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Paleobotany |
2Laboratory of Systematic and Evolutionary Botany, and Department of Palaeobotany, Institute of Botany, Chinese Academy of Sciences, Xiangshan, Beijing 100093, P. R. China 3Department of Geology, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK 4Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK
Received for publication March 28, 2000. Accepted for publication January 5, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: atmospheric CO2 • cuticles • Ginkgo • plant fossils • stomatal density • stomatal index • stomatal ratio
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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investigated the stomatal density of eight species of forest trees by observations on herbarium records covering a 200-yr period and showed a 40% reduction in relation to a rise in atmospheric CO2 of 60 µmol/mol over that time. Pe
uelas and Matamala (1990)
studied the stomatal density of 14 species of herbarium material collected 240 yr ago, arriving at a similar conclusion to that of Woodward (1987)
. These observations were reproduced by exposing some of the same tree species to reduced CO2 concentrations in controlled environment experiments (Woodward, 1987
; Woodward and Bazzaz, 1988
). Moreover, these experiments showed that the CO2 levels to which leaves were exposed during growth induced changes in their stomatal index (proportion of stomata to epidermal cells). The changes in turn affected stomatal conductance and WUE (water using efficiency) (Woodward and Bazzaz, 1988
; Berryman, Eamus, and Duff, 1994
; Beerling, McElwain, and Osborne, 1998
).
Following these results, studies on Late Quaternary plant fossils indicate a similar general inverse relationship between stomatal density and atmospheric CO2 concentration (Beerling and Chaloner, 1992, 1993b
; Beerling, 1993
; Paoletti and Gellini, 1993
; Van der Water, Leavitt, and Betancourt, 1994
; McElwain, Mitchell, and Jones, 1995
; Kürschner, 1996
; Kürschner et al., 1996
; Wagner, 1998
). For the Late Neogene, Van der Burgh et al. (1993)
used this relationship to make quantitative estimates of atmospheric CO2 concentration from the stomatal indices of fossil Quercus petraea leaves.
As extant species of vascular plants are generally not known in the fossil record earlier than the Late Tertiary, McElwain and Chaloner (1995)
suggested the concept and method of using nearest living equivalent (NLE) species for comparison of living and fossil stomatal parameters. These are defined as species from the present day that are of comparable ecological setting and/or structural similarity to their fossil counterparts and, as far as possible, of close affinity. Using this method, they attempted to deduce CO2 levels from stomatal density and index during Mesozoic and Paleozoic time, including Early Devonian, Late Carboniferous, Early Permian, Middle Jurassic, and Middle Eocene (McElwain and Chaloner, 1995, 1996
; McElwain, 1998
).
The only available NLE for fossil Ginkgoaceae is the dioecious species Ginkgo biloba (McElwain and Chaloner, 1995, 1996
; Beerling, McElwain, and Osborne, 1998
; McElwain, Beerling, and Woodward, 1999
). Ginkgo is an ideal taxon for this work because the foliage has a high fossilization potential, owing to its thick cuticle and deciduous habit. Furthermore, leaves of Ginkgo are abundant and range from the late Triassic to the present day, an interval of at least 200 million years. Moreover, CO2 enrichment experiments have shown that the stomatal characters of extant Ginkgo biloba leaves are sensitive to changes in atmospheric CO2 concentration (Beerling, McElwain, and Osborne, 1998
). No work has yet examined the recent historical response of stomatal density and index of G. biloba and so we studied herbarium material collected in the early 1900s, when the CO2 concentration was 
55 µmol/mol lower than today (Friedli et al., 1986
).
It is important to determine whether factors other than CO2 concentration influence the stomatal characters (density and index) of Ginkgo foliage. Here we consider five sources of possible natural variation of leaves within and between G. biloba trees from the Botanical Garden in Beijing, China: (1) timing of leaf maturation (May–November), (2) young vs. fully developed leaves, (3) short (determinate) shoots vs. long (indeterminate) shoots, (4) position in the canopy, and (5) male vs. female trees.
