Variations of biomass and carbon contents in different traits and components of herbaceous species from tropical grassland

Grasslands play a critical role in the global storage of atmospheric carbon (C). Precise estimation of C contents in different plant components is essential to formulate a strategy for mitigating the atmospheric C. Biomass (B) and C of different herbaceous plant components at species, functional group and site levels from tropical grassland locating on the campus of Banaras Hindu University, Varanasi, India were estimated. For this; 117 herbaceous species just-before flowering were harvested. B and C contents for each species and component were measured and statistically analyzed. The measured C (g plant-1) across the components varied from 0.08 to 31.12. On gm–2 basis; it varied between 29 (leaf) and 49 (root). Plant components, species and functional groups in isolation caused significant differences in the measured C. In the present study; the C content of stem was greater than the leaf and root. The perennial, erect, leguminous and native traits had greater C than the others. Therefore, this observation revealed that the perennial, erect, leguminous and native plants could be a better option for reducing the atmospheric CO2 by capturing it and then converting into B through photosynthesis. Further, the fitted regression equation between the root and shoot for B and C could be used for the extrapolation of B and C of the root component based on the shoot component. The conservative field measurement methods may give precise data on B and C but are destructive to grassland, difficult, time-consuming, and costly to cover at large scale. Hence, the present work could be substantial for the estimation of root C based on shoot component.


