Foliar Carbon and Nitrogen Content and Stable Isotopic Composition of Selected Philippine Flora

Home , Adenium, Dracaena (plant)

Philippine Journal of Science 150 (S1): 539-550, Special Issue on Biodiversity ISSN 0031 - 7683 Date Received: 04 Oct 2020

Roland V. Rallos*, Gerald P. Dicen, Andrea Luz G. Nery, and John Leonard R. Labides

Agriculture Research Section Philippine Nuclear Research Institute Department of Science and Technology (DOST-PNRI) Commonwealth Ave., Diliman, Quezon City National Capital Region 1101 Philippines

The elemental carbon and nitrogen content, as well as the stable isotopic composition of plants, can provide information on their nutrient dynamics and physiological characteristics. With applications ranging from paleoecology to ecological modeling, this information is critical in biodiversity conservation and management especially for highly dynamic ecosystems such as the Philippines’. Here, we determined the carbon and nitrogen content and their stable isotopic ratios in selected Philippine flora across different classifications and photosynthetic pathways. Fully developed leaves from different species of grasses, succulents, shrubs, and trees were collected and analyzed using isotope ratio mass spectrometry for carbon and nitrogen concentration and stable isotopic composition. Our results showed that trees had the highest carbon and nitrogen content, while grasses that utilized the C4 photosynthetic pathway were most efficient in terms of nutrient utilization as evidenced by their high C/N ratios. Foliar stable carbon isotopic composition of the surveyed Philippine flora was an excellent measure for distinguishing among photosynthetic pathways. The stable nitrogen isotopic composition was not distinct across plant classifications and showed indications of being sensitive to environmental factors, thus limiting its use for phylogenetic tracing. Our findings thus indicate that foliar carbon and nitrogen content and stable isotopic composition provide insights that cannot be easily achieved with other measurements.

Keywords: carbon, isotope ratio mass spectrometry, nitrogen, photosynthesis, Philippine flora, stable isotopes

INTRODUCTION development, and biochemical functioning. In addition, plant C and N are also critical for ecological processes Carbon (C) and nitrogen (N) are essential elements in all such as energy flow and nutrient cycling, while the C/N living organisms, and their metabolism is closely linked ratio is an indicator of N use efficiency (NUE) and an (Raven et al. 2004). For plants, C and N are crucial for input variable for many ecological and ecosystem models fundamental cellular activities (Coruzi and Bush 2001; (Zhang et al. 2020). Zheng 2009), which make them essential for plant growth, Information on plant isotopic compositions is essential in *Corresponding Author: [email protected] predicting future patterns of many ecosystem processes

