Cavity construction by reintroduced populations of Red‐cockaded Woodpeckers (Dryobates borealis)

Although species reintroduction attempts are now common, monitoring of reintroduction attempts rarely extends beyond initial population establishment. This short timespan likely fails to document long‐term population stability, subtle changes in behavior, and the potentially larger effects that some reintroduced species may have on other species. The Red‐cockaded Woodpecker (RCW; Dryobates borealis) is an important habitat specialist and ecosystem engineer that excavates cavities in living trees. Excavation of natural RCW cavities can take years to complete, but they also persist for many years and are used by many other species. We quantified characteristics of cavity trees excavated by RCWs (n = 44) in two populations that were reintroduced to unoccupied areas more than 10 years earlier. We measured features associated with heartwood rot and used generalized linear mixed effects regression to determine whether these features differed for trees selected for cavity excavation compared with random neighboring trees. We also assessed population trends for cavity‐nesting species that commonly used RCW cavities on one of the sites. Height of first live limb was the only factor distinguishing natural RCW cavity trees from control trees. Four of six cavity‐nesting species monitored increased significantly following RCW establishment. The increases may relate to the many natural and artificial cavities created during the reintroduction effort. Future reintroductions of the RCW should lead to successful natural cavity excavation if sufficiently large trees with smaller live crowns are present. Future efforts may also benefit the broader community of cavity‐nesting birds.     

Beyond carbon and nitrogen: guidelines for estimating three‐dimensional isotopic niche space

Isotopic niche has typically been characterized through carbon and nitrogen ratios and most modeling approaches are limited to two dimensions. Yet, other stable isotopes can provide additional power to resolve questions associated with foraging, migration, dispersal and variations in resource use. The ellipse niche model was recently generalized to n‐dimensions. We present an analogous methodology which incorporates variation across three stable dimensions to estimate the significant features of a population's isotopic niche space including: 1) niche volume (referred to as standard ellipsoid volume, SEV), 2) relative centroid location (CL), 3) shape and 4) area of overlap between multiple ellipsoids and 5) distance between two CLs. We conducted a simulation study showing the accuracy and precision of three dimensional niche models across a range of values. Importantly, the model correctly identifies differences in SEV and CL among populations, even with small sample sizes and in cases where the absolute values cannot precisely be recovered. We use these results to provide guidelines for sample size in conducting multivariate isotopic niche modeling. We demonstrate the utility of our approach with a case study of three bottlenose dolphin populations which appear to possess largely overlapping niches when analyzed with only carbon and nitrogen isotopes. Upon inclusion of sulfur, we see that the three dolphin ecotypes are in fact segregated on the basis of salinity and find the stable isotope niche of inshore bottlenose dolphins significantly larger than coastal and offshore populations.     

Changes in feeding niche widths of perch and roach following biomanipulation, revealed by stable isotope analysis

1. We made an empirical test of a recent proposal that feeding niche widths might be determined as variance of stable isotope values. We determined δ ¹³C and δ ¹⁵N values of perch ( Perca fluviatilis ), roach ( Rutilus rutilus ) and their prey from a biomanipulated lake, when the mass removal of fish led to reduced inter- and intra-specific competition and increases in zooplankton abundance and body size.
2. After the first fish removals, both perch and roach mean δ ¹³C values decreased and mean δ ¹⁵N values increased, indicating a greater diet contribution from pelagic sources.
3. Variances of both δ ¹³C and δ ¹⁵N values first increased in both fish populations, indicating a wider food spectrum and expanded feeding niche width following reduced fish abundances. Observed changes were greater for the perch population than for roach.
4. In 2006, the perch population abruptly changed its diet so that most individuals were primarily consuming the abundant young-of-the-year fish, and this was reflected in significantly reduced variances of both δ ¹³C and δ ¹⁵N values.
5. We conclude that isotopic variance can indeed reflect changes in feeding niche width and offers a promising way to study such general ecological concepts.     

