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AOBPreview originally published online on November 30, 2007
Annals of Botany 2008 101(3):435-446; doi:10.1093/aob/mcm296
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© The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Timing of Canopy Closure Influences Carbon Translocation and Seed Production of an Understorey Herb, Trillium apetalon (Trilliaceae)

Takashi Y. Ida* and Gaku Kudo

Graduate School of Environmental Science, Hokkaido University, Sapporo, 060-0810, Japan

* For correspondence: E-mail id{at}ees.hokudai.ac.jp

Received: 22 August 2007    Returned for revision: 17 September 2007    Accepted: 9 October 2007    Published electronically: 30 November 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

Background and Aims: The light availability on a temperate, deciduous-forest floor varies greatly, reflecting the seasonal leaf dynamics of the canopy trees. The growth and/or reproductive activity of understorey plants should be influenced by the length of the high-irradiance period from snowmelt to canopy closure. The aim of the present study was to clarify how spring-blooming species regulate the translocation of photosynthetic products to current reproduction and storage organs during a growing season in accordance with the changing light conditions.

Methods: Growth pattern, net photosynthetic rate, seed production, and shoot and flower production in the next year of Trillium apetalon were compared between natural and experimentally shaded conditions. Furthermore, translocation of current photosynthetic products within plants was assessed by a labelled carbon-chase experiment.

Key Results: During the high-irradiance period, plants showed high photosynthetic ability, in which current products were initially used for shoot growth, then reserved in the rhizome. Carbon translocation to developing fruit occurred after canopy closure, but this was very small due to low photosynthetic rates under the darker conditions. The shading treatment in the early season advanced the time of carbon translocation to fruit, but reduced seed production in the current year and flower production of the next year.

Conclusions: Carbon translocation to the storage organ had priority over seed production under high-irradiance conditions. A shortened bright period due to early canopy closure effectively restricts carbon assimilation, which greatly reduces subsequent reproductive output owing to low photosynthetic products for fruit development and small carbon storage for future reproduction. As populations of this species are maintained by seedling recruitment, acceleration of canopy closure timing may influence the maintenance and dynamics of populations.

Key words: 13C labelling, canopy closure, carbon translocation, deciduous forest, light availability, photosynthesis, spring-bloomer, Trillium apetalon


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Light availability within the deciduous forest understorey changes greatly from spring to early summer due to the leaf emergence of canopy trees. Such seasonal decline in light conditions is a predictable event for understorey plants, and it is thought to be a major selective force in the life-history evolution of understorey plants (Schemske et al., 1978; Lubbers and Christensen, 1986). For example, the life history of spring ephemerals is characterized by early emergence soon after snowmelt, early flowering and short growing season within a high-irradiance period before canopy closure (Muller, 1978; Kawano, 1985; Lapointe, 2001). Although photosynthetic production of spring-blooming species largely depends on the high-irradiance period, only a few studies have focused on how variations in leafing phenology of canopy trees influence the reproductive output of understorey plants (e.g. Routhier and Lapointe, 2002; Kudo et al., 2008). The growth initiation of spring-blooming species is strongly determined by snowmelt timing (Fitter et al., 1995; Diekmann, 1996; Kudo et al., 2008), while leafing phenology of canopy trees is largely determined by air temperature in early spring (Gordo and Sanz, 2005; Richardson et al., 2006) and/or the number of days below freezing point in winter (Raulier and Bernier, 2000). Contrasts between the determinant factors between spring-bloomers and canopy foliage may cause yearly fluctuations of the high-irradiance period from snowmelt to canopy closure. Kudo et al. (2008) reported that the period from snowmelt to bud burst of canopy trees varied from 26 to 49 d during 8 years in northern Japan. Such fluctuations should affect the carbon assimilation and reproductive success of understorey herbs.

If there is a trade-off in resource investment between current reproduction and future growth or reproduction, reproductive success in a single season does not reflect the lifetime fitness of a plant. For instance, an increase in carbon translocation to a reproductive organ may result in a decrease in carbon translocation to other organs. The decrease in resource translocation to growth and/or storage due to the resource investment in current reproduction is defined as a direct cost of reproduction (reviewed in Obeso, 2002). In addition, consideration of the long-term effects of reproduction, i.e. the indirect cost of reproduction, is important to understand fully the reproductive strategy of polycarpic plants (Newell, 1991; Ashman, 1992; Nicotra, 1999). The indirect cost is expressed as the decrease in subsequent growth and/or reproduction after the occurrence of a reproductive event (Newell, 1991; Ashman, 1992). Plants may regulate their reproductive output in a single season to cope with external and/or own resource conditions by changing resource allocation between current reproduction and storage for the future.

