\nLines 261 – 263: I am not convinced that using hypervolumes is the most appropriate approach to assessing the effects of IV on competition since some traits, but not others, can be responsible for competitive outcomes among species. See Kraft et al. 2015 PNAS https://doi.org/10.1073/pnas.141365011 and Funk & Wolf 2016 Ecology https://esajournals.onlinelibrary.wiley.com/doi/full/10.1002/ecy.1484 for examples. If the authors choose to retain these analyses, justification for using hypervolumes or other aggregate measures of multi-dimensional trait variability in relation to competition ought to be mentioned in the introduction.\nLine 309: How and why are results combined from from the heterogenous environments? Do species vary in traits that allow them to be more or less successful in these different treatments? If yes, could this bias results?\nLines 466 – 478: I’m wary of the discussion related to the directionality of changes in traits since the results presented do not mention directionality of trait changes, just changes in IV. The discussion ought to more directly relate to the results presented.\nLines 487 – 4892: Given that competition intensity does not necessarily translate to long-term coexistence, I think this conclusion is a bit of a stretch here and throughout the manuscript.", "reviewer_2": "Overall comments\nThis manuscript seeks to clarify how intraspecific trait variation can mediate competitive species interactions (and coexistence), and how IV might vary in response to the environment. This is an important area of research that is largely overlooked, and the authors use an extensive dataset of seedlings to address their questions. I struggled to follow the paper conceptually, primarily due to the vagueness of the text and the lack of clear examples. The main hypothesis, that inferior species should have more IV than superior species, is not an intuitive one, and the authors should explain the mechanism in more detail as it would seem that the amount of variation should depend on the trait in question.\nFor example, when there is strong competition for light, I would expect strong directional selection for taller stature in the superior species (and possibly less IV), whereas I would expect no influence on IV in height among the inferior species. The inferior species may instead grow laterally, or make thicker leaves with more photosynthetic tissue, or alter the leaf angle, such that there is no impact on height. I was looking for more explanation and examples of this sort within the text. But it seems as thought it was written in a very broad way so as to describe trait variation in general, which I don’t think worked well.\nApart from the conceptual issues, I had many questions about the methods and the experimental design, which was also lacking some detail. I thought the figures were very well made as were the tables, but again, I really struggled with the conceptual side of this paper, and I wasn’t fully convinced that IV should be higher in the inferior species, at least not without more examples from their own study and from previous studies. The one section of the paper where they do provide examples using below- and aboveground traits is also incorrect in that they incorrectly assert that higher root tissue density means a more acquisitive strategy, when it is actually the opposite. And there’s one short sentence that notes that the patterns of variation in IV will depend on the trait in question.\nLine by line edits\nLine 35 How is an inferior or superior species defined? I did go to the appendix eventually but it seems it should be better defined early on.\nLine 37: how is a competitive response evaluated? In terms of a reduction in plant size? For example?\nLine 39: is it a greater increase in IV or the amount of IV itself? If it’s the former, then that’s slightly different from the theory and perhaps needs clarification.\nLine 43: incomplete sentence\nLine 63: this sentence is a bit unclear. When two species have the same IV, the superior species is defined as that which benefits more from having a wider trait space (or possibly ecological niche?). I don’t understand how the increased per capita seed production comets into play. Because a wider trait space would mean that some individuals have lower trait values and some have higher trait values, in other words often a greater spread of values. I suppose the key point here is that it’s not just about the mean trait values but also the spread around those values that determines the strength and direction of the interaction. I think this paragraph could be clearer.\nLine 78: between competing species or between species in general? It’s an important distinction as far as theory.\nLine 79: suppression in terms of plant size? Survival? Reproduction? I just want to know how this is defined.\nLine 90: I didn’t follow this sentence; can you explain further?\nLine 95: unclear why 3 years was used as an example. What trait was measured in these example studies that are referenced? And some species don’t even live 3 years so again, the example seems arbitrarily chosen. Some traits change within seconds to minutes.\nLine 98: unclear what is meant by different changes in IV between competing species. Do you mean the sensitivity of IV could vary across species? That’s my interpretation based on the following sentence.\nLine 101: I don’t see how one results from the other. I don’t follow the logic here. Just because a species is an inferior competitor doesn’t mean it’s going to experience more (or less) trait variation. More explanation is needed to substantiate this argument.\nLine 103: vague\nLine 107: can you provide an example? For example, plant height is a trait that would vary in response to increasing abundance/density of neighbouring plants (whether the same species or different species). But height wouldn’t necessarily become more variable, if there is strong directional selection to become taller in response to more competition.\nLine 144: please add the country\nLine 163: approximately how large were the seedlings at the time of transplant? I think the seedling size at the beginning should have a strong impact on future competitive ability. Were these species reared in high light, high moisture, high nutrients? There’s almost no information about the environmental conditions the plants experienced.\nLine 168: how far apart were the seedlings? If the seedlings were quite small, they would have weak competition but if one was larger than the other, it would be asymmetric. Note: this information is presented on line 205 and should definitely be moved up sooner.\nLine 181: insufficient detail regarding the environmental conditions. What was the mean and maximum for each environmental property? Providing the minimum light doesn’t tell us much about potential light stress. I see that an appendix is referenced but I don’t think we should need to see the reference for this very basic information about growth conditions. Even just a few sentences would be useful.\nLine 196: can we also know the range of variation in the environmental variables over the course of a day or months? The term homogeneous is somewhat subjective as it depends on the time frame.\nLine 222: it would have been helpful to know earlier that competitive effects were based on biomass\nLine 225 and line 236: the authors say this is to avoid confusion yet it is very confusing. The definition of RII is defined in terms of biomass when a species is within a pair, yet this line says that you use the term competitiveness even when the species is not in a pair. So does this mean that when I see the terms superior or inferior, that this species may not necessarily be in a pair? That it could be on its own? Because then later you note how the characterization of a species as being superior or inferior depends on the identity of the other species within the pair.\nLine 230: and by resource availability\nLine 279: is it possible to also calculate the amount of variance around the mean hypervolume? Based on Figure 2 it looks like you have the standard error\nLine 407: I disagree that this is the case for all traits. That’s my main argument. That some traits will tend to be quite variable within species even in the absence of competition or heterogeneity. For example, leaf size is hugely variable in some species due to leaf ontogeny.\nLine 418: optimal being defined in terms of max biomass?\nLine 418: a concave down trait performance curve but for which trait(s)? I don’t see how you can generalize so broadly.\nLine 420: why would an individual produce a suboptimal trait value in response to a cue? Are you suggesting that an inferior species in low moisture, might have some individuals that make deeper roots and others that make shallower roots, even if the more shallow-rooted individuals perish? Is this part of the reason why these species are inferior in the first place? Are you suggesting this is maladaptive? It seems that there should be more discussion about alternative resource use strategies and performance landscapes (sensu Laughlin).\nA concave down trait performance curve versus a concave down doesn’t tell us anything about the extent of variation though. They could have different shapes but the exact same amount of variation, so the sentence that follows from this (Line 424) doesn’t exactly follow the logic (in my opinion).\nLine 469: negative or positive change? Add + or -\nLine 475: it does not make sense to me why a species would be conservative above and acquisitive below. I think the use of those terms is not helpful in this context. Higher LDMC could be associated with longer leaf lifespan, and higher stem density usually helps to ameliorate water stress, so the higher root tissue density should be a drought adaptive response, not evidence of a more resource acquisitive strategy. Root tissue density also typically increases with a reduction in soil nutrients, so again this isn’t evidence of being more acquisitive on bottom and conservative on top.", "reviewer_3": "This study explores differences in intraspecific variability between competitively inferior vs superior tree species in order to test for an interspecific mean-variance trade-off. The authors use an experimental approach that clearly involved a massive amount of work and is impressive in its scope. The results provide novel experiment evidence of the mean-variance trade-off, which is relevant for species coexistence. Overall, the study makes a nice contribution to our understanding of the role of intraspecific variation in structuring diverse communities.\nMy main concern about the study is that the authors provide no information about where or how the seeds used in the experiment were collected (e.g., over what spatial extent, how many source trees were used, etc). This information is critical for any study of intraspecific variability (IV). If competitively inferior species were rarer in the community and/or produced fewer seeds than competitively superior species, then there might be the need to collect seeds of inferior species from more source trees and over a larger spatial extent, which could bias estimates of IV.\nOn a related note, the authors appear to be using the term IV to mainly refer to phenotypic plasticity. However, IV can arise due to genetic variation and phenotypic plasticity (and there can also be genetic variation in phenotypic plasticity). For the authors’ first question about IV in a competition-free, homogenous environment, then the IV would be the result of genetic variation (since you would not expect to see plasticity under uniform conditions). For questions 2 and 3, about IV under competition and in heterogenous abiotic environments, then they are talking about phenotypic plasticity. The authors need to be clearer about the underlying drivers of IV (eg genetic variation and phenotypic plasticity). This is related to my comment above about seed collection, since that will influence how much genetic variation exists in the seed pool used in the experiment as well as how plastic the collected individuals are expected to be (eg if some species were collected from more heterogenous environments than other species).\nMy other concern is about the influence of growth form on the results. Specifically, the two deciduous species included in the experiment were the most competitively superior in both Phase 1 and Phase 2 experiments. Deciduous species employ a different ecological strategy than evergreen species, which typically means that they differ in other traits as well. Do the observed relationships remain qualitatively similar if removing those two species from the analyses? (I don’t mean whether they are still statistically significant because they probably won’t be just due to the smaller sample size, but rather whether the trend is the same.)\nSome additional comments:\n-Line 65: Why just “per capita seed production”? Wouldn’t any increase in fitness have this effect?\n-Line 69: add “the” before “same”\n-Line 71: add “the” before “Advantage”\n-Line 104: It is not clear here why this would lead to large IV in only inferior species (and not also in superior species)\n-Line 106-107: I don’t follow the logic here – why would it deviate from its optimal trait value. If IV is increased to enhance a species advantage, presumably it is achieving the optimal trait value for a given environment.\n-Line 126: do these 10 tree species coexist in nature? Are they all native?\n-Line 144: indicate the country where the site occurs (China)\n-Line 149: What was the rationale for selecting both evergreen and deciduous (and 11 of one type but only 6 of the other type)? Was there some expectation about how they would differ?\n-Line 151: As mentioned above, there needs to be a lot more detail about seed collection (how many mother trees, over what geographic area and types of environments, were all species collected at the same sites, etc).\n-Line 158: What kind of homogenous environment? What resources were the species likely competing for in that environment (e.g., where soil nutrients limited, was there limited light or water, etc)?\n-Line 180: It is not clear here or anywhere in the main text or supplement how the 9 different environments were selected. It was not all combinations of the 3 variables, so why were some combinations excluded?\n-Line 198: “populations of two competing species” is confusing here because competition was not pairwise in Phase II\n-Line 277: Was this done separately for competition-free and competition treatments?\n-Line 279: The hypervolume values were averaged separately for each species under each scenario (e.g. competition-free homogenous, competition-free heterogenous, etc)?\n-Line 280: State how many individuals had missing trait data\n-Line 288: was the change in IV calculated in absolute or relative (ie % difference) terms? Looking at the graphs, it appears to be absolution differences. Do the results chance if using % difference instead?\n-Line 292-294: Why was this only done for Phase II?\n-Line 303-305: Why wasn’t species included as a random effect? It sounds like you have multiple values for each species in the model (eg one for each treatment), but are not accounting for the fact that values from the same species are not independent of each other.\n-Line 308: Was this done using data from treatments with or without competition?\n-Line 312: Using Phase I or Phase II data (or both)?\n-Line 310-314: Why didn’t you model IV as a function of competition, RII, and RII x competition interaction (with species as a random effect)? That would be a more straightforward test of 1) whether competition affects IV and 2) whether the relationship between RII and IV changes under competition. If you model it that way, are the results consistent?