Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants

Abstract

A two-marker combination of plastid rbcL and matK has previously been recommended as the core plant barcode, to be supplemented with additional markers such as plastid trnH-psbA and nuclear ribosomal internal transcribed spacer (ITS). To assess the effectiveness and universality of these barcode markers in seed plants, we sampled 6,286 individuals representing 1,757 species in 141 genera of 75 families (42 orders) by using four different methods of data analysis. These analyses indicate that (i) the three plastid markers showed high levels of universality (87.1-92.7%), whereas ITS performed relatively well (79%) in angiosperms but not so well in gymnosperms; (ii) in taxonomic groups for which direct sequencing of the marker is possible, ITS showed the highest discriminatory power of the four markers, and a combination of ITS and any plastid DNA marker was able to discriminate 69.9-79.1% of species, compared with only 49.7% with rbcL + matK; and (iii) where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in some samples for 45.2% of the sampled genera (for genera with more than one species sampled). This finding highlights the importance of both sampling multiple individuals and using markers with different modes of inheritance. In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful backup because it is easier to amplify and sequence this subset of the marker. We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants.

Fig. 1.

Comparison of the performance of four barcoding markers (I, ITS; M, matK; P, trnH–psbA; R, rbcL). (A) Universality assessment for PCR and sequencing success. PCR success was based on 6,286 samples representing 1,757 species (5,897 angiosperm samples and 389 gymnosperm samples); sequencing success was based on 5,412 samples for ITS, 5,702 samples for trnH–psbA, 5,732 samples for matK, and 5,957 samples for rbcL. (B) Assessment of species discrimination success and sequence quality based on 3,011 individuals representing 765 species, where at least 2 species were sampled per genus and all four markers were successfully sequenced. Assessment of sequence quality with QV of ≥30 (see Materials and Methods for trace-quality criteria).

Fig. 2.

Comparison of discrimination success for the four markers (plus ITS2, the partial sequence of ITS) and all 2- to 4-marker combinations based on 3,011 individuals representing 765 species, where at least 2 species were sampled per genus and all four markers were successfully sequenced (I, ITS; I2, ITS2; M, matK; P, trnH–psbA; R, rbcL).

Fig. 3.

Discrimination success at the ordinal level (1 order of gymnosperms, 23 orders of angiosperms) for four markers (plus ITS2, the ITS partial sequence) and all possible 2- to 4-marker combinations, based on 3,011 individuals representing 765 species, where at least 2 species were sampled per genus and all four markers were successfully sequenced (I, ITS; I2, ITS2; M, matK; P, trnH–psbA; R, rbcL). Sequence of angiosperm orders is according to the Angiosperm Phylogeny Group (APG) III (42).