*3.6. Daily Carbon Gain under Actual and Simulated Conditions*

The obtained results differed between the two sites and between weather conditions (Table 2). In the clearing, the simulated change from the actual clearing leaves into understory leaves greatly

(>40%) reduced the daily net photosynthesis on both the sunny day (from A to B in Figure 10a; mean: (A) 0.745 and (B) 0.430 mol m−<sup>2</sup> day−1; A vs. B: *p* < 0.01) and on the overcast day (from A to B in Figure 10b; (A) 0.682 and (B) 0.415 mol m−<sup>2</sup> day<sup>−</sup>1; A vs. B: *p* < 0.01). This was because on both days, the reduction in gross photosynthesis (from C into D in Figure 10a,b) was much larger in magnitude than that of respiratory loss (from E into F in Figure 10a,b).


**Table 2.** Daily carbon exchange per unit area of leaf. The mean value for each item is shown.

<sup>1</sup> Daily leaf respiration rates were calculated for all the 12 leaves (6 in each site) for which dark respiration rates were measured, and the mean value at each site is shown in Table 2 and used in the leaf-exchange simulation. The same daily respiration rates were used for the two days. Among these 12 leaves, daily net- and gross-photosynthetic rates were measured or simulated for seven or eight selected leaves.

**Figure 10.** Daily carbon exchange per unit area of leaf in the clearing on (**a**) a sunny day (3 July 2020) and (**b**) an overcast day (24 June 2020). Results of the same leaf simulated in different scenarios appear in the same color. A: Estimated actual net photosynthesis. B: Simulated net photosynthesis when the photosynthetic parameters were hypothetically changed to those of the understory. C: Estimated actual gross photosynthesis. D: Simulated gross photosynthesis when the photosynthetic parameters were hypothetically changed to those of the understory. E: Estimated actual daily respiration. F: Simulated daily respiration when the leaves were changed to the understory leaves. In simulation F, all three or four leaves were assumed to have the same respiration rate as the mean value of the understory leaves.

By contrast, in the understory, the simulated change from actual understory leaves to the clearing leaves did not greatly reduce the daily net carbon gain on the sunny day (Table 2; from A to B, Figure 11a; (A) 0.145 and (B) 0.138 mol m−<sup>2</sup> day−1; A vs. B: *p* = 0.082). This is because on the sunny day, during which frequent sunflecks were observed (Figure 9b), the increment of gross daily photosynthesis (from C into D in Figure 11a) had approximately the same magnitude as the increment of respiratory loss (from E to F in Figure 11a). Those two effects offset each other. On the overcast day, during which few sunflecks were observed (Figure 9d), the simulated change from the actual understory leaves to the clearing leaves reduced the daily net photosynthesis (from A to B, Figure 11b; mean: (A) 0.075 and (B) 0.024 mol m−<sup>2</sup> day<sup>−</sup>1; A vs. B: *p* < 0.01).

**Figure 11.** Daily carbon gain per unit area of leaf in the understory on (**a**) a sunny day (3 July 2020) and (**b**) an overcast day (24 June 2020). Results of the same leaf simulated in different scenarios appear in the same color. A: Estimated actual net photosynthesis. B: Simulated net photosynthesis when the leaves were hypothetically changed to clearing leaves. C: Estimated actual gross photosynthesis. D: Simulated gross photosynthesis when the leaves were changed to the clearing leaves. E: Estimated actual daily respiration. F: Simulated daily respiration when the leaves were changed to the clearing leaves. In simulation F, all four leaves were assumed to have the same respiration rate as the mean value of the clearing leaves.

#### *3.7. Simulation: Understory without Sunflecks*

We further examined whether the obtained differences could be explained by the effect of sunflecks. We simulated the net daily carbon gain in the understory on the same sunny day (July 3) under a hypothetical situation in which all sunflecks' PPFD values (>200 μmol m−<sup>2</sup> s−1) were replaced by a fixed value 200 μmol m−<sup>2</sup> s−1. Without sunflecks, the actual understory leaves indeed performed slightly better than the simulated clearing leaves in the understory (Figure 12; mean: (A) 0.134 and (B) 0.106 mol m−<sup>2</sup> day<sup>−</sup>1; A vs. B: *p* < 0.01).

**Figure 12.** Simulated daily carbon gain without sunflecks on the sunny day (3 July 2020), in which sunflecks (>200 μmol m−<sup>2</sup> s<sup>−</sup>1) were replaced by the fixed value 200 μmol m−<sup>2</sup> s<sup>−</sup>1. A: Net daily photosynthesis calculated with actual leaves in the understory. B: Net daily photosynthesis when the leaves were hypothetically changed to the clearing leaves. The same leaf simulated under the two different scenarios appears as the same color.

