We used molecular sequence data from the nuclear ribosomal internal transcribed spacers and from the intron and spacer of the trnL-trnF chloroplast region to study phylogenetic relationships within the large (ca. 2000 species), wide-ranging, and taxonomically difficult tribe Justicieae (Acanthaceae). Analysis of the combined data set provides a highly resolved hypothesis of relationships, much of it strongly supported. Justicieae are strongly supported as monophyletic; within the tribe, five lineages and one paraphyletic grade are related as follows: [Pseuderanthemum Lineage (Isoglossinae {Tetramerium Lineage [multiple clades of Old World "justicioids" (Diclipterinae + New World "Justicioids")]})]. Many aspects of this phylogenetic hypothesis are supported by morphological and cytological data, and some conform to earlier classifications of the group. There are, however, a number of novelties. Notably, the large genus Justicia (ca. 700 species) is not monophyletic; the Old World members form a grade and the New World members are monophyletic only if a number of other genera are included. The strongly supported sister group relationship between Diclipterinae and the New World "Justicioid" Lineage is novel, and we cannot identify non-molecular synapomorphies confirming this relationship. Rhinacanthus, a "justicioid" (i.e., Justicia and morphologically similar genera) by all but phylogenetic criteria, is strongly supported as the basal member of Diclipterinae and cytological evidence confirms this placement. The Pseuderanthemum Lineage is only modestly supported as monophyletic and may represent a series of basal lineages. These plants are marked by having four staminal elements (four stamens or two stamens plus two staminodes), a plesiomorphic condition for all Acanthaceae. Additional evidence (both taxa and characters) will be necessary to resolve this uncertainty, as well as to determine the phylogenetic status of Old World "justicioids." Our analysis also provides considerable resolution within lineages.

Key words: Acanthaceae, chromosomes, Justicieae, Lamiales, molecular sequence data, morphology