\n-Line 332: change “survival” to “surviving”\n-Line 337: specify that “significant suppression” is referring to reduced biomass.\n-Line 369-370: There is a typo here in the numbers – they do not match up with what is on the graphs (and also how can you have an R-square value of -8.7).\n-Line 380: When it says “individual traits”, it makes it sound like you ran separate models for each trait. But instead, it seems like this is just a single model for all traits. This needs to be better explained both here and in the methods.\n-Line 382: Is this in the homogenous environment only? (Both here and throughout the Results, make sure to specify whether you are referring to homogenous vs heterogenous environment when referring to competition treatment results. It is often not clear.)\n-Line 387: Specify “greatest” compared to what (ie other traits measured).\n-Line 398: change “seedlings stage” to “seedling stage”\n-Line 407-409: How exactly are determining that the traits are optimal? It seems like you are assuming that they are because of the low IV, but low IV does not necessarily mean optimal trait values! Do you also see lower interspecific variation? The fact that you find low IV in a homogenous environment just means that there is little genetic variation in the trait in your seed pool, and that increased IV under heterogenous environments and competition is resulting from phenotypic plasticity.\n-Line 411-413: This makes no sense to me. If IV is selected for by the environment, then presumably individuals would be accurately perceiving the environmental stresses and adjusting trait values accordingly to increase fitness.\n-Line 414 and Line 420: specify what trade-off you are referring to\n-Line 417-422: This section is confusing and hard to follow for anyone who is not already very familiar with the papers being reference. Please rephrase to explain this more clearly.\n-Line 431: remove the word “only”. Previous studies have also found that IV is related to genetic variation, species life history strategies, dispersal modes, etc.\n-Line 460-461: rephrase as “which could allow invasive species to survive in the face of…”. As written, it makes it sound like invasive species are actively choosing this strategy, rather than high plasticity being one of the reasons they become invasive.\n-Line 466: was this higher IV in root traits compared to leaf and stem traits found in all environments? What about if you just look at environments that differ in light only? I would expect above ground traits to response more to differences in light, and root traits to respond more to differences in soil moisture and nutrients. Given that 2 of the 3 abiotic variables being manipulated in the experiment were below ground variables, it is not surprising that root traits would appear to have higher IV. You need to consider the specific abiotic variable when comparing the different traits.\n-Figure 1: again, it is not clear how the 9 environments were chosen since it is not all combinations of the 3 variables.\n-Figure 2 and Figure 3: Include exact P values for significant relationships, not just P<0.05\n-Figure 2, line 692: Do you mean “orange circles, b-d” here? I think it is a typo because only d has orange circles.\n-Figure 3: something is off with the figure legends. For example panel b legend has orange and blue lines for homogenous environment, but there are no orange and blue lines in the graph. Also in panel A, it is not clear what the difference is between blue and purple.\nFigure 4: Is this from a homogenous environment only? Specify in the legend."}, "author_response": "1 Response letter to the reviewers’ comments\n2\n5 variance trade off” that has been explored with theoretical studies emerges in empirical\n6 work. The size of the experiment is impressive and the theoretical framework for this\n7 investigation is solid. The results are noteworthy because the authors do in fact\n8 demonstrate this trade-off with their experimental work.\n10\n12 and how specific approaches were taken and wary of the conclusions made in relation\n13 to coexistence. Considerable restructuring and clearer justification for specific\n14 approaches and experimental decisions is needed to make the manuscript\n15 understandable and appealing to a broad ecological audience. My main concerns apply\n16 throughout all sections of the manuscript and include:\n17 1. Confusion about what exactly the “interspecific mean-variance trade-off” is. This\n18 trade-off needs to be more clearly defined. In providing this information in the\n19 introduction, it would be helpful to provide information about how this trade-off can be\n20 explored and tested empirically prior to introducing specific hypotheses associated with\n21 the study.\n25 conspecifics of a species, while ‘variance’ refers to trait variability (e.g., variance of\n26 functional traits) of the species. The interspecific mean-variance trade-off indicates that\n27 species with the lower mean competitive ability (i.e., inferior species) tend to exhibit\n28 greater intraspecific trait variation. This trade-off was first introduced by Begon & Wall\n29 (Begon & Wall 1987) and formally defined by Lichstein et al. (2007). It has\n30 subsequently gained theoretical support from several theoretical studies (Crawford et\n31 al. 2019; Des Roches et al. 2018; Feniova et al. 2013; Hart et al. 2016; Milles et al.\n32 2020; Uchmański 2021; Uriarte & Menge 2018).\n35 system by growing seedlings of different species together in pots and comparing their\n36 biomass between competition and competition-free treatments (see lines 463-470 and\n37 476-479 for details). These approaches enable us to identify species as superior or\n38 inferior based on a relative competition index (RII) calculated for seedling biomass.\n40 space and individual trait variability, respectively. Lines 505-511 and 535-537 illustrate\n41 these methodological details.\n42\n43 2. The experimental design is confusing to me. Specifically, how and why are the\n44 heterogenous environments included? Why would someone expect the trade-off to\n1\n45 emerge in heterogenous environments but not in a homogenous one? Are the specific\n46 treatments (i.e, light, soil moisture, phosphorous) in the heterogenous environment\n47 comparable and is grouping these results together reasonable? More information for\n48 this experimental decision and associated analyses is warranted.\n50 individuals of the same tree species often grow across diverse abiotic conditions. Such\n51 heterogeneity can alter resource availability and the niche space of the species,\n52 potentially changing the values of many functional traits and the magnitude of ITV\n53 (Banitz 2019; Uriarte & Menge 2018). Heterogeneous environments could also shift\n54 competitive hierarchies among species compared to homogeneous environments\n55 (Wang & Callaway 2021). Elucidating these intricate effects of heterogeneity on the\n56 trade-off is a big challenge, with unclear theoretical expectations. Thus, one aim of our\n57 study was just to explore whether relative ITV magnitude among competing species\n58 differs between homogeneous and heterogeneous environments. We did not expect the\n59 trade-off to exist only in heterogeneous environments. We have now clarified the\n60 rationale for incorporating heterogeneous environments in lines 98-104 in the\n61 introduction and discussed potential mechanisms underlying the observed trade-off\n62 difference between homogeneous and heterogeneous environments (lines 260-273).\n64 abiotic conditions (see Fig. S10 and Table S6 for details). For instance, pots in block 8\n65 had low soil moisture and phosphorus under medium light, while pots in block 1 had\n66 high soil moisture, low soil phosphorus and high light. These distinct pot conditions\n67 mimicked conspecific individuals experiencing diverse heterogeneous environments\n68 across our study area. We have reworded the heterogeneity manipulation description\n69 (lines 414-417, 419-445) to more clearly articulate the methodology.\n71 comparable across different abiotic environments. Our study did not intend to make\n72 such cross-environment comparisons. Instead, following previous studies (e.g.,\n74 one population and calculated their species-level ITV and competitiveness (RII). We\n75 now add a new Fig. S10 that builds on Fig. 1 to further illustrate details about the abiotic\n76 environmental conditions, competitive treatments, and species definitions in both\n77 homogeneous and heterogeneous environments in phase II experiment. We then\n78 explored the relationship between species-level RII and ITV under competition and\n79 competition-free treatments, respectively. This allowed us to test the interspecific\n80 mean-variance trade-off within spatial heterogeneous environments. A more detailed\n81 method description has been added in lines 414-417.\n82\n83 3. I am skeptical that the experimental design and results of this work actually inform\n84 our understanding of coexistence even though implications for coexistence are implied\n85 throughout the manuscript. Justification for why competition studies with the specific\n86 performance metrics used (i.e., biomass) are reasonable to infer coexistence is needed.\n88 proven metrics that can be claimed to genuinely measure competition. Biomass (or\n2\n89 productivity), species abundance, and demographic (growth and mortality) rates are\n90 among those that are often used to measure competition in the literature. Our study\n91 follows this common practice by using biomass to represent performance of the plants,\n92 but admittedly we cannot guarantee biomass is indeed the best measure. Another\n93 uncertainty is that it is difficult to know how long the experiment has to take for\n94 assessing competition of tree species. Because of these issues, we focused our study on\n95 ascertaining whether there are trait ITV trade-offs between inferior and superior species.\n97 coexistence. Thus, the existence of the trade-off would just suggest the possibility of\n98 coexistence. Following this comment, we have now discussed this issue (lines 315-319),\n99 explaining the reason why seedling biomass was used to measure competitiveness (see\n101 coexistence throughout the text.\n102\n103 4. Finally, I find parts of the manuscript tangential to the core ideas of this work. For\n104 example, IV resulting from adaptations vs. plasticity are discussed in the introduction\n105 but not mentioned again in the manuscript. I recommend the authors narrow in on the\n106 core questions, core results, and eliminate text related to ideas that cannot be tested and\n107 do not add to the overarching message of the work.\n109 removing the discussion about adaptation and evolution. We have revised the study to\n110 focus on our central questions and findings (lines 65-73).\n111\n113 5. Lines 37 – 38: What is the competitive response of a trait? Because this term is\n114 frequently used in competition literature in regard to performance outcomes of\n115 competition, its use here is confusing. To me, if a trait is responding to competition,\n116 this may be assessed as IV resulting from plastic responses to a stressor (i.e.,\n117 competition in this case), but it is not a “competitive response” in performance\n118 outcomes.\n120 response\" (line 27).\n121\n122 6. Lines 53 – 56: Given the IV is a population or species-level metric, I am unclear\n123 about how it affects competitiveness of *individual* members of a species?\n125 revised on line 43.\n126\n127 7. Here and throughout the manuscript, I think it is important to note that outcomes of\n128 pairwise or group competition trails may not directly translate coexistence outcomes.\n131 coexistence outcome. We have added relevant evidence of ITV on long-term population\n3\n132 growth on line 44 and discussed this limitation of our experiment on lines 315-319.\n133\n134 8. Lines 63 – 66: This is a bit unclear. Is the IV for a specific trait being measured, is it\n135 multi-dimensional, or does it apply to a performance outcome as suggested here (i.e.,\n136 seed production)? This ought to be defined early in the manuscript.\n138 defined as variation of a single model parameter such as competitive sensitivity (line\n139 53). In our study, the definition of ITV was based on empirically measured trait values\n140 from both one-dimensional and multidimensional trait space (lines 113-115).\n141\n142 9. Line 68: As far as I can see, Bolnick et al. 2011 does not refer to differences in\n143 competing species IV as the “interspecific mean-variance trade-off”. Because of this\n144 omission, I am having a tough time conceptualizing this trade-off. Please explicitly\n145 define the trade-off and provide citations for the use of this term that readers can refer\n146 to.\n148 variance trade-off in the revision in lines 52-61 and provided citations on line 59.\n149\n150 10. Lines 70 – 71: The second part of this sentence seems tangential. If stressful\n151 conditions are to be explicitly tested this ought to discussed to a greater extent in the\n152 introduction and related to the expected patterns.\n154 relationship between ITV and species competition. Thus, discussions related to stressful\n155 abiotic conditions have been removed.\n156\n157 11. Lines 75 – 76: Is the effect or impact of this trade-off for coexistence also absent?\n160 coexistence is absent. However, answering this question is beyond the capacity of the\n161 current experiment, because it would need a more complex design (e.g., response\n162 surface design) and longer experiment for tree species. Therefore, we only focus on the\n163 existence of the trade-off itself but have highlighted this unresolved issue as a direction\n164 for future research in the Discussion (lines 317-319).\n165\n166 12. Lines 77 - 110: I appreciate the discussion of adaptations vs. plastic responses\n167 resulting in IV. But, within the present study the authors do not determine whether IV\n168 results from adaptations (i.e., genetic differences among individuals within a\n169 population), maternal effects (i.e., differences in traits resulting from the maternal\n170 environment), or plasticity. I also do not this it is possible within the context of the\n171 study. If this text is to be included, it ought to be more directly related to the current\n172 study and the implication of these difference origins of IV relation to competition and\n173 coexistence ought to be discussed.\n4\n176 the Introduction section.\n177\n178 13. Lines 129 – 132: This is actually two questions and I think they ought to be listed\n179 individually. Also, please clarify when the environment is homogenous vs.\n180 heterogenous for each of these questions.\n182\n183 14. Lines 132 (and above): Please discuss how IV resulting from heterogenous\n184 environments vs. competition are related.\n186 determining ITV magnitude. Specifically, competition may induce plastic trait changes\n187 that increase ITV, while the abiotic environment may constrain these changes by\n188 filtering out unfit trait values induced by competition. Heterogeneous environments are\n189 expected to have less constraint on traits than homogeneous environments. In addition,\n190 heterogeneous environments may alter the intensity of competition between species,\n191 which in turn has the potential to indirectly affect competition-induced trait changes\n192 and ITV. We have added this information on lines 98-104.\n193\n194 15. Line 137: Trade-off is misspelled.