Introduction

Understanding survival and acclimation mechanisms of canopy tree saplings under contrasting irradiance has important implications for forest regeneration and management (Mitchell and Arnott 1995; Kimmins 1997; Valladares and Niinemets 2008; Čater et al. 2014). Photosynthesis, carbon allocation, and biomass partitioning play key roles in the survival and adaption of saplings to various irradiance levels (Givnish 1988; Kitajima 1994; Canham et al. 1999; Johnson and Smith 2005). For example, saplings under high irradiance levels exhibit higher photosynthetic capacity to maximize carbon absorption; in contrast, leaves under lower irradiance levels show reduced photosynthetic capacity and decreased dark respiration rates (R d ) to maintain low photosynthetic light compensation points (Koike 1986; Kitajima 1994; Kenzo et al. 2011). Carbon allocation also changes with irradiance levels; carbon is preferentially allocated to growth under high irradiance, whereas allocation to reserves such as root starch increases under low irradiance, particularly within the forest understory (Kozlowski 1992; Kobe 1997; Canham et al. 1999). Under low irradiance, saplings rely on stored carbon to meet their energy demands during periods of negative carbon balance (Myers and Kitajima 2007; Dietze et al. 2014); thus, species with high relative root biomass (high root-to-shoot ratios) exhibit high survival under low irradiance (Kitajima 1994).

The Japanese fir (Abies firma Sieb. et Zucc.) generally forms mixed forests together with various deciduous broad-leaved canopy trees and Tsuga sieboldii Carr. between the warm temperate and cool-temperate zones in southwestern Japan (Yamanaka 1962; Horikawa 1972; Ando et al. 1977). The Japanese fir tree is a key species within the forest canopy, and mature trees can reach 40 m in height and 2 m in diameter (Suzuki 1980; Suzuki and Tsukahara 1987). Fir saplings grow at various irradiance levels, from the forest understory to open sites, exhibiting distinct variation in growth rate, crown shape, and needle photosynthetic traits across irradiance levels (Nakao 1985; Kenzo et al. 2000, 2014). Saplings have high shade tolerance and can survive 20–60 years with very slow growth rates under closed-canopy conditions (Yuruki and Aragami 1973; Suzuki 1980; Aragami 1987). Shaded saplings found in the understory with relative irradiance typically less than 5% alter their crown to an umbrella shape to capture weak irradiance efficiently (Nakao 1985; Kenzo et al. 2014). Needles also display a large plasticity in response to changes in irradiance. The needle photosynthetic rate at light saturation (P max) and needle mass per area (LMA) strongly decreases under low irradiance levels (relative irradiance 5–10%) after changing crown shape (Kenzo et al. 2000). However, in saplings under high irradiance levels, annual height growth can reach 40 cm, and crowns change to a conical shape (Aragami 1987; Kenzo et al. 2014). Furthermore, sun needles develop under high irradiance and exhibit higher P max and a thicker palisade layer in the needle lamina compared with shade needles under lower irradiance (Nakao 1985; Kenzo et al. 2000). Similar light acclimation via changes in morphological and physiological properties of the crown and needles has been reported in many evergreen conifer saplings, including several Abies species such as A. alba Mill., A. balsamea (L.) Mill., and A. mariesii Mast. (Mailly and Kimmins 1997; Kohyama 1980; Duchesneau et al. 2001; Grassi and Bagnaresi 2001; Landhäusser and Lieffers 2001; Robakowski et al. 2003).

