The its Region of Nuclear Ribosomal DNA: A Valuable Source of Evidence on Angiosperm Phylogeny

The internal transcribed spacer (ITS) region of 18S-26S nuclear ribosomal DNA (nrDNA) has proven to be a useful source of characters for phylogenetic studies in many angiosperm families. The two spacers of this region, ITS-1 and ITS-2 (each <300 bp), can be readily amplified by PCR and sequenced using universal primers, even from DNAs of herbarium specimens. Despite high copy numbers of both spacers, the near uniformity ofITS paralogues, attributed to rapid concerted evolution, allows direct sequencing of pooled PCR products in many species. Divergent paralogues, where detected, require cloning, but may offer a means of obtaining multiple estimates of organismal relationships and of determining placement of the root in a phylogeny independent of outgroup considerations. In reported studies, variation between ITS sequences is mostly attributable to point mutations. A relatively minor proportion of sites is affected by insertions or deletions (indels) among sequences that are similar enough to have retained sufficient signal for phylogenetic analysis. Within these limits, sequence alignment is generally unambiguous except in small regions of apparently lower structural constraint. Phylogenetic analyses of combined, data sets from both spacers, where examined, yield trees with greater resolution and internal support than analyses based on either spacer alone. This beneficial effect of simultaneous analysis is not surprising based on the low number of useful characters in each spacer. This effect also suggests high complementarity of spacer data, in accord with similarity in size, sequence variability, and G + C content of ITS-1 and ITS-2 in most investigated groups of closely related angiosperms. Nonindependent evolution of ITS sites involved in intraspacer RNA base-pairing may occur, given possible functional constraints, but preliminary secondary structure analyses of ITS-2 in Calycadenia (Asteraceae) show no definite evidence of compensatory spacer mutations. As expected, levels of ITS sequence variation suitable for phylogenetic analysis are found at various taxonomic levels within families, depending on the lineage. The apparent rates of ITS molecular evolution are roughly correlated with plant life-form, as with chloroplast DNA (cpDNA) data, but reasons for this observation are unclear. ITS characters have improved our understanding of angiosperm phylogeny in several groups by (1) corroborating earlier unexpected findings, (2) resolving conflicts between other data sets, (3) improving resolution of species relationships, or (4) providing direct evidence of reticulate volution. Hybridization or sorting of ancestral polymorphism ina lineage can complicate interpretation ftrees based on any type of evolutionary evidence, including ITS or cpDNA sequences, particularly in the absence of at least one independent phylogenetic data set from the same organisms. The need for phylogenetic markers from the nuclear genome, to complement he rapidly growing body of cpDNA data, makes the ITS region a particularly valuable resource for plant systematists. Until recently, most plant systematists reserved DNA sequencing for phylogenetic analyses of taxa with sequences too divergent to be easily interpreted by restriction site mapping. Consequently, only moderately to slowly evolving DNA sequences have been used widely in plant phylogenetics (e.g., rbcL, see Chase et al., 1993; 18S and 26S nuclear ribosomal DNA, see Bult et al., 1995; Nickrent & Soltis, 1995). With the recent advent of polymerase chain reaction (PCR) technology, however, DNA 1 Support was provided in part by the NSF (BSR-9002260 to BGB, BSR-9106226 to CSC, and BSR-8822658 to MJD); Duke University (to BGB); University of Nevada, Reno (to MJS); and University of Arizona (to MJD). We thank Pam Soltis, Jonathan Wendel, and an anonymous reviewer for helpful comments on the manuscript. BGB also thanks Doug and Pam Soltis for inviting him to participate in the symposium at which some of this material was presented. 2 Department of Botany, Box 90338, Duke University, Durham, North Carolina 27708-0338, U.S.A. Author for correspondence. 3New address: Jepson Herbarium and Department of Integrative Biology, University of California, 1001 Valley Life Sciences Building # 2465, Berkeley, California 94720-2465, U.S.A. 4Department of Biology, University of Nevada, Reno, Nevada 89557, U.S.A. 5Rancho Santa Ana Botanic Garden, 1500 North College Avenue, Claremont, California 91711, U.S.A. 6 Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, U.S.A. 7Department of Plant Biology and Pathology, University of Maine, Orono, Maine 04469, U.S.A. 8 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138, U.S.A. ANN. MISSOURI BOT. GARD. 82: 247-277. 1995. 248 Annals of the Missouri Botanical Garden