The resource budget for current reproduction and storage varies not only among species but also over the season within a single species. In the perennial herb Primula veris, for instance, the effects of herbivory on current and future reproduction have been shown to vary depending on the reproductive stage when herbivory occurred (Garcia and Ehrlen, 2002). This is because foliar photosynthetic products during the flowering period are used for current reproduction, while those during the fruiting period are used for future reproduction in this species. This indicates the importance of the timing of external events affecting carbon assimilation in terms of the resource allocation strategy. In deciduous forests, the seasonal decline of light availability and yearly fluctuations in duration of the high-irradiance period should determine the total carbon assimilation and influence the allocation pattern of understorey herbs. There are four options for carbon translocation patterns responding to the seasonal decline of photosynthetic products: (1) plants may enhance the relative carbon translocation to current reproduction at the expense of translocation to a storage organ (compensative response for reproduction); (2) plants may enhance the relative carbon translocation to a storage organ at the expense of seed production; (3) plants may not change the translocation pattern between reproduction and storage; or (4) plants may separate the time of translocation to a storage organ and current reproduction, resulting in independent responses to the resource limitation between them.

The present study investigated the patterns of growth and carbon translocation over a growing season under natural and shade conditions in a spring-blooming species, Trillium apetalon, which is a polycarpic perennial species growing in deciduous forests. Flowering of this species occurs in early May before canopy closure, while fruits develop under decreasing light conditions and seeds are dispersed in mid-July. To understand the life-history strategy of spring-bloomers, it is crucial to know how seasonal changes in light availability influence the carbon translocation pattern. Modification of canopy closure timing via a shading treatment is a useful method in this regard. The following questions were investigated. (1) How are photosynthetic products allocated to individual organs within a growing season? (2) How do the temporal changes in light availability influence the translocation pattern? (3) Does the timing of canopy closure influence the current seed production and the reproductive activity in the next year?


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Study site and species
This study was conducted in a deciduous forest of Nopporo Forest Park (43°20'N, 141°31'E) in Hokkaido, northern Japan. The major canopy trees are Fraxinus mandshurica var. japonica and Ulmus davidiana var. japonica. Spring-blooming species appear soon after snowmelt, which usually occurs in early April. Leaf emergence of overstorey trees usually occurs in mid-May and canopy closure completes by mid-June (Tani and Kudo, 2006).

Trillium apetalon Makino (Trilliaceae) is a self-compatible spring-blooming species growing in deciduous forests of Japan. It is a polycarpic perennial and reproduces exclusively by seeds (Ohara and Kawano, 1986a). Flowering begins in late April and continues toward mid-May in Hokkaido. The flowering period of individual plants is about 1 week; fruits develop over 50–60 d and mature fully by early July (Fig. 1). Leaf senescence starts in early July and is completed just before seed dispersal. Thus, the length of the growing season of this species is longer than that of typical spring ephemerals, the growing season of which is commonly completed by canopy closure. The study species has high fruiting ability without pollinator visits due to a self-compatible mating system (Ohara et al., 2001).


Figure 1
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  		FIG. 1. Schema of reproductive phenology of Trillium apetalon: (A) fraction of absorbed photosynthetically active radiation by canopy trees (fAPAR), and (B) daily maximum photosynthetic photon flux density (PPFD) on forest floor and daily mean temperature in 2006. The horizontal line represents the foliage period, and circles represent dates of observations.

 
Meteorological data
Meteorological data of Tomakomai Experimental Forest, Hokkaido University (42°40'N, 141°36'E), were used because the light and temperature conditions on the forest floor there were similar to those of Nopporo Forest Park (Tani and Kudo, 2006). Data for air temperature and photon flux density (PFD) at 2 m height and above the canopy were automatically recorded at 1-h intervals. The fraction of absorbed photosynthetically active radiation by canopy trees (fAPAR) was calculated as follows: (PFD above canopy – PFD at 2 m height)/PFD above canopy. Light availability on the forest floor was high before canopy closure, decreased from late May to early June due to leaf expansion of canopy trees, and the low-light level continued throughout the summer (Fig. 1).

