Abstract
The utility of DNA barcoding for identifying representative specimens of the circumpolar tree genus Fraxinus (56 species) was investigated. We examined the genetic variability of several loci suggested in chloroplast DNA barcode protocols such as matK, rpoB, rpoC1 and trnH-psbA in a large worldwide sample of Fraxinus species. The chloroplast intergenic spacer rpl32-trnL was further assessed in search for a potentially variable and useful locus. The results of the study suggest that the proposed cpDNA loci, alone or in combination, cannot fully discriminate among species because of the generally low rates of substitution in the chloroplast genome of Fraxinus. The intergenic spacer trnH-psbA was the best performing locus, but genetic distance-based discrimination was moderately successful and only resulted in the separation of the samples at the subgenus level. Use of the BLAST approach was better than the neighbor-joining tree reconstruction method with pairwise Kimura's two-parameter rates of substitution, but allowed for the correct identification of only less than half of the species sampled. Such rates are substantially lower than the success rate required for a standardised barcoding approach. Consequently, the current cpDNA barcodes are inadequate to fully discriminate Fraxinus species. Given that a low rate of substitution is common among the plastid genomes of trees, the use of the plant cpDNA “universal” barcode may not be suitable for the safe identification of tree species below a generic or sectional level. Supplementary barcoding loci of the nuclear genome and alternative solutions are proposed and discussed.
Highlights
Over the past decade, several protocols for identifying species from short orthologous DNA sequences, known as DNA barcodes, have been proposed
Kimura 2-parameter (K2P) pairwise substitution rates calculated for each dataset showed very low sequence divergence values (Table 2) and the lack of the typical barcode gap, a trend that indicated a large overlap between intraspecific and interspecific pairwise distances (Fig. 1)
The best case scenario was obtained with the BLAST approach applied to trnH-psbA intergenic sequences for the expanded dataset, where 32% of the species could be retrieved in the three first hits
Summary
Several protocols for identifying species from short orthologous DNA sequences, known as DNA barcodes, have been proposed. They have been promoted as useful for the rapid identification and discovery of species and applied to biodiversity studies [1]. Barcoding relying on the mitochondrial gene coding for cytochrome c oxidase (cox or co1) has been used successfully to identify species in various animal taxa, including birds [3,4], butterflies [5,6,7], bats [8], and fish [9]. Frequent hybridisation, polyploidy, and apomixis in plants make the identification of an ideal barcode locus more difficult than in animals [12]
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