Food choice of Antarctic soil arthropods clarified by stable isotope signatures

Antarctic soil ecosystems are amongst the most simplified on Earth and include only few soil arthropod species, generally believed to be opportunistic omnivorous feeders. Using stable isotopic analyses, we investigated the food choice of two common and widely distributed Antarctic soil arthropod species using natural abundances of ¹³C and ¹⁵N and an isotope labelling study. In the laboratory we fed the isotomid springtail Cryptopygus antarcticus six potential food sources (one algal species, two lichens and three mosses). Our results showed a clear preference for algae and lichens rather than mosses. These results were corroborated by field data comparing stable isotope signatures from the most dominant cryptogams and soil arthropods (C. antarcticus and the oribatid mite Alaskozetes antarcticus). Thus, for the first time in an Antarctic study, we present clear evidence that these soil arthropods show selectivity in their choice of food and have a preference for algae and lichens above mosses.     

Nitrogen and carbon isotope variability in the green‐algal lichen Xanthoria parietina and their implications on mycobiont–photobiont interactions

Stable isotope patterns in lichens are known to vary largely, but effects of substrate on carbon and nitrogen stable isotope signatures of lichens were previously not investigated systematically. N and C contents and stable isotope (δ¹⁵N, δ¹³C) patterns have been measured in 92 lichen specimens of Xanthoria parietina from southern Bavaria growing on different substrates (bark and stone). Photobiont and mycobiont were isolated from selected populations and isotopically analyzed. Molecular investigations of the internal transcribed spacer of the nuclear ribosomal DNA (ITS nrDNA) region have been conducted on a subset of the specimens of X. parietina. Phylogenetic analysis showed no correlation between the symbionts X. parietina and Trebouxia decolorans and the substrate, isotope composition, or geographic origin. Instead specimens grown on organic substrate significantly differ in isotope values from those on minerogenic substrate. This study documents that the lichens growing on bark use additional or different N sources than the lichens growing on stone. δ¹⁵N variation of X. parietina apparently is controlled predominantly by the mass fraction of the mycobiont and its nitrogen isotope composition. In contrast with mycobionts, photobionts of X. parietina are much more ¹⁵N‐depleted and show less isotopic variability than mycobionts, probably indicating a mycobiont‐independent nitrogen acquisition by uptake of atmospheric ammonia.     

Stable isotopes of Lithosiini and lichens in Hong Kong show the biodindicator potential of lichenivorous moths

Urban landscapes provide unique environments for a wide variety of plants and animals, but their suitability may be limited by anthropogenic impacts such as pollution. We examined the potential utility of lichen and lichen-feeding moths as biodindicators of air pollution in Hong Kong by comparing carbon (C) and nitrogen (N) stable isotope values in lichens, lichenivorous and non-lichenivorous moths (Lepidoptera: Erebidae) and a moth outgroup (Lepidoptera: Geometridae). Our results show that stable isotope values for C and N were similar for lichens and lichen feeding moths, while non-lichen feeding moths formed a distinct group. In addition, we found consistent δ¹³C and δ¹⁵N values across moth body parts, indicating that any portion of the specimen is suitable for isotopic fingerprinting. Our results highlight that lichen feeding moths may be useful for integrating signals of atmospheric nitrogen pollution and could therefore have utility in monitoring and quantifying air quality over time and space.     

Mechanistic studies of sesquiterpene cyclases based on their carbon isotope ratios at natural abundance

During the process of terpene biosynthesis, C–C bond breaking and forming steps are subjected to kinetic carbon isotope effects, leading to distinct carbon isotopic signatures of the products. Accordingly, carbon isotopic signatures could be used to reveal the ‘biosynthetic history’ of the produced terpenoids. Five known sesquiterpene cyclases, regulating three different pathways, representing simple to complex biosynthetic sequences, were heterologously expressed and used for in vitro assays with farnesyl diphosphate as substrate. Compound specific isotope ratio mass spectrometry measurements of the enzyme substrate farnesyl diphosphate (FDP) and the products of all the five cyclases were performed. The calculated δ¹³C value for FDP, based on δ¹³C values and relative amounts of the products, was identical with its measured δ¹³C value, confirming the reliability of the approach and the precision of measurements. The different carbon isotope ratios of the products reflect the complexity of their structure and are correlated with the frequency of carbon–carbon bond forming and breaking steps on their individual biosynthetic pathways. Thus, the analysis of carbon isotopic signatures of terpenes at natural abundance can be used as a powerful tool in elucidation of associated biosynthetic mechanisms of terpene synthases and in future in vivo studies even without ‘touching’ the plant.     