\n196\n197 16. Lines 148 – 155: I am concerned about the potential for maternal effects with this\n198 design. Plants could express traits related to the maternal environments in which they\n199 are grown, so assessing whether IV results from adaptations vs. plasticity is difficult if\n200 seeds from a common environment are not used in competition studies. If the origin of\n201 trait differences is not relevant or does not affect results, this ought to be mentioned.\n203 ability to precisely distinguish the effects of adaptations and plasticity on ITV.\n206 that said, we believe maternal effects was not strong in our experiment, because all\n207 seeds for each species were collected from a single healthy mother tree and all mother\n208 trees were located within a 1 km2 area with similar abiotic environments. We have\n209 added more details about seed collection on lines 349-353.\n210\n211 17. Line 171: Given that this section also describes the heterogenous environment\n212 component of the study, I think this ought to be reflected in the section heading.\n214\n215 18. Lines 194 – 195: This is confusing given the various abiotic treatments described\n216 above. Please clarify.\n5\n218\n219 19. Lines 239 – 241: Were both aboveground and belowground biomass collected?\n221 limitations here and throughout.\n223 seedling. We have revised the text (lines 483-487) to clarify that total seedling biomass\n224 (aboveground + belowground) was used for calculating relative competitive index. In\n225 addition, we stated the above information earlier on lines 113-115.\n226\n227 20. Lines 261 – 263: I am not convinced that using hypervolumes is the most\n228 appropriate approach to assessing the effects of IV on competition since some traits,\n229 but not others, can be responsible for competitive outcomes among species. See Kraft\n230 et al. 2015 PNAS https://doi.org/10.1073/pnas.141365011 and Funk & Wolf 2016\n232 examples. If the authors choose to retain these analyses, justification for using\n233 hypervolumes or other aggregate measures of multi-dimensional trait variability in\n234 relation to competition ought to be mentioned in the introduction.\n236 one should use a multidimensional or one-dimensional trait approach to study ITV\n237 responses to competition (Bennett et al. 2016; Bittebiere et al. 2019; Carmona et al.\n238 2019; Funk & Wolf 2016; Kraft et al. 2015; Laughlin 2014). For example, Funk and\n240 length, low root-shoot ratio, and low leaf nitrogen content in determining a species'\n241 ability to compete at high levels, while Kraft (2015) found that individual trait was more\n242 highly correlated with fitness differences between species, and combinations of\n243 multidimensional traits were more highly correlated with niche differences, which\n244 provides experimental support for species coexistence in multiple dimensions and\n245 challenges the simple use of one-dimensional traits to infer community assembly\n246 processes. We argue that both one- and multidimensional trait approaches have their\n247 unique strengths. We thus assessed the experimental results using both\n248 multidimensional and unidimensional measures. Our results show that both approaches\n249 yielded consistent results, suggesting that the choice of methods has little impact on our\n250 main conclusions.\n252 dimensional measures in Methods section (lines 505-510), and also briefed the two\n253 approaches in Introduction (lines 113-115).\n254\n255 21. Line 309: How and why are results combined from the heterogenous environments?\n257 treatments? If yes, could this bias results?\n259 environments, conspecific seedlings across abiotic conditions were combined as a\n260 population, as conspecifics of a species in nature often exist in spatial heterogeneous\n261 habitats. We have clarified the methodology in lines 414-417.\n6\n263 them differentially successful across distinct environments. Aggregating conspecific\n264 across environments should not bias but rather provide more realistic estimations of\n266 because conspecifics in nature often experience heterogeneous environments.\n267\n268 22. Lines 466 – 478: I’m wary of the discussion related to the directionality of changes\n269 in traits since the results presented do not mention directionality of trait changes, just\n270 changes in IV. The discussion ought to more directly relate to the results presented.\n272 focused on the changing patterns of ITV and their possible drivers (lines 290-301) in\n273 the revised ms.\n274\n275 23. Lines 487 – 492: Given that competition intensity does not necessarily translate to\n276 long-term coexistence, I think this conclusion is a bit of a stretch here and throughout\n277 the manuscript.\n279 coexistence in our conclusion (lines 330-332) and as well as other sections of the text\n280 (lines 315-319).\n7\n283 1. This manuscript seeks to clarify how intraspecific trait variation can mediate\n284 competitive species interactions (and coexistence), and how IV might vary in response\n285 to the environment. This is an important area of research that is largely overlooked, and\n286 the authors use an extensive dataset of seedlings to address their questions. I struggled\n287 to follow the paper conceptually, primarily due to the vagueness of the text and the lack\n288 of clear examples. The main hypothesis, that inferior species should have more IV than\n289 superior species, is not an intuitive one, and the authors should explain the mechanism\n290 in more detail as it would seem that the amount of variation should depend on the trait\n291 in question.\n293 directional selection for taller stature in the superior species (and possibly less IV),\n294 whereas I would expect no influence on IV in height among the inferior species. The\n295 inferior species may instead grow laterally, or make thicker leaves with more\n296 photosynthetic tissue, or alter the leaf angle, such that there is no impact on height. I\n297 was looking for more explanation and examples of this sort within the text. But it seems\n298 as thought it was written in a very broad way so as to describe trait variation in general,\n299 which I don’t think worked well.\n301 our study. Sorry for the vagueness of the original text regarding the trade-off hypothesis.\n303 mechanisms in greater detail with clearer examples (lines 79-87).\n305 discussion of this caveat in Methods (lines 505-510 and 535-537) and Discussion (lines\n306 302-322). However, we did find that ITV decreased in many superior species (Table\n308\n309 2. Apart from the conceptual issues, I had many questions about the methods and the\n310 experimental design, which was also lacking some detail. I thought the figures were\n311 very well made as were the tables, but again, I really struggled with the conceptual side\n312 of this paper, and I wasn’t fully convinced that IV should be higher in the inferior\n313 species, at least not without more examples from their own study and from previous\n314 studies. The one section of the paper where they do provide examples using below- and\n315 aboveground traits is also incorrect in that they incorrectly assert that higher root tissue\n316 density means a more acquisitive strategy, when it is actually the opposite. And there’s\n317 one short sentence that notes that the patterns of variation in IV will depend on the trait\n318 in question.\n320 to make the experimental designs and methods understandable to the general reader. In\n321 the revision, we took great effort to revise the method section (see sections ‘Study site\n322 and species selection’ and ‘Experimental design’). The change is so substantial that the\n323 section has been expanded from 4 pages to 6 pages. We also recognized the need for\n324 more empirical evidence of the interspecific mean-variance trade-off. Unfortunately,\n8\n325 we were unable to find any study that directly tests the trade-off, because no prior work\n326 simultaneously quantifies both ITV and competitiveness between competing species.\n328 in addressing this research gap. That said, we do find two types of indirect evidence\n329 that suggest ITV may be higher in inferior species. First, the ITV might increase under\n330 competition through plastic trait responses. For example, Gruntman et al. (2017) found\n331 the clonal plant Potentilla reptans displayed plastic responses like ‘confrontational’\n332 vertical growth, shade tolerance, and lateral-avoidance when competing with neighbors\n333 varying heights and densities. These responses often vary substantially in direction and\n334 magnitude among conspecifics (Novoplansky 2009). This potentially increase the ITV\n335 of the clonal plant. Indeed, Bittebiere et al. (2019) found competition led to greater ITV\n336 (using a multidimensional trait space based on ramet and connect traits) of two clonal\n337 plants. Second, inferior species may have greater trait plasticity sensitivity to\n338 competition. Bennett et al. (2016) found after 6 months of competition, the magnitude\n339 of specific leaf area (SLA) plasticity in herbs rose proportionally with the competitive\n340 suppression. This implies inferior species experiencing more competitive suppression\n341 would undergo larger competition-induced trait changes. Longuetaud et al. (2013) also\n342 showed that inferior Quercus species had higher crown plasticity, while superior Fagus\n343 sylvatica exhibited lower plasticity in Western European mixed forests. Combining\n344 these two types of evidence leads us to expect that inferior species have larger ITV than\n345 superior species. We have reviewed these studies on lines 76-90.\n347 competition treatment exhibited a trend towards greater ITV for most individual traits\n348 (Fig. S6). Specifically, root traits such as root tissue density (RTD, slope = -0.51, P=\n349 0.018) and specific root length (SRL, slope = -0.46, P = 0.023) both were significantly\n350 and negatively correlated with the species competitiveness. In other words, the greater\n351 the competitive pressure on the species, the greater ITV in these root traits. These\n352 findings are included in our revised manuscript (lines 290-301).\n354 vary across individual traits. For instance, leaf toughness (LTO) in inferior species\n355 displayed lower conspecific variability versus superior species (Fig S6a, b). We also\n356 observed a positive relationship between conspecific variability of LTO and species\n357 competitiveness (Fig. S6). Interspecific competition even contributed to 2.74%\n358 decreased LTO variability (Fig. 4). These observations align with the notion that plants\n359 may adopt specific trait strategies, such as lower LTO, to enhance resource utilization\n360 efficiency and photosynthesis, ultimately supporting faster growth (Kitajima & Poorter\n361 2010). We have expanded the discussion about how conspecific variability differs\n362 among individual traits on lines 302-314.\n363\n365 3. Line 35 How is an inferior or superior species defined? I did go to the appendix\n366 eventually but it seems it should be better defined early on.\n368 in lines 84-85 and 113-115.\n9\n369\n370 4. Line 37: how is a competitive response evaluated? In terms of a reduction in plant\n371 size? For example?\n373 have replaced it with the more precise term \"plastic response\" (line 27), which was\n374 quantified as the change in the species' ITV from competition-free to competition\n375 treatments.\n376\n377 5. Line 39: is it a greater increase in IV or the amount of IV itself? If it’s the former,\n378 then that’s slightly different from the theory and perhaps needs clarification.\n380 corrected it in lines 24-25. We also found inferior species exhibited a greater increase\n381 in ITV when comparing between competition-free and competitive environments (Fig.\n382 2). We also have clarified this point (lines 30-32).\n383\n384 6. Line 43: incomplete sentence\n386\n387 7. Line 63: this sentence is a bit unclear. When two species have the same IV, the\n388 superior species is defined as that which benefits more from having a wider trait space\n389 (or possibly ecological niche?). I don’t understand how the increased per capita seed\n390 production comets into play. Because a wider trait space would mean that some\n391 individuals have lower trait values and some have higher trait values, in other words\n392 often a greater spread of values. I suppose the key point here is that it’s not just about\n393 the mean trait values but also the spread around those values that determines the\n394 strength and direction of the interaction. I think this paragraph could be clearer.\n396 relative magnitude of ITV between species is critical for competition outcomes. In Hart\n397 et al.'s (2016), superior species were characterized by low competitive sensitivity, and\n398 per capita seed production depends nonlinearly on competitive sensitivity in Hart’s\n399 model. So, variation, not just the mean, of the competitive sensitivity among\n400 conspecifics will change the population’s mean of per capita seed production based on\n402 competition outcomes (Bjørnstad & Hansen 1994). Hart et al. (2016) found that\n403 incorporating equal ITV into competitive sensitivity of competing species reduces\n404 coexistence, mainly because superior species benefit more in fitness than inferior\n405 species from increased ITV. ITV promotes coexistence only when inferior species\n406 exhibit much higher ITV, because the greater ITV in inferior species causes some\n407 individuals with equal or lower competitive sensitivity versus superior species. This\n408 bolsters the realized population growth rate of inferior species and enables species\n409 coexistence.\n411 mathematical theories like Jensen’s inequality, marring the understanding of general\n10\n412 readers. In the revised manuscript, we provide a simpler explanation on lines 52-61 for\n413 why the relative magnitude of ITV is important for species coexistence.\n414\n415 8. Line 78: between competing species or between species in general? It’s an important\n416 distinction as far as theory.\n418 competitiveness rankings between species can vary in different competition contexts.\n420 in Quercus chenii - Hovenia acerba pairs but became superior in Cyclobalanopsis\n421 myrsinifolia - Quercus chenii pairs. Thus, we classified species as inferior or superior\n422 based on the observed competitive intensities they experienced. We then examined the\n423 relative ITV magnitude between these real competing species.\n424\n425 9. Line 79: suppression in terms of plant size? Survival? Reproduction? I just want to\n426 know how this is defined.\n428 referred the term \"suppression\" to the reduction of seedling biomass due to the presence\n429 of competing species. We added detailed information in the revision in lines 113-115.\n430\n431 10. Line 90: I didn’t follow this sentence; can you explain further?\n433 magnitude of ITV is not simply linearly correlated with the width of species’ resource\n434 niche. Species with greater ITV do not necessarily have wider resource niche, and vice\n435 versa. This lack of a one-to-one relationship makes it difficult to predict the relative\n436 magnitude of ITV between competing species from a resource niche perspective alone.\n438 when introducing the expected relationships between ITV and species competitiveness.\n440 resource niche width and simplified the sentence in lines 73-74 to make it more concise.\n441\n442 11. Line 95: unclear why 3 years was used as an example. What trait was measured in\n443 these example studies that are referenced? And some species don’t even live 3 years so\n444 again, the example seems arbitrarily chosen. Some traits change within seconds to\n445 minutes.\n447 they can change quickly. We thus removed the temporal information that is not\n448 contextualized here and added the corresponding temporal information in subsequent\n449 examples (lines 81 and 82).