DOI: 10.25829/bexis.31627-10
title: Litter and fertilizer effects on seedling traits (two-leaf biomass, seedling height and specific leaf area) as obtained in a common garden experiment for 75 grassland species used in the seed addition plots of the REX experiment

description: Agricultural grasslands play an important role in conserving the biodiversity of the European cultural landscape. Both, litter cover and soil nutrient availability are well-known factors that change with grassland management, but it is not well-studied how the germination and seedling growth of multiple grassland species are influenced by their single or combined effects.<br>We studied the effects of fertilization (100 kg N per year and ha) and litter cover (250 gdw per m2) on seedling traits: time until two leaves developed, seedling height, SLA and biomass at the two-leaf stage. For this, we examined 75 temperate grassland species (16 grass species, 51 forb species, 8 legume species) which were sown as single species in a full factorial microcosm experiment. The microcosm experiment was part of the project SeedDiv within the framework of the DFG-funded joint project Biodiversity Exploratories (https://www.biodiversity-exploratories.de).
description: Study design: In total, 75 grassland species were used for the experiment, which represented three functional groups (16 grass species, 51 non-legume forb species, and 8 legume species) and which were used in the seed addition treatments of the land-use reduction experiment (REX) of the Biodiversity Exploratories. Seeds were obtained from commercial suppliers (Rieger-Hofmann GmbH, Blaufelden-Raboldshausen, Germany, and Wildsamen-Insel, Temmen-Ringenwalde, Germany) and represented regional provenances for the three regions of the Biodiversity Exploratories (North-eastern Germany, Central Germany and South-western Germany). Seeds were stored in a freezer (at -20°C) until they were prepared for the experiment.<br>The experiment had a full factorial design with fertilizer addition (two factor levels: no fertilization, and fertilization(+F)) and litter addition (two factor levels: no litter addition, and litter addition(+L)). This resulted in four treatments (C, +F, +L, +F+L) for each of the 75 species which were replicated four times in four experimental blocks resulting in a total of 1200 microcosms. Pots (11 x 11 cm, 12 cm depth) were filled with commercial potting substrate (Fruhstorfer Erde®, Type P70, Industrie-Erdenwerke Archut GmbH, Lauterbach, Germany) which we mixed with 25 vol% mineral sand. The mixture contained 38.41 mg kg-1 plant-available phosphorus, and 255.99 mg kg-1 plant-available potassium. Soil carbon concentration was 62.3 mg g-1, and total soil nitrogen concentration was 1.3 mg g-1. The pH (measured in a CaCl2 solution) was 5.9. <br>The experiment was conducted at the Experimental Field Station of the Helmholtz Centre for Environmental Research (UFZ) in Bad Lauchstädt (Saxony-Anhalt, Germany). It was sown between 1st and 8th November 2021 in the experimental field station in Bad Lauchstädt near Halle (Saale). Each pot was sown with 60 seeds of a single species. When provenances of different regions were available, seed origins were mixed in equal proportions. <br>For the fertilization treatment, the fertilizer was added in two portions, the first directly after sowing, and the second one at the start of the growing season (1st March 2022). Either 20 ml of water or fertilizer solution (3.024 mg N per ml) was added to the pot with a dispenser, resulting in a total nitrogen addition of 100 kg N ha-1 per year. Nitrogen was added as calammonium nitrate (76% NH4NO3, 24% CaCO3) (Triferto Fertilizers, Kalkammonsalpeter N27)<br>For the litter treatment, 3 gdw litter, which resembles 248 gdw m-2, was added to the respective pots after the first fertilization and it was lifted for the second portion of nitrogen. The litter was manually chopped to pieces of approx. 3 cm in length. The C and N concentration of the litter was 453.4 mg gdw-1 and 10.6 mg gdw-1, respectively. Pots were dug into the ground in beds filled with bark mulch to a depth of approx. 7 cm organized in four experimental blocks, each containing one replicate of each species x treatment combination. Beds were covered with pond nets (15 mm mesh size) to prevent loss of litter through wind or damage by birds. Pots received water through precipitation and were not additionally watered during winter. On 28th February 2022, pots were brought into an open greenhouse with ambient temperatures and a glass roof that closes during rain. The pots were placed on greenhouse tables arranged in four experimental blocks as before. In the greenhouse, all pots were watered two times per week with tap water.|Sampling design: When seedlings reached the stage of two fully expanded true leaves (or two leaf pairs in the case of species with opposite leaves), two experimental blocks were used to harvest four to five seedlings of each species x treatment combination. Seedlings were cut at ground level and transported in moistened tissue paper into the laboratory. Because the temporal development of the seedlings varied among treatments. Thus, our first biomass harvest represents a certain developmental stage of the seedlings.|Sample preparation: In the lab, the stretched height of each seedling was measured with a ruler. After that, the leaves were scanned with a flatbed scanner (Epson Perfection V550 Photo, EPSON) at 300 dpi. The scanned leaves and the remaining parts (stem, cotyledons) of the seedlings were separately dried at 70°C for 48 h and then weighed to obtain seedling biomass in the two-leaf stage (= two-leaf-biomass).|Sample analysis: Images were analyzed with the software Image J (Schneider et al. 2012) to obtain leaf area. The leaf area divided by the dry mass of the scanned leaves resulted in specific leaf area (mm2 mgdw-1; SLA).|Equipment: flatbed scanner (Epson Perfection V550 Photo, EPSON)|Data preparation: The data set does contain the values of all measurements after checking the raw data for plausibility.|Literature references: Schneider, C. A., Rasband, W. S., Eliceiri, K. W. (2012). NIH image to ImageJ: 25 years of image analysis. Nature Methods, 9(7), 671–675. https://doi.org/10.1038/nmeth.2089|Data references: Image J 1.53e, Wayne Rasband and contributors, National Institutes of Health, USA, http://imagej.nih.gov/ij, Java 1.8.0_172 (64-bit)