Seasonal changes in temperature and irradiance may also substantially affect sapling carbon balance and survival (Kimura 1969; Lassoie et al. 1983; Lei and Koike 1998; Miyazawa and Kikuzawa 2005). Particularly in mixed forests with deciduous canopy trees, irradiance at the forest understory can change drastically with the leaf phenology of the canopy layer. Although trees in boreal forests show low or negligible photosynthetic capacity during very cold winters (Bourdeau 1959; Schaberg et al. 1995, 1998; Verhoeven et al. 1999; Öquist and Huner 2003), the saplings of evergreen tree species in the understory of temperate mixed forests exhibit large carbon gains during canopy leaf fall in the winter and early spring (Hashimoto and Shirahata 1995; Katahata et al. 2005; Miyazawa and Kikuzawa 2005; Hitsuma et al. 2012). The carbon gains during canopy leaf fall (i.e., the dormant season) may be stored in plant structures for later allocation to growth and survival during the growing season, although the proportions of allocation may vary with irradiance level (Kozlowski 1992). Increases in stored carbon such as starch, which is the main storage form for carbohydrates in conifers (Gower et al. 1995), occur in the needles, stems, and roots in several evergreen conifer trees, including Abies species, during the dormant season (Miyake 1902; Kimura 1969; Schaberg et al. 2000). Despite the importance of the seasonal light regime to understand the mechanisms of sapling acclimation to forest environments with different irradiance levels, few studies have examined the dynamics of photosynthesis, carbon balance, and stored carbon throughout an entire year. In this study, we focus on seasonal changes in needle carbon gain and stored starch in A. firma in a mixed deciduous forest. We hypothesized that substantial carbon gain occurs during canopy leaf fall (dormant season) and carbon storage (i.e., starch content) increases in the season; that stored carbon is decreased during summer by growth and the need to maintain the carbon balance; and that biomass partitioning to needle and root components varies with the irradiance level. To test these hypotheses, we measured seasonal changes in needle photosynthesis, daily carbon balance, growth, biomass partitioning, and starch content in A. firma saplings growing under different irradiance levels in a temperate mixed forest in southwestern Japan.

Materials and methods

Study site

The study was conducted in a mixed deciduous-broadleaf and evergreen conifer forest in Komenono, Ehime, Japan (132°54′E, 33°53′N, 700 m a.s.l.). Annual precipitation in the region is approximately 1800 mm, and the mean annual, monthly minimum, and maximum air temperatures are 12.3, − 2.3, and 27.4 °C, respectively (Fig. 1). Soils were mainly classified as brown forest soils based on the Japanese forest soil classification standard, and the parent material was strongly weathered granite (Sanquetta et al. 1994). The canopy layer consisted of A. firma and various deciduous broad-leaved trees such as Carpinus laxiflora (Sieb. et Zucc.) Blume, Castanea crenata Sieb. et Zucc., and Quercus serrata Murray. The canopy comprised approximately 25% A. firma trees and 70% deciduous broad-leaved trees (Sanquetta et al. 1994). Dwarf bamboo [Sasa borealis (Hack.) Makino et Shibata] and some evergreen shrubs such as Camellia japonica L. and Eurya japonica Thunb. occurred at low density in the understory. Leaf fall in the canopy layer begins in late November or early December, and canopy leaf flush occurs during May (Kenzo et al. 2014).

Fig. 1
figure 1

Seasonal changes in monthly average, maximum and minimum temperature (a), monthly precipitation (b), and relative irradiance in saplings for needle photosynthesis measurements for three irradiance levels (c high, intermediate, and low). Different letters associated with relative irradiance indicate significant differences among months at the same irradiance level (Bonferroni test, P < 0.05). ‘ns’ indicates not significant. The results of the statistical analysis are shown in Table 1. Asterisks indicate significant differences among irradiance levels in same the month (ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001)

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Table 1 Summary of the statistics for the Type III test of fixed effects in the generalized linear mixed model for relative irradiance, P max, R d , daily carbon balance, nighttime carbon loss, root starch contents, and pool size
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Plant material and irradiance levels

We randomly selected 36 saplings of A. firma under different irradiance levels for the study. All sapling heights were about 1 m. Irradiance was measured using an illumination meter (Digital Illumination Meter T-1H, Konica-Minolta, Japan) at a height of 150 cm above the ground on a cloudy day, with ten replicates for each sapling. We conducted the measurements from 1100 to 1330. Simultaneously, we measured the irradiance under open conditions and calculated the relative irradiance [relative irradiance = (sapling irradiance/irradiance at open condition) × 100] for each sapling. Relative irradiance was recorded every 2–3 months during 1.5 years. Using the relative irradiance in August, we divided the saplings into three relative irradiance categories: more than 40% irradiance was considered high irradiance; 5–40% was intermediate irradiance; and less than 5% was considered low irradiance. The relative irradiance of the saplings ranged from 0.5 to 92% in August. The relative irradiance increased during canopy leaf fall (December to April), varying from 12 to 98%.

