DOI: 10.5061/dryad.mgqnk9955
title: Data from: Earthworms do not increase greenhouse gas emissions (CO2 and N2O) in an ecotron experiment simulating a realistic three-crop rotation system

description: Earthworms are known to stimulate soil greenhouse gas (GHG) emissions, but
 the majority of previous studies have used simplified model systems or
 lacked continuous high-frequency measurements. To address this, we
 conducted a two-year study using large lysimeters (5 m2 area and 1.5 m
 soil depth) in an ecotron facility, continuously measuring ecosystem-level
 CO2, N2O, and H2O fluxes. We investigated the impact of endogeic and
 anecic earthworms on GHG emissions and ecosystem water use efficiency
 (WUE) in a simulated agricultural setting. Although we observed transient
 stimulations of carbon fluxes in the presence of earthworms, cumulative
 fluxes over the study indicated no significant increase in CO2 emissions.
 Endogeic earthworms reduced N2O emissions during the wheat culture
 (-44.6%), but this effect was not sustained throughout the experiment. No
 consistent effects on ecosystem evapotranspiration or WUE were found. Our
 study suggests that earthworms do not significantly contribute to GHG
 emissions over a two-year period in experimental conditions that mimic an
 agricultural setting. These findings highlight the need for realistic
 experiments and continuous GHG measurements.
description: The
 CO<sub>2</sub> concentration was measured at the inlet and
 outlet of each dome (every 12 min) using a multiplexer system
 coupled with two LI-7000 CO<sub>2</sub>/H<sub>2</sub>O analysers (LI-COR Biosciences, Lincoln, NE,
 USA). The Reichstein et al. (2005) C flux partitioning algorithm was used
 to estimate the daytime ecosystem respiration based on an exponential
 regression model (Lloyd et al., 1994). This allowed for the estimation of
 ecosystem respiration over 24 h (Reco = Reco_night + Reco_day) and gross
 primary production (GPP = NEE_day – Reco_day). 
 Ecosystem-level
 N<sub>2</sub>O fluxes were measured continuously as an open
 system using a TILDAS Compact Single analyser (N<sub>2</sub>O
 Aerodyne Research, Inc., USA). The analyzer was coupled to a multiplexer
 system allowing N<sub>2</sub>O fluxes measurement every 72 min
 for each Macrocosm. Evapotranspiration (ET) was computed as the lysimeter
 weight difference between two consecutive days. Four shear beam load cells
 per lysimeter (CMI-C3, Precia-Molen, Privas CEDEx France), with an
 accuracy of ±200 g, were used to measure the changes in mass. Ecosystem
 WUE was estimated as the ratio of GPP to ET derived from measurements by
 lysimeter weight changes over 24h.
 Earthworms were sampled by
 utilizing the non-invasive octet electric method (Schmidt et al., 2001),
 which enabled sampling of a 1 m² surface area for a duration of 50 minutes
 using a customized version of the octet device manufactured by
 Electrotechnik Schuller (Darmstadt, Germany). To achieve coverage of the
 entire 5 m² surface area, five devices were simultaneously
 deployed. References: Reichstein, M. <em>et al</em>. On the separation of net ecosystem exchange
 into assimilation and ecosystem respiration: review and improved
 algorithm. <em>Glob.
 Change Biol</em>. <strong>11</strong>, 1424–1439 (2005).
 Lloyd, J. & Taylor, J.
 A. On the Temperature Dependence of Soil Respiration.
 <em>Funct.
 Ecol. </em><strong>8</strong>, 315–323 (1994).
 Schmidt, O. Appraisal of the
 electrical octet method for estimating earthworm populations in arable
 land.<em> </em><em>Ann. Appl. Biol.</em>
 <strong>138</strong>, 231–241 (2001).