Introduction
In current Anthropocene epoch; increased concentration of atmospheric Carbon dioxide (CO 2 ) is perceived as a major driver for global climate change events because among the greenhouse gases; CO 2 alone has the potential to contribute a total of 60% of the global warming (Broecker, 1975;and IPCC, 2014).As a consequence, we are observing an increase in earth's temperature, spatiotemporal changes in precipitation patterns, extreme weather events and shifting of seasons under the umbrella of global climate change events (IPCC, 2014).The fossil records suggested that  55.9 m.y.a, the release of natural CO 2 in high amount, rose 5 °C global temperature and caused strong warming (Alley, 2016).The events which took place at that time (dwarfing of large animals, ecosystem disruptions, soil degradation, hydrological variability, larger and more intense storms, loss of vegetation, biodiversity and soil fertility, poleward migration of terrestrial species, species extinction, increased leaf damage by insects and invasion of new species) are continuing and predicted to be repeated more intensively (Alley, 2016).
Looking into above challenges, the world scientific communities are primarily concern for estimating the total worldwide Carbon (C) storage in terrestrial vegetation (Odiwe et al., 2016) and to identify the potential sinks of C required to curb the increasing rate of atmospheric CO 2 (IPCC, 2014;Lu et al., 2015;and UNFCCC, 2015).Thereafter, total terrestrial C pools have been estimated in the range of 2477 PG C (IPCC, 2003) and 3120 PG C (Lal, 2010).Current estimate revealed 1912.2PG C for 1-m depth soil and 1415.7 PG C stored in above and below ground plant biomass.Unfortunately, a big debate is persisting about the ecological research on the amount, uncertainty and accuracy of C stored in the terrestrial ecosystems (Le et al., 2012) due to differences in sampling periods, intensity and spatial resolution of the soil profile databases, in addition to variations in estimation methods (Mokany et al., 2006;Le et al., 2012;and Russell et al., 2015).Therefore, the estimation of C present in missing or uncertain form from diverse terrestrial vegetations is needed for a better understanding of global atmospheric CO 2 mitigation.
The ecological research on C portioning in different plant components is not only important for the assessment of competitive fitness, reproduction, and growth of the plants against the environmental change (Dickson, 1989) but also precisely contribute in global total C estimation and prediction (Mokany et al., 2006;and Russell et al., 2015).The studies on the C storage in above-ground plant components from diverse ecosystems are extensively documented (Bunker et al., 2005) while root received little attention although it contributes almost 30% of the total terrestrial soil C stock and C present in uncertain form is totally ignored (Mokany et al., 2006).As the root-B is difficult (digging or uprooting) and expensive to measure; therefore, it is further challenging to reduce the uncertainty for the total C stock prediction in different plant components of the global terrestrial biosphere (Mokany et al., 2006;Le et al., 2012;and Russell et al., 2015).Similarly; the plant functional trait being a key component of the global C cycle, regulates the storage and fluxes of soil C and facilitates in developing strategies to mitigate the elevated level of atmospheric C (De Deyn et al., 2008).Hence, assessment of plant C based on herbaceous plant functional trait (which is an uncertain or missing component) perspective is equally essential as partitioning in different plant components as for C mitigation and storage is concerned.
Given that tropical forests account for two-thirds of all terrestrial B and exhibit more C stock per unit area than any other land cover type (Pan et al., 2013).Many large-scale forest experiments from the tropics have improved our understanding of CO 2 emission and storage from different terrestrial C pools of the world (Peng et al., 2013).Unfortunately, grasslands being under the uncertain component of the total C budget have been ignored while they cover nearly one-fifth of the world's land surface area (Leith, 1978;Singh et al., 2006).They harbor more than 90% plant species of the forest (Gilliam, 2007), represent more than 20% of global total net primary productivity (Hall and Scurlock, 1991;and Grace et al., 2006), contribute 50% calories consumed worldwide (Irving, 2015) and store roughly 34% of the global terrestrial C pool (White et al., 2000).These species also substantially contribute to the soil C sequestration (Dinakaran et al., 2014) because of higher root production and relatively slower rates of C turnover (Fisher et al., 1994).They have tremendous potential to influence the global C reserves (San Jose´ et al., 1998) and CO 2 reduction from the atmosphere (Minami et al., 1993).
The regression equations, C: B and root (R): shoot (S) ratios are commonly used non-destructive methods for B and C computation in different vegetations (Bollinder et al., 2002;Frank et al., 2004;UNFCCC, 2015;Sainju et al., 2017).Moreover, the regression equations for measuring B and C of grasses have not been developed while C: B and R: S ratios for estimating the B or C is very rare (Sainju et al., 2017).However, different methods have been employed to estimate C storage in the different components of grasslands, including remote sensing (Piao et al., 2004) modeling and field investigation data (Yang et al., 2010), but even precise estimation of total C in different plant components of herbaceous vegetation at cheap cost is lacking.The use of the IPCC default R: S value for estimating the root B or C of grasslands locating in different ecological conditions may cause the discrepancy in the prediction of total B or C (Barbosa et al., 2012), while the use of the vegetation-specific R: S ratios improve the accuracy in estimating the root B or C for the purpose of C accounting at global level (Mokany et al. 2006).Thus, it is urgent to find out the suitable and efficient methods for C estimation from the tropical grassland which could be useful in predicting the global C budget.
In the majority of local, regional, and global assessments, the C content has been assumed to vary between 35 and 65 % of the dry weight, while 50 % of the dry weight has been marked for the quantification of tree C (Lewis et al., 2009;Blanc et al., 2009;and Thomas and Martin, 2012), unfortunately such needful quantification for tropical grassland is disappointing.The massive degradation (20-25% of the world's native grasslands have been degraded), reduction of per capita grasslands and loss of herbage B and organic C due to rigorous anthropogenic events (Sala and Paruelo, 1997;and Sagar et al., 2017) further mound the pressure for the C estimation under the global climate change crisis in the Anthropocene era.
Looking into above problems the objectives of the present study were: (1) to estimate C content in different components of tropical grasslands, and (2) to develop regression equations between root and shoot (R:S ratios) C useful for the estimation of root C based on shoot C. Such an approach could be helpful in reliable estimation of global terrestrial C for formulating a suitable strategy required to mitigate the rate of increasing C.

Study Area
The study was conducted on the campus of Banaras Hindu University (25.3176°N latitude and 82.9739° E longitude and 80.71 m above sea level), Varanasi, India, during January 2014 -December 2015.The climate is a tropical monsoon.The cold winter (November to February), hot summer (April to June) and warm rainy (July to September) are distinct seasons.The months of October and March are transitional months between rainy and winter, and between winter and summer seasons, respectively (Verma et al. 2015).During the study period, the mean maximum temperature was 30.19 °C (range 8-44.6 °C) while the mean minimum temperature was 20.27 °C (range 6.4-30.8°C).The soil is alluvial, well-drained, pale brown, silty loam and inceptisol.In general, it is moderately fertile being low in available nitrogen and medium in available phosphorus and potassium with neutral to alkaline soil pH (Sagar et al. 2008).

