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.).本研究采用凯杰（Qiagen）DNeasy植物微量基因组DNA提取试剂盒（购自美国加利福尼亚州瓦伦西亚），依照制造商操作规程，从采自D. praestans模式产地的馆藏等模式标本（编号Woytkowski 5118，存放于GH标本馆）中提取总基因组DNA。此前针对其余5份标本的DNA提取尝试均告失败，推测原因是标本年代久远且保存质量不佳。采用聚合酶链式反应（polymerase chain reaction, PCR）进行扩增，扩增反应体系组成如下：3 μL基因组DNA、2.5 μL 10×PCR缓冲液（含MgCl₂）、0.75 μL 50 mM氯化镁、0.5 μL 10 μM脱氧核糖核苷三磷酸、终浓度10 μM的上下游引物各0.5 μL、0.2 μL Platinum Taq DNA聚合酶，以及17.05 μL无菌去离子水。针对rbcL基因，我们使用引物ESRBCL1F和ESRBCL1361R（Schuettpelz & Pryer, 2007）完成扩增；同时配备引物ES645F与ES663R（Schuettpelz & Pryer, 2007）用于后续测序反应。对于trnL-trnF基因间隔区，采用引物Fern-1（Trewick et al., 2002）与f（Taberlet et al., 1991）；针对rps4-trnS基因间隔区，则使用引物rps4F2（Hennequin et al., 2003）与trnS（Souza-Chies et al., 1997/2004）。上述两类基因间隔区的扩增引物均被用于后续测序。所有三类分子标记的扩增均在艾本德（Eppendorf）Mastercycler pro S热循环仪上完成。三类标记的PCR反应程序分别为：1. rbcL基因：94℃初始变性4 min，随后35个循环（94℃变性1 min、52℃退火1 min、72℃延伸2 min），最终72℃终延伸10 min；2. trnL-trnF基因间隔区：94℃初始变性2 min，随后35个循环（94℃变性15 s、48℃退火30 s、72℃延伸1.5 min），最终72℃终延伸3 min；3. rps4-trnS基因间隔区：94℃初始变性4 min，随后35个循环（94℃变性1 min、55℃退火1 min、72℃延伸1.5 min），最终72℃终延伸10 min。PCR扩增产物经1%琼脂糖凝胶电泳结合溴化乙锭染色进行可视化检测，并按照制造商说明书使用ExoSAP-IT（USB，美国俄亥俄州克利夫兰）完成纯化。将纯化后的PCR产物与对应标记的引物一并送往肯塔基州路易斯维尔的欧陆菲定基因组学（Eurofins Genomics）公司进行桑格测序（Sanger sequencing）。使用Biomatters有限公司开发的Geneious Prime 2019.2.1软件内置的MAFFT（Katoh & Standley, 2013）插件，对每一类分子标记的测序序列分别进行序列比对。