DOI: 10.5061/dryad.sj3tx96g5
title: Data from: Nitrogen-transforming microorganisms potentially facilitate the invasion of Moso bamboo (Phyllostachys edulis) into evergreen broadleaf forests

description: Moso bamboo (Phyllostachys edulis) invasions into broadleaf forests can
 cause serious ecological problems, such as reducing biodiversity and
 disrupting community succession. An elevated soil NH4+/NO3- ratio has been
 identified as a potential mechanism that facilitates Moso bamboo invasion.
 However, it remains unclear whether and how this change is the result of
 the modification of soil nitrogen-transforming microorganisms by
 bamboo. Here, we collected soil from evergreen broadleaf forests
 that were either uninvaded or heavily invaded by Moso bamboo. We then
 compared the effects of soil origin on: (1) the growth of two common
 broadleaf tree species,Castanopsis fissa and Schima superba; (2) the soil
 nitrogen content and nitrogen transformation rates; and (3) the key
 microbial functional genes involved in nitrogen fixation, nitrification,
 and denitrification. By showing the significantly increased NH4+/NO3-
 ratio in bamboo-invaded forest and the negative impact of this increase on
 the growth of the study plants, our study offers the first comprehensive
 synthesis of the positive feedback mechanisms of Moso bamboo invasion,
 with a focus on the instrumental role of nitrogen transformation
 processes.
description: <em><strong>Study area and site
 description</strong></em> Our study site
 was located at Nankun Mountain Nature Reserve, Guangdong, China (23°30′N,
 114°38′E). The area has a typical subtropical monsoon climate with an
 annual temperature of 12.4–28.5 ℃ and a mean annual precipitation of 2163
 mm. The soil in the sampling area was acrisol. The region is covered
 primarily by a native evergreen broadleaf forest dominated by
 <em>Schima superba, Castanopsis fissa, Castanopsis eyrei,
 Castanopsis carlesii, Machilus breviflora,</em> and
 <em>Castanopsis fabri</em>. In the study area, Moso bamboo
 (<em>Phyllostachys edulis</em>) can expand as much as 16
 ha/year to adjacent forests following anthropogenic disturbances (e.g.,
 forest product harvesting). The intercept between Moso bamboo and
 broadleaf forests was obscure despite the astonishing speed of bamboo
 invasion, suggesting a rapid competitive replacement of broadleaf tree
 species by Moso bamboo. In the present study, we selected a broadleaf
 forest that was heavily invaded, a Moso bamboo forest with very few
 remnant broadleaf plants (referred to as Moso bamboo forest (MBF)) and an
 adjacent secondary evergreen broadleaf forest that had been preserved
 since the 1950s as our study sites (referred to as evergreen broadleaf
 forest (EBF)). <em><strong>Soil
 sampling</strong></em> We conducted three
 separate soil sample collections for three distinct purposes: determining
 of soil nitrogen-transforming functional genes, measuring soil nitrogen
 transformation rates, and preparing the growth media for controlled
 experiments. To obtain soil samples for microbial DNA
 high-throughput sequencing and soil nitrogen transformation rate
 measurement, we established two parallel transects in each forest type
 (MBF and EBF) in June 2021. Within each transect, four plots (10 m × 10 m)
 were set up at 20-m intervals, resulting in a total of 16 plots for soil
 sampling (2 forest types × 8 replicates). In June 2021 and June 2024, we
 collected soil samples for DNA sequencing and soil nitrogen transformation
 rate measurements, respectively. For each collection, we used an auger to
 extract soil (20 cm depth × 10 cm diameter) from five randomly selected
 points within each plot, ensuring that the litter had been thoroughly
 removed beforehand. The soil from each plot was then mixed to create a
 composite sample and sieved through a 2-mm sieve to remove stones, roots
 and leaf litter. The soil samples were stored at -20℃ until further
 use. With respect to the soil samples used in the
 controlled experiments, we revisited 5 of the 8 replicate plots for each
 forest type in July 2021 and used an auger to collect surface soil (20 cm
 depth × 10 cm diameter) after thorough litter removal. For each plot, at
 least 20 kg of soil (equivalent to 15 soil cores) was collected, mixed and
 sieved through a 2-mm sieve to form a composite sample. We stored a 2-kg
 subsample of each plot at 4℃ for the greenhouse experiment.
