Soil organic matter, rather than temperature, determines the structure and functioning of subarctic decomposer communities.

The impacts of climate change on ecosystem structure and functioning are likely to be strongest at high latitudes due to the adaptation of biota to relatively low temperatures and nutrient levels. Soil warming is widely predicted to alter microbial, invertebrate, and plant communities, with cascading effects on ecosystem functioning, but this has largely been demonstrated over short-term (<10 year) warming studies. Using a natural soil temperature gradient spanning 10-35°C, we examine responses of soil organisms, decomposition, nitrogen cycling, and plant biomass production to long-term warming. We find that decomposer organisms are surprisingly resistant to chronic warming, with no responses of bacteria, fungi, or their grazers to temperature (fungivorous nematodes being an exception). Soil organic matter content instead drives spatial variation in microorganism abundances and mineral N availability. The few temperature effects that appear are more focused: root biomass and abundance of root-feeding nematodes decrease, and nitrification increases with increasing soil temperature. Our results suggest that transient responses of decomposers and soil functioning to warming may stabilize over time following acclimation and/or adaptation, highlighting the need for long-term, ecosystem-scale studies that incorporate evolutionary responses to soil warming.

Temperature effects on the temporal dynamics of a subarctic invertebrate community.

Climate warming is predicted to have major impacts on the structure of terrestrial communities, particularly in high latitude ecosystems where growing seasons are short. Higher temperatures may dampen seasonal dynamics in community composition as a consequence of earlier snowmelt, with potentially cascading effects across all levels of biological organisation. Here, we examined changes in community assembly and structure along a natural soil temperature gradient in the Hengill geothermal valley, Iceland, during the summer of 2015. Sample collection over several time points within a season allowed us to assess whether temperature alters temporal variance in terrestrial communities and compositional turnover. We found that seasonal fluctuations in species richness, diversity and evenness were dampened as soil temperature increased, whereas invertebrate biomass varied more. Body mass was found to be a good predictor of species occurrence, with smaller species found at higher soil temperatures and emerging earlier in the season. Our results provide more in-depth understanding of the temporal nature of community and population-level responses to temperature, and indicate that climate warming will likely dampen the seasonal turnover of community structure that is characteristic of high latitude invertebrate communities.

Genotypic traits and tradeoffs of fast growth in silver birch, a pioneer tree.

Fast-growing and slow-growing plant species are suggested to show integrated economics spectrums and the tradeoffs of fast growth are predicted to emerge as susceptibility to herbivory and resource competition. We tested if these predictions also hold for fast-growing and slow-growing genotypes within a silver birch, Betula pendula population. We exposed cloned saplings of 17 genotypes with slow, medium or fast height growth to reduced insect herbivory, using an insecticide, and to increasing resource competition, using naturally varying field plot grass cover. We measured shoot and root growth, ectomycorrhizal (EM) fungal production using ergosterol analysis and soil N transfer to leaves using 15N-labelled pulse of NH4+. We found that fast-growing genotypes grew on average 78% faster, produced 56% and 16% more leaf mass and ergosterol, and showed 78% higher leaf N uptake than slow-growing genotypes. The insecticide decreased leaf damage by 83% and increased shoot growth, leaf growth and leaf N uptake by 38%, 52% and 76%, without differences between the responses of fast-growing and slow-growing genotypes, whereas root mass decreased with increasing grass cover. Shoot and leaf growth of fast-growing genotypes decreased and EM fungal production of slow-growing genotypes increased with increasing grass cover. Our results suggest that fast growth is genotypically associated with higher allocation to EM fungi, better soil N capture and greater leaf production, and that the tradeoff of fast growth is sensitivity to competition, but not to insect herbivory. EM fungi may have a dual role: to support growth of fast-growing genotypes under low grass competition and to maintain growth of slow-growing genotypes under intensifying competition.

Leaf N resorption efficiency and litter N mineralization rate have a genotypic tradeoff in a silver birch population.

