Energy sources for aquatic animals in the Orinoco River floodplain: evidence from stable isotopes

Summary Stable carbon and nitrogen isotope ratios in autotrophs, aquatic invertebrates and fishes from the Orinoco River floodplain of Venezuela reveal that microalgae, including both phytoplankton and epiphytic (attached) forms, are predominant energy sources for many aquatic animals, even though aquatic vascular plants are much more abundant. Floating mats of the grass Paspalum repens and the water hyacinth Eichhornia spp. harbor particularly high densities of aquatic animals, but isotopic evidence indicates that few species are dependent on organic carbon originating from these plants. The stable isotopic evidence for the trophic importance of algae contradicts traditional interpretations of food webs in freshwater wetlands, which are generally thought to be based largely on detritus originating from vascular plants.     

Diffuse migratory connectivity in two species of shrubland birds: evidence from stable isotopes

Connecting seasonal ranges of migratory birds is important for understanding the annual template of stressors that influence their populations. Brewer’s sparrows (Spizella breweri) and sagebrush sparrows (Artemisiospiza nevadensis) share similar sagebrush (Artemisia spp.) habitats for breeding but have different population trends that might be related to winter location. To link breeding and winter ranges, we created isoscapes of deuterium [stable isotope ratio (δ) of deuterium; δ ²H] and nitrogen (δ ¹⁵N) for each species modeled from isotope ratios measured in feathers of 264 Brewer’s and 82 sagebrush sparrows and environmental characteristics at capture locations across their breeding range. We then used feather $\delta^{2} {\text{H}}_{\text{f}}$ and $\delta^{15} {\text{N}}_{\text{f}}$ measured in 1,029 Brewer’s and 527 sagebrush sparrows captured on winter locations in southwestern United States to assign probable breeding ranges. Intraspecies population mixing from across the breeding range was strong for both Brewer’s and sagebrush sparrows on winter ranges. Brewer’s sparrows but not sagebrush sparrows were linked to more northerly breeding locations in the eastern part of their winter range. Winter location was not related to breeding population trends estimated from US Geological Survey Breeding Bird Survey routes for either Brewer’s or sagebrush sparrows. Primary drivers of population dynamics are likely independent for each species; Brewer’s and sagebrush sparrows captured at the same winter location did not share predicted breeding locations or population trends. The diffuse migratory connectivity displayed by Brewer’s and sagebrush sparrows measured at the coarse spatial resolution in our analysis also suggests that local environments rather than broad regional characteristics are primary drivers of annual population trends.     

Differential host use in two highly specialized ant-plant associations: evidence from stable isotopes

Carbon and nitrogen stable isotopes were used to examine variation in ant use of plant resources in the Cecropia obtusifolia / Azteca spp. association in Costa Rica. Tissue of ants, host plants and symbiotic pseudococcids were collected along three elevation transects on the Pacific slope of Costa Ricas Cordillera Central, and were analyzed for carbon and nitrogen isotopic composition. Worker carbon and nitrogen signatures were found to vary with elevation and ant colony size, and between Azteca species groups. Ants in the A. constructor species group appear to be opportunistic foragers at low elevations, but rely more heavily on their host plants at high elevations, whereas ants in the A. alfari species group consume a more consistent diet across their distribution. Further, isotope values indicate that both ant species groups acquire more nitrogen from higher trophic levels at low elevation and when ant colonies are small. Provisioning by the host is a substantial ecological cost to the interaction, and it may vary, even in a highly specialized association. Nonetheless, not all specialized interactions are equivalent; where interaction with one ant species group appears conditional upon the environment, the other is not. Differential host use within the Cecropia-Azteca association suggests that the ecological and evolutionary benefits and costs of association may vary among species pairs.     

Trophic niche partitioning in communities of African annual fish: evidence from stable isotopes

Annual killifish of the genus Nothobranchius often co-occur in temporary savannah pools. Their space- and time-limited environment does not allow for any substantial habitat or temporal segregation. Coexisting species are therefore predicted to have well separated trophic niches to avoid intense food competition. Although in a previous “snapshot” study using stomach content analysis (SCA), the trophic niches of three sympatric species (N. furzeri, N. orthonotus, and N. pienaari) were found to vary among species, the difference was relatively weak and inconsistent across different sites. Here, we used the time-integrative capacity of stable isotope analysis to test whether the trophic niches of sympatric Mozambican Nothobranchius are more distinct over a long-term period. Analysis of carbon and nitrogen stable isotopes separated the trophic niche and trophic position of N. pienaari but failed to find any difference between N. furzeri/N. kadleci and N. orthonotus. No segregation was found at the sites with low prey diversity. In contrast, SCA identified N. orthonotus as the species with the most distinct trophic niche. We discuss the effect of prey diversity and different sensitivities of stomach content and stable isotope analysis in general and conclude that the trophic niches of the three sympatric Nothobranchius species are well separated.     

