DOI: 10.5061/dryad.7d7wm380b
title: Tree seedling shade tolerance arises from interactions with microbes and is mediated by functional traits

description: Shade tolerance is a central concept in forest ecology and strongly
 influences forest community dynamics. However, the plant traits and
 conditions conferring shade tolerance are yet to be resolved. We propose
 that shade tolerance is shaped not only by responses to light but also by
 a species’ defense and recovery functional traits, soil microbial
 communities, and interactions of these factors with light availability. We
 conducted a greenhouse experiment for three temperate species in the genus
 Acer that vary in shade tolerance. We grew newly germinated seedlings in
 two light levels (2% and 30% sun) and controlled additions of microbial
 filtrates using a wet-sieving technique. Microbial filtrate treatments
 included: &lt;20 µm, likely dominated by pathogenic microbes; 40-250
 µm, containing arbuscular mycorrhizal fungi (AMF); combination, including
 both filtrate sizes; and sterilized combination. We monitored survival for
 nine weeks and measured fine root AMF colonization, hypocotyl phenolics,
 stem lignin, and stem+root nonstructural carbohydrates (NSC) at three-week
 intervals. We found that differences in seedling survival between low and
 high light only occurred when microbes were present. AMF colonization,
 phenolics, and NSC generally increased with light. Phenolics were greater
 with &lt;20 µm microbial filtrate, suggesting that soil-borne
 pathogens may induce phenolic production and NSC was greater with 40-250
 µm filtrate, suggesting that mycorrhizal fungi may induce NSC production.
 Across species, microbe treatments, and light availability, survival
 increased as phenolics and NSC increased. Therefore, shade tolerance can
 be explained by interactions among soil-borne microbes, seedling traits,
 and light availability, providing a more mechanistic and trait-based
 explanation of shade tolerance and thus forest community dynamics.
description: We conducted a fully factorial greenhouse experiment, including:
 3 temperate tree species (<em>Acer negundo</em>, <em>A.
 rubrum</em>, and <em>A. saccharum</em>) X 4 soil
 microbial filtrate treatments (none/control, pathogens in &lt;20µm
 filtrate, arbuscular mycorrhizal fungi in 40-250µm filtrate, and a
 combination of the &lt;20µm and 40-250µm filtrates) X 2 light levels
 (2% and 30% full sun). We monitored survival for 9 weeks and harvested a
 subset of seedlings at 3, 6, and 9 weeks to measure phenolics, lignin,
 nonstructural carbohydrates, and root colonization by arbuscular
 mycorrhizal fungi. Detailed methods for each step can be found in the
 methods section of the manuscript. Provided data
 include: species, light level, microbial filtrate, adult tree from which
 soil was collected, bench in the greenhouse, when the seedling was
 harvested for trait measurement, the time that the seedling was no longer
 monitored, event (0 = harvested or no longer monitored after experiment
 ended, 1 = dead), the amount of arbuscular mycorrhizal fungi observed,
 phenolics, lignin, nonstructural carbohydrates, and imputed values for
 each trait (used for trait ~ survival analysis). An
 additional file provides the model used for analyzing seedling
 survival. 
description: We performed all analyses in R 3.5.1 (R Core Team, 2020). We used
 the rjags package (Plummer, 2019) to fit survival models and to run
 predicted survival and contrast simulations. We used the built-in “lmer”
 function to fit linear mixed effects models and tested significance of
 main effects using the “Anova” function in the car package (Fox &
 Weisberg, 2019). Model selection for linear mixed effects models was
 determined with the “step” function in the lmerTest package (Kuznetsova et
 al., 2017). Post-hoc Tukey pairwise comparisons of significant main
 effects were made using the “emmeans” and “joint_tests” functions in the
 multcomp package (Hothorn et al., 2008; Lenth, 2020).