Spatial,seasonal and individual variation in the diet of White‐tailed Eagles Haliaeetus albicilla assessed using stable isotope ratios

Many raptor species are considered to be generalists, taking a range of prey species. However, longitudinal dietary records are often scarce, although necessary for characterizing niche width of species at population and individual levels. Quantifying raptor diets at large spatio‐temporal scales is often necessary for refining conservation efforts, although it can be particularly difficult and may involve a great effort by conventional means. Therefore, we adopted the analysis of stable isotopes in tissues of predators and their potential food sources as a complementary methodology for assessing animals' diet. We examined the isotopic composition (δ¹³C and δ¹⁵N) of White‐tailed Eagles Haliaeetus albicilla from Germany, Finland and Greenland to detect patterns of dietary variation and quantify diet composition. The isotopic analysis included liver and muscle samples from Eagles of the three populations together with 16 potential food sources in the German population. Our results suggested dietary differences between German and Greenlandic Eagles, in accordance with the availability of freshwater and marine habitats in each population. Within the German population, we found seasonal shifts in isotopic ratios, suggesting the birds responded to temporal changes in food availabilities, and age‐related isotopic differences, indicating different diets in adults and juveniles. Isotopic values of liver and muscle tissues collected from the same animal showed intra‐individual short‐term changes in the German and Finnish but not Greenlandic population. This suggests that local feeding niches of this generalist predator may vary with local food supplies, which determines the niche width (from generalist to specialist) at the individual level. Our results also revealed that game mammal carcasses constitute an important food source (29.5% of diet) for the German Eagle population during the winter half‐year corresponding to the hunting season. This result is of relevance to management and conservation because the White‐tailed Eagle and other raptor species are affected by the ingestion of lead ammunition from shot mammalian carcasses.     

The effects of preservation on fish tissue stable isotope signatures

The effects of formalin and ethanol preservation on the δ¹³C and δ¹⁵N isotope signatures of Arctic charr Salvelinus alpinus muscle tissue were examined. The lipid content of the tissue samples studied ranged from 3·6 to 6·1% and was not correlated with the magnitude of observed isotopic shifts in preserved samples. Ethanol and formalin significantly depleted and enriched, respectively, the δ¹³C isotope signatures of preserved tissues when compared to control samples. Ethanol did not significantly enrich δ¹⁵N signatures in comparison to controls, whereas formalin did. A meta-analysis of multiple species effects further demonstrated significant preservation effects in fish tissue. Statistical analysis of data obtained by correcting preserved tissue isotope signatures with literature, bootstrapped or meta-analysis derived correction factors demonstrated significant differences between corrected and control sample isotope signatures or failure to produce a unity slope when the data sets were regressed against one another. Species-specific, bootstrapped linear correction models resulted in no such errors. Results suggest that species-specific correction methods should be used for fishes because of the known wide variation in fish tissue lipid content and composition. Accordingly, the use of pilot studies will be required to develop correction factors that properly adjust for preservation effects when interpreting temporal patterns in historic analyses of food webs.     

Stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG)

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG) technique for the comparative and quantitative analysis of total N‐glycans using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N‐glycans using a model glycoprotein (bovine fetuin). Moreover, the i‐QTaG using MALDI‐TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of ¹³C₆/¹²C₆‐2‐aminobenzoic acid‐labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N‐glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N‐glycan peaks from i‐QTaG method showed a good linearity (R² > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2‐AA labeled N‐glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up‐regulation of the Lewis antigen (～82%) in sera from prostate cancer patients. In this proof‐of‐concept study, we demonstrated that the i‐QTaG method, which enables to achieve a reliable comparative quantitation of total N‐glycans via MALDI‐TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:840–848, 2015     

Isotopic niche differs between seal and fish‐eating killer whales (Orcinus orca) in northern Norway

