Nutrient and Stoichiometric Characteristics of Underground Fissures Soil in Karst Rocky Desertication Regions

Aims Underground ssure soil is an important soil resource in karst rocky desertication area, but there are few reports on its physical and chemical properties and soil ecological stoichiometry. This study aims at investigating the physicochemical properties, nutrient content and stoichiometric characteristics of subsurface ssured soils in karst areas, which will help to understand their intrinsic linkage mechanisms and provide a scientic basis for making full use of ssured soils. Methods Typical karst ssures were selected through a combination of eld investigation and in-laboratory analysis, and soil physicochemical properties were determined through eld sampling and indoor tests. Results We found that as the depth of the ssure soil layer increases, the silt content and clay content increase and the sand content and soil water content continue to decrease. The ssure soil is basically weakly alkaline. The TN content does not change much from the surface to the lower part of the ssure, and the AN, TP and AP contents in the ssure generally decrease. In general, SOC, nitrogen and phosphorus are mainly concentrated in the surface soil compared to the other soil layers in the ssure; the C/N, N/P and C/P ratios are higher in the upper part of the ssure; C/P and N/P are higher and C/ N is lower in SL, while the opposite is true in AL.


Introduction
It is reported that karst landforms account for 12% of the total global land area, with an area of about 22 million square kilometres (Ford and Williams et al.,2013). However, in the fragile karst geological and ecological environment, karst rocky deserti cation caused by soil erosion and unreasonable and intensive land use has become a serious environmental and social problem in the world (Wang et al., 2004).
The southwestern China karst area centred on Guizhou Province is the world's largest continuous karst area (Li et al., 2017b;Peng et al., 2017) .and the special ecological geological characteristics make this area a typical fragile ecological environment (Wang et al.,2004b). With the intensi cation of stone deserti cation, soil erosion in karst areas is becoming increasingly serious ((Febles-González et al., 2014), and the ability to support plant growth is severely weakened, leading to vegetation degradation, which in turn leads to the destruction of the basic structure of karst soils, soil infertile, water-holding capacity reduced, and plants subjected to frequent deteriorating environmental stress, leading to the inability of vegetation to grow normally or eventually to die, increasing the risk of karst stone deserti cation and further leading to the collapse of karst ecosystems, all of which directly or indirectly cause serious challenges to the productivity and sustainability of karst soil resources (Smith et al., 2013) .And the special surface-subsurface dichotomy of the Tongkat region leads to soil erosion in two directions. During the runoff-driven downward loss of soil through the ssure system, part of the soil is temporarily stored in the shallow karst ssures, forming the ssure soil system. Studies have shown that the ssure soil system has become one of the important plant habitats in karstic stone desert slopes, which can provide water, nutrients and the spatial basis for plant growth, and is a good place to promote vegetation restoration projects (Waltham et al.,2008;Xiong et al.,2018). Soil organic carbon (SOC), nitrogen (N) and phosphorus (P) are important indicators of soil fertility and quality (Lal et al.,2004;Andrews and Karlen et al.,2004). Soil organic carbon has an important role and signi cance in in uencing soil physical and chemical properties, regulating soil structure, re ecting land productivity and environmental health functions, and nitrogen signi cantly increases primary productivity in most terrestrial ecosystems (Hua et al.,2015), Phosphorus levels can directly affect plant growth and development (Ruttenberg et al.,2003), In summary, the cycling of carbon, nitrogen and phosphorus in soils is closely related (Vitousek et al.,2004) and is an essential nutrient for the restoration and growth of karst vegetation. The soil conditions of the shallow karst subsurface ssures are different from those of the surface, and therefore the physical and chemical properties and nutrients of the soil are also different from those of the surface, so the study of ssured soils in this area can be of great scienti c importance for the implementation of revegetation and improvement of soil quality.
At present, many scholars have mainly focused on the physicochemical properties of soils under different rock-deserti cation classes (Ma et al., 2020) or the nutrient and stoichiometric characteristics of soils, the topsoil under different land use practices (Han et al., 2015;Wang et al., 2018b;Wang et al., 2020), or the nutrient characteristics of different karst topographies (peaks, depressions, etc.) ( Liu et al., 2014;Yang et al., 2019;Zhang et al., 2013), the in uence of karst lithology on nutrients (Wang et al., 2018b), while some researchers have focused on the nutrient dynamics of degraded agricultural soils (Li et al., 2017a;Wen et al., 2016). However, most studies have focused on the physicochemical properties of surface soils, such as organic carbon and nitrogen cycling, and less research has been conducted on the physicochemical properties, nutrient and ecological stoichiometric characteristics of karst ssure soils. Therefore, this study aims to investigate the physicochemical properties, nutrient content and stoichiometric characteristics of subsurface ssure soils in karst areas, which will help to understand their intrinsic linkage mechanisms and provide a scienti c basis for making full use of ssure soils. The results of the study can provide a certain basis for the restoration of vegetation and the rational use of soil resources in karst stone deserti cation areas, and have certain guiding signi cance for the ecological restoration and management of karst stone deserti cation areas.

