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1. Dietary protein content and digestibility influences discrimination of amino acid nitrogen isotope values in a terrestrial omnivorous mammal

   https://doi.org/10.1002/rcm.9073  

   Whiteman, John P. ; Rodriguez Curras, Mauriel ; Feeser, Kelli L. ; Newsome, Seth D. ( April 2021 , Rapid Communications in Mass Spectrometry)

   Ecologists increasingly determine the δ¹⁵N values of amino acids (AA) in animal tissue; “source” AA typically exhibit minor variation between diet and consumer, while “trophic” AA have increased δ¹⁵N values in consumers. Thus, trophic‐source δ¹⁵N offsets (i.e., Δ¹⁵N_T‐S) reflect trophic position in a food web. However, even minor variations in δ¹⁵N_{source AA}values may influence the magnitude of offset that represents a trophic step, known as the trophic discrimination factor (i.e., TDF_T‐S). Diet digestibility and protein content can influence the δ¹⁵N values of bulk animal tissue, but the effects of these factors on AA Δ¹⁵N_T‐Sand TDF_T‐Sin mammals are unknown.

   We fed captive mice (Mus musculus) either (A) a low‐fat, high‐fiber diet with low, intermediate, or high protein; or (B) a high‐fat, low‐fiber diet with low or intermediate protein. Mouse muscle and dietary protein were analyzed for bulk tissue δ¹⁵N using elemental analyzer‐isotope ratio mass spectrometry (EA‐IRMS), and were also hydrolyzed into free AA that were analyzed for δ¹⁵N using gas chromatography‐combustion‐IRMS.

   As dietary protein increased, Δ¹⁵N_{Consumer‐Diet}slightly declined for bulk muscle tissue in both experiments; increased for AA in the low‐fat, high‐fiber diet (A); and remained the same or decreased for AA in the high‐fat, low‐fiber diet (B). The effects of dietary protein on Δ¹⁵N_T‐Sand on TDF_T‐Svaried by AA but were consistent between variables.

   Diets were less digestible and included more protein in Experiment A than in Experiment B. As a result, the mice in Experiment A probably oxidized more AA, resulting in greater Δ¹⁵N_{Consumer‐Diet}values. However, the similar responses of Δ¹⁵N_T‐Sand of TDF_T‐Sto diet variation suggest that if diet samples are available, Δ¹⁵N_T‐Saccurately tracks trophic position. If diet samples are not available, the patterns presented here provide a basis to interpret Δ¹⁵N_T‐Svalues. The trophic‐source offset of Pro‐Lys did not vary across diets, and therefore may be more reliable for omnivores than other offsets (e.g., Glu‐Phe).

    

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2. Meta‐analysis of primary producer amino acid δ 15 N values and their influence on trophic position estimation

   https://doi.org/10.1111/2041-210X.13678  

   Ramirez, Matthew D. ; Besser, Alexi C. ; Newsome, Seth D. ; McMahon, Kelton W. ( August 2021 , Methods in Ecology and Evolution)

   Compound‐specific stable isotope analysis of individual amino acids (CSIA‐AA) has emerged as a transformative approach to estimate consumer trophic positions (TP_CSIA) that are internally indexed to primary producer nitrogen isotope baselines. Central to accurate TP_CSIAestimation is an understanding of beta (β) values—the differences between trophic and source AA δ¹⁵N values in the primary producers at the base of a consumers’ food web. Growing evidence suggests higher taxonomic and tissue‐specificβvalue variability than typically appreciated.

   This meta‐analysis fulfils a pressing need to comprehensively evaluate relevant sources ofβvalue variability and its contribution to TP_CSIAuncertainty. We first synthesized all published primary producer AA δ¹⁵N data to investigate ecologically relevant sources of variability (e.g. taxonomy, tissue type, habitat type, mode of photosynthesis). We then reviewed the biogeochemical mechanisms underpinning AA δ¹⁵N andβvalue variability. Lastly, we evaluated the sensitivity of TP_CSIAestimates to uncertainty in meanβ_Glx‐Phevalues and Glx‐Phe trophic discrimination factors (TDF_Glx‐Phe).