Plants enhance N use efficiency by resorbing N from senescing leaves. This can affect litter N mineralization rate due to the C:N-ratio requirements of microbial growth. We examined genotypic links between leaf N resorption and litter mineralization by collecting leaves and litter from 19 Betula pendula genotypes and following the N release of litter patches on forest ground. We found significant genotypic variation for N resorption efficiency, litter N concentration, cumulative three-year patch N-input and litter N release with high broad-sense heritabilities (H2  = 0.28-0.65). The genotype means of N resorption efficiency varied from 46% to 65% and correlated negatively with the genotype means of litter N concentration, cumulative patch N-input and litter N release. NH4+ yield under patches had a positive genotypic correlation with the cumulative patch N-input. During the first year of litter decomposition, genotypes varied from N immobilization (max 2.71 mg/g dry litter) to N release (max 1.41 mg/g dry litter), creating a genotypic tradeoff between the N conserved by resorption and the N available for root uptake during the growing season. We speculate that this tradeoff is one likely reason for the remarkably wide genotypic range of N resorption efficiencies in our birch population.

Temperature and soil fertility as regulators of tree line Scots pine growth and survival-implications for the acclimation capacity of northern populations.

The acclimation capacity of leading edge tree populations is crucially important in a warming climate. Theoretical considerations suggest that adaptation through genetic change is needed, but this may be a slow process. Both positive and catastrophic outcomes have been predicted, while empirical studies have lagged behind theory development. Here we present results of a 30-year study of 55,000 Scots pine (Pinus sylvestris) trees, planted in 15 common gardens in three consecutive years near and beyond the present Scots pine tree line. Our results show that, contrary to earlier predictions, even long-distance transfers to the North can be successful when soil fertility is high. This suggests that present northern populations have a very high acclimation capacity. We also found that while temperature largely controls Scots pine growth, soil nutrient availability plays an important role-in concert with interpopulation genetic variation-in Scots pine survival and fitness in tree line conditions. These results suggest that rapid range expansions and substantial growth enhancements of Scots pine are possible in fertile sites as seed production and soil nutrient mineralization are both known to increase under a warming climate. Finally, as the ontogenetic pattern of tree mortality was highly site specific and unpredictable, our results emphasize the need for long-term field trials when searching for the factors that control fitness of trees in the variable edaphic and climatic conditions of the far North.

The Arctic Plant Aboveground Biomass Synthesis Dataset.

Plant biomass is a fundamental ecosystem attribute that is sensitive to rapid climatic changes occurring in the Arctic. Nevertheless, measuring plant biomass in the Arctic is logistically challenging and resource intensive. Lack of accessible field data hinders efforts to understand the amount, composition, distribution, and changes in plant biomass in these northern ecosystems. Here, we present The Arctic plant aboveground biomass synthesis dataset, which includes field measurements of lichen, bryophyte, herb, shrub, and/or tree aboveground biomass (g m-2) on 2,327 sample plots from 636 field sites in seven countries. We created the synthesis dataset by assembling and harmonizing 32 individual datasets. Aboveground biomass was primarily quantified by harvesting sample plots during mid- to late-summer, though tree and often tall shrub biomass were quantified using surveys and allometric models. Each biomass measurement is associated with metadata including sample date, location, method, data source, and other information. This unique dataset can be leveraged to monitor, map, and model plant biomass across the rapidly warming Arctic.

Genotypic variation in yellow autumn leaf colours explains aphid load in silver birch.

• It has been suggested that autumn-migrating insects drive the evolution of autumn leaf colours. However, evidence of genetic variation in autumn leaf colours in natural tree populations and the link between the genetic variation and herbivore abundances has been lacking. • Here, we measured the size of the whole aphid community and the development of green-yellow leaf colours in six replicate trees of 19 silver birch (Betula pendula) genotypes at the beginning, in the middle and at the end of autumn colouration. We also calculated the difference between green leaf and leaf litter nitrogen (N) and estimated the changes in phloem sap N loading. • Autumn leaf colouration had significant genetic variation. During the last survey, genotypes that expressed the strongest leaf reflectance 2-4 wk earlier had an abundance of egg-laying Euceraphis betulae females. Surprisingly, the aphid community size during the first surveys explained N loss by the litter of different birch genotypes. • Our results are the first evidence at the tree intrapopulation genotypic level that autumn-migrating pests have the potential to drive the evolution of autumn leaf colours. They also stress the importance of recognizing the role of late-season tree-insect interactions in the evolution of herbivory resistance.

