The successful application of DNA barcoding as a tool for species discrimination and identification depends on the adequacy of barcode reference libraries of well identified species. Next generation sequencing (NGS) methods have proven to be more promising especially for herbarium specimen containing degraded DNA than conventional barcoding. To make a reference that can serve as a benchmark for identifying palms (Arecaceae), we demonstrated the practical applicability of genome skimming, target capture and k-mer NGS methods on the genus Pinanga of Borneo using genetic distance, similarity (BLAST), and phylogenetic approaches. Our result showed that the nuclear ribosomal ITS as a standard barcode reference performs better than the chloroplast regions (matK and rbcL) selected for this study. ITS was recovered for 34 out of 36 species (94%) in genome skimming dataset, and 23 (15/17 fresh species, and 8/14 herbarium species) out of 31 species (74%) in target capture dataset. K-mer reference contained all species for the two datasets. We used the standard barcode and k-mer references to identify three out of seven unknown Pinanga species. Standard barcoding method gave similar result with k-mer method for species discrimination, which shows that k-mer-based approach is a promising bioinformatics tool that can be applied in all barcoding studies. The conformity of species placement by barcoding with morphology in our studies further reveals the practicability of DNA barcoding for species discrimination.
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