DOI: 10.5061/dryad.dz08kps0p
title: How nitrogen and phosphorus supply to nutrient-limited autotroph communities affects herbivore growth: testing stoichiometric and co-limitation theory across trophic levels

description: Primary producer communities are often growth-limited by essential
 nutrients such as nitrogen (N) and phosphorus (P). The magnitude of
 limitation and whether N, P, or both elements are limiting autotroph
 growth depends on the supply and ratios of these essential nutrients.
 Previous studies identified single, serial or co-limitation as predominant
 limitation outcomes in autotroph communities by factorial nutrient
 additions. Little is known about potential consequences of such scenarios
 for herbivores and whether their growth is primarily affected by changes
 in autotroph quantity or nutritional quality. We grew a community of
 phytoplankton species differing in various food quality aspects in
 experimental microcosms at varying N and P concentrations resulting in
 three different N:P ratios. At carrying capacity, N, P, both nutrients or
 none were added to reveal which nutrients were limiting. The nutrient
 supplied communities were fed to the generalist herbivorous rotifer
 Brachionus calyciflorus to investigate how changing phytoplankton biomass
 and community composition affect herbivore abundance. We found
 phytoplankton being growth-limited either by N alone (single limitation)
 or serially, i.e. primarily by N and secondarily by P, altering available
 food quantity for rotifers. Rotifer growth showed a different response
 pattern compared to phytoplankton, suggesting that apart from food
 quantity food quality aspects played a substantial role in the transfer
 from primary to secondary production. The combined addition of N and P to
 phytoplankton had generally a positive effect on herbivore growth, whereas
 adding non-limiting nutrients had a rather detrimental effect probably due
 to stoichiometrically imbalanced food in terms of nutrient excess. Our
 experiment shows that adding various nutrients to primary producer
 communities will not always lead to increased autotroph and herbivore
 growth, and that differences between autotroph and herbivore responses
 under co-limiting conditions can be partly well explained by concepts of
 ecological stoichiometry theory.
description: Details on how the data were collected and analysed are given in
 the methods section of the publication. The experiment
 was set up in two time periods, a first pre-growing phase, in which
 phytoplankton communities grew on media with different N and P
 concentrations until they reached the stationary phase (i.e. were nutrient
 limited), and a second phase, in which growth of the communities was
 monitored after the addition of different amounts of N, P or both
 nutrients. Shortly after nutrient additions to phytoplankton communities,
 each microcosm was subsampled and distributed into wells previously
 stocked with asexually reproducing females of rotifer <em>Brachionus
 calyciflorus</em>, and their population growth was measured over
 time. In the first phase, we composed a phytoplankton
 community consisting of 6 species of different nutritional quality
 aspects. We grew this community on 3 different COMBO media (N&P
 reduced, N reduced or P reduced) and monitored  the biomass development in
 the microcosms using absorbance as proxy. When the stationary phase was
 reached (all nutrients taken up) we subsampled the microcosms to assess
 community composition. In the second phase, we addedd
 N, P, both or none to the microcosms and further measured biomass
 development. When the stationary phase was reached (all nutrients taken
 up) we subsampled the microcosms to assess again community composition. In
 addition, shortly after the nutrient addition, we started a parallel
 expeirment using phytoplankton from each microcosm as food for rotifer
 <em>B. calyciflorus</em>. We stocked rotifers in wells, fed
 them daily an aliquot from the micorcosms, and tracked rotifers abundance
 over several days. Then, rotifers were fixed and counted again.
