Natural Abundance Stable Isotope Research Articles

Quantifying seasonal precipitation using high-resolution carbon isotope analyses in evergreen wood

High-resolution natural abundance stable carbon isotope analyses across annual growth rings in evergreen trees reveal a cyclic increase and decrease in the measured carbon isotopic composition (δ 13C), but the causes of this pattern are poorly understood. We compiled new and published high-resolution δ 13C data from across annual growth rings of 33 modern evergreen trees from 10 genera and 15 globally distributed sites to quantify the parameters that affect the observed δ 13C pattern. Across a broad range of latitude, temperature, and precipitation regimes, we found that the average, measured seasonal change in δ 13C (Δδ 13C meas, ‰) within tree rings of evergreen species reflects changes in the carbon isotopic composition of atmospheric carbon dioxide (Δδ 13C CO2) and changes in seasonal precipitation (Δ P) according to the following equation: Δδ 13C meas = Δδ 13C CO2 – 0.82(Δ P) + 0.73; R 2 = 0.96. Seasonal changes in temperature, pCO 2, and light levels were not found to significantly affect Δδ 13C meas. We propose that this relationship can be used to quantify seasonal patterns in paleoprecipitation from intra-ring profiles of δ 13C measured from non-permineralized, fossil wood.

The role of cyanobacteria in Southern California salt marsh food webs

AbstractUnderstanding wetland food webs is critical for effective habitat management, restoration and conservation. Microalgae are recognized as key food sources for marsh invertebrates but the importance of different groups under various conditions is rarely examined. We tested the hypothesis that faunal utilization of microalgae, and cyanobacteria in particular, is significant in Southern California created and natural salt marshes but varies with habitat type (creek bank versus marsh interior) and season (spring versus autumn). We used stable isotope analysis and mixing models (IsoSource) to compare food webs in adjacent young (created) and mature (natural) salt marshes. Isotopic values of some primary producers, macrofauna, epifauna, and fish demonstrated significant differences between the adjacent salt marshes. δ13C and δ34S values of the benthic microalgal community varied with taxonomic composition (diatoms versus cyanobacteria) and to a lesser extent with season. Depleted δ15N values of benthic diatoms and macroalgae indicated that N2 fixed within algal mats was recycled within the benthic algal community. Marsh fauna, including most major macrofauna taxal, Cerithidea, and Fundulus, also exhibited seasonal differences in isotopic composition, and Cerithidea and selected macrofauna (oligochaetes, polychaetes) from the marsh interior were more enriched in 13C and depleted in 15N than those from the creek bank. In the young marsh, the cyanobacteria contributed a minimum of 17–100% of the primary production in food webs supporting macrofauna, and cyanobacteria contributed at least 40% of the primary production included in Cerithidea and Fundulus food webs. A wider range of primary producers contributed to food webs in the mature marsh. Cyanobacteria were a greater source of trophic support for macrofauna from the marsh interior than the creek bank, whereas Spartina was a more important food source for creek bank macrofauna in both marshes. Insect larvae largely consumed cyanobacteria, whereas polychaetes exhibited greater utilization of Spartina. Phytoplankton was the primary food resource for mussels in both marshes. Although the spatial and temporal complexity of food webs has traditionally been collapsed into the study of relatively simplified food webs, isotope signatures reveal fine‐scale patterns in food web structure that may be used to make more accurate assessments of ecosystem state. Accurate interpretation of marsh trophic structure using natural abundance stable isotopes requires fine‐scale resolution in space and time, a large number of samples, and a high level of taxonomic resolution.