Experimental design
In spring 2006, 121 individuals at flowering were randomly selected and marked. All experimental plants had only a single flowering stem and all flowers were supplementally hand-pollinated with pollen from at least two donor plants. Among them, 39 individual plants were shaded by fine-meshed spectrally neutral cloth, which reduces photosynthetic photon flux density (PPFD) to 17 % under natural conditions. Nineteen of these plants were used for the measurement of current reproductive output and performance in the next year, and the remaining 20 plants were used for the measurement of biomass in each organ and the 13C labelling experiment. The shading treatment was applied for individual plants independently from soon after flowering (15 May) until fruit maturation. The other 82 plants selected remained under natural light conditions. Twenty-nine of them were used for the measurement of current reproductive output and performance in the next year, and the remaining 53 plants were used to measure biomass allocation among individual organs and for the 13C labelling experiment.

Growth condition, photosynthetic rate and dry mass allocation were measured three times using these plants during the reproductive period as discussed further below. The first measurement was conducted on 7–8 May in the flowering period under high-irradiance conditions, the second measurement was on 24–25 May in the early-fruiting period when the light level was still high, and the third measurement was conducted on 11–12 June in the middle-fruiting period under closed canopy (Fig. 1). Seed production was measured as a proxy for current reproductive success.

Growth pattern
Dry weights of above-ground parts and rhizome were measured by harvesting 10–20 plants in each treatment (full light vs. shading) and reproductive period (flowering, early-fruiting and middle-fruiting). Individual sampled plants were taken back to the laboratory and separated into four organs: flower or fruit, stem, leaves and rhizome. Each sample was oven-dried at 70 °C for 72 h and weighed. Roots were not measured because their mass was negligibly small. The ratio of above-ground shoot mass (flower or fruit + stem + leaf) to rhizome mass was calculated for individual plants.

As leaf characters, leaf mass, area and leaf mass per area (LMA) were assessed or individual plants were sampled in each period. Seasonal changes in total leaf area were also measured for 30 plants under natural conditions and 16 plants under shade conditions in each reproductive period. Leaf area was calculated non-destructively by using the allometric relationship obtained by an elliptic approximation, which was preliminarily examined using leaf samples from 30 plants in early and mid-May, 2006. Leaf samples were optically scanned into a computer, and length (L, cm), width (D, cm) and leaf area (S, cm2) were measured with image analysis software (Image J version 1·34; National Institutes of Health, Bethesda, MD, USA) by finding the optimal binary threshold for individual leaves. The following allometric relationship was obtained by a quadratic regression: S = 0·764(L/2)2(D/2)2{pi}+ 1·498, n = 30, R2 = 0·99. Because every plant had three leaves of similar size, as in other Trilium species, the total leaf area per plant was calculated as 3S.

Before statistical analysis, data were log-transformed to improve normalities. The shoot-to-rhizome ratio, mass of individual organs and LMA under natural conditions were compared among reproductive periods by one-way ANOVAs followed by Turkey's least significant difference test. For the comparison of growth conditions between treatments, the mass of each organ, shoot-to-rhizome ratio and LMA were analysed with repeated-measures ANOVAs using data of the early- and middle-fruiting periods.

Photosynthesis
We selected seven of the experimental plants in which light-responses of photosynthetic rates were measured three times during the reproductive period, i.e. flowering, early-fruiting and middle-fruiting period, using a portable closed gas-exchange system (LI6400; Li-Cor, Lincoln, NB, USA). Four of the target plants grew under natural light conditions, while three of them were shaded from the beginning to the end of the fruiting period [relative photosynthetically active radiation (PAR) = 17 %]. Twelve light conditions (1500, 1000, 800, 500, 300, 200, 100, 60, 40, 20, 10 and 0 µmol m–2 s–1) of PAR were provided in the flowering and early-fruiting periods, and nine conditions (1500, 1000, 800, 500, 300, 100, 50, 10 and 0 µmol m–2 s–1) in the middle-fruiting period using a red–blue LED light source at constant temperature (20 °C). Ambient CO2 concentration was maintained at 350 µL L–1 by controlling the humidity of incoming air at 1·1 vapour pressure deficit (VPD, hPa). The light-saturated rate of photosynthesis (pmax) was calculated in each plant as a mean value of photosynthetic rates under high-light conditions (>1000 µmol m–2 s–1) in which light-response curves were saturated (see Fig. 3 below). Dark respiration rate at 0 µmol m–2 s–1 (Rd) was also obtained for individual plants. The net photosynthetic rate (p) as a function of photon irradiance is accurately described by a non-rectangular hyperbola as follows;


Formula

where pmax is the light-saturated rate of photosynthesis (µmol m–2 s–1), {alpha} is the photochemical efficiency of photosynthesis at low light intensity (initial slope of hyperbola: µmol m–2 s–1), I is the irradiance incident on a leaf (intensity of PAR: µmol m–2 s–1), {theta} is the degree of curvature of the line (dimensionless) and Rd is the rate of dark respiration (µmol m–2 s–1) (Marshall and Biscoe, 1980). Data obtained for individual plants in each period were fitted to this equation by non-linear least-squares estimates of the parameters.

