Ecol Res (2012) 27: 933–943 DOI 10.1007/s11284-012-0973-1
O R I GI N A L A R T IC L E
Edgard A. Bontempo e Silva • Shigeaki F. Hasegawa Kiyomi Ono • Akihiro Sumida • Shigeru Uemura Toshihiko Hara
Differential photosynthetic characteristics between seedlings and saplings of Abies sachalinensis and Picea glehnii, in the field Received: 21 October 2011 / Accepted: 3 July 2012 / Published online: 27 July 2012 The Ecological Society of Japan 2012
Abstract Abies sachalinensis and Picea glehnii are codominant tree species and major components of the forests of Hokkaido, Japan. Recent work suggests that a reversal in potential competitive superiority at different developmental stages could be important to explain their coexistence. Such shifts in competitive advantage can be mechanistically understood by studying the corresponding physiological differences between distinct life stages. Accordingly, our objective was to investigate in the field the photosynthesis of shade-growing juveniles of these species from two different size-classes, seedlings and saplings. Our results show that seedlings of both species had higher concentrations of photoprotective xanthophylls than saplings, especially in spring, and suggest that seedlings have a lower threshold of stress tolerance than saplings. Photosynthetic capacity per needle area and lateral shoot extension rate decreased from the seedling to the sapling stage in A. sachalinensis, while in P. glehnii, both increased from the seedling to the sapling stage. Abies sachalinensis had higher photosynthetic rates at the seedling stage but lower rates at the sapling stage than P. glehnii. Nevertheless, A. sachalinensis had a higher lateral shoot extension rate than P. glehnii at both stages. Our physiological results support previous ecological observations that A. sachalinensis is a superior competitor to P. glehnii in the understory, and Electronic supplementary material The online version of this article (doi:10.1007/s11284-012-0973-1) contains supplementary material, which is available to authorized users. E. A. Bontempo e Silva (&) Æ S. F. Hasegawa Æ K. Ono Æ A. Sumida Æ T. Hara Institute of Low Temperature Science, Hokkaido University, Kita-19, Nishi-8, Kita-ku, Sapporo 060-0819, Japan E-mail:
[email protected] Tel.: +81-011-7067660 Fax: +81-011-7067660 S. Uemura Forest Research Station, Field Science Center for Northern Biosphere, Hokkaido University, Nayoro, Japan
show that its competitive advantage is higher at the seedling stage than at the sapling stage. Keywords Life-history strategies Æ Photoadaptation Æ Photosynthetic pigments Æ Photosynthetic rate Æ Physiological ecology
Introduction Spruces and firs dominate many of the sub-boreal, boreal, and alpine forests of the world, which hold more than 25 % of the carbon stored in terrestrial ecosystems and are likely to be affected by global climate change (Easterling et al. 2007). The sub-boreal forests of Hokkaido, in northern Japan, are ecologically similar to strict-sense boreal forests even though they are well below the boreal latitudes (Kojima 1991; McCarthy 2001). Abies sachalinensis (Fr. Schm.) Masters and Picea glehnii Masters are dominant canopy trees in many of Hokkaido’s forests (Kojima 1991; Uemura 1994) and their coexistence and co-dominance in these forests have received considerable attention (Kubota and Hara 1995, 1996; Takahashi 1997; Takahashi and Kohyama 1999). Although both species are acknowledged to be shadetolerant and to share regeneration sites, A. sachalinensis seems to regenerate more vigorously than P. glehnii on logs or forest ground (Takahashi 1994). Abies sachalinensis generally grows faster and has a shorter longevity than P. glehnii (Uemura 1994; Umeki 2001). It has recently been proposed that a reversal in competitive superiority at different life stages plays an important role in the coexistence of these species (Nishimura et al. 2010). In their work, Nishimura et al. (2010) observed that A. sachalinensis is a superior competitor to P. glehnii at the sapling stage but that the superiority is reversed in adult trees at the upper-canopy level. To understand such shifts in competitive superiority and growth patterns of trees, it is necessary to study the corresponding physiological changes that take place at
