All fungal specimens were collected on Crown Lands for which no permission is required. Specimens of fresh, field-collected mushroom fruiting bodies were either air-dried at a temperature of 30–35 °C or frozen at − 80 °C until processing, and air-dried voucher specimens have been deposited at the Dr. Laurie L. Consaul Herbarium, London, Canada (UWO) and the National Mycological Herbarium of Canada, Ottawa (DAOM) (Table 1).
DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from air-dried specimens following Thorn et al.9. Primers ITS1 and ITS6R were used to amplify the ITS region, LS1 and LR3 to amplify ~ 650 bases of the 5’-LSU region and Canth-ef1a983-F and Canth-ef1a-1567-R to amplify Tef-19,41,42,43,44. The PCR products were checked using gel electrophoresis and successful products were cleaned using Bio Basic EZ-10 Spin Column PCR Products Purification Kit. Cleaned PCR products were submitted to the sequencing facility of Robarts Institute (University of Western Ontario) to obtain sequences through Sanger sequencing with amplification primers, and internal sequencing primers CanthITS1_Internal-R, 5.8S-R-Canth, and ITS86R-Canth for the ITS region9. New sequences produced for this study were deposited in GenBank as accessions MN181445–MN181461 and MN206911–MN206945.
Phylogenetic analyses
Sequences of the ITS, LSU and Tef-1 regions were cleaned and assembled with SeqEd v.1.03, then, together with sequences of related species downloaded from GenBank, each region was aligned separately with MAFFT v.745 under the G-INS-i strategy, with “leave gappy regions” selected. The draft Cantharellus cibarius genome (QOWL00000000.1)18 was searched by BLASTn for ribosomal and Tef-1 sequences. The full-length match to our Tef-1 sequences was found in a single scaffold (QOWL01010594_RC), but no ITS sequences and only partial LSU sequences were found (in QOWL01010930_RC, QOWL01012415, QOWL01007530_RC, and QOWL01005980_RC). Alignments were imported into MEGA X46,47, trimmed and concatenated into a single ITS-LSU-Tef-1 dataset, then optimized manually. Phylogenetic trees were constructed with maximum likelihood (ML), with 1000 bootstrapping replicates in MEGA X. Analyses were repeated with Bayesian inference using MrBayes 3.2.6 with 4 chains and 5 million generations, discarding the first 25% of trees, when the average standard deviation of split frequencies had stabilized below 0.0148. Tree topologies were compared, and Bayesian prior probabilities transferred to the ML bootstrap tree in Adobe Acrobat.
Genetic analysis of carotenoid synthesis genes
In order to design primers to amplify portions of the Al-1 and Al-2 genes in white and golden chanterelles, these genes were first located in three published Cantharellus genome sequences18 using tBLASTn49 to query the genomes with protein sequences from N. crassa (PRJNA132; Al-1 XM_959620.2 and Al-2 XM_960632.3)19. Candidate gene sequences from Cantharellus appalachiensis (QLPK00000000.1; Al-1 Scaffold 4647: QLPK01003932.1 and Al-2 Scaffold 1419: QLPK01001208.1), C. cibarius (QOWL00000000.1; Al-1 Scaffold 15338: QOWL01009792.1 and Al-2 Scaffold 1560: QOWL01001169.1), and C. cinnabarinus (QLPJ00000000.1; Al-1 Scaffold 1057: QLPJ01000880.1 and Al-2 Scaffold 2474: QLPJ01001998.1) were annotated in Geneious using a discontiguous megablast against GenBank to search for homologous motifs50. Based on these alignments, putative ORFs were annotated and aligned, and an overlapping set of PCR primers for AL-1 and AL-2 were designed in Geneious (Table 5). Designed primers were tested for specificity using the BLAST algorithm against the three Cantharellus genomes18. PCR amplified products were assessed for quality, cleaned, sent for sequencing, and assembled as above. Assembled sequences of white and gold samples were compared, along with their putative amino acid products determined using ExPASy51, guided by the translations of the Neurospora crassa Al-1 (XM_959620.2) and Al-2 (XM_960632.3) genes. Partial sequences of the Al-1 and Al-2 genes of gold and white variants have been deposited in GenBank as MW442833–MW442836.