Daily photosynthetic carbon gain was simulated under the assumption that photosynthetic rates were determined only by light conditions and the concentrations of CO2 and air temperature were constant (350 µL L–1 and 20 °C, respectively) over the growing season. First, the temporal transition of light availability was estimated for each period and treatment based on the photon flux density at 1-h intervals. PAR data during May were used for the flowering and early-fruiting periods, and data during June were used for the middle-fruiting period. Next, net photosynthetic rates per hour were estimated using the photosynthetic parameters obtained and the mean total leaf area per plant in each period. By summing the results, the daily photosynthetic carbon gain per plant was calculated.

Before statistical analysis, data were log-transformed to improve normality. To reveal seasonal changes in photosynthetic characteristics under natural conditions, each photosynthetic parameter (pmax and Rd) was analysed by one-way repeated-measures ANOVA. For the comparison of photosynthetic parameters among periods and treatments during the fruiting period, individual parameters were analysed by two-way repeated-measures ANOVA.

13CO2 tracing
The seasonal pattern of carbon translocation and the shading effect on it were measured by supplying 13CO2 to plants under natural (flowering, early-fruiting and middle-fruiting periods, n = 10, 9 and 9, respectively) and shade conditions (early- and middle-fruiting periods, n = 10 and 10, respectively). 13C level was also measured in 25 individuals under natural conditions as a control. The shading treatment was as mentioned earlier (see ‘Experimental design’ above). For the application of 13CO2, all the leaves of each plant were enclosed in a 40 x 30-cm sealed nylon bag. The 13CO2 labelling was carried out when there was sufficient sunlight to cause net CO2 uptake, on two successive sunny days. Injections of 13CO2 were made in early morning on the first and second days. A cylinder containing 30 mL of lactic acid and two tubes containing 150 mg of 99·9 at. % Ba13CO3 (Isotec Inc., Miamisburg, OH, USA) were sealed in each bag. Barium carbonate was added to lactic acid, releasing 13CO2 into the bag. It is known that translocation of a fixed carbon isotope from leaves to other organs usually occurs within 1 d in herbs or grasses (Gordon et al., 1977; Miller and Rose, 1992). Therefore, plants were exposed to the 13CO2-enriched atmosphere for 2 d in each period. 13CO2 was then injected into the chamber at a concentration of CO2 equivalent to that of normal air (about 360 p.p.m.). Plants were harvested after a 2-d labelling period. After harvesting, individual plants were separated into four organs (flower or fruit, stem, leaf and rhizome) and oven-dried at 70 °C for 72 h. After weighing each organ they were ground in a mortar.

The combined system of an elemental analyser (Flush EA; Thermo Electron, Bremen, Germany) and an isotope ratio mass spectrometer (Delta V Plus; Thermo Electron) was used to measure {delta}13C. Each approximately 1-mg sample was packaged in a tin container and carbon isotopic composition was analysed.

Excess 13C was calculated according to procedures described by Simard et al. (1997) as follows: each {delta} 13C value was first converted to the absolute isotope ratio of the sample (Rsample):


Formula

where Rstd = 0·0112372, the carbon isotope ratio of the international VPDB standard. The 13C abundance ratio (A) was calculated as


Formula

Total 13C content of the sample was calculated from the 13C abundance ratio (A) and total carbon weight of each organ. The 13C in excess of natural abundance (mg 13Cna) was calculated as


Formula

where mg 13Cna was calculated from the {delta}13C value of each organ in each period from non-labelled plants. Excess mg 13C in each organ (excess mg 13Corgan) was calculated as a product of excess mg 13Csample (per mg of sample) and the biomass of each organ (mg). Excess mg 13C of the whole plant (excess mg 13Cplant) was determined by summing the excess mg 13Corgan of all organs. As the amount of 13CO2 supplied was different among individuals, carbon translocation was expressed as a percentage of mg 13Corgan against mg 13Cplant as follows:


Formula

Before the statistical analysis, data were arcsin-transformed to improve normality. The 13C percentages in each organ were analysed by multivariate analyses of variance (MANOVA) in which reproductive periods (flowering, early- and middle-fruiting) and treatment (shading and natural light conditions) were considered as fixed effects. When MANOVA revealed significant effects, excess 13C in each organ under natural conditions was compared among periods (flowering, early- and middle-fruiting) by one-way factorial ANOVA followed by Turkey's least significant difference test. For the comparison of carbon translocation patterns between treatments, excess 13C in each organ was analysed by two-way factorial ANOVA in which period (early- and middle fruiting) and treatment were considered as fixed effects.

