How does Dryobalanops aromatica supply carbohydrate resources for reproduction in a masting year?
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- Ichie, T., Kenzo, T., Kitahashi, Y. et al. Trees (2005) 19: 704. doi:10.1007/s00468-005-0434-3
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The effect on reproduction of the dynamics of resource allocation was studied in an emergent and masting tree species, Dryobalanops aromatica (Dipterocarpaceae), in a lowland dipterocarp forest in Sarawak, Malaysia. Girdling of the reproductive shoots (5 mm diameter) caused an increase in abortion during the flowering period, but did not affect the fruit set at the middle or final stages of seed maturation. In contrast, 50% defoliation significantly affected fruit setting, but had little effect on flowering. The total leaf area of reproductive shoots was significantly correlated with final fruit set and total fruit mass. Control of the carbohydrate supply to reproductive shoots by girdling and defoliation made no difference to fruit size, but the fruit number was highly sensitive to carbohydrate availability. Total non-structural carbohydrate (TNC) decreased during the flowering period mainly in the branch (P<0.05), but fluctuated little in any organs during fruit maturation. Leaf nitrogen and photosynthetic capacity of the reproductive shoots were not significant variables for reproduction. Our results suggest that D. aromatica uses current photosynthates in the leaves of reproductive shoots as a carbon source during fruit development, but requires stored assimilates in the branch for flowering. However, since TNC was still present in all organs even after flowering, our study also suggests that storage of carbohydrate resources might not be the decisive factor in the occurrence or frequency of flowering in this species.
KeywordsDipterocarpaceaeGirdling50% Leaf removalMastingReproductive allocation
Plant reproduction sometimes involves a high carbon cost, because of the high concentration of reproductive structures (Bloom et al. 1985). In particular, masting trees, which reproduce prolifically at one time within a period of several years, need large amounts of resources in a masting year (Kelly 1994). Some researchers have predicted that masting trees need several years to accumulate reserves prior to reproduction, using mathematical models (Yamauchi 1996; Isagi et al. 1997) and experimental studies (Sork and Bramble 1993; Koenig et al. 1994). The need to accumulate is an important hypothesis concerning the immediate cause of masting events (see Kelly 1994).
Within allocation to reproduction, the relative contributions of stored assimilates and current photosynthates differs substantially across species (Dickson 1991; Lambers et al. 1998). Carbohydrate resources for reproduction are supplied mainly from assimilates of reproductive shoots in Aesculus californica (Newell 1991), Styrax obassia (Miyazaki et al. 2002), and Ilex aquifolium (Obeso 1998), or from the wood trunk in Macadamia integrifolia (Stephenson et al. 1989a, b). In contrast, these resources are mainly from current photosynthates in leaves adjacent to a reproductive shoot in Persoonia rigida (Trueman and Wallace 1999) and Alnus hirsuta var. sibirica (Hasegawa et al. 2003). Other studies have found that for some trees the photosynthetic capacity of leaves on reproductive shoots, during reproduction, is greater than that of leaves on non-reproductive shoots (Kazaryan et al. 1965; Fujii and Kennedy 1985; Ben Mimoun et al. 1996; Laporte and Delph 1996). The source of reproductive resources for masting remains unclear.
In the lowland mixed-dipterocarp forests of South-East Asia, synchronous mass flowering and consequent mass fruiting involving various species and families occurs at irregular intervals of 2–10 y at the community level (Ashton et al. 1988; Appanah 1993; Sakai et al. 1999; Sakai 2002). More than 70% of emergent trees, represented by the family Dipterocarpaceae which dominates the tropical rain forests of South-East Asia, are involved in mass flowering events, but seldom reproduce in other years (Sakai et al. 1999). Curran et al. (1999) reported that dipterocarp species have a very high density of fruits in a masting year. Moreover, since dipterocarp species produce relatively large fruit, and since total non-structural carbohydrates (hereafter denoted TNC) and/or lipids are the main resources in dipterocarp seeds (Ichie et al. 2001; Nakagawa 2003), dipterocarp species clearly need great carbon resources for reproduction in a masting year. Supposing that stores are important for reproduction, it would be possible to detect the depletion of stored resources when the plant switches to reproductive mode (Chapin et al. 1990). Very few studies have quantified resource allocation for masting of dipterocarp species, or even quantified the usual allocation pattern of tropical trees (Janzen and Wilson 1974; Bullock 1992; Tissue and Wright 1995; Marquis et al. 1997; Newell et al. 2002).