539 Philippine Journal of Science Rallos et al.: Stable Isotopic Composition Vol. 150 No. S1, Special Issue on Biodiversity of Philippine Flora and functions (e.g. primary productivity, C and N characterizing plant functional groups and photosynthetic sequestration, nutrient fluxes). Likewise, it is also used pathways (Descolas-Gros and Schölzel 2007; Cernusak to trace the plant's geographic origin and is an important et al. 2013), stable isotopes analysis can also be used to tool in forensic settings from an ecological perspective determine the diet of animals (Dalerum and Angerbjörn (Chesson et al. 2018). The stable isotopic composition 2005; Hopkins and Ferguson 2012) to make sense of their of plants depends on the source of their nutrition and movement and habitat (Hobson 1999, 2007; Bowen et al. the conditions involved in their uptake. To some extent, 2005) and is, therefore, useful in biodiversity conservation information on the processes involved in the transport and management (Walter et al. 2014). In the soil, being a and transformation of plant-derived materials is preserved huge repository of dead organic materials, stable isotopes in their stable isotope ratios (Marshall et al. 2007). For are a useful proxy for studying both long-term and short- this reason, the stable isotopes in organic materials term biogeochemical dynamics in the environment. For – in conjunction with their corresponding elemental example, the stable C and N isotope ratios can be used composition – can be considered as natural environmental to provide a mechanistic understanding of the long- tracers (Michener and Lajtha 2007). Thus, stable isotope term C storage in mangroves (Dicen et al. 2019) and in measurement has become one of the indispensable tools elucidating the effect of burning on soil organic matter used in ecological studies. biodegradability (Dicen et al. 2020). However, in all the applications, prior data on the isotopic composition of Leaf stable C isotope ratios vary based on how different relevant flora must be available as they are central to the photosynthetic pathways discriminate against heavier inter-trophic cycling of elements, especially C and N. C atoms (Farquhar et al. 1989; Cernusak et al. 2013). The C3 photosynthetic pathway tends to discriminate Studying tropical ecosystems, which are extremely diverse heavier CO2 molecules as compared with the C4 and dynamic, such as the Philippines’ can greatly benefit photosynthetic pathway – as a factor of the ratio of its from stable isotopes measurement. Consolidated stable intracellular and ambient CO2 concentration, water use isotope data from this study and other similar reports can efficiency, coordination between stomatal conductance then be used to generate isotope landscape, or isoscapes, and photosynthesis, and leaf area adjustment (Cernusak which is a very useful tool for data interpretation in et al. 2013). ecological and forensic studies. However, no study has been done on the stable isotope composition of diverse On the other hand, stable N isotope ratios in plants are Philippine flora to date. In this study, we determined reflective of the short-term dynamics in N cycling within the C and N content and their stable isotopic ratios in ecosystems (Ometto et al. 2006; Craine et al. 2015). For selected Philippine flora, across different classifications instance, microbial activities spell the difference in leaf and photosynthetic pathways. In addition, we explored stable isotope composition between temperate and tropical patterns that emerged from the relationships among plants (Martinelli et al. 1999), as well as the difference measured parameters. between leguminous and non-leguminous species (Boddey et al. 2000). Spatial, temporal, and climatic factors can also have subtle but otherwise significant effects on the stable C and N isotopic composition of plants (Liu et al. MATERIALS AND METHODS 2008; Ma et al. 2012; Xu et al. 2017). Earlier studies indicated that the measurement of C and N stable isotopic About 10–15 fully developed leaf samples of selected compositions in plants would provide an understanding Philippine flora – classified into grass (n = 17), shrub of the cycling of these nutrients. For example, Garten et (n = 22), succulent (n = 24), and tree (n = 25) species – al. (2008) and Craine et al. (2009) have shown that with were obtained around the Greater Manila Area (GMA), increasing N availability and potential N mineralization Philippines. GMA is the contiguous developed zones through natural N supply or N availability gradients, plant surrounding Metro Manila, including the adjacent stable N isotope ratios have increased. Moreover, stable C provinces of Cavite, Laguna, Batangas, Rizal, Bulacan, and N ratios have also been used to investigate the rate of and Pampanga (Figure 1). Grass species were further C sequestration and N cycling under different vegetations classified according to their photosynthetic pathways, and land use (Baldos and Rallos 2019). with n = 4 for C4 grasses and n = 13 for C3 grasses. Soils in the study area were classified under different series Variation in environmental conditions and other with varying fertility levels. The soil in Pampanga area biogeochemical processes can also have a strong influence is under Angeles series with low fertility level, Quingua over the natural abundances of stable C and N isotopes series in Bulacan area, Novaliches series and San Manuel in plants. Knowledge of the different factors that cause series in Metro Manila area, Tagaytay series in Cavite area, variations in stable isotope ratios can, thus, be explored and Calumpang series in Laguna area – which all have for several ecological research applications. Aside from

540 Philippine Journal of Science Rallos et al.: Stable Isotopic Composition Vol. 150 No. S1, Special Issue on Biodiversity of Philippine Flora

Figure 1. Map of the sampled area.

moderate fertility levels (PhilRice 2010, 2013; Carating by the presence of the catalyst Cr2O3, which was used as et al. 2014). The study area’s mean annual precipitation packing material in the combustion reactor. (MAP) and mean annual temperature (MAT) based on the WorldClim database (Fick and Hijmans 2017) were 2047 Following combustion, the resulting gaseous products mm and 27.3 ºC, respectively. Leaf samples were collected were allowed to pass through a reduction furnace from the middle part of tree and shrub canopies. Sampled maintained at 650 °C. The reduction column contains trees and shrubs have fully good vigor while sampled copper where the excess oxygen was scavenged by succulents and grasses were not foraged obviously by the reduced copper, and NOX is reduced to N2 gas. An large mammals. Sampling was done from May–June 2017. additional water trap was set up to remove the water produced from the combustion process before the final dry The leaf samples were washed thoroughly and dried at gaseous products enter the chromatographic column with 60 °C for 48–72 h. Dried leaf samples were then ground the help of ultra-high purity helium (99.999%) as carrier and made to pass through a 0.5-mm sieve. About 3–4 gas. The separated gases eluted at different retention times mg of ground and finely pulverized leaf samples were and introduced into the isotope ratio mass spectrometer weighed into tin capsules. The tin capsules were then (IRMS) (Sercon 20-22, Sercon Limited, UK) for final crimped and introduced from the autosampler into the quantification of total C and N and their corresponding combustion reactor maintained at 1000 °C, where an stable isotopic ratios. instantaneous and complete oxidation process takes place 13 15 using a flash combustion elemental analyzer (EA-GSL, The stable C and N isotope ratios (δ C and δ N, Sercon Limited, UK). This instantaneous combustion was respectively) expressed as per mil (‰) were calculated as: achieved by the optimum timing for the introduction of a pulse of ultra-high purity oxygen gas (99.999%) when the tin capsule reaches the hot zone in the combustion reactor. The C and N contained in the sample are converted to where Rsample and Rstandard are the ratios between stable CO2 and NOX, respectively. This reaction was facilitated

