All plant parts were oven‐dried at 70°C for 48 h. Dry weight of the parts was then recorded, and samples were ground to powder using a micro ball mill (MM200, Retsch GmbH, Germany). Prior to isotopic analysis, 1.5–3 mg was weighed into tin cups. The δ13C and carbon content (%) were then measured using an elemental analyser (Vario Isotope Select, Elementar, Germany) coupled to an isotope ratio mass spectrometer (IRMS) (isoprime 100, Elementar, Germany), located in the FAO/IAEA Soil and Water Management & Crop Nutrition (SWMCN) Laboratory. Normalization of δ13C values compared to the international Vienna‐Pee Dee Belemnite standard (V‐PDB) was done using two calibrated in‐house standards: a sugar beet standard (δ13CVPDB = −26.07‰) and a sugarcane standard (δ13CVPDB = −10.95‰). Both in‐house standards were calibrated against IAEA‐CH‐6 (δ13CVPDB = −10.449 ± 0.033‰) and IAEA‐CH‐7 (δ13CVPDB = −32.151 ± 0.050‰).
Vario isotope select
Manufactured by Elementar
Sourced in Germany
The Vario Isotope Select is a laboratory instrument designed for the precise measurement of stable isotope ratios. It is capable of analyzing a wide range of sample types, including solids, liquids, and gases, to determine the isotopic composition of elements such as carbon, nitrogen, oxygen, and hydrogen.
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6 protocols using vario isotope select
1
Stable Carbon Isotope Analysis of Plant Parts
2
Isotope Analysis of Marine Organisms
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Samples collected in 2019 (n = 10) were composited while those collected in 2020 were analyzed independently (n = 25) (Figure 2 ; Table S2 ). All animal and plant samples were rinsed with deionized water prior to processing. Ilyanassa obsoleta tissues were separated from the shell and tissues of the left cheliped of C. sapidus were isolated. Fundulus spp. were fileted to remove bones and scales. To remove inorganic carbon, aliquots of Fundulus spp., C. sapidus, Uca spp. and soil samples were fumigated prior to analysis [48 ].
Samples were analyzed using an Elementar Pyrocube interfaced with an Elementar Isoprime100 Isotope Ratio Mass Spectrometer (at ANS) or with an Elementar Vision and Elementar Vario Isotope Select (at the EPA). Isotope values were calculated based on reference standards and in-house working standards which have a precision at or better than ±0.40‰ (N) and ±0.14‰ (C) based on long term replication.
Shells of museum and modern specimens were powdered with a handheld Dremel tool with a diamond bit. Values of δ15N from shell-bound carbonate were measured at ANS with the instrumentation detailed above, however for this analysis, the Pyrocube moisture trap was retrofitted to include a section of NaOH to remove evolved CO2 followed by sicapent® for the removal of evolved H2O, allowing N2 to enter the isotope ratio mass spectrometer [32 (link)].
Samples were analyzed using an Elementar Pyrocube interfaced with an Elementar Isoprime100 Isotope Ratio Mass Spectrometer (at ANS) or with an Elementar Vision and Elementar Vario Isotope Select (at the EPA). Isotope values were calculated based on reference standards and in-house working standards which have a precision at or better than ±0.40‰ (N) and ±0.14‰ (C) based on long term replication.
Shells of museum and modern specimens were powdered with a handheld Dremel tool with a diamond bit. Values of δ15N from shell-bound carbonate were measured at ANS with the instrumentation detailed above, however for this analysis, the Pyrocube moisture trap was retrofitted to include a section of NaOH to remove evolved CO2 followed by sicapent® for the removal of evolved H2O, allowing N2 to enter the isotope ratio mass spectrometer [32 (link)].
3
Radiocarbon Dating of Collagen
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To measure 14C, collagen was prepared and graphitized using the methods described by Omori et al. (86 ). An elemental analyzer (vario ISOTOPE select, Elementar Analysensysteme GmbH) was used to combust the samples and isolate pure CO2 from the combusted gas (86 ). Graphite was then produced by the catalytic reduction of the sample CO2 with H2 gas and Fe powder. The radiocarbon content of the graphite was measured using an accelerator mass spectrometer at the University Museum of the University of Tokyo. The radiocarbon dates were calibrated using OxCal (87 ) and IntCal20 (88 ).
4
Detritusphere Soil Sampling and 13C Analysis
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To analyse the detritusphere soil, the 2 cylinders of soil constituting the cores were separated from the meshes containing the residues, and the remaining residues between the two meshes were removed and stored at -20°C before lyophilization.
Slices 2 mm thick were cut out of the 2 cylinders of each core from the residue bed (6.63 g of dry soil per slice). Thus, 4 samples corresponding to 0-2 mm, 2-4 mm, 4-6 mm and 12-14 mm (as distant soil) away from the residues were obtained. The slices of the 2 cylinders corresponding to the same sampling distances were pooled to increase the soil mass available for analysis and stored at -20°C before lyophilization.