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the plant’s various developmental stages, from the seedling to the senescent stage (Ryan et al. 1997; Bond et al. 2007; Thomas 2010). Moreover, since in temperate and sub-boreal forests the core components of the plant population remain mostly the same from the undergrowth layer until the upper canopy layer (Archibold 1994; McCarthy 2001), physiological and morphological changes in trees at different life stages will influence greatly any inter-specific relations between coexistent species. In fact, it has been argued that species coexistence in northern coniferous forests could be partially maintained by differences in the ecological traits at various growth stages (Parish and Antos 2006; Mori and Komiyama 2008). Furthermore, knowledge of such physiological shifts and their possible influences on the intra- and inter-specific relationship of these species, should also facilitate the estimation of many possible responses of these, and of similar, plant communities to climate change (e.g., Kolb et al. 1997; Phillips et al. 2008; Valladares and Niinemets 2008). We opted to use physiological ecology techniques to provide mechanistic support for the observed ecological patterns of community and population dynamics (Beyschlag and Ryel 2007). Although Nishimura et al. (2010) dealt with shifts in the relative competitive potential between large saplings and adult trees, we opted to study the physiological and morphological characteristics of much younger individuals of the same species. This was done considering that it should help in completing the understanding of the competitive potential relationship between these two representatives of the Spruce–Fir associations in northeastern Asia, since juveniles in the understory have been frequently underestimated in their importance (Nilsson and Wardle 2005). The objectives of this study were: (1) to compare the photosynthetic characteristics of seedlings and saplings of both species in the field; and (2) to compare how these characteristics change in response to seasonal environmental variations at the plant level. Photosynthetic pigments analysis was chosen to investigate possible variations in photosynthesis throughout the growing season. Microclimate variables were measured to assess the influence of climate variation at the plant level. Lateral shoot extension rate in the three previous years was measured to characterize growth. Finally, steadystate photosynthetic rates and chlorophyll fluorescence were measured in the late summer of 2010 and 2011 to analyze photosynthetic performance and to characterize the state of the photosynthetic apparatus.
340 m a.s.l. The soil in the region is prevailingly Inceptisol (acidic brown forest soil) and tertiary andesite bedrock. Hokkaido’s climate is classified as Dfb (humid continental climate with warm summers) after Ko¨ppen (McKnight and Hess 2000). Annual total precipitation ranges from 900 to 1,400 mm (Kojima 1991). The forest canopy is locally dominated by both P. glehnii and A. sachalinensis, with Picea jezoensis Maxim. also present in a smaller number. The lower canopy is composed of deciduous species like Acer mono Maxim. and Quercus crispula Blume, while the undergrowth is dense and mostly dominated by the dwarf bamboo Sasa senanensis Rehder.
Methods
Canopy coverage and microclimate
Study site
The canopy coverage over each individual was studied by taking three hemispherical photographs from above the crown of each plant, at noon on a cloudy day, in July 2010 (Nikon Coolpix E5400 with Nikon FC-E8 FishEye Converter Lens). These photographs were then
The study site was a deciduous conifer–hardwood mixed forest located in the Uryu Experimental Forest of Hokkaido University (4419¢19¢¢N, 14215¢41¢¢E), at
Plants Ten shade-growing individuals of each species were selected and divided into two size-classes: seedlings and saplings. Therefore, the plants can be assigned to four conceptual groups with five plants each: A. sachalinensis saplings; P. glehnii saplings; A. sachalinensis seedlings; and P. glehnii seedlings. The size-classes were defined based on their height in relation to the dwarf bamboo in the understory. We defined saplings as the juveniles of each species that surpassed considerably the height of the surrounding dwarf bamboo, and seedlings as those shorter than the dwarf bamboo undergrowth. Height and basal diameter were measured on July 9, 2009. The mean height of seedlings of both species was 52.7 cm (SE 3.1 cm), and the mean height of saplings of both species was 125.1 cm (SE 5.5 cm), whereas the height of the surrounding Sasa senanensis was mostly in between. Therefore, due to the shading effect of adjacent dwarf bamboo, we expected that saplings and seedlings were subjected to slightly different light conditions, even though all individuals, from both size-classes, were under upper canopy coverage. Seedlings and saplings growing clustered on fallen logs were avoided. Consequently, the chosen individuals were scattered through the area, without any apparent association between their location and the size-classes or species. Since age and size are often uncorrelated in juvenile trees under canopy cover, care was also taken to avoid choosing individuals that were suppressed longer than the majority by counting whorls and bud scales for the seedlings and measuring basal stem diameter for all. We also checked the initial height and basal diameter of the plants between the two size-classes to assess the homogeneity of the size-classes, independently of the species.
