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Abstract
DNA barcoding can be useful for species identification and phylogenetic analysis, but its effectivity has not been verified in most neotropical cloud forest plants. We tested three plastid barcodes, rbcLa, matK, and trnH-psbA, in selected pteridophytes, a well-represented group in these forests, from a little-explored area in Oaxaca, Mexico, applying the CBOL criteria for barcoding. We used BLASTn, genetic distance, and monophyly tree-based analyses employing neighbor-joining (NJ), maximum likelihood (ML), and Bayesian inference methods. Universal primers for rbcLa and trnH-psbA were successfully amplified and bi-directionally sequenced, but matK could not be amplified for most species. rbcLa showed the highest species discrimination in BLASTn (66.67%). trnH-psbA exhibited higher significant interspecific divergence values than rbcL and rbcLa + trnH-psbA (two-sample sign test, P value < 2.2e−16). Using NJ and ML phylogenetic trees, monophyletic species were successfully resolved (100%), differing only in support values and displaying full agreement with the most recent fern classification. ML trees showed the highest mean support value (80.95%). trnH-psbA was the only barcode that could detect the Elaphoglossoideae subfamily. Species discrimination did not increase using rbcLa + trnH-psbA. rbcLa is useful for fern barcoding, trnH-psbA is most helpful for phylogenetic analyses, and matK may not work as a universal barcoding marker.
Highlights
DNA barcoding can be useful for species identification and phylogenetic analysis, but its effectivity has not been verified in most neotropical cloud forest plants
The purpose of this study is to evaluate the performance of three plastid barcodes: partial gene rbcLa, matK, and the intergenic spacer trnH-psbA, using standard primers and under the three Consortium for the Barcode of Life (CBOL) criteria[5] to build a barcode library of pteridophytes in the Mixteca highlands, Oaxaca, Mexico
Using universal primers from Canadian Center for DNA Barcoding (CCDB) of rbcL and trnHpsbA, fern DNA was successfully amplified in most cases (96.77%)
Summary
DNA barcoding can be useful for species identification and phylogenetic analysis, but its effectivity has not been verified in most neotropical cloud forest plants. Universal primers for rbcLa and trnH-psbA were successfully amplified and bi-directionally sequenced, but matK could not be amplified for most species. RbcLa is useful for fern barcoding, trnH-psbA is most helpful for phylogenetic analyses, and matK may not work as a universal barcoding marker. A sine qua non requirement for species identification using DNA barcoding is the existence of a published trustable sequence. The Consortium for the Barcode of Life’s (CBOL) plant working group evaluated seven-candidate plastid DNA regions based on universality, sequence quality, and species discrimination. Other studies suggest using additional loci, including non-coding plastid regions, such as the intergenic spacer trnH–psbA6–8 and the nuclear marker ITS9,10 Chloroplast DNA región /primer name rbcLa rbcLa-F rbcLa-R matK MatK-1RKIM-f MatK-3FKIM-r FERmatK fEDR FERmatK rAGK trnH-psbA trnHf_05 psbA3_f
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