Phytolith Content In Larix Gmelinii Forest Soils And Its Relations With Soil Properties

Phytolith occluded carbon (PhytOC) has become one of the important mechanisms of long-term carbon sequestration in soil. Ecosystems with higher latitude in the northern hemisphere are expected to face the largest loss of soil carbon due to global warming. The contributions of phytoliths storage in soil layers of cold temperate forest need to be studied in depth. Methods We examined soil phytolith contents and other soil physicochemical properties of Larix gmelinii forest soils in Daxing’anling. ANOVA was employed to analyze differences in the quantitative characteristics of phytoliths. Bivariate correlation, regression analysis, principal component analysis and redundancy analysis were employed to analyze the relations between soil phytolith and soil properties.


Introduction
In recent decades, greenhouse gases (GHGs) mediated global warming and climate change have become one of the major global environmental issues (Prajapati 2016; Rajendiran et al. 2012). Terrestrial carbon sequestration is fundamental to global carbon cycle and being utilized to cope with CO 2 increases  Yang et al. 2018). Forest soil has the largest carbon pool in all terrestrial ecosystems (Jin et al. 2000), which is 2 ~ 3 times of terrestrial vegetation carbon pool. A great impact will be observed on atmospheric CO 2 concentration, which then potentially be fed back on climate change, even small changes occur in forest soil carbon pool (Heimann and Reichstein 2008;Kirschbaum 2004). Therefore, characteristics of soil carbon pool are an important prerequisite for understanding the mechanism of carbon cycles in terrestrial ecosystems (Qiao et al. 2014).
Considering global warming, it is urgent to nd a long-term carbon sequestration way to reduce atmospheric CO 2 concentration. Terrestrial biogeochemical carbon sequestration is estimated to counteract about 30% of the total anthropogenic CO 2 emissions (Law and Harmon 2011) and thus believed to be a promising way (Parr et al. 2010;Song et al. 2012a). Phytolith, also known as plant opal, is a hydrated silica (SiO 2 ·nH 2 O) mineral (Parr and Sullivan 2011;Piperno 1988), which is the monosilicic acid (H 4 SiO 4 ) absorbed by plants from soil solution (Epstein 1994;Hodson 2016;Wilding 1967), and precipitates in the form of amorphous silicon inside cells and cell walls of plant bodies through transpiration (Ma 2003;Neumann 2003; Wang and Lv 1993). Phytoliths are formed in plants, and the content of phytoliths in soil depends on the amount of biological litter, which is the main way for soil to accumulate phytoliths (Han et al. 2018). However, the distribution of phytoliths in different regions is different due to the differences of vegetation, hydrology, climate, geology and other environmental conditions. Due to the fact that phytoliths are abundant, durable and distinctive, it is used to deduce historic vegetation patterns and human uses across the elds of archeology, paleoethnobotany, During the formation process of phytoliths in plants, approximately 0.1% ~ 6% organic carbon could be occluded within phytoliths to form phytolith-occluded carbon (PhytOC) (Jones and Milne 1963; Parr and Sullivan 2011; Zuo and Lü 2011). When plants die or fall, PhytOC will accumulate in soil or sediment (Bartoli 1983). Due to the strong resistance of phytoliths to decomposition, PhytOC is more stable than other fractions of organic carbon in soil and even preserved for thousands of years ( (Parr and Sullivan 2005), which has become one of the important mechanisms of long-term carbon sequestration in soil ). This long-term form of biogeochemical carbon sequestration is believed to have great potential in reducing atmospheric CO 2 content and mitigating greenhouse effects (Piperno 2002;Wu and Wang 2009). However, Santos and Alexandre (2017) advances that PhytOC in phytolith cavities would participate only to a limited extent in long-term atmospheric CO 2 sequestration, and phytOC uxes must be quanti ed to accurately estimate the ux of atmospheric CO 2 sequestered by soil phytoliths. As the PhytOC should be rapidly oxidized when phytoliths start to dissolve and cavities become open, after phytolith deposition in litter, soil or sediment. Therefore, the potential of carbon sequestration in phytoliths has become a hot topic in biogeochemical carbon sequestration research (Li et al. 2013b;Parr and Sullivan 2011;Parr et al. 2010;Song et al. 2012a).
The content of phytolith closely relates to the accumulated silicon in plants. In general, silicon content is the highest in gramineae, followed by conifer and deciduous broadleaf. Therefore, for PhytOC sequestration, previous work mainly focused on bamboo (Parr et al. 2010) and crops, i.e. sugarcane (Parr et al. 2009), millet (Zuo and Lü 2011), wheat (Parr and Sullivan 2011) and rice (Li et al. 2013b), in which their reported PhytOC production uxes ranged from 0.04 to 0.71 t CO 2 ha − 1 yr − 1 . While few studies focus on the production uxes, distributions and storage processes of phytoliths and PhytOC in forest ecosystems, despite great advances in estimating production uxes of PhytOC for some terrestrial ecosystems (e.g. grasslands, wetlands, and croplands) ( Daxing'anling, located in Northeast China, is the largest primary forest area with the only cold temperate coniferous forest in China, which is also one of the most sensitive regions in response to global climate change. Where Larix gmelinii is the dominant tree species with Betula platyphylla as the associated species. Accordingly, the forest ecosystem of Daxing'anling plays an irreplaceable role in cold temperate forest carbon sequestration. We hypothesized phytolith distribution to be controlled by vegetation and to be in uenced by environmental conditions. To test our hypothesis, we used samples from Larix gmelinii soils, (i) to examine the phytoliths content in Larix gmelinii forest soil. (ii) to observe the morphotypes of phytoliths in Larix gmelinii forest soil; (iii) to discuss the effects of soil physicochemical indexes on phytoliths preservation in the cold-temperate forest soil, such as pH and nutrients. This work will offer a scienti c reference for the global long-term carbon sequestration practices such as afforestation and management.