Current reproduction, and shoot performancein the next year
To evaluate the shading effects on current reproductive success, fruit set, seed set, seed number and seed weight were compared between the natural and shade conditions by marking 29 and 19 individuals, respectively, which were randomly selected and hand-pollinated (see Experimental design, above). Mature fruits were harvested and the number of mature seeds, undeveloped seeds and unfertilized ovules was counted. Then, seed-set ratio was calculated for each fruit as a seed-to-ovule ratio. Mature seeds were oven-dried at 70 °C for 72 h and weighted. Seed weight was determined by measuring the first ten seeds selected randomly and mean mass per seed was calculated in each plant. In the next year, we checked whether target plants produced aerial shoots and, subsequently, flowers.

To assess the effects of the shading treatment on current fruit and seed production, shoot emergence and flower production in the next year, a generalized linear model (GLM), comprising a logit link function and a binomial error distribution, was established for the fruit- and seed-set ratio, proportions of shoot emergence, and flower production. Seed number and individual seed weight were compared between the control and shaded plants by the Mann–Whitney U-test. An open source system, R version 2·4·1, was used for all statistical analyses.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Growth pattern
The mass of individual organs and leaf characteristics varied significantly within the reproductive season under natural conditions (one-way ANOVA; Table 1). Plant biomass, shoot-to-rhizome ratio and leaf area per plant increased significantly from the flowering to early-fruiting period (Fig. 2). During the fruiting period, fruit mass increased, but leaf mass decreased. In contrast, LMA decreased as time progressed.


Figure 2
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  		FIG. 2. Seasonal patterns of (A) above-ground shoot mass, (B) flower or fruit mass, (C) stem mass, (D) rhizome mass, (E) shoot-to-rhizome ratio, (F) leaf mass, (G) total leaf area per plant, and (H) leaf mass per area (LMA). Fl = flowering period, E-Fr = early-fruiting period, M-Fr = middle-fruiting period. White and grey boxes mean natural and shade conditions, respectively. Box-and-whiskers plot represents 75th, 50th and 25th percentiles, top whisker ranges from the 75th to 90th percentile, and the bottom whisker from 25th to 10th percentile. Different lower-case letters above symbols under natural conditions indicate a significant difference: P < 0·05 by Turkey's multiple range test following one-way ANOVA.

 


Figure 3
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  		FIG. 3. Relationships between the net photosynthetic rate and photosynthetically active radiation (PAR; 0–1500 µmol m–2 s–1) for leaves under natural conditions (circles) and shade conditions (triangles) in the flowering (A), early-fruiting (B) and middle-fruiting periods (C).

 


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  		TABLE 1. Results of one-way ANOVA for plant growth parameters among reproductive periods (flowering, early- and middle fruiting) under natural conditions, and two-way ANOVA between early- and middle-fruiting periods under natural and shade conditions

 
The effects of the shading treatment were negative for fruit mass and LMA, but not significant for other traits (two-way ANOVA; Table 1, Fig. 2). Seasonal variations were also significant in above-ground biomass, fruit mass, leaf mass and LMA between the early- and middle-fruiting periods when the shading treatment was included in the analyses.

Photosynthesis
Light-response photosynthetic curves in each period are shown in Fig. 3. Plants in the flowering period showed highest pmax values (8·65–10·93 µmol m–2 s–1), which decreased significantly as time progressed (F2,6 = 38·38, P < 0·001). Dark respiration rate did not vary significantly throughout the reproductive period (F2,6 = 2·12, P > 0·1). The shading treatment caused a significant decrease in pmax during the fruiting period (F1,5 = 11·64, P < 0·05), in addition to seasonal reduction of pmax (F1,6 = 9·49, P < 0·05), while dark respiration rate did not vary significantly between the early- and middle-fruiting periods (F1,6 = 2·62, P > 0·05), nor between treatments (F1,5 = 0·52, P > 0·05) when natural and shade treatments were analysed together.

