DOI: 10.5061/dryad.59zw3r2jm
title: Data from: Soil moisture mediates the effect of plant belowground carbon allocation on the decomposition of root litter in a subtropical forest

description: Trees allocate carbon belowground to fuel the functioning of roots and
 mycorrhizal fungi, which affect litter decomposition, but the direction
 and magnitude of this effect are variable. While tree mycorrhizal type is
 often suggested to mediate this aboveground-belowground linkage, previous
 studies yield mixed results. In this study we investigated how absorptive
 root traits, soil conditions and litter type influence the response of
 litter decomposition to altered belowground carbon allocation, both within
 and across mycorrhizal types. We girdled transport roots of seven
 subtropical tree species to eliminate carbon allocation to distal
 absorptive roots. We monitored leaf and root litter decomposition
 surrounding girdled and un-girdled root branches. We found that girdling
 generally slowed leaf litter decomposition. However, the effect of
 girdling on root litter decomposition depended on soil moisture,
 stimulating decomposition in dry soil but suppressing it in moist soil.
 Absorptive root traits did not influence the girdling effect on either
 leaf or root litter decomposition. These findings suggest that disturbance
 in carbon allocation can impact litter decomposition, with the outcome
 largely contingent on litter type and soil moisture. Synthesis. Our
 findings highlight the importance of accounting for local soil variability
 in understanding the relationship between aboveground and belowground
 carbon dynamics. This study underscores a critical need for comprehensive
 assessment of belowground ecosystem responses to aboveground disturbances,
 as it is essential for accurately predicting future forest carbon and
 nutrient cycles.
description: We conducted our study in a subtropical forest at Tianmu Mountain
 (30°18′ N, 119°26′ E). We selected 5 AM tree species (<em>Aphananthe
 aspera</em>, <em>Phoebe chekiangensis</em>,
 <em>Cunninghamia lanceolata</em>, <em>Cryptomeria
 japonica</em> var. <em>sinensis</em>
 Miquel,<strong> </strong>and <em>Torreya
 grandis</em>)<em> </em>and 2 EM tree species<em>
 </em>(<em>Cyclobalanopsis gilva</em>,
 <em>Pseudolarix amabilis</em>) . Three individuals of each
 species were randomly selected, with about 100m elevational distance
 between two adjacent replicates. We used leaf and root litter homogenized
 from a variety of local tree species, including but not limited to the
 selected ones. Leaf litter was collected from litter traps (1 m × 1 m)
 near the selected trees from November 2020 to January 2021. Root material
 was excavated from soil monoliths (1 m × 1 m, 10 cm depth) near the litter
 traps. After field sampling, leaf and root litter were prepared in the
 laboratory. The twigs and seeds were excluded from the collected leaf
 litter. Leaf litter was air-dried at room temperature. The water content
 of the air-dried leaf litter was determined by oven-drying a subsample at
 50°C for 48 hours. Roots were gently washed. Absorptive roots
 (1<sup>st</sup>-3<sup>rd</sup> order) were
 dissected and oven-dried at 50°C for 48 hours. Dried leaf and root litter
 were carefully homogenized. Leaf litter (5 g oven-dry weight equivalent)
 and root litter (1 g oven-dry weight) were placed in 10 × 10 cm litter
 bags. Litter bags were made by heat-sealing two pieces of nylon mesh (10 ×
 10 cm). The leaf litter bags had a nylon mesh aperture of 2 mm on both
 sides, while the root litter bags had a mesh aperture of 2 mm on top and
 0.15 mm on the bottom to reduce matter from escaping the bags.
