Computational protocol: Revision of series Gravesiana (Adiantum L.) based on morphological characteristics, spores and phylogenetic analyses

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[…] Total DNA was extracted from silica-gel-dried leaf materials using a modified CTAB DNA extraction protocol []. Six pairs of primers “ESATPF412F and ESTRNR46F”, “ESATB172F and ESATPE45R”, “1F and 1379R”, “f and pl”, “trnS and rps4.5”, “Adn matK fIHS* and FER matK rAGK” were used to amplify the chloroplast gene regions atpA, atpB, rbcL, trnL-F, rps4-trnS, and matK, respectively [, , , , , , , ]. PCR reactions were performed in 30 μL reaction volumes, including 1.0–2.4 μL of each primer (5p), 17–60 ng sample DNA, 1.5 U of Taq DNA polymerase, 10 × buffer (including Mg2+), 0.25 mmol·L-1 dNTP, and ultrapure water. The PCR products were purified and sequenced with an ABI 3730XL by Majorbio Company.The sequences were assembled with Sequencher v. 4.14, aligned using the program Clustal X v. 2.0 [] and then edited manually through Bioedit v.7.1.3 []. Phylogenetic trees of each marker and the combined markers (atpA, atpB, rbcL, trnL-F, rps4-trnS and matK) were constructed using maximum parsimony (MP) and Bayesian Markov chain Monte Carlo inference (BI). The maximum parsimony analyses were performed with PAUP* 4.0b10 [], treating gaps as missing data and using the heuristic search options with 1000 random replicates and tree-bisection-reconnection (TBR) branch swapping. All characteristics were unordered and equally weighted. Through MrModeltest2 v. 2.3 [], GTR+I+G was selected as the best fit molecular evolution model for the MP and Bayesian analyses. For Bayesian inference, trees were generated for 1,000,000 generations with sampling every 100 generations. Four chains were used with a random initial tree. For each of the individual data partitions and the combined dataset, the first 2500 sample trees were discarded as burn-in to ensure that the chains reached stationarity. Nodes receiving bootstrap support (BS) of < 70% in the MP analyses or PP of < 0.95 in the BI analyses were not considered to be well supported. [...] Thirteen characters of the 78 individuals including the height of the plant (H), the length of pinna stalk (LR), pinna aligned forms (FP), number of pinna (NP), pinna size (S) and shape (SP), pinna margin (M), number of veins flabellate at base (NV), veins tendency near upper margin (TV), sori number and shape per pinna (NSS), pinna texture (T), powder-covered or not on the abaxial surface of the pinna (P), and scale shape (SS) were measured before DNA extraction. The morphologic data were maintained in a spreadsheet and then compiled into a digital matrix. Then, we mapped them onto the phylogenetic tree of the series Gravesiana constructed with six combined chloroplast genes, atpA, atpB, rbcL, trnL-F, rps4-trnS and matK, using the program Mesquite version 2.71 [] under maximum parsimony. Original measured values of the morphological characters were presented in . The classifications of the characters mapped are listed below:Height of the plant (H): (0) H ≤5 cm, (1) 5 cm13 cmLength of the pinna stalk (LR): (0) 0 mm9;Pinna size (S: length × width, unites/cm): (0) 2.0–6.0 × 2.0–5.0, (1) 6.0–15.0 × 5.0–17.0, (2) 6.0–15.0 × 2.0–5.0Pinna shape (SP): (0) obovate, (1) obdeltoid, (2) subround, (3) othersPinna margin (M): (0) entire, (1) dentateNumber of veins flabellate at base (NV): (0) NV≤4(5), (1) NV>4(5)Vein tendency near upper margin (TV): (0) straight up to terminal, (1) curve closed to marginSori number and shape per pinna (NSS): (0) 1, orbicular; (1) 1, reniform; (2) 1 to many, reniform; (3) 1 to many, transversally linear and truncate at false indusia terminal; (4) 1, shape same as (3)Pinna texture (T): (0) membranous, (1) coriaceousPowder-covered or not on abaxial surface of pinna (P): (0) no, (1) yes […]

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