Estimated daily photosynthetic carbon gain per plant was largest in the early-fruiting period (Table 2), owing to high irradiance (Fig. 1) and large leaf area (Fig. 2G). After canopy closure in the middle-fruiting period, daily photosynthetic carbon gain was only 17 % that of the highest period. Shading treatment drastically reduced the daily photosynthetic carbon gain from 4175 and 694 to 1282 and –194 µmol CO2 per plant d–1 in the early- and middle-fruiting period, respectively. The negative value under shade conditions in the middle-fruiting period means that the cost of respiration was greater than the photosynthetic carbon gain.


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  		TABLE 2. Estimated daily photosynthetic carbon gain per plant

 
Isotope analysis
MANOVA for the percentages of 13C label in each organ revealed significant differences among reproductive periods (pseudo-F8,82 = 9·706, P < 0·001; ‘pseudo-F’ represents the F-approximation to the MANOVA test statistic) and between treatments (pseudo-F8,82 = 4·680, P < 0·01). One-way ANOVAs conducted for plants under natural conditions revealed that the 13C percentages in leaf and stem were highest in the flowering period, while the 13C percentages in flower (fruit) and rhizome were lowest in the flowering period in which the former was highest in the middle-fruiting period and the latter was highest in the early-fruiting period (Table 3, Fig. 4). These results indicate that most of the carbon assimilated by leaves (93·6 %) remained in leaves and stem in the flowering period, and that plants then increased carbon translocation to rhizome prior to fruit.


Figure 4
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  		FIG. 4. Percentages of 13C label to different organs of individuals harvested in the flowering (A–D), early-fruiting (E–H) and middle-fruiting periods (I–L). Natural light conditions and shading treatments are indicated. Different lower-case letters above symbols under natural conditions indicate a significant difference among periods in each organ: P < 0·05 by Turkey's multiple range test following one-way ANOVA.

 


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  		TABLE 3. Results of one-way ANOVA for excess 13C among reproductive periods (flowering, early- and middle-fruiting) under natural conditions, and two-way ANOVA between early- and middle-fruiting periods under natural and shade conditions

 
Two-way ANOVAs conducted for intact and shaded plants in the early- and middle-fruiting periods revealed that the 13C percentages in fruit, leaf and rhizome varied significantly between periods, treatments or their interactions, whereas the 13C percentages in stem were similar (Table 3, Fig. 4). The shading treatment increased the 13C percentages in fruit and leaf but decreased in rhizomes, although the percentages in leaves and stem were similar in the early-fruiting period. In the middle-fruiting period, the 13C percentages in leaves were significantly larger in shaded plants than in the intact plants (P < 0·05). As most 13C remained in leaves under shade conditions, the 13C percentage in rhizome was reduced in comparison with natural conditions. By contrast, the shading treatment did not seem to influence the 13C distribution in fruit and stem in this period.

Current reproduction and shoot performancein the next year
In order to examine the effects of the shading treatment on current reproduction, fruit and seed production per plant were compared between the natural and shade conditions (Table 4). The fruit- and seed-set ratios of shaded plants were about two-thirds and one-third those of unshaded plants, respectively. Fruit abortion seemed to occur just before fruit maturation given that most plants still had fruits in the middle-fruiting period (mid-June). GLM analysis revealed that fruit-set and seed-set success decreased by the shading treatment (P < 0·05 and P < 0·0001, respectively). Similarly, seed number and seed weight of shaded plants were smaller than those of unshaded plants (P < 0·001 and P < 0·001, respectively). The following year, the proportion of plants with flowers among the shaded plants was 36 % of what was observed in unshaded plants, while the proportion of shoot emergence was similar. Therefore, light conditions during the fruiting period highly influenced both current seed production and reproductive performance in the next year.


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  		TABLE 4. Effect of the shading treatment on fruit and seed output in current year and shoot and flower production in the next year

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Seasonal pattern of photosynthetic carbon translocation
Both aerial shoot and rhizome biomass increased from the flowering to early-fruiting periods before canopy closure, while apparent biomass increment of these plants was not observed during the fruit-developing period under closed canopy. Thus, the major vegetative growth of this species might be completed by the canopy closure. In contrast, fruit development gradually progressed under decreasing light conditions in early summer. This means that resource translocation to the rhizome occurs prior to fruit development.