Chemical examinations of some non-vascular Paleozoic plants

The organic chemical constituents of compression fossils ofNematothallus,Spongiophyton, Orestovia andEohostimella are identified and compared with those isolated from living and fossil forms ofBotryococcus (a green alga) andTaeniocrada (a vascular plant fossil). The range and maxima in the carbon numbers observed in the normal, saturated acids isolated fromNematothallus, Orestovia, andSpongiophyton are similar to those of fossilBotryococcus, while those acids contained within compression fossils ofEohostimella are similar to the hydrocarbon composition ofTaeniocrada. Isoprenoid, branched hydrocarbons and steroids identified fromNematothallus, Orestovia, andSpongiophyton suggest these genera have algal affinities, while the presence of thick cuticles and in some cases cutin-like compounds appear to show adaptation to a terrestrial environment. Phenolic compounds retained within rock matrices associated withEohostimella are similar to those isolated fromTaeniocrada suggesting chemical, as well as morphological parallels with the land plant habit. These data are interpreted as indicating an early polyphyletic exploitation of the terrestrial habitat during the Paleozoic.     

Geochemical study of vertebrate fossils from the Upper Cretaceous (Santonian) Csehbánya Formation (Hungary): Evidence for a freshwater habitat of mosasaurs and pycnodont fish

The diverse vertebrate remains from the Upper Cretaceous freshwater settings at Iharkút, Hungary, contain two fossil groups, Pycnodontiformes fish and Mosasauridae that are almost exclusively known from marine palaeo-environments. Hence, their appearance in alluvial sediments is very unusual. Trace element and isotope compositions of the remains have been analyzed to investigate the taphonomy and the ecological differences among the different fossil groups present at Iharkút.All examined fossils have undergone post-depositional diagenetic alteration, which resulted in high concentrations of REE, U, and Fe, together with almost complete homogenization of δ¹⁸O_CO3 values. Similar REE patterns in different fossils suggest a common origin for all remains, hence the discovered species most likely lived in the same local ecosystem. Despite partial diagenetic overprinting, the δ¹⁸O_PO4 values of the fossils indicate sufficient taxon-specific isotopic diversity to permit some broad conclusions on the palaeo-environment of the fossils. In particular, it is apparent that the isotopic composition of the Pycnodontiformes fish and Mosasauridae remains is most compatible with a freshwater palaeo-habitat and incompatible with a marine palaeo-environment. In addition, the Sr concentration and isotope data indicate that the Pycnodontiformes and Mosasauridae likely lived predominantly in a freshwater environment and were not simply occasional visitors to the Iharkút river ecosystem.Regarding other fossil groups, high δ¹⁸O_PO4 values of Alligatoroidea and Iharkutosuchus teeth suggest that these small crocodile species might have inhabited swamps and ponds where the water was relatively rich in ¹⁸O due to evaporation.     

Geochemically distinct carbon isotope distributions in Allochromatium vinosum DSM 180T grown photoautotrophically and photoheterotrophically

Anoxygenic, photosynthetic bacteria are common at redox boundaries. They are of interest in microbial ecology and geosciences through their role in linking the carbon, sulfur, and iron cycles, yet much remains unknown about how their flexible carbon metabolism—permitting either autotrophic or heterotrophic growth—is recorded in the bulk sedimentary and lipid biomarker records. Here, we investigated patterns of carbon isotope fractionation in a model photosynthetic sulfur‐oxidizing bacterium, Allochromatium vinosum DSM180^T. In one treatment, A. vinosum was grown with CO₂ as the sole carbon source, while in a second treatment, it was grown on acetate. Different intracellular isotope patterns were observed for fatty acids, phytol, individual amino acids, intact proteins, and total RNA between the two experiments. Photoautotrophic CO₂ fixation yielded typical isotopic ordering for the lipid biomarkers: δ¹³C values of phytol > n‐alkyl lipids. In contrast, growth on acetate greatly suppressed intracellular isotopic heterogeneity across all molecular classes, except for a marked ¹³C‐depletion in phytol. This caused isotopic “inversion” in the lipids (δ¹³C values of phytol < n‐alkyl lipids). The finding suggests that inverse δ¹³C patterns of n‐alkanes and pristane/phytane in the geologic record may be at least in part a signal for photoheterotrophy. In both experimental scenarios, the relative isotope distributions could be predicted from an isotope flux‐balance model, demonstrating that microbial carbon metabolisms can be interrogated by combining compound‐specific stable isotope analysis with metabolic modeling. Isotopic differences among molecular classes may be a means of fingerprinting microbial carbon metabolism, both in the modern environment and the geologic record.     