Vegetation Sampling and Analyses
During the study period; through the intensive survey, a total of 117 mature herbaceous species (at the onset of the flowering stage) were collected from the entire campus of the University.For each species; 3-10 healthy individuals (depending upon the availability) just before the flowering stage were harvested.Each harvested plants were separated into root, leaf, and stem components (Poorter and Bergkotte, 1992).For our convenience, the term 'stem' is used throughout the ms instead of 'culm' (generally used for the grasses).The plant fractions were oven-dried at 80 °C to constant weighed.The oven dried plant material was used to determine the ash content.Ash content was measured after combustion of the sample in a muffle furnace at 550°C for 6 h (Singh et al., 2011).C content was determined by using the loss on ignition method (Mcbrayer and Cromack, 1980) which is approximately 50% of ash-free weight (van Soest, 1963).The plant functional attributes were studied at life form (forbs, grasses, legumes, and sedges), growth form (erect, prostrate, procumbent and decumbent), lifespan (annual, biennial and perennial), and origin or distribution (native, non-native and cosmopolitan) levels (Verma et al., 2015).These traits were determined with the help of Flora of Raipur, Durg, and Rajnandangaon (Verma et al., 1985) and Flora of the upper Gangetic plain (Duthie, 1903).
The study also estimated B and C contents on three sites differing in anthropogenic perturbations (less/ site-1, medium/site-2, and high/site-3) on the campus of Banaras Hindu University.For each site; 15, 1×1m plots were used for the sampling of plants for their B and C measurement.Hence, a total of 45, 1×1 m plots (3 sites × 15 replicate) were used for the validation of C content.For each 1 × 1 m plot, above ground peak, live B of all species was clipped at the soil surface in mid-October of the sampling year.All samples were oven-dried at 80°C to constant mass and weighed.This oven dried B was used for the determination of C content.

Statistical Analyses
Analysis of variance (ANOVA) procedure of SYSTAT package ver. 13 (SYSTAT, 2009) was used to see the effects of species, components, and traits on the measured B and C contents.A Tukey's HSD test was used to understand variations of C content among the different plant components and sites.Regression analysis was used to see the relationships of B contents between roots and shoots as well as C contents between roots and shoots components with the help of SYSTAT software ver.13 (SYSTAT, 2009).

Species Composition
A total of 117 herbaceous species belonging to 95 genera and 31 families were recorded from the campus of Banaras Hindu University.The family Poaceae had the highest species number (19) and fifteen families had single species.Among all the families, the four families (Poaceae, Asteraceae, Fabaceae, and Cyperaceae) were species-rich (Appendix 1).On the basis of dry B (g plant -1 ); Aurobindo donex, Anisomelos indica and Hyptis suveolens were dominant species in the study area (Appendix 1).
Across the species and components; maximum B (g plant -1 ) for leaf (46.04), stem (65.49), and shoot (11.53) components as well as for total plant (133.85)were represented by A. donex, while maximum root B was exhibited by Rauwolfia serpentina (25.58).Conversely; the minimum B for leaf (0.19), stem (0.08) and shoot (0.16) components were showed by Polygonum barbatum, Lippia alba, Tanacetum parthenium, respectively.Further, Lindernia anagallis had minimum root (0.01) as well as total plant (0.37) B (Appendix 1).Interestingly, these species also exhibited similar patterns regarding the maximum and minimum values for C content on g plant -1 basis.For instance; the observed C for leaf, stem, shoot, root and total plant varied from 0.08 (P.barbatum) to 20.20 (A.donex), 0.03 (L.alba) to 31.12 (A.donex), 0.07 (T.parthenium) to 51.31 (A. donex)  five times greater B than the roots (Figure 1).More or less similar trend was also exhibited by C portioning into different plant components (Figure 1).The patterns of B, as well as C across the plant components, significantly varied in the order of stem>leaf>root as suggested by the Tukey's HSD analysis (Figure 1).

