DOI: 10.5061/dryad.83bk3jb3z
title: Direct and indirect trade-offs between resistance, growth, and reproduction in the Japanese stinging nettle Urtica thunbergiana

description: Most studies of trade-offs between defence, growth, and reproduction have
 examined pairwise correlations between these processes, used ratio-based
 measures for defence allocation such as allelochemical concentration and
 trichome density, and estimated resource allocation to growth and
 reproduction in terms of biomass. However, for statistical and biological
 reasons, it may be preferable to analyse these processes holistically, to
 use absolute amounts of resistant traits and leaf mass/area, and to
 measure growth and reproduction in terms of nodes with or without
 flowers.  We aimed to identify how leaf stinging hairs as
 resistant structures and leaf area as a functional trait affected growth
 and reproduction in the Japanese stinging nettle Urtica
 thunbergiana.  We conducted a greenhouse experiment with nettles
 derived from a population that has been historically exposed to heavy
 browsing by Sika deer (Cervus nippon) in Nara Park, Japan. We analysed
 causal relationships between stinging hair number, leaf area, growth rate,
 growth performance, and reproductive output using structural equation
 modelling (SEM). In this analysis we adopted newly developed indices for a
 plant’s stinging hair number and leaf area, and measures of growth and
 reproductive traits in terms of nodes instead of biomass.  There
 was a significant covariation between stinging hair number and leaf area.
 Stinging hair number had direct negative effects on growth rate and
 performance, while leaf area had positive direct effects on growth rate,
 growth performance and reproductive output. The growth rate had a
 significant direct positive effect on reproductive output, resulting in a
 significant indirect negative effect of stinging hair number on
 reproductive output. This indicates that there is not only a trade-off
 between resistance and growth, but also an indirect trade-off between
 resistance and reproduction through reduced growth rate and suggests
 that U. thunbergiana sacrifices growth rather than reproduction
 to increase resistance. Our study provides future work on trade-offs
 between defence, growth, and reproduction with a new methodological
 framework that can assess indirect as well as direct trade-offs, together
 with the effects of leaf area as a functional trait on these processes.
description: <strong>Data analyses</strong>
   <strong>Hierarchical linear
 modelling analysis for relationship between leaf area and stinging hair
 number</strong> In our data set, the sampling
 unit of leaf was nested within plant. We therefore constructed a two-level
 hierarchical linear model (HLM) to examine the relationship between leaf
 area and stinging hair number at the within-plant and between-plant
 levels: Within-plant level Yij =
 β<sub>0j</sub> + β<sub>1j</sub> Xij +
 r<sub>ij</sub> Between-plant
 level β0j = γ<sub>00</sub> +
 γ<sub>01</sub> G<sub>j</sub> +
 u<sub>0j</sub> β1j =
 γ<sub>10</sub> + γ<sub>11</sub> G<sub>j
 </sub>+ u<sub>1j</sub>
 where Y<sub>ij </sub>= stinging
 hair number for leaf i of plant j
 X<sup><sub>ij</sub> </sup>= leaf area for
 leaf i of plant j G<sub>j </sub>= mean leaf
 area of plant j β<sub>0j</sub> =
 plant-specific intercept β<sub>1j</sub> =
 plant-specific slope γ<sub>00</sub> =
 overall mean intercept (fixed effect)
 γ<sub>10</sub> = overall mean slope (fixed linear
 effect of leaf area) γ<sub>01</sub> =
 regression coefficient associated with mean leaf area of plant relative to
 plant-specific intercept (fixed linear effect of mean leaf area of
 plant) γ<sub>11</sub> = regression
 coefficient associated with mean leaf area of plant relative to
 plant-specific slope (fixed effect of the interaction between leaf area
 and mean leaf area of plants)
 u<sub>0j</sub> = random effect of plant j on
 plant-specific intercept u<sub>1j </sub>=
 random effect of plant j on plant-specific slope
 r<sub>ij </sub>= residual In this
 model, X<sub>ij</sub> was centred at
 G<sub>j</sub>, while G<sub>j </sub>was centred at
 the grand mean value. The parameters γ<sub>10</sub> and
 γ<sub>11</sub> represent the effects of leaf area at the
 within- and between-plant levels, respectively. We then tested the
 statistical significance of the fixed and random effects. In this
 procedure, the parameters were estimated by maximum likelihood and robust
 standard errors were calculated. The parameters
 γ<sub>00</sub>, γ<sub>01</sub>,
 γ<sub>10</sub>, and γ<sub>11</sub> and the
 variance components of u<sub>0j</sub> and
 u<sub>1j</sub> were tested using t-test and likelihood ratio
 test, respectively. <strong>Structural equation
 modelling analysis (SEM)</strong> We conducted
 SEM to examine the effects of leaf area and stinging hair number on growth
 rate, growth performance, and reproductive output as follows. Firstly, we
 built the full model under the hypotheses that (1) leaf area and stinging
 hair number covary; (2) both leaf area and stinging hair number affect the
 growth and reproductive components; (3) growth rate affects both growth
 performance and reproductive output; and (4) growth performance also
 affects reproductive output. Secondly, we constructed multiple models by
 leaving or removing insignificant (P &gt; 0.05) paths from the initial
 model. Next, we computed the χ2 measure of goodness of fit (except for the
 full model due to lack of degrees of freedom) and the corrected Akaike
 information criterion for small sample sizes (AICC) for the full and each
 improved model. We then selected the model with an insignificant χ2 and
 the lowest AICC as the best model that fit the data better than any of the
 other models. Finally, we evaluated the relationships between the
 variables on the basis of the path coefficients of the best
 model.