Study area and sites
The study was conducted from July 2017 to August 2017 in the Xinfa Small Watershed, located in Zhenning County, Anshun City, Guizhou Province, China, at latitude 26°06′-26°07′ North and longitude 105°49′-105°50′ East, in a typical karst region of Guizhou Province, China (Fig. 1). The region has a humid Page 4/19 subtropical monsoon climate, with an average temperature of 14.03°C and an accumulated temperature (≥ 10°C) of 4116°C, annual average rainfall of about 1193 mm, 960-1300 hours of sunshine, an average frost-free period of 294 days and annual average land evaporation of 550 mm. The soil type is predominantly limestone, derived from Triassic limestone. Broad-leaved subtropical evergreen forests are the dominant zonal vegetation type.

Soil sampling
In this study, A 50 m x 50 m grid is laid over the study area and 60 10m x 10 m sample points are set up at the grid points, within each sample point three typical ssures are selected and sample points, ssure information and soil samples are collected. The soil samples are homogeneously mixed at four levels, 0-20 cm, 20-40 cm, 40-60 cm, 60-80 cm, at the same level and labelled U1, U2, U3, U4, in that order. Finally, the survey selected 60 typical underground shallow-layer ssures in four vegetation types: grassland, fallow land, shrubland and tree woodland: 9, 8, 29 and 14 ssures ( Table 1).

Measurement of soil physical properties and nutrients
The natural soil water content (SWC) was determined via the drying method, the soil pH was measured by extracting the liquid from a soil and water mixture (ratio of 1:5) (Institute of Soil Science of Chinese Academy of Sciences, 1978), and the soil particle distribution was determined by the pipette method. The soil organic carbon (SOC) contents were determined by oxidation with potassium dichromate under external heating, total nitrogen (TN) was measured by the Kjeldahl method, and the alkali hydrolysable nitrogen (AN) was determined by the Conway method, and the total phosphorus (TP) was measured with the Mo-Sb colourimetric method, and available phosphorus (AP) was extracted with 0.5 mol/L NaHCO3 (pH = 8.5) and then determined by colourimetric analysis.

