Variations in the Foliar D C and C3/C4 Species Richness in the Japanese ﬂora of Poaceae Among Climates and Habitat Types Under Human Activity

Ecol Res (2010) 25: 213–224 DOI 10.1007/s11284-009-0652-z

ORIGINAL ARTICLE

Yuko T. Hanba • Tsuyoshi Kobayashi Takashi Enomoto 13 Variations in the foliar d C and C3/C4 species richness in the Japanese ﬂora of Poaceae among climates and habitat types under human activity

Received: 7 January 2009 / Accepted: 28 August 2009 / Published online: 17 November 2009 The Ecological Society of Japan 2009

Abstract For 383 Poaceae species harvested over the the sites with high human activity. Leaf d-value was de- Japanese islands and stored as herbarium specimens creased with sampling year, and it was higher in the along several decades, we determined C3 and C4 types of densely inhabited district for both C3 and C4. They are photosynthesis from leaf stable carbon isotope ratio probably due to a historical decrease in the atmospheric 13 (d C). Then, we sought the relationships between C4 d-value via increasing human activity, and high gas species richness and climatic factors or habitat types. emission at the districts of high human density. Except for the two Panicum species (P. lanuginosum and P. scoparium) having the possibility of C3–C4 interme- Keywords Photosynthesis Æ Water-use efficiency Æ diate, 227 and 154 species were classified into C3 and C4. Atmospheric CO2 Æ Urbanization Æ Carbon isotope ratio The C4 species richness increased from northern to southern islands in Japan, positively correlated with mean annual air temperature. Greater C4 species richness Introduction in the seashore habitats, and smaller C4 species richness in the shaded, wet and highland habitats would be related A monocot group Poaceae includes approximately 700 to the photosynthetic responses to local environmental genera and 10000 species (Takematsu and Ichizen 1997), factors such as irradiance level and temperature regime. comprising one of the largest groups of flowering plants. No difference of leaf d-value of C3 Poaceae was obtained Species of Poaceae distributes widely over boreal to between the habitats with different soil water availability, tropical zone in the world. In Japan, approximately 120 suggesting the less importance of soil water availability genera and 440 native species are recognized in Poaceae on leaf water-use efficiency in C3 Poaceae species in Ja- (Osada 1993; Suzuki 1996). Poaceae species occurs in pan having humid climate. Additionally, possible effects various types of habitats such as highland, seashore, of human activity around the harvested time or site on forests, marsh and ruderal places of high human density leaf d-value were estimated, because the habitat includes (Osada 1993; Kitamura et al. 2004), suggesting that the members of Poaceae are adaptive to diverse environments. In paddy fields, similar shoot structure and Electronic supplementary material The online version of this article (doi:10.1007/s11284-009-0652-z) contains supplementary material, phenology of weedy Poaceae grass to rice plants make which is available to authorized users. them a strong competitor to rice in Japan (Morita 2002), so investigating the abundance and distribution pattern & Y. T. Hanba ( ) of Poaceae species will be helpful for efficient plant Kyoto Institute of Technology, Saga Ippongi-cho 1, Ukyo-ku, Kyoto 616-8354, Japan production and weed management. Poaceae is one of the E-mail: [email protected] largest families among the plants invaded into Japan Tel.: +81-75-8610888 (Shimizu et al. 2002). Clarifying the functional compo- Fax: +81-75-8818418 sition and abundance of the Poaceae species and their T. Kobayashi distribution pattern in relation to climatic factors, hab- Faculty of Agriculture, Kagawa University, itat types, and human effects on ecosystems will be Ikenobe 2393, Miki-cho, Kagawa 761-0795, Japan helpful for conservation of ecosystems and biodiversity (Yamashita 2002). T. Enomoto Research Institute for Bioresources, Photosynthetic response to environment is one of the Okayama University, Chuo 2-20-1, most important factors in determining plant growth, Kurashiki 710-0046, Japan reproduction, and survival. Therefore, the photosyn- 214 thetic types of the Poaceae species should strongly affect CAM species was found in Poaceae. On the other hand, their ecophysiological responses to environmental fac- some species of the three genera of Poaceae such as tors and their distribution patterns among the habitats. Alloteropsis, Neurachne, and Panicum show C3–C4 Although there have been extensive studies for C3 and intermediate characteristics (Sage et al. 1999a). In Pan- C4 identification in relation to the phylogeny of Poaceae icum, leaf d-values should be C3-like for C3–C4 inter- (e.g., Hattersley and Watson 1992; Giussani et al. 2001; mediate species (von Caemmerer and Hubick 1989). Grass Phylogeny Working Group 2001), the works for Thus, except Panicum species, we can use leaf d-value as the East Asia flora are limited (Tateoka 1959; Takeda decisive indicator to identify C3 or C4 species in Poaceae. et al. 1985; Okuda and Furukawa 1990), especially in Identification of C3 and C4 using leaf d-values is useful foliar stable d-value-based identification such as Wang because (1) leaf d-values can be determined on the stored (2007). Takeda et al. (1985) identified C3 and C4 species plant samples and (2) stable isotope measurement is for 299 Poaceae grasses in Japan, assuming that all of semi-automated, which provides a large amount of the members within a certain genus have a common samples analyzed in a relatively short period of time. photosynthetic pathway except a genus Panicum. How- The d-value of the atmospheric CO2 (da) decreases ever, a tribe Arundinelleae and a genus Eragrostis in- when there is significant CO2 contribution from (1) clude both C3 and C4 species even in a taxonomic unit plant and/or soil respired CO2 with low d-values (e.g., Sage et al. 1999a). Thus, much screening for Jap- (Ehleringer et al. 2000), and/or (2) gas exhaust by anese flora using additional basis must be necessary to fossil-fuel combustion with low d-value (Pataki et al. distinguish C3 and C4 species in Japan. 2007). In the cool-temperate closed forests, da near the The abundance and richness of C3 and C4 species in forest floor is significantly lower than the open air a grass flora varies greatly among the regions of the (Hanba et al. 1997). The da near the ground was À10& world (Lloyd and Farquhar 1994; Sage et al. 1999b). in suburban area, while it was À11.6& in the central The distribution analyses of C3 and C4 grasses along city of Kyoto, Japan, showing strong human activity latitudinal and altitudinal gradients in Japan have effects on the da (Hanba et al. 2009). Furthermore, showed significant effects of climatic variables on their historical effects should be considered when the plant relative abundance and/or richness (Takeda et al. 1985; samples are collected over a long period, because da in Okuda 1987; Okuda and Furukawa 1990; Mo et al. the troposphere decreases by 0.03&/year from 1985 to 2004). However, the composition and richness of C3 1991 over the Japanese islands due to anthropogenic and C4 species also would vary with habitat types, effects (e.g., Gamo et al. 1995). which have different local micro-environmental condi- Within the plants having the same photosynthetic tions, such as irradiance level, soil water availability pathway, plant ecophysiological responses to environ- and salinity, rather than the regional macro-environments can be inferred from the variation in the leaf D. mental conditions, such as climatic variables (annual Carbon isotope discrimination in C3 plants is deter- precipitation and mean temperature) as shown in Jap- mined mainly by the ratio of intercellular CO2 partial anese sedge flora (Cyperaceae, Ueno, and Takeda pressure to that of atmospheric CO2 (Ci/Ca), because 13 1992). The occurrences of Japanese Poaceae species in low Ci/Ca causes less discrimination against CO2 the natural habitats are described in relation to the (Farquhar et al. 1989). The alteration in the balance local environmental characteristics of the habitats, e.g., between stomatal conductance and CO2 assimilation, wet, ruderal, or sunny (Osada 1993; Kitamura et al. which is caused by the variations in the environmental 2004). However, there are few studies for Poaceae conditions such as soil water availability and irradiance species in Japan concerning the relationship between levels in the habitats, involves changes in Ci/Ca, and photosynthetic types and habitat types. Extensive thus, alters leaf D in C3 plants (Hanba et al. 1997, 2000; studies are needed for the identification and distribu- Kume et al. 2003). Although the variation range is much tion pattern of the C3 and C4 grasses in Eastern Asia smaller than C3 plants, carbon isotope discrimination in with respect to regional as well as local environments. C4 plants relates to irradiance levels and water supply The carbon isotope ratio (d13C) of leaves has been (Buchmann et al. 1996). used as a good criterion on the identification of C and 3 The objectives of the present work are the: C4 plants since the classic studies (e.g., Bender and Smith 1973). Leaf d-value of higher plants (dp)is 1. Determination of leaf d-value and reconsideration on approximated by (1) carbon isotope discrimination the identification of C3 and C4 species of Poaceae in during photosynthesis, D, and (2) d-value of the atmo- the Japanese flora using a set of herbarium speci- spheric CO2 surrounding the leaf, da, where dp = da mens, because the Research Institute for Biore- D. D of C3 plants are much higher (20&) than C4 sources, Okayama University has a herbarium plants (4&, O’Leary 1988; Farquhar et al. 1989). We specimen set containing a large part of Poaceae spe- should note that succulents, epiphytes, and ferns grow- cies in Japan. ing in arid regions with CAM photosynthetic pathway 2. Analysis of C3 and C4 species richness in relation to have D values from 2 to 22& (Griffiths 1992), which climatic gradient and habitat types. varies depending on the proportion of CO2 fixed during 3. Estimation of the effects of changes in atmospheric d- day and night (Winter and Holtum 2002). However, no value via human activity on the leaf d-value. 215