Effects of experimental warming on Betula nana epidermal cell growth tested over its maximum climatological growth range.

Numerous long-term, free-air plant growth facilities currently explore vegetation responses to the ongoing climate change in northern latitudes. Open top chamber (OTC) experiments as well as the experimental set-ups with active warming focus on many facets of plant growth and performance, but information on morphological alterations of plant cells is still scarce. Here we compare the effects of in-situ warming on leaf epidermal cell expansion in dwarf birch, Betula nana in Finland, Greenland, and Poland. The localities of the three in-situ warming experiments represent contrasting regions of B. nana distribution, with the sites in Finland and Greenland representing the current main distribution in low and high Arctic, respectively, and the continental site in Poland as a B. nana relict Holocene microrefugium. We quantified the epidermal cell lateral expansion by microscopic analysis of B. nana leaf cuticles. The leaves were produced in paired experimental treatment plots with either artificial warming or ambient temperature. At all localities, the leaves were collected in two years at the end of the growing season to facilitate between-site and within-site comparison. The measured parameters included the epidermal cell area and circumference, and using these, the degree of cell wall undulation was calculated as an Undulation Index (UI). We found enhanced leaf epidermal cell expansion under experimental warming, except for the extremely low temperature Greenland site where no significant difference occurred between the treatments. These results demonstrate a strong response of leaf growth at individual cell level to growing season temperature, but also suggest that in harsh conditions other environmental factors may limit this response. Our results provide evidence of the relevance of climate warming for plant leaf maturation and underpin the importance of studies covering large geographical scales.

Reduction of odorants in swine manure by carbohydrate and bacterial amendments.

Malodors from pig manure storage pits are a problem lacking a cost-efficient solution particularly for small pig (Sus domestica) farms. The objective of this study was to reduce emissions of noxious odorants by changing the conditions in the pig manure to favor an altered microbial community. Sugars (maltose, lactose, and saccharose), carbohydrate-rich waste (maltose syrup and wheat flour), and bacterial amendments (Lactobacillus plantarum and L. amylophilus) were tested for their effect on manure pH, bacterial community, and gaseous emissions. In laboratory experiments, a permanent pH reduction was achieved with all carbohydrates when added to a 5% final concentration. Maltose significantly decreased emissions of sulfur-containing compounds, whereas bacterial amendments had little effect on odorants. Lactobacilli were found in manure receiving carbohydrate amendments alone, but Clostridiales, Bacteroidetes, and Enterobacteriaceae were observed in all treatments (including the control). At the mesocosm (60 L) level, maltose syrup and wheat flour amendments caused clear pH reductions in manure, whereas L. plantarum and L. amylophilus had no additional effect. The addition of maltose syrup and wheat flour to a manure storage pit (600 m(3)) lowered the pH slightly. When the manure was spread onto the fields, the observed reduction in odor was found to be reversible, and the release of malodors was delayed rather than eliminated. We conclude that these methods require further development to produce a reliable technical application.

Olive oil-based method for the extraction, quantification and identification of microplastics in soil and compost samples.

Microplastics (MPs) have become a pressing environmental concern over the past few years and their extraction from solid samples is a scientific challenge that needs to be faced and solved. Standardized and validated protocols for MPs extraction are lacking and the existing methodology, such as density separation, is often unable to separate high density polymers. The aim of our research was to develop a non-density based, inexpensive, simple and safe method to extract MPs from soil and compost samples. We tested an oil-based extracting technique exploiting the oleophilic properties of plastics. For validating the method, soil and compost samples were spiked with six different micro-polymers: polyethylene, polystyrene, polyvinyl chloride, polycarbonate, polyethylene terephthalate and polyurethane. The obtained results are promising, and the polymer density had only a small role in the recovery rate: low, medium and high density polymers reached a mean recovery rate of 90% ±2%, 97% ± 5% and 95% ± 4%, respectively.

Insect herbivory dampens Subarctic birch forest C sink response to warming.

