**1. Introduction**

Humus has been known as the major component of soil organic carbon, which can improve soil fertility and soil physical and chemical structure [1–3]. Litter humification is an important pathway for the formation of soil humus and plays an important role in maintaining soil fertility and carbon sequestration in nature ecosystems [4–7]. Soil fauna are not only decomposers during litter humification, but also act as boosters throughout the formation of soil organic matter by producing and transferring humic substances [8–11]. The roles of soil fauna on the process of decomposition have been wildly documented, and the effect of soil fauna on litter decomposition can be elucidated from different functional groups and feeding habits [12–14]. However, their impacts on litter humification have not been well understood. Therefore, there is still a lack of unified cognition on the role of soil fauna in the accumulation of humus during litter humification.

**Citation:** Tan, Y.; Yang, K.; Xu, Z.; Zhang, L.; Li, H.; You, C.; Tan, B. The Contributions of Soil Fauna to the Accumulation of Humic Substances during Litter Humification in Cold Forests. *Forests* **2022**, *13*, 1235. https://doi.org/10.3390/f13081235

Academic Editor: Choonsig Kim

Received: 20 June 2022 Accepted: 2 August 2022 Published: 4 August 2022

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Generally, the litter humification by soil fauna works through nesting, constructing shuttle, foraging fragmentation, and regulating microorganism's structure [15]. Additionally, these feces and dead bodies of soil fauna are the main sources of soil humus [16,17]. Moreover, some species of soil fauna can weaken microbial activities and feed humic substances, displaying negative effects on the accumulation of humic substances [18]. Including other factors, microclimate and litter quality are widely considered as vital constraints regulating the relationship between soil fauna and litter humification [19–21]. Humic substances are more sensitive and unstable in cold forests than those are in subtropic/tropical forests, while stable temperature and humidity are more likely to promote the formation of humic substances [22–24]. The feeding of soil fauna may be limited under low temperature conditions, thereby affecting the formation of humic substances [25]. Nevertheless, litter quality and its interaction with living conditions of soil fauna could control the humus accumulation by regulating the community structure of soil fauna [22], leading to currently inconsistent online information.

The cold forest on the eastern edge of the Qinghai–Tibet Plateau is an important part of the ecological barrier in the upper reaches of the Yangtze River [26]. The forests have served as prominent strategic positions in regional climate regulation, biodiversity conservation, water conservation, and river runoff regulation [27,28]. However, the cold forests are also fragile and irreversible, since they could be affected by geology and long-term snow cover and seasonal freeze–thaw cycles [29–31]. As an important indicator of soil fertility, soil humus is one of the essential factors in keeping forest soil productivity [32,33]. However, the processes of litter humification and its relationships with soil fauna have not been well addressed in the cold regions. Therefore, a field incubation has carried out to investigate the contributions of soil fauna in the accumulation of humus for the foliar litter of four dominated tree species. The following hypothesis is proposed: (1) soil fauna could promote the process of humidification depending on species and key times in this region; (2) the individual density of soil fauna is related to the degree of humification of leaf litter.

#### **2. Materials and Methods**

## *2.1. Site Description*

The study site was conducted at the Long-term Research Station of Alpine Forest Ecosystem of Sichuan Agricultural University (31◦14–31◦19 N, 102◦53–102◦57 E, 2458–4619 m a.s.l), which is located in the eastern Tibet Plateau [34,35]. The average annual temperature is 2 ◦C, and the average annual rainfall in this site is 850 mm. The seasonal freeze–thaw period is from November to April and lasts for 5 to 6 months each year. The experimental sites are located at two elevations, where the higher elevation (3593 m) is a primary natural forest that is dominated with Willow (*Salix paraplesia* Schneid) and Cypress (*Juniperus saltuaria* (Rehd. et Wils) Cheng et W. T. Wang), whereas the other site (3028 m) is a plantation that dominated with Birch (*Betula albosinensis* Burkill) and Fir (*Abies fargesii* var. *faxoniana* (Rehder & E. H. Wilson) Tang S. Liu) trees. The soil is a Cambic Umbrisol (IUSS Working Group WRB, 2006), and more information on soil properties can be found in Tan et al. (2020) [36].