4. Estimation of the effect of habitat types on leaf d- the other 31 species were identified. In the present study, value through the photosynthetic response to micro- Bambusoideae is treated as a subfamily of Poaceae, environments. containing 40 species having woody (30) or grass (10) life form. The number of species (383) collected in the present study covered 87% of the Poaceae species in Japanese islands (440). Nomenclature and classifica- Materials and methods tion of subfamilies followed by Osada (1993), based on Clayton and Renvoize (1986). Plant specimens

Plant samples of Poaceae were collected from 347 sam- Geographic origins of the specimens and their climates pling sites over the Japanese islands (Fig. 1). Sampling year, names of the sampling sites, altitude, longitude and The specimens were collected from four main Japanese altitude of the sampling sites, and habitat types (e.g., islands Hokkaido, Honshu, Shikoku, and Kyushu, and roadside, grassland, seaside) were recorded. All of the three remote islands such as Ryukyu, Satsunan and mature healthy plants were collected from 1930 to 2003, Ogasawara (Fig. 1). The number of species collected slipped between absorbent papers and dried at room from each island is shown in Table 1. Potential numbers temperature. These herbarium specimens were stored at of species distributed in each island was estimated fol- room temperature at the Research Institute for Biore- lowing the literatures of grass ﬂora (Table 1; Osada sources, Okayama University. Number of species was 1993; Takematsu and Ichizen 1997; Kitamura et al. 383 in this set of the herbarium specimens, where 258 2004). Cultivated plants and a part of the alien plants native species, 72 alien species, 22 cultivated species and were eliminated from the potential distribution analysis.