541 Philippine Journal of Science Rallos et al.: Stable Isotopic Composition Vol. 150 No. S1, Special Issue on Biodiversity of Philippine Flora isotopes (13C/12C and 15N/14N) of the sample and standard, Philippine flora, foliar C/N ratios can be fitted with a log- respectively. The isotopic composition of standards used log relationship with N content with a very high coefficient was expressed relative to the stable isotopic composition of regression (R2 = 0.94; p < 0.0001) (Figure 2), indicating of Pee Dee Belemnite for C and atmospheric air for N. the highly interlinked C and N metabolism in plants. The analytical precision of measurement for both δ13C and δ15N was higher than 0.2‰. Plots and statistical analyses in this study were carried out in JMP (version 11; SAS Institute, Cary, NC, USA) software package. Significant differences in C and N concentrations, δ13C and δ15N values, and C/N ratios were tested using one-way analysis of variance. Post hoc multiple comparisons were made using Tukey’s honestly significant difference (HSD) test for significantly different groups. The level of significance was set at α = 0.05.

RESULTS AND DISCUSSION

Elemental C and N Content Figure 2. Scatterplot of the C/N ratios vs. N concentrations of Table 1 presents the summary of the elemental C and N selected Philippine flora fitted with a logarithmic content, C/N ratio, and stable isotopic composition of regression line. Blocked and red-colored symbols indicate leguminous species (Family Fabaceae). the surveyed Philippine flora (see Appendix Table I for values corresponding to each species). Shrubs and trees significantly (p < 0.05) had the highest C content, while succulents had the lowest one. The C content of C grasses Plants with higher C/N ratios have higher NUEs to ensure 4 their survivability under N-limited conditions, while those and C3 grasses were comparable (p > 0.05) to the three classifications. In terms of N content, shrubs and trees still with lower C/N ratios can only grow efficiently under significantly (p < 0.05) had the highest mean values, while high N supplies (Reich and Oleksyn 2004). Generally, C4 grasses are the most efficient in terms of N utilization, C4 grass species had the lowest; the N content of C3 grass and succulent species were not significantly different (p > assimilating 24 C per N, while the least efficient N users 0.05) from the rest. The C/N ratios among classifications are the C3 grass and tree species, only assimilating 17–19 were also statistically the same (p > 0.05), with values C per N (Table 1). Our results are consistent with the ranging from 16.9 (± 2.8) to 23.2 (± 7.1). previous report that C4 plants such as maize and sugarcane have higher photosynthetic efficiency than those of C3 The assimilation of C in plant biomass is carried out by plants such as rice (Kajala et al. 2011). Foliar C/N ratio N compounds through photosynthesis; thus, the rate of also dictates the quality of the litter that will be returned in C assimilation increases as a function of N in leaf (Reich soil and a good predictor for decomposition (Kim 2007). et al. 2006). This also suggests that C accumulates more A low C/N ratio increases net N mineralization rate and in plants in relation to N content, which makes the C/N stimulates plant growth, while the opposite occurs in a ratio a valuable expression and indicator for primary high C/N ratio (Tateno and Chapin 1997; Lukac et al. production and nutrient dynamics. From the surveyed 2010). Thus, our data on leaf C/N ratios may indicate

Table 1. Photosynthetic pathways, mean elemental C and N content, δ13C and δ15N isotopic composition, and C/N ratio across classification. Photosynthetic Classification C (%) N (%) δ13C (‰) δ15N (‰) C/N ratio pathway

ab b a C4 Grass (n = 12) C4 45.0 ± 2.5 2.0 ± 0.5 –13.8 ± 1.2 2.7 ± 3.3 23.9 ± 7.3 ab ab c C3 Grass (n = 4) C3 44.4 ± 3.0 2.7 ± 0.3 –30.2 ± 1.5 2.8 ± 2.6 16.9 ± 2.8 a a c Shrub (n = 22) C3 47.5 ± 3.5 2.8 ± 0.9 –31.3 ± 1.5 5.1 ± 4.1 19.1 ± 9.1 Succulent (n = 24) CAM 42.4 ± 3.7b 2.2 ± 0.7ab –24.0 ± 7.3b 3.8 ± 3.6 22.2 ± 10.8

a a c Tree (n = 25) C3 47.3 ± 3.3 2.8 ± 0.9 –30.4 ± 1.6 5.2 ± 4.0 19.2 ± 7.0 Means within column followed by the same letters are not significantly different at 5% probability level using Tukey’s HSD.

