Transformation of soil organic phosphorus along the Hailuogou post-glacial chronosequence, southeastern edge of the Tibetan Plateau

Geoderma 352 (2019) 414–421

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Transformation of soil organic phosphorus along the Hailuogou post-glacial chronosequence, southeastern edge of the Tibetan Plateau ⁎

T

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Jun Zhoua,d, , Yanhong Wua, , Benjamin L. Turnerb,d, Hongyang Suna, Jipeng Wangc, Haijian Binga, Ji Luoa, Xiaoli Hea, He Zhua, Qingqing Hea a Key Laboratory of Mountain Surface Processes and Ecological Regulation, Institute of Mountain Hazards and Environment, Chinese Academy of Sciences, Chengdu 610041, China b Smithsonian Tropical Research Institute, Apartado 0843-03092, Balboa, Ancon, Panama c State Key Laboratory of Geohazard Prevention and Geoenvironment Protection, Chengdu University of Technology, Chengdu 610059, China d School of Biological Sciences, The University of Western Australia, Perth, WA 6001, Australia

A R T I C LE I N FO

A B S T R A C T

Handling Editor: Jan Willem Van Groenigen

Organic phosphorus (P) accumulates in soil during pedogenesis, yet information on the composition and transformation of organic P during the early stages of soil development remains scarce. We studied the top 5 cm of mineral soil immediately beneath the organic horizon from six sites (0, 35, 45, 57, 85 and 125 years) along the Hailuogou glacier foreland chronosequence, on the southeastern edge of the Tibetan Plateau. Phosphorus compounds in the soils were determined by NaOH–EDTA extraction and solution phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy. Extractable P was dominated by phosphomonoesters (up to 51.5%) and orthophosphate (37.9–44.6%) throughout the chronosequence. The phosphomonoesters were mainly hydrolysis products of RNA and phospholipids and followed a unimodal pattern with soil age, with maximum concentration at the 57-year-old site. myo-Inositol hexakisphosphate was not detected, although scyllo-inositol hexakisphosphate accounted for 4.7–9.3% of the extracted P and D-chiro- and neo-inositol hexakisphosphates occurred small amounts in a few soils (1.1–3.3% of the extracted P). DNA accounted for 4.0–8.3% of extracted P and increased continuously along the chronosequence, associated with increased inputs of plant and microbial residues and stronger sorption to soil surfaces during the rapid decline in soil pH. Pyrophosphate, an inorganic polyphosphate, occurred in small concentrations (up to 5.0%) that fluctuated with soil age. We conclude that organic P compounds accumulate rapidly in the top mineral soil during the early stages of pedogenesis, predominantly as relatively labile compounds from plant and microbial residues.

Keywords: 31 P NMR Inositol hexakisphosphate Phosphomonoester Phosphodiesters Available phosphorus Microbial biomass phosphorus

1. Introduction According to biogeochemical theory, long-term pedogenesis on stable land surfaces involves a decline in inorganic phosphorus (P) in primary minerals, and an accumulation of organic P and occluded P within secondary minerals (Walker and Syers, 1976). This model has now been verified by observations along soil chronosequences on a variety of parent materials and climate zones (e.g. Crews et al., 1995; Parfitt et al., 2005; Selmants and Hart, 2010; Turner et al., 2012a, 2012b; Chen et al., 2015). The long-term changes in soil P have important ecological consequences, leading to P limitation and a decline in biomass and productivity on old soils (Vitousek and Farrington, 1997; Wardle et al., 2004; Coomes et al., 2013). At the same time, plant

diversity increases as P becomes limiting on old soils (Laliberté et al., 2013, 2014), with a parallel increase in the diversity of plant nutrient acquisition strategies (Zemunik et al., 2015). These findings point to the increasing importance of organic P in the nutrition of plants and microbes during long-term ecosystem development (Turner et al., 2007). The transformation of soil organic P composition with soil age has been investigated by solution phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy along a small number of long-term natural soil chronosequences (e.g. McDowell et al., 2007; Turner et al., 2007, 2014; Vincent et al., 2013). Along the Reefton and Franz Josef chronosequences in New Zealand, concentrations of phosphomonoesters and phosphodiesters increased in young soils and then declined with soil age (McDowell et al., 2007; Turner et al., 2007). In contrast,

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Correspondence to: J. Zhou, Key Laboratory of Mountain Surface Processes and Ecological Regulation, Institute of Mountain Hazards and Environment, Chinese Academy of Sciences, Chengdu 610041, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (J. Zhou), [email protected] (Y. Wu). https://doi.org/10.1016/j.geoderma.2019.05.038 Received 18 November 2018; Received in revised form 22 May 2019; Accepted 24 May 2019 Available online 04 June 2019 0016-7061/ © 2019 Elsevier B.V. All rights reserved.