Fig. 1 Map showing the 48 location of the Japanese islands where Poaceae plant samples were collected. Names of the four main islands (Hokkaido, Hokkaido island Honshu, Shikoku, and Kyushu) N and three remote islands consist of several small islands 44 Sapporo (Ryukyu, Satunan, and Ogasawara) were shown as bold italic. Sampling sites (n = 347) were shown as cross symbols. Square symbols show the name of the city and locations of the 40 meteorological stations for each of the islands Honshu island

Latitude (°N) Tokyo Fukuoka

Shikoku 32 Kyushu island island

Satsunan Islands Amami 28 Chichijima Naha Ogasawara Islands Ryukyu Islands

24 126 130 134 138 142 146 150 Longitude (°E) 216

The climates of the islands were estimated from the Kitamura et al. 2004). Then, analysis of C3 and C4 spe- data at the representative meteorological stations at each cies richness in relation to potential habitats was per- island (National Astronomical Observatory 2008; formed, assuming that variations in microenvironments Table 1). Note, however, that the local climates, pre- among sampling sites and d-value in atmospheric CO2 cipitation in particular, were different within each of the did not affect the identification of C3 and C4. Cultivated islands. Comparison of C3 and C4 species richness in plants and a part of the alien plants were eliminated from relation to regional climatic gradient in the Japanese the analysis. We distinguish the six criteria to determine Islands was performed for Hokkaido, Honshu–Shikoku, the potential habitat types, based on the possible micro- Kyushu and Ryukyu, assuming that microenvironment environmental factors affecting leaf photosynthesis and in sampling sites and d-value in atmospheric CO2 did not leaf d-value (Table 2). Firstly, irradiance level, water affect the identification of C3 and C4 based on leaf availability, salinity, altitude and human effects were d-value. considered. Then, the habitat types with strong industrial and auto-exhaust activities were identified, because anthropogenic CO2 has low d-value. Additionally, the Potential habitats of species habitats that have high levels of respired CO2 with low d- value were distinguished. Some of the species potentially The identification of the potential natural habitats of distributed in the multiple habitats. Poaceae species was performed following the literature The identifications of the habitats where plant spec- on grass flora (Osada 1993; Takematsu and Ichizen 1997; imens were collected, i.e., sampling habitats, were per-

Table 1 Attributes of the main islands and the remote islands in Japan

Islands Number of specimens Potential number Annual climate collected of species from literature Meteorological Temperature Precipitation station (N/E) (C) (mm/year)

Main island Hokkaido 38 132 Sapporo (43/141) 8.5 1128 Honshu, Shikoku 311 246 Tokyo (36/140) 15.9 1467 Kyushu 8 200 Fukuoka (34/130) 16.6 1632 Remote islands Ryukyu 38 120 Naha (26/128) 22.7 2037 Satsunan 31 – Amami (28/129) 21.5 2914 Ogasawara 13 – Chichijima (27/142) 22.9 1277

Location map of each island is shown in Fig. 1. Names of the islands, the number of specimens collected, and potentially habited numbers of the Poaceae based on the literatures describing the natural distribution area of Japanese Poaceae species (Osada 1993; Kitamura et al. 2004) are shown in the ﬁrst three columns. Potentially habited numbers of the Poaceae were not estimated for the Satsunan and Ogasawara Islands. There were species distributed over some islands. The ﬁfth and sixth columns show mean annual air temperature and cumulative annual precipitation in the representative meteorological stations for each island. Climate data was averaged from 1971 to 2000 (Japan Meteorological Agency)

Table 2 Six criteria for the potential/sampling habitats of the Poaceae species in Japan identiﬁed from the literatures describing the types of habitats of Japanese Poaceae species (Osada 1993; Kitamura et al. 2004) and descriptions of the sampling habitats, considering the eﬀects on leaf photosynthesis and leaf carbon isotope ratio (see text)

Potential/sampling Description Possible environmental Number of species Number of specimens habitat type factors aﬀecting in the potential in the sampling leaf d-value habitat habitat and photosynthesis

Ruderal Open wasteland, roadside, town, High light and drying, high 76 114 and empty lot in the urban area gas exhausts by humans Seashore Sand dune or bank of the sea, High light, saline substrate 29 31 salt marsh Mesic Natural grassland, grazed Moderate light and drying 87 39 grassland, river bed Shady Forest floor, valley Low light, high respired CO2 64 71 Wet Freshwater marsh, paddy field High water availability 62 52 Highland High altitude (above 1000 m Low temperature and 18 15 elevation in the sampling sites) low gas exhausts by humans Total 336a 317 aGross total; some of the species were distributed in the multiple potential habitats 217 formed using the above criteria for the potential habi- spatial anthropogenic effects and historical effects. The tats, following the descriptions of the each of the sam- spatial anthropogenic effects and microenvironmental pling habitat (Table 2). Most of the species were effects for the selected data sets were analyzed using collected from the same criteria of the sampling habitat ANOVA, tested by Tukey–Kramer honestly significant as that of the potential habitat (64–79%, data not difference (HSD) test using JMP software (SAS institute, shown). NC, USA).