Origin of positive δ 13C of emitted CO 2 from soils after application of biogas residues

Bioenergy production from renewable organic material is known to be a clean energy source and therefore its use is currently much promoted in many countries. Biogas by-products also called biogas residues (BGR) are rich in partially stable organic carbon and can be used as an organic fertilizer for crop production. However so far, many environmental issues relevant when BGR are applied to agricultural land (soil C sequestration, increased denitrification and nutrient leaching) still have to be studied. Therefore a field experiment was set up to investigate the degradation of BGR and its impact on the decomposition of native soil organic matter based on a natural abundance stable isotope approach. Maize, a C 4 plant has been used as bioenergy crop, therefore the δ 13C of total C in BGR was −16.0‰ PDB and soil organic matter was mostly derived from C 3 plant based detritus, SOM thus showed a δ 13C of −28.4‰ PDB. Immediately after BGR application, soil-emitted CO 2 showed unexpectedly high δ 13C of up to +23.6‰ PDB, which has never been reported earlier. A subsequent laboratory scale experiment confirmed the positive δ 13C of soil-emitted CO 2 after BGR addition and showed that obviously, the added BGR led to a consumption of dissolved inorganic C in soils. Additionally, it was observed that the δ 13C of CO 2 driven from inorganic C of BGR (BGR-IC) by acid treatment was +35.6‰ PDB. Therefore, we suggest that also under field conditions the transformation of BGR-IC into CO 2 contributed largely to CO 2 emissions in addition to the decomposition of organic matter, which affected both the amount and the carbon isotope signature of emitted CO 2 in the initial period after BGR application. Positive δ 13C of inorganic C contained in BGR was attributed to processes with strong fractionation of C isotopes during anaerobic fermentation in the biogas formation process.

Association of δ 13C in Fingerstick Blood with Added-Sugar and Sugar-Sweetened Beverage Intake

A reliance on self-reported dietary intake measures is a common research limitation, thus the need for dietary biomarkers. Added-sugar intake may play a role in the development and progression of obesity and related comorbidities; common sweeteners include corn and sugar cane derivatives. These plants contain a high amount of 13C, a naturally occurring stable carbon isotope. Consumption of these sweeteners, of which sugar-sweetened beverages are the primary dietary source, might be reflected in the δ 13C value of blood. Fingerstick blood represents an ideal substrate for bioassay because of its ease of acquisition. The objective of this investigation was to determine if the δ 13C value of fingerstick blood is a potential biomarker of added-sugar and sugar-sweetened beverage intake. Individuals aged 21 years and older (n=60) were recruited to attend three laboratory visits; assessments completed at each visit depended upon a randomly assigned sequence (sequence one or two). The initial visit included assessment of height, weight, and dietary intake (sequence one: beverage intake questionnaire, sequence two: 4-day food intake record). Sequence one participants completed a food intake record at visit two, and nonfasting blood samples were obtained via routine fingersticks at visits one and three. Sequence two participants completed a beverage intake questionnaire at visit two, and provided fingerstick blood samples at visits two and three. Samples were analyzed for δ 13C value using natural abundance stable isotope mass spectrometry. δ 13C value was compared to dietary outcomes in all participants, as well as among those in the highest and lowest tertile of added-sugar intake. Reported mean added-sugar consumption was 66±5 g/day, and sugar-sweetened beverage consumption was 330±53 g/day and 134±25 kcal/day. Mean fingerstick δ 13C value was −19.94‰±0.10‰, which differed by body mass index status. δ 13C value was associated (all P<0.05) with intake of total added sugars (g, r=0.37; kcal, r=0.37), soft drinks (g, r=0.26; kcal, r=0.27), and total sugar-sweetened beverage (g, r=0.28; kcal, r=0.35). The δ 13C value in the lowest and the highest added-sugar intake tertiles were significantly different (mean difference=−0.48‰; P=0.028). Although there are several potential dietary sources for blood carbon, the δ 13C value of fingerstick blood shows promise as a noninvasive biomarker of added-sugar and sugar-sweetened beverage intake based on these findings.