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2. Materials and methods

along the Västerbotten chronosequence developed on uplift terraces in boreal forest, two forms of organic P in organic soils (deoxyribonucleic acid (DNA) and 2-aminoethyl phosphonic acid) showed a unimodal trend, but inositol hexakisphosphate (IP6) fluctuated with age (Vincent et al., 2013). In a Holocene chronosequence of coastal dunes, concentrations of phosphomonoesters increased during the first 800 years of pedogenesis and then declined, while phosphodiesters in the organic horizon increased as a proportion of the extracted P with age (Turner et al., 2014). In addition, two studies found a continual increase in organic P with pedogenesis: the concentrations of all forms of organic P and their contributions to the extracted P always increased with soil age along the Manawatu chronosequence (McDowell et al., 2007), while the concentrations of phosphomonoesters and phosphodiesters increased along a natural revegetation chronosequence in Northwestern Russia (Celi et al., 2013). These divergent results indicate that more studies are needed to elucidate the transformation of soil organic P composition with development of soil (George et al., 2018; Huang et al., 2017). The above studies all found that the most rapid changes in soil organic P composition occurred in the early stages of pedogenesis (first few hundred years), involving a marked increase in the concentrations and richness of organic P forms. However, changes in certain forms of organic P with soil age in the initial stages of soil development and the underlying mechanisms remain unknown. For example, inositol phosphates were detected in the youngest soils (< 100 years) along the Manawatu, Franz Josef and Västerbotten chronosequences, but not in soil with an age of 181 years in the Haast chronosequence. In addition, variations in inositol phosphates were associated with simultaneous changes in amorphous metal oxides at Franz Josef (Turner et al., 2007) and Manawatu (McDowell et al., 2007), but not in two relatively young landscapes (Turner et al., 2014; Vincent et al., 2013). Variations in organic P composition have also been related to changes in plant and microbial community composition and soil properties during the early stages of pedogenesis (Celi et al., 2013; Vincent et al., 2013). Further research is required to understand the origins, transformations and drivers of organic P during the early stages of pedogenesis. A soil chronosequence and a vegetation primary succession following the retreat of the Hailuogou glacier in SW China has been forming since ~1890, allowing us to investigate the transformation of organic P during the initial stages of pedogenesis. Previous studies along the 125-year-old chronosequence found high rates of apatite weathering (Zhou et al., 2018), a rapid decrease in inorganic P and accumulation of organic P, fluctuation of exchangeable P with soil age (Zhou et al., 2013) and considerable loss of P from topsoil (Wu et al., 2015). The contribution of organic P to total P in topsoil was > 40% in the oldest two sites where conifers dominate the tree community (Prietzel et al., 2013; Zhou et al., 2013). However, it is unclear how abiotic and biotic factors influence the transformation of organic P along the chronosequence. For example, soil pH decreased markedly from 8.5 at the terminal moraine to 4.8 at the 125-year-old site, while amorphous metals (Al and Fe) and clay concentrations increased over the same period (Zhou et al., 2016). The ratio of fungi to bacteria increased with pedogenesis, which was correlated with the decrease in total P and pH in soil (Jiang et al., 2018). Vegetation succession also occurred rapidly, with five stages ending in the climax of conifers at the 125-year-old site. To elucidate the transformations of organic P compounds and the effects of edaphic and biological factors on the variations during the initial stage of pedogenesis, soil samples were collected from the Hailuogou chronosequence and analyzed by 31P NMR spectroscopy. Our objective was to determine the pattern of organic P composition along the Hailuogou chronosequence and investigate the factors controlling transformations of organic P compounds as pedogenesis proceeds.