 In late May 2021, we carefully excavated two root branches
 (including the 1st to 6th order roots) in the 0-10 cm soil near each
 target tree, one to be girdled and the other to serve as the control. We
 traced the roots to the base of the stem to confirm the tree. The paired
 root branches were of comparable size and were directly connected to a
 7th-order root. We inserted each root branch into a nylon mesh root pocket
 (20 × 35 cm, 0.15 mm aperture) through a zipper edge. We then filled all
 root pockets with nearby sieved soil (2-mm mesh) to a thickness of 3 cm,
 and placed three pairs of root litter and leaf litter bags within each
 root pocket. Leaf litter bags were closed to the upper side of the root
 pocket, while root litter bags were in the bottom. We modified the
 insertion position and angle of the 6th order root to maximize the spatial
 occupancy of the distal absorptive roots within the root pocket. The root
 pockets were then zipped up, reburied at <em>ca.</em> 10 cm
 depth and covered with the original duff layer. We waited two months for
 the roots to get over initial disturbance before the girdling
 treatment. We conducted a separate, accompanying study to investigate the
 response of microbial decomposers to girdling. For this study, we
 installed two additional root pockets with each of the trees used in the
 decomposition study. Two months after litter-bag
 incubation and prior to girdling, the first set of leaf and root litter
 bags were collected from both the decomposition and microbial studies. We
 opened the zipper of the root pocket and used long forceps to carefully
 remove the litter bags from the root pocket without disturbing the
 remaining litter bags. We placed all collected litter bags on ice in a
 cooler and transported them to the laboratory within a few hours for
 further processing. We then applied root girdling immediately after the
 litter bag harvest. We girdled the transport root (6th order) by removing
 a 1-cm ring of phloem close to the root pockets. Varying depth of root
 tissue was removed, depending on the phloem thickness of the target
 species. (Fig. S2). Four months after installation (i.e. two months after
 girdling), we harvested the second set of litter bags from the litter
 decomposition study only. Six months after installation (i.e. four months
 after girdling), we harvested all root pockets and litter bags.
 For litter bags collected from the decomposition study, we
 carefully brushed and wiped off as much adhering soil as possible.
 Infrequent encroaching roots were removed. We then oven-dried the litter
 at 50°C for 48 hours and weighed it. We also quantified the nitrogen (N)
 concentration of the initial and harvested litter samples to investigate
 the litter N dynamics in response to girdling. The N concentration were
 determined with an elemental analyzer (FLASH 2000 CHNS/O Elemental
 Analyzer, Thermo Scientific, USA). The litter samples collected for
 microbial study were stored at -80°C for later processing.
 At the time of root pocket installation, as well as after root
 pocket harvest, we collected soil samples in each root pocket to determine
 the soil properties. The soils were sieved (2-mm mesh) to remove coarse
 roots and large detritus. Soil pH was measured using a pH meter
 (Metter-FE28, Mettler Toledo, Greifensee, Switzerland) after suspending
 the soil in water (soil: water ratio was 1:2.5) and shaking for 2 minutes.
 Soil moisture was quantified by oven-drying the soil at 105 °C for 24
 hours. The dried soil was ground and analyzed for total C and nitrogen (N)
 concentration with the FLASH 2000 elemental analyzer.
 At the time of root pocket installation, we collected living
 absorptive roots from each target tree near the root pocket. We carefully
 washed the roots in water and scanned them on a desktop scanner at 300 DPI
 (Epson 12000XL, Epson America, Inc., California, USA). Scanned images were
 processed with WinRHIZO (Regent Instruments, Inc., Quebec, Canada) to
 determine the root diameter and total root length. The scanned roots were
 oven-dried (50 °C for 48 h) and weighed. Specific root length, the ratio
 between total root length and root dry weight, was calculated. Root tissue
 density was calculated as the root mass divided by turgid tissue volume
 determined by WinRHIZO. Dried root samples were ground and root tissue
 nitrogen concentration (N) was determined using the FLASH 2000 elemental
 analyzer. For the recovered root pockets, the
 1<sup>st</sup>-3<sup>rd</sup> order roots were
 dissected from the inserted root branch and distinguished into living and
 dead roots, according to their color, morphological characteristics,
 tissue density and strength. All living and dead roots were washed,
 scanned and weighed. The morphological traits were determined only on
 living roots. Using the litter bags collected 4 months
 after the girdling treatment, we compared the microbial biomass of
 saprotrophic fungi and bacteria with and without girdling. First, we
 collected approximately 0.5 g of fresh litter (well-mixed, with adherent
 soil) from each litter bag for DNA extraction. Each subsample was placed
 in a 15 ml centrifuge tube with 10 ml of nuclease-free water. We vortexed
 each subsample at top speed for 10 seconds and centrifuged it at 4000x g
 for 2 minutes to separate the litter from the adherent soil. The clean
 litter in the supernatant was collected. We extracted DNA from litter
 using the DNeasy PowerSoil Pro Kit (QIAGEN, Valencia, CA, USA) following
 the manufacturer's instructions. Quantitative polymerase chain
 reaction (qPCR) was used to quantify the abundance of fungi and bacteria
 (i.e., DNA copy numbers) in litter. Mass-specific abundance of bacteria
 and saprotrophic fungi in litter was calculated as the corresponding qPCR
 copy numbers divided by the litter dry weight. To calculate the abundance
 of saprotrophic fungi, we sequenced the fungal community and estimated the
 saprotrophic proportion. We used primer sets ITS1F-ITS2R to amplify the
 ITS region of the fungal communities. Successful PCR products were
 sequenced on the Illumina Novaseq platform (2 x 250bp). Detailed protocols
 for the PCR are provided in supplementary methods. The fungal amplicon
 sequences were analyzed using the EasyAmplicon pipeline . The pipeline
 first merged and filtered pair-end reads using the VSEARCH algorithm .