Biofilm recovery in a wastewater treatment plant‐influenced stream and spatial segregation of ammonia‐oxidizing microbial populations

We monitored the effects of wastewater treatment plant (WWTP) inputs on the recovery of stream biofilms after a large flood event that eroded most of the former biofilm communities. We monitored biomass recovery, chlorophyll a, nitrogen content, and stable isotope natural abundance (15N) over 8 weeks in light‐ and dark‐exposed biofilms upstream and downstream from WWTP inputs, respectively, as well as the abundance of ammonia oxidizers by quantitative polymerase chain reaction. Biomass and chlorophyll a recovered quickly (&lt; 2 weeks), and were significantly higher for light‐ than for dark‐exposed biofilms. There was no consistent effect of WWTP inputs on these parameters, except for the biomass on dark‐exposed biofilm that was higher at the WWTP‐influenced sites. The influence of the WWTP inputs on stream‐water ammonium concentration and its isotopic 15N signature increased as the flood receded. Biofilm 15N downstream of WWTP increased over time, tracking the increase in 15N‐ammonium from the WWTP waters. Bacterial and archaeal ammonia oxidizers were present within the biofilm assemblages from early stages of postflood recovery. However, spatial distribution of these two clades was clearly segregated among sites and between light‐ and dark‐exposed biofilms, probably related to ammonium availability and the development of photoautotrophic organisms.

Stable isotope signatures confirm carbon and nitrogen gain through ectomycorrhizas in the ghost orchid Epipogium aphyllum Swartz*

Epipogium aphyllum is a rare Eurasian achlorophyllous forest orchid known to associate with fungi that form ectomycorrhizas, while closely related orchids of warm humid climates depend on wood- or litter-decomposer fungi. We conducted (13) C and (15) N stable isotope natural abundance analyses to identify the organic nutrient source of E. aphyllum from Central Norway. These data for orchid shoot tissues, in comparison to accompanying autotrophic plants, document C and N flow from ectomycorrhizal fungi to the orchid. DNA data from fungal pelotons in the orchid root cortex confirm the presence of Inocybe and Hebeloma, which are both fungi that form ectomycorrhizas. The enrichment factors for (13) C and (15) N of E. aphyllum are used to calculate a new overall average enrichment factor for mycoheterotrophic plants living in association with ectomycorrhizal fungi (ε(13) C ± 1 SD of 7.2 ± 1.6 ‰ and ε(15) N ± 1 SD of 12.8 ± 3.9 ‰). These can be used to estimate the fungal contribution to organic nutrient uptake by partially mycoheterotrophic plants where fully mycoheterotrophic plants are lacking. N concentrations in orchid tissue were unusually high and significantly higher than in accompanying autotrophic leaf samples. This may be caused by N gain of E. aphyllum from obligate ectomycorrhizal fungi. We show that E. aphyllum is an epiparasitic mycoheterotrophic orchid that depends on ectomycorrhizal Inocybe and Hebeloma to obtain C and N through a tripartite system linking mycoheterotrophic plants through fungi with forest trees.

13C isotope fractionation during rhizosphere respiration of C3 and C4 plants

Stable carbon isotopes are used extensively to partition total soil CO2 efflux into root-derived rhizosphere respiration or autotrophic respiration and soil-derived heterotrophic respiration. However, it remains unclear whether CO2 from rhizosphere respiration has the same δ13C value as root biomass. Here we investigated the magnitude of 13C isotope fractionation during rhizosphere respiration relative to root biomass in six plant species. Plants were grown in a carbon-free sand-perlite medium inoculated with microorganisms from a farm soil for 62 days inside a greenhouse. We measured the δ13C value of rhizosphere respiration using a closed-circulation 48-hour CO2 trapping method during 40~42 and 60~62 days after sowing. We found a consistent depletion in 13C (0.9~1.7‰) of CO2 from rhizosphere respiration relative to root biomass in three C3 species (Glycine max L. Merr., Helianthus annuus L. and Triticum aestivum L.), but a relatively large depletion in 13C (3.7~7.0‰) in three C4 species (Amaranthus tricolor L., Sorghum bicolor (L.) Moench and Zea mays L. ssp. mays). Overall, our results indicate that CO2 from rhizosphere respiration is more 13C-depleted than root biomass. Therefore, accounting for this 13C fractionation is required for accurately partitioning total soil CO2 efflux into root-derived and soil-derived components using natural abundance stable carbon isotope methods.

