**1. Introduction**

The initial litter quality, mainly referred to as the initial carbon (C) quality, which is commonly present in newly shed litter, is a critical factor controlling the processes of litter decomposition and soil C sequestration [1–4]. Generally, litter with abundant labile components has been widely documented to be more decomposable and to undergo rapid decomposition [5,6]. Simultaneously, as the main carrier of soil organic C, recalcitrant components mainly manage the accumulation and stabilization of soil organic matter [7,8]. The initial qualities of labile and recalcitrant components could be characterized by the organic

Fu, C.; Dong, Q.; Yang, Y.; Wu, Q. Initial Carbon Quality of Newly Shed Foliar Litter in an Alpine Forest from Proximate Analysis and 13C NMR Spectroscopy Perspectives. *Forests* **2022**, *13*, 1886. https://doi.org/ 10.3390/f13111886

Academic Editor: Marcin Pietrzykowski

**Citation:** Yang, J.; Mu, J.; Zhang, Y.;

Received: 13 September 2022 Accepted: 8 November 2022 Published: 10 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

C fractions [9–11], thus facilitating the evaluation and prediction of litter decomposition and subsequent soil C sequestration, but there remains a lack of comprehensive knowledge.

Traditionally, proximate C fraction analysis (PA) has been used to separate watersoluble extractives (WSE), organic-soluble extractives (OSE), and acid-soluble extractives (ASE) from acid-unhydrolyzable residues (AUR), which can indicate the C fractions of organic components in plant litter and soils [12,13]. This method provides a close approximation of the lignin content in materials such as wood containing a low tannin content [12,13]. Nevertheless, the insolubility and complicated chemical structures of AUR fractions make it difficult to be characterized via this method, especially the AUR in foliar litter which generally includes substantial contributions of tannins and aliphatic C (cutin and, surface waxes) [14]. Solid-state 13C cross polarization magic angle spinning magnetic resonance (CPMAS-NMR) spectroscopy has been proven to be an effective method that allows direct and nondestructive characterization of newly shed plant materials and litter and provides more specific information on the C quality, especially in terms of the AUR in foliar litter, which can be better defined [3,15,16]. Based on this method, previous studies demonstrated that O-alkyl C, which ranges from 60 to 95 ppm chemical shifts, is the most labile component originating from decomposable carbohydrates. Aliphatic C (0–45 ppm) is commonly referred to as a biologically stable structure, and it mainly contains long-chain aliphatic compounds, cutin and waxes [2,16–18]. Likewise, aromatic C (110–140 ppm) and phenolic C (140–165 ppm) have been determined as chemically recalcitrant fractions [2,18,19]. In addition, studies have found that the ratio of aliphatic C to O-alkyl C (A/O-A) or hydrophobic C to hydrophilic C (HB/HI) ((0–45 ppm + 110–165 ppm)/(45–110 ppm + 165–190 ppm)) could reflect the enhancement in chemical structures against degradation, and the higher these two ratios are, the more stable the chemical structures against degradation [20].

