Trophic niche differentiation in soil microarthropods (Oribatida, Acari): evidence from stable isotope ratios (N/N)

The large number of animals that coexist in soil without any clear niche differentiation has puzzled biologists for a long time. We investigated stable isotope ratios (¹⁵N/¹⁴N) in a diverse group of soil microarthropods, oribatid mites, to evaluate trophic niche differentiation. The natural variation of the stable isotopes ¹⁵N/¹⁴N was measured in 36 species/taxa from four beech and beech-oak forests. Signatures of δ¹⁵N formed a gradient spanning over 12 δ units suggesting that (a) different species occupy different trophic niches and (b) oribatid mites span three to four trophic levels. This study for the first time documented strong trophic niche differentiation in decomposer microarthropods. The results suggest that trophic niche differentiation within taxonomic groups significantly contributes to the high diversity of soil animal taxa.     

Compartmentalization of the soil animal food web as indicated by dual analysis of stable isotope ratios (N/N and C/C)

The soil animal food web has become a focus of recent ecological research but trophic relationships still remain enigmatic for many taxa. Analysis of stable isotope ratios of N and C provides a powerful tool for disentangling food web structure. In this study, animals, roots, soil and litter material from a temperate deciduous forest were analysed. The combined measurement of δ¹⁵N and δ¹³C provided insights into the compartmentalization of the soil animal food web. Leaf litter feeders were separated from animals relying mainly on recent belowground carbon resources and from animals feeding on older carbon. The trophic pathway of leaf litter-feeding species appears to be a dead end, presumably because leaf litter feeders (mainly diplopods and oribatid mites) are unavailable to predators due to large size and/or strong sclerotization. Endogeic earthworms that rely on older carbon also appear to exist in predator-free space. The data suggest that the largest trophic compartment constitutes of ectomycorrhizal feeders and their predators. Additionally, there is a smaller trophic compartment consisting of predators likely feeding on enchytraeids and potentially nematodes.     

Dissipation of bacterially derived C and N through the meso- and macrofauna of a grassland soil

Feeding relationships between organisms may be determined by observations of behaviour in manipulative experiments or, as in more recent times, by the use of stable isotope labelling to trace the passage of ¹³C and ¹⁵N through food webs. Here we introduce living bacteria, labelled with both ¹³C and ¹⁵N into intact soil cores to understand further the movement of bacterially sourced C and N into the meso- and macrofauna of a grassland soil. We found that these groups showed a range of isotope levels which relate to their feeding strategies. Some had no label (e.g. dipterous larvae), whilst others were highly labelled which may indicate a preference for the added bacteria. This latter group included Collembola, generally perceived as being predominantly fungal feeders. This work describes a novel technique which has the potential to provide critical information about the dissipation of bacterially derived C and N through the soil food web.     

Effects of food quality, starvation and life stage on stable isotope fractionation in Collembola

Naturally occurring stable isotopes of carbon and nitrogen are powerful tools to investigate food webs, where the ratio of ¹⁵N/¹⁴N is used to assign trophic levels and of ¹³C/¹²C to determine the food source. A shift in δ¹⁵N value of 3‰ is generally suggested as mean difference between two trophic levels, whereas the carbon isotope composition of a consumer is assumed to reflect the signal of its diet. This study investigates the effects of food quality, starvation and life stage on the stable isotope fractionation in fungal feeding Collembola. The fractionation of nitrogen was strongly affected by food quality, i.e. the C/N ratio of the fungal diet. Collembola showed enrichment in the heavier isotope with increasing N concentration of the food source. Δ¹⁵N varied between 2.4‰, which assigns a shift in one trophic level, and 6.3‰, suggesting a shift in two trophic levels. Starvation up to 4 weeks resulted in an increase in the total δ¹⁵N value from 2.8‰ to 4.0‰. Different life stages significantly affected the isotope discrimination by Collembola with juveniles showing a stronger enrichment (Δ¹⁵N=4.9‰) compared to adults (Δ¹⁵N=3.5‰). Δ¹³C varied between −2.1‰ and −3.3‰ depending on the food quality, mainly due to compensational feeding on low quality diet. During starvation δ¹³C value decreased by 1.1‰, whereas the life stage of Collembola had no significant effect on isotopic ratios. The results indicate that the food resource and the physiological status of the consumer have important impact on stable isotope discrimination. They may cause differences in fractionation rate comparable to trophic level shifts, a fact to consider when analysing food web structure.     