We study here the resource allocation dynamics for reproduction of D. aromatica (Dipterocarpaceae), which is an emergent tree species having a masting habit that is found in Lambir Hills National Park, Sarawak, Malaysia. In particular, we address two questions: (1) Does D. aromatica supply the carbohydrate resource needed for reproduction from leaf photosynthates or from stored assimilates? (2) If storage assimilates are important for reproduction, which organ is the main carbon source for reproduction in this species? To answer these questions, we performed artificial defoliation to reduce the carbohydrate resource supply from leaf photosynthates, and girdling experiments to reduce the supply from stored assimilates. We also investigated the dynamics of the TNC concentration in various organs, and measured leaf photosynthesis and leaf nitrogen, which is related to photosynthetic capacity, in reproducing shoots.
Materials and methods
Study site and species
Our study was carried out in the Canopy Biology Plot (8 ha: 200×400 m) and the Crane Plot (4 ha: 200×200 m) in an intact lowland mixed-dipterocarp forest in Lambir Hills National Park, Sarawak, Malaysia (4°20′ N, 113°50′E, 150–250 m above sea level; Inoue et al. 1995). The area has a humid tropical climate, with weak seasonal changes in rainfall and temperature (Kato et al. 1995). The annual precipitation at a point approximately 3 km north-west of the study site averaged 2751 mm from 1985 to 2001. The 17-y monthly means remained above 150 mm; the area seldom experiences the reduced precipitation associated with unusual climatic events such as El Niño (Nakagawa et al. 2000, Potts 2003). The average annual temperature at Miri Airport, approximately 20 km north of the study site (average from 1968 to 2001), was 26.7°C, with monthly means varying from 25.9 in January to 27.4 in May.
We accessed the crowns of the trees using a canopy observation system, including tree towers and aerial walkways, at the Canopy Biology Plot (Inoue et al. 1995; Yumoto et al. 1996), and an 85 m canopy crane at the Crane Plot (Nakashizuka et al. 2002; Sakai et al. 2002). The canopy crane system allowed access to the entire crown of emergent trees (Sakai et al. 2002; Ozanne et al. 2003).
D. aromatica, which is distributed in the Malay Peninsula, Sumatra, and Borneo (Ashton 1982), is a large evergreen tree that grows up to 70 m tall and 2 m in diameter in lowland dipterocarp forests (Itoh 1995). This species has a very large symmetrical and hemispherical crown with large ascending branches; the branchlets are numerous and are bunched towards the ends (Ashton 1964). This is the most abundant canopy species in a 52 ha permanent plot established in the Park (Itoh et al. 1995), and often forms mono-specific dominant (mono-dominant) forests (Foxworthy 1932; Vincent 1961; Kachi et al. 1993).
The effect of leaf area reduction on various aspects of the reproductive output of D. aromatica was studied by performing an artificial defoliation experiment. To obstruct the resource supply from storage assimilates in the tree, a subset of reproductive shoots was girdled by removing a ring of bark and cambium approximately 1 cm wide from the base of the shoot, and 5 mm in diameter. This procedure interrupts phloem transport but does not affect xylem transport (Obeso 1998). All leaves of D. aromatica were attached to the shoot, having diameter less than 5 mm. There was no leaf below this point at the top of the crown. In this study, we define ‘shoot’ as comprising a twig from where it is 5 mm in diameter to its ends, including the leaves, flowers, and fruits on the twig.