Study area
The study area covers an area of 11,000 hm 2 with a forest coverage rate of 75%, which is located in Chaocha Forest Farm of Genhe Forestry Bureau, Inner Mongolia Autonomous Region, China (50°49'-50°51'N, 121°30'-121°31'E). The altitude varies from 810 m to 1100 m in this area. It belongs to the semi-humid climate zone in the cold temperate zone, with a freezing period of 6 ~ 7 months. Most of the rainfall is concentrated in July and August with annual precipitation of 438 ~ 530 mm. The main soil type in this area is brown coniferous forest soil with large areas of permafrost distributed. There is also a lot of gravel in soil below 30 cm. Larix gmelinii is the dominant tree species. The main understory plants are Rhododendron simsii, Ledum palustre, Betula fruticose, Vaccinium vitis-idaea, Pyrola incarnata, Maianthemum bifolium, Deyeuxia langsdor i.

Soil sampling
Field investigation and soil sampling were conducted during July and August of 2017. Based on the typical plot survey method, 28 sampling sites were selected within in the area of 5km×5km. Soil samples were collected from the pro les of 0-10, 10-20, 20-40 and 40-60 cm depth layer at each site. About 1 kg of soil was collected in each layer. To minimize sampling errors due to soil heterogeneity, three samples in the same layer were mixed as a mixture soil sample for further analysis. The soil samples were air-dried after being taken to the laboratory, then passed through a 2-mm sieve for soil properties determination and passed through a 0.15-mm sieve for phytolith extraction.

Soil properties determination
Soil water content (SWC) and bulk density (BD) were determined by the oven drying method. Soil pH was determined using a pH meter with a soil/water mass ratio of 1∶5 (w/v). Soil organic matter content was determined by potassium dichromate oxidation spectrophotometry method (HJ 615-2011); total phosphorus (TP) was determined by acid dissolution spectrophotometry method (Pierzynski 2009); ammonium nitrogen (NH 4 + -N), available potassium (AK) and available phosphorus (AP) were determined by combined extraction colorimetry method (NY/T 1849-2010); Mineral element contents in soils were determined by XRF analyzers (BRUKER S8 TIGER SERIES 2, Germany).