   We show that variation inβ_Glx‐Phevalues is two times greater than previously considered, with degree of vascularization, not habitat type (terrestrial vs. aquatic), providing the greatest source of variability (vascular autotroph = −6.6 ± 3.4‰; non‐vascular autotroph = +3.3 ± 1.8‰). Within vascular plants, tissue type secondarily contributed toβ_Glx‐Phevalue variability, but we found no clear distinction among C₃, C₄and CAM plantβ_Glx‐Phevalues. Notably, we found that vascular plantβ_Glx‐Lysvalues (+2.5 ± 1.6‰) are considerably less variable thanβ_Glx‐Phevalues, making Lys a useful AA tracer of primary production sources in terrestrial systems. Our multi‐trophic level sensitivity analyses demonstrate that TP_CSIAestimates are highly sensitive to changes in bothβ_Glx‐Pheand TDF_Glx‐Phevalues but that the relative influence ofβvalues dissipates at higher trophic levels.

   Our results highlight that primary producerβvalues are integral to accurate trophic position estimation. We outline four key recommendations for identifying, constraining and accounting forβvalue variability to improve TP_CSIAestimation accuracy and precision moving forward. We must ultimately expand libraries of primary producer AA δ¹⁵N values to better understand the mechanistic drivers ofβvalue variation.

    

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3. Effects of chemical preservation on bulk and amino acid isotope ratios of zooplankton, fish, and squid tissues

   https://doi.org/10.1002/rcm.8408  

   Hetherington, Elizabeth D. ; Kurle, Carolyn M. ; Ohman, Mark D. ; Popp, Brian N. ( April 2019 , Rapid Communications in Mass Spectrometry)

   It is imperative to understand how chemical preservation alters tissue isotopic compositions before using historical samples in ecological studies. Specifically, although compound‐specific isotope analysis of amino acids (CSIA‐AA) is becoming a widely used tool, there is little information on how preservation techniques affect amino acidδ¹⁵N values.

   We evaluated the effects of chemical preservatives on bulk tissueδ¹³C andδ¹⁵N and amino acidδ¹⁵N values, measured by gas chromatography/isotope ratio mass spectrometry (GC/IRMS), of (a) tuna (Thunnus albacares) and squid (Dosidicus gigas) muscle tissues that were fixed in formaldehyde and stored in ethanol for 2 years and (b) two copepod species,Calanus pacificusandEucalanus californicus, which were preserved in formaldehyde for 24–25 years.

   Tissues in formaldehyde‐ethanol had higher bulkδ¹⁵N values (+1.4,D. gigas; +1.6‰,T. albacares), higherδ¹³C values forD. gigas(+0.5‰), and lowerδ¹³C values forT. albacares(−0.8‰) than frozen samples. The bulkδ¹⁵N values from copepods were not different those from frozen samples, although theδ¹³C values from both species were lower (−1.0‰ forE. californicusand −2.2‰ forC. pacificus) than those from frozen samples. The mean amino acidδ¹⁵N values from chemically preserved tissues were largely within 1‰ of those of frozen tissues, but the phenylalanineδ¹⁵N values were altered to a larger extent (range: 0.5–4.5‰).

   The effects of preservation on bulkδ¹³C values were variable, where the direction and magnitude of change varied among taxa. The changes in bulkδ¹⁵N values associated with chemical preservation were mostly minimal, suggesting that storage in formaldehyde or ethanol will not affect the interpretation ofδ¹⁵N values used in ecological studies. The preservation effects on amino acidδ¹⁵N values were also mostly minimal, mirroring bulkδ¹⁵N trends, which is promising for future CSIA‐AA studies of archived specimens. However, there were substantial differences in phenylalanine and valineδ¹⁵N values, which we speculate resulted from interference in the chromatographic resolution of unknown compounds rather than alteration of tissue isotopic composition due to chemical preservation.