Physical and chemical properties of soil in ssures
The physical and chemical properties of karst ssured soil with different vegetation types in the same soil type (limestone soil) and rock type (limestone) are shown in Table 2. The soil pH at the same soil depth differs among the different vegetation types, ranged from 7.6 to 7.97, which was basically weak alkaline.
The water content of ssured soil of the same vegetation type was U1 > U2 > U3 > U4, and there was no signi cant difference, which indicated that the upper part of ssured soil can provide more water for plant growth.
In the ssures of the same vegetation type, with the increase of soil depth, the sand content (0.02-2 mm) decreased, while the silt content (0.02 − 0.002 mm) and the clay content (< 0.002 mm) increased (Table 2) The organic carbon content (including soils at depths U1, U2, U3 and U4) was SL > GL > FL > AL. Except for FL, soil layer U1 had the highest SOC content in the ssures of the three vegetation types.In U1 soil layer, SL had the highest SOC content (Fig. 2a.). and the SOC contents at depths of U2, U3 and U4 can be ranked as SL > GL > FL > AL, GL > SL > FL > AL, FL > AL > SL > GL, respectively. This result shows the high organic carbon content of ssured soils that support the growth of shrubs and grasslands.
ii) Changes in the nitrogen content of ssured soils As shown in Fig. 2b., the TN content ranged from 0.65 g/kg to 1.13 g/kg, with little variation and no signi cant differences in soil layers across vegetation ssures. However, the variation of AN was different from that of TN, where soil layer U1 had the highest amount of AN in the same vegetation type (Fig. 2c.). U1 of SL was not only signi cantly higher than U2, U3, and U4 but also signi cantly higher than U1 of the other three vegetation types (p < 0.05). SL had the highest AN content of all vegetation types, suggesting that shrubs can increase the AN content of ssured soils.
iii) Variation in phosphorus content of ssured soils The variation in TP and AP content of ssured soils of different vegetation types was different from the TN and AN content (Fig. 2d., Fig. 2e.).The total phosphorus content of ssure soils, including those at depths of U1, U2, U3 and U4, decreased in the order AL > GL > FL > SL,which suggested that plants in putre ed, grassland had an advantage in accumulating phosphorus in ssured soils. And among the ssures of different vegetation types, FL had the lowest AP content, while SL and AL had higher AP content.Also, the TP and AP contents of four vegetation types U1 were higher than those of U2, U3, and U4, indicating the enrichment characteristics of soil surface TP and fast-acting phosphorus, i.e., surface phosphorus was not e ciently utilized by plants.

iv) Correlation of SOC, N, P content of the soil in ssures
The content of C, N, and P in the ssured soil was positively correlated with vegetation type (P < 0.01).C and N, C and P, and N and P content were all linearly correlated and varied similarly (Fig. 3a., 3b., 3c.). The signi cant correlation coe cients for C and N and C and P were at the 0.05 level, and the correlation between N and P was highly signi cant (p < 0.01).

Changes in stoichiometric ratios of soil ssures nutrients under different vegetation types i) C/N ratio
The results showed that ssured soils (including AL, FL, SL and GL soils) C/N were ranked U1 < U2 < U3 < U4,and C/ N ratio of all ssure soils in the U1, U2, U3and U4 layers ranged from27.44 to 42.48, 31.87 to 37.98, 25.6 to 41.89, and 21.52 to 42.88, respectively (Fig. 4a.). C/ N ratio of ssure soils, including those at depths of U1, U2, U3 and U4, decreased in the order AL > GL > FL > SL with AL having the lowest (at all soil levels) C/N ratio. The C/N for U4 in FL was 42.88, which was signi cantly higher than the ssured soil layer of other vegetation types (P < 0.05).
ii) C/P ratio C/P ratios for ssure soils (including soils at U1, U2, U3 and U4 depths) are reduced in the order SL > FL > GL > AL (Fig. 4b.). The C/P ratio of ssure soils (including AL, FL, SL and GL soils) is reduced in the order U2 < U1 < U4 < U3,and C/P ratio of all ssure soils in the U1, U2, U3and U4 layers ranged from110.21 to 202.24, 83.38 to 219.57, 137.61 to 219.86, and 133.14 to 183.67, respectively. Except for the C/P ratio of AL, which increased with soil depth, none of the C/P ratios changed signi cantly for each soil layer under the ssures of the three vegetation types. The C/P of U1 and U2 of AL were signi cantly lower (P < 0.05) than that of the other vegetation types of ssured soil layers.
iii) N/P ratio The N/P ratios of ssure soils (including AL, FL, SL, and GL soils) decreased by U1 < U2 < U3 < U4 and were 3.48-5.64, 2.38-6.51, 4.00-6.82, and 3.10-8.02 for U1, U2, U3, and U4, respectively (Fig. 4c.). The N/P for U4 in FL was 3.10, which was signi cantly lower (P < 0.05) than the other vegetation types of ssured soil layers. The N/P ratios of fractured soils (including those at depths U1, U2, U3, and U4) increased in the order of AL < GL < FL < SL, with SL and FL N/P ratios higher at U2 and U3, and GL and AL N/P ratios highest at U4 and U1, respectively.
Environmental factor effects on soil properties SOC, TN and TP contents were all positively correlated with other soil nutrient contents and most of the chemometric ratios of soil nutrients, moreover, soil SOC contents showed a signi cant positive correlation with TN and AN contents, indicating that soil nitrogen was mainly present in organic matter as organic nitrogen. Additionally, the nitrogen to phosphorus ratio was highly signi cantly negatively correlated (P < 0.01) with TP and carbon to nitrogen ratio, and highly signi cantly positively correlated (P < 0.01) with carbon to phosphorus ratio (table 3).  Discussions Changes in C, N and P in ssured soils