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enhanced N-cycling in areas vegetated with trees and C4 plants and in the same sequence, but they segregate shrubs than those with grasses and succulents. Fornara the activities of the enzymes between night and day and Tilman (2008) have also observed that grassland (Marshall et al. 2007). CAM plants are further divided vegetation has high C/N ratios, slow mineralization into two classifications: obligate-CAM and facultative- and decomposition, and high N immobilization and use CAM. Obligate-CAM plants have average δ13C values of efficiency. This also makes grassland ecosystems an –11‰. Some species use the CAM pathway facultatively, important sink of C. utilizing C3 photosynthesis when conditions are favorable then shift back to CAM when drought strikes. Facultative- Across all plants, the C concentrations in the leaf CAM plants have δ13C values between –27 and –11% are more stable (35.4–53.7%) than N concentration (Marshall et al. 2007). (0.8–4.7%) (Appendix Table I). Thus, the variations in the leaf C/N ratio were more associated with changes For grasses (Family Poaceae), the majority of the samples 13 in N concentration. The relatively stable nature of C utilized the C4 photosynthetic pathway. The average δ C concentration per unit of dry biomass in a terrestrial value of the samples in the C4 grass species was –13.8 ecosystem has been demonstrated in plants exposed to ± 1.2‰, which significantly had the most enriched δ13C altered climatic regimes (He et al. 2006) and at elevated values among the classifications of surveyed Philippine CO2 levels (Luo et al. 2006). The factors that can affect flora (Figure 3). It can also be highlighted that these the plants’ C/N ratios include but are not limited to water samples could easily be distinguished on the upper part of 13 availability (Chen et al. 2015), biomass reallocation, and the figure due to the distinct range of δ C values of the C4 nutrient dilution impact in the plant-soil system (Zhang et photosynthetic pathway. The C4 photosynthetic pathway is al. 2020). Alternatively, other N sources such as biological often seen in grasses in tropical areas due to the need for N2-fixation (BNF) and mycorrhizal associations also affect the plants to adapt to dry or low-input soils (O’Leary 1988; N concentration in plants (Rogers et al. 2006; Püschel Forseth 2010; Kellogg 2013; von Caemmerer et al. 2014). et al. 2017). Values found in this study – in particular, samples in the Family Fabaceae, the legume plant family with BNF potential – had high N concentrations, hence the low C/N ratios. However, some non-legumes also have low C/N ratios, and this indicates that there is no consistent trend between legume and non-legume plants. Thus, variations in C/N ratios are likely related to water availability, soil fertility, and other available N sources. The environmental factors that affect C/N ratios explain the overlapping values across classifications and photosynthetic pathways. This implies the limitation of using this parameter for the phylogenetic tracing of plant species, which is in agreement with the recent finding of Zhang et al. (2020) who reported that the leaf C/N ratios of 2,139 species were not constrained by the plants’ phylogeny. Nevertheless, plant C/N ratio – in addition to Figure 3. Scatterplot of the stable C and N isotope ratios of selected C and N content – can be used as input parameters and Philippine flora. Blocked and red-colored symbols baselines for ecological models (Xu et al. 2018; Zhang et indicate leguminous species (Family Fabaceae). al. 2020), and this necessitates a comprehensive survey of flora from ecosystems of interest such as the one shown in this study. There were four grass species in the study – namely, rice (Oryza sativa), ribbon grass (Phalaris arundinacea), Stable C and N Isotope Composition and two bamboo species of the genera Phyllostachys Numerous studies have provided the range for δ13C and Bambusa – which had δ13C values, indicating that values of the three photosynthetic pathways in terrestrial these grasses utilized the C3 photosynthetic pathway. In 13 plants. C3 plants are characterized by δ C values ranging temperatures above 30 °C, which is the typical temperature from –37 to –20‰, C4 plants from –16 to –12‰, and in tropical areas such as the Philippines, the photosynthetic CAM (crassulacean acid metabolism) plants from –27 efficiency is reduced by up to 40% (Karki et al. 2013), to –10‰ (O’Leary 1988; Marshall et al. 2007). CAM which is a challenge for economically important crops 13 plants have similar δ C values with C4 plants because such as rice that utilize the C3 pathway. Thus, studies CAM plants rely on the same carboxylating enzymes as involving genetic engineering are being conducted, for

