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The pentatricopeptide repeat (PPR) gene family, with hundreds of members in land plant genomes, has been recognized as a tremendous resource for plant phylogenetic studies based on publicly available genomic data from model organisms. However, whether this appealing nuclear gene marker system can be readily applied to non-model organisms remains questionable, particularly given the potential uncertainties in designing specific primers to only amplify the locus of interest from the sea of PPR genes. Here we demonstrate empirically the use of PPR genes in the family Verbenaceae and the Verbena complex. We also lay out a general scheme to design locus-specific primers to amplify and sequence PPR genes in non-model organisms. Intergeneric relationships within the family Verbenaceae were fully resolved with strong support. Relationships among the closely related genera within the Verbena complex and among some species groups within each genus were also well resolved, but resolution among very closely related species was limited. Our results suggest that PPR genes can be readily employed in non-model organisms. They may be best used to resolve relationships in a spectrum from among distantly related genera to among not-so-closely related congeneric species, but may have limited use among very closely related species.

Two major impediments to infer plant phylogenies at inter- or intra- species level include the lack of appropriate molecular markers and the gene tree/species tree discordance. Both of these problems require more extensive investigations. One of the foci of this study is examining the phylogenetic utility of a combined chloroplast DNA dataset (>5.0kb) of seven non-coding regions, in comparison with that of a large fragment (ca. 3.0kb) of a low-copy nuclear gene (waxy), in a recent, rapidly diversifying group, the Verbena complex. The complex includes three very closely related genera, Verbena (base chromosome number x=7), Glandularia (x=5), and Junellia (x=10), comprising some 150 species distributed predominantly in South and North America. Our results confirm the inadequacy of non-coding cpDNA in resolving relationships among closely related species due to lack of variation, and the great potential of low-copy nuclear gene as source of variation. However, this study suggests that when both cpDNA and nuclear DNA are employed in low-level phylogenetic studies, cpDNA might be very useful to infer organelle evolutionary history (e.g., chloroplast transfer) and more comprehensively understand the evolutionary history of organisms. The phylogenetic framework of the Verbena complex resulted from this study suggests that Junellia is paraphyletic and most ancestral among the three genera; both Glandularia and Verbena are monophyletic and have been derived from within Junellia. Implications of this phylogenetic framework to understand chromosome number evolution and biogeography are discussed. Most interestingly, the comparison of the cpDNA and nuclear DNA phylogenies indicates two independent intergeneric chloroplast transfers, both from Verbena to Glandularia. One is from a diploid North American Verbena species to a polyploid North American Glandularia species. The other is more ancient, from the South American Verbena group to the common ancestor of a major Glandularia lineage, which has radiated subsequently in both South and North America. The commonly assumed introgressive hybridization may not explain the chloroplast transfers reported here. The underlying mechanism remains uncertain.