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analyzed with the free software LIA32 (version 0.377e, Yamamoto 2010). The estimated ‘‘cover percentage’’ (percentage of the sky that is not visible) was averaged from the three hemispherical photographs to one value per plant, and used in the canopy coverage analysis. To monitor microclimatic seasonal variation, a weather station was installed at the site. The station consisted of a data-logger (Hobo Micro Station, H21002, Onset Computer Corporation, USA) and four sensors: one soil water content sensor (S-SMB-M005); one air temperature and relative air humidity sensor (STHB-M008); and two sensors for photosynthetic photon flux density (PPFD sensor, S-LIA-M003), one installed at a 50-cm height and the other installed at a 120-cm height. The installation heights of the PPFD sensors reflect the average heights of the different size-classes (seedlings around 50 cm and saplings around 120 cm). The station was located at the center of the study area where most of the seedlings and saplings occurred and, therefore, under the canopy and in similar conditions as the studied plants. Data consisted of 15-s averages from all sensors and were logged once an hour, from June 1 until the September 30, 2010. Pigment analysis Needles were collected once in May, June, July, and September 2010. Three plants of each group (A. sachalinensis saplings; P. glehnii saplings; A. sachalinensis seedlings; and P. glehnii seedlings) were sampled in each month. These were first chosen at random from the available five and then alternated to include two plants that were not sampled in the previous month. From each individual, three shoots produced in the previous year (1-year-old needles) were collected from the top of the crown. Care was taken to collect shoots that were separated from each other and positioned in roughly equidistant points in the circumference of the crown. The collection was made from 12:30 to 13:30 hours on cloudy days. The shoots were frozen in liquid nitrogen immediately and stored at 80 C. Pigment analysis was based on Gilmore and Yamamoto (1991) with some modifications. The needles from the three shoots taken from each plant were grounded together with liquid nitrogen using mortar and pestle and from the resultant mass, three 50-mg samples were used for pigment extraction (three identical samples from each individual). The extraction consisted of adding 1 ml of acetone (100 %) and centrifuging for 10 min at 15,000 rpm at 4 C. The supernatant from each sample was then filtered through a 0.45-lm syringe filter (Millex-LH, Millipore) and then used in a chromatographic analysis. The analysis was made using an HPLC system from Shimadzu with a Shim-pack CLCODS column (Shimadzu, Kyoto, Japan). The solvents were acetonitrile/methanol (85:15, v/v) and methanol/ ethyl acetate (68:32, v/v). Sample injection volume was 20 ll, and the flow rate of all separations was 1 ml
min 1. Pigment peaks were identified and their amounts were quantified by using authentic standards from DHI Water and Environment (Hoersholm, Denmark). The three chromatographic results from each individual (3 pseudo-replicates) were then averaged and that value was used for statistical analysis. Photosynthetic rate Photosynthetic light response curves (net photosynthetic rate by PPFD) were made with three seedlings of each group on September 16, 2010, and on August 4, 2011. This technique was used to complement the pigment data, by providing a steady-state assessment of photosynthetic capacity of the four different groups. Measurements were made with the LI-6400 portable photosynthesis system equipped with a conifer LED chamber (LI-COR, Inc., Lincoln, NE, USA). Leaf temperature was kept at 25 C, CO2 concentration at 360 lmol mol 1 and relative humidity was kept stable at the ambient level, using the built-in humidity control system on LI-6400, to minimize discrepancies between measurements. Light curves were made following the standard settings of the light curve program built in LI6400’s operational system (OPEN 5.3.2). The used PPFD levels were, in the following order: 0, 10, 55, 100, 200, 400, 500, 750, and 1,000 lmol m 2 s 1. To evaluate the steady-state photosynthetic capacity, we chose to use the maximum measured rate of photosynthesis (Pmax) from each light curve. Leaf area had to be estimated after the measurement and used to recalculate the measured photosynthetic rate, since the measurement chamber assumes a standard of 10 cm2 of leaf area. The needles that fitted inside the gasket of the measurement chamber were cut after each measurement and photographed over a white sheet of paper, with five solid black squares of known sizes printed on it and a tag containing a code for identifying the seedling from which it came from. Afterwards, the photographs were analyzed with UTHSCSA Image Tools for Windows Version 3.00 (University of Texas Health Science Center of San Antonio) to determinate leaf area in pixels. Chlorophyll fluorescence To investigate the overall fitness of the photosynthetic apparatus and to complement the Pmax measurements, we measured dark-adapted chlorophyll fluorescence of all plants. Chlorophyll fluorescence was measured using a PAM-2000 portable pulse-modulated fluorometer (Heinz Walz GmbH, Effeltrich, Germany). Three shoots at the top of each plant were chosen for each individual measurement. Care was taken to choose only shoots that had fully expanded needles, without any distinguishable difference from the others, and were evenly distributed in the crown (i.e., not positioned in the same side of the
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crown or turned to the same direction). They were darkadapted for 20 min before the beginning of the measurement routine, which consisted of measuring minimum fluorescence yield, F0; maximum fluorescence yield, Fm; and the ratio between variable and maximum fluorescence, Fv/Fm—generally equivalent to the maximum quantum yield of Photosystem II (Maxwell and Johnson 2000). All measurements were made using lastyear’s needles (i.e., those produced in the previous spring flush), from 7:00 to 8:30 on the mornings of September 16, 2010 and August 4, 2011. Plant size and growth Finally, we also measured individual plant growth. Height and basal diameter were measured at the beginning (July 9, 2009) and at the end of the study (July 13, 2011). The mean extension rate of lateral shoots was also measured on July 13, 2011. Height, in this work, is equal to stem length since inclined or bent individuals were avoided when choosing the study plants and, therefore, all chosen individuals were erect and rooted in mostly flat surfaces. The diameter measure chosen for this work was basal stem diameter. Although these plants do not necessarily produce new shoots every year (apical or lateral), we have observed new lateral shoots every year in all individuals in the period of this study. Therefore, lateral shoot extension rate was chosen to complement height growth measurements (apical shoot extension). Lateral shoot extension rate was obtained by measuring the length of three lateral shoots from each year, per plant (nine shoots from each plant). Measured shoots were chosen from branches from the whole circumference of the crown in order to minimize any biasing effects from light environmental heterogeneity.