Phytolith extraction and determination
The air-dried soil (< 0.15 mm) was used to extract phytolith. The modi ed wet air oxidation method and heavy liquid otation method were used for separation and extraction of phytolith from soil (Li et al. respectively. The phytolith translocation increases from surface humic horizon to lower layers of soil pro le as T r increases. The larger the T r value is, the weaker the soil preservation is ( ANOVA was employed to analyze differences in the quantitative characteristics of phytoliths. Bivariate correlation and regression analysis were used to analyze the relations between soil phytolith and soil properties for Larix gmelinii forest. Principal component analysis (PCA) was used for characterization of soil samples according to their properties and phytolith contents. Partial redundancy analysis (Partial RDA) can distinguish the individual interpretation rate and its interactions of variables, which was employed to analyze contributions of grouped environmental variables to phytolith contents and was expressed by Venn diagram. Correlations between soil phytolith and selected soil properties were also examined by multiple regressions.

Descriptive statistics of soil properties
Descriptive statistics of selected soil properties were summarized in Table 1  For mineral elements (Table 2), there was a signi cant difference in Fe between 0-10 cm and 20-40 cm soil layers (p < 0.05). Other mineral elements (Na, Mg, Al, K, Ca) showed signi cant differences between 0-10 cm soil layer and other layers (p < 0.01).  (Fig. 1).

Vertical characteristics
Phytolith contents showed a broad variation in Larix gmelinii forest soil (Fig. 2).

Relations between soil phytolith and basic physicochemical indexes and nutrients
The PCA indicated relations between soil phytolith and basic physicochemical indexes, nutrients (Fig. 3). The rst principal component (PC1) explained by SOC and BD, showed relatively high loadings (32.0%) of phytolith. The second principal component (PC2) also showed relatively high loadings (18.3%) and was mainly explained by pH, SWC and AP. The PC1 positively correlated with SOC, phytolith, TP, AP, AK, SWC, TP and NH 4 + -N, and negatively correlated with pH and BD. The PC2 positively correlated with phytolith, BD, TP, AP and AK, and negatively correlated with pH, NH 4 + -N and SWC. According to the variables ordination in PCA diagrams, it appears that phytolith tends to associate to SOC, TP, AK, AP and NH 4 + -N rather than SWC, pH and BD. It is likely that the soil samples have closer relations with pH and BD. There was a negative correlation between phytolith and pH and BD, which suggested that pH and BD increasing can result in a loss of soil phytolith. In contrast, SOC, AP, AK, TP and NH 4 + -N favored the phytolith preservation in soil. At the same time, it can be seen that phytolith had a stronger correlation with phosphorus nutrients than potassium and nitrogen nutrients; and SWC is not the limiting factor of phytolith.

Relations between soil phytolith and mineral elements
Soil phytolith content was signi cantly negatively correlated with Na, Mg, Al and Fe contents (p < 0.05) (Fig. 4). Na and Mg showed a signi cant correlation with phytolith at the 0.001 level. Signi cant correlations between phytolith and Al, Fe was found at the 0.05 level. Whereas no signi cant correlation was found between soil phytolith and K, Ca.

Relations between soil phytolith and soil properties
Soil basic physicochemical properties, nutrients and mineral elements accounted for 75.5% of soil phytolith variations in Larix gmelini forest (Fig. 5). Mineral elements (c + e + f + g) had the greatest in uence on phytoliths (69.6%), and soil nutrients (b + d + e + g) had the least in uence on phytoliths (40.1%). The individual interpretation rate of each grouped soil factor to phytoliths was in the order of nutrients (8.4%) > basic physicochemical properties (5.9%) > mineral elements (1.2%). On the aspects of interaction effect, the interaction of the three factors (g) had the greatest impact on phytolith, which can explain 35.6% variation. The interaction between basic physicochemical properties and mineral elements (f) also showed an important effect on phytoliths with an explanation rate of 28.3%. On the aspects of common in uence, the interpretation rate of basic physicochemical properties and nutrients to phytolith was the highest (74.3%); the interpretation rate of basic physicochemical properties and mineral elements to phytolith was the lowest (67.1%).
(PC-basic physicochemical properties including SWC, BD and the pH; NI-nutrient indexes including AK, AP, TP, NH 4 + -N and SOC; ME-mineral elements including Na, K, Ca, Mg, Fe, Al. a, b, c indicate the individual interpretation rate of PC, NI, ME to phytoliths; d, e, f indicate the interactive interpretation rate of PC and NI, NI and ME, PC and ME to phytoliths; g indicate the interactive interpretation rate of PC, NI and ME to phytoliths.)