description: # Earthworms greenhouse gas emissions in an ecotron experiment datasets
 [https://doi.org/10.5061/dryad.mgqnk9955](https://doi.org/10.5061/dryad.mgqnk9955) The experimental setup involved applying an earthworm treatment with three levels (i) anecic earthworms, (ii) endogeic earthworms, and (iii) a control with very low earthworm biomass (as the total elimination of earthworm is unfeasible) in large lysimeters. The experiment simulated a three plant species crop rotation consisting of a succession of Triticum aestivum (winter wheat) - Sinapsis alba (winter white mustard) - Zeamays (maize). The experiment was conducted in the European Ecotron of Montpellier (Montferrier-sur-Lez, France, www.ecotron.cnrs.fr). It utilized the Macrocosms experimental platform consisting in twelve identical and independent units, each consisting of an 30 m³ aboveground compartment enclosed by a highly transparent material to light and UV radiation (250 μm thick Teflon-FEP film, DuPont, USA). Additionally, it included a belowground compartment containing a 5 m<sup>2</sup> stainless steel lysimeter hosting 14 t of soil (volume of 7.5 m<sup>3</sup>). Each treatment level was replicated four times in four separate domes. ## Description of the data and file structure Two datasets are used in this experiment. (i) The file "ew\_ghg\_gram\_day.csv" contains the measurements for all green house gases and the fresh earthworm biomass measured at the end of each crop growing season and used as a covariable in the mixed model. The columns are described in the table below : | Variable | Levels\_number | Description | Units | | -------- | ------------- | ----------- | ----- | | mac | 12 | macrocosm identity | number; 1-12 | | date | 664 | day of the measurements | yyyy-mm-dd | | Week | 96 | week of the year | number; 1-96 | | EW\_T | 3 | earthworm treatment levels: Control, Anecic, Endogeic | unitless | | crop | 5 | Cultivated crop and intercrop: wheat, mustard, maize, intercrop\_1, intercrop\_2 | unitless | | month | 12 | month of the measurements | Abbreviated month name | | EW\_BM | 36 | eartworm fresh weight biomass | g m<sup>-2</sup> | | N2O | 7018 | concentration in N<sub>2</sub>O | mg N-N<sub>2</sub>O m<sup>-2</sup> day<sup>-1</sup> | | NEE | 7297 | net ecosystem exchange; concentration in CO<sub>2</sub> | g C-CO<sub>2</sub> m<sup>-2</sup> day<sup>-1</sup> | | Reco | 7304 | ecosystem respiration; concentration in CO<sub>2</sub> | g C-CO<sub>2</sub> m<sup>-2</sup> day<sup>-1</sup> | | GPP | 7304 | gross primary production; concentration in CO<sub>2</sub> | g C-CO<sub>2</sub> m-2 day<sup>-1</sup> | | ET | 1208 | evapotranspiration | kg H<sub>2</sub>O m<sup>-2</sup>day<sup>-1</sup> | | WUE | 7940 | water use efficiency | g C-CO<sub>2</sub> kg H<sub>2</sub>O m<sup>-2</sup> day<sup>-1</sup> | | AirHR | 1189 | air relative humidity | % | | AirTemp | 1188 | air temperature | Celsius degrees | | Rg | 100 | global radiation | W m<sup>-2</sup> | | SWC\_13 | 1189 | soil water content at 13cm depth | % | | SWC\_25 | 1185 | soil water content at 25cm depth | % | | SWC\_80 | 1189 | soil water content at 80cm depth | % | | SoilTemp\_13 | 1189 | soil temperature at 13cm depth | Celsius degrees | | SoilTemp\_25 | 1189 | soil temperature at 25cm depth | Celsius degrees | | SoilTemp\_80 | 1189 | soil temperature at 80cm depth | Celsius degrees | | VPD | 1189 | vapor pressure deficit | kPa | NA values for all variables are missing values due to recording device malfunctioning. (ii) the file "ew\_extraction\_biomass.csv" contains the measurements of fresh earthworm biomass as they were measured, extracted or added at several periods during the experiment. The columns are described in the table below : | Variable | Levels\_number | Description | Units | | -------- | ------------- | ----------- | ----- | | crop | 4 | Cultivated crop and intercrop: wheat, mustard, maize, fallow | unitless | | EW\_T | 3 | earthworm treatment levels: Control, Anecic, Endogeic | unitless | | mac | 12 | macrocosm identity | number; 1-12 | | bm | 56 | eartworm fresh weight biomass | g m<sup>-2</sup> | | date | 4 | month of the measurements | Month name yyyy | | status | 2 | Status of the action on the earthworm fresh weight biomass: either measured, added or removed | NA | ## Code/Software We provide here the script ("EW\_GHG\_statistical\_analysis\_final.rmd") and the html version ("EW\_GHG\_statistical\_analysis\_final.html") of its reporting that contains the statistical analyses, plots and model output tables for the paper article titled "Earthworms do not increase greenhouse gas emissions (CO<sub>2 and N2</sub>O) in an ecotron experiment simulating a realistic three-crop rotation system". The analysis was conducted using R (version 4.2.1 (2022-06-23 ucrt)) and RStudio (version 2023.06.2 Build 561) and the following R packages: kableExtra (version 1.3), nlme (version 3.1 - 157), MASS (version 7.3-57), plotly (version 4.10.1), patchwork (version 1.1.2), plotrix (version 3.8-2), broom.mixed (version 0.2.9.4), multilevelmod (version 1.0.0), suncalc (version 0.5.1), ggtext (version 0.1.2), multcomp (version 1.4-22), gginnards (version 0.1.1), lemon (version 0.4.6), tidyverse (version 2.0.0). This script is structured in such a way that it enables the writing of the article using the html report produced by knitting the script without the need to re run parts of the script.