The observed patterns of 13C translocation among organs in the flowering period demonstrated that current foliage assimilation largely remained in leaves and stem. As leaf size and stem length continuously increased during the flowering period (T. Y. Ida, unpubl. res.), current photosynthetic products may be used to develop the aerial shoot in the beginning. Previous studies reported that the flower production and vegetative growth of most spring ephemerals rely on the stored resource in underground parts (Muller, 1978; Routhier and Lapointe, 2002). However, the present study indicates that the formation of the aerial shoot is supported not only by stored resources but also by current assimilation in this species. Although the photosynthetic rate per unit leaf area was highest in the flowering period, estimated photosynthetic carbon gain per plant was largest in the early-fruiting period because of the larger leaf area. Therefore, use of photosynthetic products to develop leaf size early in the season results in the increase in subsequent photosynthetic carbon gain even when photosynthetic rate per unit area decreases as time progresses.

The translocation of photosynthetic products to the rhizome increased after the completion of shoot development at the time when total carbon gain per plant was maximum. The increasing translocation to the rhizome was accompanied by decreasing 13C distributions in leaves and stem. This means that photosynthetic products during the physiologically most active period are stored in the rhizome for future growth and reproduction. In the middle-fruiting period when plants showed lowest carbon gain (only 17 % that of the early-fruiting period) under closed canopy, carbon translocation to the rhizome decreased but that to the fruit increased. Thus, plants shift the priority of the usage of photosynthetic products from storage to reproduction after canopy closure.

The shading experiment demonstrated that the seasonal pattern of photosynthetic carbon translocation is influenced by the seasonal dynamics of light availability. Shading in the early-fruiting period reduced translocation to the rhizome, while translocation to the fruit was advanced. Interestingly, the translocation pattern in the middle-fruiting period was similar to that of shaded plants in the early-fruiting period. This indicates that plants may change the balance of carbon translocation between storage and current reproduction in response to the ambient light conditions. In shaded plants, estimated carbon gain was negative and most photosynthetic products remained in the leaves in the middle-fruiting period. Because the shading treatment was continued even after canopy closure in the present experiment, it might cause an unrealistically low light level for the latter half of the experiment. Therefore, it is difficult to quantify the effect of canopy closure timing on carbon economy accurately. Nevertheless, the results clearly demonstrate that plants shift the priority of the usage of photosynthetic products from storage to reproduction in response to the light availability.

Irrespective of the reduction of photosynthetic carbon gain under low light conditions, shoot and rhizome biomass did not differ between control and shaded plants. However, this does not mean that the shading treatment during one season had a small impact on subsequent growth. Aerial shoot occupies 17–28 % of the total biomass, while rhizome represents 72–83 %. The extent of the fluctuation of rhizome weight was 40 % over the entire growing season. If starch represents approximately 50 % of the rhizome biomass, then approximately 80 % of the starch in rhizome seemed to be used for early growth. Therefore, the reduction of photosynthetic products during one season should have a large impact on rhizome growth. The shading treatment significantly reduced flower production in the following year. This suggests that reproductive activity is most sensitive to the restriction of carbon assimilation by the accelerated time of shading in the previous year.

Budget for fruit production
The initiation of carbon translocation to fruit was accelerated by the shading, but the shading treatment greatly reduced fruit mass and seed production. Although this species produces only one fruit having 1–4 % of the total plant mass, the amount of foliar photosynthetic products under low light conditions is insufficient to develop the fruit, resulting in fruit abortion and/or failure of seed production in the late season. There are two possibilities for the resource budget for fruit production other than foliar photosynthetic products that may enable stable seed production. One is the use of photosynthetic products by non-foliar organs, such as fruit and calyx. The importance of non-foliar photosynthesis as a primary source of fruit production has been reported in recent studies (Aschan and Pfanz, 2003; Herrera, 2005; Hoch and Keel, 2006; Horibata et al., 2007). In a typical spring ephemeral, for example Adonis ramosa, foliar photosynthetic products are not used for current reproduction, while photosynthesis by fruits is a major source of seed production (Horibata et al., 2007). Rapid fruiting under high irradiance before canopy closure enables the use of non-foliar organs for photosynthesis. In contrast, fruit development in T. apetalon initiates just before canopy closure and continues over 1 month mainly under closed canopy. This life cycle should limit the effectiveness of non-foliar photosynthesis, if any, in this species.

