Ribitol

Plant samples were harvested and immediately snap-frozen in liquid nitrogen. Samples were freeze-dried and then homogenized with 4 mm steel balls for 1 min at 25/s frequency. Dry plant powder was suspended in 400 μl of methanol containing 200 μM DL-3-aminobutyric acid (BABA) and 400 μM ribitol as internal standards and agitated at 1500 rpm for 15 min. Subsequently, 200 μl of chloroform were added and samples were agitated for five additional minutes. Finally, 400 μl of ultra-pure water were added, samples were then vigorously vortexed and centrifuged at 13 000 g for 5 min. Two aliquots of upper phase per samples were transferred to clean microtubes and dried in vacuo.
For amino acids analysis, dry residues were suspended in ultra-pure water and 10 μl of the resulting extract were sampled for amino acids derivatization according to the AccQTag Ultra Derivitization Kit protocol (Waters Corporation, Milford, MA). Amino acids were analysed using an Acquity UPLC^® system (Waters Corporation, Milford, MA) by injecting 1 μl of the derivatization mix onto an Acquity UPLC^® BEH C18 1.7 μm 2.1 × 100 mm column heated at 55°C. Amino acids were eluted at 0.7 ml.min^-1 flow with a mix of 10-fold diluted AccQTag Ultra Eluent (A; Waters Corporation, Milford, MA) and acetonitrile (B) according to the following gradient: initial, 99.9% A; 0.54 min, 99.9% A; 6.50 min, 90.9% A, curve 7; 8.50 min, 78.8% A, curve 6; 8.90 min, 40.4% A, curve 6; 9.50 min, 40.4% A, curve 6; 9.60 min, 99.9% A, curve 6; 10.10 min, 99.9% A. Derivatized amino acids were detected at 260 nm using a photo diode array detector. Amount of amino acids was expressed in μmoles per g of dry weight of sample (μmoles.g^-1 DW) making reference to BABA signal, external calibration curve of amino acids and dry weight of samples.
For GC-MS analysis, dry residues were dissolved in 50 μl of freshly prepared methoxyamine hydrochloride solution in pyridine (20 mg/ml). Samples were agitated for 90 min at 30°C, 50 μl of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA; Sigma, #394866) were then added and derivatization was conducted at 37°C for 30 min under agitation. After transfer to glass vials, samples were incubated at room temperature over-night before injection. GC-MS analysis was performed according to Roessner et al. [64 (link)]. GC-MS system consisted of a TriPlus autosampler, a Trace GC Ultra chromatograph and a Trace DSQII quadrupole mass spectrometer (Thermo Fischer Scientific Inc, Waltham, MA). Chromatograms were deconvoluted using the AMDIS software v2.65 http://chemdata.nist.gov/mass-spc/amdis/. Metabolites levels were expressed in relative units making reference to ribitol signal and dry weight of samples.

Raw data obtained from GC/MS measurements were processed by applying version 2.2 N-2013-01-15 of the in-house developed software MetaboliteDetector [49 (link)]. The peak identification was performed non-targeted with a combined compound library for each GC column applied. After processing, non-biological peaks and artefacts were eliminated by the aid of blanks. Peak areas were normalized to the corresponding internal standards (o-cresol or ribitol) and derivatives were summarized. Data were fitted sigmoidally after Boltzmann and uptake rates were determined using Origin9.0G software when applicable.
For leucine and phenylalanine fermentation products, peak areas were estimated after normalization on the relative proportion of the specific quantification ion compared to the total ion chromatogram but we cannot exclude differences of the two compounds during drying or derivatization procedures.

Stable carbon ¹³C was chosen to trace photobiont assimilated carbon. Substrate-attached sporocarps with active algal partner (Li-6400XT-proved, Figure. S1) were enabled to assimilate ¹³CO₂ enriched air in a water-sealed 3L inverse Petri dish with internal fan (See Figure. S2). The labelling device is described in detail elsewhere^{61 (link)}. [¹³CO₂] was about 1000 µmol mol⁻¹. Atmospheric CO₂ was previously replaced by CO₂ free synthetic air and then ca 3 mL of ¹³CO₂ (> 99 atom %, Sigma-Aldrich, Luis., USA) was injected. Samples assimilated for two to 18 h under about 200 µmol m⁻² s⁻¹ of white LED light. Then fungal/algal crusts were scratched down by razor and killed in boiling methanol. Homogenised and filtered metOH extracts were used for analysis of polyols and ergosterol (see Methods S1 for further details).
We employed three approaches to trace ¹³C enriched metabolites. (1) HPLC–MS to separate algal polyols (ribitol and sorbitol) from fungal mannitol and to measure their ¹³C content. Although mannitol was detected in some algal groups^{62 (link),63 (link)}, it is not produced by algae in our systems, i.e., Coccomyxa and Desmococcus^{64 (link)}. (2) GC-IRMS to separate trimethylsilyl derivatives of those polyols (TMS-ribitol, TMS-mannitol and TMS-sorbitol) and, again, to quantify their ¹³C enrichment. And (3) HPLC–MS to separate fungal specific ergosterol and to measure if it is ¹³C enriched. For more details see Method S1.

Serial dilutions (10⁻¹–10⁻³) were made with peptone water (Oxoid Ltd., Hampshire, United Kingdom). Equal volumes (200 μL) from each dilution were spread over the surface of Dicloran Rose Bengal Chloramphenicol (DRBC) (Merck, Darmstadt, Germany) and CHRomagar Candida™ agar plates (BBL International Inc., London, UK). All the plates were incubated for 48 h at 30 °C. Colony counts were performed on individual plates. Representative yeast colonies were selected and grouped by morphotype, isolated, and conserved using Saboraud Dextrose Agar (Merck) immersed in sterile mineral oil and cryopreserved in glycerol 30% (v/v). A macroscopic evaluation of colonies was performed with a stereoscope (Celestron Lab’s S10-60 Stereo, Celestron, LLC, Torrance, CA, USA) taking account of the shape, edge, surface, appearance, elevation, brightness, and consistency. All the yeasts were evaluated for sugar assimilation using the API 20C AUX system (BioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. Several carbohydrates were evaluated, including D-Glucose, Glycerol, 2-keto-Gluconate calcium, L-Arabinose, D-Xylose, Adonitol, Xylitol, D-Galactose, Inositol, D-Sorbitol, Methyl-D-Glucopyranoside, N-Acetyl-Glucosamine, D-Lactose (Bovine), D-Maltose, D-Sucrose, D-Trehalose, D-Melezitose and D-Raffinose [28 (link)]. The galleries were incubated at 30 °C for 72 h in an airtight box containing a small volume of water to create a humid atmosphere. The observation of yeast growth was considered positive. The negative control contained no carbon source, and the positive control contained glucose. The carbohydrate assimilation profile obtained for each tested isolate was interpreted using ApiwebTM software (BioMérieux, reference: 40011).