i) Effect of physicochemical properties on nutrients in ssured soils
Soil mechanical composition is a natural property for maintaining soil quality, and a proper proportion of soil mechanical composition facilitates plant root activity and absorption of water and nutrients from the soil. (Su and Zhou et al.,2018).In this study, in the same vegetation type, as the soil layer deepened, the sand content decreased, while the silt content (0.02-0.002mm) and clay content increased (Table 4), which is similar to the results of peng et al (2020),and this indicated that the surface soil tends to accumulate in cracks and that the underlying soil is buried and continuously compacted; at the rock-soil interface, the water chemistry is very strong and the soil often contains dissolved solids and sand-sized fragments of weathered rock, leading to low clay content here (Peng et al., 2020).
Among the different vegetation types, AL-supporting ssured silt (0.02 − 0.002 mm) and clay (< 0.002 mm) had the highest content and the lowest sand content (0.02-2 mm), as the AL was previously slopecultivated land that was abandoned to grow plants such as dog-tailed grass, and the increase in soil meal and clay particles indicated that the vegetation was effective in amending the soil Mechanical composition, and it is further shown that as the clay content increases, the clay provides a larger surface area as a binding site, allowing the enrichment of organic matter in the ne soil particles and thus an increase in C, N or P. (Ge et al., 2019). Additionally, the ssures supporting SL had the highest sand content ( (2019). (Ge et al., 2019) that soil nutrients are mainly concentrated in clays that provide a greater surface area as binding sites, allowing the enrichment of organic matter in ne soil particles. (Ge et al., 2019). Some studies have shown that soil particles enter well-developed karst ssures with rainfallrunoff (Nie et al., 2012). These particles interact with each other; they are ltered, rearranged and eventually piled up (Tang et al., 2016). As a result, karst soils are characterised by a basic 'loose top and tight bottom', where nutrients are concentrated in the sand in the upper part of the ssures, as well as by soil erosion, which can lead to nutrient loss and the loss of farmland on karst slopes. The results of the study suggest that karst near-surface ssures are therefore the main cause of soil nutrient loss (Peng et al., 2017).
Soil water content re ects soil moisture and wetting within the community and can have a direct impact on the exchange of matter and energy between the litter and the soil surface and on the degree of nutrient leaching from the soil. (Kang and Liu et al., 2017). Our result showed that soil water content is mostly positively correlated with nutrients and, within the same vegetation type, the distribution of soil water content gradually decreases from top to bottom, indicating that more of the water required for plant growth comes from the upper part of the ssured soil.
The effect of pH on soil nutrient characteristics is not signi cant, probably due to its small spatial heterogeneity in pH and small differences in soil pH, so that pH is not a major factor in uencing soil C, N and P. The effect of pH on soil C, N and P is not signi cant, probably due to its small spatial heterogeneity in pH and small differences in soil pH.