#### *2.2. Litterbag Experiment and Sample Collection*

A primary natural forest and a cypress plantation were selected in the study site. Fresh senesced foliar litters were collected through a 5 m × 5 m litter collector at each site in October 2013. All litter materials were air-dried at room temperature (25 ◦C) for 15 days and then weighted 10 ± 0.05 g for each sample that was placed in a 20 cm × 20 cm litterbag with one type of foliar litter. Litterbags with two mesh sizes were used to exclude and permit the access of soil fauna into the litterbags to determine fauna effects on litter humification [37]. Specifically, there were two types of litterbags, with both types having the bottoms of the litterbags with a mesh size of 0.04 mm, but the tops had two mesh sizes (0.04 mm and 3.00 mm): 0.04 mm for the treatment to exclude the entry of soil fauna (fauna exclusion) and 3.00 mm for the control to permit the entry of macro-, meso- and

microfauna [36,38]. A total of 360 litterbags (2 meshes × 4 species × 5 times × 3 replicates × 3 plots) were randomly distributed on the soil surface (with a distance between litter bags of about 5 cm) just after the litterfall peak in November 2013.

The temperature of litterbag was measured using DS1923-F5 Recorders (iButton DS1923-F5, Maxim/Dallas Semiconductor, Sunnyvale, CA, USA) every 2 h at each site and used to calculate the daily average (DAT) and total accumulated temperature (TAT, the accumulation of daily average temperature in the litterbag during each stage) during the whole experimental periods. The average daily temperature in the alpine forests was showed in Figure 1. The trend of the daily average temperature of the primary forest and the plantation forest is basically the same, but from the growing season to the leaf falling stage period, the daily average temperature of the plantation forest is higher than that of the primary forest. The water content of litter was dried into oven for 48 h until its weight was constant, then it was recorded.

**Figure 1.** The average daily temperature in the alpine forests of different sampling stages. OF: onset of freezing, DF: deep freezing stage, TS: thawing stage, GS: growing season, LF: leaf falling stage.

According to our previous research and long-term sequential temperature observations [39], litterbags were collected in different stages: onset of freezing stage (OF, December 2013), deep freezing stage (DF, March 2014), thawing stage (TS, April 2014), growing season (GS, August 2014), and leaf falling stage (LS, October 2014). Thereafter, 9 litterbags filled with each litter species were randomly collected at each sampling site at every stage. A part of the recovered litterbags was used for soil fauna collection and the determination of matrix quality. The other part was air-dried to remove impurities, then oven-dried at 65 ◦C for 48 h (DHJ 2450B, Sheng Yuan Instrument, Zhengzhou, China) and weighed (Figure 2). The air-dried fallen leaves were crushed, sieved (0.25 mm mesh), and stored in a kraft paper bag for the extraction experiment of humus.

**Figure 2.** Cumulative mass loss rates of four litter (excluded (NSF) and non-excluded soil fauna (SF)) at different sampling stages (mean ± standard error, n = 3). OF: onset of freezing, DF: deep freezing stage, TS: thawing stage, GS: growing season, LF: leaf falling stage.

#### *2.3. Soil Fauna Collection and Chemistry Analysis*

The soil fauna in the litterbags were extracted by the Tullgren funnel method over a period of 48 h at 30 ◦C, as previously described [37]. All extracted soil fauna were counted and classified under a microscope (Leica MZ 125, Leica Microsystems GmbH, Wetzlar, Germany). After one year of exposure, we collected a total number of 2005 soil fauna individuals in the litter bag, among which Collembola (*Isotomidae* and *Campodeoidea*, etc.) and mites (*Oribatida* and *Prostigmata*, etc.) were the dominant groups, accounting for 49% and 46% of the total number of soil fauna, respectively (Figure 3, Supplementary Materials Table S1).

**Figure 3.** Individual and group density of soil fauna in different sampling stage (mean ± standard error, n = 3). OF: onset of freezing, DF: deep freezing stage, TS: thawing stage, GS: growing season, LF: leaf falling stage. The letters a and b mean in the sampling stage with different letters differ significantly, *p* < 0.05.

NaOH method was used to extracted humic substances [40]. 0.5 g of litter sample was added to 100 mL of 0.1 mol/L NaOH + Na4P2O7·10 H2O mixed solutions, shaken for 10 min, and put in a water bath with a constant temperature at 100 ◦C for 1 h. The liquid was filtered and used in part to measure humic substances (HS). We took out another 20 mL, added 0.5 mol/L H2SO4 to pH 3, and put it in a water bath with a constant temperature at 80 ◦C for 30 min. The liquid was taken out and filtered, and then the precipitated part was dissolved with 0.05 mol/L NaOH solution for the determination of humic acid (HA). The humic substances and the humic acid test solution were both filtered with a 0.45 μm filter membrane, and then measured with TOC analyzer (multi N/C 2100, Analytik Jena, Thüringen, Germany).

