P1. Syst. Evol. 217:119-135 (1999) Plant Systematics and Evolution © Springer-Verlag 1999 Printed in Austria Phylogenetic utility of the nuclear rDNA ITS region in subfamily Ixoroideae (Rubiaceae): comparisons with cpDNA rbcL sequence data K. ANDREASEN, B. G. BALDWIN, and B. BREMER Received October 27, 1997; in revised version March 9, 1998 Key words: Rubiaceae, lxoroideae. - cpDNA, nrDNA, rbcL, ITS, DNA sequences, phylogeny, incongruence, combined analysis. Abstract: ITS of the nrDNA were sequenced for 21 taxa in lxoroideae and outgroups (Rubiaceae) and compared with sequences of the cp-gene rbcL. Separate and combined analyses were performed. ITS-variation was extensive and, because of alignment ambiguities, some sites were excluded from the analyses. Several topologies from the rbcL analysis that conflicted with earlier classifications are corroborated by the ITS data: 1) Posoqueria should be excluded from Gardenieae. 2) The disputed genus Bertiera, previously in Gardenieae, is basal in an extended Coffeeae, including Tricalysia. 3) Ixora should be excluded from Pavetteae. 4) Vangueria, (Antirheoideae), belongs to Ixoroideae. This affiliation of Antirheoideae tribes with Ixoroideae is also shown by new ITS and rbcL data for Alberta. Incongruities found between the two data sets may be caused by density of taxon sampling, different evolutionmy rates, phylogenetic sorting, homoplasy caused by functional constraints, or sampling of non-orthologous ITS types. Molecular studies using the internal transcribed spacer (ITS) region (consisting of ITS-l, 5.8S, and ITS-2) in 18S-26S nuclear, ribosomal DNA (nrDNA) have been applied widely to phylogenetic questions in flowering plants (see review in BALDWIN & al. 1995). These investigations have concentrated on lower4evel taxonomic problems, but subfamily (e.g. Apioideae, DowNm & KATZ-DOWNm1996) and family level (e.g. Winteraceae, Sun & al. 1993) studies also have been conducted. Furthermore, at least in ITS-2, there are regions conserved across flowering plants; HERSI4KOVn'Z & ZlMMER (1996) presented an alignment in which one-third to one-half of the ITS-2 sequence is alignable above the family level in angiosperms. Hence, the ITS region has the potential for contributing phylogenetic signal at higher taxonomic levels than were earlier anticipated. For some angiosperm groups more than one molecular region has now been investigated phylogenetically. Especially interesting are studies based on both chloroplast (cp) and nuclear (n) regions, e.g. WENDEL & al. 1991, BALDWIN 1992, WOJCmCHOWS~ & al. 1993, DOWNm & KATZ-DOWNm 1996, GIELLY & al. 1996, SOLTIS & al. 1996, and OXELMAN& al. 1997. In contrast to phylogenetic studies