Seasonal climate change regulates the C quality in litter due to the plant phenology. The consumption of decomposable C fractions is accelerated by strong rainfall and transpiration, which results from the increased temperature and solar radiation as a direct consequence of the decline in the fractions of WSE, OSE fractions and carbohydrate O-alkyl C [21,22]. Similarly, the aforementioned climatic changes could lead to the accumulation of recalcitrant C fractions [23]. Moreover, as the main fractions determining the litter quality, structurally complex C fractions notably determine and vary with the various litter decomposition stages, particularly the early stage of decomposition, which is susceptible to climatic conditions, rather than the late stage of decomposition [24–26]. A large proportion of labile C input derived from newly shed litter rapidly decreases at the early stage of decomposition [7,22], whereas the limitation on litter decomposition at the late stage of decomposition is enhanced, and more recalcitrant C fractions are formed [27]. In addition, vegetation is often considered the key factor resulting in variation in the litter C quality [28]. It has been reported that ASE and AUR were the main C fractions in foliar litter, while the content of recalcitrant AUR was higher in coniferous litter than that in broadleaf litter [26,29]. The chemical structures and compositions of AUR vary with vegetation, which results in the differences in the decomposable capacity and C fraction contents [25,30]. It has been considered that a strong signal intensity is mostly found for O-alkyl C, which indicates that newly shed foliar litter comprises labile carbohydrates. Moreover, Ono et al. [19,31] found that more aliphatic C was detected in newly shed coniferous litter (cedar and cypress) than in broadleaf litter. Conversely, broadleaved litter (beech and oak) retained more recalcitrant aromatic C, which is more decomposable than aliphatic C. In addition, it was reported that phenolic C (lignin fractions) and methoxyl C in coniferous and broadleaf species were significantly related to the accumulation of AUR at the initial stage of decomposition, suggesting that the contribution of phenolic C and methoxyl C to the AUR fraction is higher and not limited by the species at this decomposition stage [26]. Therefore, we addressed the hypothesis that (1) the contents of C fractions would vary with the species, and more labile C fractions would be released from newly shed foliar litter, while more recalcitrant C fractions might accumulate during seasons with heavy rainfall and severe temperatures,

while (2) the labile C fractions might be the major factor controlling C accumulation during litter decomposition.

To test the above hypotheses, we used PA analysis and 13C CPMAS NMR spectroscopy to detect the initial C quality of newly shed foliar litter for four dominant tree species, namely fir, spruce, willow and rosa, which were collected on a monthly basis from August 2015 to July 2016 in an alpine forest on the eastern Tibetan Plateau. In this study, we aimed to address three scientific questions: (1) How do the plant species and litter quality affect the C quality of newly shed foliar litter? (2) How does C quality of newly shed foliar litter vary seasonally? (3) How does initial C quality regulate decomposition processes? Therefore, our objective was to evaluate the capacity of the initial C input from plant litter and the initial potential of litter-derived C for C sequestration in the decomposition process in this study.

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

## *2.1. Site Description*

This study was conducted at the Long-Term Research Station of the Alpine Forest Ecosystem in the Miyaluo Nature Reserve (31◦14 N, 102◦53 E, 2982–3020 m above sea level (masl)) which is located on the eastern Tibetan Plateau. This region is a typical and transitional winter-cold zone between the Tibetan Plateau and the Sichuan Basin. The annual mean temperature is 2.7 ◦C. The maximum and minimum temperatures are 23 ◦C (July) and −18 ◦C (January), respectively. The annual mean precipitation reaches approximately 850 mm. Winter normally extends from late October until the following April with snow accumulating, and the growing season with the most concentrated litterfall extends from April until November. The forest is dominated by coniferous fir (*Abies faxoniana*) and spruce (*Picea asperata*). The main understory shrubs include willow (*Salix paraplesia*), rosa (*Rosa omeiensis*) and azalea (*Rhododendron lapponicum*). The forest soil in this area is dark brown soil and brown soil, and it is considered to be poor and barren due to the frequent natural disasters and low temperature conditions [22,32,33].

#### *2.2. Experimental Design and Sample Collection*

Based on a previous field investigation, three replicate 50 × 50 m sampling sites exhibiting similar aspects and slopes were established in a spruce-fir forest (2982–3020 m above sea level (masl)), and the three sites were at least 50 m apart from each other. Twenty litterfall funnel-shaped trap collectors were placed randomly at each site and fixed approximately 1 m above the ground at the end of July 2015.

Newly shed foliar litter was collected on a monthly basis from August 2015 to April 2016. The collected litter samples were air-dried at room temperature for one week, and foliar litter was classified as fir, spruce, willow and rosa according to the litter species. All foliar litter materials were ground and passed through a 0.25-mm sieve as subsamples for chemical analysis.