Microenvironmental and anthropogenic Measurements of leaf carbon isotope ratio eﬀects on the leaf d-value and statistical analysis

In the present data set, historical effect on leaf d-value Leaf samples (about 5 cm2) were taken from the tip of was considered; because plant specimens were collected the mature leaf blade of the specimens and then finely for several decades (from 1930 to 2003) when rapid ground using sample mill (MF10, IKA, Germany). urbanization and increasing global air CO2 concentra- Subsamples of 1–2 mg were subjected to isotope mea- tion should affect the d-value of atmospheric CO2, to- surements using a combined system of an elemental gether with the possible historical change of plant Ci/Ca. analyzer (EA1108, Carlo-Erba, Italy), an interface Additionally, to investigate the spatial effect of hu- (Finnigan MAT conflo II, Bremen, Germany), and an man activity, the sampling sites were classified into two isotope mass spectrometer (Finnigan MAT delta S, districts based on the population density of human Bremen, Germany). Measurements were performed with inhabiting the surrounding area of the site, i.e., ‘‘densely two repetitions for each sample and mean standard inhabited district (DID)’’ and ‘‘no densely inhabited deviation for the two repetitions was 0.23&. district (no-DID)’’ determined from the location (longitude and latitude) of the site and its correspondence with DID or non-DID in the map of human density (the Results Ministry of Internal Affairs and Communications, Ja- pan). DID is a district that has above 5000 individuals Identification of C3 and C4 species aggregated by some land units which have above from leaf carbon isotope ratio 4000 individuals kmÀ2. The mean level of atmospheric CO2 concentration in a DID of Japan was 377 ppmv in The typical bimodal distribution was observed for the 1993, significantly higher than that of the global mean frequency distribution of the leaf d-value in 381 Poaceae concentration (353 ppmv) in 1990 (Aikawa et al. 1995). species in Japan (Fig. 2). The plants having a negative Among the 347 sampling sites, 96 were DIDs, 246 were isotope value higher than À23.4& were classified as C3 no-DIDs, and five were not-classifiable sites. and those having negative values less than À16.0& were For the analysis of microenvironmental and anthro- classified as C4, according to O’Leary (1988). Mean pogenic effects on leaf d-value, MANOVA (multiple analysis of variance) was performed with (1) photosynthetic types (C3 or C4), (2) spatial anthropogenic effects (DID vs. no-DID), (3) habitat types (Tables 2, 4) sampling year, using JMP software (SAS institute, NC, USA). The effect of the region is not analyzed because most of the samples were collected from Honshu. Historical changes in leaf d-value and carbon isotope discrimination were analyzed for the C3 and C4 Poaceae specimens collected from 1930 to 2002. Data was selected for the samples collected from ruderal habitats to eliminate the habitat effects on leaf d-value. Leaf carbon isotope discrimination was calculated using the d-value of atmospheric CO2 obtained by Francey et al. (1999) from 1930 to 1991 and Randerson (2007) from 1992 to 2002. The spatial anthropogenic effects were compared between DID and no-DID for each of the sampling habitats. Data were selected for the samples collected after 1990 to minimize the effect of sampling year. To detect the microenvironmental effects, leaf d-value of C3 and C4 species was compared among the six criteria of Fig. 2 Frequency distribution of leaf d-value of the 381 Poaceae the sampling habitats. Data was selected for the samples species in Japan. Shaded and blank bars indicate C3 and C4 species, collected from no-DID after 1990 to minimize the respectively 218

Table 3 Classification of 47 genera of the subfamily Classification Number of C3 or C4 species for each genus Panicoideae based on C3 and C4 species richness in each genus C3 Coelachne, Cyrtococcum, Ichnanthus, Isachne(4), Oplismenus(5), Sacciolepis(2) C3,C4 Panicum (C3;1, C4;7, not identified two species having C3-like d-value) or not identified C4 Andropogon, Arthraxon, Arundinella, Bothriochloa (2), Brachiaria (2), Capillipedium (3), Cenchrus (2), Coix (3), Cymbopogon (2), Dichanthium, Digitaria (10), Dimeria, Echinochloa (7), Eremochloa, Eriochloa (2), Eulalia, The number of C3 or C4 species Hemarthria (2), Imperata, Ischaemum (3), Melinis, Microstegium(5), Miscanthus (8), in each genus is shown in Paspalum (10), Pennisetum (2), Phacelurus, Pogonatherum, Rhynchelytrum, parentheses. The genera without Rottboellia, Saccharum, Schizachyrium, Setaria (12), Sorghum (2), Spinifex, parentheses contained one Spodiopogon (3), Stenotaphrum, Themeda, Thuarea, Trichachne, Vetiveria, Zea species only d-value of C3 species was À29.2& (n = 227), where the Table 4 Correlation coefficients (r) between relative C4 species lowest value was À34.5& (Melica onoei) and the highest richness in the grass flora of Poaceae and climatic factors in the & Leymus mollis Japanese islands Hokkaido, Honshu–Shikoku, Kyushu and Ry- value À23.5 ( ). The d-value in C4 species ukyu (n =4) varied from À10.3& (Trichachne insularis)toÀ15.9& (Cleistogenes hackelii), where the mean value was Parameters r À12.5& (n = 154). There was no overlap of the carbon isotope values for C and C Poaceae species, where the Latitude (N) –0.98* 3 4 Annual climate difference between the lowest value for C4 plants and the Mean temperature (C) 0.95* highest value of C3 plants was 7.6&. Cumulative precipitation (mm/year) 0.99* Six subfamilies were identified for 383 Poaceae species, where all species of four subfamilies such as * The correlation is statistically significant (P < 0.05). Climate data was averaged from 1971 to 2000 in the representative mete- Arundinoideae (7), Bambusoideae (40), Centothecoi- orological stations in each island (Japan Meteorological Agency, deae (2) and Pooideae (163) were C3. All species of the see Table 1) subfamily Chloridoideae (45) were C4. The subfamily Panicoideae had both C3 (15) and C4 (109) species. At the genus level in the subfamily Panicoideae, Panicum had both C3 and C4 species (Table 3).