categorical explanatory variables. In this study, we concentrated on chlorophylls, carotenes, xanthophylls, and lutein, as the leaf pigments of interest. Photosynthetic rate was analyzed by species-specific ANOVAs. In these analyses, Pmax of each species was modeled as the response variable, while ‘‘size-class’’ (seedling and sapling), ‘‘month’’ (September 2010 and August 2011) and the interaction ‘‘size-class:month’’ were used as categorical explanatory variables. Chlorophyll fluorescence results were also analyzed by species-specific ANOVAs in the same design as Pmax, but using Fv/Fm of each species as the response variable. The correlation between Pmax and Fv/Fm was tested by Pearson’s product moment correlation coefficient (r) two-tailed significance test. Initial height and initial basal diameter (measured on July 9, 2009) were used as the response variables in the preliminary analysis of the size-classes and species groups. These were analyzed by ANOVAs with ‘‘species’’, ‘‘size-class’’ and the interaction ‘‘species:sizeclass’’ as the categorical explanatory variables. Height and basal diameter growth were analyzed by repeated-measures ANOVA with ‘‘Year’’ (2009 and 2011) as the categorical explanatory variable. Mean lateral shoot extension was analyzed by ANOVA. The three shoot length measures of a single plant in each of 3 years were averaged to one value, representing the mean shoot extension of that plant. That mean shoot length was used as the response variable and ‘‘species’’, ‘‘size-class’’ and the interaction ‘‘species:sizeclass’’ as the categorical explanatory variables. All ANOVAs were made using Statistica (ver. 7, Statsoft) and Pearson’s PMCC tests were made using SPSS (ver. 15, SPPS IBM).
Results Statistical analysis
Canopy coverage and microclimate
Canopy coverage was analyzed by ANOVA. We used ‘‘cover percentage’’ as the response variable with ‘‘sizeclass’’, ‘‘species’’, and the interaction ‘‘species:size-class’’ as the categorical explanatory variables. Photosynthetic photon flux density, measured at 50- and 120-cm heights, was analyzed by ANOVA. Mean daily PPFD was used as the response variable and ‘‘sensor height’’ (two levels, 50 and 120 cm) was used as a categorical explanatory variable. Pigment data were analyzed by ANOVAs using concentration in ‘‘lmol g 1 fresh weight’’, for chlorophyll, or ‘‘mol mol 1 chlorophyll’’, for all other pigments. Each pigment was modeled individually as the response variable; while ‘‘month’’ (May, June, July, and September) ‘‘species’’ (A. sachalinensis and P. glehnii), ‘‘size-class’’ (seedling and sapling) and the complete interactions between these factors (‘‘month:species’’, ‘‘month:sizeclass’’ and ‘‘month:species:size-class’’), were used as
The analysis of the hemispherical photographs showed that mean cover percentage was 81.95 % (SD 1.49). Cover percentage was not affected by ‘‘size-class’’, ‘‘species’’ or their interaction (n = 20; error df = 16; p > 0.27). July and August were the warmest months, while June and September were the coldest and presented more variable daily averages (Fig. 1a). During the study period, soil water increased from June to July, decreasing afterwards until September. Mean PPFD at both heights (50 and 120 cm) was at its highest in June, decreasing sharply in July, and from that month on, PPFD decreased slightly with the shortening of the day length until September (Fig. 1b). Solar radiation has been previously measured at this plot, and it was observed to peak from May to June, with May’s monthly mean being slightly lower than June’s (Matsumoto et al. 2008). Daily mean PPFD at 120 cm was generally higher than that at 50 cm but the difference between the
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Fig. 1 Microclimate. a Mean monthly soil water content (bars) and mean monthly air temperature (line). b Mean monthly PPFD at 50 and 120 cm above ground (bars), and mean monthly relative air humidity (line). Vertical brackets denote the standard error of the mean (±SE for the line and +SE for the bars)
measurements at both heights was not significant (p = 0.857). Leaf pigments The fresh weight contents of chlorophylls a and b (Chla and Chlb) were generally higher in A. sachalinensis than in P. glehnii (Fig. 2a). Picea glehnii had higher contents of a carotene (aCar) and lutein than A. sachalinensis (Fig. 2a). Saplings had generally higher contents of b carotene (bCar) and lutein than seedlings, but seedlings had their xanthophyll cycle pool at a more de-epoxidated state (DEPS) than saplings (Fig. 2b). Abies sachalinensis saplings and P. glehnii seedlings showed initially higher concentrations of Chla in May when compared with the other two groups (Fig. 3a). The concentration of Chla subsequently decreased in June and, afterwards, increased to its highest level in September. The same was not observed in A. sachalinensis seedlings or P. glehnii saplings, which showed only a slight decrease in Chla in June, remaining stable throughout most of the study period and increasing again in September (Fig. 3a). The levels of Chlb remained relatively stable throughout the measured months (Fig. 3a), and were only statistically affected by ‘‘species’’ and the interaction ‘‘species:size-