2.1. Site description and sampling In July 2015, six sites representing different exposure age (0, 35, 45, 57, 85 and 125 years) were selected to collect soil samples in the Hailuogou glacier retreat area chronosequence (29°34′07.83″ N, 101°59′40.74″ E) at the southeastern edge of the Tibetan Plateau, southwest China (Fig. 1). The age of the six sites was determined from historical records and tree rings (Zhong et al., 1999). The range of elevation of this chronosequence is from 2855 to 2982 m above sea level (a.s.l.) along ~2 km downstream from the mouth of the glacier. The soils are Regosols according to the World Reference Base for Soil Resources (Zhou et al., 2013). Primary vegetation succession along the chronosequence is categorized into five stages: (i) bare land, (ii) Salix rehderiana C.K. Schneid.-Hippophae rhamnoides L.-Populus purdomii Rehder, (iii) Populus purdomii Rehder, (iv) Abies fabri (Mast.) Craib-Picea brachytyla (Franch.) E. Pritz. and (v) Picea brachytyla (Franch.) E. Pritz.Abies fabri (Mast.) Craib (Li and Xiong, 1995) (Table 1). Precipitation and temperature have been recorded by the Gongga Mountain Alpine Ecosystem Observation Station, Chinese Academy of Sciences since 1987 (Fig. 1). Zhou et al. (2018) reported detailed information of the soil profiles at the six stages (Fig. S1). The thickness of organic horizon was 0, 3–6, 5–8, 5–8, 6–12 and 10–14 cm at the 0, 35, 45, 57, 85 and 125-year-old site, respectively. Six profiles with a distance of > 10 m from each other were excavated by hand at each site. The locations of these profiles were on gentle slopes and under canopies of dominant plants (except the 0-yearold site). We collected samples from organic horizons and the upper 5 cm of mineral soil, respectively. The organic horizon represents the litter layer, mainly including the Oe (fermented and shredded litter) and Oa (humifid litter) horizons. Soil samples from the six profiles were mixed well to yield a composite sample for each horizon at each site. Mineral soil samples were sieved (< 2 mm) and air dried before further analysis. 2.2. Soil properties Microbial biomass P (MB-P) was extracted from field-moist samples by the fumigation extraction method (Brookes et al., 1982). The MB-P was the difference of the NaHCO3 extractable inorganic P between fumigated and unfumigated soils. Labile P (NaHCO3-P) was extracted by 0.5 M NaHCO3 (pH = 8.5, 16 h, 25 °C), with total P determined in the extracts by persulfate digestion (121 °C). The concentrations of the MBP and the labile P were determined by the phosphomolybdate blue method (Murphy and Riley, 1962) using a UV–Vis spectrophotometer (Shimadzu UV 2450). Total soil P was quantified by digestion of refluxing with nitric acid, hydrofluoric acid and perchloric acid, with detection by inductive-coupled plasma optical emission spectroscopy (ICP-OES; Optima 8300, PerkinElmer, Inc.). 2.3. Solution

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P NMR

For quantification of organic P composition by solution 31P NMR spectroscopy we focused on the upper mineral soil samples from the 35to 125-year-old sites. We did not include the youngest site because organic P was undetectable (Zhou et al., 2013). Total P in the organic horizon samples were quantified, but we did not perform NMR spectroscopy on these samples. To measure organic P compounds in mineral soil, subsamples (1.00 ± 0.01 g) were shaken in 20 mL of a solution containing 0.25 M NaOH and 50 mM Na2EDTA (disodium ethylenediaminetetraacetate) for 16 h (Cade-Menun and Preston, 1996). Extracts were centrifuged (11,000g, 20 min, 2 °C) and the supernatant was transferred to clear centrifuge tubes, frozen at −80 °C and lyophilized. All lyophilized materials (300–400 mg powder) were redissolved in 0.1 mL D2O and 415

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Fig. 1. The location of sampling sites along the Hailuogou post-glacial chronosequence, southwest China.

2.4. Statistics

0.9 mL of a solution containing 1 M NaOH and 100 mM Na2EDTA, with0.5 mL 100 μg mL−1 methylene diphosphonic acid (MDP; SigmaAldrich, product number M9508-1G) added to each solution as an internal standard. The redissolved solution was vortexed for 2 min, centrifuged at 11,000g for 10 min, transferred to a 5 mm NMR tube and immediately analyzed by solution 31P NMR spectroscopy. The 31P NMR spectra were recorded by a Bruker Avance II-600 MHz spectrometer (Bruker, Swiss) operating at 202.456 MHz. Solutions were measured using a 6 μs pulse (45°), a delay time of 4.0 s, an acquisition time of 0.4 s and broadband proton decoupling. Around 30,000 scans were acquired for each solution. Spectra were plotted with a line broadening of 2 Hz and chemical shifts of signals were determined in parts per million (ppm) relative to an external standard of 85% H3PO4. Main P classes were identified based on chemical shifts reported in the literature (Doolette et al., 2009; Turner et al., 2003; Turner and Richardson, 2004; Turner et al., 2012a, 2012b) using the software MestReNova. The spectra were corrected for MDP at 17.4 ppm. Peaks were first ‘picked’ automatically by the software. Then those peaks that were clearly visible but not identified automatically were picked manually. Peak areas were calculated by deconvolution and integration of individual peaks. Concentrations of P compounds (mg P kg−1 soil) were calculated using the known concentration of the MDP spike and are presented on the basis of oven-dry soil. Replicate P spectra were not acquired, although previous studies suggested that error for solution 31 P NMR spectroscopy of soil extracts and similar samples was approximately 4% for phosphomonoesters and 10% for DNA (Turner, 2008). A recent study also justified the high repeatability of the results measured by the 31P NMR spectroscopy (Jarosch et al., 2015).

