Ntraspecific vs. minimum interspecific p-distances for single regions of Schisandraceae. Each

Ntraspecific vs. minimum interspecific p-distances for single regions of Schisandraceae. Each dot represents a species for which two or more individuals were sampled. Dots above the diagonal line indicate the presence of a barcoding gap. doi:10.1371/journal.pone.0125574.gidentification, incorrect identification, and no match were set according to previous Varlitinib site studies [89,90]. Character-based analysis. The search for diagnostic characters during the single-locus assessment was performed using the web-based CAOS (Characteristic Attribute Organization System) workbench (http://boli.uvm.edu/caos-workbench/caos.php) [91]. Aligned DNA sequences and ML trees were imported into Mesquite v2.76 [92] and exported as NEXUS file formats for the CAOS-Analyzer. The outputs of the CAOS-Analyzer were used for the CAOS-Barcoder in order to find `characteristic attributes’ (CAs) (character-based diagnostics), including pure characters (purchase NVP-BEZ235 existing across all members of a clade but never in any other clade) and private characters 1471-2474-14-48 (existing across some members of a clade but never in any other clade). Nucleotide positions at which pure CAs and private CAs shared in at least 80 of all members journal.pone.0174109 within a group were included in the calculation. Both simple CAs (confined to a single nucleotide position) and compound CAs (combined states at multiple nucleotide positions) were considered [93].PLOS ONE | DOI:10.1371/journal.pone.0125574 May 4,6 /DNA Barcoding for SchisandraceaeFig 2. Plots of maximum intraspecific vs. minimum interspecific p-distances for multi-locus combinations of Schisandraceae. Each dot represents a species for which two or more individuals were sampled. Dots above the diagonal line indicate the presence of a barcoding gap. doi:10.1371/journal.pone.0125574.gResults Amplification and sequence analysisThe four commonly used barcoding loci performed equally well in terms of the universality of amplification and sequencing (Table 1). There were 437 new sequences generated in this study: 108 ITS, 110 trnH-psbA, 110 matK, and 109 rbcL (S1 Table). Including the sequences from GenBank and previous studies, in grand total, 123 ITS, 114 trnH-psbA, 114 matK, and 118 rbcL sequences of Schisandraceae species were included in this study and summarized in Table 1. In addition, the multi-copy problem for ITS in plants reviewed by Nietto Rosello [94] was not present in this study. Among four commonly used barcoding loci, ITS showed the highest percentage of parsimony informative sites (24.46 ), followed by trnH-psbA (14.68 ), matK (7.26 ), and rbcL (4.02 ) (Table 1). Most of the parsimony informative sites for ITS came from the ITS1 and ITS2 regions (97.06 ), and ITS1 provided more informative sites than IT S2 (Table 1). Indels were more prevalent in trnH-psbA and ITS alignments, compared with matK and rbcL alignments (Table 1).Distance-based identificationAmong four commonly used barcoding loci, ITS had the highest average interspecific divergence (0.0988 for the whole family, 0.0247 for Schisandra and Kadsura, 0.0147 for Illicium), while trnH-psbA was at an intermediate level of variation, but higher than both matK and rbcLPLOS ONE | DOI:10.1371/journal.pone.0125574 May 4,7 /DNA Barcoding for SchisandraceaeTable 2. Wilcoxon tests for four commonly used barcoding loci based on the data from Schisandraceae. Wilcoxon signed-rank tests based on the interspecific and intraspecific p-distances among four barcoding loci W+ WW+ ITS ITS ITS trnH-psbA trnH-psbA matK ITS ITS.Ntraspecific vs. minimum interspecific p-distances for single regions of Schisandraceae. Each dot represents a species for which two or more individuals were sampled. Dots above the diagonal line indicate the presence of a barcoding gap. doi:10.1371/journal.pone.0125574.gidentification, incorrect identification, and no match were set according to previous studies [89,90]. Character-based analysis. The search for diagnostic characters during the single-locus assessment was performed using the web-based CAOS (Characteristic Attribute Organization System) workbench (http://boli.uvm.edu/caos-workbench/caos.php) [91]. Aligned DNA sequences and ML trees were imported into Mesquite v2.76 [92] and exported as NEXUS file formats for the CAOS-Analyzer. The outputs of the CAOS-Analyzer were used for the CAOS-Barcoder in order to find `characteristic attributes’ (CAs) (character-based diagnostics), including pure characters (existing across all members of a clade but never in any other clade) and private characters 1471-2474-14-48 (existing across some members of a clade but never in any other clade). Nucleotide positions at which pure CAs and private CAs shared in at least 80 of all members journal.pone.0174109 within a group were included in the calculation. Both simple CAs (confined to a single nucleotide position) and compound CAs (combined states at multiple nucleotide positions) were considered [93].PLOS ONE | DOI:10.1371/journal.pone.0125574 May 4,6 /DNA Barcoding for SchisandraceaeFig 2. Plots of maximum intraspecific vs. minimum interspecific p-distances for multi-locus combinations of Schisandraceae. Each dot represents a species for which two or more individuals were sampled. Dots above the diagonal line indicate the presence of a barcoding gap. doi:10.1371/journal.pone.0125574.gResults Amplification and sequence analysisThe four commonly used barcoding loci performed equally well in terms of the universality of amplification and sequencing (Table 1). There were 437 new sequences generated in this study: 108 ITS, 110 trnH-psbA, 110 matK, and 109 rbcL (S1 Table). Including the sequences from GenBank and previous studies, in grand total, 123 ITS, 114 trnH-psbA, 114 matK, and 118 rbcL sequences of Schisandraceae species were included in this study and summarized in Table 1. In addition, the multi-copy problem for ITS in plants reviewed by Nietto Rosello [94] was not present in this study. Among four commonly used barcoding loci, ITS showed the highest percentage of parsimony informative sites (24.46 ), followed by trnH-psbA (14.68 ), matK (7.26 ), and rbcL (4.02 ) (Table 1). Most of the parsimony informative sites for ITS came from the ITS1 and ITS2 regions (97.06 ), and ITS1 provided more informative sites than IT S2 (Table 1). Indels were more prevalent in trnH-psbA and ITS alignments, compared with matK and rbcL alignments (Table 1).Distance-based identificationAmong four commonly used barcoding loci, ITS had the highest average interspecific divergence (0.0988 for the whole family, 0.0247 for Schisandra and Kadsura, 0.0147 for Illicium), while trnH-psbA was at an intermediate level of variation, but higher than both matK and rbcLPLOS ONE | DOI:10.1371/journal.pone.0125574 May 4,7 /DNA Barcoding for SchisandraceaeTable 2. Wilcoxon tests for four commonly used barcoding loci based on the data from Schisandraceae. Wilcoxon signed-rank tests based on the interspecific and intraspecific p-distances among four barcoding loci W+ WW+ ITS ITS ITS trnH-psbA trnH-psbA matK ITS ITS.

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