Dominant factor screening
Path coe cients were calculated through stepwise regression analysis (Fig. 6). Na, Mg and K were screened from soil indexes. The simple correlation coe cients of Na, Mg and phytolith were − 0.660 and − 0.577 (p < 0.001); the simple correlation coe cient between K and phytolith was 0.314 (p < 0.05), indicating these three variables played an important role on soil phytoliths in Larix gmelinii forest. All three factors had a negative direct effect on phytolith content followed by descending order of Na, Mg and K. Through the analysis of the indirect path coe cient, it was found that K had the greatest indirect in uence on phytolith through Na, and the indirect path coe cient was 0.328.

Discussion
Eight types of phytoliths were recorded in Larix gmelinii forest soils (Fig. 1), which similar to the SEM images of epidermal cell wall phytoliths in Klein and Geis's studies (Klein and Geis 1978). Elongate was the dominant phytolith shape in Larix gmelinii forest soils, while the dendritic-elongate, papillate-elongate phytoliths maybe re ect that isolated phytoliths from Larix gmelinii forest soils not only come from the Larix gmelinii trees but also affected by understory grasses and shrubs.
The carbon sequestration potential of phytoliths not only depends on the encapsulating e ciency of organic matter during the formation of phytoliths (Parr and Sullivan 2011; Parr et al. 2010), but also on the phytolith yield during plant growth ). Accordingly, the signi cant positive correlations between soil phytolith contents and carbon content occluded in phytolith, phytolith carbon content in soil were also found in Larix gmelinii forest (p < 0.01, Fig. 7), indicating that the phytolith content increasing would promote the carbon sequestration capacity of phytolith. Almost all of the silica in larix needles is located in the epidermal and hypodermal cell walls (Sangster et al. 2001). Therefore, the Larix phytoliths all seem to be cell wall types and deposited on a carbohydrate matrix, which will result in potentially high in carbon. However, the speci c in uence mechanism on carbon sequestration of Larix gmelinii remains to be studied further. Combined with previous studies (He 2016;Lin 2015), phytolith storages in soil from different climatic zones of China were calculated (Fig. 8). An obvious latitude trend from south to north in China was found: tropical zone (15.9 t ha − 1 ) < subtropical zone (21.6 t ha − 1 ) < cold temperate zone (41.0 t ha − 1 ). Phytolith contents in soil distributed in different regions are closely related to the regional geographical conditions and vegetation (Wang and Lv 1993). Theoretically, the soil is rapidly desalinated and desiliconized in the high temperature and humidity area (Alexandre et al. 1997). Thus, the biogeochemical stability of soil phytoliths in tropical areas is lower than that in subtropical areas  . As tropical soils are mostly highly weathered, where silicon supply for plants is much lower than in other ecosystem soils. Therefore, our results showed that the phytolith content in soil from Larix gmelinii forest was obviously higher than those in other areas.
However, some recent studies have shown that phytoliths most probably re ect only the minor part of phytogenic silica in plants and soils (

Conclusions
In conclusion, phytolith is an important way for long-term sequestration of soil organic carbon. The phytolith storage in cold temperate zone (41.0 t ha − 1 ) is obviously higher than in tropical and subtropical zones. Soil physicochemical properties have a certain impact on soil phytolith content in Larix gmelinii forest, such as soil organic carbon, pH value, nutrients and mineral elements. However, the acid-base level of soil is more important for phytolith preservation. The effects of hydrothermal conditions on the yield and stability of phytoliths on a large spatial scale need to be studied in future. The phytolith structures and the effect of silicon availability on phytoliths will allow us to fully understand phytolith dynamics.
Declarations Figure 1 Optical microscope images of phytoliths extracted from Larix gmelinii forest soils  Path coe cients of soil phytolith in Larix gmelinii forest