Trophic shift of stable isotopes and fatty acids in Collembola on bacterial diets

Fatty acid (FA) analysis is a promising tool to study trophic relationships in soil food webs. We determined FA biomarkers to trace bacterial food sources (Bacillus megaterium, Pseudomonas putida, Enterobacter aerogenes) of Collembola (Heteromurus nitidus, Protaphorura fimata, Folsomia candida). In addition, δ¹⁵N, δ¹³C, C/N ratio, body weight and NLFA/PLFA ratio (neutral lipid/phospholipid fatty acids) of Collembola were assessed. These measures indicated that P. putida ranked first, B. megaterium second and E. aerogenes third in food quality. FAs specific for bacteria were found in the NLFAs of the Collembola reflecting the respective bacterial diet. Biomarker FAs for gram-positive bacteria were methyl branched i14:0, i15:0, a15:0 and i17:0. Consumption of gram-negative bacteria was reflected by the cyclic form cy17:0 (E. aerogenes, P. putida) and by 16:1ω5 (P. putida).     

C and N natural abundance of the soil microbial biomass

Stable isotope analysis is a powerful tool in the study of soil organic matter formation. It is often observed that more decomposed soil organic matter is ¹³C, and especially ¹⁵N-enriched relative to fresh litter and recent organic matter. We investigated whether this shift in isotope composition relates to the isotope composition of the microbial biomass, an important source for soil organic matter. We developed a new approach to determine the natural abundance C and N isotope composition of the microbial biomass across a broad range of soil types, vegetation, and climates. We found consistently that the soil microbial biomass was ¹⁵N-enriched relative to the total (3.2 ‰) and extractable N pools (3.7 ‰), and ¹³C-enriched relative to the extractable C pool (2.5 ‰). The microbial biomass was also ¹³C-enriched relative to total C for soils that exhibited a C3-plant signature (1.6 ‰), but ¹³C-depleted for soils with a C4 signature (−1.1 ‰). The latter was probably associated with an increase of annual C3 forbs in C4 grasslands after an extreme drought. These findings are in agreement with the proposed contribution of microbial products to the stabilized soil organic matter and may help explain the shift in isotope composition during soil organic matter formation.     

Organic matter in density fractions of water-stable aggregates in silty soils: Effect of land use