description: # Direct and indirect trade-offs between resistance, growth, and
 reproduction in the Japanese stinging nettle Urtica thunbergiana We
 collected nettle seedlings in Nara Park in early spring and raised them in
 a greenhouse near the park. For each plant that survived in early
 November, we counted the stinging hairs on the upper surface of the leaves
 along the main axis and measured the leaf area (cm^2). We also counted the
 total number of nodes and the number of inflorescence-producing nodes. ##
 Description of the data and file structure This file contains
 characteristic data for each nettle used for hierarchical linear model
 analysis of the relationship between leaf area and stinging hair number
 and structural equation model analysis of trade-offs between resistance,
 growth and reproduction. Leaf area and stinging hair number were corrected
 with reference to undamaged part when leaves were damaged by insects and
 viruses. ### Sheet Leaves **"Leaf traits"** * Leaf code * NA_ID:
 Plant ID * Node #: Node number along the main axis from the base * Leaf #:
 Leaf number of a pair of leaves on the node * Leaf area (cm^2) * Sting
 hair number: The number of stinging hairs on the upper surface of the leaf
 * Density (/cm^2): The density of stinging hairs per cm^2 ### Sheet
 Node\_production **"Tables for estimation of growth rate, consisting
 of the number of nodes and the number of nodes with inflorescences along
 the main axis on each census date (MM/DD in 2021)"** ### Sheet
 Number\_of\_nodes **"Tables for estimation of growth performance and
 reproductive output"** * The number of primary shoots * The number of
 secondary and thirdly shoots along the primary shoots * The total number
 of nodes along the all shoots (This was used as growth performance.) * The
 number of nodes along the primary shoots * The number of nodes along the
 secondary and thirdly shoots * Total number of flower nodes: The total
 number of nodes with inflorescences (This was used as reproductive
 output.) * Sheet Growth_rate: Tables for calculation of the growth rate
 for each plant (i.e., number of nodes at the 1st inflorescence emergence /
 days from 5th April to 1st inflorescence emergence day), consisting of the
 date of emergence of true leaves and inflorescences at each node along the
 main axis. ### Sheet Data\_for\_HLM **"Table for conducting
 hierarchical linear modelling analysis"** * 21NA ID: Plant ID * Node
 #: Node number along the main axis from the base * Leaf #: Leaf number of
 a pair of leaves on the node * Leaf area (cm^2) * Stinging hair number:
 The number of stinging hairs on the upper surface of the leaf * Plant mean
 LA (LA_m): Plant mean leaf area * Centered at LA_m (LA(wc)): Leaf area
 centered at plant mean leaf area * Centered at grand mean LA: Leaf area
 centered at grand mean leaf area * Plant mean SHN: Plant mean number of
 stinging hairs * Plant mean LA: Plant mean leaf area * Plant mean LA_(gm):
 Plant mean leaf area centered at grand mean leaf area Sheet Data_for_SEM
 **Table for conducting structural equation modelling analysis** * Plant ID
 * Stinging hair number index* * Leaf area index* * Growth rate: see Sheet
 Growth_rate: * Growth performance: see Sheet Number_of_nodes * Reroductive
 output: Sheet Number_of_nodes * *: We computed the indices as follows. To
 begin with, for each plant, we calculated the mean area (or mean number of
 stinging hairs) of the two leaves at each node along the main axis (when
 only one leaf was collected, its value was used). We then combined the
 data from all plants for each node and obtained the first and third
 quartiles of each sample. Next, for each plant, we assigned a score of +1
 to a node if the leaf area at that node was greater than the third
 quartile, 0 if it was between the first and third quartiles, and −1 if it
 was less than the first quartile. Finally, for each plant, we calculated
 the value by dividing the total score by the number of nodes and used this
 as an index of a plant’s leaf area (or stinging hair number). ## Authors
 **Yuzaki Inori** Department of Biological Sciences, Faculty of Science,
 Nara Women's University, Nara, 630-8263 JAPAN
 [inori.yuuzaki.cr@gmail.com](mailto:inori.yuuzaki.cr@gmail.com) **Minami
 Oishi** Department of Biological Sciences, Faculty of Science, Nara
 Women's University, Nara, 630-8263 JAPAN
 [minami-needs-ranko@ezweb.ne.jp](mailto:minami-needs-ranko@ezweb.ne.jp)
 **Hiroaki Sato** Department of Biological Sciences, Faculty of Science,
 Nara Women's University, Nara, 630-8263 JAPAN
 [scarab@cc.nara-wu.ac.jp](mailto:scarab@cc.nara-wu.ac.jp)