The complete external transcribed spacer of 18S-26S rDNA: Amplification and phylogenetic utility at low taxonomic levels in Asteraceae and closely allied families

For molecular phylogenetic reconstruction of some intrageneric groups of pl ants, a DNA region is needed that evolves more rapidly than the internal tr anscribed spacer (ITS) of the 18S-26S nuclear ribosomal DNA (nrDNA) repeat. If the region identified is nuclear, it would also be desirable for it to undergo rapid concerted evolution to eliminate problems with coalescence. T he external transcribed spacer (ETS) of the nrDNA repeat has shown promise for intrageneric phylogenetic reconstruction, but only the 3' end of the re gion has been utilized for phylogenetic reconstruction and "universal" prim ers for PCR amplification have been elusive. We present a method for reliab ly amplifying and sequencing the entire ETS throughout Asteraceae and some closely allied families. We also show that the ETS is more variable and phy logenetically informative than the ITS in three disparate genera of Asterac eae-Argyranthemum (tribe Anthemideae), Asteriscus (tribe Inuleae), and Heli anthus (tribe Heliantheae). The full ETS was amplified using a primer (ETS1 f) within the intergenic spacer in combination with a primer (18S-2L) in th e 5' end of the highly conserved 18S gene. ETS1f was designed to correspond to a highly conserved region found in Helianthus and Crepis, which are in separate subfamilies of Asteraceae, ETS1f/18S-2L primed in all of the tribe s of Asteraceae as well as exemplar taxa from Campanulaceae, Goodeniaceae, and Calyceraceae, For both Argyranthemum and Asteriscus, we were able to di rectly sequence the ETS PCR products when a single band was produced. When multiple bands were produced, we gel-purified and occasionally cloned the b and of interest before sequencing. Although PCR produced single bands for H elianthus species, it was necessary to clone Helianthus amplifications prio r to sequencing due to multiple intragenomic ETS repeat types. Alignment of FTS sequences for Argyranthemum and Asteriscus was straightforward and una mbiguous despite some subrepeat structure in the 5' end. For Helianthus, di fferent numbers of large tandem subrepeats in different species required an alysis of the orthology of the subrepeats prior to alignment. In all three genera, the ETS provided more informative variation for phylogenetic recons truction and allowed better resolution of relationships than the ITS, Altho ugh cloned sequences from Helianthus differed, intragenomic clones consiste ntly formed clades, This result indicated that concerted evolution was proc eeding rapidly enough in ETS that species-specific phylogenetic signal was retained. It should be now be possible to use the entire ETS for phylogenet ic reconstruction of recently diverged lineages in Asteraceae and at least three other families (approximately 26,000 species or about 8% of all angio sperms). (C) 2000 Academic Press.