 <em><strong>Soil physiochemical
 properties</strong></em> Soil pH was
 measured in the field by a five-point sampling method with a soil pH
 detector (PHSJ-4F). Soil organic matter content was determined using an
 elemental analyser (model CHN-ORAPID, Heraeus Co., Hanau, Germany).
 Ammonium nitrogen (NH<sub>4</sub>^+<sup>-N) and nitrate
 nitrogen (NO<sub>3</sub></sup>-<sup>-N) were
 determined using a San</sup>++^ continuous flow chemistry analyser
 (Skalar Analytica I.B.V, Netherlands) via the nitrogen diffusion method
 (Verdouw et al., 1978). Available phosphorus (P) was determined by the
 Mo‒Sb colorimetric method on the basis of the the Bray procedure (Bray and
 Kurtz, 1945). All the soil properties were expressed on an oven-dried
 weight basis. Because there was a three-year time lag
 between the soil sampling for DNA sequencing and nitrogen transformation
 measurement, to ensure the compatibility of the data derived from the soil
 samples from the two different periods, we measured the soil
 NH<sub>4</sub>^+<sup>-N content and
 NO<sub>3</sub></sup>-^-N content for both periods and
 conducted paired t tests for the two periods.
 <em><strong>Greenhouse
 experiment</strong></em> From July 2021 to
 October 2021, we conducted a controlled experiment in a greenhouse at Sun
 Yat-sen University,Guangdong, China to explore the potentially different
 effects of soil from Moso bamboo forests and evergreen broadleaf forests
 on the growth performance of two dominant broadleaf species in southern
 China, i.e., <em>Castanopsis fissa</em> and<em>Schima
 superba</em>. For each species, fifty 2-month-old seedlings of
 similar sizes (i.e., approximately 10 cm in height) were purchased from a
 local plantation. We chose a random subset of 10 seedlings of each species
 to quantify the initial height and biomass for these two species and
 selected another 15 random seedlings for the greenhouse
 experiment. For the growth medium, we filled each 3-L
 pot with 3500 g of γ-ray-sterilized base soil (radiation dose: 25
 kilogray), which was collected from the non-rhizospheric area in the
 evergreen broadleaf forest in July 2021. We subsequentlyinoculated 300 g
 of live soil inoculum from either the Moso bamboo forest or the evergreen
 broadleaf forest described in <em>Section 2</em>.
 One seedling of each species was transplanted to a pot, with 5
 replicates for each combination of species and the origin of the soil
 inoculum, resulting in a total of 20 pots (Fig. 1b). All the pots were
 watered every other day. After 3 months of cultivation, all the plants
 were harvested, and their heights and biomasses were measured after 72 h
 of oven-drying. <em><strong>Soil nitrogen
 transformation rate measurement</strong></em>
 Nitrogen fixation rates were measured by the changes in total
 nitrogen before and after cultivation. To do this, the field water holding
 capacity and soil water content of each sample were assessed. A 20 g
 equivalent of fresh soil (oven-dried basis) was placed into a 150 ml
 flask. To provide a sufficient carbon source, 1 mg C/g dry soil of glucose
 and disodium malate were added separately. Additional distilled water was
 added to achieve 60% of the water holding capacity. The flasks were sealed
 with filter paper and incubated at 25°C for 10 days in the dark, with
 moisture promptly replenished as needed. The Kjeldahl digestion method was
 employed to measure the change in total nitrogen content, thereby
 indicating nitrogen-fixing activity (Li et al., 2010; Sainju et al.,
 2017). Nitrification rates were inferred from the
 changes in NO<sub>3</sub>^-<sup>-N content before and
 after cultivation. A 20-g equivalent of fresh soil (oven-dried basis) was
 placed into a 150-ml flask. We added glucose (0.5 mg C/g dry soil),
 glutamic acid (0.5 mg C/g dry soil), and 4 ml of