Nitrogen stable isotopes in recirculating aquaculture for super-intensive shrimp production: Tracing the effects of water filtration on microbial nitrogen cycling

To understand how filtration affected microbial community nitrogen (N) cycling, natural abundance stable isotope ratios of suspended particles and nitrate (NO3−) were measured in recirculating aquaculture systems (RAS) for the super-intensive culture (425shrimpm−2) of Pacific white shrimp, Litopenaeus vannamei. Samples were collected throughout a trial during which triplicate RAS were equipped with either a foam fractionator (FF) or a propeller-washed bead filter (BF) to determine solids removal and inorganic N cycling efficacy of each filtration technique. Shrimp performance was unaffected by filtration technique. The time course of DIN concentration was consistent with rapid full nitrification (NH4+ to NO2− to NO3−) in the BF treatment. Persistent, elevated NO2− suggested a slower onset of NO2− oxidation in the FF treatment. However, NO3− began to accumulate at approximately the same time in both treatments and NO3− accumulated more rapidly in the FF treatment even though feed N input to the FF treatment was slightly lower than in the BF treatment. Consistently depleted δ15N–NO3− in the FF treatment illustrated isotopic fractionation of the NH4+ pool attributable to NH4+ oxidation. After an initial depletion, δ15N–NO3− in the BF treatment remained significantly enriched relative to the FF treatment, consistent with isotopic fractionation due to denitrification where the filtration mechanism promoted sub-oxic conditions. The latter is supported by significantly lower NO3− and N budget analyses which indicate the potential for N loss. Together these results suggest that, despite identical inocula, the microbial community responded differently to the filtration technique and as a result natural abundance isotopes revealed distinct dominant N cycle pathways.

Quantifying Nitrogen Process Rates in a Constructed Wetland Using Natural Abundance Stable Isotope Signatures and Stable Isotope Amendment Experiments

This study describes the spatial variability in nitrogen (N) transformation within a constructed wetland (CW) treating domestic effluent. Nitrogen cycling within the CW was driven by settlement and mineralization of particulate organic nitrogen and uptake of NO3-. The concentration of NO3- was found to decrease, as the delta15N-NO3- signature increased, as water flowed through the CW, allowing denitrification rates to be estimated on the basis of the degree of fractionation of delta15N-NO3-. Estimates of denitrification hinged on the determination of a net isotope effect (eta), which was influenced byprocesses that enrich or deplete 15NO3- (e.g., nitrification), as well as the rate constants associated with the different processes involved in denitrification (i.e., diffusion and enzyme activity). The influence of nitrification on eta was quantified; however, it remained unclear how eta varied due to variability in denitrification rate constants. A series of stable isotope amendment experiments was used to further constrain the value of eta and calculate rates of denitrification, and nitrification, within the wetland. The maximum calculated rate of denitrification was 956 +/- 187 micromol N m(-2) h(-1), and the maximum rate of nitrification was 182 +/- 28.9 micromol N m(-2) h(-1). Uptake of NO3- was quantitatively more important than denitrification throughoutthe wetland. Rates of N cycling varied spatially within thewetland, with denitrification dominating in the downstream deoxygenated region of the wetland. Studies that use fractionation of N to derive rate estimates must exercise caution when interpreting the net isotope effect. We suggest a sampling procedure for future natural abundance studies that may help improve the accuracy of N cycling rate estimates.

Carbon and nitrogen flows through the benthic food web of a photic subtidal sandy sediment

Carbon and nitrogen flows within the food web of a subtidal sandy sediment were studied using stable isotope natural abundances and tracer addition. Natural abundances of 13 C and 15 N stable isotopes of the consumers and their potential ben- thic and pelagic resources were measured. δ 13 C data revealed that consumers did not feed on the bulk microphytobenthos (MPB) but rather were selective in their food uptake, preferring either benthic diatoms (-16‰), or benthic cyanobacteria (-20‰). MPB was labelled through a pulse-chase experi- ment with 13 C-bicarbonate and 15 N-nitrate. The fate of MPB was followed in the different heterotrophic compartments. Transfer of 13 C and 15 N to consumers was fast, although only a small fraction of total label was transferred to the heterotrophic compartments within the 4 d of the experiment. Heterotrophic bac- teria were responsible for most of the total hetero- trophic incorporation of 13 C. Within the metazoan community, the incorporation of 13 C by the meio- fauna was more than 2-fold that of the macrofauna, despite a significantly lower biomass. The dual labelling also revealed differential feeding or assim- ilation strategies in meio- and macrofauna. The low 13 C: 15 N ratios of the meiofauna (the smaller organ- isms) seemed to indicate that they preferentially assimilated N or specifically grazed on N-rich resources. However, the macrofauna (larger organ- isms) seemed to feed on bulk sediment, consistent with high 13 C: 15 N ratios. This dual approach, which combined natural abundance and a pulse-chase addition of stable isotopes, revealed crucial informa- tion on the key role of MPB in structuring benthic communities in sandy sediments.