Ecological diversity has been reported for killer whales (Orcinus orca) throughout the North Atlantic but patterns of prey specialization have remained poorly understood. We quantify interindividual dietary variations in killer whales (n = 38) sampled throughout the year in 2017–2018 in northern Norway using stable isotopic nitrogen (δ¹⁵N: ¹⁵N/¹⁴N) and carbon (δ¹³C: ¹³C/¹²C) ratios. A Gaussian mixture model assigned sampled individuals to three differentiated clusters, characterized by disparate nonoverlapping isotopic niches, that were consistent with predatory field observations: seal‐eaters, herring‐eaters, and lumpfish‐eaters. Seal‐eaters showed higher δ¹⁵N values (mean ± SD: 12.6 ± 0.3‰, range = 12.3–13.2‰, n = 10) compared to herring‐eaters (mean ± SD: 11.7 ± 0.2‰, range = 11.4–11.9‰, n = 19) and lumpfish‐eaters (mean ± SD: 11.6 ± 0.2‰, range = 11.3–11.9, n = 9). Elevated δ¹⁵N values for seal‐eaters, regardless of sampling season, confirmed feeding at high trophic levels throughout the year. However, a wide isotopic niche and low measured δ¹⁵N values in the seal‐eaters, compared to that of whales that would eat solely seals (δ_N‐measured = 12.6 vs. δ_N‐expected = 15.5), indicated a diverse diet that includes both fish and mammal prey. A narrow niche for killer whales sampled at herring and lumpfish seasonal grounds supported seasonal prey specialization reflective of local peaks in prey abundance for the two fish‐eating groups. Our results, thus, show differences in prey specialization within this killer whale population in Norway and that the episodic observations of killer whales feeding on prey other than fish are a consistent behavior, as reflected in different isotopic niches between seal and fish‐eating individuals.     

Characterization of 11 microsatellite marker loci in the Malagasy big‐headed turtle (Erymnochelys madagascariensis)

The Malagasy big‐headed turtle (Erymnochelys madagascariensis) is the only Erymnochelys species living in lakes, rivers and watersheds of western Madagascar. This species is endangered due to over harvesting of natural populations for human consumption. Eleven nuclear microsatellite loci were isolated from a genomic DNA library derived from a free‐ranging Malagasy big‐headed turtle from the Beroboka Classified Forest, Madagascar. Population genetic parameters were estimated on 10 individuals sampled from Ampijoroa and Andranohobaka River, Madagascar, to determine marker utility and as preliminary baseline values to study future populations in these locations.     

Carbon and nitrogen parasitism by a xylem‐tapping mistletoe (Tapinanthus oleifolius) along the Kalahari Transect: a stable isotope study

The present study explores the xylem‐tapping parasitism by mistletoe (Tapinanthus oleifolius) on native tree species along the Kalahari Transect (KT) using the stable isotopes of carbon and nitrogen. Mistletoe‐host pairs were collected at three geographical locations along the KT rainfall gradient in the 2005 and 2006 wet seasons. Foliar total carbon, total nitrogen and their stable isotope compositions (δ¹³C and δ¹⁵N) were measured. Heterotrophy (H) was calculated using foliar δ¹³C values of mistletoes and their hosts as an indicator of proportion of carbon in the mistletoes derived from host photosynthate. Based on the mistletoe H‐value and relationship between the mistletoe foliar δ¹⁵N and their host foliar δ¹⁵N, the results showed that mistletoes along the KT derived both nitrogen and carbon from their hosts. Mistletoes may regulate water use in relation to nitrogen supply. The proportion of carbon in the mistletoes derived from host photosynthate was between 35% and 78%, and the degree of heterotrophy was species‐specific with only limited annual variation. The study emphasizes the importance of incorporating parasitic associations in future studies on studying carbon, water and nutrient cycling along the Kalahari.     

Combining stable isotope analysis and conventional techniques to improve knowledge of the diet of the European Roller Coracias garrulus

Diet studies are crucial for understanding the ecology and evolution of species, as well as for establishing appropriate conservation and management strategies. However, they remain methodologically challenging due to variation between seasons, sites, sexes or age groups and even variation between individuals. Due to method‐specific characteristics and biases, a combination of existing techniques can overcome the inherent limitations of each technique and provide a more accurate and broad picture of species’ food preferences. Here, we examine diet information obtained using three different assessment methods to better understand the trophic ecology of the European Roller Coracias garrulus, a species targeted by conservation measures in Europe. First, we analysed regurgitated pellets and video‐recordings to report the diet composition of adult and nestling Rollers, respectively. Secondly, we used stable isotope analysis (SIA) to investigate adult sexual diet segregation as well as to confirm the main findings regarding adult and nestling diets obtained through conventional methods. Based on the analysis of pellets, the diet of adult Rollers was dominated by Coleoptera, whereas camera images revealed that the diet of nestlings was dominated by Orthoptera, mainly grasshoppers and bush crickets. Blood isotopic signatures of adult and nestling Rollers confirmed the results obtained through pellet and video‐recording techniques. Of the three methods, pellet analysis provided the most comprehensive trophic information regarding the detectable prey spectrum and prey species contribution, as well as basic diet information for the SIA. Our results also highlight the potential of SIA for assessing intraspecific variation in diet by sampling individuals of known age and sex, which is often unfeasible through conventional approaches. SIA analysis showed no differences in δ¹³C and δ¹⁵N ratios of blood between males and females and a high degree of overlap amongst isotopic niches, suggesting no sex‐specific partitioning in resource use. Overall, we showed that the combination of different methods could be used to gain new and clearer insights into avian trophic ecology that are essential for informing habitat management aiming to improve availability of foraging resources.     

