DOI: 10.5061/dryad.gtht76hm1
title: A reassessment of the little-known Amazonian fern Diplazium praestans based on molecular and morphological evidence

description: Family- and genus-level circumscription of ferns in the suborder
 Aspleniineae (eupolypods II) has long been controversial, due in part to
 confusion about the relationship among the families Aspleniaceae and
 Athyriaceae.  Recent studies have demonstrated that character
 states traditionally used to infer a close relationship between these two
 families are either symplesiomorphic or homoplastic, and re-examination of
 numerous taxa has led to the recircumscription of several clades, and the
 description of several new families and genera.  In light of
 these findings, we re-evaluated the taxonomic affinities of Diplazium
 praestans, a little-known fern from western Amazonia that is
 morphologically disparate to the remainder of Neotropical Diplazium.
  Using sequence data from three chloroplast markers and analysis
 of eight morphological characters, we demonstrate that Diplazium praestans
 was erroneously placed in that genus and instead is a Hymenasplenium.
  We place it in a phylogenetic context, reassess its morphology
 in light of our findings, evaluate its conservation status under IUCN
 criteria, and provide a new combination: Hymenasplenium praestans.
  We also provide an updated key to the Neotropical species of
 Hymenasplenium and discuss unresolved taxonomic problems in the genus.
description: 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.).