 (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> solution (200 µg N/g dry soil) separately. The water content was adjusted to 60% of the water holding capacity. The flasks were incubated at 25°C for 24 h in the dark. NO<sub>3</sub></sup>-^-N was extracted and its concentration was measured to determine the nitrifying activity (Patra et al., 2006; Li et al., 2024). Denitrification rates were inferred from the changes in NH<sub>4</sub>^+<sup>-N before and after cultivation. A 15 g equivalent of fresh soil (oven-dried basis) was placed into a 150 ml flask. We added glucose (0.5 mg C/g dry soil), glutamic acid (0.5 mg C/g dry soil), and 5 ml of KNO<sub>3</sub> (200 µg N/g dry soil) separately. The water content was adjusted to 100% of the water holding capacity. The flasks were sealed with rubber stoppers and incubated at 25°C for 24 h in the dark. NH<sub>4</sub></sup>+^-N was extracted, and its concentration was measured to determine its denitrifying activity (Luo et al., 1996). <em><strong>DNA extraction, high-throughput sequencing and bioinformatic analysis</strong></em> Total DNA was extracted from fresh samples (0.5-g dry soil basis) using a TGuide S96 Soil DNA Extra Kit (TiangenBiotech, Beijing) following the manufacturer’s instructions. DNA quality and concentration were measured with a synergy HTX microplate reader (GeneCompang Limited). A total of seven nitrogen-transforming functional genes (<em>nifH</em>, archaeal <em>amoA</em>, bacterial <em>amoA</em>, <em>nirS/K</em>, <em>narG</em> and <em>nosZ</em>) were amplified using 7 pairs of primers (POI F and POI R, amoA26F and amoA417R, amoA-1F and amoA-2R, Cd3aF and R3cdR, nirKF and nirKR, narG-F and narG-R, nosZ-F and nosZ-R) using a Veriti 96-well Thermal Cycler (Applied Biosystems,Thermo Fisher) (Braker et al., 1998; Bru et al., 2007; Park et al., 2008; Poly et al., 2001; Rösch et al., 2002; Rotthauwe et al., 1997) (Table S1). The PCR conditions were as follows: initial denaturation at 95 °C for 5 min; 25 cycles of denaturation at 95°C for 30 s; annealing at 50°C for 30 s; elongation at 72°C for 40 s and a final 7 min extension at 72°C. Individual reactions consisted of 50 ng of extracted DNA, 0.3 μL of upstream and downstream primers, 5 μL of KOD FX Neo Buffer (TOYOBO) (Biolink Biotechnology Co. Ltd, Beijing, China), 2 μL of dNTPs (2 mM each), and 2 μL of KOD FX Neo and ddH<sub>2</sub>O diluted in a final volume of 10 μL.The PCR primers used are listed in Table S1. The PCR products were examined with a 1.8% agarose gel. The bands with the correct sizes were excised and then purified with an OMEGA DNA purification kit (Omega Bio-Tek Inc., GA, USA)and recycled with a Monarch DNA Gel Extraction kit (T1020L, New England BioLabs Inc., MA, USA). The functional gene community composition was assessed using the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA), following standard protocols. The raw read data obtained from sequencing were trimmed primarily by Trimmomatic (version 0.33) to omit low-quality reads (Bolger et al., 2014), and the paired-end reads were assembled by FLASH (version 1.2.11) with a minimum 10 bp overlap and a &lt;0.2 allowable error ratio (Magoč and Salzberg, 2011). Using Cutadapt (version 1.9.1) (Martin, 2011)to identify and extract primer sequences, a clean sequence without primer sequences was obtained. Then, Usearch (version 10) (Edgar, 2010) was used to splice the clean sequences of each sample, and UCHIME (version 4.2) (Edgar, 2016) was used to identify and remove the chimeric sequences, resulting in the final valid high-quality data sequence. The high-quality sequences were subsequently clustered at the 97% similarity level using the Usearch UPARSE algorithm to generate operational taxonomic units (OTUs) (Edgar, 2013). The taxonomic database of each functional gene was established using the corresponding gene sequences derived from the Functional Gene Pipeline and Repository (Fish et al., 2013).