Evaluation of a Novel Isotope Biomarker for Dietary Consumption of Sweets

Carbon isotopic signatures ("δ¹³C") might reflect consumption of corn- and cane-based sweeteners. The authors hypothesized that the δ¹³C value of human serum is higher for individuals with high versus low intakes of corn- and cane-based sweeteners (measured as sweetened beverage intake). They conducted a cross-sectional study within the Atherosclerosis Risk in Communities Magnetic Resonance Imaging study (Maryland, 2005-2006). Diet was assessed by food frequency questionnaire, and blinded serum samples were assayed by natural abundance stable isotope mass spectroscopy. Studied were 186 participants (53% male; mean age, 71 years; mean body mass index, 30 kg/m²). Serum δ¹³C values for individuals with high sweetened beverage intakes were significantly higher than for those with low intakes (-19.15‰ vs. -19.47‰, P < 0.001). Serum δ¹³C value increased 0.20‰ for every serving/day of sweetened beverages (P < 0.01). The association between sweetened beverages and serum δ¹³C value remained significant after adjustment for confounding by corn-based product intake (P < 0.001). Serum δ¹³C values were also associated with waist circumference, body mass index, and waist-to-hip ratio. This study provides the first known evidence that the δ¹³C value of human serum differs between persons consuming low and high amounts of sweets. Within the proper framework, serum δ¹³C value could be developed into an objective biomarker promoting more reliable assessment of dietary sweets intake.

Isotopic evidence of partial mycoheterotrophy in the Gentianaceae: Bartonia virginica and Obolaria virginica as case studies

An estimated 10% of plant species have evolved to steal C from their symbiotic fungal partners (mycoheterotrophy), and while physiological evidence for full and partial mycoheterotrophy is well developed in the Orchidaceae and Ericaceae, it is lacking for the majority of other mycoheterotrophic taxa. The family Gentianaceae not only contains several lineages of achlorophyllous mycoheterotrophs, but also contains species that are putative partially mycoheterotrophic. The North American genera Bartonia and Obolaria (Gentianaceae) are green but have leaves reduced to scales or foliose bracts and so have ambiguous mycoheterotrophic status. • We investigated the natural abundance (13)C and (15)N profiles of both genera along with total N and chlorophyll content and investigated mycorrhizal infection using light microscopy. • The shoots of B. virginica were significantly more enriched in (15)N than the surrounding vegetation but not in (13)C. In contrast, the shoots of O. virginica are not enriched in (15)N compared to the surrounding vegetation but were significantly enriched in (13)C. Total N concentrations were significantly higher than the surrounding vegetation in B. virginica, while the collaroid roots of both species were infected by arbuscular mycorrhizal fungi. • This microscopic evidence coupled with the natural abundance stable isotope profiles strongly suggests that both species are partially mycoheterotrophic. However, differences in the root-shoot stable isotopic patterns relative to surrounding vegetation between B. virginica and O. virginica are suggestive of the utilization of different physiological pathways or extent of commitment to mycoheterotrophic C gain.