description: This repository contains the data for "How nitrogen and
 phosphorus supply to nutrient-limited autotroph communities affects
 herbivore growth: testing stoichiometric and co-limitation theory across
 trophic levels" by Redoglio, Andrea; Radtke, Kassandra; Sperfeld,
 Erik, 2022, Oikos, doi:10.1111/oik.09052. This
 repository contains the raw data and R scripts for "How nitrogen and
 phosphorus supply to nutrient-limited autotroph communities affects
 herbivore growth: testing stoichiometric and co-limitation theory across
 trophic levels" by Redoglio, Andrea; Radtke, Kassandra; Sperfeld,
 Erik, 2022, Oikos.<br><br>1. Data files: definitions of column
 headers, explanations of abbreviations and units
 used<br><br>A) "Data_AlgaeCounts.csv" - Datafile
 used for R Script
 "R-script_Fig.4&S2_Stats-Fig.4&S2.R"<br><br>Orientation: columns<br><br>Column name: Medium<br>Definition: Medium categories at the beginning of the experiment<br>Code: N&P low = nitrogen and phosphorus reduced; N low = nitrogen reduced; P low = phosphorus reduced <br>Missing: no<br>Type: text<br><br>Column name: Day<br>Definition: day of the experiment when phytoplankton samples were collected and fixed for biovolume determination<br>Code: 22 = experimental day 22; 49 = experimental day 49 <br>Missing: no<br>Type: numeric<br>Unit: unitless<br><br>Column name: Algae<br>Definition: species of phytoplankton in the community<br>Code:  Acutodesmus = Acutodesmus obliquus; Monoraphidium = Monoraphidium minutum; Synechococcus = Synechococcus elongatus; Anabaena = Anabaena variabilis; Cryptomonas = Cryptomonas ovata; Nannochloropsis = Nannochloropsis limnetica<br>Missing: no<br>Type: text<br><br>Column name: Dilution<br>Definition: dilution coefficient of Lugol-fixed phytoplankton samples <br>Code: 10 = 1:10 dilution; 100 = 1:100 dilution<br>Missing: no<br>Type: numeric<br>Unit: unitless<br><br>Column name: Biovol_mm3<br>Definition: average single cell biovolume (measured in mm^3) of the respective species  <br>Missing: no<br>Type: numeric<br>Unit: cubic millimeters<br><br>Column(s) name: C1 - C2  - C3<br>Definition: number of phytoplankton cells in one quadrant of the Sedgewick-Rafter counting chamber in treatment CONTROL replicate 1, replicate 2 and replicate 3, respectively<br>Missing: no<br>Type: numeric<br>Unit: unitless<br><br>Column(s) name: N1 - N2  - N3<br>Definition: number of phytoplankton cells in one quadrant of the Sedgewick-Rafter counting chamber in treatment NITROGEN replicate 1, replicate 2 and replicate 3, respectively<br>Missing: no<br>Type: numeric<br>Unit: unitless<br><br>Column(s) name: P1 - P2  - P3<br>Definition: number of phytoplankton cells in one quadrant of the Sedgewick-Rafter counting chamber in treatment PHOSPHORUS replicate 1, replicate 2 and replicate 3, respectively<br>Missing: no<br>Type: numeric<br>Unit: unitless<br><br>Column(s) name: NP1 - NP2  - NP3<br>Definition: number of phytoplankton cells in one quadrant of the Sedgewick-Rafter counting chamber in treatment NITROGEN&PHOSPHORUS replicate 1, replicate 2 and replicate 3, respectively<br>Missing: no<br>Type: numeric<br>Unit: unitless<br><br>B) "Data_OD.csv" - Datafile used for R Script "R-script_Fig.2&3_Stats-Tab.2,Fig. 3.R"<br><br>Orientation: columns <br><br>Column name: Medium<br>Definition: Medium categories at the beginning of the experiment<br>Code: N&P low = nitrogen and phosphorus reduced; N low = nitrogen reduced; P low = phosphorus reduced <br>Missing: no<br>Type: text<br><br>Column name: Treatment<br>Definition: Treatment categories from the 2nd phase of the experiment<br>Code: Ctrl = no nitrogen and phosphorus added; N = nitrogen added; P = phosphorus added; N&P =  nitrogen and phosphorus added <br>Missing: no<br>Type: text<br><br>Column name: Replikat<br>Definition: replicates (flasks) of phytoplankton communities<br>Code: 1 = replicate 1, 2 = replicate 2 and 3 = replicate 3<br>Missing: no<br>Type: numeric<br>Unit: unitless<br><br>Columns name: 1 to 49<br>Definition: day of the experiment when the OD measurement was taken, at an interval of 3 days (every 3rd day)<br>Type: numeric<br>Unit: absorbance (unitless)<br><br>C) "Data_RotiferAbundance_OD_Biovolume.csv" - Datafile used for R Script "R-scrpt_Fig.6&S3_Stats-Fig.6&S3,Tab.3,Tab.S2,Tab.S3.R" and "R-script_Fig.S1.R"<br><br>Orientation: columns<br><br>Column names: Medium<br>Definition: Medium categories at the beginning of the experiment<br>Code: N&P low = nitrogen and phosphorus reduced; N low = nitrogen reduced; P low= phosphorus reduced<br>Missing: no<br>Type: text<br><br>Column names: Treatment<br>Definition: Treatment categories from the 2nd phase of the experiment<br>Code: Ctrl = no nitrogen and phosphorus added; N = nitrogen added; P = phosphorus added; N&P =  nitrogen and phosphorus added <br>Missing: no<br>Type: text<br><br>Column names: Replikat<br>Definition: replicate of phytoplankton community used to feed rotifers microcosms<br>Code: 1 = replicate 1, 2 = replicate 2 and 3 = replicate 3<br>Missing: no<br>Type: numeric<br>Unit: unitless<br><br>Column names: 1 to 35<br>Definition: day of the experiment when the number of rotifers was counted, at an interval of 1 day (everyday)<br>Type: numeric<br>Missing: no<br>Unit: rotifer abundance per well (unitless)<br><br>Column names: Well_ID<br>Definition: identity of a replicate of micorcosms (wells with rotifers)<br>Code: 1 = replicate 1, 2 = replicate 2, 3 = replicate 3 and 4 = replicate 4<br>Type: numeric<br>Missing: no<br>Unit: unitless<br><br>Column names: OD<br>Definition: optical density of the phytoplankton community given as food measured on day 49 (final day of the experiment) <br>Type: numeric<br>Missing: no<br>Unit: absorbance (unitless)<br><br>Column names: d22_Acu<br>Definition: biovolume of Acutodesmus obliquus on day 22<br>Type: numeric<br>Missing: no<br>Unit: mm3/ mL (cubic millimeters per milliliter)<br><br>Column names: d22_Mon<br>Definition: biovolume of Monoraphidium minutum on day 22<br>Type: numeric<br>Missing: no<br>Unit: mm3/ mL (cubic millimeters per milliliter)<br><br>Column names: d22_Syn<br>Definition: biovolume of Synechococcus elongatus on day 22<br>Type: numeric<br>Missing: no<br>Unit: mm3/ mL (cubic millimeters per milliliter)<br><br>Column names: d22_Ana<br>Definition: biovolume of Anabaena variabilis on day 22<br>Type: numeric<br>Missing: no<br>Unit: mm3/ mL (cubic millimeters per milliliter)<br> <br>Column names: d22_Cry<br>Definition: biovolume of Cryptomonas ovata on day 22<br>Type: numeric<br>Missing: no<br>Unit: mm3/ mL (cubic millimeters per milliliter)<br><br>Column names: d22_Nan<br>Definition: biovolume of Nannochloropsis limnetica on day 22<br>Type: numeric<br>Missing: no<br>Unit: mm3/ mL (cubic millimeters per milliliter)<br><br>Column names: d49_Acu<br>Definition: biovolume of Acutodesmus obliquus on day 49<br>Type: numeric<br>Missing: no<br>Unit: mm3/ mL (cubic millimeters per milliliter)<br><br>Column names: d49_Mon<br>Definition: biovolume of Monoraphidium minutum on day 49<br>Type: numeric<br>Missing: no<br>Unit: mm3/ mL (cubic millimeters per milliliter)<br><br>Column names: d49_Syn<br>Definition: biovolume of Synechococcus elongatus on day 49<br>Type: numeric<br>Missing: no<br>Unit: mm3/ mL (cubic millimeters per milliliter)<br><br>Column names: d49_Ana<br>Definition: biovolume of Anabaena variabilis on day 49<br>Type: numeric<br>Missing: no<br>Unit: mm3/ mL (cubic millimeters per milliliter)<br> <br>Column names: d49_Cry<br>Definition: biovolume of Cryptomonas ovata on day 49<br>Type: numeric<br>Missing: no<br>Unit: mm3/ mL (cubic millimeters per milliliter)<br><br>Column names: d49_Nan<br>Definition: biovolume of Nannochloropsis limnetica on day 49<br>Type: numeric<br>Missing: no<br>Unit: mm3/ mL (cubic millimeters per milliliter)<br><br>Column names: OD_d35<br>Definition: optical density of the phytoplankton community given