Fig. 2 Mean leaf pigment content: a in each species; b in each sizeclass; ‘‘Chla’’ is chlorophyll a and ‘‘Chlb’’ is chlorophyll b (lmol g 1 fresh weight); ‘‘b Car’’ is b carotene per total chlorophyll, and ‘‘VAZ’’ is the sum of violaxanthin (V) + antheraxanthin (A) + zeaxanthin (Z) content, per total chlorophyll (mol mol chlorophyll 1); DEPS is the de-epoxidation state of the xanthophyll cycle pigments: (0.5A + Z) (V + A + Z) 1 (mol mol 1); ‘‘aCar’’ is a carotene per total chlorophyll and ‘‘Lutein’’ is lutein per total chlorophyll (mol mol chlorophyll 1). Asterisk indicates that the effect ‘‘species’’ or ‘‘size-class’’ is significant in the correspondent ANOVA (see Tables 1, 2, 3). Vertical brackets denote the standard error of the mean (+SE)
class’’ (p < 0.05 for both factors, please see the electronic supplementary material—ESM). The contents of aCar and bCar, expressed as values relative to total chlorophyll content (the sum of Chla and Chlb), varied significantly throughout the study period. In all plants, aCar decreased sharply from May to June but, in saplings of both species, bCar increased from May to June and decreased thereafter (Fig. 3b). Mean aCar was affected by ‘‘species’’ while bCar was affected by ‘‘size-class’’ (p < 0.05 for both factors, please see the ESM). The ratio between chlorophylls a and b (Chl a/b) increased throughout the measured months and reached its maximum observed values in September (Fig. 4). From all the effects modeled in the ANOVA, only ‘‘sizeclass’’ and the interaction ‘‘species:size-class’’ were not significant for Chl a/b (p < 0.05 for all other factors,
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Fig. 3 Monthly changes in chlorophylls and carotenes. a Fresh weight content of chlorophylls a and b; b content of a and b carotene per total chlorophyll. Vertical brackets denote the standard error of the mean (±SE) Fig. 5 Monthly changes in carotene derivatives. a Xanthophyll pool—violaxanthin (V) + antheraxanthin (A) + zeaxanthin (Z) content, per total chlorophyll; b de-epoxidation state of the xanthophyll cycle pigments: (0.5A + Z) (V + A + Z) 1; c lutein content per total chlorophyll. Vertical brackets denote the standard error of the mean (±SE)
Fig. 4 Monthly changes in the ratios of chlorophylls a and b, and a and b carotenes. Vertical brackets denote the standard error of the mean (±SE)
please see the ESM). The ratio between a and b carotenes (a/b Car) showed a pronounced decrease in June for all groups (Fig. 3). It was also highly affected by
‘‘month’’ and ‘‘species’’, but contrary to what was observed in Chl a/b, the ratio of a to b carotene was affected by ‘‘species:size-class’’ (please see the ESM). The sum of the xanthophyll cycle pigments (VAZ)—violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z)—in relation to total chlorophyll content was affected by ‘‘size-class:month’’ and independent of ‘‘species’’ (p < 0.05 for both factors, please see the ESM). Consequently, the de-epoxidation state of the xanthophyll cycle pigments, DEPS = (0.5 · A + Z)/(V + A+Z), followed a similar pattern but was more pronouncedly affected by ‘‘size-class’’ (Table 3). In seedlings of both species, mean DEPS decreased from May to July and increased again in September but in A. sachalinensis that increase was significantly higher (Fig. 5b). The amounts and variation patterns of relative lutein content were also more similar among plants of the same size-class than among plants of the same species (Figs. 2b, 5c and ESM). The decrease in aCar observed
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Photosynthetic rate and chlorophyll fluorescence We observed a reversal in Pmax between the different size-classes inside each of the species, and also between the two species. Mean Pmax of P. glehnii saplings was greater than that of its seedlings and, in A. sachalinensis, mean Pmax of seedlings was greater than that of its saplings (Fig. 6a). The categorical factors ‘‘size-class’’ and ‘‘month’’ were significant for Pmax of both species (Table 1). Mean Fv/Fm of P. glehnii was affected by the categorical factor month (Table 1) but, all individuals presented Fv/Fm above 0.8 in both month’s measurements. Pmax and Fv/Fm were correlated in P. glehnii only, but uncorrelated in any other categories (Table 2).