\n450\n451 12. Line 98: unclear what is meant by different changes in IV between competing\n452 species. Do you mean the sensitivity of IV could vary across species? That’s my\n453 interpretation based on the following sentence.\n11\n456\n457 13. Line 101: I don’t see how one results from the other. I don’t follow the logic here.\n459 more (or less) trait variation. More explanation is needed to substantiate this argument.\n461 to exhibit greater ITV in lines 79-87.\n462\n463 14. Line 103: vague\n465 competing species (especially inferior species) is that conspecifics of that species have\n466 different directions and magnitudes of trait changes in the competitive environments.\n468\n469 15. Line 107: can you provide an example? For example, plant height is a trait that\n470 would vary in response to increasing abundance/density of neighbouring plants\n471 (whether the same species or different species). But height wouldn’t necessarily\n472 become more variable, if there is strong directional selection to become taller in\n473 response to more competition.\n475 for illustrating variable plastic responses to competition, it was not measured in our\n476 study. As a result, we were unable to provide empirical evidence for height variability\n477 in the subsequent Results and Discussion sections. Instead, we used stem-specific\n478 density (SSD), which was measured in our study, as a representative example to\n479 illustrate the concept. Specifically, taller plants often adopt rapid vertical elongation\n480 strategy to maximize light access above the canopy (Gruntman et al. 2017) and usually\n481 became superior species at seedling stage. This directional selection can result in lower\n486 discussions and specific examples of different ITV responses in superior species on\n487 lines 246-258 in order to simplify the logic flow of the Introduction and allow for a\n488 fuller discussion of this trait-specific pattern later on.\n489\n490 16. Line 144: please add the country\n492\n493 17. Line 163: approximately how large were the seedlings at the time of transplant? I\n494 think the seedling size at the beginning should have a strong impact on future\n495 competitive ability. Were these species reared in high light, high moisture, high\n496 nutrients? There’s almost no information about the environmental conditions the plants\n497 experienced.\n499 conditions. Seedling heights at transplantation were approximately 6-25 cm, with\n12\n500 deciduous species averaging 12-25 cm and evergreen species averaging 6-13 cm. We\n501 listed seedling initial heights for each species in Table S5 and lines 364-366. Following\n502 your suggestion, we constructed a mixed-effects model to explore the relationship\n503 between first-measured height (two months after germination) and competitiveness for\n504 all seedlings in the second phase of the experiment, with species as a random effect.\n506 0.018, P < 0.001), indicating that taller seedlings were more competitive (higher RII\n507 values). We have now included this information in the revision (lines 352-353).\n509 germination stage. The soil used for rearing was topsoil (15-30 cm) collected from the\n510 forest containing the mother trees, with 0.61 g/kg total carbon, 0.04 g/kg total nitrogen,\n511 0.45 g/kg total phosphorus and a pH value of 5.21. We have added this information on\n512 lines 356-362.\n513\n514 18. Line 168: how far apart were the seedlings? If the seedlings were quite small, they\n515 would have weak competition but if one was larger than the other, it would be\n516 asymmetric. Note: this information is presented on line 205 and should definitely be\n517 moved up sooner.\n519 the 15-20 cm pot spacing to minimize between-pot interference. Additionally, to ensure\n520 fair comparisons, the initial size of the conspecific seedlings was kept consistent for\n521 each species among pots, as indicated in Table S5. We have moved the above\n522 information forward from Phase II to Phase I in Methods (lines 391-394). There were\n523 inherent differences in the initial heights between the species, likely resulting in\n524 asymmetric competition. We have now included this information earlier in the\n525 manuscript (lines 364-368).\n526\n527 19. Line 181: insufficient detail regarding the environmental conditions. What was the\n528 mean and maximum for each environmental property? Providing the minimum light\n529 doesn’t tell us much about potential light stress. I see that an appendix is referenced but\n531 growth conditions. Even just a few sentences would be useful.\n533 added in Methods (lines 419-445).\n534\n535 20. Line 196: can we also know the range of variation in the environmental variables\n536 over the course of a day or months? The term homogeneous is somewhat subjective as\n537 it depends on the time frame.\n539 environment across pots within a given environmental block, rather than temporal\n540 invariance. Abiotic factors like light intensity naturally vary over time. We have\n541 redefined homogeneous environment in more detail in lines 399-409 and 413-414.\n542\n13\n543 21. Line 222: it would have been helpful to know earlier that competitive effects were\n544 based on biomass\n546 based on seedling biomass in our study.\n547\n548 22. Line 225 and line 236: the authors say this is to avoid confusion yet it is very\n549 confusing. The definition of RII is defined in terms of biomass when a species is within\n550 a pair, yet this line says that you use the term competitiveness even when the species is\n551 not in a pair. So does this mean that when I see the terms superior or inferior, that this\n552 species may not necessarily be in a pair? That it could be on its own? Because then later\n553 you note how the characterization of a species as being superior or inferior depends on\n554 the identity of the other species within the pair.\n556 if species i has greater value of RII when competing with species j, then species i and j\n557 are called superior and inferior species for that species group, respectively. These\n558 competitiveness labels are also retained for that species group in the competition-free\n559 environment. This design simplifies comparing ITVs between species in that group\n560 across competition and competition-free treatments.\n562 in different groups of species, as competitive ability is not a constant attribute of a\n563 species but can change depending on whom the species compete with and by resource\n564 availability. This is particularly relevant in our study, as Phase I includes eight different\n565 groups of species, and a species may be included in more than one species group. We\n566 revised these text in lines 468-479.\n567\n568 23. Line 279: is it possible to also calculate the amount of variance around the mean\n569 hypervolume? Based on Figure 2 it looks like you have the standard error\n571 each species. See detailed methods in lines 526-529.\n572\n573 24. Line 230: and by resource availability\n575\n576 25. Line 407: I disagree that this is the case for all traits. That’s my main argument.\n578 competition or heterogeneity. For example, leaf size is hugely variable in some species\n579 due to leaf ontogeny.\n581 competition-free environment. For example, in our study, the chlorophyll content (Chl)\n582 of seedlings under competition-free homogeneous environment showed higher ITV\n583 (1.18 in Bao's CV) than the mean ITV (1.16 in Bao’s CV) under competitive\n584 homogeneous environment. Therefore, what we intended to convey is that most traits\n585 have lower ITV in a competition-free homogeneous environment compared to\n14\n586 heterogeneous or competitive environments. To accurately express the above meaning,\n587 we have added that most traits are consistent with the overall pattern (lines 216-217).\n588\n589 26. Line 418: optimal being defined in terms of max biomass?\n591 maximum biomass and seed production, as well as low death rate. In our 3-years study,\n592 most seedlings were still alive and far from mature to produce seeds, so for our study,\n593 ‘optimal’ just refers to maximum biomass. We have added the above information in\n594 lines 216-221.\n595\n596 27. Line 418: a concave down trait performance curve but for which trait(s)? I don’t\n597 see how you can generalize so broadly.\n599 different traits and environments (Li et al. 2022; Siefert & Laughlin 2023; Stears et al.\n600 2022). Thus, it is an overgeneralization to apply theoretical trait-performance curves\n601 (Hart et al. 2016; Stump et al. 2022) in our experimental study. Furthermore, we\n602 realized these curve discussions do not actually explain the differential ITV changes\n603 between competing species. So we removed that content and instead added text on\n604 potential mechanisms driving the observed distinct ITV changes between competing\n605 species in lines 229-245.\n606\n607 28. Line 420: why would an individual produce a suboptimal trait value in response to\n608 a cue? Are you suggesting that an inferior species in low moisture, might have some\n609 individuals that make deeper roots and others that make shallower roots, even if the\n610 more shallow-rooted individuals perish? Is this part of the reason why these species are\n611 inferior in the first place? Are you suggesting this is maladaptive? It seems that there\n612 should be more discussion about alternative resource use strategies and performance\n613 landscapes (sensu Laughlin).A concave down trait performance curve versus a concave\n614 down doesn’t tell us anything about the extent of variation though. They could have\n615 different shapes but the exact same amount of variation, so the sentence that follows\n616 from this (Line 424) doesn’t exactly follow the logic (in my opinion).\n618 why individuals of superior species display suboptimal trait values under competition.\n620 trait-performance curves had some limitations. We therefore removed those texts and\n621 added new explanations covering possible drivers behind the distinct ITV changes\n622 between competing species in lines 229-245.\n623\n624 29. Line 469: negative or positive change? Add + or –\n626\n627 30. Line 475: it does not make sense to me why a species would be conservative above\n628 and acquisitive below. I think the use of those terms is not helpful in this context. Higher\n15\n630 helps to ameliorate water stress, so the higher root tissue density should be a drought\n631 adaptive response, not evidence of a more resource acquisitive strategy. Root tissue\n632 density also typically increases with a reduction in soil nutrients, so again this isn’t\n633 evidence of being more acquisitive on bottom and conservative on top.\n635 about conservative and acquisitive strategies and instead focused on adaptive responses\n636 of traits in lines 290-314.\n637\n16\n640 inferior vs superior tree species in order to test for an interspecific mean-variance trade-\n641 off. The authors use an experimental approach that clearly involved a massive amount\n642 of work and is impressive in its scope. The results provide novel experiment evidence\n643 of the mean-variance trade-off, which is relevant for species coexistence. Overall, the\n644 study makes a nice contribution to our understanding of the role of intraspecific\n645 variation in structuring diverse communities.\n647\n648 2. My main concern about the study is that the authors provide no information about\n649 where or how the seeds used in the experiment were collected (e.g., over what spatial\n650 extent, how many source trees were used, etc). This information is critical for any study\n651 of intraspecific variability (IV). If competitively inferior species were rarer in the\n652 community and/or produced fewer seeds than competitively superior species, then there\n653 might be the need to collect seeds of inferior species from more source trees and over\n654 a larger spatial extent, which could bias estimates of IV.\n656 collection in lines 351-353. In brief, seeds for each species were meticulously collected\n657 from one healthy, mature parent tree within a 1 km2 area of our study region. This\n658 ensured the low genetic variation among conspecific seeds and minimized maternal\n659 effect differences among species. However, it suffers a common experimental\n660 limitation by reducing representation of our ITV for broadly distributed species. We\n661 acknowledge this limitation in lines 352-353.\n662\n663 3. On a related note, the authors appear to be using the term IV to mainly refer to\n664 phenotypic plasticity. However, IV can arise due to genetic variation and phenotypic\n665 plasticity (and there can also be genetic variation in phenotypic plasticity). For the\n666 authors’ first question about IV in a competition-free, homogenous environment, then\n667 the IV would be the result of genetic variation (since you would not expect to see\n668 plasticity under uniform conditions). For questions 2 and 3, about IV under competition\n669 and in heterogenous abiotic environments, then they are talking about phenotypic\n670 plasticity. The authors need to be clearer about the underlying drivers of IV (eg genetic\n671 variation and phenotypic plasticity). This is related to my comment above about seed\n672 collection, since that will influence how much genetic variation exists in the seed pool\n673 used in the experiment as well as how plastic the collected individuals are expected to\n674 be (eg if some species were collected from more heterogenous environments than other\n675 species).\n678 cannot precisely distinguish ITV sources, as neither genetic variation nor phenotypic\n679 plasticity was fully controlled in our study. In fact, controlling the drivers of ITV\n680 remains challenging for trees. Thus, we have narrowed our study scope by removing\n681 the discussion on adaptation and evolution from the introduction and just focused on\n17\n682 our core questions and findings. Meanwhile, in the discussion section (lines 220-226),\n683 we thoroughly considered potential mechanisms producing the observed trade-off\n684 given our experimental design.\n685\n686 4. My other concern is about the influence of growth form on the results. Specifically,\n687 the two deciduous species included in the experiment were the most competitively\n688 superior in both Phase 1 and Phase 2 experiments. Deciduous species employ a different\n689 ecological strategy than evergreen species, which typically means that they differ in\n690 other traits as well. Do the observed relationships remain qualitatively similar if\n691 removing those two species from the analyses? (I don’t mean whether they are still\n692 statistically significant because they probably won’t be just due to the smaller sample\n693 size, but rather whether the trend is the same.)\n695 of our study. Following this comment, we conducted an additional analysis by\n696 excluding deciduous species. The results (Fig. S5) remained qualitatively similar.\n698 greater ITV, as shown by the significant negative slopes of -7.57 (Fig. S5e) and -24.39\n699 (Fig. S5f), respectively. Analyzing competition-free heterogeneous environment\n700 showed a similar pattern, with a significant slope of -15.42 (Fig. S5c). In the pairwise\n701 competition under homogeneous environment, the number of species pairs dropped\n702 from 8 to 3 without deciduous species. While the overall pattern was not significant due\n703 to low sample size exactly as you suspected, a weaker decreasing trend was observed\n704 (Fig. S5d). These results together support the presence of the interspecific mean-\n705 variance trade-off in our study. We have included this information in Methods (lines\n706 561-563) and Results (Lines 163-164).