Abstract
BackgroundThe chloroplast trnH-psbA spacer region has been proposed as a prime candidate for use in DNA barcoding of plants because of its high substitution rate. However, frequent inversions associated with palindromic sequences within this region have been found in multiple lineages of Angiosperms and may complicate its use as a barcode, especially if they occur within species.Methodology/Principal FindingsHere, we evaluate the implications of intraspecific inversions in the trnH-psbA region for DNA barcoding efforts. We report polymorphic inversions within six species of Gentianaceae, all narrowly circumscribed morphologically: Gentiana algida, Gentiana fremontii, Gentianopsis crinita, Gentianopsis thermalis, Gentianopsis macrantha and Frasera speciosa. We analyze these sequences together with those from 15 other species of Gentianaceae and show that typical simple methods of sequence alignment can lead to misassignment of conspecifics and incorrect assessment of relationships.Conclusions/SignificanceFrequent inversions in the trnH-psbA region, if not recognized and aligned appropriately, may lead to large overestimates of the number of substitution events separating closely related lineages and to uniting more distantly related taxa that share the same form of the inversion. Thus, alignment of the trnH-psbA spacer region will need careful attention if it is used as a marker for DNA barcoding.
Highlights
DNA barcoding, the use of short, standardized orthologous DNA sequences to identify species, promises a rapid and efficient method to explore the dimensions of biodiversity
The inversion region of all taxa sampled is flanked on both sides by a minimum of 18 bp of reverse complementary, or palindromic, sequences (Figure 1)
Our data show that intraspecific inversions can lead to an overestimate of divergence among conspecific sequences and misleading estimates of relationships among closely related species
Summary
DNA barcoding, the use of short, standardized orthologous DNA sequences to identify species, promises a rapid and efficient method to explore the dimensions of biodiversity. The chloroplast trnH-psbA spacer has been proposed as one such barcode for plants, either alone or in conjunction with other sequences [4,5,6]. We explore a complication of using trnH-psbA that has been overlooked in the plant barcode literature: frequent inversions in a region of trnHpsbA that is flanked by inverted repeats. The chloroplast trnH-psbA spacer region has been proposed as a prime candidate for use in DNA barcoding of plants because of its high substitution rate. Frequent inversions associated with palindromic sequences within this region have been found in multiple lineages of Angiosperms and may complicate its use as a barcode, especially if they occur within species
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