DOI: 10.5061/dryad.z08kprrb2
title: Stoichiometric traits (N:P) of understory plants contribute to reductions in plant diversity following long-term nitrogen addition in subtropical forest

description: Nitrogen enrichment is pervasive in forest ecosystems, but its influence
 on understory plant communities and their stoichiometric characteristics
 is poorly understood. We hypothesize that when forest is enriched with
 nitrogen (N), the stoichiometric characteristics of plant species explains
 changes in understory plant diversity. A 13 year field experiment was
 conducted to explore the effects of N addition on foliar carbon (C): N:
 phosphorus (P) stoichiometry and understory plant species richness in a
 subtropical Chinese fir forest. Four levels of N addition were applied: 0,
 6, 12, and 24 g m-2 yr-1. Individual plant species were categorized into
 resistant plants, intermediate resistant plants, and sensitive plants
 based on their response to nitrogen addition. Results showed that N
 addition significantly decreased the number of species, genera and
 families of herbaceous plants. Foliar N:P ratios were greater in sensitive
 plants than resistant or intermediate resistance plants, while intrinsic
 water use efficiency showed an opposite trend. However, no relationship
 was detected between soil available N and foliar N, and soil N:P and
 foliar N:P ratios. Our results indicated that long-term N addition
 decreased the diversity of understory plants in a subtropical forest.
 Through regulating water use efficiency with N addition, sensitive plants
 change their N:P stoichiometry and have a higher risk of mortality, while
 resistant plants maintain a stable N:P stoichiometry, which contributes to
 their survival. These findings suggest that plant N:P stoichiometry plays
 an important role in understory plant performance in response to
 environmental change of N.
description: Plant investigation and sampling analyses
 Understory plant communities were investigated in September 2011,
 7 years after the start of the N addition treatment (Wu et al. 2013). One
 5 m × 5 m subplot was established in each 20 x 20 m plot and all plants
 taller than 5 cm were recorded. Plant richness (family, genera and
 species) and percentage cover were evaluated within each subplot.
 Understory plant species were divided into two functional groups: woody
 plants (including tree seedlings, shrubs and woody vines) and herbaceous
 plants. In September 2016 (13 years after N addition started), the
 understory plants were re-assessed using the same methods as in 2011 (Wu
 et al. 2013). We divided the understory species into three resistance
 types: resistant plants (RP), intermediate resistant plants (IRP) and
 sensitive plants (SP) based on the mean presence and absence of the plant
 species recorded in 2011. Briefly, plant species found in plots with any
 of the three N addition treatments and in the control (N0) plots, were
 classified as RP. Plant species found in the N0, N1 and N2 treatment
 plots, but not in the N3 treatment plots, were categorized as IRP.
 Finally, plant species found only in N0 treatment plots were categorized
 as SP. After the second plant community assessment
 (species presence and abundance in each treatment plot) in 2016, plant
 leaves were immediately collected in each plot to measure leaf C, N, P and
 their C:N:P stoichiometric traits. Leaves from 22 plant species were
 collected from the twelve plots of four treatments for foliar chemical
 analysis. For each individual plant, 5 mature leaves were sampled and
 bulked to make a composite sample. The fresh plant leaves were oven-dried
 at 60℃ then ball milled prior to analysis of <sup>13</sup>C
 and <sup>15</sup>N natural abundances, and total C, N and P.
 Foliar P was analysed by persulfate oxidation followed by colorimetric
 analysis (Bao 2000), whereas foliar C and N were analyzed by Elemental
 Analyzer (Flash 200 EA-HT, Thermo Fisher Scientific, Inc., USA). Foliar
 δ<sup>13</sup>C and δ<sup>15</sup>N was analyzed
 by Isotope Ratio Mass Spectrometer (Deata V Advantage, Thermo Fisher
 Scientific, Inc., USA). The standards for foliar
 <sup>13</sup>C and <sup>15</sup>N were Pee Dee
 Belemnite and atmospheric N<sub>2</sub>. The calculation as
 the following: δ<sup>13</sup>C
 (δ<sup>15</sup>N) =
 (<i>R</i><sub>sample</sub>/<i>R</i><sub>standard</sub>—1) × 1000 (‰), where <i>R</i> represents the isotope ratio (<sup>13</sup>C/<sup>12</sup>C or <sup>15</sup>N/<sup>14</sup>N) from samples or standards. The analytical precision for δ<sup>13</sup>C and δ<sup>15</sup>N were better than 0.1‰ and 0.2‰, respectively. Based on the value of δ<sup>13</sup>C, we calculated the intrinsic water use efficiency (iWUE) according to the descriptions from (Lu et al. 2018).