ii) Changes in C, N and P in ssured soils
In this study, the distribution pattern of carbon, nitrogen and phosphorus content in the ssured soils under different vegetation types shows a certain similarity, i.e. the distribution of the upper layer is high and the lower layer is low, which is linear. These data showed a general decreasing trend in soil SOC content of different vegetation types with increasing soil depth, which is similar to what many authors have found when studying land-use types associated with karst regions. ( Huang and Zhou et al., 2017;Wang and Sheng et al., 2016). In general, apoplastic matter can provide large amounts of organic matter directly to the surface layer, leading to differences in organic matter input between the surface soil and the subsoil (Fröberg et al., 2007),the organic carbon in the surface layer of the ssured soil comes from plant litter and root inputs and is the richest in nutrients (Jackson et al., 1996;Leeand Fitzgerald et al., 2014 ) as the soil layer deepens, the organic matter content decreases, the quality is poorer and the supply of oxygen may be limited (Bai et al., 2015;Schrumpf et al., 2013) and the degradability of soil organic matter components varies under the action of microbial decomposition, with di cult-to-degrade substances gradually increasing and easy-to-degrade substances gradually decreasing (Wang and Sheng et al.,2016), moreover, the addition of the litter increases the respiration rate of soil micro-organisms, which accelerates the decomposition and release of existing SOCs from the soil (Liao and Long et al.,2016), the main reason for the highest SOC content in SL ssured soils may be the rapid rate of vegetation turnover in scrubland, which allows for the migration of ssured soils downwards of apoplastic material etc. to form nutrient accumulations, similar results were obtained by Chen et al, Fu et al and Chen et al, who reported that shrubland was more effective in increasing SOC during vegetation restoration. (Chen et al., 2007;Chen et al., 2012;Fu et al., 2010) and the upward trend in FL in U4 are probably since the plants are rooted in deep ssure soils, where roots and litter replenish the organic matter or the ssure soils have higher water content, lower temperature variability and slower decomposition of the underlying organic matter.
In this study, despite the small annual temperature differences and abundant rainfall in the ssured soils of the karst region, the TN content did not vary much and was not signi cantly different in all vegetation ssures and in all soil layers. The hydrolysed nitrogen content also decreased with increasing soil depth in the same vegetation type, indicating that nitrogen nutrients are more concentrated in the ssured soil surface layer and are less susceptible to leaching in the ssured soil.
TP and AP are mainly concentrated in the ssured soil surface layer (U1), which is characterised by "surface aggregation". However, because soil phosphating is a sedimentary mineral, the migration rate in the soil is very low (Filippelli, 2008),the vertical variation of TP and AP with the deepening of the soil layer is small, which is consistent with the ndings of Peng et al(2016)., Lv et al(2017)., .
Additionally, the surface layer of the ssured soil contains a high concentration of powders and clay particles, which are strongly adsorbed by soil TP and AP (Hassink et al., 1994;John et al., 2005).