Measurements of needle photosynthetic traits

We measured the needle gas exchange for 9 of the 36 saplings. Measurements were taken for three saplings for each irradiance level. The photosynthetic rate at light saturation (P max) and dark respiration rate (R d ) of current-year needles attached at top of the crown were measured using a portable photosynthesis meter (SPB-H4, ADC, UK) in August and October 1999 and in January, April, May, July, and October 2000. P max was measured at ambient temperature and carbon dioxide (CO2) concentration (ca. 360–390 ppm). Photosynthetic photon flux density (PPFD) was maintained at 1000 μmol m−2 s−1, which was saturated light intensity for this species (Kenzo et al. 2000). All measurements were conducted from 0800 to 1100 to avoid midday depression of photosynthesis (Hodges 1967). We also measured R d after 30–60 min of dark acclimation. To calculate the daily needle carbon balance, we conducted diurnal measurements of the needle gas exchange in March, August, October 1999, and January 2000 for the same saplings used for the above photosynthesis measurements. The gas exchange was measured on a fully expanded current-year needle at the upper crown surface. The rate of gas exchange was recorded every 5–10 min at an interval of about 15–24 h including daylight hours (approximately 0700–1800). The diurnal needle carbon balances (daily carbon balance) were computed from the diurnal courses in net needle CO2 exchange rate [e.g., Total CO2 = (A 1 × t 1) + (A 2 × t 2) + … (A n  × t n ), where A is the needle net CO2 assimilation rate, and t is the time to the next measurement point; Kenzo et al. 2003]. We divided the carbon balance during daytime (determined as measurement time greater than PAR > 1 μmol photon m−2 s−1) and nighttime (nighttime carbon loss). When the nighttime respiration rate was not measured throughout the entire night, we calculated it by assuming that the average value of the measured nighttime rate continued until sunrise. All environmental conditions were ambient, and an open-top chamber was used. We conducted measurements on 3 days that had mostly sunny conditions. All needles used for the gas exchange measurements were collected after the measurements to determine the needle area as a basis for calculating the area-based CO2 assimilation rate. The area of fresh needles was calculated using a leaf area meter (Area Meter MK2, Delta Devices).

Measurements of starch content and sapling growth

We collected the fresh roots (about 1 g, 3–5-mm diameter) of 16 saplings from all selected saplings in the morning (at approximately 0700–0900), including those for which photosynthesis measurements were taken, to avoid daily fluctuations in starch content (Kenzo et al. 2013). Measurements were made for five saplings at high irradiance, six saplings at intermediate irradiance, and five saplings at low irradiance; these were conducted in April, August, and October 1999, and March 2000. We also collected 1-year-old needles and branches in April and August. We also measured the height of all sampled saplings in March 1999 and 2000. The relative height growth rates (RGRh) of the saplings were calculated using the formula,

$${\text{RGRh}}=\frac{{\ln (H{t_2}) - \ln (H{t_1})}}{{{t_1} - {t_2}}}$$

where Ht 1 and Ht 2 are the height (in cm) at the early (t 1) and later inventory (t 2), and t (time) is measured in years.

All samples were stored in a compact freezer immediately after collection. In the laboratory, all samples were dried at 60 °C for 3 days, after which dry mass was measured (Kenzo et al. 2013). Then, the dried samples were ground to a fine powder. Starch concentrations were determined by extracting 0.1 g of aliquots of dry sample in perchloric acid, and then combining the solubilized starch with iodine (Pucher et al. 1948; Pate et al. 1990). We also prepared blanks and varying concentrations of known starch standards with iodine to calculate a calibration curve. The fractional transmittance was read using a UV/VIS spectrophotometer (Shimadzu UV-1400, Kyoto, Japan) at 620 nm. The proportion of starch remaining in roots from April to August was calculated as the starch content in August divided by the starch content in April. Reports on the growth phenology of saplings of A. firma indicated that shoot elongation and the stem increment of most saplings started in late April and stopped by late August at the study site (Kenzo et al. 2014).

Determination of biomass partitioning of saplings

All 36 saplings under different irradiance levels were excavated and divided into needle, stem, branch, and root components in the field. All fresh weights were measured using an electronic balance in the laboratory. All samples were dried in an oven at 105 °C for 80 h until they reached constant mass, after which dry mass was measured. The root-to-shoot ratio (root dry mass/all aboveground dry mass) and the needle mass ratio (needle dry mass/non-needle organs dry mass) were calculated. We also calculated the root starch pool size from the root biomass and root starch content.

