A reassessment of the little-known Amazonian fern Diplazium praestans based on molecular and morphological evidence

Total genomic DNA was extracted from the isotypea collection from the type locality of D. praestansan herbarium specimen of D. praestans ( (Woytkowski 5118, GH)) of D. praestans using the Qiagen DNeasy plant mini kit (Valencia, California, U.S.A.) following the manufacturer’s protocol. Previous attempts to extract DNA from five other specimens had failed, likely due to their old age and the poor quality of the specimens. Amplification was performed using the polymerase chain reaction (PCR) using 3 μL of genomic DNA, 2.5 μL of 10× PCR buffer (MgCl2), 0.75 μL of 50 mM MgCl2, 0.5 μL of 10 μM deoxyribonucleotide triphosphates, 0.5 μL of each primer at 10 μM concentration, 0.2 μL of Platinum Taq DNA polymerase, and 17.05 μL of purified water. For the rbcL gene, we used the primers ESRBCL1F and ESRBCL1361R (Schuettpelz and Pryer 2007) for amplification; the primers ES645F and ES663R (Schuettpelz and Pryer 2007) were also sent with the amplification primers for sequencing. For the intergenic spacer trnL-trnF we used the primers Fern-1 (Trewick et al. 2002) and f (Taberlet et al. 1991), and for the spacer rps4-trnS we used the primers rps4F2 (Hennequin et al. 2003) and trnS (Souza-Chies et al. 19972004); the same primers used for amplification of the intergenic spacers were sent for sequencing. Amplification of all three markers was performed on an Eppendorf Mastercycler pro S thermocycler. The PCR conditions for rbcL consisted of an initial denaturation cycle of 4 min at 94°C and then 35 cycles of 1 min at 94°C, 1 min at 52°C, 2 min at 72°C, followed by a final extension period of 10 min at 72°C. PCR conditions for trnL-trnF consisted of an initial denaturation cycle of 2 min at 94°C and then 35 cycles of 15 s at 94°C, 30 s at 48°C, 1.5 min at 72°C, followed by a final extension period of 3 min at 72°C. PCR conditions for rps4-trnS consisted of an initial denaturation cycle of 4 min at 94°C and then 35 cycles of 1 min at 94°C, 1 min at 55°C, 1.5 min at 72°C, followed by a final extension period of 10 min at 72°C. The PCR products were electrophoresed and visualized on 1% agarose gel with ethidium bromide and purified using ExoSAP-IT (USB, Cleveland, Ohio) following manufacturer protocols. Purified PCR products and primers for each marker were sent to Eurofins Genomics (Louisville, Kentucky) for sanger sequencing. Sequence alignment was performed for each marker separately using the MAFFT (Katoh and Standley 2013) plugin for Geneious Prime 2019.2.1 (Biomatters Ltd.).