2.6. Analyses of 13 C content of residues and soil 13 C contents in residues and in the different slices of soil were analysed at different sampling times using an elemental analyser (Vario Isotope Select, Elementar, Hanau, Germany) coupled to an isotopic mass spectrometer (Precision, Elementar UK Ltd, Cheadle, UK). The 13 C contents of the intermediate soil slices (between 6 and 12 mm) that were not analysed were estimated by linear regression.
Slices 2 mm thick were cut out of the 2 cylinders of each core from the residue bed (6.63 g of dry soil per slice). Thus, 4 samples corresponding to 0-2 mm, 2-4 mm, 4-6 mm and 12-14 mm (as distant soil) away from the residues were obtained. The slices of the 2 cylinders corresponding to the same sampling distances were pooled to increase the soil mass available for analysis and stored at -20°C before lyophilization.
2.6. Analyses of 13 C content of residues and soil 13 C contents in residues and in the different slices of soil were analysed at different sampling times using an elemental analyser (Vario Isotope Select, Elementar, Hanau, Germany) coupled to an isotopic mass spectrometer (Precision, Elementar UK Ltd, Cheadle, UK). The 13 C contents of the intermediate soil slices (between 6 and 12 mm) that were not analysed were estimated by linear regression.
5
Carbon Isotope Analysis of Plant Samples
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For carbon isotope analysis, 2.6 ± 0.3 mg (bulk), 0.5 ± 0.1 mg (soluble sugar), 0.22 ± 0.03 mg (starch) and 0.14 ± 0.04 mg (cellulose) of sample were weighed into tin capsules. δ13C values of these samples were determined with an elemental analyzer (Vario Isotope Select, Elementar, Germany) coupled to an isotope ratio mass spectrometer (isoprime 100, Elementar, Germany). All δ13C values were normalized on the Vienna-Pee Dee Belemnite scale by using two in-house calibrated standards (δ13CVPDB = -26.07 ± 0.08‰; δ13CVPDB = -10.95 ± 0.06‰), containing sucrose from sugar beet and sugarcane respectively. In-house standards were calibrated with IAEA-CH-6 (δ13CVPDB = -10.449 ± 0.033‰) and IAEA-CH-7 (δ13CVPDB = -32.151 ± 0.050‰). Repeated extraction and measuring of a quality control sample, consisting of cassava leaf material, resulted in δ13CVPDB values of -24.62 ± 0.15‰ (n = 7, mean ± 1 standard deviation) for cellulose, -27.56 ± 0.02‰ (n = 10) for soluble sugar and -27.13 ± 0.10‰ (n = 10) for starch.
6
Alpha-cellulose Isotopic Analysis Protocol
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About 2–4 mg of alpha-cellulose were weighed and packed in tin foil capsules (Elemental Microanalysis Ltd. 5 × 3.5 mm #D1015). Packed samples were combusted in an Elemental Analyser (Elementar vario ISOTOPE select) linked to an Isotope Ratio Mass Spectrometer (Elementar AMS precision IRMS) and an Automated Graphitization Equipment (Ionplus AGE 3). A fraction of CO2 resulting from sample combustion in Elemental Analyser is analyzed in the IRMS and the rest is graphitized over iron powder by AGE 3. For δ13C measurements, samples were analyzed along with the NBS SRM-4990C oxalic acid II and IAEA-C3 cellulose63 (link) standards, and the precision for these measurements was 0.2 ‰.
For radiocarbon analysis, graphitized samples were pressed into aluminium targets. The targets containing samples and standards are then analyzed in the 500 kV NEC Accelerator Mass Spectrometer at DANGOOR Research Accelerator Mass Spectrometry (D-REAMS) Laboratory in the Weizmann Institute of Science64 (link). To check the accuracy of the radiocarbon measurements, VIRI D65 (link) and VIRI M66 (link) standards were analyzed along with the samples. VIRI D and VIRI M yielded an average value of 2856 ± 18 BP and 73.884 ± 0.072 pMC respectively, which are in very good agreement with the consensus value (VIRI D: 2836 ± 3.3 BP; VIRI M: 73.900 ± 0.0322 pMC)65 (link),66 (link) of these standards.
For radiocarbon analysis, graphitized samples were pressed into aluminium targets. The targets containing samples and standards are then analyzed in the 500 kV NEC Accelerator Mass Spectrometer at DANGOOR Research Accelerator Mass Spectrometry (D-REAMS) Laboratory in the Weizmann Institute of Science64 (link). To check the accuracy of the radiocarbon measurements, VIRI D65 (link) and VIRI M66 (link) standards were analyzed along with the samples. VIRI D and VIRI M yielded an average value of 2856 ± 18 BP and 73.884 ± 0.072 pMC respectively, which are in very good agreement with the consensus value (VIRI D: 2836 ± 3.3 BP; VIRI M: 73.900 ± 0.0322 pMC)65 (link),66 (link) of these standards.
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