Mycorrhizae transfer carbon from a native grass to an invasive weed: evidence from stable isotopes and physiology

Invasive exotic weeds pose one of the earth's most pressing environmental problems. Although many invaders completely eliminate native plant species from some communities, ecologists know little about the mechanisms by which these exotics competitively exclude other species. Mycorrhizal fungi radically alter competitive interactions between plants within natural communities, and a recent study has shown that arbuscular mycorrhizal (AM) fungi provide a substantial competitive advantage to spotted knapweed, Centaurea maculosa, a noxious perennial plant that has spread throughout much of the native prairie in the northwestern U.S. Here we present evidence that this advantage is potentially due to mycorrhizally mediated transfer of carbon from a native bunchgrass, Festuca idahoensis, to Centaurea. Centaurea maculosa, Festuca idahoensis (Idaho fescue, C₃), and Bouteloua gracilis (blue gramma, C₄) were grown in the greenhouse either alone or with Centaurea in an incomplete factorial design with and without AM fungi. Centaurea biomass was 87–168% greater in all treatments when mycorrhizae were present in the soil (P < 0.0001). However, Centaurea biomass was significantly higher in the treatment with both mycorrhizae and Festuca present together than in any other treatment combination (P < 0.0001). This high biomass was attained even though Centaurea photosynthetic rates were 14% lower when grown with Festuca and mycorrhizae together than when grown with Festuca without mycorrhizae. Neither biomass nor photosynthetic rates of Centaurea were affected by competition with the C₄ grass Bouteloua either with or without mycorrhizae. The stable isotope signature of Centaurea leaves grown with Festuca and mycorrhizae was more similar to that of Festuca, than when Centaurea was grown alone with mycorrhizae (P = 0.06), or with Festuca but without mycorrhizae (P = 0.09). This suggests that carbon was transferred from Festuca to the invasive weed. We estimated that carbon transferred from Festuca by mycorrhizae contributed up to 15% of the aboveground carbon in Centaurea plants. Our results indicate that carbon parasitism via AM soil fungi may be an important mechanism by which invasive plants out compete their neighbors, but that this interaction is highly species-specific.     

Fractionation of stable carbon isotopes by phosphoenolpyruvate carboxylase from c(4) plants

The active species of "CO(2)" and the amount of fractionation of stable carbon isotopes have been determined for a partially purified preparation of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) from corn (Zea mays) leaves. The rates of the enzyme reactions, using substrate amounts of HCO(3) (-), CO(2) or CO(2) plus carbonic anhydrase, show that HCO(3) (-) is the active species of "CO(2)" utilized by PEP carboxylase. The K(m) values for CO(2) and HCO(3) (-) are 1.25 mm and 0.11 mm, respectively, which further suggest the preferential utilization of HCO(3) (-) by PEP carboxylase. The amount of fractionation of stable carbon isotopes by PEP carboxylase from an infinite pool of H(12)CO(3) (-) and H(13)CO(3) (-) was -2.03 per thousand. This enzyme fractionation (delta), together with the fractionation associated with absorption of CO(2) into plant cells and the equilibrium fractionation associated with atmospheric CO(2) and dissolved HCO(3) (-) are discussed in relation to the fractionation of stable carbon isotopes of atmospheric CO(2) during photosynthesis in C(4) plants.     

Holocene changes in the ecology of northern fur seals: insights from stable isotopes and archaeofauna