Functional Group Effects on B and C
ANOVA showed that B and C contents of different plant components differed substantially due to variations in the functional groups related to nativity and growth forms (Table 1).The plant components of life form functional groups also changed notably due to variations in their B and C contents, except B as well as C of root component and whole plant B. For lifespan functional group; only B and C of root component as well as whole plant varied statistically (Table 1).Results showed that the perennials, native, grasses and erect functional group categories represented greater B and C in their stems compared to other components of the functional groups (Table 2).Their corresponding S: R ratios for B and C ranged from 4.3 to 4.7, 4.2 to 4.5, 5.3 to 5.8, and 5.4 to 5.9.Annual, nonnative, legume and decumbent functional group categories had 6.8 and 7.5, 6.5 and 7.2, 5.5 and 6.3, and 6.4 and 6.9 times higher shoot B and C than their root components.Interestingly; perennial, cosmopolitan, sedge, and prostate functional groups represented the lowest shoot to root (highest root to shoot) ratios for B and C in their respective functional groups (Table 2).

Effects of site on B and C contents (g m -2 )
Statistical analysis showed that the sites caused significant difference in the herbaceous species (F2, 42: 33.06, P  0.001) which varied from 9 (site-3) -15 (site-2).Analysis further revealed that sites substantially influenced the B and C contents of leaf (F2, 42: 9.31, P  0.001 and F2, 42: 8.92, P  0.001), shoot (F2, 42: 10.56, P  0.001 and F2, 42: 10.64, P  0.001), root components (F2, 42: 13.96, P  0.001 and F2, 42: 13.50, P  0.001) and whole plant (F2, 42: 25.09, P  0.001 and F2, 42: 23.90, P  0.001).For example, across the sites; the B and C contents of leaf, stem, shoot, root components, and whole plant ranged from 48 to 102 and 21 to 42, 70 to 94 and 31 to 42, 118 to 195 and 51 to 85, 73 to 184 and 29 to 75, and 192 to 380 and 81 to 160, respectively (Table 3).The trend showed that the values of these variables for different plant components were lowest at highly disturbed location (site-3) and highest at less disturbed (site-1) location (Table 3).The mean C: B ratios for different plant components varied between 0.41 (root) and 0.45 (stem).Root had comparatively larger variability (0.36 to 0.50) than the other traits, while the whole plant expressed least (0.39-0.47) variability in the C: B ratios (Table 4).Thus, the results suggested that the C prediction based on C: B ratio for the whole plant will be more consistent.

Relationships Between Root and Shoot Components Related to B and C Contents
Various significant regression equations between shoot (X) and root (Y) based on B and C contents considering species as data points are shown in Figure 2.Among these significant equations; the considerable R 2 (determination coefficient) varied from 0.27 (logarithmic equation for C content) to 0.52 (power equation for B).Similarly, the Standard Error of Estimate (SEE) ranged between 1.12 (power equation for C) and 3.15 (logarithmic equation for B).In both the cases; next, to the power equation, the maximum R2 (0.46; for B, and 0.45; for C content) and minimum SEE (2.72; for C content and 1.15; for B) was exhibited by the linear equation (Figure 2).

