Length variation in the nuclear ribosomal DNA internal transcribed spacer region of non-flowering seed plants

The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region was PCR-amplified in 32 genera of non-flowering seed plants. Length of the ITS region was determined by restriction site mapping of PCR products and nucleotide sequences were obtained from the ITS-2 and 5.8S rDNA of selected genera. In contrast to the relatively narrow range of ITS region lengths reported from angiosperms [565-700 base pairs (bp)], substantial length variation (975-3125 bp) is observed in the ITS region of Coniferales, Cycadales, Ginkgoales, and Gnetales. Restriction site analyses indicate that the 5.8S rDNA + ITS-2 ranges from 375-450 bp, while the ITS-1 is responsible for most of the length variation found in gymnosperm ITS regions. The representatives of Pinaceae exhibit the greatest variation in IIS region length (1550-3125 bp), while those of sampled members of Cupressaceae, Taxodiaceae, Cephalotaxaceae, and Taxaceae are relatively stable (975-1125 bp). The observed ITS region lengths in Sciadopityaceae (1250 bp) and Araucariaceae (1325-1350 bp) are somewhat larger than those of Cupressaceae, Taxodiaceae, Cephalotaxaceae, and Taxaceae, while those of sampled Podocarpaceae (2000-2100 bp) fall into the range observed in the Pinaceae. Outside of the Coniferales, ITS region length is similar among Cycadales (1150- 1450 bp), Ginkgoales (1200 bp), and two of the three members of Gnetales, Ephedra (1500 bp) and Gnetum (1200 bp). In contrast, the TTS region of Welwitschia is only 750 bp long, ca. 50 bp longer than the longest known angiosperm ITS region. Levels of nucleotide sequence variation as estimated by restriction site mapping suggest that phylogenetic analysis of the ITS region will be informative at the intrageneric level in most non-flowering seed plants. In Cupressaceae, intergeneric comparisons may also be feasible. Because the ITS region is relatively long, it may also permit population-level phylogenetic analysis in many non-flowering seed plants.