Pigment analysis
Field-collected mushroom fruiting bodies were weighed while fresh, wrapped in aluminum foil, and frozen at − 80 °C for pigment analyses, and other samples were weighed fresh and then dried to obtain a conversion for fresh to dry weight. Pigments were extracted with ice-cold 100% acetone at 4 °C and dim light. The supernatant was filtered through a 0.22 µm syringe filter and samples were stored at − 80 °C until analysed. Pigments were separated and quantified by high-performance liquid chromatography (HPLC) as described previously20, with some modifications. The system consisted of a Beckman System Gold programmable solvent module 126, diode array detector module 168 (Beckman Instruments, San Ramon, California, USA), CSC-Spherisorb ODS-1 reverse-phase column (5 mm particle size, 25 × 0.46 cm I.D.) with an Upchurch Perisorb A guard column (both columns from Chromatographic Specialties Inc., Concord, Ontario, Canada). Samples were injected using a Beckman 210A sample injection valve with a 20 μL sample loop. Pigments were eluted isocratically for 6 min with a solvent system of acetonitrile:methanol:0.1 M Tris–HCl (pH 8.0), (72:8:3.5, v/v/v), followed by a 2 min linear gradient to 100% methanol:hexane (75:25, v/v) which continued isocratically for 4 min. Total run time was 12 min. Flow rate was 2 mL min−1. Absorbance was detected at 440 nm and peak areas were integrated by Beckman System Gold software. Retention times and response factors of Chl a, Chl b, lutein and ß-carotene were determined by injection of known amounts of pure standards purchased from Sigma (St. Louis, MO, USA). The retention times of zeaxanthin, antheraxanthin, violaxanthin and neoxanthin were determined by using pigments purified by thin-layer chromatography as described by Diaz et al.52.
Extraction and analysis of chanterelle lipids
Samples of each chanterelle species were homogenized to fine powder in a cryomill (Reitch, Germany) and 100 mg of the homogenized powder mixed with 1 mL methanol (MeOH), 1 mL chloroform (CHCl3) and 0.8 mL water following Pham et al.53. The sample mixture was thoroughly vortexed, then centrifuged (Sorvall Legend XT/XF centrifuge; ThermoFisher Scientific, Mississauga, Ontario) at 2500 rpm for 15 min. The organic layer was transferred to new vials, dried under nitrogen and then reconstituted in 1 mL chloroform:methanol (1:1 v/v). Aliquots were then used for either gas chromatography with mass spectrometric and flame ionization detection (GC–MS/FID) or ultra-high-performance liquid chromatography with heated electrospray ionization high resolution accurate mass tandem mass spectrometric analysis (UHPLC-HESI-HRAM/MS–MS) for fatty acids and intact lipids analysis, respectively.
For GC–MS/FID analysis, chanterelle fatty acids were converted to fatty acid methyl esters (FAMEs) as follows: To 300 µL aliquot of the lipid extract, 50 μL of C18:0 alkane (1 mg mL−1 in chloroform: methanol 1:1 v/v) was added as internal standards and the samples dried under nitrogen and the fatty acids esterified by adding 400 µL methanolic HCl (1.5 N). The samples were then incubated in a pre-heated oven at 60 °C for 30 min. After incubation, 0.8 mL of distilled water was added to the cooled samples and the FAMEs extracted with 3 aliquots each of 500 μL of hexane. The fractions were combined, dried under N2, re-suspended in 50 μL hexane, and the FAMEs analyzed using a Trace 1300 gas chromatograph coupled to a Flame Ionization Detector and TSQ 8000 mass spectrometer (Thermo Fisher Scientific). The FAMEs were separated on a BPX70 high-resolution column (10 m × 0.1 mm ID × 0.2 μm, Canadian Life Science, Peterborough, Ontario) using helium as the carrier gas at a flow rate of 1 mL min−1. One μL of each sample was injected in split mode (1:15) using a Tri-plus auto-sampler (Thermo Fisher Scientific). The operation conditions were as follows: initial oven temperature set at 50 °C for 0.75 min, increased to 155 °C at 4 °C min−1, ramped to 210 °C at 6 °C min−1, then 240 °C at 15 °C min−1 and final temperature held for 2 min. Methylated fatty acids were determined by comparison with retention times and mass spectra obtained from commercial standards (Supelco 37 component mix, Supelco PUFA No. 3, Sigma Aldrich, Oakville, Ontario) and the NIST database (Thermo Fisher Scientific). Standard curves were employed to determine the amount of individual fatty acids, and values are presented as nmole%.