as food measured on day 35 (final day of the rotifers experiment) <br>Type: numeric<br>Missing: no<br>Unit: absorbance (unitless)<br><br>D) "Data_Excluded rotifers wells.csv" - Datafile used for R Script "R-script_Fig.S1.R"<br><br>Orientation: columns<br><br>Column name: medium<br>Definition: Medium categories at the beginning of the experiment<br>Code: N&P low = nitrogen and phosphorus reduced; N low = nitrogen reduced; P low= phosphorus reduced<br>Missing: no<br>Type: text<br><br>Column name: treatment<br>Definition: Treatment categories from the 2nd phase of the experiment<br>Code: Ctrl = no nitrogen and phosphorus added; N = nitrogen added; P = phosphorus added; N&P =  nitrogen and phosphorus added <br>Missing: no<br>Type: text<br><br>Column name: excluded<br>Definition: number of wells (microcosms with rotifers) excluded from the statistical analysis<br>Missing: no<br>Type: numeric<br>Unit: unitless<br><br>2. Statistics and data analyses<br><br>Statistical analysis and visualization of the data can be found in the R scripts.<br><br>All statistical analyses and tests of their assumptions were performed using the statistical software R, version 4.0.2. Data were visualized using the ggplot2 package. General linear models (lm function) were used to test the effects of medium and treatment on phytoplankton biomass on days 21 and 49, estimated by OD measurements. Tukey’s HSD post-hoc tests were used to assess differences between treatments within each media group. Post-hoc tests were applied for each medium separately to readily identify predicted factorial limitation scenarios in phytoplankton (Fig. 1). For the analyses of rotifer abundance, we excluded wells in which the rotifers died out or did not grow (exclusion criterion: cumulative daily counts were less than the number of counting days multiplied by the starting density, i.e. cumulative counts &lt; 24 = 12 d × 2 rotifers, Fig. S1a) to avoid introducing a potential bias likely caused by stochastic extinction events (see above). Furthermore, there was no clear relationship between the number of excluded wells and media or treatment (Fig. S1b), suggesting no systematic effects of medium or treatment on mortality. Linear mixed-effects (LME) models were applied to test the fixed effects of phytoplankton growth medium and nutrient addition treatment on rotifer abundance counted at day 34 (alive) and at day 35 (Lugol-fixed) using the lmer function of the lme4 package (model 1). Phytoplankton replicate identity was used as random effect to account for potential non-treatment effects by using multiple rotifer wells per phytoplankton community replicate. P-values for ANOVA tables were computed using the anova function of the lmerTest package. Marginal and conditional R2 were calculated using the r2_nakagawa function of the performance package to assess the variance explained by fixed effects and the entire model, respectively. Multiple comparisons between treatments within each media category were conducted using the glht function of the multcomp package with p-values adjusted according to Holm, using log-transformed abundance data to mitigate heteroscedasticity. We applied additional LME models to investigate whether phytoplankton biomass and biovolume of particular phytoplankton species have a significant effect on rotifer abundance and how much variance these fixed variables explained. We added OD measured at the end of the rotifer experiment (day 35) as sole explanatory variable or together with the fixed factor medium to the models (model 2 and 3, respectively). We explored the role of biovolume of particular phytoplankton species with LME models. Because only green algae showed substantial treatment differences in the second phase after nutrient addition, we used the sum of biovolumes of the two green algae (A. obliquus and M. minutum) on day 49 as fixed variable in the model together with the fixed factor medium (model 4).