\n18\n707\n709 error, SD7, cube-root transformation) and competitiveness of evergreen species (points)\n710 under competition-free (hollow points, a-c), pairwise competition in homogeneous\n711 environment (orange circles, d, N=3) and multispecies competition in both\n712 homogeneous (blue squares, e, N=5) and heterogeneous (purple triangles, f, N=5)\n713 environments. In a and d, the gray circles represent inferior and superior species in the\n714 3 evergreen species pairs, and the orange circles represent their mean values.\n716 and orange lines represent the pairwise competition relationship and other solid (P <\n717 0.05) and dashed (P > 0.05) lines are simple linear regression lines with a 95%\n718 confidence interval. NS means non-significant and black asterisks indicate significant\n719 levels (•:<0.1; *: <0.05; **: <0.01; ***: <0.001).\n720\n722 5. Line 65: Why just “per capita seed production”? Wouldn’t any increase in fitness\n723 have this effect?\n725 metric discussed in Hart et al. (2016) study we cited. We have revised the sentence to\n726 reflect broader potential fitness impacts from ITV (lines 52-56).\n727\n728 6. Line 69: add “the” before “same”\n730\n19\n731 7. Line 71: add “the” before “Advantage”\n733\n734 8. Line 104: It is not clear here why this would lead to large IV in only inferior species\n735 (and not also in superior species)\n737\n738 9. Line 106-107: I don’t follow the logic here – why would it deviate from its optimal\n739 trait value. If IV is increased to enhance a species advantage, presumably it is achieving\n740 the optimal trait value for a given environment.\n742 the Introduction, we have removed these texts about suboptimal trait values. Instead,\n743 we added a more comprehensive explanation of why superior species can display\n744 distinct ITV changes in lines 249-253.\n745\n746 10. Line 126: do these 10 tree species coexist in nature? Are they all native?\n748 information in Line 109.\n749\n750 11. Line 144: indicate the country where the site occurs (China)\n752\n753 12. Line 149: What was the rationale for selecting both evergreen and deciduous (and\n754 11 of one type but only 5 of the other type)? Was there some expectation about how\n755 they would differ?\n757 chosen based on previous knowledge about species composition in the study area while\n758 constrained by seed availability. Specifically, forests in our study area are subtropical\n759 evergreen broad-leaved forests with noticeable deciduous elements (Yang et al. 2010).\n761 (Wang et al. 2020; Yang et al. 2021). Furthermore, only those species with large mother\n762 trees within a 1 km2 area were included to ensure adequate seed sourcing (lines 350-\n763 353).\n765 in our forest, because our previous experiment revealed deciduous tree species often\n766 exhibit faster growth and superior seedling competitiveness (Yang et al. 2021). We\n767 have added these considerations in Methods (lines 344-351 and 364-368).\n768\n769 13. Line 151: As mentioned above, there needs to be a lot more detail about seed\n770 collection (how many mother trees, over what geographic area and types of\n771 environments, were all species collected at the same sites, etc).\n773 of each species from a mature, healthy adult tree within our study area. We have added\n774 detailed description about seed collection in Method (lines 349-353).\n20\n775\n776 14. Line 158: What kind of homogenous environment? What resources were the species\n777 likely competing for in that environment (e.g., where soil nutrients limited, was there\n778 limited light or water, etc)?\n780 abiotic conditions both within and between pots in the same environment treatment. To\n781 ensure this spatial homogeneity, 3.5 kg of thoroughly mixed soil collected from the\n782 study area was added to each pot. Additionally, light intensity was kept consistent by\n783 using the same black netting above all pots, with monthly pot rotation to minimize\n784 potential micro-variation in light exposure.\n786 treatments. In terms of resource competition, our study primarily focused on\n787 aboveground light resources and belowground water and nutrient resources. Different\n788 environmental treatments may vary in resource availability. For instance, under low\n789 light conditions in certain blocks, plants may intensify competition for light, while\n790 under low phosphorus or limited water conditions, they may compete more intensely\n791 for soil nutrients or water, respectively. It is worth noting that plants may face multiple\n792 constraints from the aforementioned abiotic factors simultaneously.\n794 and limited resources in the Methods (lines 399-409; lines 420-430).\n795\n796 15. Line 180: It is not clear here or anywhere in the main text or supplement how the 9\n797 different environments were selected. It was not all combinations of the 3 variables, so\n798 why were some combinations excluded?\n800 levels each, with the same 40 replications per combination in our current experiment,\n801 would have required 9,720 pots and 17,010 seedlings in total, far exceeding our\n802 experimental capacity. To balance feasibility and representation, we selected a subset\n803 of 9 environments using a 3x3 orthogonal design table, enabling the exploration of\n804 environment combinations with available resources. We have added this rationale for\n805 our environment selection approach in Methods, lines 414-430.\n806\n807 16. Line 198: “populations of two competing species” is confusing here because\n808 competition was not pairwise in Phase II\n810 and moved \"populations of two competing species\" to lines 414-417 in the revised\n811 manuscript to improve clarity.\n812\n813 17. Line 277: Was this done separately for competition-free and competition treatments?\n815\n816 18. Line 279: The hypervolume values were averaged separately for each species under\n817 each scenario (e.g. competition-free homogenous, competition-free heterogenous, etc)?\n21\n819 scenario. We made it clear in lines 526-529.\n820\n821 19. Line 280: State how many individuals had missing trait data\n823 individuals with missing trait values in Phase 1 and in Phase II, respectively. We have\n824 added this information in Methods (lines 396-398 and 456-457).\n825\n826 20. Line 288: was the change in IV calculated in absolute or relative (ie % difference)\n827 terms? Looking at the graphs, it appears to be absolution differences. Do the results\n828 chance if using % difference instead?\n830 competition-free to competition treatments and they exhibit consistent trends.\n832 numbers displayed on the right side of each point indicate the relative ITV change. We\n833 have clarified this in lines 538-541 and 565-568.\n834\n835 21. Line 292-294: Why was this only done for Phase II?\n838 comparable between phases. To verify the selection of traits did not bias Phase II, we\n839 added analyses using all traits. We have clarified this point in lines 543-547.\n840\n841 22. Line 303-305: Why wasn’t species included as a random effect? It sounds like you\n842 have multiple values for each species in the model (eg one for each treatment), but are\n843 not accounting for the fact that values from the same species are not independent of\n844 each other.\n846 that the same species is not independent between the two competition treatments? In\n847 this case, we have made revision to clarify the modeling approach in Methods (lines\n848 552-561). Specifically, we constructed separate mixed-effects models for competition-\n849 free and competition treatments, each with a random intercept and slope. Because they\n850 were modeled separately, the non-independence of trait values among conspecifics\n851 could not bias our results.\n853 environmental blocks, we need to build new models with both the species and\n854 environmental blocks as two cross-over random factors. Results (Fig. S4) were\n855 consistent with our previous findings, i.e., it still shows significant negative\n856 relationships between species competitiveness and ITV in 7-dimensional (Slope = -3.68,\n859 above information to Methods (lines 558-561) and Results (lines 161-163).\n860\n22\n861\n863 standard error, cube-root transformation) and competitiveness of species (points) under\n864 competition-free (colored hollow points, a, c) and multispecies competition (colored\n865 hollow points, b, d) in 9 homogeneous environments. The top right panel shows the\n866 relationship between intraspecific trait variability and competitiveness within each\n867 abiotic environment. Intraspecific trait variability was quantified by the hypervolume\n868 of 7 traits. Intraspecific trait variability was quantified by hypervolume of the raw 7\n869 traits (a-b) and the reduced dimension first 3 principal axis traits (PC1-PC3, c-d),\n870 respectively. The solid (P < 0.05) and dashed (P > 0.05) lines are simple linear\n871 regression lines with a 95% confidence interval.\n872\n873 23. Line 308: Was this done using data from treatments with or without competition?\n875 and competition-free treatments. We have clarified it in lines 550-563 and 567-571.\n876\n877 24. Line 312: Using Phase I or Phase II data (or both)?\n879\n23\n880 25. Line 310-314: Why didn’t you model IV as a function of competition, RII, and RII\n881 x competition interaction (with species as a random effect)? That would be a more\n882 straightforward test of 1) whether competition affects IV and 2) whether the\n883 relationship between RII and IV changes under competition. If you model it that way,\n884 are the results consistent?\n886 employed new mixed-effects models assessing the relationships between competitive\n887 treatments (competition-free and competition), competitiveness (RII), and their\n888 interaction on species' ITV. We ran these analyses separately for homogeneous and\n889 heterogeneous environments, with species as a random factor. The new results (Table\n891 incorporated these new models and results in pages 26-27 and 7-9). However,\n892 interpreting results of models with a binary variable and an interaction of binary and\n893 continuous variables is not straightforward and needs advanced statistical knowledge.\n895 for each competition treatment), we also retained previous visualizations like Fig. 2 to\n896 intuitively examine the interspecific mean-variance trade-offs for the different\n897 competition treatments.\n898\n900 relationships involving competitive treatments (competition-free and competition,\n901 abbreviated as ‘CompTreat’), competitiveness (RII), and their interaction concerning\n902 species’ ITV within the context of multispecies experiment. These analyses were\n903 separately conducted in both homogeneous (specifically, environmental block 1, which\n904 corresponds to the Phase I environment) and heterogeneous environments, with species\n905 as a random factor. Values in bold indicate statistically significant effects (P < 0.05).\nPredictors Estimates CI P\nCompTreat_competition-free 0.67 -2.08 – 3.41 0.591\nMultispecies CompTreat_competition -0.37 -3.11 – 2.38 0.766\nexperiment in a -0.81 -5.43 – 3.81 0.696\ncompetitiveness\nhomogeneous\nenvironment CompTreat_competition* -8.03 -14.56 – -1.49 0.022\ncompetitiveness\nPseudo R2 0.879\nCompTreat_competition-free -0.10 -3.35 – 3.16 0.947\nMultispecies CompTreat_competition 0.32 -2.93 – 3.57 0.825\nexperiment in a\ncompetitiveness -8.70 -14.42 – -2.98 0.008\nheterogeneous\nenvironment CompTreat_competition*\n-6.26 -11.99 – -0.54 0.036\nPseudo R2 0.900\n906\n907 26. Line 332: change “survival” to “surviving”\n24\n909\n910 27. Line 337: specify that “significant suppression” is referring to reduced biomass.\n912\n913 28. Line 369-370: There is a typo here in the numbers – they do not match up with what\n914 is on the graphs (and also how can you have an R-square value of -8.7).\n916 regression line. We have adjusted our presentation to avoid such confusion.\n917\n918 29. Line 380: When it says “individual traits”, it makes it sound like you ran separate\n919 models for each trait. But instead, it seems like this is just a single model for all traits.\n922 models that considered all traits collectively and separate models for each individual\n923 trait. We have made this clear in the methods (lines 565-571) and the results (lines 188-\n924 195, Fig. S6).\n925\n926 30. Line 382: Is this in the homogenous environment only? (Both here and throughout\n927 the Results, make sure to specify whether you are referring to homogenous vs\n928 heterogenous environment when referring to competition treatment results. It is often\n929 not clear.)\n931 (corresponding to Fig. 2), including all seedlings in homogeneous two-species\n932 competition, homogeneous multispecies competition and heterogeneous multi-species\n933 competition. We have clarified these on lines 538-541.\n934\n935 31. Line 387: Specify “greatest” compared to what (ie other traits measured).\n937 been added in lines 197-199.\n938\n939 32. Line 398: change “seedlings stage” to “seedling stage”\n941\n942 33. Line 407-409: How exactly are determining that the traits are optimal? It seems like\n943 you are assuming that they are because of the low IV, but low IV does not necessarily\n944 mean optimal trait values! Do you also see lower interspecific variation? The fact that\n945 you find low IV in a homogenous environment just means that there is little genetic\n946 variation in the trait in your seed pool, and that increased IV under heterogenous\n947 environments and competition is resulting from phenotypic plasticity.\n949 due to low intraspecific genetic variation, and this has been added in lines 220-226.\n951 distributions. Specifically, if the distribution of traits in the plant community is in part\n25\n952 the result of selective pressure exerted by environmental conditions favoring species\n953 with specific functional traits, the environment should then select traits that are optimal\n954 or at least labile to the current environment, at which point the species should have low\n955 interspecific variability and exhibit a narrow range of trait values. This is also known\n956 as the trait optimization hypothesis (McGill et al. 2006). In our study, we found some\n957 evidence supporting this hypothesis in homogeneous environments free of competition\n958 because the ITV of most experimental species (Figs. 2a-b and S2a-b) and interspecific\n959 trait dissimilarities of 62.5% species pairs (Table S6) were relatively lower in such a\n960 simple environment compared to heterogeneous or competitive environments. We have\n961 clarified and expanded this concept in the revised manuscript to provide a more\n962 comprehensive explanation (lines 215-226).\n963\n964 34. Line 411-413: This makes no sense to me. If IV is selected for by the environment,\n965 then presumably individuals would be accurately perceiving the environmental stresses\n966 and adjusting trait values accordingly to increase fitness.\n968\n969 35. Line 414 and Line 430: specify what trade-off you are referring to\n971\n972 36. Line 417-422: This section is confusing and hard to follow for anyone who is not\n973 already very familiar with the papers being reference. Please rephrase to explain this\n974 more clearly.\n976 models and theoretical assumptions about trait-performance curves. The complexity\n977 and lack of empirical evidence led us to remove those previous discussions. Instead, we\n978 have provided a new explanation with clearer logic and empirical evidence for the\n979 drivers of interspecific mean-variance trade-offs in lines 229-258.\n980\n981 37. Line 431: remove the word “only”. Previous studies have also found that IV is\n982 related to genetic variation, species life history strategies, dispersal modes, etc.