Climate warming is anticipated to make high latitude ecosystems stronger C sinks through increasing plant production. This effect might, however, be dampened by insect herbivores whose damage to plants at their background, non-outbreak densities may more than double under climate warming. Here, using an open-air warming experiment among Subarctic birch forest field layer vegetation, supplemented with birch plantlets, we show that a 2.3 °C air and 1.2 °C soil temperature increase can advance the growing season by 1-4 days, enhance soil N availability, leaf chlorophyll concentrations and plant growth up to 400%, 160% and 50% respectively, and lead up to 122% greater ecosystem CO2 uptake potential. However, comparable positive effects are also found when insect herbivory is reduced, and the effect of warming on C sink potential is intensified under reduced herbivory. Our results confirm the expected warming-induced increase in high latitude plant growth and CO2 uptake, but also reveal that herbivorous insects may significantly dampen the strengthening of the CO2 sink under climate warming.

Species-specific effects of live roots and shoot litter on soil decomposer abundances do not forecast plant litter-nitrogen uptake.

Plant species produce litter of varying quality and differ in the quality and quantity of compounds they release from live roots, which both can induce different decomposer growth in the soil. To test whether differences in decomposer growth can forecast the amount of N species acquire from plant litter, as suggested by theory, we grew individuals of three grassland plants-Holcus lanatus, Plantago lanceolata and Lotus corniculatus-in soils into which (15)N-labelled litter of either Holcus, Plantago or Lotus was added. We measured the effects of live roots and litter of each species on soil microbes and their protozoan and nematode feeders, and to link decomposer growth and plant nutrient uptake, we measured the amount of N taken up by plants from the added litter. We hypothesised that those species that induce the highest growth of microbes, and especially that of microbial feeders, will also take up the highest amount of N from the litter. We found, however, that although numbers of bacterial-feeding Protozoa and nematodes were on average lower after addition of Holcus than Plantago or Lotus litter, N uptake was higher from Holcus litter. Further, although the effects on Protozoa and bacterial- and fungal-feeding nematodes did not differ between the live plants, litter-N uptake differed, with Holcus being the most efficient compared to Plantago and Lotus. Hence, although microbes and their feeders unquestionably control N mineralization in the soil, and their growth differs among plant species, these differences cannot predict differences in litter-N uptake among plant species. A likely reason is that for nutrient uptake, other species-specific plant traits, such as litter chemistry, root proliferation ability and competitiveness for soil N, override in significance the species-specific ability of plants to induce decomposer growth.

Effects of a glyphosate-based herbicide on soil animal trophic groups and associated ecosystem functioning in a northern agricultural field.

Despite an increasing concern of consequences of using vast amounts of glyphosate-based herbicides in agroecosystems, their potential effects on non-target soil organisms and soil functioning are mostly unknown. It has also been argued that fields in northern latitudes should be under special surveillance as the short active period of decomposers may restrict glyphosate degradation. We investigated the effects of a glyphosate-based herbicide, Roundup, on the abundance of enchytraeids and nematodes, both essential groups in decomposer food webs, and plant litter mass loss and soil availability of mineral N in a two-year agricultural field setting in south-west Finland. Our experiment consisted of (1) non-treated weed plots, (2) plots, where weeds were killed by hoeing, and (3) plots treated with both Roundup and hoeing. We found that killing plants by hoeing had drastic effects on soil fauna and functioning, and apparently, distinguishing these effects from direct glyphosate effects is profoundly important when evaluating glyphosate risks in soils. In contrast, the effects of Roundup on soil fauna and functioning were minor and transient and no glyphosate remains were found in the soil at the end of the experiment. These results suggest that side-effects can be minor and glyphosate degradation effective also in soil under northern climatic conditions.

Intrapopulation Genotypic Variation of Foliar Secondary Chemistry during Leaf Senescence and Litter Decomposition in Silver Birch (Betula pendula).

