Successful DNA sequencing of a 75 year-old herbarium specimen of Aspicilia aschabadensis (J. Steiner) Mereschk.

Improved DNA extraction methods have allowed increasing use of herbarium specimens for taxonomic and phylogenetic studies. Although DNA of historical specimens is often highly degraded, 250–800 bp long sequences have been obtained, for instance, from over 200 year-old specimens of angiosperms (Ames & Spooner 2008; Andreasen et al. 2009), from over 100 year-old oomycetes (Telle & Thines 2008), and from a 74 yearold fungus (O’Gorman et al. 2008). Due to low concentration and poor quality of DNA, amplification of short, c. 300 bp, overlapping fragments has proven a most successful method (Drábková et al. 2002; O’Gorman et al. 2008). The results are in striking contrast with lichen-forming fungi, which, in our experience, specimens older than 5–10 years usually tend either to fail or give poor quality sequences. Here we report a successful amplification and sequencing of nuclear ribosomal ITS regions from a 75 year-old herbarium specimen of Aspicilia aschabadensis (J. Steiner) Mereschk (Turkmenistan, Central part of Kopetdag district, next to the border of Iran, stony part of mountain, to the SW of Solukli, 12 vi 1934, A Borisova [LE]). Using the same methods as described below, we have repeatedly obtained sequences from rather old Aspcilia specimens, viz. Aspicilia changaica (20 years old), A. transbaicalica (25 years old), A. jussuffii (29 years old), A. lacunosa (39 years old) and A. “desertorum” (39 years old). However, DNA extraction from an 80 year old specimen of Aspicilia tominii was not successful. The phylogenetic position of the genus Aspicilia is still not well established. According to recent studies, the genus, as currently delimited, belongs to two families. In higher level phylogenetic studies by Schmitt et al. (2006) and Lumbsch et al. (2007) the inclusion of Aspicilia in the family Megasporaceae was well supported. However, in the study by Miądlikowska et al. (2006), Aspicilia was nested within the family Pertusariaceae (see Sohrabi & Ahti 2010). Total DNA of the sample was extracted using Qiagen’s DNAeasy Blood and Tissue Kit. For DNA extraction, c. 1 × 1 mm piece of medulla was used. We followed the instructions given by the kit manufacturer, except for the initial grinding and eventual elution, in which 160 l of ATL (40 l +120 l) buffer was used instead of 180 l suggested by the manufacturer. ITS regions of the nuclear ribosomal DNA were amplified using the following primers: ITS1F (Gardes & Bruns 1993) and ITS 4 (White et al. 1990). The PCR amplification was performed with Ready-To-Go PCR beads (Pharmatica Biotech) by adding 4 l undiluted DNA, 1 l of each primer (ITS1F/ITS4) (10 M) and sterile water to a total volume of 19 l for dissolving the beads. The Gene Amp PCR system 9700 (PerkinElmer) PTC-100 and PTC-200 Thermocyclers (MJ Research) were used under the following conditions: initial denaturation for 5 min at 95°C, followed by 5 cycles of 30 s at 95°C, 30 s at 58°C, and 1 min at 72°C; in the remaining 30 or 35 cycles the annealing temperature was decreased to 56°C; in the last cycle a final extension for 7 min at 72°C was included. An alternative