Changes in C, N and P stoichiometry ratios in ssured soils
Soil C/N, C/P and N/P ratios are important parameters for measuring soil quality and organic matter composition and reveal the relative availability of soil nutrients (Wang et al.,2008), the changes in C/N and N/P of ssured soils (including AL, FL, SL and GL soils) under different vegetation types in our study were U1 < U2 < U3 < U4 and C/P: U2 < U1 < U4 < U3. In contrast to Qiao et al.'s ndings (Qiao et al., 2020), C/N and N/P decrease progressively as the soil layer deepens, probably due to the different spatial distribution of roots in deep karstic ssures and hence the different utilisation of soil N elements. In the 60-80 cm ssured soils of SL and FL, a large number of plant roots are distributed and take up a large amount of N from the soil, thus making soil C/N and N/P larger than the surface layer.
Additionally, the amount of nitrogen affects the spatial heterogeneity of soil carbon, nitrogen and phosphorus ratios by in uencing the physical and chemical properties of the soil, nutrient levels (e.g. C, N, P) and microbial activity. As can be seen from (Figure xx), the lower the nitrogen amount, the higher the soil C/N and the lower the N/P; the higher the nitrogen amount, the lower the soil C/N and the higher the N/P.
Overall, C/N, C/P and N/P are AL < FL < SL < GL, AL < GL < FL < SL and AL < GL < FL < SL, respectively. C/P, C/N and N/P for AL are minimum and C/P and N/P for SL are maximum. Compared to other types of fractured soils, most of the nutrients in these soils have been absorbed by crops, affecting their C, N and P storage content, so soil C/N, C/P and N/P are lower in AL than in other land-use types. The herbaceous succession under SL, the apoplastic and root succession of the regenerating stands and the biological xation result in a large input of C and N (Gundale et al., 2011;Johnson and Curtis, 2001;Yuan and Chen, 2010), the nutrient accumulation is high, so C/P, N/P is maximum. Zhou et al. (Zhou et al., 2018) also found that the spatial pattern of soil carbon to nitrogen ratios across the pro le was not strongly related to vegetation cover. And C and N are structural components whose accumulation and consumption are relatively constant (Cleveland and Liptzin et al.,2007).
Our results show that the mean soil C: N, C: P and N: P ratios in the study area range from 28.39 to 35.99, 146.05 to 192.15 and 4.74 to 5.76, respectively, and that it has been found that the karst region has the highest C: N (9.22-19.56), C: P (40.36-92.6) and N: P (3.45-98) ratios in the study area. (6.80), both higher than China's C: N (11.9) and C: P (61.0) and global C: N (14.3) and C: P (186.0), and lower than China's N: P (5.2) and global N: P (13.1). High C: N, C:P in the study area means a slow mineralisation rate of these nutrients (Cleveland and Liptzin et al.,2007),Furthermore, the signi cant correlation between soil SOC content and the carbon to nitrogen, carbon to phosphorus ratio (Table 5) also con rms that changes in soil SOC content in the study area are highly constrained by changes in soil SOC content. Soil N: P is signi cantly higher than in the other areas, suggesting that there may be a relative de cit of soil P in the study area or a relative surplus of soil N (Wang and Wang et al.,2018).

Conclusions
The physicochemical properties, nutrient and stoichiometric characteristics of karstic rift soils were evaluated and the results showed that soil pH at the same soil depth varies with the type of vegetation and is basically weakly alkaline. The water content of the soil also decreases as the depth of the soil increases, and the soil in the upper part of the ssure gives more water for plant growth. Under the same vegetation type, the silt content (0.02 − 0.002 mm) and clay content (< 0.002 mm) increased and the sand content (0.02-2 mm) decreased with increasing soil depth, with AL and GL showing better soil structure.
There was little change in TN content from the surface layer of the ssure to the lower layer, and a general decrease in AN, TP and AP content in the ssure. In general, SOC, nitrogen and phosphorus were mainly concentrated in the surface soil compared to the other soil layers of the ssure. SL has the highest SOC, AN and AP content, especially in soil layer U1, which shows high fertility, but low TP content. The C/N, N/P and C/P ratios of ssured soils overall decrease with increasing soil depth. SL has higher C/P and N/P and lowers C/ N, while the opposite is true for AL. The higher C/N, C/P and N/P variations indicate that the karstic rift soils are severely de cient in N and P and therefore some fertiliser, such as N and P, can be applied. Rift soils are one of the key conditions for the implementation of vegetation restoration in karstic stone deserti cation areas. The low nutrient content and high ecological stoichiometry of karstic rift soils indicate that rift soils in karstic areas need more soil nutrient management, improvement of quality conditions. Therefore, based on our results, it is recommended that priority be given to the selection of herbaceous plants to improve the structure of ssured soils and enhance soil nutrients.