DOI: 10.5061/dryad.fttdz092r
title: Warming, nitrogen deposition and provenance shift above-belowground insect interactions and host compensatory growth

description: Above-belowground insect herbivore interactions and plant compensatory
 growth are crucial for reshaping the fitness of invasive plants, and it is
 likely that climate warming, nitrogen (N) deposition, and plant provenance
 influence this interaction and growth in a complex way. We performed an
 experiment with Solidago canadensis from home and introduced ranges,
 leaf-chewing Spodoptera litura, and root-feeding Protaetia brevitarsis
 under climate warming and N deposition, and addressed how these abiotic
 stressors and plant provenance jointly shaped the reciprocal effects
 between S. litura and P. brevitarsis and the compensatory growth of S.
 canadensis after herbivory. Under ambient conditions, S. litura and P.
 brevitarsis inhibited each other on the basis of growth; warming, N
 addition or warming plus N addition shifted or even reversed this
 competition depending on provenance. While the survival-based
 above-belowground interactions differed from growth-based ones, warming or
 warming plus N addition also shifted or even reversed the neutralism or
 amensalism detected under ambient conditions depending on provenance.
 Solidago canadensis from its home range was more tolerant of herbivory
 than from its introduced range under ambient conditions; warming, N
 addition or warming plus N addition decreased the plant compensatory
 growth of native S. canadensis, but increased that of invasive S.
 canadensis relative to ambient conditions. These findings suggest that
 climate warming and N deposition could enhance positive above-belowground
 insect interactions, increasing insect pressures on S. canadensis, and
 that plant provenance might be important in mediating climate change
 effects on insect interactions and host compensatory growth under plant
 invasions.
description: A study platform (i.e., a long-term experiment) was established
 at the Chengdu field site (30.67ºN, 104.06ºE) in June 2012. The purpose of
 this experiment was to provide identical climate scenarios and seeds for
 subsequent experiments and to minimize maternal effects. The study site
 belongs to a subtropical climate, where the mean annual precipitation is
 918 mm and the mean annual temperature is 18.6 °C, and the soil is a
 ferralsol (Peng et al., 2019). The long-term experiment involved warming,
 N addition, and plant provenances, each with two levels. Experimental
 warming was achieved by heating with MSR-2420 infrared radiators, and
 the heater was suspended 1.5–2.0 m above the soil surface, roughly
 increasing air temperatures by 2 °C (Peng et al., 2019). Nitrogen
 enhancement was achieved by adding ammonium nitrate
 (NH<sub>4</sub>NO<sub>3</sub>) on the soil
 surface: adding a pulse of 1 g N m<sup>–2</sup> as an aqueous
 ammonium nitrate solution on four occasions per year. Plant seeds were
 collected from the native and introduced ranges. A detailed description
 regarding the long-term experimental design has been given previously
 (Peng et al., 2019; Zhou and He, 2022). <em>Study
 species</em> <em>Solidago
 canadensi</em>s L. (Asteraceae) is native to North America and an
 invader worldwide (Weber, 2001). This species has invaded several
 provinces in southeastern China, and is expanding rapidly (Peng et al.,
 2019). We selected two provenances of <em>S. canadensi</em>s:
 one from its home range (i.e., Montana, USA) and the other from its
 introduced range (i.e., Zhejiang, China). We collected seeds from five
 populations in North America and China in 2011. We also selected
 <em>Spodoptera litura</em> (Fabricius) (Lepidoptera:
 Noctuidae) as a leaf-feeding insect (hereafter leaf insect) because it is
 a generalist herbivore and its larvae feed on plant leaves (Zhang et al.,
 2021), and<em> Protaetia brevitarsis</em> (Coleoptera)
 (Scarabaeidae: Cetoniinae) as a root-feeding insect (hereafter root
 insect) because it is a generalist herbivore and its larvae feed on plant
 roots (Yang et al., 2020; Wang et al., 2022).