Plant size and growth
Fig. 6 Photosynthetic variables. a Maximum measured photosynthetic rate, Pmax; b ratio between variable and maximum chlorophyll fluorescence after dark adaptation, Fv/Fm; Data are presented in standard box-plots: with the box being delimited, below and above, by the lower and upper quartiles; the thick middle-line, representing the median; and the lower and upper whiskers, representing the sample minimum and maximum, respectively
in June in all species (Fig. 3b) was accompanied by a general increase in lutein (Fig. 5c). Nevertheless, the increase in lutein averages observed in June was more pronounced in saplings than seedlings. All samples showed similar decreases in the relative concentrations of both pigments from July to September (Figs. 3b, 5c).
The analysis of initial height and initial basal diameter (measured in July 2009) of the different size-classes and species shows that among ‘‘size-class’’ (sapling and seedling), ‘‘species’’ (A. sachalinensis and P. glehnii) and the interaction ‘‘species:size-class’’, only the ‘‘size-class’’ was significant (ANOVA; n = 20; error df = 15; effect df = 2; F = 57.121; p < 0.01). Furthermore, initial height was positively correlated with initial diameter (r = 0.6; p < 0.01). No significant height or basal diameter growth was observed between July 9, 2009, and July 13, 2011 (ANOVA; n = 40; error df = 37; effect df = 2; F = 0.676; p = 0.515). In P. glehnii, lateral shoot extension of saplings tended to be greater than that of seedlings, while in A. sachalinensis, the opposite relationship was observed. Saplings of both species showed more variation in the length of lateral shoot extension than seedlings (Fig. 7). Mean lateral shoot extension was different between the species but not significantly different between the sizeclasses (Table 3).
Table 1 Species-specific ANOVAs of: maximum measured photosynthetic rates (Pmax) and ratios between variable and maximum chlorophyll fluorescence after dark adaptation (Fv/Fm); ANOVAs include data from only one species at a time Effect
Specific Pmax
Specific Fv/Fm
df
MS
F
p
df
MS
A. sachalinensis Size-class Month Size-class:month Error
1 1 1 8
4.1067 2.5208 0.0040 0.2705
15.185 9.321 0.015
0.005* 0.016* 0.906
1 1 1 8
0.0000 0.0003 0.0000 0.0001
0.000 3.815 0.078
1.000 0.087 0.787
P. glehnii Size-class Month Size-class:month Error
1 1 1 8
3.9331 6.1490 0.7651 0.3756
10.4705 16.3697 2.0368
0.012* 0.004* 0.191
1 1 1 8
0.0000 0.0011 0.0001 0.0000
0.009 30.153 1.568
0.926 0.001* 0.246
F
Size-classes are ‘‘seedling’’ and ‘‘sapling’’, and months are September 2010, and August 2011. For each ANOVA n = 12 * Denotes statistically significant effects
p
940 Table 2 Pearson correlation tests of Pmax and Fv/Fm on the following levels: all data; Abies sachalinensis only; Picea glehnii only; seedlings only; and saplings only Levels
n
Correlation (Pmax and Fv/Fm)
All data A. sachalinensis P. glehnii Seedlings Saplings
24 12 12 12 12
0.259 0.262 0.580 0.113 0.398
Significance (two-tailed) 0.222 0.410 0.048* 0.726 0.200
Fig. 7 Lateral shoot extension rate in standard box-plots: with the box being delimited, below and above, by the lower and upper quartiles; the thick middle-line, representing the median; and the lower and upper whiskers, representing the sample minimum and maximum, respectively
Table 3 ANOVA of lateral shoot extension rate of the last 3 years Effect
df
MS
F
p
Species Size-class Species:size-class Error
1 1 1 16
7.465 0.105 0.631 0.366
20.401 0.287 1.724
0.000* 0.600 0.208
Species are Abies sachalinensis and Picea glehnii; size-classes are ‘‘seedling’’ and ‘‘sapling’’. n = 20 * Denotes statistically significant effects
Discussion Leaf pigments The decrease in Chla observed in June (Fig. 3a1, a4), during what could be deemed the spring recovery period (from May to June), is similar to other observations made with seedlings of P. glehnii (Kitao et al. 2004). The subsequent increase in Chla content and consequently Chl a/b ratio, observed in the following months can probably be attributed to the maturation of the needles produced in the preceding year (2009).