    

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4. Nitrogen stable isotope turnover and discrimination in lizards

   https://doi.org/10.1002/rcm.9030  

   Warne, Robin W. ; Wolf, Blair O. ( January 2021 , Rapid Communications in Mass Spectrometry)

   Nitrogen stable isotope ratio (δ¹⁵N) processes are not well described in reptiles, which limits reliable inference of trophic and nutrient dynamics. In this study we detailed δ¹⁵N turnover and discrimination (Δ¹⁵N) in diverse tissues of two lizard species, and compared these results with previously published carbon data (δ¹³C) to inform estimates of reptilian foraging ecology and nutrient physiology.

   We quantified¹⁵N incorporation and discrimination dynamics over 360 days in blood fractions, skin, muscle, and liver ofSceloporus undulatusandCrotaphytus collaristhat differed in body mass. Tissue samples were analyzed on a continuous flow isotope ratio mass spectrometer.

   Δ¹⁵N for plasma and red blood cells (RBCs) ranged between +2.7 and +3.5‰; however, skin, muscle, and liver did not equilibrate, hindering estimates for these somatic tissues.¹⁵N turnover in plasma and RBCs ranged from 20.7 ± 4 to 303 ± 166 days among both species. Comparison with previously published δ¹³C results for these same samples showed that¹⁵N and¹³C incorporation patterns were uncoupled, especially during winter when hibernation physiology could have played a role.

   Our results provide estimates of¹⁵N turnover rates and discrimination values that are essential to using and interpreting isotopes in studies of diet reconstruction, nutrient allocation, and trophic characterization in reptiles. These results also suggest that somatic tissues can be unreliable, while life history shifts in nutrient routing and metabolism potentially cause¹⁵N and¹³C dynamics to be decoupled.

    

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5. Amino acid carbon isotope fingerprints are unique among eukaryotic microalgal taxonomic groups

   https://doi.org/10.1002/lno.12350  

   Stahl, Angela R. ; Rynearson, Tatiana A. ; McMahon, Kelton W. ( April 2023 , Limnology and Oceanography)

   Eukaryotic microalgae play critical roles in the structure and function of marine food webs. The contribution of microalgae to food webs can be tracked using compound‐specific isotope analysis of amino acids (CSIA‐AA). Previous CSIA‐AA studies have defined eukaryotic microalgae as a single functional group in food web mixing models, despite their vast taxonomic and ecological diversity. Using controlled cultures, this work characterizes the amino acidδ¹³C (δ¹³C_AA) fingerprints—a multivariate metric of amino acid carbon isotope values—of four major groups of eukaryotic microalgae: diatoms, dinoflagellates, raphidophytes, and prasinophytes. We found excellent separation of essential amino acidδ¹³C (δ¹³C_EAA) fingerprints among four microalgal groups (mean posterior probability reclassification of 99.2 ± 2.9%). We also quantified temperature effects, a primary driver of microalgal bulk carbon isotope variability, on the fidelity ofδ¹³C_AAfingerprints. A 10°C range in temperature conditions did not have significant impacts on variance inδ¹³C_AAvalues or the diagnostic microalgalδ¹³C_EAAfingerprints. Theseδ¹³C_EAAfingerprints were used to identify primary producers at the base of food webs supporting consumers in two contrasting systems: (1) penguins feeding in a diatom‐based food web and (2) mixotrophic corals receiving amino acids directly from autotrophic endosymbiotic dinoflagellates and indirectly from water column diatoms, prasinophytes, and cyanobacteria, likely via heterotrophic feeding on zooplankton. The increased taxonomic specificity of CSIA‐AA fingerprints developed here will greatly improve future efforts to reconstruct the contribution of diverse eukaryotic microalgae to the sources and cycling of organic matter in food web dynamics and biogeochemical cycling studies.

    

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