Study site and sample collection
The study was conducted at a 6-ha permanent plot in Ogawa Forest Reserve, which is an old-growth deciduous forest in central Japan (37ºN, 140ºE, 620–670 m in elevation). The mean annual temperature and precipitation from 1986 to 1995 were 10.7°C and 1910 mm (Mizoguchi, Morisawa & Ohtani 2002). The dominant tree species are Quercus serrata, Fagus crenata and F. japonica. The canopy height is ca. 20 m, and tall trees reach 30 m (Table 1; Tanaka & Nakashizuka 1997; Aiba & Nakashizuka 2009). A detailed description of the forest composition is available in Masaki et al. (1992) and of the forest dynamics in Nakashizuka et al. (1992).
Table 1. The tree species studied and their maximum heights in Ogawa Forest Reserve (OFR), Japan
| Acer mono ||ACM||23.3|
| Acer amoenum ||ACA||18.9|
| Carpinus cordata ||CRC||14.6|
| Carpinus japonica ||CRJ||14.0|
| Carpinus tschonoskii ||CRT||20.9|
| Castanea crenata ||CSC||22.9|
| Fagus crenata ||FGC||24.6|
| Fagus japonica ||FGJ||24.0|
| Quercus serrata ||QRS||27.0|
| Styrax obassia ||STO||15.9|
Ten of the principal canopy tree species were chosen for this study (Table 1). We looked only at canopy trees, because it is possible that lower trees absorbed not only atmospheric 14C but also 14C of older origin, from contamination stagnant at the forest floor arising originally from plant and soil respiration (Bazzaz & Williams 1991; Kumagai et al. 2006; Sun et al. 2007). Seeds of the studied species were collected by seed and litter traps having a mouth area of 0.5 m2 set at each point of a 7 × 7 m grid in a 1-ha subplot (100 m × 100 m) located at the centre of the 6-ha plot (221 traps in all) from 1987 to 1988; in a 1.2-ha subplot (100 × 120 m) (263 traps) from 1989 to 1991; and at every 10 × 10 m grid point in a 1.2-ha subplot from 1992 (143 traps) (Tanaka & Nakashizuka 2002). Seeds that had fallen into the traps were collected every two or four weeks and were identified and classified into sound seeds (attaining a mature size and having a sound embryo) and the rest (Shibata, Tanaka & Nakashizuka 1998). The mature seeds were air-dried and kept in the warehouse of the Forestry and Forest Products Research Institute, Tsukuba, Japan.
Fluctuation in annual seed production
To examine the intensity of the masting habit in each species, we used the CV of the number of sound seeds per unit area (m−2) falling into the traps in the plot from 1987 to 2000; this was taken as an index of the annual fluctuation of seed production in a population (Table 2; Shibata et al. 2002).
Table 2. Annual variations of the number of sound seeds (m−2) in the studied species in OFR
Some species in this study, such as Q. serrata, continually produce a relatively constant number of seeds (Table 2; Shibata et al. 2002). In other species, including two Fagus and three Carpinus species, there is a large annual fluctuation in seed production (Table 2). The minimum (Q. serrata) and maximum (F. crenata) species differed by a factor of more than 4.5. In general, the intervals between mast seeding years in F. crenata are 5–7 years (Maeda 1988; Hiroki & Matsubara 1995; Suzuki, Osumi & Masaki 2005; Masaki et al. 2008).
We used the seeds, including the seed coat and the endosperm, collected from the traps set for the 10 species at the study site from 1990 to 1996 (Table 3). The studied seeds were collected in different years in each species, because there were different masting years in these species and because most of the collected seeds had been used in other studies (Tanaka 1995; Shibata, Tanaka & Nakashizuka 1998; Shibata et al. 2002). All seed samples were powdered to finer than 40 mesh, and about 2 mg of the resulting powder (to produce about 1 mg C) underwent combustion for 2 h in evacuated and sealed Vycor tubes with CuO, Cu and Ag wire at 850°C. After cooling, the tubes were cracked on a vacuum line, and the CO2 was cryogenically purified and then graphitized by using Fe catalysis for 6 h at 650°C (Kitagawa et al. 1993). For measurement of radiocarbon by AMS, the graphite samples were sent to Rafter Radiocarbon Laboratory, Institute of Geological and Nuclear Sciences, New Zealand. The radiocarbon value is shown as Δ14C (‰), which is the deviation in parts per thousand from the activity of nineteenth-century wood, and has been corrected for fractionation by using the stable C isotope ratio of the sample (Stuiver & Polach 1977). The Δ14C signatures were reported for each analysis together with an analytical error (‰), which was 2.3‰ on average (2.5‰ at the maximum). These values were more accurate than those in previous studies (5.5‰ on average in Hyodo, Tayasu & Wada 2006; Hyodo et al. 2008). Because the ‘bomb’-14C of atmospheric CO2 has exponentially decreased since the Nuclear Test Ban Treaty of 1963 (Levin & Kromer 1997), analytical accuracy can be greater when, after the treaty, the Δ14C value is higher and the year is closer to 1963. Therefore, it is suggested that we can estimate the age of carbon with greater accuracy.