Geographical distribution and potential habitats of C3 and C4 species

The relative C4 species richness of the islands in Japan was negatively correlated with their latitude (Table 4). Mean annual temperature and annual precipitation (see Table 1) were positively correlated to the relative C4 species richness. The relative C4 species richness in the potential habitats was greatest at the seashore habitat (66% in the Fig. 3 Relative richness of C3 (solid bar) and C4 (dotted bar) species grass flora, Fig. 3). In the ruderal and mesic habitats, in the grass flora of Poaceae in the six types of potential habitats in relative C4 species richness was 49 and 44%, respec- Japan. Descriptions of the potential habitats are shown in Table 2 tively. Relative C4 species richness was significantly lower than that of the C3 in the shaded and wet habitats (37 and 28%). In highlands, C3 species only were dis- Effects of anthropogenic CO2 and habitat tributed. types on leaf d-value Both of the C3 and C4 alien species prefer ruderal habitat compared to the native species (Table 5). Only The results of MANOVA for the all of the data set 10% of the 138 native C3 species were distributed in the showed that photosynthetic types (C3 or C4), spatial ruderal habitat, while 68% of the 37 alien C3 species anthropogenic effects (DID or no-DID), habitat types were distributed in the ruderal habitat. Similarly, 25% of (ruderal, seashore, mesic, wet, shaded or highland), and the 91 native C4 species were distributed in the ruderal sampling year had significant effects on leaf d-value habitat, while 67% of the 18 alien C4 species were found (Table 6). For the C3 species, habitat types and sampling in the ruderal habitat. The C4 species richness in the year had significant effects on leaf d-value. For the C4 ruderal habitat was 62% (23/37) for native species and species, spatial anthropogenic effects and habitat types 32% (12/37) for alien species. had significant effects on leaf d-value. 219

d-value of global atmospheric CO2 decreased with sampled in the other habitats by <0.8& (Fig. 6b), year for the selected data set of ruderal habitats (Fig. 4 although the difference was not statistically significant. a). The leaf d-value of the C3 plants significantly decreased with year from 1930 to 2002 (Fig. 4b). The leaf d-value of the C4 plants slightly decreased with year, Discussion although the trend was not statistically significant. The historical changes of leaf carbon isotope discrimination Identification of C3 and C4 from leaf d-value of the C3 and C4 plants were small (Fig. 4c). containing samples in districts with high The spatial effect of anthropogenic CO2 on leaf d- anthropogenic effects value was estimated from the comparison between the districts with high human density (DIDs) and no-DIDs The bimodal distribution pattern for the 381 Poaceae for each type of the sampling habitats (Fig. 5). In C3 species in the Japanese islands (Fig. 2) is in line with the grasses, the mean leaf d-value in DIDs was lower than report of Vogel (1993) for the 351 Poaceae grass species, that in no-DIDs by 0.4–0.6& in the ruderal, mesic and showing that the difference of leaf carbon isotope ratio wet habitats, although the difference between DIDs and between C3 and C4 is held for the plant specimens con- no-DIDs was not statistically significant (Fig. 5a, taining samples collected from the districts with high P > 0.1). In C4 plants, the mean leaf d-value in DIDs anthropogenic effects. The districts with high anthropo- was more negative than those in no-DIDs in the ruderal genic effects have more negative d-value of atmospheric habitat by 1.1& (Fig. 5b, P < 0.05). CO2, while they were only 2& more negative in the The mean leaf d-value of the grasses was compared streets with heavy traffic than in the open country in among six types of sampling habitats (Fig. 6). For C3 Kyoto and Shiga, Japan (Hanba et al. 2009). In Paris, species, the lowest leaf d-value was observed for the France, d-value of atmospheric CO2 was À11.9& in the species collected from the shaded habitats (–30.2&, streets of Paris, whereas it was from À9.4& in the open Fig. 6a). C3 species sampled in the wet and highland country on average (Widory and Javoya 2003). These habitats had significantly less negative leaf d-value differences in d-value of atmospheric CO2 between sam- compared to those sampled in the shaded habitats pling sites are much smaller than that between highest leaf (P < 0.05), where the highest value was À28.0& in the d-value of C3 and lowest leaf d-value of C4 plants (7.54&). highland habitats. Similar to the C3 species, C4 species The photosynthetic types in the subfamily and the sampled in the shaded habitats tended to have more tribe levels are consistent with the review by Sage et al. negative leaf d-value (À12.8&) compared to those (1999a), where Bambusoideae, Pooideae and Centoth- ecoideae (which is a tribe in Bambusoideae in Sage et al. 1999a) are recognized as C3, Chloridoideae is all C4, and the tribe Arundineae in Arundinoideae is C3. The Jap- anese C4 genus list of the subfamily Panicoideae in the Table 5 Species numbers of the native or alien plants having C3 or C4 photosynthetic pathway and their potential habitats present study is in line with Okuda and Furukawa (1990). Potential habitats Number of species More than 30 species in seven families are known to be C –C intermediates for the terrestrial higher plants C3 native C4 native C3 alien C4 alien 3 4 (Sage et al. 1999b), where the genus Panicum is in- Ruderal 14 23 25 12 cluded (Brown et al. 1985; Oguro et al. 1985). C3–C4 Seashore 9 17 1 1 intermediates of Panicum have C3-like leaf d-values Mesic 35 32 13 4 when ambient CO partial pressure is 330 lbar (von Wet 37 18 1 3 2 Shaded 45 18 1 0 Caemmerer and Hubick 1989; von Caemmerer 1992). Highland 18 0 0 0 C3–C4 intermediates of Panicum have PEP carboxylase Total 156 102 45 26 having similar properties to the enzyme from C3 Pan- icum species (Holaday and Black 1981), although they Some of the species have multiple potential habitats, and potential habitats are not determined for some of the species, so the sum of partition many organelles to bundle sheath cells as each column does not correspond to the total number of species large as that in C4 Panicum plants (Brown and

