DOI: 10.5061/dryad.vhhmgqnrb
title: Isotope analyses of amino acids in fungi and fungal feeding Diptera larvae allow differentiating ectomycorrhizal and saprotrophic fungi-based food chains

description: 1- Both ectomycorrhizal (ECM) and saprotrophic fungi are fundamental to
 carbon and nutrient dynamics in forest ecosystems; however, the relative
 importance of these different fungal functional groups for higher trophic
 levels of the soil food web is virtually unknown. 2- To explore
 differences between fungal functional groups and their importance for
 higher trophic levels, we analysed isotopic composition of nitrogen and
 carbon in amino acids (AAs) and bulk tissue of leaf litter, fungi, and
 fungal-feeding Diptera larvae. 3- By accounting for isotopic variability
 of utilized substrates, compound-specific isotope analyses of nitrogen in
 AAs yielded more realistic results for the trophic position of fungi than
 bulk isotope analyses, with converging trophic positions of saprotrophic
 and ECM fungi. 4- Saprotrophic and ECM fungi possessed different AA δ13C
 signatures separating fungal functional groups and their consumers in
 fingerprinting approaches, thereby allowing to trace energy fluxes from
 these basal resources to higher trophic levels. 5- A pronounced isotopic
 fractionation even in essential/source AAs of fungal-feeding Diptera
 larvae necessitates further studies on tissue-/compound-specific isotopic
 differences in fungi and on potential supplementation by gut
 microorganisms. 6- The results highlight the potential of
 compound-specific isotope analysis of amino acids to identify and
 integrate contributions of different fungal functional groups to higher
 trophic levels in soil food webs.
description: Fungal sporocarps (cups/gulls) and Diptera larvae were collected
 along with fresh leaf litter on September 2016 at Malinki Biological
 Station, south of Moscow, Russia (55.4595°N, 37.1794°E). Samples were
 collected within few hectares of an over-ripe coniferous 150- to
 160-year-old forest dominated by Norway spruce and Scots pine. Replicate
 samples of fungi and Diptera larvae were taken from individual sporocarps
 (Table 1). Leaf litter was collected close to the respective
 fungi. We
 analyzed bulk isotopic composition (δ<sup>13</sup>C,
 δ<sup>15</sup>N) and AA isotopic composition
 (δ<sup>13</sup>C, δ<sup>15</sup>N) of four species
 of ECM fungi, i.e. <i>Amanita muscaria</i> (L.) Lam.,
 <i>Leccinum scabrum</i> (Bull.) Gray, <i>Lactarius
 flexuosus</i> (Pers.) Gray, and <i>Gomphidius
 glutinosus</i> (Schaeff.) Fr., and four species of saprotrophic
 fungi, including the humus saprotrophic fungi <i>Agaricus
 arvensis</i> Schaeff. and <i>Macrolepiota procera</i>
 (Scop.) Singer, and the litter saprotrophic fungi <i>Lycoperdon
 perlatum</i> Pers. and <i>Mycena pura</i> (Pers.) P.
 Kumm. Fungal functional groups were assigned according to Agerer (1987),
 Ingold & Hudson (1993), and Kohzu et al. (1999).
 Diptera larvae were collected from within the fungal sporocarps.
 Larvae of the crane fly <i>Metalimnobia quadrimaculata</i>
 (Linnaeus, 1760) (Limoniidae) were collected in sporocarps of <i>L.
 flexuosus</i>. <i>Pegomya</i> sp. Robineau-Desvoidy,
 1830 (Anthomyidae) was collected in sporocarps of <i>A.
 arvensis</i>. The fungus gnat larvae of <i>Mycetophila
 fungorum</i> (De Geer, 1776) (Mycetophilidae) were collected in
 sporocarps of <i>A. muscaria</i>. Undetermined Mycetophilidae
 larvae were collected from sporocarps of <i>M. procera</i>.
