Fungal and Algal DNA Barcoding Protocol

Fresh specimens were used for DNA extraction of both fungal and algal partners. DNA was extracted with a cetyltrimethylammonium bromide (CTAB)-based protocol⁵⁴ . ITS nrDNA locus was found to be a useful fungal barcode sequence, easily amplifiable, and moreover with a sufficient number of references in the NCBI database. In the case of algae, we sequenced nrDNA ITS and 18S regions and the rbcL gene. The latter was found as best amplified and therefore selected for barcoding and phylogenetic analysis. Polymerase chain reactions were performed in a reaction mixture containing master mix consisting of 2.5 mmol/L MgCl₂, 0.2 mmol/L of each dNTP, 0.3 μmol/L of each primer, 0.5 U Taq polymerase in the manufacturer’s reaction buffer (Top-Bio, Praha, Czech Republic), and milli-Q water to make up a final volume of 10 μL. The primers used for PCR and the cycling conditions are summarized in Table S2. Successful amplifications were sent for Sanger sequencing (GATC Biotech, Konstanz, Germany). Sequences were edited using BioEdit v.7.0.9.0⁵⁵ and Geneious Prime 2022.0 (https://www.geneious.com).
Sequences of our specimens were supplemented by relevant sequences from GenBank—NCBI database. Sequences were aligned by MAFFT v.7^{56 (link)}; available online at http://mafft.cbrc.jp/alignment/server/) using the Q-INS-i algorithm and adjusted manually. The best-fit model of sequence evolution was selected using the Akaike information criterion calculated in jModelTest v.0.1.1^{57 (link)}. Relationships were assessed using Bayesian inference as implemented in MrBayes v.3.1.2^{58 (link)}. Two runs starting with a random tree and employing four simultaneous chains each (one hot, three cold) were executed. The temperature of a hot chain was set empirically to 0.1, and every 100th tree was saved. The analysis was considered to be completed when the average standard deviation of split frequencies dropped below 0.01. The first 25% of trees were discarded as the burn-in phase, and the remaining trees were used for construction of a 50% majority consensus tree.