Explaining geographical variation in the isotope composition of mouse lemurs (Microcebus)

Aim We sought to quantify geographical variation in the stable isotope values of mouse lemurs (Microcebus) and to determine whether this variation reflects trophic differences among populations or baseline isotopic differences among habitats. If the latter pattern is demonstrated, then Microcebus can become a proxy for tracking baseline habitat isotopic variability. Establishing such a baseline is crucial for identifying niche partitioning in modern and ancient communities. Location We studied five species of Microcebus from eight distinct habitats across Madagascar. Methods We compared isotopic variation in C₃ plants and Microcebus fur within and among localities. We predicted that carbon and nitrogen isotope values of Microcebus should: (1) vary as a function of abiotic variables such as rainfall and temperature, and (2) covary with isotopic values in plants. We checked for trophic differences among Microcebus populations by comparing the average difference between mouse lemur and plant isotope values for each locality. We then used multiple regression models to explain spatial isotope variation in mouse lemurs, testing a suite of explanatory abiotic variables. Results We found substantial isotopic variation geographically. Ranges for mean isotope values were similar for both Microcebus and plants across localities (carbon 3.5–4.0‰; nitrogen 10.5–11.0‰). Mean mouse lemur and plant isotope values were lowest in cool, moist localities and highest in hot, dry localities. Rainfall explained 58% of the variation in Microcebus carbon isotope values, and mean plant nitrogen isotope values explained 99.7% of the variation in Microcebus nitrogen isotope values. Average differences between mouse lemur and plant isotope values (carbon 5.0‰; nitrogen 5.9‰) were similar across localities. Main conclusions Isotopic data suggest that trophic differences among Microcebus populations were small. Carbon isotope values in mouse lemurs were negatively correlated with rainfall. Nitrogen isotope values in Microcebus and plants covaried. Such findings suggest that nitrogen isotope values for Microcebus are a particularly good proxy for tracking baseline isotopic differences among habitats. Our results will facilitate future comparative research on modern mouse lemur communities, and ecological interpretations of extinct Holocene communities.     

Acid treatment effects on the stable isotopic signatures of fossils

Abstract: Prior to geochemical analyses, fossil bones and teeth are often extracted from any surrounding lithified sediments using chemical techniques such as immersion in acid. As stable isotope analysis becomes more commonplace in palaeoecological investigations, it is important to consider what effects these chemical preparation techniques may have on any subsequent isotopic data and to constrain these effects as quantitatively as possible. This study aims to elucidate these effects, as it is vital that variability in a data set should not be introduced as a result of protocols used during sample preparation; in addition, it defines the most effective and viable method of carbonate removal for processing bulk fossil samples without causing alteration of their stable isotopic signatures. Various strengths of two weak acids commonly used during palaeontological preparation were tested to evaluate their effects on the δ¹⁵N and δ¹³C_org isotopic signatures of the vertebrae of a large Eocene fossil fish. Changes in the isotopic values occurred over time regardless of which acid was used, each causing a variable response in both δ¹⁵N and δ¹³C_org isotopic values. Without careful monitoring of the acidification process in a controlled environment, any resulting data could therefore confound interpretation. Based on these experiments, it is recommended that 2 m acetic acid be used for the pretreatment of fossils prior to the acquisition of N and C isotope data where carbonate removal is necessary.     