The location of soil organic matter (SOM) within the soil matrix is considered a major factor determining its turnover, but quantitative information about the effects of land cover and land use on the distribution of SOM at the soil aggregate level is rare. We analyzed the effect of land cover/land use (spruce forest, grassland, wheat and maize) on the distribution of free particulate organic matter (POM) with a density <1.6 g cm⁻³ (free POM_<1.6), occluded particulate organic matter with densities <1.6 g cm⁻³ (occluded POM_<1.6) and 1.6-2.0 g cm⁻³ (occluded POM_1.6-2.0) and mineral-associated SOM (>2.0 g cm⁻³) in size classes of slaking-resistant aggregates (53-250, 250-1000, 1000-2000, >2000 μm) and in the sieve fraction <53 μm from silty soils by applying a combined aggregate size and density fractionation procedure. We also determined the turnover time of soil organic carbon (SOC) fractions at the aggregate level in the soil of the maize site using the ¹³C/¹²C isotope ratio. SOM contents were higher in the grassland soil aggregates than in those of the arable soils mainly because of greater contents of mineral-associated SOM. The contribution of occluded POM to total SOC in the A horizon aggregates was greater in the spruce soil (23-44%) than in the grassland (11%) and arable soils (19%). The mass and carbon content of both the free and occluded POM fractions were greater in the forest soil than in the grassland and arable soils. In all soils, the C/N ratios of soil fractions within each aggregate size class decreased in the following order: free POM_<1.6>occluded POM_<1.6-2.0>mineral-associated SOM. The mean age of SOC associated with the <53 μm mineral fraction of water-stable aggregates in the Ap horizon of the maize site varied between 63 and 69 yr in aggregates >250 μm, 76 yr in the 53-250 μm aggregate class, and 102 yr in the sieve fraction <53 μm. The mean age of SOC in the occluded POM increased with decreasing aggregate size from 20 to 30 yr in aggregates >1000 μm to 66 yr in aggregates <53 μm. Free POM had the most rapid rates of C-turnover, with residence times ranging from 10 yr in the fraction >2000 μm to 42 yr in the fraction 53-250 μm. Results indicated that SOM in slaking-resistant aggregates was not a homogeneous pool, but consisted of size/density fractions exhibiting different composition and stability. The properties of these fractions were influenced by the aggregate size. Land cover/land use were important factors controlling the amount and composition of SOM fractions at the aggregate level.     

Carbon and nitrogen degradation on molecular scale of grass-derived pyrogenic organic material during 28 months of incubation in soil

The present study focuses on the microbial recalcitrance of pyrogenic organic material (PyOM) on a molecular scale. We performed microcosm incubation experiments using ¹³C- and ¹⁵N-enriched grass-derived PyOM mixed with a sub soil material taken from a Haplic Cambisol. Solid-state ¹³C and ¹⁵N NMR studies were conducted to elucidate the humification processes at different stages of PyOM degradation. The chemical structure of the remaining PyOM after incubation was clearly different from the initial pyrogenic material. The proportion of O-containing functional groups was increased, whereas that of aryl C and of N-containing heterocyclic structures had decreased, probably due to mineralisation and conversion to other C and N groups. After 20 months of incubation the aryl C loss reached up to 40% of the initial amount and up to 29% of the remaining PyOM C was assigned to carboxyl/carbonyl C and O-aryl C. These reactions alter the chemical and physical properties of the char residue and make it more available for further microbial attack but also for adsorption processes. Our study presents direct evidence for the degradation of N-heterocyclic domains in charred plant remains adding new aspects to the understanding of the N cycling in fire-affected ecosystems.     

A GC/MS method for the assessment of N and C incorporation into soil amino acid enantiomers

Identifying the transformation process of amino acid enantiomers was essential to probe into the fate, turnover and aging of soil nitrogen due to their important roles in the biogeochemical cycling. If this can be achieved by differentiating between the newly biosynthesized and the inherent compounds in soil, then the isotope tracer method can be considered most valid. We thereby developed a gas chromatography/mass spectrometry (GC/MS) method to trace the ¹⁵N or ¹³C isotope incorporation into soil amino acid enantiomers after being incubated with ¹⁵NH₄⁺ or U-¹³C-glucose substrates. The most significant fragments (F) as well as the related minor ions were monitored by the full scan mode and the isotope enrichment in amino acids was estimated by calculating the atom percentage excess (APE). ¹⁵NH₄⁺ incorporation was evaluated according to the relative abundance increase of m/z F+1 to F for neutral and acidic amino acids and F+2 to F (mass 439) for lysine. The assessment of ¹³C enrichment in soil amino acids was more complicated than that of ¹⁵N due to multi-carbon atoms in amino acid molecules. The abundance ratio increment of m/z F+n to F (n is the original skeleton carbon number in each fragment) indicated the direct conversion from the added glucose to amino acids, but the total isotope incorporation from the added ¹³C can only be calculated according to all target isotope fragments, i.e. the abundance ratio increment summation from m/z (Fa+1) through m/z (Fa+T) represented the total incorporation of the added ¹³C (Fa is the fragment containing all original skeleton carbons and T is the carbon number in the amino acid molecule). This method has a great advantage especially for the evaluation of high-abundance isotope enrichment in organic compounds compared with GC/C/IRMS. And in principle, this technique is also valid for amino acids besides enantiomers if stereoisomers are not concerned. Our assessment approach could shine a light on investigating the biochemical mechanism of microbial transformation of N and C in soils of terrestrial ecosystem.     