 <em>Insect feeding experiment</em>
 To test the effects of climate warming, N deposition, and plant
 provenance on the bi-directional interactions between above- and
 belowground insect herbivores and host-plant compensatory growth, we
 conducted an experiment at the Chengdu field site (30.67ºN, 104.06ºE) in
 May–November 2021. This experiment involved four factors: plant
 provenance, temperature, N, and feeding modes; the first three factors had
 two levels and the last one had four levels, thereby yielding 32
 combinations (2 provenances × 2 temperatures × 2 N levels × 4 feeding
 modes). We first randomly collected seeds from three
 <em>S. canadensis</em> populations per combination in the
 long-term experiment (see above) in December 2020. Next, we cultivated
 <em>S. canadensis</em> seedlings in May 2021 in a greenhouse,
 in which the air temperature and relative humidity ranged from 15–25
 °C and 60–80%. We collected the same local topsoil as the long-term
 experiment, and then sifted it free from rocks. A mixture of local topsoil
 and sand (1:1 volume) was filled into plastic trays (42 cm length × 42 cm
 width × 5 cm depth). The seeds were planted in the trays, which were
 placed on benches in the greenhouse and watered as required. Three
 similar-sized seedlings were transplanted into 0.5-L pots filled with a
 mixture of local topsoil and sand (1:1 volume), and exposed to each of the
 eight combinations consisting of warming, N addition, and plant provenance
 in June 2021. It should be noted that all seedlings were subject to the
 same environments as maternal plants. For example, the seedlings were
 grown under warming if their maternal plants were previously exposed to
 warming. In addition, the warming and N treatments were identical between
 this experiment and the long-term experiment. All seedlings per pot had
 grown for five months before insect herbivory to provide enough leaf food
 for insects, and were thinned to only one similar-sized seedling among
 pots prior to insect feeding. We used four different
 herbivory treatments consisting of control (no herbivory), leaf herbivory
 only (LHO), root herbivory only (RHO), and leaf and root herbivory (LRH).
 Each herbivory was replicated 10 times, and there were 960 pots (8
 maternal combinations × 4 feeding modes × 3 populations per combination ×
 10 replicates). Larvae of <em>S. litura</em> and <em>P.
 brevitarsis</em> were obtained from the Henan Jiyuan Keyun Company,
 and they arrived at the third instar on diet trays. For leaf herbivory
 only, a <em>S. litura</em> larva was weighed and placed on the
 leaves grown under eight combinations; for root herbivory only, a
 <em>P. brevitarsis</em> larva was weighed and placed on the
 roots grown under eight combinations; for leaf and root herbivory, a
 <em>S. litura</em> larva and a <em>P.
 brevitarsis</em> larva were weighed and simultaneously placed on the
 leaves and roots grown under eight combinations. Each pot was covered with
 an insect net to prevent insects from escaping and external interference.
 The insect feeding duration lasted for 14 days, in which <em>S.
 litura</em> survival was observed every day and <em>P.
 brevitarsis</em> survival was observed at the end of this
 experiment. Here, we focused on the relative growth
 rate and survival rate of insects. At the end of this experiment, we
 recorded the number of surviving leaf and/or root insects, and weighed
 them. The relative growth rate was calculated as larval mass gain per
 initial mass per unit time (mg
 g<sup>–</sup><sup>1</sup> d<sup>–</sup><sup>1</sup>), and the survival rate was calculated as follows: the final larvae / the initial larvae feeding on a given host-plant population × 100%. Ten days after ending the insect herbivory, we harvested all plants, separated them into shoots and roots, oven-dried them at 65 °C for 48 h, and measured their biomass. The whole-plant biomass (g) was defined as the sum of shoot dry biomass and root dry biomass. The biomass of shoots, roots, and whole plants at the end of the experiment were selected to gauge their compensatory potential after insect herbivory damage. <em>Data analysis</em> <em>Performance of above- and belowground insect herbivores</em> Here, insect performance parameters included relative growth and survival rates. Before data analysis, we first tested whether our data met the assumptions of normality and homoscedasticity. Based on the residuals from the models, the Gaussian family was appropriate for our data. The data of relative growth were log-transformed to meet the assumptions of normality. We then performed linear mixed-effect models (i.e., four-way analysis of variance (ANOVA)) with plant populations as a random factor to test the fixed effects of warming, N addition, provenance and feeding, and their interaction effects on the relative growth rate of <em>S. litura</em> or <em>P. brevitarsis</em>. We used four-way ANOVA to test the fixed effects of warming, N addition, provenance and feeding, and their interaction on the survival rate of <em>S. litura </em>or <em>P. brevitarsis</em>. Please note that there were only two levels for feeding in these analyses: leaf herbivory only and leaf and root herbivory for<em> S. litura</em>, and root herbivory only and leaf and root herbivory for <em>P. brevitarsis</em>. Importantly, these analyses revealed unidirectional effects between above- and belowground insects. To unravel the bi-directional effects between above- and belowground insects, we calculated the difference between the treatment and control groups as the response variable, and treated leaf and root herbivory as the treatment group and leaf herbivory only or root herbivory only as the control group when quantifying the reciprocal effects of above- and belowground insects. Accordingly, we could analyze the type and outcome of interactions between above- and belowground insects. Here, we defined the effect of the direction from the initial insect herbivore to the subsequent insect herbivore as a plus (+) when the presence of the initial herbivore allows the subsequent herbivore to perform better compared to the absence of the effector herbivore, the effect as a minus (–) when the presence of the initial herbivore allows the subsequent herbivore to perform worse compared to the absence of the effector herbivore, and the effect as a neutral (0) when the presence of the initial herbivore has no effect on the subsequent herbivore. For a specific environment (ambient, warming, N addition, and warming plus N addition), the differences in relative growth and survival rates among feeding modes were tested using the “TukeyHSD function”. All analyses were performed using the package “lme4” (Bates et al., 2015) and the package “multcomp” (Hothorn et al., 2008) in R 4.0.2 (R Core Team, 2020). <em>Compensatory growth of the host plant S. canadensis after insect herbivore damage</em> To address compensatory growth, we selected the ultimate biomass as a response variable when analyzing data. We used linear mixed-effect models (i.e., four-way ANOVA) with plant populations as a random factor to test the fixed effects of warming, N addition, provenance and feeding, and their interaction effects on the biomass of <em>S. canadensis</em> shoots, roots, and whole plants. For each plant provenance, the differences in shoot, root, and whole-plant biomass among four feeding modes under a given environment were tested using the “TukeyHSD function”. We also considered leaf herbivory only, root herbivory only, and leaf and root herbivory as a whole. In this case, we used linear mixed-effect models, where plant populations and three feeding modes nested in feeding were treated as random factors, to test the fixed effects of warming, N addition, provenance and feeding, and their interaction effects on the shoot, root, and whole-plant biomass of <em>S. canadensis</em>. For a specific environment, the differences in shoot, root, and whole-plant biomass between the control and each feeding were tested using the “TukeyHSD function”. All analyses were performed using the package “lme4” (Bates et al., 2015) and the package “multcomp” (Hothorn et al., 2008) in R 4.0.2 (R Core Team, 2020).
description: # Warming, nitrogen deposition and provenance shift above-belowground
 insect interactions and host compensatory growth
 [https://doi.org/10.5061/dryad.fttdz092r](https://doi.org/10.5061/dryad.fttdz092r) ## Description of the data and file structure ### Files and variables #### File: Zhou\_\_\_He\_Dataset.rar **Description:** Note that there were codes in this dataset: l  T0: unwarming l  T1: warming l  N0: no N addition l  N1: N addition l  W: warming l  N: N deposition l  LHO: leaf herbivory only l  RHO: root herbivory only l  LRH: leaf and root herbivory. Metadata for datasheet_insect relative growth rate column entry value unit explanation A provenance categorical populations from North America or China B warming categorical T0 unwarming,T1 warming C nitrogen categorical N0 no nitrogen addition, N1 nitrogen addition D environment categorical four combinations consisting of warming and nitrogen, W warming, N N deposition E feeding categorical LHO leaf herbivory only, RHO root herbivory only, LRH leaf and root herbivory F herbivore categorical leaf insect, root insect G population categorical different populations H replication numerical replicate I relative growth rate numerical mg g-1 d-1 biomass gain per initial biomass per unit time Metadata for datasheet_insect survival rate column entry value unit explanation A provenance categorical populations from North America or China B warming categorical T0 unwarming,T1 warming C nitrogen categorical N0 no nitrogen addition, N1 nitrogen addition D environment categorical four combinations consisting of warming and nitrogen, W warming, N N deposition E population categorical different populations F treatment categorical LHO leaf herbivory only, RHO root herbivory only, LRH leaf and root herbivory G herbivore categorical leaf insect, root insect H replication numerical replicate I survival rate numerical % final