Combining meteorology, eddy fluxes, isotope measurements, and modeling to understand environmental controls of carbon isotope discrimination at the canopy scale

Estimates of terrestrial carbon isotope discrimination are useful to quantify the terrestrial carbon sink. Carbon isotope discrimination by terrestrial ecosystems may vary on seasonal and interannual time frames, because it is affected by processes (e.g. photosynthesis, stomatal conductance, and respiration) that respond to variable environmental conditions (e.g. air humidity, temperature, light). In this study, we report simulations of the temporal variability of canopy‐scale C₃ photosynthetic carbon isotope discrimination obtained with an ecophysiologically based model (ISOLSM) designed for inclusion in global models. ISOLSM was driven by half‐hourly meteorology, and parameterized with eddy covariance measurements of carbon and energy fluxes and foliar carbon isotope ratios from a pine forest in Metolius (OR). Comparing simulated carbon and energy fluxes with observations provided a range of parameter values that optimized the simulated fluxes. We found that the sensitivity of photosynthetic carbon isotope discrimination to the slope of the stomatal conductance equation (m, Ball–Berry constant) provided an additional constraint to the model, reducing the wide parameter space obtained from the fluxes alone. We selected values of m that resulted in similar simulated long‐term discrimination as foliar isotope ratios measured at the site. The model was tested with ¹³C measurements of ecosystem (δ_R) and foliar (δ_f) respiration. The daily variability of simulated ¹³C values of assimilated carbon (δ_A) was similar to that of observed δ_f, and higher than that of observed and simulated δ_R. We also found similar relationships between environmental factors (i.e. vapor pressure deficit) and simulated δ_R as measured in ecosystem surveys of δ_R. Therefore, ISOLSM reasonably simulated the short‐term variability of δ_A controlled by atmospheric conditions at the canopy scale, which can be useful to estimate the variability of terrestrial isotope discrimination. Our study also shows that including the capacity to simulate carbon isotope discrimination, together with simple ecosystem isotope measurements, can provide a useful constraint to land surface and carbon balance models.     

Trophic Discrimination Factors of Stable Carbon and Nitrogen Isotopes in Hair of Corn Fed Wild Boar

Stable isotope measurements are increasingly being used to gain insights into the nutritional ecology of many wildlife species and their role in ecosystem structure and function. Such studies require estimations of trophic discrimination factors (i.e. differences in the isotopic ratio between the consumer and its diet). Although trophic discrimination factors are tissue- and species- specific, researchers often rely on generalized, and fixed trophic discrimination factors that have not been experimentally derived. In this experimental study, captive wild boar (Sus scrofa) were fed a controlled diet of corn (Zea mays), a popular and increasingly dominant food source for wild boar in the Czech Republic and elsewhere in Europe, and trophic discrimination factors for stable carbon (Δ¹³C) and nitrogen (Δ¹⁵N) isotopes were determined from hair samples. The mean Δ¹³C and Δ¹⁵N in wild boar hair were –2.3 ‰ and +3.5 ‰, respectively. Also, in order to facilitate future derivations of isotopic measurements along wild boar hair, we calculated the average hair growth rate to be 1.1 mm d^-1. Our results serve as a baseline for interpreting isotopic patterns of free-ranging wild boar in current European agricultural landscapes. However, future research is needed in order to provide a broader understanding of the processes underlying the variation in trophic discrimination factors of carbon and nitrogen across of variety of diet types.     

Isotopic and chemical investigations of Fe (III) reduction by Shewanella putrefaciens strain 200R

Experiments were conducted using the Fe⁺³‐reducing bacterium Shewanella putrefaciens strain 200R to determine the stable carbon isotope fractionation during dissimilatory Fe (III) reduction and associated lactate oxidation at circum‐neutral pH. Previous studies used equilibrium fractionation factors (~14.3‰) between bacterial biomass and synthesized fatty acids to identify the predominant carbon fixation pathways for some of the most frequently isolated microbes including Shewanella under anaerobic conditions. We investigated the carbon isotope disproportionation among organic carbon substrate (lactate), biomass and respired carbon dioxide at the lag to stationary phase of the growth curve. Ferric citrate and sodium lactate were used as electron acceptor and donor, respectively. Sodium bicarbonate or potassium phosphate was used as buffering agent. Iron (II), iron (III), dissolved inorganic carbon (DIC) and carbon isotope ratios were measured for both bicarbonate‐ and phosphate‐buffered systems. Carbon isotope ratio measurements were made on the respired CO₂ (as DIC) and microbial biomass for both buffering conditions. The fraction of lactate consumed was estimated using DIC as a proxy and was verified by direct measurement using HPLC. Our result showed that bicarbonate‐buffered system has an enhancing effect in the reduction process compared to the phosphate system. Both systems resulted in carbon isotope fractionations between the lactate substrate and DIC that could be modelled as a Rayleigh process. The biomass produced under both buffer conditions was depleted on average by ~2‰ relative to the substrate and enriched by ~5‰ relative to the DIC. This translates to an overall isotopic fractionation of 10–12‰ between the biomass and respired CO₂ in both buffering systems.     