The correlations between organic P compounds and soil properties were determined by Pearson's correlation coefficients. All the variables were tested for normality (Shapiro-Wilk) before the correlation analysis was applied. A significance level of P < 0.05 was used in this study (except where noted). 3. Results 3.1. Phosphorus extraction in NaOH–EDTA Concentrations of NaOH-EDTA extractable P (NaOH-EDTA P) in the top 5 cm of mineral soil immediately beneath the organic horizon (top mineral soil) tended to increase from the 35-year-old to the 57-year-old site and then declined at the last two sites, showing a similar temporal trend with the total P (Table 2). Recovery of NaOH-EDTA P varied between 25 and 46% of the total P, with a mean recovery of 36.8 ± 7.2% (Table 2). The NaOH-EDTA P consisted of 51–57% organic P and 43–49% inorganic P (Table 2). In addition, the P not extracted by NaOH-EDTA, accounted for the majority (54–75%) of total P in the top mineral soil along the Hailuogou chronosequence (Table 2). 3.2. Phosphorus determination by solution

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P NMR spectroscopy

A variety of inorganic and organic P compounds were detected by solution 31P NMR spectra of NaOH-EDTA extracts in the Hailuogou chronosequence (Fig. 2). The inorganic compounds included orthophosphate (Ortho-P) and pyrophosphate (Pyro-P). Except for the

Table 1 Selected site information and chemical-physical properties in the top 5 cm of mineral soil along the Hailuogou chronosequence, SW China. Soil age

pHa

Feoxa

SOCa

TNb

(g kg−1)

(years) 0 35 45 57 85 125

Aloxa

8.5 5.6 6.4 5.5 4.4 4.8

0.4 1.5 2.2 3.4 3.5 4.5

NaHCO3-P

Microbial P

C:Nc

C:Pc

N:Pc

(t ha−1)

(P kg-1) 1.5 5.6 5.7 7.9 7.3 8.0

1.9 75 61 65 139 178

4.5 4.2 4.8 8.7 10.0

0.3 23.9 26.0 37.0 29.1 27.0

12.0 26.3 37.8 38.9 46.8

19.4 16.9 15.8 18.6 20.8

4 252 167 161 433 605

12.9 9.8 10.2 23.2 29.2

- not detected. The detection limit of TN is 0.018 g kg−1. a Data are taken from Zhou et al. (2016). Alox and Feox: concentration of Al and Fe extracted by NH4-oxalate solution. SOC: soil organic carbon. b Data are taken from Zhou et al. (2018). TN: total nitrogen. c Molar ratios. The concentration of total P is shown in Table 2. d Data are taken from Luo et al. (2004). 416

Biomassd

48.9 110.8 184.7 308.0 382.3

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Table 2 Concentration of total phosphorus in the organic horizon and concentration of phosphorus fractions in the top 5 cm of mineral soil along the Hailuogou chronosequence, SW China. Soil age

Total Pa O horizon

Mineral soil

NaOHEDTA Pb

NaOHEDTA Poc

NaOHEDTA Pid

Unextracted Pe

– 265 (34) 233 (25) 422 (40) 387 (46) 296 (39)

– 152 (20) 131 (14) 236 (22) 207 (25) 152 (20)

– 113 (14) 102 (11) 186 (18) 180 (21) 144 (19)

– 509 (66)f

−1

(Years)

mg kg

0 35

– 941

1363 774

45

910

949

57

1279

1053

85

1048

838

125

946

765

716 (75) 631 (60) 451 (54) 469 (61)

a Determined by ICP-OES detection after nitric acid, hydrofluoric acid and perchloric acid digestion. b Determined by solution 31P NMR using an internal standard (see Materials and methods). c Sum of NaOH-EDTA extractable orthophosphate monoesters, diesters, and phosphonates (see Fig. 4). d Sum of NaOH-EDTA extractable inorganic orthophosphate, pyrophosphate, and polyphosphate (see Fig. 4). e Difference between total P and NaOH-EDTA P. f Values in parentheses are concentrations expressed as a proportion (%) of total soil P.