In August 2001, which was during the period of flower bud formation of D. aromatica in that year (Fig. 1), we selected three flowering trees for study. All were emergent specimens (more than 40 m tall) in the study plots, that flowered across their entire crown. Thirty-six shoots per trees were selected haphazardly from several large branches on each individual, and were tagged with numbered plastic tape where they were 5 mm in diameter. After measuring the number of leaves on these twigs, 18 tagged shoots per tree were selected for defoliation, and half of the length of the lamina in all leaves of the tagged shoots was clipped by scissors. Nine shoots from each of the defoliated and undefoliated treatments were then girdled where they were 5 mm in diameter. Other tagged shoots, that were neither defoliated nor girdled, acted as controls.
The numbers of flowers and fruits remaining on the tagged shoots were counted so as to determine the fruit set in mid-August 2001 (the period of flower bud formation), in mid-September (the green fruit period just after flowering, hereafter called initial fruit set), in mid-November (the middle stages of seed maturation, hereafter called middle of fruit set), and at the beginning of January 2002 (the final stages of seed maturation, hereafter called final fruit set). At the end of November, when the seed wings had begun to turn from green to brown, the shoots were covered by a fishing net. The net had at least 98% light transmittance. Tagged shoots with ripe fruits were harvested at the beginning of January 2002, and we measured the number of mature fruits and leaves, leaf area, and dry mass of each part (fruit, leaf, and shoot). Dried leaves were then used for nitrogen analysis.
Measurement of storage assimilates
To study the concentrations of storage resources, woody tissues were sampled from canopy branches of diameter 10 cm located above the crown, and also from the main stem approximately 5 m above the ground and from roots of diameter 10 cm at distances up to 1 m from the base of the tree. At each of these locations, a single core per tree, of length 5 cm (measured from branch/stem/root surface including inner bark) and diameter 1 cm, was taken using a woodworking drill. The root cores were taken after rinsing of the root bark to remove all soil. After sampling, the holes were filled with wood chips and were coated with a preservative to protect the trees from insects and pathogens. Three terminal and reproductive shoots on the top of the crown were also cut where their diameter was 0.5 cm, and adjoining shoots (diameter 0.5 cm) were taken from each individual and separated into leaves, twigs, flowers, and fruits.
All samples were stored in a freezer at −20°C from immediately after measurement of the fresh mass until the chemical analyses were performed. Dry ice was used to keep the samples cool during transport from the study site to the laboratory. In the laboratory, all samples were dried at 60°C for 3 days and their dry mass was measured. The dried samples were then ground to a fine powder using a wonder blender (Osaka Chemical Co. Ltd. WB-1) prior to carbohydrate analysis.
The TNC concentration was calculated from the total content of sugar and starch. Total sugar was determined using the phenol-sulphuric acid method of Dubois et al. (1956), as modified by Ashwell (1966). About 100 mg of dried powder sample was used for each analysis, and was extracted overnight at 90°C using 80% ethanol. The fractional transmittance was read using UV/VIS spectrophotometers (Shimadzu UV-1400, Kyoto, Japan) at 490 nm. Starch was determined using a modified version of the method of Lawrence et al. (1990) and Newell et al. (2002). Following total sugar extraction, the residue was oven-dried at 60°C for 2 days. We weighed approximately 50 mg of the dried samples and then added these samples to 2.0 ml of 0.2 M sodium acetate-acetic acid buffer (pH 5.0). The samples were then incubated in a steam bath at 100°C for 1 h. After cooling to room temperature we added 0.5 ml buffer containing amyloglucosidase (14 U mg−1) and α-amylase (1000 U mg−1); the enzyme solution was run into dialysis tubing under running deionized water for at least 6 h to remove remaining sugars and other impurities, and the solution was then filtered through Whatman no. 1 filter paper. The samples with enzyme solution were incubated for at least 10 h at 37°C, and were cleared by centrifuging at 3000 rpm for 5 min. Enzyme blanks and starch standards were included with each set of samples. Glucose levels were determined by the phenol-sulfuric acid method, as for total sugar analysis. At least two replicates of each sample were analyzed for both total sugar and starch.