A diagenetic control on the Early Triassic Smithian–Spathian carbon isotopic excursions recorded in the marine settings of the Thaynes Group (Utah,USA)

In the aftermath of the end‐Permian mass extinction, Early Triassic sediments record some of the largest Phanerozoic carbon isotopic excursions. Among them, a global Smithian‐negative carbonate carbon isotope excursion has been identified, followed by an abrupt increase across the Smithian–Spathian boundary (SSB; ~250.8 Myr ago). This chemostratigraphic evolution is associated with palaeontological evidence that indicate a major collapse of terrestrial and marine ecosystems during the Late Smithian. It is commonly assumed that Smithian and Spathian isotopic variations are intimately linked to major perturbations in the exogenic carbon reservoir. We present paired carbon isotopes measurements from the Thaynes Group (Utah, USA) to evaluate the extent to which the Early Triassic isotopic perturbations reflect changes in the exogenic carbon cycle. The δ¹³C_carb variations obtained here reproduce the known Smithian δ¹³C_carb‐negative excursion. However, the δ¹³C signal of the bulk organic matter is invariant across the SSB and variations in the δ³⁴S signal of sedimentary sulphides are interpreted here to reflect the intensity of sediment remobilization. We argue that Middle to Late Smithian δ¹³C_carb signal in the shallow marine environments of the Thaynes Group does not reflect secular evolution of the exogenic carbon cycle but rather physicochemical conditions at the sediment–water interface leading to authigenic carbonate formation during early diagenetic processes.     

Stable isotopes (δD and δ13C) are geographic indicators of natal origins of monarch butterflies in eastern North America

Wing membranes of laboratory and field-reared monarch butterflies (Danaus plexippus) were analyzed for their stable-hydrogen (δD) and carbon (δ¹³C) isotope ratios to determine whether this technique could be used to identify their natal origins. We hypothesized that the hydrogen isotopic composition of monarch butterfly wing keratin would reflect the hydrogen isotope patterns of rainfall in areas of natal origin where wings were formed. Monarchs were reared in the laboratory on milkweed plants (Asclepias sp.) grown with water of known deuterium content, and, with the assistance of volunteers, on native milkweeds throughout eastern North America. The results show that the stable hydrogen isotopic composition of monarch butterflies is highly correlated with the isotopic composition of the milkweed host plants, which in turn corresponds closely with the long-term geographic patterns of deuterium in rainfall. Stable-carbon isotope values in milkweed host plants were similarly correlated with those values in monarch butterflies and showed a general pattern of enrichment along a southwest to northeast gradient bisecting the Great Lakes. These findings indicate that natal origins of migratory and wintering monarchs in Mexico can be inferred from the combined δD and δ¹³C isotopic signatures in their wings. This relationship establishes that analysis of hydrogen and carbon isotopes can be used to answer questions concerning the biology of migratory monarch butterflies and provides a new approach to tracking similar migratory movements of other organisms. Received: 1 July 1998 / Accepted: 11 November 1998     

SOURCES OF INORGANIC CARBON FOR PHOTOSYNTHESIS BY THREE SPECIES OF MARINE DIATOM1

The utilization of inorganic carbon by three species of marine diatom, Skeletonema costatum (Grev.) Cleve. Ditylum brightwellii (West) Grun., and Chaetoceros calcitrans Paulsen was investigated using an inorganic carbon isotopic disequilibnum technique and inorganic carbon dose-response curves. Stable carbon isotope data of the diatoms are also presented. Observed rates of photosynthetic oxygen evolution were greater than could be accounted for by the theoretical rate of CO₂ supply from the uncatalyzed dehydration of HCO₃^? in the external medium, suggesting use of HCO₃^? as an inorganic carbon source. Data from the isotopic disequilibrium experiment demonstrate the use of both HCO₃^? and CO₂ for photosynthesis. Carbon isotope discrimination values support the use of HCO₃^? by the diatoms.     