Regardless of the observed variations in the content of either individual chlorophyll, the seasonal variations in Chl a/b ratio between the four groups is very similar and it suggests that these species maintain similar-sized antenna complexes in both size-classes (Fig. 4a). Considering the results from the analysis of hemispherical photographs (‘‘Results’’ section, ‘‘Canopy coverage and microclimate’’ subsection) and PPFD at the seedling and sapling levels (Fig. 1b), we did not expect any significant differences in the de-epoxidation state of the xanthophyll cycle among the four groups. Nevertheless, we observed that while the saplings of both species showed almost no zeaxanthin in May, the seedlings had nearly half of their xanthophyll pool (VAZ) converted to zeaxanthin at that time (Fig. 5a). Furthermore, although the increase in the relative amount of the de-epoxidated xanthophylls, zeaxanthin and antheraxanthin, observed in September seedlingsamples was relatively small (better visualized in its effect on DEPS, Fig. 5b) and apparently did not influence photosystem II (PSII) efficiency, as it can be inferred from the mean Fv/Fm results of seedlings and saplings (Fig. 6b), it is similar to the differences observed in DEPS between seedlings and saplings at the beginning of the growing season. The observed differences in zeaxanthin and antheraxanthin contents between seedlings and saplings, at the beginning and end of the growing season, could not have been caused by differences in exposure to environmental conditions due to the following considerations. Since seedlings are shorter than saplings and, therefore, more deeply immersed in the Sasa senanensis (dwarf bamboo) undergrowth, we do not believe that seedlings were more exposed to light than saplings. Also, by being shorter than saplings, seedlings are likely less exposed to cold air since air temperature is well correlated to stand density in temperate forests (Nunez and Bowman 1986; Ball et al. 1997; Langvall and Ottosson Lo¨fvenius 2002). Therefore, since all plants from both species and sizeclasses were similarly exposed to variations in climate conditions but showed such different responses to those variations, we can infer that seedlings of both species are more sensitive to abiotic stress than saplings. A decrease in sensitivity to combined sources of abiotic stresses, from the seedling to the sapling stage, may in part be attributed to the continuing development of the root system (Dawson 1996; Drake et al. 2009), since root growth greatly increases access to water and nutrients. Furthermore, since non-structural carbon reserves generally increase with increasing plant size, saplings would also be able to maintain a positive carbon balance through a longer period of unfavorable climatic conditions than seedlings (Niinemets 2010). Therefore, since saplings are likely to have a better developed root system and larger non-structural carbon reserves than seedlings, they have a higher tolerance threshold to the interactive effects of multiple abiotic stresses (Drake et al. 2009; Niinemets 2010).
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A lower threshold of stress tolerance in seedlings than in saplings and the timing of the observed variations in the needle content of photoprotective pigments suggest that seedlings must invest in winter-time slow-relaxing photoprotective mechanisms (Demmig-Adams and Adams 2006) at an earlier time than saplings and must also sustain the winter photoinactivated state (Adams et al. 2004) for a longer period than saplings. Photoinhibition tendency at bud-break Increased sensitivity to photoinhibition at low temperatures has been observed in 1-year-old needles of P. glehnii seedlings, just before bud-break (Kitao et al. 2004). This momentary photoinhibition event has been attributed by Kitao et al. (2004) to a possible feedback limitation on photosynthesis, caused by high starch cellular content (Pammenter et al. 1993). This was supported by the fact that starch has been observed to accumulate in needles of Picea rubens (Red Spruce) concomitantly with bud-break (Schaberg et al. 2000). Such feedback limitation at the time when temperature is low and daily mean PPFD is high would, in turn, stimulate an increase in the concentration of xanthophylls associated with dynamic photoprotection by thermal dissipation (zeaxanthin, antheraxanthin and lutein). We believe that a similar feedback limitation of photosynthesis-driven starch accumulation has affected seedlings and sapling of both species in our study and that it may have been the cause of the relative increase in DEPS observed in saplings of both species in June (Fig. 5b). This is substantiated by the fact that budbreak in all plants occurred between June and July’s leaf sampling, having been clearly observed in the field and later confirmed in the frozen needle samples. It is also supported by the observed increase in lutein in June (Fig. 5c), since lutein is reportedly necessary for the rapid rise of xanthophyll thermal dissipation (Pogson et al. 1998). The observed increase in lutein content in June, higher in saplings than in seedlings, might have been necessary to increase the thermal dissipation capacity of saplings to a similar level as it was observed in seedlings in that month. Seedlings still maintained considerably high levels of de-epoxidated xanthophylls in June, even though their DEPS decreased from May to July (Fig. 5b). Such negative feedback event, with accumulated photosynthates limiting the photosynthetic activity at the onset of bud-break, may result in photoinhibitory damage for juveniles of these species growing more exposed to high light intensity or in other circumstances of aggravated abiotic stress. Photosynthetic and growth rates Measurements of shoot extension in the last 3 years apparently agree with the Pmax results, in each species.
