7 (0?.87) 0.11 (0?.30) 1.47 (0?.86) 0.26 (0?.10)trnH-psbA Yes 100 100 33 (114) 8 579 85 94 41 (1?6) 6.96 (0?4.12) 0. 09 (0?.84) 2.44 (0?.05) 0.15 (0?.84) 1.04 (0?.26) 0.02 (0?.13)matK Yes 100 100 33 (114) 5 826 60 65 2 (6) 2.84 (0?.91) 0.04 (0?.34) 0.86 (0?.85) 0.06 (0?.34) 0.13 (0?.49)rbcL Yes 99.09 100 32 (118) 5 672 27 29 0 1.41 (0?.80) 0.02 (0?.11) 0.66 (0?.19) 0.03 (0?.11) 0.13 (0?.30) 0.01 (0?.10)33 (123) 1 299 98 104 26 (1?3) 15.09 (0?9.47) 0.15 (0?.51) 3.28 (0?.98) 0.15 (0?.51) 2.10 (0?.73) 0.13 (0?.41)The purchase FT011 distance based on the data from Schisandraceae. The distance based on the data from Schisandra and Kadsura. The distance based on the data from Illicium.doi:10.1371/journal.pone.0125574.tDNA extraction, amplification and sequencingTotal genomic DNA was extracted from specimens by grinding silica-gel dried-leaf tissue in liquid nitrogen, and then using the CTAB procedure [68]. Total genomic DNA was dissolved in TE buffer (10 mM Tris Cl, pH 8.0, 1 mM EDTA) to a final concentration of 30?0 ng/L. Polymerase chain reaction (PCR) amplification of targeted DNA regions was performed using 2 aq PCR MasterMix (Biomed, Imatinib (Mesylate) site Beijing, China), which containing 0.05 u/L of Taq DNA Polymerase, 4 mM MgCl2, 0.4 mM of dNTP and reaction buffer. The PCR mix included 12.5 L 2 aq PCR MasterMix, 2 L each primer (5 M), 1? L template DNA and enough distilled deionized water to give a final volume of 25 L. The primer information and optimal PCR conditions are displayed in S2 Table [69?4]. PCR products were examined electrophorectically using 0.8 agarose gels. The PCR products were purified using BioMed multifunctional DNA fragment purification recovery kits (Beijing, China), and then were sequenced using the amplification primers. The bidirectional sequencing was completed using the ABI 3730 DNA Sequencer (Applied Biosystems, Carlsbad, California, USA).Sequence alignmentThe quality estimation and assembly for the newly generated sequences were performed with ContigExpress 6.0 (Invitrogen, Carlsbad, California, USA). All the newly acquired sequences were confirmed via BLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi) against the online nucleotide database and further deposited in GenBank. The accession numbers of new sequences and published sequences included in this study are provided in S1 Table. The sequence alignment for each locus was initially performed by using MUSCLE [75], and then manually edited in GeneDoc 2.7.0 [76]. The number of indel events for each dataset was inferred by deletion/insertion polymorphisms (DIP) analysis in DnaSP v5 [77]. In the DIP analysis, indels of different lengths, even in the same position of the alignment, are treated as different events. Because ofPLOS ONE | DOI:10.1371/journal.pone.0125574 May 4,4 /DNA Barcoding for Schisandraceaethe high divergence of ITS sequences among different plant families, only the 5.8S rDNA from the outgroup species could be aligned with ingroup sequences. Since the trnH-psbA sequences of other family are too divergent to be aligned with the sequences of Schisandraceae, the trnHpsbA sequences of the outgroup species were not used in the analysis. In further analyses, both family-level and genus-level assessments of the discriminatory power for single regions and their combinations were included. For the genus-level assessment, Illicium and Schisandra/ Kadsura were analyzed independently, because Illicium is quite different from Schisandra and Kadsura according to species morphology [37?9] and s.7 (0?.87) 0.11 (0?.30) 1.47 (0?.86) 0.26 (0?.10)trnH-psbA Yes 100 100 33 (114) 8 579 85 94 41 (1?6) 6.96 (0?4.12) 0. 09 (0?.84) 2.44 (0?.05) 0.15 (0?.84) 1.04 (0?.26) 0.02 (0?.13)matK Yes 100 100 33 (114) 5 826 60 65 2 (6) 2.84 (0?.91) 0.04 (0?.34) 0.86 (0?.85) 0.06 (0?.34) 0.13 (0?.49)rbcL Yes 99.09 100 32 (118) 5 672 27 29 0 1.41 (0?.80) 0.02 (0?.11) 0.66 (0?.19) 0.03 (0?.11) 0.13 (0?.30) 0.01 (0?.10)33 (123) 1 299 98 104 26 (1?3) 15.09 (0?9.47) 0.15 (0?.51) 3.28 (0?.98) 0.15 (0?.51) 2.10 (0?.73) 0.13 (0?.41)The distance based on the data from Schisandraceae. The distance based on the data from Schisandra and Kadsura. The distance based on the data from Illicium.doi:10.1371/journal.pone.0125574.tDNA extraction, amplification and sequencingTotal genomic DNA was extracted from specimens by grinding silica-gel dried-leaf tissue in liquid nitrogen, and then using the CTAB procedure [68]. Total genomic DNA was dissolved in TE buffer (10 mM Tris Cl, pH 8.0, 1 mM EDTA) to a final concentration of 30?0 ng/L. Polymerase chain reaction (PCR) amplification of targeted DNA regions was performed using 2 aq PCR MasterMix (Biomed, Beijing, China), which containing 0.05 u/L of Taq DNA Polymerase, 4 mM MgCl2, 0.4 mM of dNTP and reaction buffer. The PCR mix included 12.5 L 2 aq PCR MasterMix, 2 L each primer (5 M), 1? L template DNA and enough distilled deionized water to give a final volume of 25 L. The primer information and optimal PCR conditions are displayed in S2 Table [69?4]. PCR products were examined electrophorectically using 0.8 agarose gels. The PCR products were purified using BioMed multifunctional DNA fragment purification recovery kits (Beijing, China), and then were sequenced using the amplification primers. The bidirectional sequencing was completed using the ABI 3730 DNA Sequencer (Applied Biosystems, Carlsbad, California, USA).Sequence alignmentThe quality estimation and assembly for the newly generated sequences were performed with ContigExpress 6.0 (Invitrogen, Carlsbad, California, USA). All the newly acquired sequences were confirmed via BLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi) against the online nucleotide database and further deposited in GenBank. The accession numbers of new sequences and published sequences included in this study are provided in S1 Table. The sequence alignment for each locus was initially performed by using MUSCLE [75], and then manually edited in GeneDoc 2.7.0 [76]. The number of indel events for each dataset was inferred by deletion/insertion polymorphisms (DIP) analysis in DnaSP v5 [77]. In the DIP analysis, indels of different lengths, even in the same position of the alignment, are treated as different events. Because ofPLOS ONE | DOI:10.1371/journal.pone.0125574 May 4,4 /DNA Barcoding for Schisandraceaethe high divergence of ITS sequences among different plant families, only the 5.8S rDNA from the outgroup species could be aligned with ingroup sequences. Since the trnH-psbA sequences of other family are too divergent to be aligned with the sequences of Schisandraceae, the trnHpsbA sequences of the outgroup species were not used in the analysis. In further analyses, both family-level and genus-level assessments of the discriminatory power for single regions and their combinations were included. For the genus-level assessment, Illicium and Schisandra/ Kadsura were analyzed independently, because Illicium is quite different from Schisandra and Kadsura according to species morphology [37?9] and s.