\n984\n985 38. Line 460-461: rephrase as “which could allow invasive species to survive in the\n986 face of…”. As written, it makes it sound like invasive species are actively choosing this\n987 strategy, rather than high plasticity being one of the reasons they become invasive.\n989 removing the active tone and improved clarity (lines 284-286).\n990\n991 39. Line 466: was this higher IV in root traits compared to leaf and stem traits found in\n992 all environments? What about if you just look at environments that differ in light only?\n994 traits to respond more to differences in soil moisture and nutrients. Given that 2 of the\n995 3 abiotic variables being manipulated in the experiment were below ground variables,\n26\n996 it is not surprising that root traits would appear to have higher IV. You need to consider\n997 the specific abiotic variable when comparing the different traits.\n999 could stem from including more soil environmental variables. Unfortunately, because\n1000 our experiment was not a full factorial design (explained in lines 419-422), we were\n1001 unable to isolate the specific effect of individual environmental factor on each trait. We\n1002 added your point about higher root trait variation in Discussion (lines 297-301).\n1003\n1004 40. Figure 1: again, it is not clear how the 9 environments were chosen since it is not\n1005 all combinations of the 3 variables.\n1007 added this information into Methods (lines 399-409) and a brief explanation in the\n1008 figure caption (lines 801-804).\n1009\n1010 41. Figure 2 and Figure 3: Include exact P values for significant relationships, not just\n1013\n1014 42. Figure 2, line 692: Do you mean “orange circles, b-d” here? I think it is a typo\n1015 because only d has orange circles.\n1017\n1018 43. Figure 3: something is off with the figure legends. For example panel b legend has\n1019 orange and blue lines for homogenous environment, but there are no orange and blue\n1020 lines in the graph. Also in panel A, it is not clear what the difference is between blue\n1021 and purple.\n1023\n1024 44. Figure 4: Is this from a homogenous environment only? Specify in the legend.\n1026 (corresponding to Fig. 2), including all seedlings in homogeneous two-species\n1027 competition, homogeneous multispecies competition and heterogeneous multi-species\n1028 competition. Being clarified in both the legend and lines 538-541.\n1029\n27\n1034 architecture indirectly affects coexistence among competitors in the understory.\n1037 biodiversity in disturbed, heterogeneous environments. Oikos, 128, 1478–1491.\n1041 (2013). Increased competition does not lead to increased phylogenetic\n1042 overdispersion in a native grassland. Ecology Letters, 16, 1168–1176.\n1044 relationship between competition and intraspecific trait variation. Journal of\n1047 trait space responses to competition in two clonal plant species. Functional\n1052 hierarchies and intraspecific variability drive competitive interactions in\n1055 trait variation increases species diversity in a trait-based grassland model. Oikos,\n1056 128, 441–455.\n1058 et al. (2018). The ecological importance of intraspecific variation. Nature\n1061 variability on the outcome of competition between cladoceran species. Inland\n1064 functional traits predict competitive outcomes? Ecology, 97, 2206–2211.\n1066 plants under competition. Nature Communication, 8, 2235.\n1068 affects species coexistence. Ecology Letters, 19, 825–838.\n1070 moyennes. Acta Mathematica, 30, 175–193.\n1072 predicts sapling leaf lifespan and shade tolerance of tropical tree species. New\n28\n1075 multidimensional nature of species coexistence. Proceedings of the National\n1078 community assembly. Journal of Ecology, 102, 186–193.\n1080 trait–performance relationships of tree seedlings along experimentally\n1081 manipulated light and water gradients. Ecology, 103, e3703.\n1083 reduces inter-tree competition in a mixed broadleaved forest. European Journal\n1084 of Forest Research, 132, 621–634.\n1086 ecology from functional traits. Trends in Ecology & Evolution, 21, 178–185.\n1088 personality-related movement behavior promotes coexistence. Oikos, 129,\n1089 1441–1454.\n1093 fitness across species and environments. Methods in Ecology & Evolution, 14,\n1094 1035–1048.\n1096 et al. (2022). Water availability dictates how plant traits predict demographic\n1097 rates. Ecology, 103, e3799.\n1104 coexistence. Ecology Letters, 21, 1496–1504.\n1106 plant competition increases soil labile organic carbon and nitrogen contents.\n1109 and water availability. Ecology, 102, e03361.\n1111 broadleaved forest in Tiantong’s 20 ha dynamic plot, Zhejiang Province, eastern\n1114 of incorporating the responses of species mean trait values in trait-based\n1115 community assembly. Ecological Indicators, 130, 108095.\n1116\nGeneral comments\nThis manuscript seeks to clarify how intraspecific trait variation can influence competitive interactions\nin superior (dominant) versus inferior species, considering resource availability. I appreciate that the\nauthors have included much more information about the methods, and have tried to clarify the\nintroduction and discussion in several places. However, I am still finding this paper hard to follow,\nunfortunately. I understand the big picture but the explanation of the details still needs work.\nIn reading the response to reviewer 1, I thought it was helpful to see a more formal definition of the\nmean-variance trade-off. However, I still don’t think that the mechanistic explanation is clear enough\nfor a broad audience that is unfamiliar with this work. Reading the main text (lines 52-63), it is still\nconfusing, and written at a high level rather than being written at such a level that anyone could\nunderstand it. For example, the term competitive sensitivity is never defined, demographic rates are\nnever defined. The phrase “incorporating equal ITV into the demographic parameters…” does not make\nsense to me, even after reading it several times. I would strongly urge the authors to revise the text\nagain for clarity and perhaps to focus on simplifying the writing and making it more direct, with less\njargon and undefined terminology. I suppose the main hurdle to overcome is explaining the existing\ntheoretical framework satisfactorily.\nMy understanding of the theory is that whereas dominant species may occupy only a few ecological\nniches with abundant resources (certainly there are some dominant species that occupy regions of low\nresource availability that inferior species can’t utilize. So I don’t understand the focus on high resource\navailability), inferior species may use ITV as a means to capitalize on/occupy the remaining open\nniches. But isn’t it also possible that inferior species are inferior because they have a limited ability to\nexpress variation in their traits (a low acclimation potential and low ITV?). The authors say that in a\nsecond scenario (when dominant species occupy most of the niches) that inferior species may be\nconstrained to low ITV but I don’t think you can assign causality in this way, for the reasons I\ndescribed above. In short, a more nuanced explanation of the theory (with examples and a description\nof exceptions) would be particularly useful, and is what I’d recommended in the last revision. I\nunderstand that the authors weren’t able to find empirical examples, but they could still put forth\nsome examples even if there aren’t published studies to go along with them. Maybe even a nice\nconceptual diagram would help. I understand that they are simply describing an existing body of work\nand not putting forth this theory for the first time, but if readers don’t understand it then the empirical\ntests won’t make sense either.\nGoing back to the response letter, you say that there is indirect evidence that ITV is greater in inferior\nspecies, yet you provide little detail here and don’t explain the causal mechanisms. The example of\nPotentilla doesn’t mention that this species is inferior. The following example is also for a clonal\nspecies, which is a bit odd, and then you only say that competition led to greater ITV but you still\nhaven’t explained how an inferior species would have higher ITV. Only one sentence in this rebuttal\nparagraph specifies the use of an inferior species, and that was for the Quercus. In short, I wasn’t\nlooking for more explanation about how ITV influences competition or vice versa, I was looking for\nclear examples of an inferior species having less ITV.\nAnother example in the response letter where I don’t think you’ve provided a clear explanation\n(Line 405): “ITV promotes coexistence only when inferior species exhibit much higher ITV,\nbecause the greater ITV in inferior species causes some individuals with equal or lower\ncompetitive sensitivity versus superior species. This bolsters the realized population growth rate\nof inferior species and enables species coexistence.” Greater ITV in all traits or traits that reflect\nresource use? Or ITV in life history traits? It seems like a big stretch to say greater ITV in general.\nAnd when you say much higher ITV, do you mean higher relative to superior species in the same\nenvironment or just generally speaking? Can you be more specific?\nSpecific comments:\nLine 78: be careful with English grammar. I found several mistakes (Lines 46, 63, 67, 95). Here\nit should probably say “phenotypic plasticity resulting from competitive interactions can increase\nITV, resulting in complex interactions between ITV and competition.” And then here you really\nneed an example: “For example, when the roots of neighboring plants come into contact with one\nanother, one species may subsequently produce deeper roots in order to penetrate deeper into the\nsoil and reduce competition. In other cases, both species may produce deeper roots at the same\ntime, resulting in even stronger competition.” The example you provided about the ramet and\nconnectance traits is vague and therefore not particularly helpful. And I also don’t know what a\nconnectance trait is in the first place.\nLine 86: in other words, there is asymmetry in the effect of competitive interactions for ITV and\nvice versa\nIn response to reviewer 1’s comment and your reply: when you grew the plants in competition\nand then alone, was the latter specifically meant to identify the fundamental niche?\nLine 95: I don’t understand the explanation for why the inferior species would not adjust ITV.\nAbove you said that it would and not you are saying it won’t because there are other even more\ninferior species interacting with it.\nLine 97: what do you mean by higher order interactions?\nLine 103: I am still confused by the use of a homogeneous environment. If there’s heterogeneity, it\nsimply means that there could be high resource patches mixed with low resource patches, therefore,\nenvironmentally driven ITV (due to plasticity or local adaptation) is likely to have some influence on\nspecies interactions. In other words, the environment and presence of a competitor together influence\nthe amount of ITV in the inferior species. When all patches are the same though, it should matter\nwhether we are talking about a high resource or a low resource environment. If low resources, then\nthere could be filtering of species as you say, but I don’t see how a filter would necessarily restrict the\nrange of means and also the amount of ITV. Can you comment further on how the relationship\nbetween ITV and competition would differ with resource availability (not heterogenity)?\nLine 104: across a gradient of resource availability or across spatially heterogeneous environments?\nEnvironmental conditions is vague.\nLine 117: here you should just say what method you used. Whether it was looking at SD or CV.\nLine 493 of response letter: 12-25 cm is a large amount of variation in seedling height. That’s more\nthan double at the initiation of the study. I’m glad you include the results from the mixed effects\nmodel but shouldn’t initial plant size be included in all the other models if it had a significant impact on\ncompetition? This seems like a big deal.\nLine 344 of main text: remove the word “slightly”, these are acidic soils\nFigure 2. are the orange lines in panels a and d the mean change when comparing inferior to\nsuperior?\nLine 210: rather than just keep repeating the interspecific mean variance trade-off, you should use\nplain English to summarize. In other words, in a homogeneous environment with no competition,\nthere was no evidence that inferior species (with lower mean trait values) also had higher ITV.\nLine 234: same comment. Instead of competitive suppression just say what you mean in plain English\nrather than with lots of terms and jargon.\nLine 251: grammar. Become not became.\nLine 316: grammar. “of its importance” not on\nLine 318: response surfaces\nReviewer #3 (Remarks to the Author):\nThe authors have address all of my concerns with the previous version, and the revised text is\nmuch clearer. I think this study will make a nice contribution to the field. I have only a few minor\nsuggestions:\nLine 28-32: Results should be reported in the past tense (e.g., No trade-off WAS found…)\nLine 42: remove “previous unrecognized”. It has been recognized for over a decade – see Clark 2010\nScience and Clark et al. 2010 Ecol Monographs (you should look at these closely and see if you\nshould cite them here or elsewhere since they seem highly relevant)\nLine 67 – change ‘rest’ to ‘remaining’\nLine 68 – remove ‘have to’ or else rephrase because it is not grammatically correct\nLine 84 – ‘increases’ should be ‘increased’\nLine 129 – ‘greater increase’, it is not clear an increase over what. I think you mean in a competitive\nor heterogenous environment compared to a competitive-free homogenous environment, but you\nshould make that clear.\nLine 135 – it will be unclear to readers what Phase I and II are since you have not yet explain it\nin the main text. I suggest adding a few words to make it clear what they mean.\nLine 163 – add ‘the’ before ‘two’, otherwise it sounds like there were more than 2 deciduous species\nand you just chose to remove 2 of them\nLine 331 – add ‘potential’ before ‘importance’. As pointed out by another reviewer, you cannot\nprove its importance for coexistence with this study.\nLine 547 – change “was” to “were”\nResponse letter to the reviewers’ comments\nBlack: reviewer comments; Blue: our responses; Purple Italic: our revisions\nReviewer #2 (Remarks to the Author):\nGeneral comments\n1.This manuscript seeks to clarify how intraspecific trait variation can influence competitive\ninteractions in superior (dominant) versus inferior species, considering resource availability. I\nappreciate that the authors have included much more information about the methods, and have\ntried to clarify the introduction and discussion in several places. However, I am still finding this\npaper hard to follow, unfortunately. I understand the big picture but the explanation of the details\nstill needs work. In reading the response to reviewer 1, I thought it was helpful to see a more formal\ndefinition of the mean-variance trade-off. However, I still don’t think that the mechanistic\nexplanation is clear enough for a broad audience that is unfamiliar with this work. Reading the\nmain text (lines 52-63), it is still confusing, and written at a high level rather than being written at\nsuch a level that anyone could understand it. For example, the term competitive sensitivity is never\ndefined, demographic rates are never defined. The phrase “incorporating equal ITV into the\ndemographic parameters…” does not make sense to me, even after reading it several times. I would\nstrongly urge the authors to revise the text again for clarity and perhaps to focus on simplifying\nthe writing and making it more direct, with less jargon and undefined terminology. I suppose the\nmain hurdle to overcome is explaining the existing theoretical framework satisfactorily.