Picea glehnii saplings showed higher photosynthetic rates than its seedlings (Fig. 6a), and mean shoot extension was also greater in saplings than in seedlings of that species (Fig. 7). Conversely, A. sachalinensis seedlings had higher Pmax (Fig. 6a) and a greater mean shoot extension than its saplings (Fig. 7). Measured maximum photosynthetic rate per needle area was directly related to shoot extension in each species but not across species probably because of differences between the growth characteristics of each species. Abies spp. have been shown to have higher leaf area ratio (LAR) than co-occurring Picea spp. (Messier et al. 1999; Machado et al. 2003) and the shoot extension results of this study show that A. sachalinensis’ shoots grew more than those of P. glehnii, especially at the seedling stage. Although the competitive superiority reversal between these two species presented in Nishimura et al. (2010) happened at later developmental stages than those studied here, the results of this study already show a distinct tendency, observable in the differences measured between individuals in the seedling and saplings stages, for P. glehnii to surpass A. sachalinensis as the superior competitor. Furthermore, although seedlings of both species had higher DEPS than saplings at the time when Pmax was measured, photosynthetic rates of A. sachalinensis seedlings were higher than that of its saplings. These observations show that there was no noticeable trade-off between investment in photoprotection and photosynthetic capacity, in mid- and late summer for A. sachalinensis seedlings but, such a trade-off relationship between DEPS and Pmax in seedlings and saplings might have been present in the case of P. glehnii. Nevertheless, this study is inconclusive in that regard since a more careful monitoring of the photosynthetic performance throughout the growing season, along with a study of the seasonal variation of the content of photosynthesis proteins, should be necessary to verify this.
Conclusions This study shows that the photosynthetic capacity and the seasonal variation in the content of photosynthetic pigments of A. sachalinensis and P. glehnii were different between saplings and seedlings, highlighting important ontogenetic changes that must happen between these two size-classes. However, while some of these variables presented similar dynamics in each of the two studied species (shoot extension, a carotene and ratio of chlorophyll a to b) or in each of the two studied size-classes (b carotene, DEPS and lutein), others showed mixed results that suggest a complex interaction between both factors, species and size-class (Pmax, chlorophylls a and b, and the ratio of a and b carotenes). Seedlings were shown to have higher DEPS than saplings if exposed to similar environmental conditions, suggesting a higher sensitivity to combined abiotic
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stresses. Nevertheless, the observed differences in the levels of photoprotection between seedlings and saplings did not influence the photosynthetic capacity of these individuals. Furthermore, a previously described photoinhibition tendency at bud-break with important consequences for juveniles of these species, and possibly to juveniles of other evergreen species, was observed in the field for the first time in this study. The morphological and physiological results of this study support the ecological observation of Nishimura et al. (2010) that A. sachalinensis is a superior competitor to co-occurring P. glehnii in the understory. Our results also suggest that this superiority decreases from the seedling to the sapling stage. We believe that more studies, addressing older individuals should follow to expand the knowledge of the photosynthetic characteristics of Spruce–Fir associations. Acknowledgments We thank Cynthia Emi Ouchi and Takumi Fujibe for their invaluable help in the field, Dr. Azusa Tabata and the reviewers for their insightful comments on this manuscript and the staff of the Research Station of Uryu Experimental Forest of Hokkaido University, for their assistance and lodging in the field. We also thank Drs. Takashi Kohiyama and Yuji Kodama for their advice and support throughout this study. This research was funded by the Institute of Low Temperature Science and the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).
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