Table 6 Result of MANOVA (multivariate analysis of variance) for the leaf carbon isotope ratio with photosynthetic types (C3 vs. C4), spatial anthropogenic eﬀects (DID vs. no-DID), habitat types (ruderal, seashore, mesic, wet, shaded or highland), and sampling year

Data set/factor Overall C3/C4 DID/no-DID Habitat type Sampling year

All ** ** * ** ** C3 ** – NS ** ** C4 ** – ** * NS

Size of the data set was n = 381 for the all of the data, n = 227 for the C3 plants and n = 154 for the C4 plants. The signiﬁcance was tested by F test, where * and ** indicate that P-value was <0.05 and <0.01, and NS indicates not signiﬁcant 220

-6 (a) (a) (‰) wet 2

-7 mesic

-8

atmospheric CO ruderal n isotope ratio of global Carbon -27 -28 -29 -30 -31

-9 Potential habitats

-10 (b) (b) a -14 ruderal b -18 -10 -11 -12 -13 -14 -22 Leaf carbon isotope ratio (‰) -26 Fig. 5 Difference in the mean leaf d-value of C3 a or C4 b Japanese -30 Poaceae plants between two districts divided by the population density of human inhabiting the surrounding area of the sampling Leaf carbon isotope ratio (‰) -34 site, i.e., densely inhabited district (DID, shaded columns) and no densely inhabited district (no-DID, blank columns). Data were 26 selected for the samples collected after 1990. Bars indicate standard (c) errors. Different letters beside the bars indicate statistically 22 significant difference using ANOVA (P < 0.05). The descriptions of the types of the sampling habitats were shown in Table 2 18

14 -27 10 (a) 6 -28 2 1920 1940 1960 1980 2000 Leaf carbon isotope discrimination (‰) Sampling year -29

Fig. 4 Historical effects on a d-value of atmospheric CO2 obtained by Francey et al. (1999) and Randerson (2007), b leaf d-value of C3 -30 and C4 specimens, where regression line is d-value 2 (&) = 29.1 À 0.0295 · year, r = 0.24, P < 0.05, n = 23 for Leaf carbon isotope ratio (‰) a ab abc bc abc c C3 (solid line) and regression line is not statistically significant for -31 C4 (dashed line, n = 17, P > 0.1), and c leaf carbon isotope -10 discrimination of the C3 and C4 specimens, where regression lines (b) (dashed lines) are not statistically significant (P > 0.1). Each point represents averaged d-value for the sampling year. Specimens were -11 selected for the Poaceae plants from ruderal habitats from 1930 to 2002 -12 Hattersley 1989). Among 11 Panicum species, three species such as P. bisulcatum, P. lanuginosum, and P. -13 scoparium were identified as C3 plants from the leaf d- value in the present study, but they have a possibility Leaf carbon isotope ratio (‰) aaa aa -14 exhibiting C3-C4 intermediate photosynthesis. Panicum shaded ruderal mesic wet seashore highland bisulcatum has been identified as a C plant, because it 3 Habitat types of sampling sites has no chloroplasts in bundle sheath (Oguro et al.

1985; Takeda et al. 1985) and has a high compensation Fig. 6 The mean leaf d-value of C3 (a, closed) and C4 (b, open) point (Waller and Lewis 1979; Takeda et al. 1985). For Poaceae species collected from the six types of sampling habitats in the other two species, P. lanuginosum and P. scoparium, Japan. Data were selected for the samples collected from no densely inhabited districts (no-DIDs) after 1990. Dashed lines show the further anatomical and physiological analysis should be mean values for the C3 species and C4 species. Different letters needed to distinguish whether they are C3-C4 interme- indicate statistically significant difference using ANOVA diate or C3. (P < 0.05) 221