Priming effects: Interactions between living and dead organic matter

In this re-evaluation of our 10-year old paper on priming effects, I have considered the latest studies and tried to identify the most important needs for future research. Recent publications have shown that the increase or decrease in soil organic matter mineralization (measured as changes of CO₂ efflux and N mineralization) actually results from interactions between living (microbial biomass) and dead organic matter. The priming effect (PE) is not an artifact of incubation studies, as sometimes supposed, but is a natural process sequence in the rhizosphere and detritusphere that is induced by pulses or continuous inputs of fresh organics. The intensity of turnover processes in such hotspots is at least one order of magnitude higher than in the bulk soil. Various prerequisites for high-quality, informative PE studies are outlined: calculating the budget of labeled and total C; investigating the dynamics of released CO₂ and its sources; linking C and N dynamics with microbial biomass changes and enzyme activities; evaluating apparent and real PEs; and assessing PE sources as related to soil organic matter stabilization mechanisms. Different approaches for identifying priming, based on the assessment of more than two C sources in CO₂ and microbial biomass, are proposed and methodological and statistical uncertainties in PE estimation and approaches to eliminating them are discussed. Future studies should evaluate directions and magnitude of PEs according to expected climate and land-use changes and the increased rhizodeposition under elevated CO₂ as well as clarifying the ecological significance of PEs in natural and agricultural ecosystems. The conclusion is that PEs - the interactions between living and dead organic matter - should be incorporated in models of C and N dynamics, and that microbial biomass should regarded not only as a C pool but also as an active driver of C and N turnover.     

Savanna-derived organic matter remaining in arable soils of the South African Highveld long-term mixed cropping: Evidence from C and N natural abundance

Sustainable agriculture requires the formation of new humus from the crops. We utilized ¹³C and ¹⁵N signatures of soil organic matter to assess how rapidly wheat/maize cropping contributed to the humus formation in coarse-textured savanna soils of the South African Highveld. Composite samples were taken from the top 20 cm of soils (Plinthustalfs) cropped for lengths of time varying from 0 to 98 years, after conversion from native grassland savanna (C4). We performed natural ¹³C and ¹⁵N abundance measurements on bulk and particle-size fractions. The bulk soil δ¹³C values steadily decreased from −14.6 in (C4 dominated) grassland to −16.5‰ after 90 years of arable cropping. This δ¹³C shift was attributable to increasing replacement of savanna-derived C by wheat crop (C3) C which dominated over maize (C4) inputs. After calculating the annual C input from the crop yields and the output from literature data, by using a stepwise C replacement model, we were able to correct the soil δ¹³C data for the irregular maize inputs for a period of about one century. Within 90 years of cropping 41-89% of the remaining soil organic matter was crop-derived in the three studied agroecosystems. The surface soil C stocks after 90 years of the wheat/maize crop rotation could accurately be described with the Rothamsted Carbon Model, but modelled C inputs to the soil were very low. The coarse sand fraction reflected temporal fluctuations in ¹³C of the last C₃ or C₄ cropping and the silt fraction evidenced selective erosion loss of old savanna-derived C. Bulk soil ¹⁵N did not change with increasing cropping length. Decreasing δ¹⁵N values caused by fertilizer N inputs with prolonged arable cropping were only detected for the coarse sand fraction. This indicated that the present N fertilization was not retained in stable soil C pool. Clearly, conventional cropping practices on the South African highlands neither contribute to the preservation of old savanna C and N, nor the effective humus reformation by the crops.     