\nResponse: We sincerely appreciate your constructive feedback and encouragement regarding our\nmost recent revision. Following your suggestions, we have streamlined this mechanistic\nexplanation of the theory by eliminating unnecessary jargon and focusing directly on the essence\nof the matter (see lines 52-66). For further clarity, we have incorporated an example related to seed\nquality to better elucidate this theoretical framework for a broad audience.\nLines 52-66:\n“Such is the case of tree species whose seed quality strongly correlates with their\ncompetitive ability at the seedling stage13, a high diversity of seed qualities can allow inferior\nspecies to produce some highly competitive individuals. However, any competitive advantage\ngained by an inferior species through such variation is outweighed if the superior species has\nsimilarly high ITV, and this outweighing occurs because the superior species has an inherently\nelevated mean seed quality. Equivalent ITV thus fails to provide the inferior species with a foothold\nover the superior species due to this baseline discrepancy in seed quality12. The coexistence is\npromoted only when the inferior species exhibits greater trait variation among its conspecifics\nthan the superior species10. This phenomenon is known as the interspecific mean-variance trade-\noff12. When this trade-off occurs, some individuals of the inferior species have the same or greater\ntrait value (i.e., seed quality) than do those of the superior species14, thus bolstering the persistence\nof their populations11,15.”\n2. My understanding of the theory is that whereas dominant species may occupy only a few\necological niches with abundant resources (certainly there are some dominant species that occupy\nregions of low resource availability that inferior species can’t utilize. So I don’t understand the\nfocus on high resource availability), inferior species may use ITV as a means to capitalize\non/occupy the remaining open niches. But isn’t it also possible that inferior species are inferior\nbecause they have a limited ability to express variation in their traits (a low acclimation potential\nand low ITV?). The authors say that in a second scenario (when dominant species occupy most of\nthe niches) that inferior species may be constrained to low ITV but I don’t think you can assign\ncausality in this way, for the reasons I described above. In short, a more nuanced explanation of\nthe theory (with examples and a description of exceptions) would be particularly useful, and is\nwhat I’d recommended in the last revision. I understand that the authors weren’t able to find\nempirical examples, but they could still put forth some examples even if there aren’t published\nstudies to go along with them. Maybe even a nice conceptual diagram would help. I understand\nthat they are simply describing an existing body of work and not putting forth this theory for the\nfirst time, but if readers don’t understand it then the empirical tests won’t make sense either.\nResponse: Thanks for your alternative explanation of the diminished ITV in inferior species. We\nhave incorporated this compelling possibility into our revised exposition of the theoretical\nframework, including explicative examples and examinations of exceptions (see lines 72-78).\nAccordingly, we expanded our discussion on scenarios wherein inferior species exhibit low ITV\n(see lines 78-80). To further visualization these explanations, we have additionally constructed\nand included a new conceptual diagram (Fig. S1) in the Supplementary Information. We hope\nthese changes and additions are helpful to illustrate the theoretical framework of our study.\nFig. S1 | Two distinct scenarios delineate the relative magnitude of intraspecific trait variability\n(ITV) in superior (green curve) and inferior (red curve) species. In scenario (a), the inferior\nspecies has greater ITV than the superior competitor. This could manifest, using root as an\nexample, by the following mechanism – superior species occupy a few underground resource\nniches while the inferior species actively adapt or passively exploit the remaining accessible\nresource niches. Occupying multiple niches culminates in a multimodal trait distribution,\ntranslating to amplified ITV for root depth in the inferior species. Conversely, scenario (b) depicts\nthe inferior species possessing less ITV than the superior counterpart. Contributing factors may\nbe inherent constraints in trait plasticity or adaptability that contribute to their competitive\ndisadvantages. The colored solid lines depict trait distribution examples for both inferior and\nsuperior species under two scenarios.\nLines 72-78:\n“For example, while superior species may benefit from deep roots to monopolize soil\nresources, inferior species might adapt either by extending their roots deeper22 or by spreading\nthem laterally to access shallow soil resources23, resulting in a multipeaked root depth trait\ndistribution with higher ITV (Fig. S1a). Conversely, if the superior species already occupies most\nof the niche space24,25, the inferior species may compress its trait values to maximize its survival\nin the remaining limited niche space. This may result in a smaller ITV in the inferior species.”\nLines 78-80:\n“Alternatively, inferior species might inherently face limitations in expressing trait\nvariability26,27, which would lead to low ITV (Fig. S1b).”\n3. Going back to the response letter, you say that there is indirect evidence that ITV is greater in\ninferior species, yet you provide little detail here and don’t explain the causal mechanisms. The\nexample of Potentilla doesn’t mention that this species is inferior. The following example is also\nfor a clonal species, which is a bit odd, and then you only say that competition led to greater ITV\nbut you still haven’t explained how an inferior species would have higher ITV. Only one sentence\nin this rebuttal paragraph specifies the use of an inferior species, and that was for the Quercus. In\nshort, I wasn’t looking for more explanation about how ITV influences competition or vice versa,\nI was looking for clear examples of an inferior species having less ITV.\nResponse: Thank you for specifying the information that would be most pertinent. We found an\nexample in Carmona et al. (2019) demonstrating competitively inferior species displayed higher\nITV than superior species. Compared to traits measured in absence of competition, the traits of the\ninferior species became more similar to those of the superior when the two species grew in pairwise\ncompetition. As a result, competitive hierarchies were reduced, favoring the persistence of the\ncompetitively inferior species. This example has been incorporated into the revised text (see lines\n99-101), replacing the previous instance involving clonal plants.\nAdditionally, in response to your request for an example of an inferior species with less\nITV, we referred to Ashton et al. (2010). Specifically, despite a superior competitor exploiting\nincreased availability of certain nitrogen forms through phenotypic plasticity, the inferior species\ndid not show a parallel shift in its nitrogen absorption profile when grown in competition.\nLines 99-101:\n“Similarly, in an experiment with Mediterranean annual plant species, the inferior species\nexperienced greater changes in trait values when competing with superior species, resulting in\nreduced competitive hierarchies and favoring coexistence7.”\n4. Another example in the response letter where I don’t think you’ve provided a clear explanation\n(Line 405): “ITV promotes coexistence only when inferior species exhibit much higher ITV,\nbecause the greater ITV in inferior species causes some individuals with equal or lower\ncompetitive sensitivity versus superior species. This bolsters the realized population growth rate\nof inferior species and enables species coexistence.” Greater ITV in all traits or traits that reflect\nresource use? Or ITV in life history traits? It seems like a big stretch to say greater ITV in general.\nAnd when you say much higher ITV, do you mean higher relative tos superior species in the same\nenvironment or just generally speaking? Can you be more specific?\nResponse: We apologize for the lack of clarity in our previous response. While the concept of\n\"greater ITV\" is highly generalizable across contexts and operationalized differently per study (e.g.\nHart et al. 2016; Lichstein et al. 2007), herein we refer specifically to greater variability in traits\ndirectly linked to competitive ability (e.g. seed quality) within inferior species versus superior\nspecies given similar environmental conditions. For enhanced precision, we have made the term\nmore specific in our manuscript at lines 53-61 and 63-66.\nLines 53-61:\n“Such is the case of tree species whose seed quality strongly correlates with their\ncompetitive ability at the seedling stage13, a high diversity of seed qualities can allow inferior\nspecies to produce some highly competitive individuals. However, any competitive advantage\ngained by an inferior species through such variation is outweighed if the superior species has\nsimilarly high ITV, and this outweighing occurs because the superior species has an inherently\nelevated mean seed quality. Equivalent ITV thus fails to provide the inferior species with a foothold\nover the superior species due to this baseline discrepancy in seed quality12.”\nLines 63-66:\n“When this trade-off occurs, some individuals of the inferior species have the same or\ngreater trait value (i.e., seed quality) than do those of the superior species14, thus bolstering the\npersistence of their populations11,15.”\nSpecific comments:\n5. Line 78: be careful with English grammar. I found several mistakes (Lines 46, 63, 67, 95). Here\nit should probably say “phenotypic plasticity resulting from competitive interactions can increase\nITV, resulting in complex interactions between ITV and competition.” And then here you really\nneed an example: “For example, when the roots of neighboring plants come into contact with one\nanother, one species may subsequently produce deeper roots in order to penetrate deeper into the\nsoil and reduce competition. In other cases, both species may produce deeper roots at the same\ntime, resulting in even stronger competition.” The example you provided about the ramet and\nconnectance traits is vague and therefore not particularly helpful. And I also don’t know what a\nconnectance trait is in the first place.\nResponse: Thank you for your constructive feedback. We have meticulously revised the\nmanuscript to address all grammatical errors. Additionally, we have enlisted the Premium Editing\nservices of American Journal Experts (AJE), with the verification code 9B3D-052B-155A-EFCE-\nC97P, aiming to refine the manuscript's clarity and appeal to diverse audiences.\nMoreover, in response to your suggestion, we have incorporated an example explaining\nhow root phenotypic plasticity and associated influences shape ITV into the manuscript (lines\n89-92).\nLines 89-92:\n“For example, when the roots of neighboring plants come into contact, one species may\nrespond by producing deeper roots, mitigating competition22. Alternatively, both species may\nsimultaneously exhibit root elongation, intensifying competition33.”\n6. Line 86: in other words, there is asymmetry in the effect of competitive interactions for ITV and\nvice versa.\nResponse: Yes, we have clarified and emphasized this point in lines 101-103.\nLines 101-103:\n“These findings imply that the impact of competition on ITV might be asymmetrical\nbetween superior and inferior species34, triggering the interspecific mean-variance trade-off.”\n7. In response to reviewer 1’s comment and your reply: when you grew the plants in competition\nand then alone, was the latter specifically meant to identify the fundamental niche?\nResponse: When allowed to grow alone, the abiotic conditions of seedlings did approximate their\nfundamental niche (Ashton et al. 2010). However, given the constrained type and range of\nenvironmental variables in our study, alone growth conditions likely did not encapsulate the full\nspectrum of abiotic environments permissible to each species' survival without competition. Our\nprimary objective in utilizing alone and mixed-species plantings was to investigate how\ncompetition shapes ITV.\n8. Line 95: I don’t understand the explanation for why the inferior species would not adjust ITV.\nAbove you said that it would and not you are saying it won’t because there are other even more\ninferior species interacting with it.\nResponse: Apologies for any confusion. We recognize that our discussion regarding potential\nadjustments of ITV in inferior species under multispecies interactions lacked clarity. Earlier in the\nmanuscript, we looked at how inferior species can change their ITV in response to pairwise\ncompetition. Then we discussed that inferior species may respond differently under more complex\nmultispecies interactions for two key reasons: 1) Ecological niche saturation: In competitive\nenvironments with higher species number, ecological niches become widely occupied, resulting in\nfewer unoccupied niche spaces. This occupancy saturation limits opportunities for inferior species\nto effectively utilize ITV adjustments to access open niche dimensions. 2) Complex competitive\ndynamics: Multispecies systems elicit more complex interactions between species, similar to\n“rock-paper-scissors” cycles. Although an inferior species could alter traits to gain an advantage\nover one competitor, such ITV shifts may inadvertently handicap it against another species. These\ncomplex interactions constrain the degree to which species can substantially modify traits\nconferring multilateral competitors. We have revised the relevant text in lines 110-119 to\naccurately reflect this feedback.\nLines 110-119:\n“Unlike simple pairwise competition, multispecies interactions involve more than two\nspecies, thus possibly reducing unoccupied niche space as the total number of species increases37.\nHowever, heterogeneous abiotic environments can expand the overall available niche space,\npotentially increasing unoccupied niches36,38. The uncertainty surrounding vacant niches in such\necosystems makes it difficult to predict how inferior species might adapt their ITV to access\nuntapped resources and respond to competition. Furthermore, abiotic environments may also\naffect the magnitude of ITV by either directly filtering out individuals with unfit trait values20,39 or\nindirectly adjusting interspecific competition, which in turn alters the extent and direction of\nplastic responses of traits40.”\n9. Line 97: what do you mean by higher order interactions?\nResponse: Higher-order interactions are ecological scenarios where a species' direct impact on\nanother is influenced by the presence of additional species, an occurrence primarily noted in multi-\nspecies systems (Levine et al. 2017). This dynamic unfolds as one species undergoes trait plasticity\nchanges in response to another, which in turn modifies its direct effects on a third party.\nConsequently, these are often termed as trait-mediated indirect interactions (Padilla et al. 2013).