description: # **Dryad dataset** Dataset
 DOI: [10.5061/dryad.sj3tx96g5](file:///Users/huixuanliao/Desktop/Work/%E5%AE%9E%E9%AA%8C%E5%AE%A4%E5%AE%9E%E9%AA%8C%E8%AE%BE%E8%AE%A1%E5%92%8C%E8%AE%BA%E6%96%87/%E5%8D%9C%E6%B4%9B%E7%90%B0/Functional%20Ecology/Data%20Archive/10.5061/dryad.sj3tx96g5) **Description of the data and file structure** This dataset includes five data sheets and one R script for statistical analyses. **Files and variables** **File: Dataset1-SoilProperties.csv** **Description:** This data sheet contains data of the contents of soil organic matter, ammonium (NH4+-N), nitrate (NO3--N), ammonium-nitrate ratio, available phosphorous, and soil pH of the soils sampled from the Moso bamboo forest (MBF) and the evergreen broadleaf forest (EBF).  **Variables** * ID: IDs of soil samples * ForestType: the type of forest community where the focal soil sample was taken [evergreen broadleaf forest (EBF) vs. Moso bamboo forest (MBF)] * SOM: soil organic matter content (g·kg^-1^) * NH4: ammonium (NH4^+^-N) content (mg·kg^-1^) * NO3: nitrate (NO3^-^-N) content (mg·kg^-1^) * NH4_NO3: NH4^+^/NO3^-^ ratio * Available_P: available phosphorous content (mg·kg^-1^) * pH: soil pH (acidity) **File: Dataset2-GrowthPerformance-Greenhouse.csv** **Description:** This data sheet contains data of the biomass, height, and relative growth rate of the two studied broadleaf tree species, *Castanopsis fissa and Schima superba*, that were grown under the greenhouse condition. **Variables** * Species: Names of the studied broadleaf tree species - Inocula: Soil inocula origin [evergreen broadleaf forest (EBF) vs. Moso bamboo forest (MBF)] - Biomass: total biomass (g) - Height: plant height (cm) - RGR_Biomass: relative growth rate measured by biomass increase (g·g-1·day-1) - RGR_Height: relative growth rate measured by biomass increase (cm·cm-1·day-1) **File: Dataset3-Ntransformation.csv** **Description:** This data sheet contains data of the nitrogen transformation rates of the soils sampled from MBF vs. EBF communities, with the contents of total nitrogen, ammonium (NH4+-N), and nitrate (NO3--N) corresponding with the focal soil sample. **Variables** * ID: IDs of soil samples * ForestType: the type of forest community where the focal soil sample was taken [evergreen broadleaf forest (EBF) vs. Moso bamboo forest (MBF)] * N_fixation: nitrogen fixation rate (g·kg^-1^) * Nitrification: nitrification rate (mg·kg^-1^) * Denitrification: denitrification rate (mg·kg^-1^) * Total_N: total nitrogen content (mg·kg^-1^) * NH4: ammonium (NH4^+^-N) content (mg·kg^-1^) * NO3: nitrate (NO3^-^-N) content (mg·kg^-1^) **File: Dataset4-OUT_Relative_Abundance-Genus_Level.csv** **Description:** This data sheet contains data of the relative abundance of OTUs associated with the seven studied gene families, *nifH*, archaeal *amoA*, bacterial *amoA*, *nirK*, *nirS*, *narG*, *nosZ*. **Variables** * Gene_family: the gene family studied (*nifH*, archaeal *amoA*, bacterial *amoA*, *nirK*, *nirS*, *narG*, or *nosZ*) * Genus: microbial genus detected under the focal gene family * SK1: the relative abundance of the focal genus in the 1st soil sample from the evergreen broadleaf forest (EBF) * SK2: the relative abundance of the focal genus in the 2nd soil sample from the evergreen broadleaf forest (EBF) * SK3: the relative abundance of the focal genus in the 3rd soil sample from the evergreen broadleaf forest (EBF) * SK4: the relative abundance of the focal genus in the 4th soil sample from the evergreen broadleaf forest (EBF) * SK5: the relative abundance of the focal genus in the 5th