Technical Note: Calcium and carbon stable isotope ratios as paleodietary indicators

Calcium stable isotope ratios are hypothesized to vary as a function of trophic level. This premise raises the possibility of using calcium stable isotope ratios to study the dietary behaviors of fossil taxa and to test competing hypotheses on the adaptive origins of euprimates. To explore this concept, we measured the stable isotope composition of contemporary mammals in northern Borneo and northwestern Costa Rica, two communities with functional or phylogenetic relevance to primate origins. We found that bone collagen δ¹³C and δ¹⁵N values could differentiate trophic levels in each assemblage, a result that justifies the use of these systems to test the predicted inverse relationship between bioapatite δ¹³C and δ⁴⁴Ca values. As expected, taxonomic carnivores (felids) showed a combination of high δ¹³C and low δ⁴⁴Ca values; however, the δ⁴⁴Ca values of other faunivores were indistinguishable from those of primary consumers. We suggest that the trophic insensitivity of most bioapatite δ⁴⁴Ca values is attributable to the negligible calcium content of arthropod prey. Although the present results are inconclusive, the tandem analysis of δ⁴⁴Ca and δ¹³C values in fossils continues to hold promise for informing paleodietary studies and we highlight this potential by drawing attention to the stable isotope composition of the Early Eocene primate Cantius. Am J Phys Anthropol 154:633–643, 2014. © 2014 Wiley Periodicals, Inc.     

Elemental formula annotation of polar and lipophilic metabolites using (13) C, (15) N and (34) S isotope labelling, in combination with high-resolution mass spectrometry

The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.     

Climate controls on C3 vs. C4 productivity in North American grasslands from carbon isotope composition of soil organic matter

We analyzed the δ¹³C of soil organic matter (SOM) and fine roots from 55 native grassland sites widely distributed across the US and Canadian Great Plains to examine the relative production of C₃ vs. C₄ plants (hereafter %C₄) at the continental scale. Our climate vs. %C₄ results agreed well with North American field studies on %C₄, but showed bias with respect to %C₄ from a US vegetation database (statsgo ) and weak agreement with a physiologically based prediction that depends on crossover temperature. Although monthly average temperatures have been used in many studies to predict %C₄, our analysis shows that high temperatures are better predictors of %C₄. In particular, we found that July climate (average of daily high temperature and month's total rainfall) predicted %C₄ better than other months, seasons or annual averages, suggesting that the outcome of competition between C₃ and C₄ plants in North American grasslands was particularly sensitive to climate during this narrow window of time. Root δ¹³C increased about 1‰ between the A and B horizon, suggesting that C₄ roots become relatively more common than C₃ roots with depth. These differences in depth distribution likely contribute to the isotopic enrichment with depth in SOM where both C₃ and C₄ grasses are present.     

Plant functional types broadly describe water use strategies in the Caatinga,a seasonally dry tropical forest in northeast Brazil

1. In seasonally dry tropical forests, plant functional type can be classified as deciduous low wood density, deciduous high wood density, or evergreen high wood density species. While deciduousness is often associated with drought‐avoidance and low wood density is often associated with tissue water storage, the degree to which these functional types may correspond to diverging and unique water use strategies has not been extensively tested.
2. We examined (a) tolerance to water stress, measured by predawn and mid‐day leaf water potential; (b) water use efficiency, measured via foliar δ¹³C; and (c) access to soil water, measured via stem water δ¹⁸O.
3. We found that deciduous low wood density species maintain high leaf water potential and low water use efficiency. Deciduous high wood density species have lower leaf water potential and variable water use efficiency. Both groups rely on shallow soil water. Evergreen high wood density species have low leaf water potential, higher water use efficiency, and access alternative water sources. These findings indicate that deciduous low wood density species are drought avoiders, with a specialized strategy for storing root and stem water. Deciduous high wood density species are moderately drought tolerant, and evergreen high wood density species are the most drought tolerant group.
4. Synthesis. Our results broadly support the plant functional type framework as a way to understand water use strategies, but also highlight species‐level differences.