The other possibility is the use of stored resources for seed production. Lapointe (1998) reported that stored carbohydrates in stem during the high-light period were sufficient to develop fruit in a congeneric species, Trillium erectum, even when leaves were removed after canopy closure. This indicates the low contribution level of photosynthesis after canopy closure. The contribution of stem as a temporary resource reservoir in T. apetalon was not assessed in the current study. Because the shaded plants showed negative carbon assimilation in the middle-fruiting period, carbon supply by photosynthesis to the fruit may be negligible in these plants. Nevertheless, fruits with undeveloped seeds in the shaded plants that ultimately did not set seeds remained until the seed dispersal period. The delay of fruit abortion after the severe shading stress may suggest the function of stem as a temporary resource reservoir also in T. apetalon. However, restriction of carbon assimilation in the early-fruiting period by the shading treatment might restrict resource storage in stem. Therefore, seed production of this species is carbon-limited under shade conditions and highly sensitive to the light conditions in the fruit-developing period.

Life-history strategy of T. apetalon
Under seasonally fluctuating conditions on the floor of deciduous forests, various types of light resource utilization exists in forest herbs (e.g. Uemura, 1994). Spring ephemerals exploit the short, bright period from snowmelt to canopy closure by the simultaneous production of leaves having high photosynthetic activity and short longevity (Rothstein and Zak, 2001). Summer-green herbs with continuous leaf production effectively use the period of decreasing light conditions during canopy closure by the regulation of leaf mass (Tani and Kudo, 2006). Trillium apetalon has an intermediate strategy between these types; this species uses both bright and shade periods without additional leaf production but with increasing leaf size, thereby inducing shade acclimation of the leaves.

Temporal separation in carbon translocation to rhizome and seed production in T. apetalon indicates the absence of direct trade-off between resource storage and reproduction. Preferential resource investment in rhizome in the high-irradiance period prior to canopy closure indicates a conservative strategy for survival and future growth. Most spring herbs growing in deciduous forests are potentially long-lived polycarpic perennials (Struik, 1965; McKenna and Houle, 2000). In T. apetalon, more than 10 years are required to reach a sexually mature stage (Ohara and Kawano, 1986b), and individual plants in the reproductive stage survive and flower continuously over many years (Ohara et al., 2001). As this species has high selfing ability without pollinator visit, stable high reproductive success is expected if resource limitation does not occur (Kudo et al., 2008). In contrast, Lubbers and Lechowicz (1989) demonstrated that the translocation of foliar photosynthetic products to current reproduction occurred at the expense of storage for survival and future reproduction in a congeneric species, Trillium grandiflorum, following partial defoliation. They concluded that such a resource investment strategy should reflect the unpredictable pollinator availability as seed production of this species was limited by pollen supply. In the present study, the resource usage pattern responding to the shading treatment was evaluated. Because defoliation and shading treatments may cause different responses even in the same species (e.g. Mikola et al., 2000), however, variations in resource investment between the Trillium species should be carefully interpreted.

Yearly fluctuations in environmental conditions, such as snowmelt time, temperature during the flowering period and canopy closure time, may affect reproductive success of spring bloomers. Kudo et al. (2008) demonstrated that seed set of T. apetalon varied highly among individual plants in a year with a short bright period before canopy closure. According to the results herein, this might be because late-flowering individuals within a population failed in seed production due to shading stress in the early-fruiting period. The conservative resource allocation to ensure survival and future growth may enhance the lifetime fitness for long-lived forest herbs, because the seasonal decline of light availability is a predictable trend on the floor of deciduous forests (Schemske et al., 1978; Lubbers and Christensen, 1986). Thus, the timing of canopy closure influences current seed production, while its effect on storage for future growth may be small if canopy closure does not occur extremely earlier than usual. The population structure of this species commonly shows a decreasing number of plants with increasing growth stage, i.e. a typical sigmoid-shaped stage structure (Ohara and Kawano, 1986b), indicating that continuous seedling recruitment maintains the population. If acceleration of canopy closure continuously occurs due to climate warming (Menzel, 2002; Gordo and Sanz, 2005), for instance, reduction of the high-irradiance period may influence the maintenance and dynamics of T. apetalon populations.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We are grateful to T. Tani and Y. Kameyama for their discussions on this study. We thank A. Sugimoto, S. Hirakawa and S. Hasegawa for their assistance with stable isotope analysis, E. Nabeshima for help with photosynthesis measurements, A. Koyama for help with the field survey, and anonymous reviewers for their critical reading and valuable comments. This work was partly supported by JSPS Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists and Grants-in-Aid from the Japanese Society for the Promotion of Science for Scientific Research (nos. 16370007 and 1840501007).


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

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