Record of archaeal activity at the serpentinite‐hosted Lost City Hydrothermal Field

Samples of young, outer surfaces of brucite–carbonate deposits from the ultramafic‐hosted Lost City hydrothermal field were analyzed for DNA and lipid biomarker distributions and for carbon and hydrogen stable isotope compositions of the lipids. Methane‐cycling archaeal communities, notably the Lost City Methanosarcinales (LCMS) phylotype, are specifically addressed. Lost City is unlike all other hydrothermal systems known to date and is characterized by metal‐ and CO₂‐poor, high pH fluids with high H₂ and CH₄ contents resulting from serpentinization processes at depth. The archaeal fraction of the microbial community varies widely within the Lost City chimneys, from 1–81% and covaries with concentrations of hydrogen within the fluids. Archaeal lipids include isoprenoid glycerol di‐ and tetraethers and C₂₅ and C₃₀ isoprenoid hydrocarbons (pentamethylicosane derivatives – PMIs – and squalenoids). In particular, unsaturated PMIs and squalenoids, attributed to the LCMS archaea, were identified for the first time in the carbonate deposits at Lost City and probably record processes exclusively occurring at the surface of the chimneys. The carbon isotope compositions of PMIs and squalenoids are remarkably heterogeneous across samples and show highly ¹³C‐enriched signatures reaching δ¹³C values of up to +24.6‰. Unlike other environments in which similar structural and isotopic lipid heterogeneity has been observed and attributed to diversity in the archaeal assemblage, the lipids here appear to be synthesized solely by the LCMS. Some of the variations in lipid isotope signatures may, in part, be due to unusual isotopic fractionation during biosynthesis under extreme conditions. However, we argue that the diversity in archaeal abundances, lipid structure and carbon isotope composition rather reflects the ability of the LCMS archaeal biofilms to adapt to chemical gradients in the hydrothermal chimneys and possibly to perform either methanotrophy or methanogenesis using dissolved inorganic carbon, methane or formate as a function of the prevailing environmental conditions.     

Dual‐tracer‐based isotope turnover rates in a highly invasive mysid Limnomysis benedeni from Lake Constance

Understanding the ecological patterns of invasive species and their habitats require an understanding of the species’ foraging ecology. Stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope values provide useful information into the study of animal ecology and evolution, since the isotope ratios of consumers reflect consumer's dietary patterns. Nevertheless, the lack of species‐ and element‐specific laboratory‐derived turnover rates could limit their application. Using a laboratory‐based dual stable isotope tracer approach (Na¹⁵NO₃ and NaH¹³CO₃), we evaluated the δ¹⁵N and δ¹³C isotope turnover rates in full‐grown adult invasive Limnomysis benedeni from Lake Constance. We provide δ¹⁵N and δ¹³C turnover rates based on nonlinear least‐squares regression and posterior linear regression models. Model precisions and fit were evaluated using Akaike's information criterion. Within a couple of days, the δ¹⁵N and δ¹³C of mysids began to change. Nevertheless, after about 14 days, L. benedeni did not reach equilibrium with their new isotope values. Since the experiment was conducted on adult subjects, it is evident that turnover was mainly influenced by metabolism (in contrast to growth). Unlike traditional dietary shifts, our laboratory‐based dual stable isotope tracer approach does not shift the experimental organisms into a new diet and avoids dietary effects on isotope values. Results confirm the application of isotopic tracers to label mysid subpopulations and could be used to reflect assimilation and turnover from the labeled dietary sources. Field‐based stable isotope studies often use isotopic mixing models commonly assuming diet‐tissue steady state. Unfortunately, in cases where the isotopic composition of the animal is not in equilibrium with its diet, this can lead to highly misleading conclusions. Thus, our laboratory‐based isotopic incorporation rates assist interpretation of the isotopic values from the field and provide a foundation for future research into using isotopic tracers to investigate invasion ecology.     