紫茎泽兰在中国的气候生态位稳定且其分布范围仍有进一步扩展的趋势

明确入侵物种气候生态位的时空动态格局, 不仅有助于理解入侵物种生态位的变化趋势, 还可以使物种分布模型更真实地预测物种的扩散动态。本文使用恶性入侵植物紫茎泽兰(Ageratina adenophora)在原产地及我国的分布点和环境因子数据, 结合生态位分析及物种分布模型, 分析了紫茎泽兰入侵至我国后其气候生态位的时空变化特征、地理分布格局变化及其限制因子。结果表明: (1)紫茎泽兰入侵至我国后其气候生态位在时间和空间上虽略有扩张, 但仍保持较高的稳定性。尤其是入侵的初始阶段(1980年之前)稳定性最高(NS = 0.995), 此后其气候生态位的稳定性逐渐降低, 具体表现为该植物在中国更倾向于分布在潮湿(高最暖季降水量)、寒冷(低最干季平均温)且平均日较差低的生境。(2)过去50年里紫茎泽兰的适宜分布范围显著增加(151.77%-271.14%), 且其适宜分布区的重心在2010年之前表现出向东北(贵州西南部、四川南部)扩散的趋势, 2010年以后表现出向西南(云南北部、贵州北部)扩散的趋势。在扩散过程中, 该物种的气候生态位稳定性也表现出随着纬度的增加而逐渐降低的趋势。(3)未来气候变化情景下, 紫茎泽兰的适宜分布范围将进一步增加(111.97%), 到2040年该物种基本可以扩散至其整个潜在适宜分布区, 仅在藏东南地区有小范围的适宜生境受扩散限制无法达到。     

Trophic niche partitioning and diet composition of sympatric fin (Balaenoptera physalus) and humpback whales (Megaptera novaeangliae) in the Gulf of Alaska revealed through stable isotope analysis

Fin and humpback whales are large consumers that are often sympatric, effectively sharing or partitioning their use of habitat and prey resources. Stable carbon and nitrogen isotopes in the skin of fin and humpback whales from two regions in the western Gulf of Alaska, Kodiak, and Shumagin Islands, were analyzed to test the hypothesis that these sympatric baleen whales exhibit trophic niche partitioning within these regions. Standard ellipse areas, estimated using Bayesian inference, suggested that niche partitioning between species is occurring in the Kodiak region but not in the Shumagin Islands. Isotopic mixing models based on stable isotopes from whales and local prey samples, were used to estimate possible diet solutions for whales in the Kodiak region. Comparison of isotopic niches and diet models support niche partitioning, with fin whales foraging primarily on zooplankton and humpback whales foraging on zooplankton and small forage fish. The results of this study show that niche partitioning between sympatric species can vary by region and may be the result of prey availability, prey preferences, or both.     

Taxon-specific variation in the stable isotopic signatures (δ13C and δ15N) of lake phytoplankton