internal standards MDP at 17.40 ppm, the organic phosphates consisted of phosphomonoesters (Mono-P), DNA, phospholipids and phosphonates. Of the inorganic P compounds, Ortho-P and Pyro-P occurred at δ = 6.10 ± < 0.01 ppm (mean ± standard deviation of five spectra) and δ = −4.22 ± 0.01 ppm, respectively (Fig. 2). Of the organic P compounds, a complex group of signals between 3.8 and 6.8 ppm were identified as Mono-P (excluding the Ortho-P signal at 6.10 ppm). A broad signal at δ = −0.34 ± 0.03 ppm was identified as DNA. Small signals between δ = 0.5 and 1.0 ppm were assigned to phospholipids not degraded in the alkaline extraction. In addition, phosphonates gave small signals at δ = 20.54 ± < 0.01 ppm in three sites (Fig. 2). Within the Mono-P pool, myo-IP6 was not detected, while D-chiroand neo-IP6 were identified at δ = 6.46 ± 0.01 and 6.70 ± 0.01 ppm, respectively (Fig. 3). The prominent signal at δ = 3.98 ± < 0.01 ppm was assigned to scyllo-IP6. The major signals between 3.8 and 6.0 ppm in the Mono-P region were assigned to products from hydrolyzed phospholipids and ribonucleic acid (RNA), including prominent signals from α- and β-glycerophosphate at δ = 5.11 ± 0.01 and 4.77 ± 0.01 ppm, respectively (Fig. 3).

Fig. 2. Solution 31P NMR spectra of NaOH-EDTA extracts of soils from the Hailuogou post-glacial chronosequence, southwest China. The signals were assigned as follows: Pn: phosphonate (δ = 20.54 ppm), MDP: methylene diphosphonic acid (δ = 17.40 ppm), internal standard, Or: orthophosphate (δ = 6.10 ppm), M: phosphomonoester (δ = 3.8–6.8 ppm, excluding the orthophosphate signal at 6.10 ppm), Pyr: pyrophosphate (δ = −4.22 ppm), DNA: δ = −0.34 ± 0.03 ppm and Pld: phospholipid (δ = 0.5–1.0 ppm).

percentages of the P extracted by NaOH-EDTA solution across the Hailuogou chronosequence (Table 3). The Mono-P was the largest proportion of the NaOH-EDTA P in the first four sites, while the Ortho-P became the largest proportion at the oldest site. The proportion of the Mono-P decreased with soil age, while the proportion of the Ortho-P increased. The largest proportion (55.5–59.2%) of the Mono-P was αand β-glycerophosphate (i.e. degradation products of phospholipids formed during the alkaline extraction and analysis). DNA contributed the third largest proportion of NaOH-EDTA P (7.1–16.1%) in all sites except the 35-year-old site and showed an increasing temporary trend. The proportions of Pyro-P, phospholipid and phosphonate did not display an obvious trend with soil age (Table 3).

3.3. Changes in P composition along the Hailuogou chronosequence The concentration of the Ortho-P increased from 100.6 mg kg−1 at the 35-year-old site to its maximum (168.9 mg kg−1) at the 57-year-old site and then decreased to 131.8 mg kg−1 at the oldest site (Fig. 4). The concentration of the Pyro-P fluctuated between 6.9 and 16.8 mg kg−1 across the five sites. The concentration of the Mono-P showed a similar parabolic trend to the Ortho-P with the maximum of 216.0 mg kg−1 at the 57-year-old site. In contrast, the concentration of DNA increased continuously soil age. At the 125-year-old site, DNA was almost two times of that at the 35-year-old site. Both phospholipids and phosphonates did not show any temporal trend because of their low concentration and absence in some sites. Indeed, phospholipids were only detected at the 45- and 85-year-old sites, where phosphonates were not detected (Fig. 4). The Mono-P and the Ortho-P accounted for the two largest

3.4. Relationship between P compounds and soil properties We found no significant correlations between soil properties and total inorganic P in NaOH-EDTA extraction (Table 4). Total organic P and Mono-P concentrations were strongly positively correlated with labile P (NaHCO3-P) and total P in the organic horizons. In addition, phosphodiesters were strongly negatively correlated with soil pH, and positively correlated with oxalate extractable Fe, soil organic C, total N, microbial biomass P and plant biomass (Table 4).