In this work, we also test the earlier suggestion that the stomatal density and index of Mesozoic Ginkgo leaves were both lower relative to modern values due to growth in a postulated high-CO2 environment (Beerling, McElwain, and Osborne, 1998
). The Jurassic material from China provides an independent test of previous results on British Middle Jurassic leaves, while the Lower Cretaceous fossils extend the temporal range of samples to test the duration of the high-CO2 Mesozoic "greenhouse" climate (Berner, 1994, 1998
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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).
Ginkgo huttoni (Sternberg) Heer was from the Scalby Formation of Middle Jurassic, Scalby Ness, Yorkshire, UK (Harris, Millington, and Miller, 1974
).
Ginkgo yimaensis Zhou was from the Yima Formation of Middle Jurassic in Henan Province, China (Zhou and Zhang, 1989
).
Ginkgo obrutschewii was from the Dzungaria Formation of Early Jurassic in Xinjiang Province, China (Seward, 1911
).
Modern Ginkgo
In 1998, fresh leaves of G. biloba (maidenhair tree) were collected from trees 46 yr old in the botanical garden, Institute of Botany, Chinese Academy of Science, Beijing (PE). The leaves for comparison of male and female trees were collected in 1999 in the same garden. With each sampling, only large leaves at each collecting time were selected for measurement. But leaves of all sizes were collected when the relationship between leaf area and stomatal density and index was investigated. Herbarium specimens of G. biloba were from the herbarium in the same institute (PE) originally collected from Shanxi Province, China by Harry Smith in 1924 (specimen number 5538).
Cuticle preparation
A small piece (0.5–1 cm2) of modern material was taken from the middle part of a leaf and processed for several hours in a 10% CrO3 solution (chromic acid) to remove the mesophyll layer. This material was then rinsed in water. The leaf fragments were subsequently separated into abaxial and adaxial cuticle and the remains of mesophyll and vascular tissue removed with forceps. The fossil material was macerated in a Schulze's solution (Kerp, 1990
) for 30 min to several hours according to the material. All cuticle samples were mounted either in dilute glycerin or glycerine jelly and sealed with Canada balsam.
Replica preparation
In order not to damage the herbarium specimens, we made replicas of all material used, prepared using clear nail polish applied to the abaxial leaf surface. When dry, the film of polish was pulled from the leaf and mounted in water for examination by transmitted light microscopy. The outlines of epidermal cells were obscure using the replica technique; however, the stomata were clearly visible and easily counted (see Fig. 1).
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Stomatal counts
Computer-aided determination of stomatal density and index were performed on a Leica Q500IW Image Analysis System. When the image of the epidermis cuticle was on the screen each cell (as judged by the observer) was scored for the count. This means that there was a subjective element in the recognition of each cell counted. Stomatal density and index of extant materials are mean values for 25 (field area 0.231 mm2 at 20 x 10 x 0.32 magnification) or 50 (field area 0.121 mm2) counted images from five leaves per sample. Counts were made only within the stomatal bands (intervein bands) for both modern and fossil leaves.
Stomatal density is defined as the number of stomata per square millimeter of leaf surface.
Stomatal index was calculated using the equation of Salisbury (1927)
[stomatal index = number of stomata x 100/(number of stomata + number of epidermal cells)].
Stomatal ratio is a ratio of the stomatal index of the NLE species divided by that of the fossil species (Chaloner and McElwain, 1997
).
Statistics
All of the data were analyzed using analysis of variance (ANOVA), after first checking for homogeneity of variance and additivity (Sokal and Rohlf, 1981
). Transformation was not required in any set of data. For the statistical comparisons we report the F value together with the degrees of freedom of the main effect and the residual, respectively, and its significance.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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), only the stomatal density of G. biloba leaves was insensitive to changes in temperature of 12°C, as estimated from the long-term climatic data for Beijing between May and November (Müller, 1982
).
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The effect of canopy position on leaf stomatal characters
Leaves of G. biloba were collected (September, 1998) from three layers within the canopy (7.0–7.5 m from the ground surface, 4.5–5.0 m, and 2–2.5 m) on a female tree with a height of 7.8 m; the depth of canopy was 5.8 m (7.8–2 m above the ground). Mature leaves were collected from each height, all from short shoots. ANOVA indicated that there was no significant effect of height on stomatal density (F2,57 = 0.22, P > 0.05), but a significant effect on stomatal index (F2,57 = 5.52, P = 0.01) (see Fig. 11). The female tree sampled was growing in a closely spaced line of trees, and so this "sun vs. shade" difference is probably only an approximation of a natural closed woodland. We note that the effect of height, and therefore irradiance, on stomatal density in our G. biloba canopy was considerably less than that seen between sun and shade leaves of Alnus glutinosa (Poole et al., 1996
).