Abundant secondary metabolites, such as condensed tannins, and their interpopulation genotypic variation can remain through plant leaf senescence and affect litter decomposition. Whether the intrapopulation genotypic variation of a more diverse assortment of secondary metabolites equally persists through leaf senescence and litter decomposition is not well understood. We analyzed concentrations of intracellular phenolics, epicuticular flavonoid aglycones, epicuticular triterpenoids, condensed tannins, and lignin in green leaves, senescent leaves and partly decomposed litter of silver birch, Betula pendula. Broad-sense heritability (H2) and coefficient of genotypic variation (CVG) were estimated for metabolites in senescent leaves and litter using 19 genotypes selected from a B. pendula population in southern Finland. We found that most of the secondary metabolites remained through senescence and decomposition and that their persistence was related to their chemical properties. Intrapopulation H2 and CVG for intracellular phenolics, epicuticular flavonoid aglycones and condensed tannins were high and remarkably, increased from senescent leaves to decomposed litter. The rank of genotypes in metabolite concentrations was persistent through litter decomposition. Lignin was an exception, however, with a diminishing genotypic variation during decomposition, and the concentrations of lignin and condensed tannins had a negative genotypic correlation in the senescent leaves. Our results show that secondary metabolites and their intrapopulation genotypic variation can for the most part remain through leaf senescence and early decomposition, which is a prerequisite for initial litter quality to predict variation in litter decomposition rates. Persistent genotypic variation also opens an avenue for selection to impact litter decomposition in B. pendula populations through acting on their green foliage secondary chemistry. The negative genotypic correlations and diminishing heritability of lignin concentrations may, however, counteract this process.

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.

Biostimulation proved to be the most efficient method in the comparison of in situ soil remediation treatments after a simulated oil spill accident.

The use of in situ techniques in soil remediation is still rare in Finland and most other European countries due to the uncertainty of the effectiveness of the techniques especially in cold regions and also due to their potential side effects on the environment. In this study, we compared the biostimulation, chemical oxidation, and natural attenuation treatments in natural conditions and pilot scale during a 16-month experiment. A real fuel spill accident was used as a model for experiment setup and soil contamination. We found that biostimulation significantly decreased the contaminant leachate into the water, including also the non-aqueous phase liquid (NAPL). The total NAPL leachate was 19 % lower in the biostimulation treatment that in the untreated soil and 34 % lower in the biostimulation than oxidation treatment. Soil bacterial growth and community changes were first observed due to the increased carbon content via oil amendment and later due to the enhanced nutrient content via biostimulation. Overall, the most effective treatment for fresh contaminated soil was biostimulation, which enhanced the biodegradation of easily available oil in the mobile phase and consequently reduced contaminant leakage through the soil. The chemical oxidation did not enhance soil cleanup and resulted in the mobilization of contaminants. Our results suggest that biostimulation can decrease or even prevent oil migration in recently contaminated areas and can thus be considered as a potentially safe in situ treatment also in groundwater areas.

Genotype × herbivore effect on leaf litter decomposition in Betula Pendula saplings: ecological and evolutionary consequences and the role of secondary metabolites.

Plant genetic variation and herbivores can both influence ecosystem functioning by affecting the quantity and quality of leaf litter. Few studies have, however, investigated the effects of herbivore load on litter decomposition at plant genotype level. We reduced insect herbivory using an insecticide on one half of field-grown Betula Pendula saplings of 17 genotypes, representing random intrapopulation genetic variation, and allowed insects to naturally colonize the other half. We hypothesized that due to induced herbivore defence, saplings under natural herbivory produce litter of higher concentrations of secondary metabolites (terpenes and soluble phenolics) and have slower litter decomposition rate than saplings under reduced herbivory. We found that leaf damage was 89 and 53% lower in the insecticide treated saplings in the summer and autumn surveys, respectively, which led to 73% higher litter production. Litter decomposition rate was also affected by herbivore load, but the effect varied from positive to negative among genotypes and added up to an insignificant net effect at the population level. In contrast to our hypothesis, concentrations of terpenes and soluble phenolics were higher under reduced than natural herbivory. Those genotypes, whose leaves were most injured by herbivores, produced litter of lowest mass loss, but unlike we expected, the concentrations of terpenes and soluble phenolics were not linked to either leaf damage or litter decomposition. Our results show that (1) the genetic and herbivore effects on B. pendula litter decomposition are not mediated through variation in terpene or soluble phenolic concentrations and suggest that (2) the presumably higher insect herbivore pressure in the future warmer climate will not, at the ecological time scale, affect the mean decomposition rate in genetically diverse B. pendula populations. However, (3) due to the significant genetic variation in the response of decomposition to herbivory, evolutionary changes in mean decomposition rate are possible.