The effect of climates and microenvironments The high C4 species richness in the seashore habitats on C3/C4 species richness and their leaf d-values (Fig. 3) would be partly ascribed to a high water-use efficiency of the C4 species, because high water-use effi- Following the report of Takeda et al. (1985), Okuda and ciency involves a low transpiration rate, allowing C4 Furukawa (1990) suggests that C4 Poaceae species in plants to generate low water potential that is necessary Japan is generally more adaptive to higher temperature, for extracting water from saline soil, and to diminish the which is in line with the increasing relative C4 species uptake of sodium and chloride ions from saline soil richness in Poaceae in Japanese islands with regional (Long and Mason 1983). Additionally, a greater poten- mean annual air temperature (Table 4) or regional mean tial productivity of C4-type photosynthesis in higher light monthly air temperature in a growing season (Okuda and temperature (von Caemmerer and Furbank 1999; 1987). No C4 plants were observed at the highlands in Long 1999) may be involved in the high C4 species the present study (Fig. 3), confirming less adaptive nat- richness in the seashore habitats, because seashore open ure of C4 plants to lower temperature. In the Kirigamine habitats have high irradiance and temperature levels. The grasslands in the subalpine zone in the Honshu Island in CO2-saturated photosynthesis of C4 on a CO2-responce Japan, the coverage of C4 plants in the vegetation de- curve rises with temperature, up to the thermal optimum clined while that of C3 plants increased with increasing of typically between 38 and 45C (Sage and Kubien altitude, depending on mean annual air temperature 2007). C4 representation decreases in response to shading (Nishimura et al. 1997; Mo et al. 2004). and become absent in deep understory where incident On the other hand, a positive relationship between C4 radiation ranges from below 10–20% (Pearcy 1990). species richness and annual precipitation was obtained The possible small effect of soil water availability on (Table 4). In North and South America where mean Poaceae plants is also suggested from the leaf d-value of annual precipitation varies from 100 to 1500 mm/ the C3 plants. Leaf d-value of the C3 plants from the wet year, precipitation and its seasonal distribution are habitat was similar to those from the ruderal and sea- important for C3 and C4 distributions (Paruelo and shore habitats where water availability should be occa- Lauenroth 1996; Paruelo et al. 1998; Murphy and sionally limited (Fig. 6a), suggesting that in the humid Bowman 2007). However, in the humid climate in Japan climate in Japan, the difference in local soil water (Table 1), water availability may not be a decisive factor availability hardly affects leaf water-use efficiency in C3 in the geographical distribution of grasses, and thus, it Poaceae grass species. In supporting this view, leaf d- may not be a primary factor affecting the proportion of value of a C3 sedge Kobresia pygmaea was negatively C3 and C4 grasses, as suggested from the study on the correlated to annual precipitation in the habitats of low Kirigamine grasslands in Japan (Mo et al. 2004). The annual precipitation (from 40 to 1000 mm/year) in the positive relationships between C4 species richness and Tibetan Plateau, China, while leaf d-value of a conifer- annual precipitation in the present study and in the ous Abies fabri tree was not correlated with precipitation previous studies in Japan (Takeda et al. 1985; Ueno and in the habitats having high annual precipitation (from Takeda 1992) may be affected by the correlation be- 1750 to 2300 mm/year, Song et al. 2008). tween regional annual precipitation and annual tem- The lowest leaf d-value at the shaded habitats for perature in the Japanese islands. both C3 and C4 Poaceae species (Fig. 6a, b) should be The fact that C3 species richness is similar to that of partly due to a low d-value of atmospheric CO2 as a C4 in ruderal habitat including lowered air and soil result of plant and soil respiration (Francey et al. 1985; humidity habitats, again suggests that water availability Broadmeadow and Griffiths 1993; Hanba et al. 1997), may not be a primarily factor for C3 or C4 dominance because the shaded habitats were often in the forests (Fig. 3). The significant difference in phenology be- where d-value of atmospheric CO2 was by 2.2& lower tween C3 and C4 in open habitat may diminish the than that at the top of the canopy (Hanba et al. 1997). dominance of C4 over C3 in the open habitat in Japan Additionally, for the C3 species, high Ci/Ca at the low (Mishio and Kawakubo 2000), which may be related to irradiance level causes large leaf carbon isotope dis- the similar species richness between C3 and C4 in the crimination (Zimmerman and Ehleringer 1990), result- ruderal habitat in the present study. Otherwise, the ing more negative leaf d-value by 2.6& in the cool- dominance of C3 over C4 was obtained in the wet temperate forest (Hanba et al. 1997). The changes in Ci/ habitats (Fig. 3). However, soil nutrients, mainly Ca may also involve the less negative leaf d-value of the nitrogen rather than soil water availability, may play a C3 species at the higher altitude (Fig. 6a; Ko¨rner et al. role in the C3 dominance, because the wet habitats in 1991, 1988). A small leaf internal CO2 conductance in Japan are often neighbored by paddy fields (Osada the thick leaves having thick mesophyll cell walls at 1993; Kitamura et al. 2004) where application of fer- higher altitude may also involve the less negative leaf d- tilizer should increase soil nutrient levels significantly. value (Kogami et al. 2001). In humid temperate grasslands, experimental nitrogen The variation in leaf d-value among the habitat types additions favored C3 over C4 grasses and forbs because was less in C4 plants than that in C3 plants in Japan plant production increases less in C4 than C3 by in- (Fig. 6), partly because of the less sensitivity of leaf d- creased nitrogen, caused by low nitrogen use efficiency value in the C4 plants to the variation in Ci/Ca compared in C4 (Wedin and Tilman 1996). to the C3 plants (Henderson et al. 1992; Kuba´sek et al. 222