For the UHPLC-HESI-HRAM/MS–MS analysis, a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an automated Dionex UltiMate 3000 UHPLC system was used to analyze the intact chanterelle lipids according to our previously published method53. Briefly, the intact lipids were resolved using an Accucore C30 column (150 mm × 2 mm I.D., particle size: 2.6 µm, pore diameter: 150 Å) and the following solvent systems: (i) Solvent A consisted of acetonitrile:water (60:40 v/v) containing 10 mM ammonium formate and 0.1% formic acid and (ii) Solvent B consisted of isopropanol:acetonitrile:water (90:10:1 v/v/v) with 10 mM ammonium formate and 0.1% formic acid. The conditions used for separation were 30 °C (column oven temperature), flow rate of 0.2 mL min−1, and 10 µL of sample injected. The gradient system used was as follow: solvent B increased to 30% in 3 min; 43% in 5 min, 50% in 1 min, 90% in 9 min, 99% in 8 min, and finally maintained at 99% for 4 min. The column was re-equilibrated for 5 min before each new injection. Full scans and tandem MS acquisitions were performed in both negative and positive modes using the following parameters: sheath gas: 40, auxiliary gas: 2, ion spray voltage: 3.2 kV, capillary temperature: 300 °C; S-lens RF: 30 V; mass range: 200–2000 m/z; full scan at 70,000 m/z resolution; top-20 data-dependent MS/MS resolution at 35,000 m/z, collision energy of 35 (arbitrary unit); injection time of 35 min for C30RP chromatography; isolation window: 1 m/z; automatic gain control target: 1e5 with dynamic exclusion setting of 5.0 s. The instrument was externally calibrated to 1 ppm using electrospray ionization (ESI); negative and positive calibration solutions (Thermo Fisher Scientific) were used to calibrate the instrument at 1 ppm. Tune parameters were optimized using PC 18:1(9Z)/18:1(9Z), Cer d18:1/18:1(9Z), PG 18:1(9Z)/18:1(9Z), sulfoquinovosyl diacylglycerols [SQDG] 18:3(9Z,12Z,15Z)/16:0, monogalactosyl diglyceride [MGDG] 18:3(9Z,12Z,15Z)/16:3(7Z,10Z,13Z), and digalactosyldiacylglycerol [DGDG] 18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z) lipid standards (Avanti Polar Lipids, Alabaster, AL, USA) in both negative and positive ion modes. The data were processed using either X-Calibur 4.0 (Thermo Fisher Scientific) or LipidSearch version 4.1 (Mitsui Knowledge Industry, Tokyo, Japan) software packages.
Phenolics analysis by GC–MS
Reagent grade phenolic acid standards including benzoic acids, p-hydroxybenzoic acid, vanillic acid, gallic acid, 3,4-dihydroxybenzoic acid, syringic acid, gentisic acid, veratric acid, salicylic acid, cinnamic acid, o-coumaric acid, m-coumaric acid, p-coumaric acid, ferulic acid, sinapic acid, caffeic acid, sodium hydroxide, N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA-TCMS) were purchased from Sigma Aldrich. Methanol, ethyl acetate, and hydrochloric acid (36% w/v) were purchased from VWR (Mississauga, Ontario, Canada). For alkaline hydrolysis of powdered chanterelles, 100 µL of aqueous 3,4-dihydroxybenzoic acid solution (0.2 mg mL−1) was added to a mixture containing 4 g of sample in 8 mL 1 M sodium hydroxide. The resultant mixture was incubated in the dark for 24 h at 25 °C on an orbital shaker (50 rpm). The pH of the reaction mixture was adjusted to 2.0–2.5 using concentrated HCl then vortexed. The organic components were extracted four times with 4 mL methanol: ethyl acetate (1:3 ratio) into pre-weighed vials. The solvent was evaporated under nitrogen at 35 °C to determine the crude extraction yield. The extracts were resuspended in 1 mL ethyl acetate, vortexed, then 300 µL of extract transferred into a pre-weighed vial, dried under nitrogen, and 50 µL of BSTFA-TCMS and 50 µL of pyridine added. The resultant mixture was incubated at 70 °C in darkness for 30 min then transferred to GC vials for GC–MS analysis. Standard solutions were derivatized in a similar manner.