#### **4. Discussion**

#### *4.1. Carbon Gain or Saving via Acclimation*

It is frequently discussed that photosynthetic acclimation increases daily net photosynthesis [3,6,17,24,48]. Supporting this theory, net daily carbon photosynthesis per unit leaf area in the clearing was higher for the actual sun leaves than for the simulated shade leaves (Figure 10). This was because during the daytime hours, PPFD on the leaves was always higher than the critical values, irrespective of the diurnal changes or weather (Figure 9a,c). By contrast, in the understory, our results did not always support the same theory; when sunflecks are present, photosynthetic shade acclimation may not always increase net daily photosynthesis. The understory leaves performed better than the clearing leaves in the understory on the overcast day, but not on the sunny day (Figure 11). Lower dark respiration rates in shade-acclimated leaves indeed resulted in lower LCPs (Table 1), but this does not necessarily imply that the shade leaves had higher net carbon gain than the sun leaves in the understory. In the understory, the simulated sun leaves frequently outperformed the shade leaves during the sunflecks (Figure 9b). When these sunflecks were hypothetically removed (Figure 12), or on the overcast day during which few sunflecks were observed (Figure 9d), such shade-acclimated understory leaves indeed had higher net carbon gain than the clearing leaves in the understory light environment. These results suggest that the observed difference was caused by sunflecks. Our results therefore support the notion [23] that under a diurnally fluctuating light environment, information on the static photosynthetic parameters and daily averaged light environment may not be sufficient to evaluate shade acclimation in forest understories. The implication may therefore be that laboratory experiments under controlled light [6,20] or field experiments using shade cloths [19,30,33], in which sunflecks were not taken into consideration, may not provide an accurate estimate of carbon gain in the understory. In this regard, it is possible to obtain better estimates through experiments using plants grown in natural conditions, as observed in [2,60,62] and the present study; those using natural canopy shading [18]; or those using advanced techniques that allow the rapid fluctuation of artificial light intensity in the case of a laboratory experiment [51,80].

For the present case, the observed reduced LMA in the understory can instead be interpreted as an effective cost-saving strategy [24,25,81] rather than as maximizing net daily photosynthesis in low-light environments. LMA was approximately twofold larger in the clearing than in the understory, whereas mass-based photosynthetic capacity (*P*g\_max\_mass) and respiration rate (*R*mass) were similar between the sites. Consequently, both light-saturated photosynthetic capacity and respiration rate per unit area of the leaves (*P*g\_max\_area and *R*area) were approximately twofold higher in the clearing. Higher investment of photosynthetic apparatus per unit area results in a higher LMA and *P*g\_max\_area [24]. Additionally, a greater leaf thickness increases the internal surface area available for the diffusive transfer of CO2 within a leaf [24,28,82–85]. Our results therefore confirm the findings of previous studies on other species that within-species variation in LMA and leaf thickness explain the variation of area-based photosynthetic traits across different light environments [4,28,29,34,35,45,86]. The lower LMA and lower photosynthetic capacity of shade-acclimated leaves incur a lower carbon cost [23–25,27,29,82] and lower nitrogen costs [31,37,41,42,87–89] to produce a unit area of leaf. In the case of this species, having lower LMA associated with lower *P*g\_max\_area in the understory may therefore have reduced leaf construction cost per unit area, in support of the cost-saving hypotheses [24,25,81]. Such reduced LMA, or equivalently, increased leaf area per unit mass (specific leaf area, SLA), increases whole-plant leaf area and light capture with a given amount of resources as a method of acclimation to low-light environments [5,6,18,19].

#### *4.2. Morphological Acclimation*

Leaf laminae in the shaded understory were flatter and therefore more horizontally displayed, whereas laminae in the clearing were more upright to decrease excessive irradiance and maximize leaf area per unit ground (Figure 5b). Similar changes in leaf three-dimensional structures (i.e., flatter in the shade) to maximize light capture in low light environments have been reported for a different species of a forest herb [55], for other within-canopy variation of lamina morphology for broadleaved trees [36,67,68], and for the three-dimensional arrangement of conifer needles [90–92]. This result is consistent with several previous findings that the leaves in well-lit places are more vertically upright, while leaves in shaded places are more horizontally displayed to maximize light capture [36,45–47,60]. However, we did not focus on the consequence of the morphological acclimation in the present study. As predicted by the game theory [93], leaf angle is determined not only on the basis of optimal light capture but also on the competition [93] and/or the contact [94] with neighboring plants. Further study is therefore needed to evaluate the consequence of morphological acclimation by taking the existing competition into consideration. Additionally, in the present study, we made a simplified assumption that the leaf 3D structure was approximated by a cone (Figure 1c). However, the shape of the actual leaves was much more complex and was trumpet-shaped; a lamina was more horizontal at the edge of each leaf and gradually was more vertical towards the center (see photographs in Figure 1). In the present study, we measured photosynthetic rate only at the edge of each leaf with the LI-6400 (Figure 3); environmental heterogeneity within a single leaf [95] was not investigated. More detailed studies that model complex 3D structure [68,95] are needed for this species.

Our study had several additional limitations. First, we examined only leaves. Although investigating leaves is equivalent to investigating the entire above-ground part of individual ramets for this species (Figure 1), the importance of the whole-plant carbon economy, including roots, has long been recognized [25]. Although leaf respiration rate is positively correlated with the respiration rates of roots [96] and the entire plant [97], further studies on whole-plant respiration rates [97,98] and whole-plant biomass allocation patterns [5,6,18,20,99] are needed for this species. Second, we ignored photosynthetic induction time and instead estimated the instantaneous photosynthetic rate using steady-state photosynthetic light response curves. Efficiency of photosynthesis may differ between steady-state and short-sunfleck conditions [49,56,58] due to stomatal [42,100–106], mesophyll [42], and biochemical [102,104,107] limitations. In our dataset, however, understory leaves frequently received sunflecks during the day (Figure 9b). Leaves of forest understory plants that are induced once maintain an induced condition for a relatively long time [49,58], so the magnitude of this overestimation might not be very large. Induction times were reported to be similar between shade-tolerant and shade-intolerant species [108]. Currently, however, there is little information on within-species differences in induction time between sunlit and shaded leaves. Third, the diurnal course of the photosynthetic rate depends not only on light but also on other environmental factors (i.e., humidity, temperature, VPD, etc.) [42,109,110] in addition to whole-plant water availability [4,111–115]. Therefore, the effect of midday depression due to stomatal closure [60,110,112,115] and photoinhibition [60,112] also would significantly alter the daily carbon gain of leaves. Furthermore, the strength of such effects may differ between sun and shade leaves [60,112,115]. Further detailed studies are therefore needed to reconfirm our findings before generation.