soil sample from the evergreen broadleaf forest (EBF) * SK6: the relative abundance of the focal genus in the 6th soil sample from the evergreen broadleaf forest (EBF) * SK7: the relative abundance of the focal genus in the 7th soil sample from the evergreen broadleaf forest (EBF) * SK8: the relative abundance of the focal genus in the 8th soil sample from the evergreen broadleaf forest (EBF) * SZ1: the relative abundance of the focal genus in the 1st soil sample from the Moso bamboo forest (MBF) * SZ2: the relative abundance of the focal genus in the 2nd soil sample from the Moso bamboo forest (MBF) * SZ3: the relative abundance of the focal genus in the 3rd soil sample from the Moso bamboo forest (MBF) * SZ4: the relative abundance of the focal genus in the 4th soil sample from the Moso bamboo forest (MBF) * SZ5: the relative abundance of the focal genus in the 5th soil sample from the Moso bamboo forest (MBF) * SZ6: the relative abundance of the focal genus in the 6th soil sample from the Moso bamboo forest (MBF) * SZ7: the relative abundance of the focal genus in the 7th soil sample from the Moso bamboo forest (MBF) * SZ8: the relative abundance of the focal genus in the 8th soil sample from the Moso bamboo forest (MBF) * EBF: mean relative abundance across the eight replicative soil samples from the evergreen broadleaf forest (EBF) * MBF: mean relative abundance across the eight replicative soil samples from t the Moso bamboo forest (MBF) **File: Dataset5-MicrobialCommunityCharacteristics.csv** **Description:** This data sheet contains data of the total relative abundance of OTUs, gene diversity (Chao1), and community structure of the seven studied gene families, *nifH*, archaeal *amoA*, bacterial *amoA*, *nirK*, *nirS*, *narG*, *nosZ*. **Variables** * Metric: the metric of community characteristics (abundance, Chao1, and NMDS) * ID: IDs of soil samples * ForestType: the type of forest community where the focal soil sample was taken [evergreen broadleaf forest (EBF) vs. Moso bamboo forest (MBF)] * nifH: the gene family *nifH* * Arch_amoA: the gene family *amoA* of archaea * Bact_amoA: the gene family *amoA* of bacteria * nirK: the gene family *nirK* * nirS: the gene family *nirS* * narG: the gene family *narG* * nosZ: the gene family *nosZ* **Code/software** **Description of R script:** **20250331-Data_Analyses.R** The R script contains seven sections for data analyses: section 1 shows the code for loading all necessary R packages; section 2 shows the code for the t test for the difference in soil properties between MBF vs. EBF communities using Dataset 1; section 3 shows the codes for the two-way ANOVA analysis testing the effects of soil inocula (MBF vs. EBF soil) and species on the growth of the two studied broadleaf tree species under greenhouse conditions, using Datasets 2; section 4 shows the code for the t test for the difference in soil nitrogen transformation rates between MBF vs. EBF communities using Dataset 3; section 5 shows the code for the non-metric multi-dimensional scaling (NMDS) of the nitrogen-transforming gene families using Dataset 4 and the t test for the difference in the community characteristics of nitrogen-transforming gene families between MBF vs. EBF communities using Dataset 5; section 6 shows the code for Mantel's test for the correlation between nitrogen-related genes, nitrogen transformation, and nitrogen content using Datasets 3 and 5. **Software:** R version 4.2.1. Running under macOS Sequoia 15.3.1. Attached packages: base, methods, datasets, stats, graphics Loaded packages: dplyr, emmeans, vegan