Members of Botrychium s.s., commonly called moonworts, have a nearly worldwide distribution with a center of diversity in the mountains of western North America. Many moonwort species are difficult to distinguish morphologically, and identification of species, as well as understanding their evolutionary relationships, has been problematic. A cpDNA phylogeny of 20 moonwort species based on the rbcL gene revealed three main diploid species clades and identified the diploid progenitor of eight polyploid species. However, low levels of sequence divergence among species limited resolution of species relationships and did not provide strong internal support for relationships among species clades. We used the cpDNA trnL-F intergenic spacer region to establish a second cpDNA sequence data set of moonwort species. Analysis of trnL-F sequences produced a phylogeny with a topology identical to that of the rbcL tree, but with higher DNA sequence divergence among species and more internal support for relationships among diploid species clades. Placement of polyploid species was identical to that in the rbcL tree. In addition, sequences for three species not included in the original rbcL study were obtained. Botrychium boreale was placed in the lanceolatum clade and may represent a polyploid derivative of B. lanceolatum. The diploid B. pallidum was placed in the simplex-campestre clade, separate from its putative tetraploid derivative B. minganense. The tetraploid B. gallicomontanum was placed in the simplex-campestre clade near one of its presumed diploid parents, B. campestre.

Key words: Botrychium s.s., cpDNA, moonwort, Ophioglossaceae, polyploidy