Protected wading bird species threaten relict centenarian cork oaks in a Mediterranean Biosphere Reserve: A conservation management conflict

Conservation management conflicts frequently arise when an overpopulation of a protected organism has negative effects on other valuable elements in the same ecosystem. We studied the interactions between a colony of protected tree-nesting wading birds and a remnant population of centenarian cork oaks that was part of the formerly dominant forests in the Doñana Biological Reserve (SW Spain). A significant increase in the tree mortality rates has been recorded in areas that are yearly influenced by the bird colony.We analysed a cohort of surviving trees using a gradient of nesting bird influence. Tree-nesting history, bird isotopic signature (δ¹⁵N), tree health-related parameters (defoliation, δ¹³C and leaf surface coverage by faeces) and several soil variables were evaluated. Bird influence was related to increased soil salinity. This increase correlated to increased water-use efficiency for the leaves and to crown defoliation, suggesting that the heavily occupied trees are under higher stress and in poorer health condition than the unoccupied ones. We tested structural equations models (SEM) that were based on hypothesised bird effects on the health of the trees. Soil-mediated effects of the nesting birds best explained the symptoms of the declining health of the trees, whereas the percent of leaves’ surface that was covered by faeces did not improve the fitted SEM model.For the reserve’s managers, a challenging trade-off exists between preserving the relict trees, which have a high genetic diversity and a key ecological role in these savannah-like ecosystems, and maintaining the current nesting area for these protected, but expanding, wading birds.     

Organic C and N stabilization in a forest soil: Evidence from sequential density fractionation

In mineral soil, organic matter (OM) accumulates mainly on and around surfaces of silt- and clay-size particles. When fractionated according to particle density, C and N concentration (per g fraction) and C/N of these soil organo-mineral particles decrease with increasing particle density across soils of widely divergent texture, mineralogy, location, and management. The variation in particle density is explained potentially by two factors: (1) a decrease in the mass ratio of organic to mineral phase of these particles, and (2) variations in density of the mineral phase. The first explanation implies that the thickness of the organic accumulations decreases with increasing particle density. The decrease in C/N can be explained at least partially by especially stable sorption of nitrogenous N-containing compounds (amine, amide, and pyrrole) directly to mineral surfaces, a phenomenon well documented both empirically and theoretically. These peptidic compounds, along with ligand-exchanged carboxylic compounds, could then form a stable inner organic layer onto which other organics could sorb more readily than onto the unconditioned mineral surfaces (“onion” layering model).To explore mechanisms underlying this trend in C concentration and C/N with particle density, we sequentially density fractionated an Oregon andic soil at 1.65, 1.85, 2.00, 2.28, and 2.55 g cm⁻³ and analyzed the six fractions for measures of organic matter and mineral phase properties.All measures of OM composition showed either: (1) a monotonic change with density, or (2) a monotonic change across the lightest fractions, then little change over the heaviest fractions. Total C, N, and lignin phenol concentration all decreased monotonically with increasing density, and ¹⁴C mean residence time (MRT) increased with particle density from ca. 150 years to >980 years in the four organo-mineral fractions. In contrast, C/N, ¹³C and ¹⁵N concentration all showed the second pattern. All these data are consistent with a general pattern of an increase in extent of microbial processing with increasing organo-mineral particle density, and also with an “onion” layering model.X-ray diffraction before and after separation of magnetic materials showed that the sequential density fractionation (SDF) isolated pools of differing mineralogy, with layer-silicate clays dominating in two of the intermediate fractions and primary minerals in the heaviest two fractions. There was no indication that these differences in mineralogy controlled the differences in density of the organo-mineral particles in this soil. Thus, our data are consistent with the hypothesis that variation in particle density reflects variation in thickness of the organic accumulations and with an “onion” layering model for organic matter accumulation on mineral surfaces. However, the mineralogy differences among fractions made it difficult to test either the layer-thickness or “onion” layering models with this soil. Although SDF isolated pools of distinct mineralogy and organic-matter composition, more work will be needed to understand mechanisms relating the two factors.