Species-wise B and C contents
A. donax showed maximum above ground B and C contents; hence, this could be used as an energy crop.It is advocated because of the greater photosynthetic capacity of A. donax in full sunlight compared to other C3 plants as suggested by Webster et al. (2016).The B production of A. donax in terms of energy and at the same time reduction of atmospheric CO 2 seems to be an interesting observation (Lewandowski et al., 2003).Further, reports indicated that A. donax may reach B yields up to 100 t ha -1 in the second or third year of cultivation under the suitable climate and irrigation (Vasconcelos et al., 2007).It forms dense stands on highly disturbed lands (Saltonstall and Bonnett, 2012), highly fire-tolerant species and having a high level of carbohydrates in its cell walls (67.85 % dry weight; Scordia et al., 2010;and Chandel et al., 2011), hence, could be a valuable species for C sequestration and a suitable substrate for ethanol production (Scordia et al., 2010;and Chandel et al., 2011).
The average total C of herbaceous species was 42.72% of their dry biomass.Similar to the present study, Davies et al. (2011) also reported 42.02% C of the dry biomass for the herbaceous species.Tumuluru (2015) reported 43.92 and 42.08% C of the dry biomass for the corn stover and switchgrass, respectively.However, in various studies, it was around 45% of the plant dry biomass (Olson et al., 1983;and Wang et al., 1999).Across the species, percent of total above-ground C on the dry-weight basis from steppe grassland of Inner Mongolia, China has been reported in a range of 15 to 41 having 29 as mean value (Sagar et al., 2017).The percent below ground C on the dry weight basis was 40.60.The NGGI Workbook 4.2 Revision 2 (1997) assumed 42% C of the root dry matter for crops and grasses.
The study showed 51 -85 (gm -2 ) above ground C. Based on 0.43 C: B ratio, other studies also reported 44 to 154 (gm -2 ) above ground C in an N-input study from the study area (Verma et al., 2013 and2015).In a steppe grassland of Inner Mongolia, China it was in a range of 7.12 to 10.72 (Sagar et al., 2017) while in other studies of USA, the value was around 140-150 (Golubiewski, 2006;and Davies et al., 2011).Thus, the above ground C in the present study fall under these reported ranges; viz: 7.12 (Sagar et al., 2017) and 150 (Golubiewski, 2006;and Davies et al., 2011).

Plant Component-wise C Content
Similar to the present study, significant variations in the root, stem and leaf B and C contents were reported by Poorter and Bergkotte (1992).The percentage above ground C was substantially higher than the below-ground C which signified the dependency of the plant growth on the supply of C from the shoots, and the nutrients and water from the roots.Thus, the assimilation of C by foliage and the acquisition of mineral nutrients and water by fine roots are balanced with the utilization of C and nutrients in the plants (Cannell and Dewar, 1994).Further, the C allocation to above ground and below ground plant parts are facilitated by nitrogen supply via regulating the cytokinins and sucrose productions (Van der Werf and Nagel, 1996).The cytokinins and sucrose productions through N supply could be other reason for allocating greater C in the stem than the other organs (Van der Werf and Nagel, 1996).Notably greater C in stem compared to root and leaf could be due to storage of greater B in stem component than the others because the stem part of the plant has been reported to contain a comparatively higher concentration of lignin than the leaves and roots (Poorter and Bergkotte, 1992).

Functional traits-wise C content
In the present study, the perennial plants stored more C and B in comparison to annual and biennial plants which could be attributable to the excess accumulation of photosynthates in form of carbohydrates, lipids and other chemical compounds (Dickson, 1989).Since the present study area experienced a marked seasonality, hence, this storage reserve material could be used by the plants for their respiration and maintenance during the dormant season (Dickson, 1989).It has been argued that in the seasonal environments, perennial plants have the ability to fix and store enough C, over the growing season to survive the winter and emerge following the year (Farrar et al., 2014) and different species incorporates different C according to their specific metabolism (Liu et al., 2017).Among all life form categories, the legumes had relatively more C compared to others.It may be further linked to their nitrogen-fixing ability in their root nodules which is utilized by such plants for their B and C production.Probably, it could be a reason for considering the leguminous plant as a key driver for C sequestration in many studies (Fornara and Tilman, 2008;and Wu et al., 2016).On the other hand, grasses showed high B (84.2 %) and C allocation above ground compared to the below ground (15.8%).Similar to our results, Irving (2015) also reported 80-85 % B in above ground and only 15 -20% in below-ground components of the grasses.
The study suggested that erect plant trait harbored greater B and C than the other traits which could be due to the availability of sufficient light to capture the atmospheric C. It is well-known fact that light is an important limiting factor for the growth and B build-up in the plants (Neufeld and Young, 2003).As observed in other studies; the tall plants were thought to be more competent in atmospheric C capturing and B build-up and outshade the short-statured species (Diekmann and Falkengren-Grerup, 2002).Thus, the erect plant species had greater B and C than the prostrate and short-statured plants due to their high C capturing ability in presence of sufficient sunlight (Sagar et al., 2012;and Verma et al., 2015).Greater uptake of atmospheric CO 2 into the B of erect trait through the photosynthesis (Cardinale et al., 2012), benefits the storage of CO 2 into the soil as soil organic-C (Fornara and Tilman, 2008;Cong et al., 2014;and Sagar et al., 2017), hence, such trait could be a strategy in sequestering the C into the soil as organic-C against the problem of global warming.
The notable changes in above ground and below ground C partitioning (R:S ratio) based on C content due to differences in species, lifespan, nativity and growth forms could be related to the deeper and denser rooting systems.The deeper, larger and denser rooting systems accumulate greater B and C in below ground than the above ground and are more beneficial for the accumulation of C into the soil (Rasmussen et al., 2010).In present study; R. serpentine, Evolvulus alsinoidis, Convolvulus prostatus, Boerhavia diffusa, Alysicarpus vaginalis, Lauania procumbence, Ruellia tuberosa and Vetiveria zizanoidies have high R:S ratio because of deeper and denser rooting systems that could have facilitated greater resource allocation in the roots (Coleman and McConnaughay, 1995;and Poorter et al., 2012).