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15 example, to explore the possibility of installing the C4 temperate leaves (Craine et al. 2015). Foliar δ N values photosynthetic pathway in rice in order for them to have were also shown to increase with increasing MAT and a higher yield, reduced water loss, and increased NUE – decreasing MAP (Szpak et al. 2013; Craine et al. 2015). especially in hot and dry areas (Sage 2000; Karki et al. 15 2013; Wang et al. 2016). The δ N signatures of the selected Philippine flora are also shown in Figure 3. In N-fixing plants, the heavier Plants in the tree and shrub categories had average δ13C stable isotope 15N is discriminated against 14N during values of –30.4 ± 1.6‰ (n = 26) and –31.3 ± 1.5‰ (n = biochemical, biogeochemical, and physiological processes 15 22), respectively. All of the species in the tree and shrub (He et al. 2009). Non-N2-fixing plants generally have δ N categories had δ13C values that were under –27‰, which values that are greater than 0‰ while plants that utilize indicates that the species utilize the C3 photosynthetic significant amounts of atmospheric N2 are characterized pathway. The average δ13C values of shrubs and trees also by comparatively low δ15N values that are approximately varied significantly from the average values of the grass equal to 0‰ (Szpak et al. 2013; Craine et al. 2015). and succulent categories. This further showed that the plants in the trees and shrub categories utilized a different In the surveyed Philippine flora, several of the samples 15 photosynthetic pathway from those in the succulent and from the Fabaceae family had δ N values ranging from grass species. –1.4 to 9.0‰, which did not follow the expected trend on δ15N on leguminous species. This was also observed in a The plants in the succulent classification had an average study by Szpak et al. (2013) where no consistent pattern δ13C value of –24.0 ± 7.3‰ (n = 24). This value varied in plant δ15N was seen with respect to leguminous trees significantly from other categories. The average value and shrubs. While some had δ15N values relatively close also fell under the CAM photosynthetic pathway. to 0‰, other samples have δ15N of up to 9.6‰. Martinelli Succulence is a characteristic of CAM plants; thus, all in et al. (1999) also suggested that in tropical ecosystems, this classification utilize the CAM photosynthetic pathway δ15N values should be above zero since the nitrogen cycle (Marshall et al. 2007). is more open – with greater losses of light nitrogen gases via fractionating pathways – leaving heavy nitrogen It was observed that several of the plants in the succulent available to internal cycling. Likewise, the enriched δ15N classification had δ13C values, which corresponded to values of some sampled plants under the Fabaceae family the C3 photosynthetic pathway. These plants fall under may indicate that these plants are not reliant on BNF the facultative-CAM category, which means that they are as the source for N nutrition or may have limited BNF able to shift back and forth between C3 and CAM-type potential (e.g. Cassia alata, C. fistula, and Caesalpinia photosynthesis depending on the conditions to which the pulcherrima). Finally, since the samples were taken from plant is exposed (Ting 1989; Pate 2001; Forseth 2010). around the GMA, which is a highly urbanized region, 13 Facultative-CAM plants will have δ C values resembling disturbance could have contributed to the high variability those of C3 plants under well-watered conditions but will in δ15N values of leguminous species. For instance, 13 have δ C values closer to C4 plants when in dry or saline frequent flooding and high mean annual temperature can environments (Pate 2001). enhance losses of N through surface runoff and leaching δ15N values are often used in assessing contributions (Peng et al. 2011), ammonia volatilization (Liu et al. of various N sources to plant N uptake in the field. 2020), and denitrification (Granger and Wankel 2016) – Variations in the natural abundance of N stable isotopes causing considerable N fractionation, such that the soil is in plant tissue include the sources of plant N, N fixation, enriched with heavy N (Wang et al. 2017). atmospheric sources of N, and mycorrhizal status (Boon and Bunn 1994; Marshall et al. 2007; Craine et al. 2015; Newton 2016). In a study by Martinelli et al. (1999), δ15N and N concentrations in leaves were positively CONCLUSION correlated (p < 0.007) in the site. However, despite the Foliar C and N content and their corresponding stable samples being taken in one location, we did not observe isotopic composition can provide information on inter- 15 this correlation between δ N and N concentration or any classification differences in nutrient use and cycling other parameter – whether the samples were classified into and physiology. The elemental C and N contents of the distinct photosynthetic pathways or not. surveyed Philippine flora indicate that C4 grass species Several studies have also been done on the effect of climate are more efficient in terms of N utilization, which is on the δ15N values of samples. Foliar δ15N declined with often one of the most limiting nutrients in an ecosystem. increasing rainfall across 97 sites. Latitude was shown to The C/N ratio and N content of the surveyed species have no effect on the δ15N values of samples but the δ15N follow a logarithmic trend, which indicates the closely values in tropical leaves were 6.5‰ higher compared to interlinked metabolism of C and N in plant leaves. For