417

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Hailuogou chronosequence (Table 2) is a result of the large proportion of phosphate in primary minerals (Zhou et al., 2018), which is poorly soluble in NaOH-EDTA – an extraction procedure designed to recover soil organic P (Bowman and Moir, 1993; Turner et al., 2005). Low extraction efficiency was found for young soil in several previous studies (e.g. McDowell et al., 2007; Turner et al., 2014). The high contribution of Ortho-P to the NaOH-EDTA P (Table 3) further reflects the nature of the young soil along the Hailuogou chronosequence where the weathering rate of primary mineral phosphates still remains high (Zhou et al., 2018). The concentrations of P forms presented in Fig. 4 might be related to different P extraction efficiency along the sequence. However, the similar increasing trend between extraction efficiency (Table 2) and concentrations of organic P in topsoil with age (Zhou et al., 2013) in the Hailuogou chronosequence indicates that the extraction efficiency is controlled mainly by the content of organic P in the soils. In addition, although the extraction efficiency for the oldest two sites was comparable to that for the 57-year-old site (Table 2), the concentrations of the two major P forms (Mono-P and Ortho-P) at the oldest two sites were lower than those at the 57-year-old site (Fig. 4). This also indicates that the extraction efficiency is unlikely a significant factor influencing the concentrations of P forms. 4.2. Phosphomonoesters 4.2.1. Composition of phosphomonoesters The NaOH-EDTA extraction procedure can hydrolyze phospholipids and RNA to Mono-P, such as nucleotides and α- and β-glycerophosphate or mononucleotides of RNA (Makarov et al., 2002; Turner et al., 2003; Doolette et al., 2009; Smernik et al., 2015). Our finding that 55.5–59.2% of the Mono-P was α- and β-glycerophosphate indicates that most of the Mono-P was derived from phospholipids and RNA along the chronosequence. By using two dimensional 1H-31P NMR spectroscopy, Vincent et al. (2013) found that most of the non-inositol Mono-P was RNA and phospholipid degradation products in a marine terrace chronosequence in Sweden. They further quantified that the contents of RNA- and phospholipid-breakdown products represented 40 ± 10% of NaOH-EDTA extractable organic P. In our study, the strong positive correlation between the labile P and Mono-P (Table 4) suggests a close relationship between Mono-P and microbial biomass in the Hailuogou chronosequence, given that Makarov et al. (2002) pointed out that phospholipids extracted with NaHCO3 probably originated from soil microbial biomass. Therefore, it is reasonable to categorize the majority of the Mono-P as the hydrolysis products of the diesters phospholipids and RNA along the Hailuogou chronosequence (Fig. 4). This can also explain the absence of phospholipids in some sites (Table 3). Phospholipids differ in the extent to which they are degraded in alkaline solution, so the phospholipids we detected were likely to be the more resistant phosphatidyl ethanolamine or phosphatidyl serine, rather than the readily degraded phosphatidyl choline (Turner et al., 2003).

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Fig. 3. Major signals shown by the solution P NMR spectra of NaOH-EDTA extracts of soils from the Hailuogoneou post-glacial chronosequence, southwest China. The signals were assigned as follows: neo: neo-inositol hexakisphosphate (δ = 6.70 ppm), D-c: D-chiro-inositol hexakisphosphate (δ = 6.46 ppm), Or: orthophosphate (δ = 6.10 ppm), α and β: α- and β-glycerophosphate (δ = 5.11 and 4.77 ppm), and S: scyllo-inositol hexakisphosphate.

4.2.2. Variations in phosphomonoesters and factors during the initial stage of pedogenesis The parabolic trend of the Mono-P in the top mineral soil during the ~125 years pedogenesis along the Hailuogou chronosequence (Fig. 4) differs from the increasing trend of Mono-P during the early stages of pedogenesis reported by previous studies (Celi et al., 2013; McDowell et al., 2007; Turner et al., 2007, 2014; Vincent et al., 2013). In contrast, this parabolic trend is similar to the trend occurred in older chronosequences where soils have developed for thousands of years (e.g. Turner et al., 2007, 2014). In these previous studies, this parabolic pattern of Mono-P abundance with soil age is only observed on older natural chronosequences but not younger ones. Several mechanisms are likely responsible for the parabolic pattern of Mono-P with soil age along the Hailuogou chronosequence. First, as

Fig. 4. Variations in the concentrations of soil organic P compounds with age along the Hailuogou post-glacial chronosequene, southwest China. Ortho-P: orthophosphate, Pyro-P: pyrophosphate, Mono-P: phosphomonoesters.