Table 3. The number of seeds analysed, seed collection dates, Δ14C and average and total carbon accumulation periods in the species studied
| Acer mono ||3||November 1995||112.1 ± 1.6||0.35 ± 0.23||1.08|
| Acer amoenum ||3||November 1995||111.1 ± 0.5||0.20 ± 0.07||0.35|
| Carpinus cordata ||3||October 1990||149.4 ± 1.9||0.04 ± 0.21||0.07|
| Carpinus japonica ||3||October 1990||149.8 ± 2.4||0.09 ± 0.26||0.18|
| Carpinus tschonoskii ||3||October 1990||152.2 ± 0.7||0.35 ± 0.08||1.06|
| Castanea crenata ||3||October 1995||114.1 ± 0.4||0.53 ± 0.06||1.18|
| Fagus crenata ||3||October–November 1993||127.6 ± 4.5||0.43 ± 0.62||1.13|
| Fagus japonica ||3||October–November 1993||128.5 ± 2.1||0.56 ± 0.25||1.25|
| Quercus serrata ||3||November 1996||107.8 ± 1.3||0.67 ± 0.20||1.38|
| Styrax obassia ||3||September 1993||127.1 ± 0.6||0.30 ± 0.08||0.97|
To determine the Δ14C of background atmospheric CO2 at the study site during the study periods, we also analysed the leaf litter of Betula grossa (Betulaceae), which is a deciduous and canopy species at the site. Litter was collected by using litter traps in 1992, 1994, 1998 and 2001. We did this because the Δ14C of atmospheric CO2 is known to vary with the latitude, region and surrounding environment (Pataki et al. 2010), mainly due to anthropogenic CO2 emission from fossil fuels (Levin & Kromer 1997; Pataki et al. 2010). Recent studies have shown that deciduous temperate tree species use current photosynthates mostly for leaf formation, because developing leaves become C-autonomous very soon after bud break (Keel & Schädel 2010; Landhäusser 2011). We can therefore use current leaves of deciduous tree species as an indicator of Δ14C of atmospheric CO2 at the study site. Betula grossa has two leaf types, early leaves and late leaves (Miyazawa & Kikuzawa 2004). Early leaves appear following spring budding, whereas late leaves appear successively and continuously after expansion of the early leaves (Jones & Harper 1987a,b; Wilson 1991; Miyazawa & Kikuzawa 2004). It is easy to separate early leaves from late leaves in this species, because the former are of significantly larger leaf size and LMA than the latter. Furthermore, the two leaf types fall at different times: early leaves do not abscise until near the end of the growing season, whereas late leaves begin to abscise around the middle of it (Miyazawa & Kikuzawa 2004). In this study, we used early leaves of B. grossa as an indicator of Δ14C of background atmospheric CO2 at the study site, as only this species met the conditions necessary for the indicator. These conditions include reaching the canopy height so as to prevent contamination of older 14C from plant and soil respiration, being pioneer species which have earlier autotrophic photosynthesis than late successional species (Lambers, Chapin & Pons 2008), and simple separation of leaves from collected litter.
We estimated the carbon accumulation period for seed production by the following method. First, Δ14C of atmospheric CO2 was taken to decrease exponentially (Eqn 1; Levin & Kromer 1997; Hyodo, Tayasu & Wada 2006):
where t represents the dominical year. We assumed that the early leaves of B. grossa had been produced by using current photosynthates from May to June in the leafing year, because they started to develop from the beginning of May and matured in approximately 2 months (Miyazawa & Kikuzawa 2004). Next,
where x1 and x2 are the start and end dates, respectively, for the supply of carbohydrate resources used to produce the early leaves, namely May 1 and July 1 in each year, and l is the period of carbon accumulation, namely 2 months. We then numerically estimated the values of a and b using the function nls in R software, version 2.15.1 (R Development Core Team 2012). It was found that a = 2073.7 and b = 16.56 (Fig. 1). We found that Δ14C of leaves of B. grossa was slightly higher than that of atmospheric CO2 recorded in the Northern Hemisphere (estimated Δ14C values of atmospheric CO2; Hua & Barbetti 2004) (Fig. 1).
Finally, mean and total carbon accumulation periods for seed production were calculated by using the following formula:
where tseed was calculated by Eqn 1.
The total carbon accumulation period was roughly estimated by the following expression:
where l is the carbon accumulation period and x is the last date of the growing season (i.e. the photosynthetic period for seed maturation) in the seed-producing year. If the estimated date x-l was during the growing season, the total carbon accumulation period was represented by l. But in contrast, if the estimated date x-l was earlier than the beginning of the growing season, the total carbon accumulation period was estimated by the following equation, which assumes the total carbon accumulation to cover over 2 years:
where d1 is the date of the beginning of the growing season in the seed-producing year, d2 is that of its end in the previous year and l2 is the carbon accumulation period in the previous year. In this case, the total carbon accumulation period for seed maturation is given by calculation, x – (d2 – l2). If this estimated date was earlier than the beginning of growing season in the previous year, this equation was expanded into an accumulation period of 3 years or more.
We consider the growing season to be a photosynthetic production period, the duration of leaf emergence. At the study site, almost all deciduous tree species begin leaf development at the beginning of May and then shed leaves at the beginning of November. We therefore assumed d1 and d2 to be May 1 and October 31 of the focal year, respectively. When seed was collected before October 31, x was assumed to be the date of the seed collection, otherwise x was assumed to be October 31.
Some individuals of the tree species indicated lower values of Δ14C in their seed than those in the atmospheric CO2 of the seed collection date (i.e. negative estimated values of l), probably due to an analytical error. To eliminate this error, we used the mean values of Δ14C in seed (‰) and the seed collection dates for the calculation in each species. Microsoft Excel Goal Seek was used for estimating the values of l.