2007). Additionally, the leakiness of CO2 from the which is equivalent to a 2.2& decrease by 72 years, sup- bundle sheath cells, which is a primarily cause in deter- posing that the decreasing ratio is 0.03&/year from 1930 mining C4 carbon isotope discrimination (Farquhar to 2002. This decreasing level corresponds well with the et al. 1982), was affected differently by light intensity and decreasing level of leaf d-value (Fig. 4b). Note, however, growth temperature (Henderson et al. 1992; Tazoe et al. that local and species effects cannot be fully excluded from 2006; Kuba´sek et al. 2007). In the natural habitats, high the present data set, because plant samples were different temperature is often accompanied by high irradiance species collected from diverse sampling sites. level, where high temperature reduces leakiness while The present study is the first report that shows a his- high irradiance either increases or reduces leakiness, torical decrease of leaf d-value for C3 plants in Japan. depending on species (Kuba´sek et al. 2007). Dongarra` and Varrica (2002) reported that the d-value of In the ruderal habitats in the present study, over 50% the tree ring of Platanus hybrida Brot. grown in Palermo, of the Poaceae species were alien plants (Table 5), Italy, decreased from 1880 to 1998 by 3.6, 0.031&/year, relating that the alien plants invade in the ruderal hab- which is also in line with our present results. The less clear itats (Crawley et al. 1997). C4 species richness of the historical trend in leaf d-value of C4 specimens may be aliens was similar to that of the natives in the ruderal partly due to small sample size before 1980; if the whole of habitats, suggesting that C4-type photosynthesis is not the C4 samples (n = 161) are plotted together, leaf d- necessarily advantageous for colonization of the alien value decreased by 0.027& per year (data not shown). plants in the ruderal habitats in Japan. In agreement Leaf d-values of the plants collected from DIDs was with this view, the relative abundance of alien or culti- by 1& more negative than those collected from the no- vated plants in C4 species in the semi-natural grasslands DIDs both for C3 and C4 species (Fig. 5), although the in south-western Japan was only 31.3% (46/147) in the difference is not significant for C3 species. The d-value of literature survey by Okuda and Furukawa (1990). For atmospheric CO2 near the soil surface was À11.6& in the C3 plants, leaf water-use efficiency should be similar the roadsides with heavy traffic and À10& in the sub- between native and alien species in the ruderal habitats, urban area of Japan (Hanba et al. 2009), which can be a because they have similar leaf d-values (data not shown). cause of the observed difference in leaf d-value for C4 species between the plants growing in DIDs and non- DIDs. For the C3 plants, the anthropogenic effect on Anthropogenic effects on leaf d-values leaf d-value is less clear than that in C4, because other in C3 and C4 species micro-environmental and/or ecophysiological effects on leaf d-values should be larger. Leaf d-value of plants For the C3 Poaceae specimens collected from ruderal near an urban Paris roadway was more negative by habitats, significant historical decrease in leaf d-value is 4.5& compared to those in a rural area, which was obtained from 1930 to 2002 (Fig. 4b). On the other caused by the fossil-caused CO2 (Lichtfouse et al. 2003). hand, the historical variation in leaf carbon isotope discrimination is small (Fig. 4c), showing that the effect of climatic change on leaf Ci/Ca for these 70 years may Conclusions be small in Japan. The present result contrasts with the result by Saurer et al. (2004) which shows a clear de- Although a considerable part of the Poaceae specimens crease of leaf carbon isotope discrimination from 1861– were collected from the districts with high human effects, 1890 to 1961–1990 in northern Eurasia, partly related to the identification C3 and C4 for 383 Poaceae species can that the effects of global warming on leaf water-use be performed using leaf d13C values, except for the two efficiency may be more significant in northern Eurasia Panicum species P. lanuginosum and P. scoparium which (59–71N) than in Japan (26–43N). have C3-like isotope values and possibility of C3-C4 In the present data set, the historical variation from intermediate. The increasing regional relative C4 species 1930 to 2002 is –2& in leaf d-value for the C3 plants richness in Poaceae in Japanese islands along with re- (Fig. 4b). The decrease of d-value in global atmospheric gional mean annual air temperature and regional annual CO2 is 1.11& from 1930 to 2002 (Fig. 4a), which partly precipitation supports the previous studies concerning explains the decrease of leaf d-value. There was a positive geographical distribution of C3 and C4 Poaceae in Ja- relationship between leaf d-value for the C3 plants and d- pan. The increasing regional relative C4 species richness value in global atmospheric CO2 (P < 0.05, data not in the grass flora from northern to southern islands, the shown). Additionally, a higher increase of anthropogenic low C4 species richness in highland habitat, and the high CO2 of Japan than that of global average should decrease C4 species richness in seashore habitat suggest that d-value of atmospheric CO2 further and thus decrease leaf habitat preferences of C4 plants in Japan are strongly d-value. The mean level of atmospheric CO2 concentra- related to the photosynthetic characteristics of C4 such tion in a DID of Japan was 377 ppmv in 1993, signifi- as high photosynthetic rate at high temperature and high cantly higher than that of the global mean concentration irradiance levels. (353 ppmv) in 1990 (Aikawa et al. 1995). The decrease of The historical analysis of leaf d-value from 1930 to 2002 d-value of atmospheric CO2 is by 0.03&/year from 1985 and comparison of leaf d-value between densely inhabited to 1991 over the Japanese islands (e.g., Gamo et al. 1995), districts (DIDs) and no-DIDs clearly showed the anthro- 223 pogenic effects on leaf d-value. Increasing urbanization Francey RJ, Allison CE, Etheridge DM, Trudinger CM, Enting IG, Leuenberger M, Langenfelds RL, Michel E, Steele LP may increase emission of gasses with low d-value, resulting 13 in historical decreases of leaf d-value especially in the (1999) A 1000-year high precision record of d C in atmospheric CO2. Tellus B 51:170–193 urbanized districts. Leaf d-value provides valuable infor- Gamo T, Tsutsumi M, Sakai H, Nakazawa T, Machida T, Honda mation for floristic analysis, plant physiological responses, H, Itoh T (1995) Long-term monitoring of carbon and oxygen as well as estimating human impacts on plants. isotope ratios of stratospheric CO2 over Japan. Geophys Res Lett 22:397–400 Giussani LM, Nchez JHC-S, Zuloaga FO, Kellogg EA (2001) Acknowledgments This work was supported by the JSPS Research Molecular phylogeny of the grass subfamily Panicoideae (Po- Fellowship for Young Scientists (12740423-00) and Ohara Foun- aceae) shows multiple origins of C4 photosynthesis. Am J Bot dation at Research Institute for Bioresources, Okayama University 88:1993–2012 (2004). Leaf d-value was measured at the Center for Ecological Grass Phylogeny Working Group (2001) Phylogeny and subfami- Research, Kyoto University. We deeply appreciate Y. Kohata, H. lial classification of the grasses (Poaceae). 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