4. Discussion 4.1. Recovery of NaOH-EDTA P The low recovery of total P in the mineral topsoil along the 418

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Table 3 Proportions (%) of phosphorus compounds determined by NaOH–EDTA extraction and solution Hailuogou glacier foreland chronosequence, SW China. Soil age (Years)

Inorganic P Ortho-phosphate

31

P NMR spectroscopy in the top mineral soil (0–5 cm) along the

Organic P Pyro-phosphate

Phosphomonoesters Sum

α-a

Phosphodiesters β-a

scyllo-

neo-

D-chiro

Other

DNA

Phospholipids

17.0 16.8 16.0 11.5 8.9

9.3 6.6 6.2 4.7 4.7

1.1 0.7 2.0 1.1 0.7

N.D. 2.3 1.2 1.0 1.1

12.5 11.7 11.5 13.0 11.7

4.7 4.0 4.3 4.7 8.3

N.D. 1.0 N.D 1.1 N.D.

Phosphonate

(% of extracted P) 35 45 57 85 125

37.9 41.0 40.0 44.3 44.6

5.1 3.0 4.0 2.1 4.1

51.5 51.0 51.2 47.8 42.6

11.5 12.9 14.3 16.6 15.5

0.8 N.D. 0.5 N.D. 0.4

Values are the proportion (%) of the NaOH-EDTA extractable total phosphorus. N.D. not detected. a α- and β-glycerophosphates are presumably degradation products of phospholipids formed during the alkaline extraction and analysis.

trees at the 85- and 125-year-old sites. This may contribute the decrease in Mono-P at the last two sites (Fig. 4, Table 3). Although the deciduous broadleaf forest produced a comparable amount of litter (2.1 kg ha−1 year−1) to the coniferous forest (2.2 kg ha−1 year−1), the N and P contents of the litter in the broadleaf forest at the 57-year-old site were higher than those in the coniferous forest (Wang, 2016). The decomposition rate of the poor-quality conifer litter was slower than that of the high-quality broadleaf litter (Gholz et al., 2000). Consequently, less organic P was returned to the mineral soil from the organic horizons at the coniferous forest compared with the deciduous forest. The change in vegetation type might therefore influence the pattern of Mono-P in the top mineral soil via the P content and quality of the litter.

Table 4 Pearson's correlation coefficients between soil properties and the concentrations of phosphorus compounds measured by solution 31P NMR spectroscopy along the Hailuogou chronosequence.

pH Alox Feox SOC Total N C:N ratio C:P ratio N:P ratio Labile P MB-P Biomass TPMS TPOH