15N and 13C natural abundance of two mycoheterotrophic and a putative partially mycoheterotrophic species associated with arbuscular mycorrhizal fungi

• In contrast to mycoheterotrophs that associate with ectomycorrhizal and saprotrophic fungi, we know little about the ecophysiology of arbuscular mycorrhizal mycoheterotrophs. Here, we identify the mycorrhizal fungi of two unrelated mycoheterotrophs and one putative partial mycoheterotroph that form arbuscular mycorrhizas, and analyse their carbon (C) and nitrogen (N) isotope signatures. • We used molecular methods to identify the mycorrhizal fungi of Dictyostega orobanchoides, Burmannia capitata (Burmanniaceae) and Voyria aphylla (Gentianaceae). Their C and N sources were investigated by analysing their stable isotope natural abundances (δ(13)C and δ(15)N). In addition, four putative partially mycoheterotrophic Burmannia species were grown ex situ. • We found that both mycoheterotrophs and a green Burmannia species are associated with nonoverlapping Glomeromycota fungi. The investigated mycoheterotrophs are significantly more enriched in (13)C than co-occurring autotrophic plants but lack significant (15)N enrichment. The green Burmannia species is not significantly enriched in (13)C and (15)N compared with surrounding plants and can grow fully autotrophically under controlled conditions. • Our results suggest that mycoheterotrophic Burmanniaceae and Gentianaceae are able to exploit arbuscular mycorrhizal fungi. Green relatives of mycoheterotrophic Burmanniaceae from high-light grassland sites also associate with arbuscular mycorrhizal fungi but we found no evidence that they receive detectable amounts of C from fungi.

Activity, abundance and diversity of nitrifying archaea and bacteria in the central California Current

A combination of stable isotope and molecular biological approaches was used to determine the activity, abundance and diversity of nitrifying organisms in the central California Current. Using (15)NH(4)(+) incubations, nitrification was detectable in the upper water column down to 500 m; maximal rates were observed just below the euphotic zone. Crenarchaeal and betaproteobacterial ammonia monooxygenase subunit A genes (amoA), and 16S ribosomal RNA (rRNA) genes of Marine Group I Crenarchaeota and a putative nitrite-oxidizing genus, Nitrospina, were quantified using quantitative PCR. Crenarchaeal amoA abundance ranged from three to six genes ml(-1) in oligotrophic surface waters to > 8.7 x 10(4) genes ml(-1) just below the core of the California Current at 200 m depth. Bacterial amoA abundance was lower than archaeal amoA and ranged from below detection levels to 400 genes ml(-1). Nitrification rates were not directly correlated to bacterial or archaeal amoA abundance. Archaeal amoA and Marine Group I crenarchaeal 16S rRNA gene abundances were correlated with Nitrospina 16S rRNA gene abundance at all stations, indicating that similar factors may control the distribution of these two groups. Putatively shallow water-associated archaeal amoA types ('Cluster A') decreased in relative abundance with depth, while a deep water-associated amoA type ('Cluster B') increased with depth. Although some Cluster B amoA sequences were found in surface waters, expressed amoA gene sequences were predominantly from Cluster A. Cluster B amoA transcripts were detected between 100 and 500 m depths, suggesting an active role in ammonia oxidation in the mesopelagic. Expression of marine Nitrosospira-like bacterial amoA genes was detected throughout the euphotic zone down to 200 m. Natural abundance stable isotope ratios (delta(15)N and delta(18)O) in nitrate (NO(3)(-)) and nitrous oxide (N(2)O) were used to evaluate the importance of nitrification over longer time scales. Using an isotope mass balance model, we calculate that nitrification could produce between 0.45 and 2.93 micromol m(-2) day(-1) N(2)O in the central California Current, or approximately 1.5-4 times the local N(2)O flux from deep water.

Decreased precipitation exacerbates the effects of sea level on coastal dune ecosystems in open ocean islands