1. The variability in the stable isotope signatures of carbon and nitrogen (δ¹³C and δ¹⁵N) in different phytoplankton taxa was studied in one mesotrophic and three eutrophic lakes in south‐west Finland. The lakes were sampled on nine to 16 occasions over 2–4 years and most of the time were dominated by cyanobacteria and diatoms. A total of 151 taxon‐specific subsamples covering 18 different phytoplankton taxa could be isolated by filtration through a series of sieves and by flotation/sedimentation, followed by microscopical identification and screening for purity. 2. Substantial and systematic differences between phytoplankton taxa, seasons and lakes were observed for both δ¹³C and δ¹⁵N. The values of δ¹³C ranged from ?34.4‰ to ?5.9‰ and were lowest in chrysophytes (?34.4‰ to ?31.3‰) and diatoms (?30.6‰ to ?26.6‰). Cyanobacteria were most variable (?32.4‰ to ?5.9‰), including particularly high values in the nostocalean cyanobacterium Gloeotrichia echinulata (?14.4‰ to ?5.9‰). For δ¹³C, the taxon‐specific amplitude of temporal changes within a lake was usually <1–8‰ (<1–4‰ for microalgae alone and <1–8‰ for cyanobacteria alone), whereas the amplitude among taxa within a water sample was up to 31‰. 3. The values of δ¹⁵N ranged from ?2.1‰ to 12.8‰ and were high in chrysophytes, dinophytes and diatoms, but low in the nitrogen‐fixing cyanobacteria Anabaena spp., Aphanizomenon spp. and G. echinulata (?2.1‰ to 1.6‰). Chroococcalean cyanobacteria ranged from ?1.4‰ to 8.9‰. For δ¹⁵N, the taxon‐specific amplitude of temporal changes within a lake was 2–6‰, (2–6‰ for microalgae alone and 2–4‰ for cyanobacteria alone) and the amplitude among taxa within a water sample was up to 11‰. 4. The isotopic signatures of phytoplankton changed systematically with their physical and chemical environment, most notably with the concentrations of nutrients, but correlations were non‐systematic and site‐specific. 5. The substantial variability in the isotopic signatures of phytoplankton among taxa, seasons and lakes complicates the interpretation of isotopic signatures in lacustrine food webs. However, taxon‐specific values and seasonal patterns showed some consistency among years and may eventually be predictable.     

Factors controlling the stable isotope composition and C:N ratio of seston and periphyton in shallow lake mesocosms with contrasting nutrient loadings and temperatures

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Effect of historical land‐use and climate change on tree‐climate relationships in the upper Midwestern United States

Contemporary forest inventory data are widely used to understand environmental controls on tree species distributions and to construct models to project forest responses to climate change, but the stability and representativeness of contemporary tree‐climate relationships are poorly understood. We show that tree‐climate relationships for 15 tree genera in the upper Midwestern US have significantly altered over the last two centuries due to historical land‐use and climate change. Realised niches have shifted towards higher minimum temperatures and higher rainfall. A new attribution method implicates both historical climate change and land‐use in these shifts, with the relative importance varying among genera and climate variables. Most climate/land‐use interactions are compounding, in which historical land‐use reinforces shifts in species‐climate relationships toward wetter distributions, or confounding, in which land‐use complicates shifts towards warmer distributions. Compounding interactions imply that contemporary‐based models of species distributions may underestimate species resilience to climate change.     

Reconstructing long‐term changes (150 years) in the carbon cycle of a clear‐water lake based on the stable carbon isotope composition (δ13C) of chironomid and cladoceran subfossil remains

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The evolution of climate tolerance in conifer‐feeding aphids in relation to their host's climatic niche

Climate adaptation has major consequences in the evolution and ecology of all living organisms. Though phytophagous insects are an important component of Earth's biodiversity, there are few studies investigating the evolution of their climatic preferences. This lack of research is probably because their evolutionary ecology is thought to be primarily driven by their interactions with their host plants. Here, we use a robust phylogenetic framework and species‐level distribution data for the conifer‐feeding aphid genus Cinara to investigate the role of climatic adaptation in the diversity and distribution patterns of these host‐specialized insects. Insect climate niches were reconstructed at a macroevolutionary scale, highlighting that climate niche tolerance is evolutionarily labile, with closely related species exhibiting strong climatic disparities. This result may suggest repeated climate niche differentiation during the evolutionary diversification of Cinara. Alternatively, it may merely reflect the use of host plants that occur in disparate climatic zones, and thus, in reality the aphid species' fundamental climate niches may actually be similar but broad. Comparisons of the aphids' current climate niches with those of their hosts show that most Cinara species occupy the full range of the climatic tolerance exhibited by their set of host plants, corroborating the hypothesis that the observed disparity in Cinara species' climate niches can simply mirror that of their hosts. However, 29% of the studied species only occupy a subset of their hosts' climatic zone, suggesting that some aphid species do indeed have their own climatic limitations. Our results suggest that in host‐specialized phytophagous insects, host associations cannot always adequately describe insect niches and abiotic factors must be taken into account.     