Impact of disturbances on B and C
Studies have suggested that grazers increase soil compaction, disturb soil aggregates and decrease the stability of soil aggregate, and change the soil structure.Compact soils show lower porosity, higher bulk density; lower moisture content due to reduced water infiltration and increased run-off/drainage reduced plant available water and reduced soil aeration.Further, biotic disturbances reduce soil organic matter, deplete clay content in the soil and reduce soil structure, increase soil erosion and reduction of topsoil, generating the denser subsoil exposed, promoting higher bulk density at the soil surface (See Sagar and Verma for greater detail) and ultimately, loss of herbaceous B and C.Moreover, reduction in B and C along the disturbance gradient may be argued because of harvesting of above-ground herbage cover which may inhibit the oxygen level in the roots, consequently, there could be a failure of reproduction and death of a certain individual (Matayaya et al., 2017).The above ground herbaceous plant component is related to the photosynthetic tissue of the plant.Hence, the removal of the above-ground herbage cover results in the reduction of the plants' photosynthetic tissue which causes loss of carbon and nutrients for growth and development of the plants (Ferraro and Oesterheld, 2002) and reduction in the herbaceous biomass (Leriche et al., 2003).Therefore, biotic pressure modulated these conditions and synergistically reduced the B and C contents of the herbaceous species.

C:B as a C estimator
In prediction analysis for selecting a suitable model, it is necessary to test all reasonable models as rigorously as possible against known standards to avoid summary judgment based on a single data set (Colwell and Coddington, 1994) because the utility of a model generally depends on its ability to predict unknown value for a given known value (Systat, 2009).Further, higher correlation coefficient, lower standard error of estimate (standard error of estimate quantifies the spread of the real data points around the fitted regression curve), least discrepancy in prediction (Sagar et al., 2003;and Systat, 2009) and wide adaptability with minimum time and cost epitomize the performance and utility the of model (Colwell and Coddington, 1994;and Xie et al. 2009).For estimation of root C using different regression equation (based on root and shoot C), the power equation had higher correlation coefficient and lowest SEE than the others, thus, C: B ratio could be an appropriate estimator of the C content.

Conclusion
Comparatively greater C in perennials, erects, legumes and native trait categories than the others suggested that the species having such traits could be used for reducing the atmospheric CO 2 by capturing it and then converting into the B through the photosynthesis.The conservative field measurement methods may give precise data on B and C, but are destructive to grassland, difficult, time-consuming, and costly to cover at large scale, hence, the C:B ratio could be used as an estimator of C at species, components, functional group and site levels in the tropical grassland.

Figure 1 :
Figure 1: Mean biomass (g plant -1 ), carbon content (g plant -1 ) and carbon to biomass ratio based on 117 herbaceous species for different plant components in the tropical grassland at Varanasi, India.Bars affixed with different letters within each component are significantly different from each other.

Figure 2 :
Figure 2: Linear and non-linear relationships (a) between shoot biomass; SB (X-axis) and root biomass; RB (Y-axis), and (b) between shoot carbon; SC (X-axis) and root carbon; RC (Y-axis) based on 117 herbaceous species in the tropical grassland at Varanasi, India.R 2 =Determination coefficient, SEE = Standard Error of Estimate, P = level of significance.