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Nitrogen (N)-fixing symbiosis is critical to terrestrial ecosystems, yet possession of this trait is known for few plant species. Broader presence of the symbiosis is often indirectly determined by phylogenetic relatedness to taxa investigated via manipulative experiments. This data gap may ultimately underestimate phylogenetic, spatial, and temporal variation in N-fixing symbiosis. Still needed are simpler field or collections-based approaches for inferring symbiotic status. N-fixing plants differ from non-N-fixing plants in elemental and isotopic composition, but previous investigations have not tested predictive accuracy using such proxies. Here we develop a regional field study and demonstrate a simple classification model for fixer status using nitrogen and carbon content measurements, and stable isotope ratios (δ 15 N and δ 13 C), from field-collected leaves. We used mixed models and classification approaches to demonstrate that N-fixing phenotypes can be used to predict symbiotic status; the best model required all predictors and was 80-94% accurate. Predictions were robust to environmental context variation, but we identified significant variation due to native vs. non-native (exotic) status and phylogenetic affinity. Surprisingly, N content-not δ 15 N-was the strongest predictor, suggesting that future efforts combine elemental and isotopic information. These results are valuable for understudied taxa and ecosystems, potentially allowing higher-throughput field-based N-fixer assessments., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Rationale: Bone lipid compound-specific isotope analysis (CSIA) and bone collagen and apatite stable isotope ratio analysis are important sources of ecological and paleodietary information. Pressurized liquid extraction (PLE) is quicker and utilizes less solvent than traditional methods of lipid extraction such as soxhlet and ultrasonication. This study facilitates dietary analysis by optimizing and testing a standardized methodology for PLE of bone cholesterol., Methods: Modern and archaeological bones were extracted by PLE using varied temperatures, solvent solutions, and sample weights. The efficiency of PLE was assessed via quantification of cholesterol yields. Stable isotopic ratio integrity was evaluated by comparing isotopic signatures (δ 13 C and δ 18 O values) of cholesterol derived from whole bone, bone collagen and bone apatite. Gas chromatography/mass spectrometry (GC/MS) and gas chromatography isotope ratio mass spectrometry (GC/IRMS) were conducted on purified collagen and lipid extracts to assess isotopic responses to PLE., Results: Lipid yield was optimized at two PLE extraction cycles of 75 °C using dichloromethane/methanol (2:1 v/v) as a solvent with 0.25-0.75 g bone sample. Following lipid extraction, saponification combined with the derivatization of the neutral fraction using trimethylsilylation yielded nearly twice the cholesterol of non-saponified or non-derivatized samples. It was also found that lipids extracted from purified bone collagen and apatite could be used for cholesterol CSIA. There was no difference in the bulk δ 13 C values of collagen extracted from bone with or without lipid. However, there was a significant depletion in 18 O of bone apatite due to lipid presence or processing., Conclusions: These results should assist sample selection and provide an effective, alternative extraction method for bone cholesterol that may be used for isotopic and paleodietary analysis. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Rationale: Stable isotope analysis is a valuable technique for dietary estimation in ecological and archaeological research, yet many variables can potentially affect tissue stable isotope signatures. Controlled feeding studies across a range of species have consistently demonstrated impacts of caloric restriction on tissue stable isotope ratios, but most have focused on juvenile, fasting, and/or starving individuals, and most have utilized soft tissues despite the importance of bone for paleodietary analyses. The goal of this study was to determine whether temporally defined, moderate food restriction could affect stable carbon and/or nitrogen isotope ratios in adult mammalian bone - a tissue that arguably reflects long-term dietary signals., Methods: Adult rats fed a standard laboratory diet were restricted to 45% of ad libitum intakes for 3 or 6 months. Relevant anatomical and physiological parameters were measured to confirm that the restriction protocol resulted in significant nutritional stress and to provide independent data to facilitate interpretation of stable isotope ratios. Femoral bone δ(13)Ccollagen, δ(15)Ncollagen, and δ(13)Capatite values were determined by isotope ratio mass spectrometry., Results: Calorie-restricted animals exhibited a small, yet significant enrichment in (15)Ncollagen compared with control animals, reflecting protein-calorie stress. While the δ(13)Ccollagen values did not differ, the δ(13)Capatite values revealed less enrichment in (13)C than in controls, reflecting catabolism of body fat. Independent anatomical and physiological data from these same individuals support these interpretations., Conclusions: Results indicate that moderate caloric restriction does not appreciably undermine broad interpretations of dietary signals in adult mammalian bone. Significant variability among individuals or groups, however, is best explained by marked differences in energy intake over variable timescales. An inverse relationship between the δ(13)Capatite and δ(15)Ncollagen values observed in this study indicates that a more robust pattern is expected with more severe or prolonged restriction and suggests this pattern may have utility as a marker of food deprivation in archaeological populations., (Copyright © 2014 John Wiley & Sons, Ltd.)