description: # Dataset for "Tree seedling shade tolerance arises from interactions
 with microbes and is mediated by functional traits" *** We conducted
 a fully factorial blocked-design greenhouse experiment at the Michigan
 State University Tree Research Center in Lansing, Michigan, USA (42.7 ºN,
 84.5 ºW). The experiment consisted of three species, four microbial
 communities (&lt;20 µm, representing pathogenic microbes; 40-250 µm,
 representing AMF; combined filtrate (both &lt;20 µm and 40-250 µm);
 and sterilized combined filtrate) and two light levels (2% and 30% full
 sun, representing shade and light gap environments). Individual pots were
 set up on six different benches (three per light level), where all
 treatment combinations were represented. We planted 80 seedlings per
 treatment combination for a total of 1,920 seedlings. We monitored
 seedlings every three days for survival, and randomly selected subsets for
 trait measurements (percent colonization by arbuscular mycorrhizal fungi,
 phenolics, lignin, and nonstructural carbohydrates) at three, six, and
 nine weeks. Data were collected 2016-2017. ## Description of the data and
 file structure There are two included files. A .csv file containing data
 and a .txt file with the survival model. In the .csv file, we include
 measured trait, survival, and imputated trait data for the greenhouse
 experiment. Detailed information about each column follows: * **No:**
 Seedling unique ID number * **Species:** 3 Temperate tree species that
 co-occur within the same forest, belong to the same genus, but vary in
 shade tolerance * Acsa = *Acer saccharum* * Acru = *Acer rubrum* * Acne =
 *Acer negundo* * **Light:** Light levels were created using shade cloth
 over greenhouse benches * Low = 2% full sunlight * High = 30% full
 sunlight * **Microbe:** Microbial filtrates were acquired through the
 wet-sieving method (see manuscript for more details) * Control =
 sterilized Combined filtrate, used only in supplementary analyses * None =
 deionized water + filter paper * Small = &lt;20µm filtrate in
 deionized water + filter paper * Large = deionized water + 40-250µm
 filtrate on filter paper * Combined = both &lt;20µm filtrate in
 deionized water + 40-250µm filtrate on filter paper * **Adult:** Number of
 the adult tree used for soil collection; used as a random effect in
 analyses; up to 4 per species * **Bench:** Number of the bench each
 seedling was grown on; used as a random effect in analyses * **Harvest:**
 Time (in weeks) that the seedling was harvested (if at all) for trait
 measurement * 3, 6, or 9 weeks * **Time:** Number of days into the
 experiment, at which time the seedling either was harvested, died, or the
 experiment ended * **Event:** Used for survival analysis to indicate
 status of each individual seedling at a given time (above) * 0 = harvested
 or experiment ended * 1 = dead * **AMF:** Calculated as AMF Count / AMF
 Intersections for percent colonization by AMF (see manuscript for detailed
 methods) * **Phenolics:** Calculated as nmol Gallic acid equivalents per
 mg dry extract (see manuscript for detailed methods) * **NSC:** Calculated
 as percent dry mass nonstructural carbohydrates (see manuscript for
 detailed methods) * **Lignin:** Calculated as percent dry mass lignin (see
 manuscript for detailed methods) * **AMF\_Imp:** Percent colonization AMF,
 imputated from measured data for analyses of trait relationships with
 survival * **PHN\_Imp:** Phenolic content, imputated from measured data
 for analyses of trait relationships with survival * **NSC\_Imp:** Percent
 dry mass nonstructural carbohydrates, imputated from measured data for
 analyses of trait relationships with survival * **LIG\_Imp:** Percent dry
 mass lignin, imputated from measured data for analyses of trait
 relationships with survivalSharing/Access information Missing data is
 coded as NA. ## Sharing/Access Information There are no licenses or
 restrictions placed on the data (public domain). All data was collected
 from this single experiment and is presented in the associated manuscript:
 Wood KEA, Kobe RK, McCarthy-Neumann S. 2023*.* Tree seedling shade
 tolerance arises from interactions with microbes and is mediated by
 functional traits. *Frontiers in Ecology and Evolution.* For any questions
 about the manuscript or dataset, including methodology and availability,
 please contact the corresponding author: Katherine EA Wood,
 woodkat7@msu.edu. ## Code/Software We performed all analyses in R 3.5.1 (R
 Core Team, 2020). We used the *rjags* package (Plummer, 2019) to fit
 survival models and to run predicted survival and contrast simulations. We
 used the built-in “lmer” function to fit linear mixed effects models and
 tested significance of main effects using the “Anova” function in the
 *car* package (Fox & Weisberg, 2019). Model selection for linear
 mixed effects models was determined with the “step” function in the
 *lmerTest* package (Kuznetsova et al., 2017). Post-hoc Tukey pairwise
 comparisons of significant main effects were made using the “emmeans” and
 “joint\_tests” functions in the *multcomp* package (Hothorn et al., 2008;
 Lenth, 2020). surv-model.txt includes the survival model to be run through
 RJags in R. Software and package versions should not matter, since the
 text file is just the model structure.