2.1 Specimen sampling
One hundred and eighty-two (182) specimens of Niebla were collected from Baja
California Norte, Mexico in December 2016. Secondary metabolites were later identified from all specimens using thin layer chromatography (TLC) following standard methods for solvent systems ‘C’ and ‘G’ (Culberson,
1972; Orange et al., 2001). Specimens were determined by R. Spjut
according to his classification (Spjut, 1996), with exception to three
additional specimens collected in the southern USA Channel Islands, San Clemente and
Santa Barbara, determined also according to Spjut (1996). From this collection, 40 specimens representing 27
species in Spjut (1996), were selected for
restriction site-associated DNA sequencing (RADSeq) with further selection within species from different localities to infer evolutionary relationships and delimit species boundaries
(Appendix 1). Additionlly, seven
specimens of the related sister genus, Vermilicinia spp., were used as the outgroup (Spjut et al., 2020). We
note that specimens were selected and sequenced before a broad molecular
phylogeny was available for Niebla (Spjut et al., 2020).
From the 40 specimens, a small portion of the thallus free of visible contamination
was excised. Total genomic DNA was then extracted using the E.Z.N.A.
Plant DNA DS Mini Kit (Omega Bio-tek), following the manufacturers’
recommendations to amplify the fungal internal transcribed spacer region (ITS), the standard DNA barcoding marker for fungi (Schoch et al., 2012) we selected for RADSeq using ZR
Fungal/Bacterial DNA MiniPrep Kit (Zymo Research, Irvine, CA, USA) as
previously described (Grewe et al., 2017). We amplified the ITS region
(ITS1, 5.8S & ITS2) using primers ITS1 (Gardes and Bruns 1993) with
ITS4 (White et al. 1990). Polymerase chain reaction (PCR) amplifications
were performed using Ready-To-Go PCR Beads (GE Healthcare, Pittsburgh,
PA, United States), with cycling parameters following a 66–56°C
touchdown reaction (Lindblom and Ekman 2006). PCR products were
visualized on 1% agarose gel and enzymatically cleaned using ExoSAP-IT
Express (USB, Cleveland, OH, United States). Complementary strands were
sequenced using the same primers used for amplifications, and sequencing
reactions were performed using BigDye 3.1 (Applied Biosystems, Foster
City, CA, United States). Products were run on an ABI 3730 automated
sequencer (Applied Biosystems) at the DNA Sequencing Center at Brigham
Young University, Provo, UT, United States.