544 Philippine Journal of Science Rallos et al.: Stable Isotopic Composition Vol. 150 No. S1, Special Issue on Biodiversity of Philippine Flora phylogenetic tracing, the stable isotopic composition of JAM, MARSHALL JD, FARQUHAR GD. 2013. leaves – particularly δ13C – is a better predictor of the Environmental and Physiological Determinants of photosynthetic pathway utilized by plants. In contrast, Carbon Isotope Discrimination in Terrestrial Plants. δ15N values were not distinct across plant classifications, New Phytologist 200(4): 950–965. possibly owing to the tropical climate of and/or the high CHEN D, WANG S, XIONG B, CAO B, DENG X. 2015. probability of disturbance in the area. Nevertheless, the Carbon/nitrogen imbalance associated with drought- stable isotopic composition of diverse Philippine flora induced leaf senescence in sorghum bicolor. PLoS can provide insights that cannot easily be obtained with ONE 10(8). other measurements. CHESSON LA, BARNETTE JE, BOWEN GJ, BROOKS JR, CASALE JF, CERLING TE, COOK CS, DOU- THITT CB, HOWA JD, HURLEY JM, KREUZER ACKNOWLEDGMENTS HW, LOTT MJ, MARTINELLI LA, GRADY SPO, PODLESAK DW, TIPPLE BJ, VALENZUELA LO, This study was funded by the DOST–Philippine Council WEST JB. 2018. Applying the principles of isotope for Agriculture, Aquatic, and Natural Resources Research analysis in plant and animal ecology to forensic sci- and Development through the establishment of an isotope ence in the Americas. Oecologia 187(4): 1077–1094. ratio mass spectrometry facility in the DOST-PNRI. The authors acknowledge the Myanmar research fellows Ms. CORUZZI G, BUSH DR. 2001. Nitrogen and Carbon Cho Cho Win, Ms. Nyein Aye Wai, Ms. Phyu Phyu Swe, Nutrient and Metabolite Signaling in Plants. Plant and Mr. Billy Ne Win for their assistance during field Physiology 125(1): 61–64. sampling and sample preparation. CRAINE JM, BROOKSHIRE ENJ, CRAMER MD, HASSELQUIST NJ, KOBA K, MARIN-SPIOTTA E, WANG L. 2015. Ecological interpretations of nitrogen isotope ratios of terrestrial plants and soils. Plant and NOTES ON APPENDICES Soil 396(1–2): 1–26. The complete appendices section of the study is accessible CRAINE JM, ELMORE AJ, AIDAR MPM, BUSTA- at http://philjournsci.dost.gov.ph MANTE M, DAWSON TE, HOBBIE EA, KAHMEN A, MACK MC, MCLAUCHLAN KK, MICHELSEN A, NARDOTO GB, PARDO LH, PEÑUELAS J, REICH PB, SCHUUR EAG, STOCK WD, TEMPLER REFERENCES PH, VIRGINIA RA, WELKER JM, WRIGHT IJ. 2009. BALDOS AP, RALLOS RV. 2019. Indications of en- Global patterns of foliar nitrogen isotopes and their hanced soil ecosystem functions in polyculture refor- relationships with climate, mycorrhizal fungi, foliar ested grassland. Annals of Tropical Research 1(C): nutrient concentrations, and nitrogen availability. New 32–44. Phytologist 183(4): 980–992. BODDEY RM, PEOPLES MB, PALMER B, DART PJ. DALERUM F, ANGERBJÖRN A. 2005. Resolving Tem- 2000. Use of the 15N Natural Abundance Technique poral Variation in Vertebrate Diets Using Naturally to Quantify Biological Nitrogen Fixation by Woody Occurring Stable Isotopes. Oecologia 144(4): 647–658. Perennials. Nutrient Cycling in Agroecosystems 57(3): DESCOLAS-GROS C, SCHÖLZEL C. 2007. Stable Iso- 235–270. tope Ratios of Carbon and Nitrogen in Pollen Grains BOON PI, BUNN SE. 1994. Variations in the stable in Order to Characterize Plant Functional Groups isotope composition of aquatic plants and their im- and Photosynthetic Pathway Types. New Phytologist plications for food web analysis. Aquatic Botany 48: 176(2): 390–401. 99–108. DICEN GP, NAVARRETE IA, RALLOS RV, SALMO BOWEN GJ, WASSENAAR LI, HOBSON KA. 2005. SG, GARCIA MCA. 2019. The Role of Reactive Iron Global Application of Stable Hydrogen and Oxygen in Long-Term Carbon Sequestration in Mangrove Sedi- Isotopes to Wildlife Forensics. Oecologia 143(3): ments. Journal of Soils and Sediments 19(1): 501–510. 337–348. DICEN GP, RALLOS RV, LABIDES JLR, NAVARRETE CARATING RB, GALANTA RG, BACATIO CD. 2014. IA. 2020. Vulnerability of Soil Organic Matter to Mi- The Soils of the Philippines. Springer Netherlands. 363p. crobial Decomposition as a Consequence of Burning. Biogeochemistry 150(2): 123–137. CERNUSAK LA, UBIERNA N, WINTER K, HOLTUM

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APPENDIX

Table I. Elemental C and N content, δ13C and δ15N isotopic composition, and C/N ratio of surveyed Philippine flora.

Common Photosynthetic N δ15N C Classification Scientific name Family δ13C (‰) C/N name pathway (%) (‰) (%)

C4 grass Andropogon citratus Tanglad Poaceae C4 1.4 1.8 46.6 –13.8 33.4

C4 grass Chrysopogon aciculatus Amorseco Poaceae C4 2.1 –0.7 44.9 –14.8 21.3

C4 grass Coix lacryma-jobi Adlay Poaceae C4 1.8 2.2 42.2 –12.3 23.3

C4 grass Cynodon dactylon Bermuda Poaceae C4 2.1 11.6 44.8 –14.8 21.4 grass

C4 grass Dactyloctenium aegyp- Damong Poaceae C4 2.9 2.0 40.8 –16.3 14.2 tium balang

C4 grass Digitaria sp. Crabgrass Poaceae C4 2.2 1.5 45.5 –13.2 20.5

C4 grass Echinochloa crus-galli Bayokibok Poaceae C4 2.9 3.1 42.7 –13.8 14.7

C4 grass Eleusine indica Paragis Poaceae C4 2.2 4.0 43.8 –14.0 19.6

C4 grass Imperata cylindrica Cogon Poaceae C4 1.6 0.3 50.4 –13.5 31.5

C4 grass Paspalum conjugatum Carabao Poaceae C4 1.2 –2.9 45.4 –12.5 38.7 grass

C4 grass Pennisetum purpureum Napier Poaceae C4 2.2 3.2 45.3 –15.4 20.7

C4 grass Rotboellia cochinchinesis Agingai Poaceae C4 2.0 4.7 44.0 –11.9 21.8

C4 grass Saccharum officinarum Tubo Poaceae C4 1.6 3.8 48.4 –13.4 29.8

C3 grass Oryza sativa Palay Poaceae C3 2.7 4.8 43.3 –31.5 15.9

C3 grass Phalaris arundinacea Reed canary Poaceae C3 2.3 –1.0 48.7 –28.5 21.1 grass

