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1. 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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2. Stable isotope fingerprinting traces essential amino acid assimilation and multichannel feeding in a vertebrate consumer

   https://doi.org/10.1111/2041-210X.13903  

   Manlick, Philip J. ; Newsome, Seth D. ( June 2022 , Methods in Ecology and Evolution)

   Animals often consume resources from multiple energy channels, thereby connecting food webs and driving trophic structure. Such ‘multichannel feeding’ can dictate ecosystem function and stability, but tools to quantify this process are lacking. Stable isotope ‘fingerprints’ are unique patterns in essential amino acid (EAA) δ¹³C values that vary consistently between energy channels like primary production and detritus, and they have emerged as a tool to trace energy flow in wild systems. Because animals cannot synthesize EAAs de novo and must acquire them from dietary proteins, ecologists often assume δ¹³C fingerprints travel through food webs unaltered. Numerous studies have used this approach to quantify energy flow and multichannel feeding in animals, but δ¹³C fingerprinting has never been experimentally tested in a vertebrate consumer.

   We tested the efficacy of δ¹³C fingerprinting using captive deer micePeromyscus maniculatusraised on diets containing bacterial, fungal and plant protein, as well as a combination of all three sources. We measured the transfer of δ¹³C fingerprints from diet to consumer liver, muscle and bone collagen, and we used linear discriminant analysis (LDA) and isotopic mixing models to estimate dietary proportions compared to known contributions. Lastly, we tested the use of published δ¹³C source fingerprints previously used to estimate energy flow and multichannel feeding by consumers.

   We found that EAA δ¹³C values exhibit significant isotopic (i.e. trophic) fractionation between consumer tissues and diets. Nevertheless, LDA revealed that δ¹³C fingerprints are consistently routed and assimilated into consumer tissues, regardless of isotopic incorporation rate. Isotopic mixing models accurately estimated the proportional diets of consumers, but all models overestimated plant‐based protein contributions, likely due to the digestive efficiencies of protein sources. Lastly, we found that δ¹³C source fingerprints from published literature can lead to erroneous diet reconstruction.

   We show that δ¹³C fingerprints accurately measure energy flow to vertebrate consumers across tissues with different isotopic incorporation rates, thereby enabling the estimation of multichannel feeding at various temporal scales. Our findings illustrate the power of δ¹³C fingerprinting for quantifying food web dynamics, but also reveal that careful selection of dietary source data is critical to the accuracy of this emerging technique.

    

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3. 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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4. Assessment of nutritional stress in famine burials using stable isotope analysis

   https://doi.org/10.1002/ajpa.24054  

   Walter, Brittany S. ; DeWitte, Sharon N. ; Dupras, Tosha ; Beaumont, Julia ( April 2020 , American Journal of Physical Anthropology)

   We compared δ¹⁵N and δ¹³C values from bone and dentine collagen profiles of individuals interred in famine‐related and attritional burials to evaluate whether individuals in medieval London who experienced nutritional stress exhibit enriched nitrogen in bone and tooth tissue. Dentine profiles were evaluated to identify patterns that may be indicative of famine during childhood and were compared with the age of enamel hypoplasia (EH) formation to assess whether isotopic patterns of undernutrition coincide with the timing of physiological stress.

   δ¹⁵N and δ¹³C isotope ratios of bone collagen were obtained from individuals (n= 128) interred in attritional and famine burials from a medieval London cemetery (c. 1120–1539). Temporal sequences of δ¹⁵N and δ¹³C isotope profiles for incrementally forming dentine collagen were obtained from a subset of these individuals (n= 21).

   Results indicate that individuals from attritional graves exhibit significantly higher δ¹⁵N values but no significant differences were found between burial types for the sexes. Analyses of dentine profiles reveal that a lower proportion of famine burials exhibit stable dentine profiles and that several exhibit a pattern of opposing covariance between δ¹⁵N and δ¹³C. EH were also observed to have formed during or after the opposing covariance pattern for some individuals.

   The results of this study may reflect differences in diet between burial types rather than nutritional stress. Though nutritional stress could not be definitively identified using bone and dentine collagen, the results from dentine analysis support previous observations of biochemical patterns associated with nutritional stress during childhood.

    

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5. 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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