We used sequence data from the nuclear ribosomal internal transcribed spacer (nr-ITS) region, both alone and in combination with data from the intron and spacer of the trnL-trnF chloroplast (cp) region, to study phylogenetic relationships within the large tropical and subtropical family Acanthaceae. Substitution rate in the nr-ITS region is nearly twice that of the trnL-trnF cp region, and more than twice the rates of other cp loci that have been sequenced for members of Acanthaceae (i.e., rbcL, ndhF). In terms of phylogenetic relationships, the hypothesis based on ITS was largely congruent with the trnL-trnF results. Exceptions are Crossandra pungens and the two Acanthus species, which are placed enigmatically by nr-ITS data. The combined analysis provides strong support for a single hypothesis of relationships among Acanthaceae sensu stricto (s.s.) and their closest relatives. 1) Elytraria (representing Nelsonioideae) is more distantly related to Acanthaceae s.s. than Thunbergia and Mendoncia. 2) These last two genera are sister taxa and together are the sister group of Acanthaceae s.s. 3) Acanthaceae s.s. are monophyletic. 4) There are at least four major monophyletic lineages within Acanthaceae s.s.: the Acanthus, Barleria, Ruellia, and Justicia lineages. 5) These four lineages are related as follows: {Acanthus lineage [Barleria lineage (Justicia Ruellia lineages)]}. 6) Within the Justicia lineage, there are at least five monophyletic sublineages, related as follows: {Odontonema sublineage [Stenostephanus sublineage (Henrya sublineage {Dicliptera New World Justicia sublineages})]}.

Communicating Editor: Jeff Rettig