Changes in leaf photosynthesis and leaf nitrogen
The net photosynthetic rate at light saturation (P max) was measured at the same time as TNC sampling for the same three trees. Measurements were made between 08:00 h and 10:00 h in order to avoid the midday depression in photosynthesis (Ishida et al. 1996; Kenzo et al. 2003). Three leaves from the reproductive shoot at the top of the crown of each tree were used for measurements. P max was measured using a portable open gas exchange measurement system (LI-6400, Li-Cor, NE, USA) at 1500 μmol m−2 s−1 PPFD, 30°C leaf temperature, and 360 ppm CO2 concentration. The light intensity was controlled by an internal LED light source (Li-640B, Li-Cor, NE, USA). We then measured the area and the dried weight of the leaves. Finally, nitrogen concentration in the leaves was measured using a C/N analyzer (SUMIGRAPH NC-900, Shimadzu, Kyoto, Japan). Nitrogen peaks were identified using acetanilide as the standard.
At the start of the experiment, the mean number of leaves on all experimental shoots (n=108) was 62.9±22.1, and the mean number of flower buds was 147.0±39.9. The number of flower buds did not correlate significantly with the number of leaves (r=0.118, P=0.369).
Effect of girdling and defoliation on fruit production
Three-factor ANOVAs: effects of trees examined, defoliation, and girdling treatment on initial, middle, and final fruit set (%) and total fruit mass. Initial, middle, and final fruit set were arcsin-transformed for ANOVAs
Source of variation
Initial fruit set
Middle fruit set
Final fruit set
Tree × girdling
Tree × defoliation
Girdling × defoliation
Tree × girdling × defoliation
Mean (± SD, n=27 shoots for each treatment) number of mature fruits, dry mass per fruit, leaf number, total leaf area, SLA, and leaf nitrogen at the defoliation and girdling treatments
Girdled + 50% defoliated
Number of mature fruit*
Dry fruit mass (g)NS
Number of leavesNS
Total leaf area (cm2)***
Leaf nitrogen (%)NS
Carbohydrate resource dynamics
Photosynthesis and nitrogen in leaves
Mean (± SD, n=9 leaves) Pmax and nitrogen concentration in leaves at each reproductive stage
Flower bud (Aug. 2001)
Initial fruit set
Middle fruit set
Final fruit set
Pmax (μmol m−2 s−1)NS
N concentration (%)NS
The carbon source for reproduction of D. aromatica was clearly different in the flowering and fruiting periods. Resource reduction induced by defoliation and girdling probably induces abortion during reproduction of D. aromatica. The effects of each treatment, however, were clearly different between flowering and fruiting periods.
In flowering, storage assimilate in the tree, especially in the branch, may be an important carbohydrate supply for reproduction in this species. The flowering periods of most dipterocarp species, including D. aromatica, last for only several weeks after years without reproductive activity (Wood 1956; Chan and Appanah 1980; Ashton 1982; Momose et al. 1996). A large amount of resources may be needed for the high respiration involved in processes such as nectar secretion and pollen production (Kozlowski 1992). We found that the initial fruit set, just after flowering, was significantly reduced by girdling. Also, storage resources in the branch decreased significantly during the flowering period. This species may therefore depend on storage assimilates in the branch as a carbon source during flowering.
Photosynthates in the reproductive shoot appear to provide most of the carbon needed for fruit development. This would explain the observation that the middle and final fruit set were affected much more by defoliation than by girdling. Moreover, storage assimilates did not decrease in any organ, if anything increasing slightly, after the period of the middle fruit set. As a result, final fruit set was strongly correlated with the leaf area of reproductive shoots. Some researchers have suggested that the seed wing elongated from the sepals, which is a characteristic of dipterocarp trees, could have photosynthetic capability (Ashton 1989; Cranbrook and Edwards 1994). However, the photosynthetic capacity of seed wings of D. aromatica is clearly lower than the respiration of the fruit itself during fruit development (Kenzo et al. 2003). It is possible that the extent of photosynthesis prevailing in the leaves of reproductive shoots is related to reproductive success in D. aromatica.