 Next, the amplicons were denoised into amplicon sequence variants (ASVs)
 in <em>de novo</em> mode. The UNITE database (Abarenkov et
 al., 2010) were used as reference databases for fungal taxonomy
 assignment, with a 0.5 confidence threshold. Finally, the FungalTraits
 database was used to determine the primary lifestyle of the fungal ASVs.
 The abundance of saprotrophic fungi was calculated as the total fungal
 abundance determined by qPCR multiplied by the saprotrophic proportion of
 the fungal community based on the FungalTraits database. 
 We calculated the simple exponential decay rate constants for
 both leaf and root litter: ln
 (M<sub>t</sub>/M<sub>0</sub>) = -kt 
 where k is the decomposition constant
 (yr<sup>-1</sup>), and M<sub>0</sub> is the
 initial dry weight of the sample. We then calculated ∆k of leaf and root
 litter as the difference between k in the girdled
 (k<sub>girdled</sub>) and un-girdled
 (k<sub>un-girdled</sub>) root pockets of the same
 tree: ∆k = k<sub>girdled</sub> -
 k<sub>un-girdled </sub>
description: # Soil Moisture Mediates the Effect of Plant Belowground Carbon Allocation
 on the Decomposition of Root Litter in a Subtropical Forest We have
 submitted our raw data (**Data_ALL.xlsx**) ### *Descriptions* ### Species
 abbreviation:  Apas = *Aphananthe aspera*, Phch =* Phoebe chekiangensis*,
 Cula = *Cunninghamia lanceolata*, Crja = *Cryptomeria japonica, *Togr* *=*
 Torreya grandis,* Cygi =* Cyclobalanopsis gilva*, Psam = *Pseudolarix
 amabilis*. ### Datasets included: **[Species and plot information]**
 species names and abbreviations; sample plot (S1,S2,S3); tree ID
 (S1-xx); mycorrhizal type (AM = arbuscular mycorrhizas, EM =
 ectomycorrhizas); root pocket ID (Dxx, the number has no ecological
 meaning) **[Litter information]** litter type (root vs. leaf); treatment
 (girdling vs. control); months of decomposition (0, 2, 4, 6
 months); Recovery_Mass (dry weight of initial or remaining litter, unit
 =gram); Fraction_of_Litter_Remaining (ranging from 0 to 1); root and leaf
 litter k values (simple decomposition constant) in either control or
 girdling treatments; delta k value (K_girdling - K_control); RetrieveBatch
 (zero=0 month, first = 2 months, second = 4 months, thrid = 6
 months); LeafLitterRemaining , RootLitterRemaining (remaining dry litter
 mass relative to initial litter mass, ranging from 0 to
 1); LeafTotalN, RootTotalN (remaining nitrogen amount relative to initial
 nitrogen amount, ranging from 0 to 1) **[Absorptive root traits, 1st - 3rd
 orders]** specific root length (SRL, m g⁻¹); root diameter (mm); root
 tissue density (RTD, g cm⁻³); live-to-dead root length ratio, live-to-dead
 root mass ratio **[Soil information, sieved at 2-mm mesh]** soil
 gravimetric water content (oven-drying the soil at 105 °C for 24
 hours); soil pH (soil: water = 1:2.5); total soil nitrogen (%), total soil
 carbon (%), determined by elemental analyzer **[Decomposer information]**
 Lifestyle includes Ectomycorrhizal, Saprotrophic (Lophiostoma/ Podospora/
 Subulicystidium/ Other Saprotroph), Plant pathogen, and Other fungal
 guilds (Others); TreeID here represents species abbreviation_litter type
 (Root vs. Leaf)_treatment(C=control, G = girdling); RelativeAbundance=the
 relative sequencing read abundance of the decomposers with different life
 styles  in leaf and root litter. Please see the main text and supporting
 information for more details in calculation.