NaOHEDTA Pi

Orthophosphate

Pyrophosphate

NaOHEDTA Po

Phosphomonoesters

Phosphodiesters

−0.68 0.62 0.82 0.29 0.40 −0.23 0.20 0.28 0.82 0.65 0.56 0.31 0.82

−0.70 0.64 0.81 0.32 0.44 −0.22 0.23 0.31 0.78 0.67 0.58 0.30 0.78

−0.06 −0.02 0.41 −0.17 −0.18 −0.15 −0.16 −0.18 0.57 0.03 −0.09 0.25 0.67

−0.44 0.37 0.64 −0.04 0.07 −0.47 −0.13 −0.06 0.89⁎ 0.40 0.24 0.51 0.94⁎

−0.35 0.28 0.55 −0.16 −0.04 −0.55 −0.24 −0.17 0.89⁎ 0.31 0.13 0.57 0.95⁎

−0.89⁎ 0.84 0.88⁎ 0.89⁎ 0.93⁎ 0.45 0.84 0.87 0.30 0.88⁎ 0.97⁎⁎ −0.29 0.23

4.2.3. Inositol phosphates myo-IP6 is abundant in nature, originating mainly from plant seeds (Raboy, 2003, 2007). In contrast, scyllo-IP6, neo-IP6, and D-chiro-IP6 are considered to be derived from soil microbial action (Turner, 2007). Thus, the absence of myo-IP6 but presence of other isomers, indicates low inputs of seeds/pollen (myo-IP6) compared to microbial synthesis (scyllo-, D-chiro- and neo-IP6). The abundance of scyllo-IP6 other than Dchiro-, neo- and myo-IP6 might also reflects the greatest resistance of the scyllo-IP6 to enzymatic hydrolysis among the four isomers (Cosgrove, 1970). The absence of myo-IP6 in young, fertile, or wetland soils has also been reported in several previous studies (Celi et al., 2013; Turner and Newman, 2005; Turner et al., 2014). Although the rapid development of vegetation in the young Hailuogou glacier retreat area produced plenty of litter, the slow decomposition rate suggests means that only small amounts of myo-IP6 can enter the mineral soils on an annual basis in the cool wet environment (Fig. 1). Amorphous metal oxides, clays, association with organic matter or precipitation with cations, could protect IP6 from biological attacks and thus facilitate the accumulation of IP6 in soils (Jørgensen et al., 2015; Yan et al., 2014). However, insufficient stabilization potential seems unlikely to explain the absence of myo-IP6, because although amorphous Al and Fe concentrations are low along the chronosequence (Table 1), this would affect all the stereoisomers equally. The slow turnover of plant-derived inputs therefore appears the most likely explanation for the undetectable concentrations of myo-IP6 along the chronosequence.

NaOH-EDTA Pi and Po: inorganic and organic P extracted by NaOH-EDTA solution, respectively. Alox and Feox: concentration of Al and Fe extracted by NH4-oxalate solution, respectively. SOC: soil organic carbon. Labile P: P extracted by NaHCO3-P solution. MB-P: microbial biomass P. Biomass: plant biomass. TPMS and TPOH: the concentration of total P in the top 5 cm of mineral soil and organic horizons, respectively. ⁎ Correlation is significant at the 0.05 level (2-tailed). ⁎⁎ Correlation is significant at the 0.01 level (2-tailed).

discussed in Section 4.2.1, most of Mono-P was the hydrolysis products from RNA and phospholipids. Because free RNA is highly unstable (Ehretsmann et al., 1992), the precursor RNA must be from live cells. And phospholipids in soil are also tightly connected to the live soil microbial biomass (Makarov et al., 2002; Turner et al., 2007). Moreover, at the 1000-year-old site of the Franz Josef chronosequence, New Zealand, the microbial biomass P accounted for 71.8% of the total biomass phosphorus (i.e. plant plus microbial), indicating the major contribution of microbial biomass to soil organic P. Thus, much of the observed Mono-P was presumably derived from microorganisms (Turner et al., 2013). Despite this, microbial biomass P did not display a parabolic pattern (Table 1), but gradually increased along the chronosequence. This indicates that other factors must influence the parabolic trend in monoesters along the chronosequence. There is a change in vegetation types along the chronosequence, from the deciduous broadleaf trees at the 57-year-old site to coniferous

4.3. Phosphodiesters The increasing trend in DNA along the Hailuogou chronoseuqence (Fig. 4) corresponds with those observed in the initial stages of older chronosequences in New Zealand and Sweden (McDowell et al., 2007; Turner et al., 2007, 2014; Vincent et al., 2013). The increase in DNA is related to the increase in microbial biomass and to the rapid succession from broadleaf to conifer forest (Table 1), according to the positive 419

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relationships between DNA and the plant biomass and microbial biomass P (Table 4). Vincent et al. (2013) attributed the increase in phosphodiesters in the early stages of a chronosequence in Sweden to the transition from Alnus- to Picea-dominated vegetation. In addition, an increase in phospholipid-derived fatty acid (PLFA) concentrations of fungi by ten times from the 35- to the 125-year-old site (Zhou et al., 2018) further indicates that the increase in microbial biomass is an important source of the accumulated DNA in soil. Because much of phospholipids was degraded during the alkaline extraction, it is hard to link the increase in PLFA to the pattern of the more resistant phospholipids we detected. Aside from its source, the stabilization condition of soil also regulates the changes in DNA. First, the acidic soil environment at the 85and 125-years-old sites dominated by conifers is beneficial to the accumulation of DNA, as shown by the observed negative correlation between the soil pH and DNA (Table 4). In particular, DNA becomes stabilized in soils below its isoelectric point around pH 5 (Condron et al., 2005). A greater proportion of DNA in acidic soils was reported in tropical forests (Turner and Engelbrecht, 2011) and temperate arable soils (Turner and Blackwell, 2013). In addition, the strong positive correlation between DNA and the soil organic C, total N and amorphous Fe further suggests that DNA is stabilized by the increasing soil organic matter or organic-mineral complexes, similar to the previous results from field observation and laboratory incubation experiments (Makarov et al., 2002; Turner et al., 2007).

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