ITS1: a DNA barcode better than ITS2 in eukaryotes?

Abstract A DNA barcode is a short piece of DNA sequence used for species determination and discovery. The internal transcribed spacer ( ITS / ITS 2) region has been proposed as the standard DNA barcode for fungi and seed plants and has been widely used in DNA barcoding analyses for other biological groups, for example algae, protists and animals. The ITS region consists of both ITS 1 and ITS 2 regions. Here, a large‐scale meta‐analysis was carried out to compare ITS 1 and ITS 2 from three aspects: PCR amplification, DNA sequencing and species discrimination, in terms of the presence of DNA barcoding gaps, species discrimination efficiency, sequence length distribution, GC content distribution and primer universality. In total, 85 345 sequence pairs in 10 major groups of eukaryotes, including ascomycetes, basidiomycetes, liverworts, mosses, ferns, gymnosperms, monocotyledons, eudicotyledons, insects and fishes, covering 611 families, 3694 genera, and 19 060 species, were analysed. Using similarity‐based methods, we calculated species discrimination efficiencies for ITS 1 and ITS 2 in all major groups, families and genera. Using Fisher's exact test, we found that ITS 1 has significantly higher efficiencies than ITS 2 in 17 of the 47 families and 20 of the 49 genera, which are sample‐rich. By in silico PCR amplification evaluation, primer universality of the extensively applied ITS 1 primers was found superior to that of ITS 2 primers. Additionally, shorter length of amplification product and lower GC content was discovered to be two other advantages of ITS 1 for sequencing. In summary, ITS 1 represents a better DNA barcode than ITS 2 for eukaryotic species.