Statistical analysis

The effects of the independent values (month and irradiance level) on dependent variables including the relative irradiance, P max, R d , daily carbon balance, nighttime carbon loss, and root starch content were evaluated by fitting generalized linear mixed models (Sokal and Rohlf 1995). The effects of individual saplings were included as random effects. Type III tests of the fixed effects (Wald-type test) were performed. Post hoc multiple comparison testing was performed using the Bonferroni method when the result was significant at P < 0.05. To test the seasonal effect on these dependent variables (e.g., P max) at the same irradiance level, we constructed generalized linear mixed models with the Wald-type test and conducted post hoc multiple comparison testing using the Bonferroni method. We performed ANOVA to test the irradiance effects on the relative growth rate, needle and branch starch contents, proportion of remaining starch, biomass partitioning, and relative irradiance in August. Tukey–Kramer’s or Tukey’s tests were used to compare means when ANOVA indicated significant differences (Sokal and Rohlf 1995). All analyses were conducted using SPSS for Windows (ver. 22; IBM, Armonk, NY, USA).

Results

Seasonal changes in irradiance levels

The irradiance levels experienced by the saplings changed dramatically with leafing phenology of upper deciduous canopy trees (Fig. 1c; Table 1), except for saplings under high irradiance levels. Leaf fall in the canopy layer began in late November to early December, and leaf flush occurred in May. The relative irradiance in saplings increased with canopy leaf fall from 2 to 15% under low irradiance levels and from 10 to 50% under intermediate irradiance levels (Fig. 1c). We also observed direct sunlight on sapling needles during the defoliated season under low and intermediate irradiance levels. By contrast, the relative irradiance did not vary greatly throughout the year in saplings under high irradiance.

Seasonal changes in net CO2 assimilation rate in needles

Clear seasonal changes in P max were observed at all irradiance levels (P < 0.05, Fig. 2; Table 1). P max decreased in summer and winter and increased in spring and autumn. P max was higher under high and intermediate irradiance compared with low irradiance levels throughout the year; values at high irradiance were approximately twice as high as those at low irradiance. R d also changed with the season (P < 0.05, Fig. 2; Table 1), declining in January and increasing in April, July, and August. Similar to P max, R d was higher under high and intermediate irradiance levels than under low irradiance.

Fig. 2
figure 2

Changes in the maximum photosynthetic rate at light saturation (P max, positive values with solid line), and dark respiration rate (R d , negative values with dotted line). Different letters indicate significant differences among months (P < 0.05). For P max, letters in italics, underlined letters, and normal font indicate high, intermediate, and low irradiance levels, respectively. For R d , the statistical results for the three irradiance levels are displayed together because the same statistical result was found for all levels. Bars indicate the standard error. The results of the statistical analysis are shown in Table 1. Asterisks indicate significant differences among irradiance levels in the same month (ANOVA; *, P < 0.05; **, P < 0.01; ns, P > 0.05). Number in the parentheses indicates number of saplings

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Seasonal changes in daily needle carbon balance and nighttime carbon loss among irradiance levels

Diurnal changes in the needle gas exchange and daily needle carbon balance showed distinct patterns across seasons and irradiance levels (Figs. 3, S1, S2, S3, and Table 1). Although the seasonal patterns of the daily needle carbon balance differed among irradiance levels, the nighttime needle carbon loss (nighttime respiration) had the same seasonal trends among irradiance levels: it was highest in August and decreased in January and March (Fig. 3; Table 2). Saplings under high irradiance received strong direct sunlight and exhibited high positive net carbon assimilation during the day, although midday photosynthetic depression occurred in August (Fig. S1D). In fact, the daily needle carbon balance in August was lower compared with the other seasons (P < 0.05, Fig. 3). The daily carbon balance for saplings under intermediate irradiance levels remained positive throughout the year (Fig. 3). Especially in March, saplings received strong direct sunlight and had a higher net needle CO2 assimilation rate due to the leaf fall of overstory trees (Fig. S2A, B). By contrast, saplings under low irradiance levels in August and October were strongly shaded with exposure to few sunflecks (Fig. S3C, E); negative net needle CO2 assimilation was even observed during the day (Fig. S3D, F). In comparison, during the leaf fall period, positive net needle CO2 assimilation was recorded throughout the daytime in January and March (Fig. 3, S3B, H). The daily needle carbon balance was also negative in August and October, whereas carbon gain significantly increased in January and March (P < 0.05, Fig. 3). Although daily PPFD did not significantly change in saplings under high irradiance (Table S1), saplings in intermediate and low irradiance received significantly large amounts of PPFD during the leaf fall season in January and March compared to August and October (P < 0.05, Table S1).