In this species, the leaf nitrogen concentration and the photosynthetic capacity of the leaves did not decrease during the flowering or fruiting periods. Many reports have stated that resorptive nitrogen from senescing is made available for reproduction (e.g. May and Killingbeck 1992; Eckstein and Karlsson 2001). Also, some deciduous masting trees in temperate forests have smaller leaves on reproductive than on non-reproductive shoots (Chapin and Moilanen 1991; Hiura et al. 1996; Miyazaki et al 2002), so that the amount of photosynthates per shoot is less in reproductive shoots, even though the photosynthetic capacity was greater for leaves on reproductive shoots (Miyazaki et al. 2002). However, D. aromatica did not display such differences in leaf area or leaf nitrogen concentration. Also, the Pmax values of leaves on reproductive shoots were similar to values in previous studies found during non-reproductive periods (Ishida et al. 1996, 1999; Kenzo et al. 2004). It is possible that this species has a different system for supplying carbon and nitrogen from these temperate and/or deciduous masting trees.
Storage of resources may not be important for masting in this species. Stevenson and Shackel (1998) showed that in horticultural pistachio, which has alternate masting for reproduction, the overall production of biomass in non-masting and masting year was similar, and then, suggested that the non-masting year is not a period of substantial storage carbohydrate accumulation. In our study, TNC concentrations in the tree decreased only during the flowering period, but non-structural carbohydrate was still present in all organs even after flowering. Although we did not examine the resources needed in the early period of the flower bud formation, some studies have found that allocation to reproduction in some dipterocarp trees was small for flower budding and was concentrated mostly in the stages of fruit development in a masting year (Ichie et al. 2005; T. Ichie unpublished data). It was previously thought that, following a masting event, dipterocarp trees need several years to accumulate an adequate assimilate level for flowering (Ashton 1982), but our study also suggests that storage of carbohydrate resources might not be the decisive factor in the occurrence or frequency of flowering in this species. Future study is still needed on the relation between the timing of masting and other internal factors, such as storage resources of mineral nutrients and/or endogenous hormones for flower bud initiation.
In D. aromatica the carbohydrate supply affected the number of fruits, rather than their size; this is in contrast to Phellodendron amurense var. sachalinense (Mizui and Kikuzawa 1991) and Persoonia rigida (Trueman and Wallace 1999). Maintenance of fruit size even under reduced carbohydrate availability may be significant for seedling establishment, since some Dryobalanops species including D. aromatica need enough reserves in the seed to set up the resulting seedling for photosynthesis after mass fruiting without seed dormancy (Itoh et al. 1995; Ichie et al. 2001).
In conclusion, D. aromatica supplied the carbohydrate resources needed for flowering over a short period mainly from storage assimilates, whereas the resources for fruiting come from current photosynthesis on reproductive shoots; fruiting is a relatively long-term event lasting approximately 4 months. Control of the carbohydrate supply affects the number of fruits rather than their size, and final fruit sets depend on the total leaf area of reproductive shoots. Our findings suggest that accumulation of assimilates is not the factor that limits mass flowering events in this species, and that the amount of photosynthates in the leaves of reproductive shoots may decide reproductive success during a masting year.
We are grateful to the Sarawak Forest Department for permission to conduct the study and for their help, and to S. Sakai for providing the data on flowering phenology. We also thank K. Kawaguchi and Y. Seki for help with the laboratory work. Valuable comments were provided by S. Kitaoka. This study was partly supported by a Project of the Research Institute for Humanity and Nature (P2-2), the Japan Science and Technology Agency, and JSPS Research Fellowships for Young Scientists for T. Ichie