Effects of microbivore species composition and basal resource enrichment on trophic-level biomasses in an experimental microbial-based soil food web.

Previous theoretical and empirical evidence suggests that species composition within trophic levels may profoundly affect the response of trophic-level biomasses to enhanced basal resources. To test whether species composition of microbivorous nematodes has such an effect in microbial-based soil food webs, I created three microcosm food webs, consisting of bacteria, fungi, bacterial-feeding nematodes (Acrobeloides tricornus, Caenorhabditis elegans), fungal-feeding nematodes (Aphelenchus avenae, Aphelenchoides sp.) and a predatory nematode (Prionchulus punctatus). The food webs differed in species composition at the second trophic level: food web A included A. tricornus and Aph. avenae, food web B included C. elegans and Aphelenchoides sp., and food web AB included all four species. I increased basal resources by adding glucose to half of the replicates of each food web, and sampled microcosms destructively four times during a 22-week experiment to estimate the biomass of organisms at each trophic level. Microbivore species composition significantly affected bacterivore and fungivore biomass but not bacterial, fungal or predator biomass. Greatest bacterivore and fungivore biomass was found in food web A, intermediate biomass in food web AB, and smallest biomass in food web B. Basal resource addition increased the biomass of microbes and microbivores but did not affect predator biomass. Importantly, microbivore species composition did not significantly modify the effect of additional resources on trophic-level biomasses. The presence of a competitor reduced the biomass of A. tricornus and Aph. avenae, in that the biomass of these species was less in food web AB than in food web A, whereas the biomass of C. elegans and Aphelenchoides sp. was not affected by their potential competitors. The biomass of Aph. avenae increased with additional resources in the absence of the competitor only, while the biomass of A. tricornus and Aphelenchoides sp. increased also in the presence of their competitors. The results imply that microbivore species composition may determine the second-level biomass in simple microbe-nematode food webs, but may not significantly affect biomass at other levels or modify the response of trophic-level biomasses to enhanced basal resources. The study also shows that even if the role of predation in a food web is diminished, the positive response of organisms to increased resource availability may still be hindered by competition.

Studying the effects of plant species richness on ecosystem functioning: does the choice of experimental design matter?

We established two parallel greenhouse experiments to empirically test the effect of experimental design on the conclusions made of the effects of plant species richness on ecosystem functioning. The experiments included an identical group of six grassland plants and were performed under identical environmental conditions, but were set up according to two different designs. In the richness design (RD), which specifically aims at testing the effect of species richness, each replicate at each richness level was chosen by a separate random draw from the total pool of species, whereas in the richness and composition design (RCD), which aims at testing the effect of both richness and composition, richness levels included deliberately replicated monocultures (at the one-species level) and mixtures (at other levels) of constituent species. When regression analysis was applied, both experimental designs found a positive effect of plant species richness on primary production (estimated using shoot mass accumulation), with species richness explaining 34% and 16% of the total variation in production in RD and RCD, respectively. Based on an overyielding analysis, this positive overall effect of species richness on primary production was in both experiments caused by the sampling effect rather than complementarity. When analysis of variance was applied, RCD also found that shoot production was greatly affected by the presence of one species, the legume Trifolium hybridum, and that 98-99% of variation in primary production within richness levels, i.e. the variation not explained by species richness, was explained by species composition. It appears that while the RD experiment suggests that plant species richness was a significant controller of primary production in our experimental grassland community, the RCD experiment suggests that primary production was mainly determined by plant species composition, i.e. the identity of the species present. Our results are consistent with earlier mathematical simulations in that experimental designs differ in their ability to discriminate the effects of species richness and composition on ecosystem functioning and may therefore lead to different conclusions of the role of species richness in functioning. We propose that this may partly explain the different relative role of plant species richness in ecosystem functioning in earlier investigations using RD and RCD.