APPENDIX 1 in The Amidella clade in Europe (Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens (E. - J. Gilbert) Bertault and the importance of taxon-specific PCR primers for identification

APPENDIX 1. — List of taxon-specific primers, their matching and positive control universal primers and annealing temperatures (Ta), used either for sequencing or as molecular markers. * refers to the site number of the 5’ terminal on the alignment used for the phylogenetic reconstruction in Figure 2.
TargetRegion ID Strand Position* SequenceTa (°C) Matching Control CommentAmanitaITSAcu If1 plus975’ AGCGGGCATGTGCACG63.5ITS2ITS1–curtipesAcu If2 plus5335’ TGCATGAAGAGGAGTTTTTGGAC54.3ITS4ITS3–LSUAcu Lr1 minus14105’ GCCAGCCCATTGAATGATCTG55.0NLC2RLR3–AmanitaITSAle If1 plus2765’ GTTTTATATGAATGGCTATTG51.0ITS4ITS3–lepiotoidesAle Ir1 minus5725’ GTCCAACAAACATCTCCAAC56.9ITS1FITS2–LSUAle Lf1 plus11955’ GAAGTCAGTAGAGTTGGCTG57.5LR3NLC2R –Ale Lr1 minus13015’ CCTTAGCTTTTCTCTAGTG57.5NLC2RLR3–AmanitaITSApo If1 plus585’ TTAAAACTCTGGCAAAG49.4ITS2ITS1–ponderosaApo If2 plus4995’ GAGTGTCATTCATATTCTC50.3ITS4ITS3–LSUApo Lr1 minus13995’ CAAAGCCCAGCCTAGTAG56.8NLC2RLR3–AmanitaITSAps Ir1 minus3395’ TTGTTCATTAACAATTGTCTTTC54.7ITS1FITS4A. curtipes producespseudovalensvery faint bands;comb. nov.Ode11 is divergentAps If1 plus5225’ AGTCTTCTTCGTGACAAG53.9ITS4ITS3Variety pseudovalenshas mismatchesAps Ir2 minus6915’ CTTATTTCATGGTGTAAAACTTTATC55.9ITS3ITS4–Aps Ir3 minus7315’ GACACAAATTCATTTAGAAAG51.5ITS3ITS4A. curtipes producesvery faint bandsLSUAps Lf1 plus8875’ GCAGCGTCTGGTTGTCC60.3NLC2LR0RA. curtipes alsopositive (only onemismatch)Aps Lr1 minus13015’ CCTTAGCTTTTTTCTACCGGTCAGAA62.9NLC2RLR3–Aps Lr2 minus14015’ AATGAATGGCCTGGCGA59.1NLC2RLR3Some A.curtipesspecimens displaya distinctive set ofslower-migratingbands