*2.4. Data Calculation and Statistical Analyses*

Soil fauna individual density (Di) and group density (Dg) were calculated as [37]:

> Individual density (Di)=Ni/M Groupdensity (Dg)=Ng/M

where, Ni, Ng, and M represent the individual number of soil fauna, the group number of soil fauna, and the mass residue of litter, respectively.

Fulvic acid concentration (FA), humification degree (HD), the contribution rate of soil fauna to the accumulation of humic substances, humic and fulvic acid, and humification degree were calculated as [30,36]:

$$\text{FA (g/Kg)} = \text{HS} - \text{HA}$$

$$\text{HD (\%)} = \text{HS/OC} \times 100\%$$

$$\text{C}\_{\text{HS}} \text{(\%)} = \text{(HS}\_{\text{(SFt)}} \times \text{M}\_{\text{(SFt)}} - \text{HS}\_{\text{(NSSF)}} \times \text{M}\_{\text{(NSSFt)}}) / \text{HS}\_{\text{(NSSFt)}} \times \text{M}\_{\text{(NSFt)}} \times 100\%$$

$$\text{C}\_{\text{HA}} \text{(\%)} = \text{(HA}\_{\text{(SFt)}} \times \text{M}\_{\text{(SFt)}} - \text{HA}\_{\text{(NSSFt)}} \times \text{M}\_{\text{(NSSFt)}}) / \text{HA}\_{\text{(NSSFt)}} \times \text{M}\_{\text{(NSSFt)}} \times 100\%$$

$$\text{C}\_{\text{FA}} \text{ (\%)} = \text{(FA}\_{\text{(SFt)}} \times \text{M}\_{\text{(SFt)}} - \text{FA}\_{\text{(NSSFt)}} \times \text{M}\_{\text{(NSSFt)}}) / \text{FA}\_{\text{(NSSFt)}} \times \text{M}\_{\text{(NSSFt)}} \times 100\%$$

$$\text{C}\_{\text{HD}} \text{ (\%)} = \text{(HD}\_{\text{(SFt)}} \times \text{M}\_{\text{(SFt)}} - \text{HD}\_{\text{(NSSFt)}} \times \text{M}\_{\text{(NSSFt)}}) / \text{HD}\_{\text{(NSSFt)}} \times \text{M}\_{\text{(NSSFt)}}$$

where HS and HA represent the concentrations of humic substances and humic acid, OC represents organic carbon concentration of litter. CHS, CHA, CFA, and CHD represent the contribution rate of soil fauna to the accumulation of humic substances, humic and fulvic acid, and humification degree, respectively. HS(SFt), HA(SFt), FA(SFt), and HD(SFt) represent the accumulation of humic substances, humic and fulvic acid, and humification degree under non-excluded soil fauna at the stage t, respectively. HS(NSFt), HA(NSFt), FA(NSFt), and HD(NSFt) represent the accumulation of humic substances, humic and fulvic acid, and humification degree under excluded soil fauna at the stage t, respectively. M(SFt) and M(NSFt) represent the mass residue of excluded soil fauna and non-excluded soil fauna at the stage t.

One-way ANOVA was used to analyze individual density and group density of soil fauna and the contribution rate of soil fauna to humic substances, humic acid, fulvic acid, and humification degree accumulation. Multivariate analysis of variance was used to analyze the individual density and group density of soil fauna, accumulation of humic substances, humic acid, fulvic acid, and degree of humification with species time, and their interaction. Pearson correlation analysis was used to calculate the correlation parameters of the contribution rate of soil fauna to the accumulation of humic substances (HS), humic acid (HA), fulvic acid (FA), and humification degree (HD) to carbon, nitrogen, phosphorus, lignin (L), cellulose (CE), total accumulated temperature (TAT), daily average temperature (DAT), litter water content (LWC), soil fauna individual density (Di), and group density (Dg) at different stages. Linear regression analysis was used for the individual and group density of soil fauna and the contribution rate of soil fauna to the degree of litter humification. All

significant differences were set at a level of *p* = 0.05. All statistical analyses were performed with SPSS 20.0 (IBM SPSS Statistics Inc., Chicago, IL, USA).