AbstractThe alteration of fresh and marine water cycling is likely to occur in coastal ecosystems as climate change causes the global redistribution of precipitation while simultaneously driving sea‐level rise at a rate of 2–3 mm yr−1. Here, we examined how precipitation alters the ecological effects of ocean water intrusion to coastal dunes on two oceanic carbonate islands in the Bahamas. The approach was to compare sites that receive high and low annual rainfall and are also characterized by seasonal distribution (wet and dry season) of precipitation. The spatial and temporal variations in precipitation serve as a proxy for conditions of altered precipitation which may occur via climate change. We used the natural abundances of stable isotopes to identify water sources (e.g., precipitation, groundwater and ocean water) in the soil–plant continuum and modeled the depth of plant water uptake. Results indicated that decreased rainfall caused the shallow freshwater table on the dune ecosystem to sink and contract towards the inland, the lower freshwater head allowed ocean water to penetrate into the deeper soils, while shallow soils became exceedingly dry. Plants at the drier site that lived nearest to the ocean responded by taking up water from the deeper and consistently moist soil layers where ocean water intruded. Towards the inland, decreased rainfall caused the water table to sink to a depth that precluded both recharge to the upper soil layers and access by plants. Consequently, plants captured water in more shallow soils recharged by infrequent rainfall events. The results demonstrate dune ecosystems on oceanic islands are more susceptible to ocean water intrusion when annual precipitation decreases. Periods of diminished precipitation caused drought conditions, increased exposure to saline marine water and altered water‐harvesting strategies. Quantifying species tolerances to ocean water intrusion and drought are necessary to determine a threshold of community sustainability.

Tracing energy flow in stream food webs using stable isotopes of hydrogen

Summary1. Use of the natural ratios of carbon and nitrogen stable isotopes as tracers of trophic interactions has some clear advantages over alternative methods for food web analyses, yet is limited to situations where organic materials of interest have adequate isotopic separation between potential sources. This constrains the use of natural abundance stable isotope approaches to a subset of ecosystems with biogeochemical conditions favourable to source separation.2. Recent studies suggest that stable hydrogen isotopes (δD) could provide a robust tracer to distinguish contributions of aquatic and terrestrial production in food webs, but variation in δD of consumers and their organic food sources are poorly known. To explore the utility of the stable hydrogen isotope approach, we examined variation in δD in stream food webs in a forested catchment where variation in δ13C has been described previously.3. Although algal δD varied by taxa and, to a small degree, between sites, we found consistent and clear separation (by an average of 67‰) from terrestrial carbon sources. Environmental conditions known to affect algal δ13C, such as water velocity and stream productivity did not greatly influence algal δD, and there was no evidence of seasonal variation. In contrast, algal δ13C was strongly affected by environmental factors both within and across sites, was seasonally variable at all sites, and partially overlapped with terrestrial δ13C in all streams with catchment areas larger than 10 km2.4. While knowledge of isotopic exchange with water and trophic fractionation of δD for aquatic consumers is limited, consistent source separation in streams suggests that δD may provide a complementary food web tracer to δ13C in aquatic food webs. Lack of significant seasonal or spatial variation in δD is a distinct advantage over δ13C for applications in many aquatic ecosystems.

Re-examining the Importance of Fish in the Diets of Stream-dwelling Crayfishes: Implications for Food Web Analyses and Conservation

We examined the diets of two species of stream-dwelling North American crayfishes (Orconectes propinquus and O. rusticus) at eight sites in the Midwestern United States both by measuring natural abundances of stable isotopes of carbon and nitrogen and by quantifying gut contents. Our goals were to test the hypothesis proposed by others that crayfish are primarily carnivorous, and in particular, to examine the frequency of fish consumption by crayfishes. Stable isotope profiles indicated that both species of crayfish were omnivorous and had a trophic position between those of other invertebrate consumers and fishes. Isotope profiles demonstrated that lower quality foods such as leaf litter, periphyton and fine particulate organic matter made up a larger proportion of the diets of both species than did animal material and that fish comprised approximately 12% of the diet of both crayfishes; this percentage was less variable than that of other food types. Gut content analysis corroborated stable isotope results in finding a similar percent occurrence of fish matter in both species. Our results suggest that the consumption of fish by crayfishes is often underestimated or ignored.