     

Isotopic fractionation with morphological change and sexual specificity in the lappet moth Euthrix potatoria

The δ¹⁵N values of adult holometabolous insects exceed those of larvae, but otherwise little information on terrestrial invertebrates has been obtained in food‐web analyses using stable isotope ratios (δ¹⁵N, δ¹³C). Changes in δ¹³C during metamorphosis and differences between males and females have not been examined. We collected the larvae and cocoons of Euthrix potatoria (L.) (Lepidoptera: Lasiocampidae) in the field and used them to assess the species’ isotopic fractionation. Each emerged moth was divided into five body parts. We conducted stable N and C isotope analyses for each body part, as well as for cocoons and exuviae, and also compared stable isotope ratios between sexes. We confirmed δ¹⁵N enrichment through metamorphosis and estimated that δ¹⁵N enrichment is accomplished by the relative concentration of ¹⁵N due to the excretion of copious meconium, which contains abundant ¹⁴N. We also observed changes in δ¹³C values through metamorphosis. Both isotope values tended to change more in males than in females. The proportion of the whole‐adult weight represented by meconium was higher in males than in females, suggesting that high meconium secretion in males contributes to the sexual difference in δ¹⁵N. These phenomena may be common in Holometabola, which require a pupal stage. For more accurate food‐web assessments, it is important to consider stable isotope changes during different life cycles, as well as sexual differences.     

Variations in stream water uptake by Eucalyptus camaldulensis with differing access to stream water

The stable isotopes ²H and ¹⁸O were used to determine the water sources of Eucalyptus camaldulensis at three sites with varying exposure to stream water, all underlain by moderately saline groundwater. Water uptake patterns were a function of the long-term availability of surface water. Trees with permanent access to a stream used some stream water at all times. However, water from soils or the water table commonly made up 50% of these trees' water. Trees beside an ephemeral stream had access to the stream 40–50% of the time (depending on the level of the stream). No more than 30% of the water they used was stream water when it was available. However, stream water use did not vary greatly whether the trees had access to the stream for 2 weeks or 10 months prior to sampling. Trees at the third site only had access to surface water during a flood. These trees did not change their uptake patterns during 2 months inundation compared with dry times, so were not utilising the low-salinity flood water. Pre-dawn leaf water potentials and leaf ¹³C measurements showed that the trees with permanent access to the stream experienced lower water stress and had lower water use efficiencies than trees at the least frequently flooded site. The trees beside the ephemeral stream appeared to change their water use efficiency in response to the availability of surface water; it was similar to the perennial-stream trees when stream water was available and higher at other times. Despite causing water stress, uptake of soil water and groundwater would be advantageous to E. camaldulensis in this semi-arid area, as it would provide the trees with a supply of nutrients and a reliable source of water. E. camaldulensis at the study site may not be as vulnerable to changes in stream flow and water quality as previously thought.     

Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest

The stable C isotope composition (δ¹³C) of CO₂ respired by trunks was examined in a mature temperate deciduous oak forest (Quercus petraea). Month-to-month, day-to-day and diurnal, measurements were made to determine the range of variations at different temporal scales. Trunk growth and respiration rates were assessed. Phloem tissue was sampled and was analysed for total organic matter and soluble sugar ¹³C composition. The CO₂ respired by trunk was always enriched in ¹³C relative to the total organic matter, sometimes by as much as 5‰. The δ¹³C of respired CO₂ exhibited a large seasonal variation (3.3‰), with a relative maximum at the beginning of the growth period. The lowest values occurred in summer when the respiration rates were maximal. After the cessation of radial trunk growth, the respired CO₂ δ¹³C values showed a progressive increase, which was linked to a parallel increase in soluble sugar content in the phloem tissue (R = 0.95; P < 0.01). At the same time, the respiration rates declined. This limited use of the substrate pool might allow the discrimination during respiration to be more strongly expressed. The late-season increase in CO₂ δ¹³C might also be linked to a shift from recently assimilated C to reserves. At the seasonal scale, CO₂ δ¹³C was negatively correlated with air temperature (R = −0.80; P < 0.01). The diurnal variation sometimes reached 3‰, but the range and the pattern depended on the period within the growing season. Contrary to expectations, diurnal variations were maximal in winter and spring when the leaves were missing or not totally functional. By contrast to the seasonal scale, these diurnal variations were not related to air temperature or sugar content. Our study shows that seasonal and diurnal variations of respired ¹³C exhibited a similar large range but were probably explained by different mechanisms.