\nWhile these interactions may have unforeseen effects on ITV, their inherent complexity makes\nthem challenging to thoroughly investigate in this context. Therefore, to maintain focus and clarity,\nwe've removed related content from the Introduction section.\n10. Line 103: I am still confused by the use of a homogeneous environment. If there’s\nheterogeneity, it simply means that there could be high resource patches mixed with low resource\npatches, therefore, environmentally driven ITV (due to plasticity or local adaptation) is likely to\nhave some influence on species interactions. In other words, the environment and presence of a\ncompetitor together influence the amount of ITV in the inferior species. When all patches are the\nsame though, it should matter whether we are talking about a high resource or a low resource\nenvironment. If low resources, then there could be filtering of species as you say, but I don’t see\nhow a filter would necessarily restrict the range of means and also the amount of ITV. Can you\ncomment further on how the relationship between ITV and competition would differ with resource\navailability (not heterogenity)?\nResponse: Thank you for highlighting the lack of clarity. Here is the explanation about the filtering\nprocess of low resource environments on species’ trait distributions (Fig. S8): With ITV,\nadaptations towards the environment vary across individuals. When no individuals demonstrate\nsuitable adaptations, the entire species faces elimination (species 4 with green trait distribution).\nHowever, if only certain individuals are maladapted, the filter selectively removes those, leaving\nadapted members (species 1 with red trait distribution). Consequently, the surviving population\nexperiences tightened ITV around a shifted mean trait value aligned with the selective pressure.\nWe have added this conceptual diagram in Discussion (lines 235-240).\nResource availability can modulate the association between ITV and competition. For\ninstance, in high-resource settings (e.g., environment block 1 with abundant light and water),\ninferior species pressures exhibited higher ITV (slope of -8.84 in Figs. 1e and S5b). Under\nresource-constrained environments (e.g., block 4 with scarce light and nutrients), inferior species\nstill exhibited elevated ITV but the relationship was substantially attenuated (slope of -2.58 in Fig.\nS5b). This aligns with the stress gradient hypothesis, whereby species in resource-limited\nenvironments experience restricted growth and dampened competition (Stein et al. 2014). While\nwe did not exhaustively investigate these nuanced relationships, we document these patterns here\n(Discussion, lines 284-293) as meriting detailed exploration in future work.\nFig. S8 | A conceptual diagram delineates abiotic environmental filtering operating at the\nindividual level across various species (colored trait distribution curves). If all individuals of a\nspecies are maladapted, the species go extinction (species 4 with green trait distribution curve).\nAlternatively, with variable ITV, if a subset of individuals are maladapted, the filter eliminates\nonly these individuals, enabling the persistence of adapted subset (species 1 with red curve). The\nresulting truncated population passing through this bottleneck exhibits reduced ITV and a changed\nmean trait value. Essentially, environmental filtering alters trait distribution via this ITV reduction\nand mean shift.\nLines 235-240:\n“In a competition-free homogeneous environment, all individuals, regardless of the status\nof the species in the competitive hierarchy, are subject to similar abiotic selection and use the\nsame set of resources (e.g., Fig. S8). This results in similar variation in most traits between inferior\nand superior species and lower levels of ITV in a competition-free homogeneous environment than\nin heterogeneous or competitive environments47.”\nLines 284-293:\n“Moreover, resource availability can also modulate the association between ITV and\ncompetition. For instance, in environments with abundant resources (e.g., environment block 1\nwith abundant light and water), inferior species exhibited higher ITVs (slope of -8.84 in Figs. 1e\nand S5b), while in resource-constrained conditions (e.g., block 4 with scarce light and nutrients),\nthis relationship weakened (slope of -2.58 in Fig. S5b). This finding aligns with the stress gradient\nhypothesis36, whereby species in resource-limited environments experience restricted growth and\ndampened competition. These findings highlight the need for further exploration of the effect of\nresource availability on the relationship between ITV and competition.”\n11. Line 104: across a gradient of resource availability or across spatially heterogeneous\nenvironments? Environmental conditions is vague.\nResponse: It is whether the trade-off still hold across spatially heterogeneous environments.\nCorrected.\n12. Line 117: here you should just say what method you used. Whether it was looking at SD or\nCV.\nResponse: We employed the CV statistic to assess ITV for a single trait, specifically using Bao's\nCV. This method has been validated for a more accurate estimation of ITV (Yang et al. 2020). We\nhave now incorporated this information into the main text for clarity (see line 135-137).\nLines 135-137:\n“We correspondingly estimated the ITVs for each species in both treatments in each\nscenario using two methods: multidimensional trait space41–43 and individual trait variability\n(Bao’s CV)6,7,44,45.”\n13. Line 493 of response letter: 12-25 cm is a large amount of variation in seedling height. That’s\nmore than double at the initiation of the study. I’m glad you include the results from the mixed\neffects model but shouldn’t initial plant size be included in all the other models if it had a\nsignificant impact on competition? This seems like a big deal.\nResponse: Thank you for your suggestion. Please note that the 12-25 cm differences in initial plant\nheight largely stem from interspecific distinctions (see Table S7). These interspecific variances\nreflect intrinsic differences in life stage development across species and are difficult to control in\nan experiment. Rather, intraspecific differences in initial height were carefully controlled for by\nselecting similarly sized conspecifics during transplanting (line 426-429 and 489-490). As such,\nintraspecific height variations are relatively minor compared to interspecific discrepancies.\nAdditionally, we conducted supplementary analyses to evaluate potential impacts of initial\nplant height on the relationship between competition and ITV. Specifically, we included the\naverage initial height of each species as a fixed effect in the mixed-effects model. The new results\nindicated that initial plant height did not exert a significant effect on the ITV of the species, both\nin homogeneous (P = 0.936) and heterogeneous (P = 0.158) environments (refer to Table R1).\nImportantly, the new results (Table R1) were consistent with our previous findings (Fig. 2 and\nTable S1), indicating that initial size differences between seedlings do not skew our detected\nconclusions. In fact, our previous model already account for the interspecific differences by\nincluding species identity as a random factor. This may explain the consistent outputs between\nmodels with and without initial plant sizes. Given the non-significant impact of initial height on\nour model's results, we opted to exclude this parameter from the final model in order to retain\nparsimony and emphasis on the variables of primary interest.\nTable R1 Summary of the mixed-effects models employed to explore the relationships involving\ncompetitive treatments (competition-free and competition, abbreviated as ‘CompTreat’),\ncompetitiveness (RII), their interaction, and average initial height of each species concerning\nspecies’ ITV within the context of multispecies experiment. These analyses were separately\nconducted in both homogeneous (specifically, environmental block 1, which corresponds to the\nPhase I environment) and heterogeneous environments, with species as a random factor. Values in\nbold indicate statistically significant effects (P < 0.05).\nPredictors Estimates CI P\nMultispecies CompTreat_competition-free 0.93 -5.40 – 7.26 0.738\nexperiment in a CompTreat_competition 0.53 -5.80 – 6.86 0.850\nhomogeneous competitiveness -0.51 -8.14 – 7.11 0.878\nenvironment\ninitial height -0.01 -0.21 – 0.20 0.936\nCompTreat_competition* -7.14 -13.97 – -0.32 0.043\ncompetitiveness\nPseudo R2 0.856\nMultispecies CompTreat_competition-free -5.96 -15.24 – 3.32 0.173\nexperiment in a CompTreat_competition -5.54 -14.83 – 3.74 0.201\nheterogeneous competitiveness -15.26 -26.42 – -4.09 0.014\nenvironment\ninitial height 0.20 -0.10 – 0.50 0.158\nCompTreat_competition* -6.26 -12.13 – -0.39 0.040\ncompetitiveness\nPseudo R2 0.918\nLines 426-429:\n“To ensure a fair comparison between treatments, the initial height and base diameter of\nthe seedlings were kept similar for each species among the pots. If a seedling died within the first\nmonth after transplanting, it was replaced with a similar-sized conspecific seedling.”\nLines 489-490:\n“Healthy seedlings of similar initial heights were selected for each species (Table S7) and\ntransplanted into the pots.”\n14. Line 344 of main text: remove the word “slightly”, these are acidic soils\nResponse: We have removed the word \"slightly\" from manuscript.\n15. Figure 2. are the orange lines in panels a and d the mean change when comparing inferior to\nsuperior?\nResponse: In panels a and d in Fig. 2, the orange dots represent the mean of ITV for eight superior\nand inferior species. The orange line segment connecting these dots indeed illustrates the\ndifference in ITV between competing species. We have updated the figure notes (lines 889-892)\nto express this more explicitly.\nLines 889-892:\n“The orange dots in a and d represent the mean ITVs of the eight superior (or inferior)\nspecies. The grey and orange line connecting these dots visually depicts the difference in ITV\nbetween competing species, and significance was tested by paired Wilcoxon rank-sum test.”\n16. Line 210: rather than just keep repeating the interspecific mean variance trade-off, you should\nuse plain English to summarize. In other words, in a homogeneous environment with no competition,\nthere was no evidence that inferior species (with lower mean trait values) also had higher ITV.\nResponse: We have revised the pertinent sections (lines 230-233) to state more directly.\nLines 230-233:\n“Under a competition-free homogeneous environment, our observations did not support\nthe hypothesis that species with lower average competitive abilities exhibit higher levels of ITV,\ncountering the assumption of an interspecific mean-variance trade-off.”\n17. Line 234: same comment. Instead of competitive suppression just say what you mean in plain\nEnglish rather than with lots of terms and jargon.\nResponse: Thanks to your constructive comment. We have replaced the term \"intensity of\ncompetition (RII), as evidenced by a relative decrease in overall biomass.\" (lines 258-259).\n18. Line 251: grammar. Become not became.\nResponse: Corrected.\n19. Line 316: grammar. “of its importance” not on\nResponse: Corrected.\n20. Line 318: response surfaces\nResponse: Corrected.\nReviewer #3 (Remarks to the Author):\nThe authors have address all of my concerns with the previous version, and the revised text is\nmuch clearer. I think this study will make a nice contribution to the field. I have only a few minor\nsuggestions:\n1. Line 28-32: Results should be reported in the past tense (e.g., No trade-off WAS found…)\nResponse: revised.\n2. Line 42: remove “previous unrecognized”. It has been recognized for over a decade – see Clark\nyou should cite them here or elsewhere since they seem highly relevant)\nResponse: Thanks for your valuable suggestion. After a thorough review of the mentioned papers,\nwe have rephrased the sentence, omitting “previous unrecognized”. Additionally, we have\naccordingly cited both references in the revised manuscript (line 43).\n3. Line 67 – change ‘rest’ to ‘remaining’\nResponse: Done.\n4. Line 68 – remove ‘have to’ or else rephrase because it is not grammatically correct\nResponse: Thanks. We have revised the sentence (lines 75-78) for clarity and correctness.\nLines 75-78:\n“Conversely, if the superior species already occupies most of the niche space24,25, the\ninferior species may compress its trait values to maximize its survival in the remaining limited\nniche space. This may result in a smaller ITV in the inferior species.”\n5. Line 84 – ‘increases’ should be ‘increased’\nResponse: Done.\n6. Line 129 – ‘greater increase’, it is not clear an increase over what. I think you mean in a\ncompetitive or heterogenous environment compared to a competitive-free homogenous\nenvironment, but you should make that clear.\nResponse: Thanks. To enhance clarity, we have rephrased the sentence (lines 149-152).\nLines 149-152:\n“We found that the emergence of the trade-off was driven by a greater increase in ITV in\ninferior species than in superior species when competing species transitioned from competition-\nfree homogeneous environments to competitive or heterogeneous environments.”\n7. Line 135 – it will be unclear to readers what Phase I and II are since you have not yet explain it\nin the main text. I suggest adding a few words to make it clear what they mean.\nResponse: Thank you for your insightful suggestion. We have incorporated a concise definition\nof Phase I and Phase II at the end of introduction section (lines 127, 130 and 132).\n8. Line 163 – add ‘the’ before ‘two’, otherwise it sounds like there were more than 2 deciduous\nspecies and you just chose to remove 2 of them\nResponse: Great point! We have added “the” before “two”.\n9. Line 331 – add ‘potential’ before ‘importance’. As pointed out by another reviewer, you cannot\nprove its importance for coexistence with this study.\nResponse: Thanks for your valuable feedback. We have rephrased this sentence (line 366-367).\nLines 366-367:\n“These findings provide the first experimental evidence for this trade-off, providing\nempirical evidence for its potential importance in species coexistence.”\n10. Line 547 – change “was” to “were”\nResponse: Done.\nReference\nAshton, I.W., Miller, A.E., Bowman, W.D. & Suding, K.N. (2010). Niche complementarity due\nto plasticity in resource use: plant partitioning of chemical N forms. Ecology, 91, 3252–\n3260.\nCarmona, C.P., de Bello, F., Azcárate, F.M., Mason, N.W.H. & Peco, B. (2019). Trait\nhierarchies and intraspecific variability drive competitive interactions in Mediterranean\nannual plants. Journal of Ecology, 107, 2078–2089.\nLevine, J.M., Bascompte, J., Adler, P.B. & Allesina, S. (2017). Beyond pairwise mechanisms of\nspecies coexistence in complex communities. Nature, 546, 56–64.\nPadilla, F.M., Mommer, L., Caluwe, H. de, Smit-Tiekstra, A.E., Wagemaker, C.A.M., Ouborg,\nN.J., et al. (2013). Early Root Overproduction Not Triggered by Nutrients Decisive for\nCompetitive Success Belowground. Plos one, 8, e55805.\nStein, A., Gerstner, K. & Kreft, H. (2014). Environmental heterogeneity as a universal driver of\nspecies richness across taxa, biomes and spatial scales. Ecology Letters, 17, 866–880.\nYang, J., Lu, J., Chen, Y., Yan, E., Hu, J., Wang, X., et al. (2020). large underestimation of\nintraspecific trait variation and its improvements. Frontiers in Plant Science, 11, 53.\nYang, J., Lu, J., Wang, R., Wang, X., Li, S. & Shen, G. (2021). Importance and benefit of\nincorporating the responses of species mean trait values in trait-based community\nassembly. Ecological Indicators, 130, 108095."}]

Decision letter