 Depending on dry weight, we used one (<i>M.
 quadrimaculata</i>) to several individuals (other Diptera larvae)
 for extraction of AAs. For CSIA of undetermined Mycetophilidae larvae and
 <i>M. quadrimaculata</i>, we had to use all available
 individuals and therefore omitted bulk isotope analysis.
 For C and N bulk isotope and C-to-N ratio analyses, fungi and
 leaf litter were dried at 60°C for 24 h and ground to powder. Appropriate
 amounts (ca. 2 mg for leaf litter and 0.5 to 1.0 mg for fungi) were
 weighed into tin capsules and stored in a dessicator until analysis.
 Diptera larvae (between 0.5 and 0.9 mg dry weight) were also weighed into
 tin capsules and dried at 60°C for 24 h. Stable isotope and C-to-N ratios
 of fungi, Diptera, and leaf litter were determined using a coupled system
 of an elemental analyzer and a mass spectrometer (Reineking, Langel,
 & Schikowski, 1993). Isotopic signatures are expressed using the δ
 notation as δX (‰) = (R<sub>sample</sub> –
 R<sub>standard</sub>)/R<sub>standard</sub> x 1000,
 with X  representing the target isotope and R the ratio of heavy to light
 isotope (<sup>13</sup>C/<sup>12</sup>C and
 <sup>15</sup>N/<sup>14</sup>N). For
 δ<sup>15</sup>N and δ<sup>13</sup>C analyses, N in
 atmospheric air and Vienna PD Belemnite served as standards,
 respectively. For CSIA, dried samples were transferred
 to Pyrex culture tubes and flushed with N<sub>2</sub> gas,
 sealed and hydrolyzed in 6 N HCl at 110°C in a heating block for 20 h
 (Larsen et al., 2013). After hydrolysis, lipophilic compounds were removed
 by adding n-hexane/DCM to the Pyrex tubes that were flushed shortly with
 N<sub>2</sub> gas and sealed before they were vortexed for 30
 s. The aqueous phase was then filtered through a Pasteur pipette lined
 with glass wool that had been pretreated at 450°C . All samples were
 transferred into 4 ml dram vials before evaporating the samples to dryness
 under a steam of N<sub>2</sub> gas at 110°C in a heating block
 for 30 min. The samples were then stored at -18°C . To volatize the AAs,
 we followed the derivatization procedure of Corr, Berstan, &
 Evershed (2007), methylating the dried samples with acidified methanol and
 subsequently acetylating them with a mixture of acetic anhydride,
 trimethylamine, and acetone (1:2:5) to produce N-acetyl methyl ester
 derivatives. To reduce oxidation of AAs during derivatization, reaction
 vials were flushed and sealed with N<sub>2</sub> prior to the
 methylation and acetylation reactions. AA
 derivatives were injected into a Thermo Trace GC coupled via a GP
 interface to a Delta Plus mass spectrometer (Thermo, Bremen, Germany). The
 GC was equipped with an Agilent J&W VF-35ms GC column (30 m x 0.32
 mm x 1.00 µm). The temperature program started with 80°C held for 1 min,
 increased by 20°C per minute to 135°C, then by 5°C per minute to 160°C and
 held for 3 min, then increased again by 8°C per minute to 300°C and held
 for 3 min. The injection temperature was 280°C and helium was used as
 carrier gas. The flow rate of helium was 2 mL
 min<sup>-1</sup>. All samples were analyzed in triplicate. To
 account for C added during derivatization and variability of isotope
 fractionation during analysis, pure AAs with known
 δ<sup>13</sup>C and δ<sup>15</sup>N values were
 also derivatized and analyzed. Nor-leucine was used as internal reference.
 The N isotopic composition of AAs in samples was expressed relative to
 atmospheric N by normalizing measured values (versus reference gas) using
 scales derived from known δ<sup>15</sup>N values of the
 reference mixture. The C isotopic composition was corrected for carbon
 added during derivatization following O’Brien, Fogel, & Boggs
 (2002) and was expressed relative to Vienna PD
 Belemnite.  
description: Please read the "ReadMe" file to use this dataset.
 Blanks are missing values.