DOI: 10.5061/dryad.000000038
title: Data from: A critical assessment of the stoichiometric knife-edge: no evidence for artefacts caused by the experimental P-supplementation of algae

description: The stoichiometric knife-edge refers to the reduced performance of
 consumers encountering food with excess phosphorus (P) relative to carbon
 (C) or nitrogen (N). Studies that provide evidence for such knife-edge in
 aquatic systems often apply phosphate supplementation to create P-rich
 food treatments. However, this method may suffer from artefacts, because
 after uptake algae may store P in a form different from the P-rich
 biomolecules typically consumed by zooplankton. Our aim was to test if P
 supplementation results in potential biases. We experimentally exposed
 populations of the herbivore rotifer species, Brachionus calyciflorus
 (Pallas), to four different food quality treatments: algae grown under
 P-saturating  (HPchem, molar C:P ratio=59.7±2.7) and P-sufficient
 (MPchem, molar C:P=116.3±5.2) conditions in chemostats, and algae grown
 under P-limiting conditions, but with molar C:P ratios equal to 
 HPchem and MPchem treatments, respectively (HPLP+P, molar C:P =59.8±0.14;
 MPLP+P, molar C:P=121.0±4.3). The latter two treatments were achieved
 through P-supplementation of P-limited algae. Results show that for
 rotifers fed algae with either excess or intermediate P content,
 population growth rates were consistently higher on algae grown in
 chemostats than algae treated with the P supplementation method.
 Importantly, growth rates were also consistently lower in HP than in MP
 treatments and the magnitude of this negative impact was independent on
 algal growth history. The latter result confirms the existence of a
 stoichiometric knife-edge, and indicates that P supplementation is a
 reliable method to study the relative effect of excess P on zooplankton
 performance in a standardized way.
description: We cultured C. reinhardtii in nine continuous 2L-chemostats at
 23±1℃ using modified WC (Woods Hole Chu-10) medium (Guillard and Lorenzen,
 1972) at a dilution rate of 0.33 day-1. More specifically, we created
 algae with contrasting C:P ratios by culturing them under different
 combinations of nutrient and light conditions: (1) high P supply (120 µmol
 P L-1, using K2HPO4 ) at 40 µmol quanta m-2 s-1 continuous light,
 resulting in phytoplankton cultures with a molar C:P  ratio of 60, further
 referred to as 'HPchem'; (2) intermediate P supply (65 µmol P
 L-1) at 40 µmol quanta m-2 s-1 of continuous light, yielding algae with a
 molar C:P of 115 (‘MPchem’); (3) low P supply (15 µmol L-1) at 120 µmol
 quanta m-2 s-1 continuous light, resulting in algae with a C:P of 600
 (‘LPchem’). During the experiment, we created two food
 quality treatments through P-supplementation. One treatment was created by
 enriching the LPchem algae with an amount of inorganic phosphate so that
 it achieved a molar C:P ratio similar to that of the MPchem algae (further
 referred to as the ‘MPLP+P’ treatment). The other treatment was created by
 enriching the LPchem algae so that they achieved a molar C:P ratio similar
 to that of the HPchem algae (‘HPLP+P’). Together with the MPchem and
 HPchem algae, the MPLP+P and HPLP+P algae provided four different food
 quality treatments with the MPLP+P and HPLP+P treatments combining a
 history of growth under P-limited conditions with an artificially elevated
 cell P content through P supplementation. The algae for HPchem and MPchem
 treatments were harvested daily from the chemostats and diluted with
 nutrient free WC medium. To create the MPLP+P and HPLP+P treatments, we
 first harvested algae from LP chemostats and then added inorganic
 phosphate (K2HP04, 0.05 mol L-1) to LP algae 90 minutes before feeding
 them to experimental rotifer cultures. Estimates of the amount of P needed
 for the algae to obtain the target molar ratios were based on the algal C
 content estimated from cell counts (Multisizer 3TM Coulter Counter,
 Beckman Coulter) and a C – biovolume regression relationship. For all the
 four food quality treatments, algae were kept in the dark for 90 minutes
 before being fed to the rotifers. Experimental rotifer
 populations were fed with the four food quality treatments and their daily
 population growth rates calculated. Elemental composition of the food
 quality treatments were also assessed: algae were collected on GF/F
 filters and dried in an oven at 60℃. Algal C and N contents were
 determined using a FLASH 2000 organic element analyzer (Interscience B.V.,
 Breda, The Netherlands), while P content was determined by a QuAAtro
 segmented flow autoanalyzer (Beun de Ronde, Abcoude, The Netherlands).
 Food C:P, N:P and C:N ratios were calculated as molar ratios.