Correcting for the effects of natural abundance in stable isotope resolved metabolomics experiments involving ultra-high resolution mass spectrometry

BackgroundStable isotope tracing with ultra-high resolution Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) can provide simultaneous determination of hundreds to thousands of metabolite isotopologue species without the need for chromatographic separation. Therefore, this experimental metabolomics methodology may allow the tracing of metabolic pathways starting from stable-isotope-enriched precursors, which can improve our mechanistic understanding of cellular metabolism. However, contributions to the observed intensities arising from the stable isotope's natural abundance must be subtracted (deisotoped) from the raw isotopologue peaks before interpretation. Previously posed deisotoping problems are sidestepped due to the isotopic resolution and identification of individual isotopologue peaks. This peak resolution and identification come from the very high mass resolution and accuracy of FT-ICR-MS and present an analytically solvable deisotoping problem, even in the context of stable-isotope enrichment.ResultsWe present both a computationally feasible analytical solution and an algorithm to this newly posed deisotoping problem, which both work with any amount of 13C or 15N stable-isotope enrichment. We demonstrate this algorithm and correct for the effects of 13C natural abundance on a set of raw isotopologue intensities for a specific phosphatidylcholine lipid metabolite derived from a 13C-tracing experiment.ConclusionsCorrection for the effects of 13C natural abundance on a set of raw isotopologue intensities is computationally feasible when the raw isotopologues are isotopically resolved and identified. Such correction makes qualitative interpretation of stable isotope tracing easier and is required before attempting a more rigorous quantitative interpretation of the isotopologue data. The presented implementation is very robust with increasing metabolite size. Error analysis of the algorithm will be straightforward due to low relative error from the implementation itself. Furthermore, the algorithm may serve as an independent quality control measure for a set of observed isotopologue intensities.

Heterotrophic carbon gain by the root hemiparasites, Rhinanthus minor and Euphrasia rostkoviana (Orobanchaceae)

Hemiparasitic plants gain virtually all mineral nutrients and water from their host plant whilst organic carbon is provided, at least in part, by their own photosynthetic activity, although their rates of assimilation are substantially lower than that found in non-parasitic plants. Hence, hemiparasites must gain at least some of their organic carbon heterotrophically from the host plant. Despite this, heterotrophic carbon gain by root hemiparasites has been investigated only for a few genera. We investigated heterotrophic carbon gain by two root hemiparasites, Rhinanthus minor L. and Euphrasia rostkoviana Hayne (Orobanchaceae), using natural abundance stable isotope (delta(13)C) profiles of both parasites attached to C(3) (wheat) and C(4) (maize) hosts coupled to a linear two-source isotope-mixing model to estimate the percentage of carbon in the parasite that was derived from the host. Both R. minor and E. rostkoviana attached to maize hosts were significantly more enriched in (13)C than those attached to wheat hosts with R. minor becoming more enriched in (13)C than E. rostkoviana. The natural abundance (13)C profiles of both parasites were not significantly different from their wheat hosts, but were less enriched in (13)C than maize hosts. Using a linear two-source isotope-mixing model, we estimated that R. minor and E. rostkoviana adult plants derive c. 50 and 25% of their carbon from their hosts, respectively. In light of these results, we hypothesise that repeatedly observed negative effect of competition for light on hemiparasites acts predominantly in early ontogenetic stages when parasites grow unattached or the abstraction of host nutrients is less effective.

Irradiance governs exploitation of fungi: fine-tuning of carbon gain by two partially myco-heterotrophic orchids.

While all members of the Orchidaceae are fully dependent on mycorrhizal fungi during their achlorophyllous juvenile stages, mature plants may remain fully myco-heterotrophic, become fully autotrophic or develop a nutritional mode where the carbon gain through photosynthesis is complemented by organic carbon from fungal partners. This so-called partial myco-heterotrophy is intriguingly complex. Current knowledge indicates a large range in the proportion of fungus-derived carbon between and within partially myco-heterotrophic plant species. However, the driving factors for this variation are so far mostly unknown. Here we show for two green species of the orchid genus Cephalanthera that light availability is the major determinant of the degree of myco-heterotrophy. Using leaf stable isotope natural abundance analysis together with time-integrated microscale light climate monitoring we could demonstrate that there is a sensitive reaction to varying light availability within forests. Low light levels result in strong myco-heterotrophy while higher irradiances successively drive the orchids towards autotrophy. Our results demonstrate that partial myco-heterotrophy in these species is not a static nutritional mode but a flexible mechanism driven by light availability which allows a balanced usage of carbon resources available in nature.