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noted the negative correlation between leaf area and stomatal density within a species. Stomatal index, however, is thought to be relatively constant with respect to leaf area. Once differentiation of the guard cells has taken place, the ratio of guard cells to epidermal cells will of course be unaltered by leaf expansion, while the absolute stomatal density will fall. Stomatal density is therefore sensitive to the state of maturation of the leaf. Stomatal index is remarkably constant, once recognizable stomata can be discerned in the epidermis, i.e., index is independent of any subsequent expansion of the leaf. However, the lack of distinction between veinal and interveinal areas in the smallest immature leaves (<2 cm2 area) means that the stomatal index of such juvenile leaves is unreliable. Based on 12 leaves from long shoots and 19 leaves from short shoots of differing leaf areas, the relationship between leaf area and stomatal density and index was investigated. The results show a general inverse relationship between stomatal density and leaf area with the smallest (i.e., youngest) leaves sampled possessing the highest stomatal densities (Fig. 12). A similar effect is seen for the relationship between stomatal index and leaf area (Fig. 12). However, this arises because the epidermal characters of young G. biloba leaves are such that there is no significant differentiation between vein and stomatal bands (Fig. 2); the encircling cells around stomata are not fully developed, but the young stomata can still be observed on the younger leaves (Fig. 4). Since it is easy to differentiate the vein zone and stomatal zone (Fig. 3) in the larger (more mature) leaves, stomatal index is then stable with increasing leaf area. For example, there was no significant correlation between stomatal index and leaf area for the last ten leaves in ascending size sequence (Fig. 12).
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The results showed that female G. biloba trees possessed higher stomatal density than the male trees (P < 0.05) (Table 2). In contrast to this result, Beerling et al. (1992) found that female plants of Salix herbacea from 2200 m above sea level had significantly (P < 0.05) lower abaxial stomatal densities than male plants from the same locality. However, no significant differences in stomatal index were detected for S. herbacea or G. biloba, which tends to confirm the view that stomatal index is more stable than stomatal density.
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) and instrumental (Keeling and Whorf, 1994
) records indicate that the global atmospheric CO2 concentration has increased by 55 µmol/mol. Collectively therefore these data indicate a response consistent with an historical CO2 effect on stomatal development at this time, an effect that has also been observed in CO2-enrichment experiments with G. biloba (Beerling, McElwain, and Osborne, 1998
).
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From these data (Table 3) we next calculated the stomatal ratios of the fossils (defined as the ratio of the fossil plant stomatal index relative to that of modern G. biloba). These were then converted to RCO2 values (defined as the ratio of atmospheric CO2 in the past relative to the preindustrial value of 300 µmol/mol) following the standardization procedure of McElwain (1998)
to allow comparison with the predictions of a long-term carbon cycle model (Berner, 1994, 1998
).
Calculated in this way, the stomatal ratios of G. coriacea from Early Cretaceous and G. yimaensis from early Middle Jurassic agree closely with Berner's CO2 curve, while the stomatal ratios of G. obrutschewii and G. huttoni from the Early and Middle Jurassic are somewhat lower (Fig. 13). Remarkably, the stomatal ratios of the last two species correspond to the troughs of atmospheric CO2 concentration at those points on the time axis of Berner's curve (Fig. 13). Earlier work on plant fossils has indicated a fourfold increase in atmospheric carbon dioxide at the Triassic–Jurassic boundary (McElwain, Beerling, and Woodward, 1999
), after which the atmospheric CO2 falls again. The stomatal ratio of Baiera spectabilis from Jameson Land (depth 20 m equal to the Early Jurassic), East Greenland is only 1.69 (McElwain, Beerling, and Woodward, 1999
), which is quite close to the stomatal ratio of G. obrutschewii (1.67) from the Early Jurassic.
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FOOTNOTES |
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5 Author for correspondence (Fax: +86-10-62593385, lics{at}public2.east.cn.net
).
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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