Fig. 3
figure 3

Changes in needle daily carbon balance (solid line) and nighttime carbon loss (dotted line). Different letters indicate significant differences among months (P < 0.05). Letters in italics, underlined, and normal font are high, intermediate, and low irradiance levels, respectively. Bars indicate the standard error. The results of the statistical analysis are shown in Table 1. Asterisks indicate significant differences among irradiance levels in the same month (ANOVA; *, P < 0.05; **, P < 0.01; ns, P > 0.05). Numbers in parentheses are the numbers of saplings

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Table 2 Relative growth rate of height (RGRh), starch contents and proportion of remaining starch in needle, branch and root in April and August
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Seasonal changes in starch content

The root starch content per tissue dry weight and starch pool size showed clear seasonality; content and pool size increased in spring, decreased sharply in August, and increased again from October to spring (Fig. 4a, b; Table 1). The needle and branch starch contents in April were also higher than those observed in August, although both starch contents were lower than that of roots in August (Table 2). The starch contents in August were less than 0.4% in needles and 0.6% in branches, whereas the values in roots were 2.0–6.4% (Table 2). The root starch content in April was higher under high and intermediate irradiance levels compared with low irradiance, whereas the content in August was highest under low irradiance (Fig. 4a; Table 2). By contrast, the needle and branch starch contents were similar among the sapling irradiance levels in both April and August (Table 2).

Fig. 4
figure 4

Changes in root starch content (a) and starch pool size (b) among sapling irradiance levels. Different letters indicate significant differences among months (P < 0.05). Letters in italic, underlined, and normal font indicate high, intermediate, and low irradiance, respectively. Bars indicate the standard error. The results of the statistical analysis are shown in Table 1. Asterisks indicate significant differences among irradiance levels in the same month (ANOVA; *, P < 0.05; **, P < 0.01; ns, P > 0.05). Numbers in parentheses are the numbers of saplings

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Relative growth rate, remaining starch content, and biomass partitioning among irradiance levels

The relative height growth rate (RGRh) in saplings was significantly higher under intermediate and high irradiance levels compared with under low irradiance (Table 2). The starch remaining in roots was significantly higher under low irradiance (38.2%) compared with intermediate (9.7%) and high irradiance (5.1%), although a similar relationship was not observed for needles or branches (Table 2). Biomass partitioning among the needle, stem (including branch), and root components differed significantly among irradiance levels (P < 0.05, ANOVA, Fig. 5). Saplings under high and intermediate irradiance had larger needle biomass compared with those under low irradiance, whereas saplings under low irradiance had a relatively large root component (Fig. 5). The root-to-shoot ratio was also higher under low irradiance, whereas high needle biomass, as indicated by the needle mass ratio, was observed in saplings under intermediate and high irradiance (Table 3).

Fig. 5
figure 5

Biomass partitioning among irradiance levels. Different letters indicate significant differences among months by ANOVA with post hoc LSD analysis (Tukey–Kramer’s HSD test, P < 0.05). Numbers in parentheses are the numbers of saplings

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Table 3 Relative irradiance in August, root shoot ratio, needle mass ratio, needle biomass, stem and branch biomass, and root biomass between irradiance levels
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Discussion