C3 grass Bambusa sp. Kawayan Poaceae C3 2.9 3.0 44.0 –31.5 15.0

C3 grass Physllostachys sp. Golden Poaceae C3 2.7 4.2 41.7 –29.4 15.7 bamboo

Shrub Acalypa sp. Chenille Euphorbiaceae C3 3.8 5.7 41.7 –35.6 10.9 plant

Shrub Antidesma bunius Bignay Euphorbiaceae C3 2.0 3.2 49.3 –32.4 25.2

Shrub Caesalpinia pulcherrima Caballero Fabaceae C3 3.3 9.0 49.0 –29.9 15.0

Shrub Cassia alata Akapulko Fabaceae C3 3.2 6.2 49.8 –32.5 15.4

Shrub Coffee arabica Kape Rubiaceae C3 2.0 4.6 51.0 –30.8 25.5

Shrub Duranta erecta Golden Verbenaceae C3 3.3 2.0 44.4 –33.2 13.4 duranta

Shrub Duranta repens Golden Verbenaceae C3 3.5 6.0 49.5 –31.8 14.2 dewdrop

Shrub Syzygium myrtifolium Eugenia Myrtaceae C3 1.1 1.6 53.7 –31.0 48.0

Shrub Ficus septica Hauli tree Moraceae C3 3.0 2.9 41.7 –34.2 13.7

Shrub Gardenia jasminoides Rosal ‎Rubiaceae C3 1.8 4.1 47.2 –31.0 26.0

Shrub Hibiscus rosa-sinensis Gumamela Malvaceae C3 2.8 1.6 41.9 –30.4 15.1

Shrub Ipomoea crassicaulis Morning Convolvula- C3 3.9 0.3 46.0 –29.1 11.7 glory ceae

Shrub Ixora coccinea Santan Rubiaceae C3 1.5 15.9 53.2 –30.5 35.9

Shrub Jasminum samba Sampaguita Oleaceae C3 3.4 2.6 46.2 –31.0 13.6

Shrub Manihot esculenta Cassava Euphorbiaceae C3 4.7 5.2 49.3 –31.0 10.5

Shrub Murraya paniculata Kamuning Rutaceae C3 3.1 5.9 51.1 –31.2 16.8

Shrub Mussaenda philippica Agboy Rubiaceae C3 2.3 13.1 49.5 –30.8 21.2

Shrub Phyllanthus virgatus Kaya-an Phyllanthaceae C3 2.1 4.0 49.1 –31.0 23.8

Shrub Physalis minima Pantug- Solanaceae C3 2.8 6.5 44.7 –30.7 16.0 pantugan

Shrub Rosa chinesis Rose Rosaceae C3 2.2 3.8 45.7 –31.6 21.3

Shrub Thevetia peruviana Yellow Apocynaceae C3 2.7 13.9 46.4 –31.0 17.2 oleander

548 Philippine Journal of Science Rallos et al.: Stable Isotopic Composition Vol. 150 No. S1, Special Issue on Biodiversity of Philippine Flora

Common Photosynthetic N δ15N C Classification Scientific name Family δ13C (‰) C/N name pathway (%) (‰) (%)

