How nitrogen and phosphorus supply to nutrient-limited autotroph communities affects herbivore growth: testing stoichiometric and co-limitation theory across trophic levels

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.

Methods
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 Brachionus calyciflorus, 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 B. calyciflorus. 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.