Seasonal changes in daily carbon balance among irradiance levels

Consistent with our hypothesis, large carbon gains occurred in saplings during the canopy leaf fall period, particularly in the spring, regardless of irradiance level, although similar large carbon gains were also observed in October in saplings under high irradiance. High photosynthetic assimilation due to an improved light environment in January and March caused high daily carbon gain in saplings, particularly under intermediate and low irradiance levels. Although P max in January was suppressed by low temperatures compared to other seasons, the decreased respiration rate at night also contributed to the large carbon gain. The nighttime carbon loss declined by 1/3 to 1/7 compared to summer because nighttime temperatures were 10–20 °C lower in winter and spring, and because Q 10 values (the proportional increase in R d with 10 °C warming) of various plant species usually increase under lower growth temperature such as winter (Amthor 1989). Similarly, high carbon gains during winter have been reported for several evergreen conifer species in both mature and sapling trees in forests with milder winter temperatures (Waring and Franklin 1979; Schaberg et al. 2000); this pattern is particularly strong for saplings growing under deciduous canopy trees (Hashimoto and Shirahata 1995; Hitsuma et al. 2012). For example, in Douglas-fir (Pseudotsuga menziesii) trees in western Oregon where winters are mild and wet, photosynthesis during the dormant season (late October through late April) accounted for as much as 55% of total annual carbon assimilation (Emmingham and Waring 1977). The positive relationship between annual diameter growth and winter temperature in A. firma observed by Takahashi and Okuhara (2012) also supports the importance of winter carbon assimilation by photosynthesis in this species.

By contrast, summer may not be the optimal season for photosynthetic production of saplings, particularly under high and low irradiance, although the mechanism driving photosynthetic limitation differed between high and low irradiance. Under high irradiance, midday depression of photosynthesis and high nighttime respiration suppressed daily carbon gain. The reduction of sapling growth in A. firma under open compared to moderately shaded conditions (Nakao 1985) also indicates limited carbon gain during summer at high irradiance. A similar midday depression of photosynthesis has also been reported in two Abies species in North America; the midday declines occurred under clear skies in open conditions, although the depression was not as obvious on cloudy days and large amounts of carbon gain occurred (Hodges 1967; Hodges and Scott 1968). Drier soil, high temperature, and high vapor pressure deficit (VPD) in summer may induce stomatal closure with high respiration rates, which may then cause midday depression of photosynthesis (Hodges 1967). In this study, the midday depression was also lower on cloudy days (data not shown), and thus the surplus carbon on cloudy days may be important for sapling carbon balance and growth during summer. In contrast to high irradiance levels, reduced light intensity due to elongated leaves in the canopy layer may cause decreased photosynthetic assimilation of saplings under intermediate and low irradiance levels. In addition, increased needle R d caused by high temperatures may also suppress needle daily carbon balance. Particularly, notable was the negative daily carbon balance in saplings under strongly shaded low irradiance levels, which was maintained until at least October. Sunflecks, which can allow relatively high photosynthetic assimilation in understory plants (Pearcy 1983; Ustin et al. 1984; Chazdon 1988) including Abies alba (Robakowski et al. 2004), occurred occasionally in the summer, and thus the contribution to daily needle carbon balance in the saplings was limited under low irradiance levels.

Stored starch, biomass partitioning, and survival strategy among irradiance levels

Saplings of A. firma adapted to different irradiance levels by altering biomass partitioning, such as the root-to-shoot ratio and use of stored carbon, which primarily originated from carbon assimilation during the leaf fall period of the canopy layer. Carbon assimilated during canopy leaf fall was stored in the various structures of saplings, particularly the roots, regardless of irradiance level. Many evergreen conifer species store assimilated carbon as starch during the dormant season (Gower et al. 1995). Schaberg et al. (2000) found that increases of starch and sugars in red spruce seedlings were driven by photosynthetic production during the dormant season. Similar increases in starch and sugar content in needles, branches, and roots just prior to the growing season have been observed in many evergreen conifer species of both mature and sapling trees, including several Abies species (Miyake 1902; Kimura 1969; Little 1970; Höll 1985; Amundson et al. 1992; Webb and Kilpatrick 1993; Schaberg et al. 2000; Hoch et al. 2003). Stored starch under high and intermediate irradiances may be mainly used for growth, whereas saplings under low irradiance may use it to maintain the carbon balance during the summer, when the carbon balance is negative. Further detailed study of the allocation of stored starch to growth versus compensation for negative carbon balance among irradiance levels and seasons will provide a better understanding of the responses and acclimation of fir saplings to the seasonal changes in irradiance in mixed deciduous forest.