A Thermo Scientific Trace 1300 gas chromatograph coupled to a Triple Quad mass spectrometer (Thermo Fisher Scientific) was used for the analysis and the compounds resolved on a ZB-5MS non-polar stationary phase column (30 m × 0.25 mm I.D., 0.25 μm film thickness, Phenomenex, Torrance, CA, USA) with helium as the carrier gas (flow rate of 0.6 mL min−1). One microliter of the standard or sample was injected in basic mode (15:0) using a Tri-plus auto-sampler. The oven temperature program was as follows: the initial oven temperature was 70 °C (held for 1 min), was increased at 12 °C min−1 to 220 °C (held for 3 min), 15 °C min−1 to reach 250 °C and held for 1 min. Identification of the phenolic acids (as trimethylsilyl ether, TMS) was based on the comparison of their retention times and mass spectra with that of the NIST library and commercial standards, with quantities calculated and expressed as nmole%.
Analysis of the volatile profile of chanterelles by SPME-GC/MS
Volatile metabolites were extracted and analysed by Solid-Phase Microextraction and Gas Chromatography/Mass Spectrometry (SPME-GC/MS) following Vidal et al.54. Briefly, 100 mg of sample powder obtained after cryo homogenization was placed in 10 mL headspace glass vials and kept at 50 °C for 5 min (sample equilibration) before volatile metabolites extraction and analysis began. A divinylbenzene/carboxen/polydimethylsyloxane (DVB/CAR/ PDMS) coated fibre (1 cm long, 50/30 μm film thickness; Supelco, Sigma-Aldrich), was inserted into the headspace of the sample vial and held there for 60 min55,56. Chanterelle volatile composition was analyzed using a Trace 1300 gas chromatograph coupled to a TSQ 8000 Triple Quadrupole mass spectrometer (Thermo Fisher Scientific). The extracted volatile compounds were separated using a ZB-5MS non-polar stationary phase column (30 m × 0.25 mm I.D., 0.25 μm film thickness; Phenomenex) with He used as the carrier at a flow rate of 1 mL min−1. After extraction the fibre was desorbed for 10 min in the injection port and the instrument operated as follows: splitless mode with a purge time of 5 min, initial oven temperature set at 50 °C (5 min hold) and increased to 290 °C at 4 °C min−1 (2 min hold). Ion source and quadrupole mass analyzer temperatures were set at 230 and 150 °C respectively, injector and detector temperatures held at 250 and 290 °C respectively, mass spectra ionization energy set at 70 eV, and data acquisition done in scan mode. After each sample desorption, the fiber was cleaned for 10 min at 250 °C in the conditioning station. Volatile compounds were identified by matching the obtained mass spectra with those of available standards, and mass spectra from commercial libraries NIST/EPA/NIH (version 2.2, Thermo Fisher Scientific) or the scientific literature55,56. Volatile compounds in the chanterelle samples were semi-quantified based on the area counts × 10−6 of the base peak. Compounds with lower abundances than 10−6 area counts were considered as traces. Although the chromatographic response factor of each compound is different, the area counts determined are useful for comparison of the relative abundance of each compound in the different samples analysed55,56.
Statistics and reproducibility
Results of analyses of lipids and phenolics are presented as means and standard errors of 4 replicates and those of head-space analyses of volatiles are based on 2 replicates (Tables 2, 3, 4). The values of all dependent variables were tested for normal distribution and homoscedasticity by Shapiro–Wilk and Levene tests, respectively. For variables with homogeneous variance, parametric one-way analysis of variance (ANOVA) was used to determine if there were significant differences between chanterelle samples. Where significance was detected, the means were compared with Fisher’s Least Significant Difference (LSD), α = 0.05. Where the assumption of normality was not met (pentanal, 3-octen-2-one, 2-nonanone, 2-undecanone, (E)-α-Ionone, 3-hydroxy-α-ionene, all phenolic acids, and some minor fatty acids 10:0, 15:0, 14:1, 16:1n5, 20:1n9 and 24:1n9), no significance was detected after the data were treated with a non-parametric Kruskal–Wallis test and significance (p < 0.05) was adjusted using a post-hoc Bonferroni correction, in some cases despite clear numerical differences and separation in the first two factors of principal component analysis (PCA). PCA was conducted using XLSTAT Premium version (Addinsoft, Paris, France) to discern similarities or differences between the variants. Figures were prepared using XLSTAT Premium version and SigmaPlot 13.0 software programs (Systat Software Inc., San Jose, CA).
Source: Ecology - nature.com