Contrasting dynamics of water and mineral nutrients in stems shown by stable isotope tracers and cryo‐SIMS

Lateral exchange of water and nutrients between xylem and surrounding tissues helps to de‐couple uptake from utilization in all parts of a plant. We studied the dynamics of these exchanges, using stable isotope tracers for water (H₂¹⁸O), magnesium (²⁶Mg), potassium (⁴¹K) and calcium (⁴⁴Ca) delivered via a cut stem for various periods to the transpiration stream of bean shoots (Phaseolus vulgaris cv. Fardenlosa Shiny). Tracers were subsequently mapped in stem cross‐sections with cryo‐secondary ion mass spectrometry. The water tracer equilibrated within minutes across the entire cross‐section. In contrast, the nutrient tracers showed a very heterogeneous exchange between xylem vessels and the different stem tissues, even after 4 h. Dynamics of nutrients in the tissues revealed a fast and extensive exchange of nutrients in the xylem parenchyma, with, for example, calcium being completely replaced by tracer in less than 5 min. Dilution of potassium tracer during its 30 s transit in xylem sap through the stem showed that potassium concentration was up‐regulated over many hours, to the extent that some of it was probably supplied by phloem recirculation from the shoot.     

On the use of stable oxygen isotope (δ18O) measurements for tracking avian movements in North America

Tracking migratory animals has benefitted using measurements of naturally occurring stable isotopes of hydrogen (δ²H) in keratinous tissues such as hair and feathers to link animal origins to continental patterns or isoscapes of δ²H in precipitation. However, for most taxa, much less information exists on the use of stable oxygen isotope ratios (δ¹⁸O) despite the fact that δ²H and δ¹⁸O are strongly linked in environmental waters through the meteoric relationship and the possibility of using both isotopes to infer greater information on origins and climatic conditions where tissues are grown. A fundamental requirement of using stable isotopes to assign individuals and populations to origins is the development of a rescaling function linking environmental food web signals to the tissue of interest and for birds, this has not been carried out. Here, we derived the relationship between H and O isotopes in known source feathers of 104 individuals representing 11 species of insectivorous passerines sampled across the strong precipitation isoscape of North America. We determined again a strong expected relationship between feather δ²H (δ²H_f) and long‐term amount‐weighted precipitation δ²H (δ²H_p; r² = 0.77), but the corresponding relationship between δ¹⁸O_f and δ¹⁸O_p was poor (r² = 0.32) for the same samples. This suggests that δ²H measurements are currently more useful for assignment of insectivorous songbirds to precipitation isoscapes but does not preclude other uses of the δ¹⁸O_f data. Currently, mechanisms responsible for the decoupling of H and O isotopes in food webs is poorly known, and we advocate a much broader sampling of both isotopes in the same keratinous tissues across precipitation isotope gradients and across taxa to resolve this issue and to increase the power of using water isotopes to track migratory animals.     

Host identification in unfed ticks from stable isotope compositions (δ13C and δ15N)

Determination of the ratios of natural stable isotopes (¹³C/¹²C and ¹⁵N/¹⁴N) in unfed Ixodes ricinus nymphs and adults, which, in their previous stage, fed on captive wild rodents (Apodemus sylvaticus and Myodes glareolus), wild birds (Parus major and Cyanistes caeruleus) or domestic ruminants (Ovis aries and Bos taurus), demonstrated that it is possible to identify each host category with confidence. First, the tick–blood spacing, which is the difference between values obtained from ticks and the blood of hosts that they had fed on in the previous stage, was consistent (152 spacings investigated from 15 host individuals in total). Second, potential confounding factors (tick age and sex) did not affect the discriminatory power of the isotope patterns, nor did different rearing conditions (room temperature vs. 4 °C) or the duration of development (maximum of 430 days). The findings that the tick–blood isotope spacings, across a diverse range of hosts, were similar and predictable, and that confounders had little or no effect on this, strongly support the usage of the isotope approach. Because each of the host categories has a different role in the population dynamics of I. ricinus and in tick‐borne pathogen ecology, the method described here has great potential for the clarification of tick and tick‐borne pathogen ecology in the field.