Shrub Vigna radiata Mungo Fabaceae C3 4.4 3.2 43.9 –29.0 10.1 Succulent Adenium obesum Kalachuchi Apocynaceae CAM 2.3 1.0 46.3 –29.0 20.5 Succulent Agave bracteosa Spider agave Asparagaceae CAM 2.6 2.3 42.8 –15.8 16.2 Succulent Agave sansevieria Snake plant Agavaceae CAM 1.8 7.6 42.0 –14.9 23.8 Succulent Aloe vera Sabila Asphodelaceae CAM 1.1 5.4 39.9 –15.3 36.0 Succulent Ananas comosus Pinya Bromeliaceae CAM 1.0 8.8 45.6 –15.4 45.5 Succulent Anthurium andraeanum Flamingo lily ‎Araceae CAM 2.7 0.3 42.1 –33.0 15.6 Succulent Billbergia pyramidalis Flaming Bromeliaceae CAM 0.8 –3.6 43.8 –16.3 52.3 torch Succulent Cereus hildmannianus Hedge cactus Cactaceae CAM 2.1 6.2 35.4 –13.7 17.2 Succulent Cheilocostus speciosus Crepe ginger Costaceae CAM 3.3 2.0 43.6 –29.7 13.4 Succulent Cordyline fruticosa Good luck Asparagaceae CAM 2.1 8.4 50.4 –28.0 24.0 plant Succulent Dendrobium sp. Dendrobium Orchidaceae CAM 2.4 13.1 45.1 –14.2 19.0 orchid Succulent Dracaena fragrans Fortune plant Asparagaceae CAM 2.9 2.9 42.9 –31.8 14.7 Succulent Dracaena surculosa Gold dust Asparagaceae CAM 2.0 2.8 45.6 –31.7 22.6 Dracaena Succulent Epipremnum aureum Devil's vine Araceae CAM 3.2 2.6 45.0 –32.1 14.2 Succulent Euphorbia milii Crown of Euphorbiaceae CAM 1.6 –0.2 39.3 –16.2 25.0 thorn Succulent Euphorbia tithymaloides Devil's Euphorbiaceae CAM 2.6 4.6 43.2 –21.0 16.9 backbone Succulent Ficus elastica Rubber plant Ficeae CAM 1.5 3.6 47.5 –30.2 31.9 Succulent Hippeastrum puniceum Easter lily Amaryllida- CAM 2.5 1.9 42.5 –27.2 16.8 ceae Succulent Hoya cornosa Hoya Apocynaceae CAM 1.2 7.3 43.2 –17.7 35.4 Succulent Peperomia obtusifolia Baby Piperaceae CAM 2.8 4.9 39.5 –30.7 14.2 rubberplant Succulent Peperomia pellucida Pansit- Piperaceae CAM 3.0 –0.1 40.0 –23.3 13.3 pansitan Succulent Talinum fruticosum Philippine Talinaceae CAM 3.4 1.1 35.7 –29.3 10.5 spinach Succulent Tradescantia spathacea Bangka- Commelinace- CAM 2.8 2.9 35.8 –29.2 12.7 bangkaan ae Succulent Zamioculcas zamiifolia Emerald Araceae CAM 1.9 4.5 40.6 –31.2 21.7 palm

Tree Albizia lebbeck Aninapla Fabaceae C3 4.4 2.5 52.2 –31.6 11.9

Tree Annona muricata Guyabano Annonaceae C3 2.6 7.4 47.2 –31.1 17.9

Tree Annona squamosa Atis Annonaceae C3 3.3 8.5 47.5 –30.2 14.3

Tree Artocarpus heterophyllus Langka Moraceae C3 1.9 1.4 42.1 –30.1 22.8

Tree Averrhoa bilimbi Kamias Oxalidaceae C3 2.2 3.7 47.5 –30.6 21.6

Tree Averrhoa carambola Balimbing Oxalidaceae C3 1.8 3.4 48.7 –27.9 27.4

Tree Azadirachta indica Neem tree Meliaceae C3 4.1 1.8 48.6 –32.9 11.8

Tree Cassia fistula Kanya pistula Fabaceae C3 2.5 6.0 48.2 –29.5 19.1

Tree Chrysophyllum cainito Caimito Sapotaceae C3 1.6 7.8 43.9 –29.7 27.1

Tree Citrus aurantiifolia Dayap Rutaceae C3 2.7 6.5 45.2 –30.0 16.9

Tree Diospyros blancoi Mabolo Ebenaceae C3 2.0 19.7 42.1 –31.8 20.7

Tree Ficus nota Tibig Moraceae C3 2.1 0.2 38.5 –30.6 18.5

Tree Gliricidia sepium Kakawate Fabaceae C3 3.9 4.0 45.3 –30.2 11.6

Tree Leucaena leucocephala Ipil-ipil Fabaceae C3 3.7 5.1 45.8 –30.8 12.4

Tree Magnifera indica Manga Anacardiaceae C3 4.6 2.2 47.0 –31.7 10.3

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Common Photosynthetic N δ15N C Classification Scientific name Family δ13C (‰) C/N name pathway (%) (‰) (%)

Tree Moringa oleifera Malunggay Moringaceae C3 2.4 2.9 47.3 –29.8 19.9

Tree Nephelium lappaceum Rambutan Sapindaceae C3 1.9 6.1 50.3 –31.7 26.5

Tree Polyalthia longifolia False ashoka Annonaceae C3 2.4 6.9 51.8 –30.8 22.0

Tree Psidium guajava Bayabas Myrtaceae C3 2.3 7.0 50.5 –31.1 21.6

Tree Pterocarpus indicus Narra Fabaceae C3 3.9 -1.4 50.8 –25.3 13.0

Tree Samanea saman Rain tree Fabaceae C3 3.6 3.6 52.3 –30.9 14.6

Tree Sandoricum koetjape Santol Meliaceae C3 1.4 8.6 50.9 –28.2 37.2

Tree Swietenia macrophylla Mahogany Meliaceae C3 3.8 7.2 45.8 –30.7 12.2

Tree Syzygium cumini Duhat or Myrtaceae C3 1.5 8.6 48.4 –33.3 33.3 lomboy

Tree Tamarindus indica Sampalok Fabaceae C3 2.8 1.8 46.5 –30.1 16.6

550