DNA isolation, PCR amplification and sequencing

Genomic DNA was extracted from freshly collected and frozen herbarium specimens. About 15 mg of visually uncontaminated lichen thallus was sampled from each specimen for molecular analyses. Frozen lichen samples were lyophilized and disrupted with a stainless steel bead in a Retsch MM2000 mill (Düsseldorf, Germany) for 2 min at 30 Hz.

Genomic DNA was extracted using the Qiagen DNAeasy Plant Kit (QIAGEN, Hilden, Germany), following the manufacturer’s Plant Tissue Mini protocol. Sequences were generated from three independent nuclear markers: the nuclear ribosomal internal transcribed spacer (ITS) region, a partial sequence of the putatively single-copy translation elongation factor-1 alpha gene (EF-1α), and an intron-containing portion of the putatively single-copy RNA polymerase II second largest subunit gene (RPB2). The fungus-specific primer ITS1F (Gardes & Bruns 1993) and the universal primer ITS4 (White et al. 1990) were used to amplify the ITS, as described in Cornejo et al. (2009). To make the PCR more specific and to amplify shorter fragments, the partial RPB2 gene was amplified and cycle sequenced with the modified primer sets Lp-RPB-R (5’-CCCATGGCTTGCTTACCCAT-3’) and Lp-RPB-F (5’-CAAACCGCGTCAACTGCATAAT-3’) designed by Cornejo & Scheidegger (2015). The partial EF-1α gene (Johannesson et al. 2000) was amplified and cycle sequenced with the modified primer sets Lp-EF-1α-F (5’-RGACAAGRCTCACATCAACGTGGT-3’) and Lp-EF-1α-R (5’-CCAGTGATCATGTTCTTGATGAAGT-3’) (Cornejo & Schei­degger 2015).

All amplifications were performed with 1 μL DNA extract in a total of 15 μm containing the JumpStartTM REDTaq® Ready MixTM PCR Reaction Mix (Sigma-Aldrich, St. Louis, MO, USA) and 100 nM of each primer. The cycling conditions for the ITS primer set designed in our lab were: (a) 2 min at 94 °C, (b) ١٠ cycles of (30 s at 94 °C, ٤٥ s at ٥٦ °C, ٤٥ s at ٧٢ °C), (c) 25 cycles of (30 s at 94 °C, ٤٥ s at ٥٢ °C, ٤٥ s at ٧٢ °C), (d) 10 min at 72 °C final extension. The cycling conditions for the RPB2 primer set designed in our lab were: (a) 2 min at 94 °C, (b) ٣٠ cycles of (60 s at 94 °C, ٦٠ s at ٤٩ °C, ٦٠ s at ٧٢ °C), (c) 10 min at 72 °C final extension. The cycling conditions for amplification of the EF-1α locus with primer designed in our lab were: (a) 2 min at 94 °C, (b) ٣٥ cycles of (30 s at 94 °C, ٣٠ s at ٥٣ °C, ٣٠ s at ٧٢ °C), (c) 10 min at 72 °C final extension. The PCR products were visualised on 1.5 % agarose gel. The PCR products were then sent to Microsynth AG (Balgach, Switzerland) for sequencing with the same primers as the original PCR amplifications. Specimens used in this study, along with voucher information from GenBank accession numbers, chemical features and type of diaspores or apothecia are listed in Table 2.

Overview of 72 green-algal Lobaria specimens arranged according to the retrieved clades in the species-tree, with the respective voucher information from GenBank. Sequences that were newly obtained in this study are indicated in bold face.