The remains of northern fur seals (Callorhinus ursinus) are among the most abundant of pinniped elements recovered from mainland coastal archaeological sites in both California and Oregon. This is surprising as all contemporary northern fur seals breed exclusively on offshore islands, primarily at high latitudes, and the species is otherwise pelagic. The vulnerability of these animals to human predation suggests that either humans were foraging much further offshore than has been presumed or alternatively that the ecology of these animals has shifted during the late Holocene. We used isotopic and archaeofaunal analysis of the remains of pinnipeds from the middle to late Holocene of central and northern California to clarify the breeding and foraging behavior, and migration patterns of these ancient animals. The carbon and nitrogen isotope compositions of ancient northern fur seals reveal that these animals fed as far offshore as they do today, and that they remained at middle latitudes throughout the year. From an archaeological site at Moss Landing, California, we identified 16 skeletal elements from at least 12 very small northern fur seal pups. From another site near Mendocino, California, we identified the remains of at least 6 pups. We estimate the size and age of 5 of the young animals using sex-specific regressions of body length on the short dentary length derived from measurements of modern specimens. Our estimates indicate these ancient pups were substantially smaller, and therefore younger, than modern 3-month-old northern fur seal pups from similar latitudes and their nitrogen isotope compositions suggest they had not been weaned. As present-day northern fur seals do not leave their rookeries until they are at least 4 months old, we consider it highly unlikely that these ancient pups swam to these mainland locations from some distant island rookery. While there are numerous nearshore rocky outcrops along the Mendocino Coast, which may have supported small breeding colonies, the Moss Landing site is centered on a 40-km-long sandy beach, and is more than 120 km from what at the time were the nearest offshore islands. We conclude that northern fur seal adult females, subadults, and pups whose remains were recovered at the Moss Landing archaeological site must have been taken at a mainland rookery. Evidence that northern fur seals once bred on the mainland at this central California location suggests that the abundant remains of these animals at numerous other archaeological sites along the California coast also reflect the presence of nearby mainland rookeries. Based on the relative abundance of their remains in ancient human occupation sites and the widespread distribution of sites where their remains have been found, it appears that northern fur seals were once the predominant pinniped throughout a region where they now only rarely occur. Furthermore, their presence along the central and northern California coasts appears to have once severely limited the distribution of other pinnipeds, which are now common to the region.     

Seasonal reliance on nectar by an insectivorous bat revealed by stable isotopes

Many animals have seasonally plastic diets to take advantage of seasonally abundant plant resources, such as fruit or nectar. Switches from insectivorous diets that are protein rich to fruits or nectar that are carbohydrate rich present physiological challenges, but are routinely done by insectivorous songbirds during migration. In contrast, insectivorous bat species are not known to switch diets to consume fruit or nectar. Here, we use carbon stable isotope ratios to establish the first known case of a temperate bat species consuming substantial quantities of nectar during spring. We show that pallid bats (Antrozous pallidus) switch from a diet indistinguishable from that of sympatric insectivorous bat species in winter (when no cactus nectar is present) to a diet intermediate between those of insectivorous bats and nectarivorous bats during the spring bloom of a bat-adapted cactus species. Combined with previous results that established that pallid bats are effective pollinators of the cardon cactus (Pachycereus pringlei), our results suggest that the interaction between pallid bats and cardon cacti represents the first-known plant-pollinator mutualism between a plant and a temperate bat. Diet plasticity in pallid bats raises questions about the degree of physiological adaptations of insectivorous bats for incorporation of carbohydrate-rich foods, such as nectar or fruit, into the diet.     

Phylogeography of the montane caddisfly Drusus discolor: evidence for multiple refugia and periglacial survival

We studied the genetic population structure and phylogeography of the montane caddisfly Drusus discolor across its entire range in central and southern Europe. The species is restricted to mountain regions and exhibits an insular distribution across the major mountain ranges. Mitochondrial sequence data (COI) of 254 individuals from the entire species range is analysed to reveal population genetic structure. The data show little molecular variation within populations and regions, but distinct genetic differentiation between mountain ranges. Most populations are significantly differentiated based on F(ST) and exact tests of population differentiation and most haplotypes are unique to a single mountain range. Phylogenetic analyses reveal deep divergence between geographically isolated lineages. Combined, these results suggest that past fragmentation is the prominent process structuring the populations across Europe. We use tests of selective neutrality and mismatch distributions, to study the demographic population history of regions with haplotype overlap. The high level of genetic differentiation between mountain ranges and estimates of demographic history provide evidence for the existence of multiple glacial refugia, including several in central Europe. The study shows that these aquatic organisms reacted differently to Pleistocene cooling than many terrestrial species. They persisted in numerous refugia over multiple glacial cycles, allowing many local endemic clades to form.     