Space‐time tradeoffs in the development of precipitation‐based isoscape models for determining migratory origin

Precipitation stable isotope patterns over continental scales provide a fundamental tool for tracking origins of migratory species. Hydrogen isotopes from rain and environmental waters are assimilated into animal tissues and may thereby reveal the location where tissues were synthesized. Predictive isotopic maps (or isoscapes) of stable hydrogen isotope values in precipitation (δ²H_p) are typically generated by time‐averaging observations from a global network of stations that have been sampled irregularly in space and time. We previously demonstrated that restricting the temporal range in δ²H_p isoscapes to biologically relevant time frames did not improve predictions of geographic origin for two migratory species in North America and Europe; rather, it decreased the accuracy of assignment. Here, we examined whether the reduction in assignment accuracy stemmed from a decrease in the number of sampling stations available to support isoscape development for shorter time periods. Multiple regression models were used to predict the hydrogen isotope composition in precipitation using isotopic measurements from each station along with a suite of independent variables. The reduction in the number of stations with δ²H_p measurements used to estimate isoscape model parameters did not alter the accuracy and precision of assignments consistently. We also examined accuracy across a range of reduced station numbers and found that mean accuracy was affected only at very low numbers of stations, indicating that the spatial isotopic patterns in precipitation that are useful for assignment applications can be characterized with data from relatively limited data stations. The number and spatial distribution of stations may have more influence when geostatistical models are used to generate isoscapes, as they incorporate spatial correlation in the dataset. The results can be used to guide future research in understanding how data availability and constraints in creating δ²H_p isoscapes may affect predictions of geographic origins.     

Discriminating geographic origins of migratory waders at stopover sites: insights from stable isotope analysis of toenails

In this study we test the potential of stable isotope analysis to reveal wintering origins of waders mixing at stopover sites, using the dunlin Calidris alpina as a case study. We determined stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope signatures of toenails of dunlins captured during winter at reference sites along the East‐Atlantic Flyway, from Mauritania to the United Kingdom. Afterwards, during spring migration, dunlins were sampled at the Tagus estuary, Portugal, and assigned to their wintering grounds according to their stable isotope signatures. Toenails from wintering dunlins at different sites had significantly different δ¹³C and δ¹⁵N signatures, despite some overlap in isotopic carbon ratios of birds from Morocco, Portugal and the UK. Among birds sampled during migration in Portugal, we found a clear bimodal pattern in δ¹³C values, corresponding to passage migrants from Mauritania (enriched δ¹³C values) and wintering birds from the Tagus estuary (depleted δ¹³C values). The first passage migrants from Mauritania appeared at the Tagus estuary by the end of March, with peak numbers during late April and early May. Our study provides evidence that isotopic signatures of toenails can play a determinant role in tracing the wintering origins of migrant dunlins at their stopover areas. Toenails, instead of feathers, can be the powerful and innovating tissue to sample in wader studies, allowing to bridge the gap in the field of migratory connectivity between sites used in different phases of the life cycle of waders.     

Tracing site‐specific isotopic signatures along a Blue Tit Cyanistes caeruleus food chain

Food chains culminating with temperate insectivorous passerines are well described, yet whether trophic webs can be site‐specific remains a largely unexplored question. In the case of site‐ or habitat‐specificity of food webs, stable isotope signatures of bird feathers may enable assignment of unmarked individuals to a site or a habitat of origin. We address this question in landscapes that include contrasting forest habitat patches with either deciduous Downy Oak Quercus humilis or evergreen Holm Oak Quercus ilex as dominant tree species. First, we examine the spatial variation across habitats and sites in the stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) along the oak leaf–Tortrix moth Tortrix viridana caterpillar–Blue Tit Cyanistes caeruleus food chain. Secondly, we assess whether the isotopic signatures allow for correct assignment of individual birds to their site or habitat of origin. At the scale of the landscape, stable isotope values enabled identification of the different components of the Blue Tit food chain: from oak leaves to Blue Tit nestlings and yearling birds. However, isotopic signatures were site‐specific (i.e. geographical) more than habitat‐specific (i.e. deciduous vs. evergreen oaks). Discriminant analyses correctly assigned 85% of nestlings and 83% of resident yearling birds, indicating a pronounced effect of site on Blue Tit feather isotopic signatures. We thereby demonstrate that isotopes reflect a stronger association of locally born birds to the local features of their habitat than that of un‐ringed yearling birds, whose plumage may have grown while in a wider geographical area. This study provides evidence of site‐specific isotopic signatures from oak leaves to Blue Tit feathers at a fine spatial scale.