larvae/initial larvae * 100% Metadata for datasheet_plant biomass column value unit explanation A provenance categorical populations from North America or China B warming categorical T0 unwarming,T1 warming C nitrogen categorical N0 no nitrogen addition, N1 nitrogen addition D environment categorical four combinations consisting of warming and nitrogen, W warming, N N deposition E feeding categorical control, LHO leaf herbivory only, RHO root herbivory only, LRH leaf and root herbivory F herbivore categorical control, insect G population categorical different populations H replication numerical I shoot biomass numerical g oven-dried mass J root biomass numerical g oven-dried mass K total biomass numerical g oven-dried mass #### File: R\_codes.R **Description:** Note that there were codes in this software : The codes contained in the ‘R_codes.R’ file was utilized for analyzing the experimental data. The analysis results were consistent with the findings presented in the tables of the manuscript. The codes include six main sections of analysis: (1) Relative growth rate of leaf-feeding insects; (2) Relative growth rate of root-feeding insects; (3) Survival rate of leaf-feeding insects; (4) Survival rate of root-feeding insects; (5) Shoot biomass, root biomass, and whole-plant biomass; and (6) Differences in shoot, root, and whole-plant biomass between the control and each feeding treatment for both North America and China. The package “MuMIn” was used for model selection, the packages “lme4” and “car” were mainly used for linear mixed-effects model analysis, and the package “multcomp” was employed for post-hoc multiple comparisons. In the "R_codes.R", based on the “Zhou_He_Dataset”, we applied linear mixed-effects models (i.e., four-way analysis of variance (ANOVA)), treating plant populations as a random factor to test the fixed effects of warming, nitrogen addition, provenance, feeding, and their interactions on the relative growth rate of leaf-feeding or root-feeding insects. We used four-way ANOVA to test the fixed effects of warming, N addition, provenance and feeding, and their interaction on the survival rate of leaf insect or root insect. We used linear mixed-effect models with plant populations as a random factor to test the fixed effects of warming, N addition, provenance and feeding, and their interaction effects on the biomass of S. canadensis shoots, roots, and whole plants. For each plant provenance, the differences in shoot, root, and whole-plant biomass among four feeding modes under a given environment were tested using the “TukeyHSD function”. Fixed factors: (1) provenance: populations from North America or China; (2) warming: unwarming,warming; (3) nitrogen:N0 no nitrogen addition, N1 nitrogen addition; (4)feeding: control, LHO leaf herbivory only, RHO root herbivory only, LRH leaf and root herbivory Random factor: population categorical different populations ## Code/software  All analyses were performed using the package “lme4” (Bates et al., 2015) and the package “multcomp” (Hothorn et al., 2008) in R 4.0.2 (R Core Team, [2020](https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2435.14019#fec14019-bib-0051)).  The analysis codes were available in a file titled 'R_codes.R'. 1\. Relative growth rate of leaf insect: library(lme4) library(car) library(multcomp) library(MuMIn) data1&lt;-read.table("Zhou_He_Ecology2024_insect relative growth rate_leaf insect.csv",header=TRUE,sep=",") data1 shapiro.test(data1$Relative.growth.rate) R9&lt;-log(data1$Relative.growth.rate+100) shapiro.test(R9) YEWR_result&lt;-lmer(R9~Provenance*Warming*Nitrogen*Feeding+(1|Population),data=data1) YEWR_result summary(YEWR_result) Anova(YEWR_result) r.squaredGLMM(YEWR_result) 2.Relative growth rate of root insect: data2&lt;-read.table("Zhou_He_Ecology2024_insect relative growth rate_root insect.csv",header=TRUE,sep=",") data2 shapiro.test(data2$Relative.growth.rate) YEWR_result2&lt;-lmer(Relative.growth.rate~Provenance*Warming*Nitrogen*Feeding+(1|Population),data=data2) YEWR_result2 summary(YEWR_result2) Anova(YEWR_result2) r.squaredGLMM(YEWR_result2) 3.Survival rate of leaf insect: data3&lt;-read.table("Zhou_He_Ecology2024_insect survival rate_leaf insect.csv",header=TRUE,sep=",") data3 S1_result&lt;-aov(Survival.rate~Provenance*Warming*Nitrogen*Treatment,data=data3) summary(S1_result) 4.Survival rate of root insect: data4&lt;-read.table("Zhou_He_Ecology2024_insect survival rate_root insect.csv",header=TRUE,sep=",") data4 S2_result&lt;-aov(Survival.rate~Provenance*Warming*Nitrogen*Treatment,data=data4) summary(S2_result) 5.Shoot biomass, root biomass, whole-plant biomass data5&lt;-read.table("Zhou_He_Ecology2024_plant biomass.csv",header=TRUE,sep=",") data5 SB_result&lt;-lmer(Shoot.biomass~Provenance*Warming*Nitrogen*Feeding+(1|Population),data=data5) SB_result summary(SB_result) Anova(SB_result) r.squaredGLMM(SB_result) TB_result&lt;-lmer(Total.biomass~Provenance*Warming*Nitrogen*Feeding+(1|Population),data=data5) TB_result summary(TB_result) Anova(TB_result) r.squaredGLMM(TB_result)