Effects of forest conversion into grassland on soil aggregate structure and carbon storage in Panama: evidence from soil carbon fractionation and stable isotopes

Land-use and land-cover strongly influence soil properties such as the amount of soil organic carbon (SOC), aggregate structure and SOC turnover processes. We studied the effects of a vegetation shift from forest to grassland 90 years ago in soils derived from andesite material on Barro Colorado Island (BCI), Panama. We quantified the amount of carbon (C) and nitrogen (N) and determined the turnover of C in bulk soil, water stable aggregates (WSA) of different size classes (<53 μm, 53–250 μm, 250–2000 μm and 2000–8000 μm) and density fractions (free light fraction, intra-aggregate particulate organic matter and mineral associated soil organic C). Total SOC stocks (0–50 cm) under forest (84 Mg C ha⁻¹) and grassland (64 Mg C ha⁻¹) did not differ significantly. Our results revealed that vegetation type did not have an effect on aggregate structure and stability. The investigated soils at BCI did not show higher C and N concentrations in larger aggregates, indicating that organic material is not the major binding agent in these soils to form aggregates. Based on δ¹³C values and treating bulk soil as a single, homogenous C pool we estimated a mean residence time (MRT) of 69 years for the surface layer (0–5 cm). The MRT varied among the different SOC fractions and among depth. In 0–5 cm, MRT of intra-aggregate particulate organic matter (iPOM) was 29 years; whereas mineral associated soil organic C (mSOC) had a MRT of 124 years. These soils have substantial resilience to C and N losses because the >90% of C and N is associated with mSOC, which has a comparatively long MRT.     

Determining biological tissue turnover using stable isotopes: the reaction progress variable

The reaction progress variable is applied to stable isotope turnover of biological tissues. This approach has the advantage of readily determining whether more than one isotope turnover pool is present; in addition, the normalization process inherent to the model means that multiple experiments can be considered together although the initial and final isotope compositions are different. Consideration of multiple isotope turnover pools allows calculation of diet histories of animals using a time sequence of isotope measurements along with isotope turnover pools. The delayed release of blood cells from bone marrow during a diet turnover experiment can be quantified using this approach. Turnover pools can also be corrected for increasing mass during an experiment, such as when the animals are actively growing. Previous growth models have been for exponential growth; the approach here can be used for several different growth models. Electronic Supplementary Material The online version of this article () contains supplementary material, which is available to authorized users.     

Uncertainty in source partitioning using stable isotopes

Stable isotope analyses are often used to quantify the contribution of multiple sources to a mixture, such as proportions of food sources in an animal's diet, or C₃ and C₄ plant inputs to soil organic carbon. Linear mixing models can be used to partition two sources with a single isotopic signature (e.g., '¹³C) or three sources with a second isotopic signature (e.g., '¹⁵N). Although variability of source and mixture signatures is often reported, confidence interval calculations for source proportions typically use only the mixture variability. We provide examples showing that omission of source variability can lead to underestimation of the variability of source proportion estimates. For both two- and three-source mixing models, we present formulas for calculating variances, standard errors (SE), and confidence intervals for source proportion estimates that account for the observed variability in the isotopic signatures for the sources as well as the mixture. We then performed sensitivity analyses to assess the relative importance of: (1) the isotopic signature difference between the sources, (2) isotopic signature standard deviations (SD) in the source and mixture populations, (3) sample size, (4) analytical SD, and (5) the evenness of the source proportions, for determining the variability (SE) of source proportion estimates. The proportion SEs varied inversely with the signature difference between sources, so doubling the source difference from 2‰ to 4‰ reduced the SEs by half. Source and mixture signature SDs had a substantial linear effect on source proportion SEs. However, the population variability of the sources and the mixture are fixed and the sampling error component can be changed only by increasing sample size. Source proportion SEs varied inversely with the square root of sample size, so an increase from 1 to 4 samples per population cut the SE in half. Analytical SD had little effect over the range examined since it was generally substantially smaller than the population SDs. Proportion SEs were minimized when sources were evenly divided, but increased only slightly as the proportions varied. The variance formulas provided will enable quantification of the precision of source proportion estimates. Graphs are provided to allow rapid assessment of possible combinations of source differences and source and mixture population SDs that will allow source proportion estimates with desired precision. In addition, an Excel spreadsheet to perform the calculations for the source proportions and their variances, SEs, and 95% confidence intervals for the two-source and three-source mixing models can be accessed at http://www.epa.gov/wed/pages/models.htm.     