Biomass partitioning among needles, stems, and roots changed greatly depending on the irradiance level of the growing saplings. Increases in needle biomass under high and intermediate irradiance levels may have contributed to increases in photosynthetic production, as a thick needle layer with high P max can optimally use high light (Sprugel et al. 1996; Kenzo et al. 2000; Landhäusser and Lieffers 2001; Kato and Yamamoto 2002). In contrast, under low light, small numbers of needles were arranged in an umbrella-shaped crown to avoid self-shading and intercepted weak irradiance at a minimal cost in needle construction and maintenance (Leverenz and Hinckley 1990; Williams et al. 1999). Kenzo et al. (2014) reported that the Leaf Area Index (LAI) under low irradiance levels (relative irradiance in summer less than 5%) of A. firma saplings with an umbrella-shaped crown decreased to approximately 1, whereas the LAI in saplings at intermediate and high irradiance reached 2–3. The relative amount of the root component increased at low irradiance; an increased root-to-shoot ratio may be advantageous for starch storage to maintain carbon balance during summer. In fact, the relative starch pool of the root component at the level reached 88% compared with the needle (1%) and stem (11%) components in summer (data not shown). In addition, higher carbon allocation to storage in saplings within a dark understory may also contribute to their ability to recover from damage to stems and needles by fallen branches, pathogens and herbivore attack, all of which frequently occur in the understory (Myers and Kitajima 2007). High storage capacity for starch and other resources in roots have been reported for many shade-adapted plants such as spring ephemerals, understory trees, and saplings with shade tolerance (Kozlowski 1992; Poorter and Kitajima 2007; Kenzo et al. 2013). Large storage capacity with a high root-to-shoot ratio allows plants to survive longer periods in the shaded understory (Kitajima 1994; Kobe 1997; Myers and Kitajima 2007). Similar increases in the root-to-shoot ratio of A. firma saplings under deeply shaded conditions (relative irradiance 1%) have also been reported in a natural fir-hemlock forest in southern Japan (Yuruki and Aragami 1973). However, previous studies have also observed increases in the relative amount of leaf biomass of tree seedlings rather than increases in root biomass especially in nursery experiments with the same species (Givnish 1988; Geiger and Servaites 1991). Nakao (1985) also reported that A. firma seedlings increased needle biomass rather than root biomass under moderate shade (relative irradiance was controlled to about 10% by shade cloth), although the experiment did not consider seasonal changes in irradiance, and examined only four growing seasons. The conflicting results may have been caused by the relatively short period of shade without significant changes in crown shape. The experiment by Nakao (1985) also did not find any changes in crown shape such as the umbrella form or branch dying up. Results of the present study indicated that the relative root biomass may increase by decreasing the needle and stem (mainly in branches) biomass as a result of living branches’ drying up to construct an umbrella shape crown under long-term suppression of deep shade. Overall, saplings of A. firma under low irradiance may be unable to reach the canopy layer due to limited carbon gains; therefore, long-term survival increases the probability of experiencing canopy gap formation, enabling them to regenerate to the canopy layer. Several demographic studies have also reported that regeneration of A. firma depends on canopy gap formation or large-scale disturbance (Suzuki 1980; Suzuki and Tsukahara 1987; Yuruki and Aragami 1987; Yuruki et al. 1987; Agetsuma 1992; Sanquetta et al. 1994). However, sapling survival under evergreen trees and shrubs may be difficult, as photosynthesis may not occur in winter and spring. In fact, sapling survival of A. firma was inhibited under the shade of evergreen dwarf bamboo (Yuruki and Aragami 1973; Aragami 1987). By contrast, sapling recruitment in deciduous forests occurred frequently compared to evergreen forests, including pure stands of A. firma (Kaji 1975). Therefore, to maintain natural regeneration of A. firma, forests should be managed as mixed forests with deciduous broad-leaved canopy trees.

Conclusions

Saplings of A. firma can establish in a wide range of irradiance levels by having different needle photosynthetic traits, starch storage, and biomass partitioning to roots and needles. Under high and intermediate irradiance levels, saplings allocated carbon to needle biomass with high P max to achieve high assimilation under strong irradiance, although the midday depression of assimilation in summer limited the daily needle carbon gain in saplings under high irradiance. In comparison, saplings under low irradiance levels depend on photosynthetic production during leaf fall under a canopy of deciduous trees, and starch stored during this period may be used to maintain the carbon balance in the summer and may permit long-term sapling survival under shaded conditions.

Author contribution statement

T.K. designed the study, interpreted results and wrote the manuscript. R.Y. and I.N. conducted field measurements and interpreted results.