Many scientific disciplines are currently experiencing a 'reproducibility crisis' because numerous scientific findings cannot be repeated consistently. A novel but controversial hypothesis postulates that stringent levels of environmental and biotic standardization in experimental studies reduce reproducibility by amplifying the impacts of laboratory-specific environmental factors not accounted for in study designs. A corollary to this hypothesis is that a deliberate introduction of controlled systematic variability (CSV) in experimental designs may lead to increased reproducibility. To test this hypothesis, we had 14 European laboratories run a simple microcosm experiment using grass (Brachypodium distachyon L.) monocultures and grass and legume (Medicago truncatula Gaertn.) mixtures. Each laboratory introduced environmental and genotypic CSV within and among replicated microcosms established in either growth chambers (with stringent control of environmental conditions) or glasshouses (with more variable environmental conditions). The introduction of genotypic CSV led to 18% lower among-laboratory variability in growth chambers, indicating increased reproducibility, but had no significant effect in glasshouses where reproducibility was generally lower. Environmental CSV had little effect on reproducibility. Although there are multiple causes for the 'reproducibility crisis', deliberately including genetic variability may be a simple solution for increasing the reproducibility of ecological studies performed under stringently controlled environmental conditions.

Many scientific disciplines currently are experiencing a "reproducibility crisis" because 57 numerous scientific findings cannot be repeated consistently. A novel but controversial 58 hypothesis postulates that stringent levels of environmental and biotic standardization in 59 experimental studies reduces reproducibility by amplifying impacts of lab-specific 60 environmental factors not accounted for in study designs. A corollary to this hypothesis is 61 that the deliberate introduction of controlled systematic variability (CSV) in experimental 62 designs can increase reproducibility. We tested this hypothesis using a multi-laboratory 63 microcosm study in which the same ecological experiment was repeated in 14 laboratories 64 across Europe. Each laboratory introduced environmental and genotypic CSV within and 65

Carbon (C) dynamics in canopy and roots influence whole-tree carbon fluxes, but little is known about canopy regulation of tree-root activity. Here, the patterns and dynamics of canopy-root C coupling are assessed in tropical trees.Large aeroponics facility was used to study the root systems of Ceiba pentandra and Khaya anthotheca saplings directly at different light intensities.In Ceiba, root respiration (R r ) co-varied with photosynthesis (A n ) in large saplings (3-to-7m canopy-root axis) at high-light, but showed no consistent pattern at low-light. At mediumlight and in small saplings (c. 1-m axis), R r tended to decrease transiently towards midday. Proximal roots had higher R r and nonstructural carbohydrate concentrations than distal roots, but canopy-root coupling was unaffected by root location. In medium-sized Khaya, no R r pattern was observed, and in both species, R r was unrelated to temperature.The early-afternoon increase in R r suggests that canopy-root coupling is based on mass flow of newly fixed C in the phloem, whereas the early-morning rise in R r with A n indicates an additional coupling signal that travels faster than the phloem sap. In large saplings and potentially also in higher trees, light and possibly additional environmental factors control the diurnal patterns of canopy-root coupling, irrespective of root location.

Despite the important role of tropical forest ecosystems in the uptake and storage of atmospheric carbon dioxide (CO2), the carbon (C) dynamics of tropical tree species remains poorly understood, especially regarding belowground roots. This study assessed the allocation of newly assimilated C in the fast-growing pioneer tropical tree species Ceiba pentandra (L.), with a special focus on different root categories. During a 5-day pulse-labelling experiment, 9-month-old (~3.5-m-tall) saplings were labelled with 13CO2 in a large-scale aeroponic facility, which allowed tracing the label in bulk biomass and in non-structural carbohydrates (sugars and starch) as well as respiratory CO2 from the canopy to the root system, including both woody and non-woody roots. A combined logistic and exponential model was used to evaluate 13C mean transfer time and mean residence time (MRT) to the root systems. We found 13C in the root phloem as early as 2 h after the labelling, indicating a mean C transfer velocity of 2.4 ± 0.1 m h−1. Five days after pulse labelling, 27% of the tracers taken up by the trees were found in the leaves and 13% were recovered in the woody tissue of the trunk, 6% in the bark and 2% in the root systems, while 52% were lost, most likely by respiration and exudation. Larger amounts of 13C were found in root sugars than in starch, the former also demonstrating shorter MRT than starch. Of all investigated root categories, non-woody white roots (NRW) showed the largest 13C enrichment and peaked in the deepest NRW (2–3.5 m) as early as 24 ± 2 h after labelling. In contrast to coarse woody brown roots, the sink strength of NRW increased with root depth. The findings of this study improve the understanding of C allocation in young tropical trees and provide unique insights into the changing contributions of woody and non-woody roots to C sink strengths with depth.

RATIONALE:Oversaturation of the Faraday cup amplifiers of isotope ratio mass spectrometers when using tracers that are highly enriched in heavier isotopes (up to 99.9%) remains a major bottleneck to obtaining high-precision measurements. The memory effect plays a key role in reducing tracer sample measurement precision and accuracy. Several sample preparation approaches are known to reduce memory effects and to improve tracer sample measurement precision. However, the potential benefits when using very high enrichment tracer samples (> +1000 mUr) have not been tested. METHODS:In this study, we test how specific sample positioning for measurements and frequent use of natural isotope abundance reference materials within the sequence affects the precision and accuracy of isotopic ratio analyses when using a Flash elemental analyser coupled to a Delta plus XP isotope ratio mass spectrometer for very high enrichment (> +22000 mUr) 15 N tracer sample measurements. Furthermore, we investigate if tracer sample dilution with natural isotope abundance materials reduces memory effects and increases measurement precision and accuracy when measurements of high-enrichment 15 N and 13 C biomass tracer samples are conducted. RESULTS:Frequent use of natural isotope abundance materials and specific positioning increased 15 N tracer sample precision, but it had a negative effect on the precision of quality control substances. 15 N and 13 C tracer sample dilution improved measurement precision by a maximum of ±0.9 mUr; however, a strong linear relationship between the original and the calculated φ values was found.Highly enriched 15 N tracer samples caused a maximum memory effect of 0.11%. High levels of 15 N abundance within the samples affected measurement accuracy by an average of 6.7%. CONCLUSIONS:We conclude that highly enriched tracer samples do not require dilution before analysis. Tracer sample precision can be improved by using a specific measurement order of expected isotope abundance and by the frequent use of natural abundance reference materials.