Estimating the timing of diet shifts using stable isotopes

Stable isotope analysis has become an important tool in studies of trophic food webs and animal feeding patterns. When animals undergo rapid dietary shifts due to migration, metamorphosis, or other reasons, the isotopic composition of their tissues begins changing to reflect that of their diet. This can occur both as a result of growth and metabolic turnover of existing tissue. Tissues vary in their rate of isotopic change, with high turnover tissues such as liver changing rapidly, while relatively low turnover tissues such as bone change more slowly. A model is outlined that uses the varying isotopic changes in multiple tissues as a chemical clock to estimate the time elapsed since a diet shift, and the magnitude of the isotopic shift in the tissues at the new equilibrium. This model was tested using published results from controlled feeding experiments on a bird and a mammal. For the model to be effective, the tissues utilized must be sufficiently different in their turnover rates. The model did a reasonable job of estimating elapsed time and equilibrial isotopic changes, except when the time since the diet shift was less than a small fraction of the half-life of the slowest turnover tissue or greater than 5–10 half-lives of the slowest turnover tissue. Sensitivity analyses independently corroborated that model estimates became unstable at extremely short and long sample times due to the effect of random measurement error. Subject to some limitations, the model may be useful for studying the movement and behavior of animals changing isotopic environments, such as anadromous fish, migratory birds, animals undergoing metamorphosis, or animals changing diets because of shifts in food abundance or competitive interactions.     

Measuring the trophic ecology of ants using stable isotopes

Ants are prominent components of most terrestrial arthropod food webs, yet due to their highly variable diet, the role ants play in arthropod communities can be difficult to resolve. Stable isotope analysis is a promising method for determining the dietary history of an organism, and has the potential to advance our understanding of the food web ecology of social insects. However, some unique characteristics of eusocial organisms can complicate the application of this technique to the study of their trophic ecology. Using stable isotopes of N and C, we investigated levels of intraspecific variation both within and among colonies. We also examined the effect of a common preservation technique on δ¹⁵N and δ¹³C values. We discuss the implications of our results on experimental design and sampling methods for studies using stable isotopes to investigate the trophic ecology of social insects. Received 4 February 2005; revised 23 June 2005; accepted 4 July 2005.     

Microbiological fractionation of stable sulfur isotopes: A review and critique

Microbiological transformations of sulfur compounds discriminate to various degrees between the stable sulfur isotopes ³²S and ³⁴S. Comparatively little is known on isotopic effects associated with sulfur‐oxidizing organisms, and the interpretation of results is complicated since the sulfur pathways are poorly defined and compounds containing two or more sulfur atoms at different oxidation states may be involved. Dissimilatory reduction of sulfate, and sulfite reduction by certain assimilatory microorganisms, causes particularly marked isotopic effects, the expression of which depends on the extent of reaction and other incompletely defined environmental conditions. Models have been proposed to account for these effects based on current knowledge of the reduction pathways. Many of the trends observed during dissimilatory sulfate reduction in the laboratory can also be found in the modern environment leaving little doubt that microbiological factors play a significant role in determining sulfur isotope distributions in nature. However, unusually large isotopic effects, rarely approached in the laboratory, are often observed in nature. The reasons for this are not entirely clear, but in sediments it is possible that diffusional isotopic effects are imposed on biological effects.     

Source partitioning using stable isotopes: coping with too many sources

Stable isotopes are increasingly being used as tracers in environmental studies. One application is to use isotopic ratios to quantitatively determine the proportional contribution of several sources to a mixture, such as the proportion of various pollution sources in a waste stream. In general, the proportional contributions of n+1 different sources can be uniquely determined by the use of n different isotope system tracers (e.g., δ¹³C, δ¹⁵N, δ¹⁸O) with linear mixing models based on mass balance equations. Often, however, the number of potential sources exceeds n+1, which prevents finding a unique solution of source proportions. What can be done in these situations? While no definitive solution exists, we propose a method that is informative in determining bounds for the contributions of each source. In this method, all possible combinations of each source contribution (0–100%) are examined in small increments (e.g., 1%). Combinations that sum to the observed mixture isotopic signatures within a small tolerance (e.g., ±0.1‰) are considered to be feasible solutions, from which the frequency and range of potential source contributions can be determined. To avoid misrepresenting the results, users of this procedure should report the distribution of feasible solutions rather than focusing on a single value such as the mean. We applied this method to a variety of environmental studies in which stable isotope tracers were used to quantify the relative magnitude of multiple sources, including (1) plant water use, (2) geochemistry, (3) air pollution, and (4) dietary analysis. This method gives the range of isotopically determined source contributions; additional non-isotopic constraints specific to each study may be used to further restrict this range. The breadth of the isotopically determined ranges depends on the geometry of the mixing space and the similarity of source and mixture isotopic signatures. A sensitivity analysis indicated that the estimated ranges vary only modestly with different choices of source increment and mass balance tolerance parameter values. A computer program (IsoSource) to perform these calculations for user-specified data is available at http://www.epa.gov/wed/pages/models.htm.