Molecular phylogenetics supports widespread cryptic species in moonworts (<i>Botrychium</i> s.s., Ophioglossaceae)

<i>Premise of the study:</i> Previous phylogenetic studies of moonworts (Botrychium sensu stricto (s.s.)) included few taxa from outside of North America. This low geographical representation limited interpretations of relationships of this group rich in cryptic species. With 18 out of 30 species in the genus being polyploid, understanding their evolutionary history remains a major challenge.<br> <i>Methods:</i> A new molecular phylogeny was reconstructed using Maximum Likelihood (ML) and Bayesian Inference (BI) analyses based on multiple accessions of the most wide- ranging Arctic taxa of Botrychium in North America and Europe using three noncoding plastid DNA regions (<i>psbA-trnH^GUG, trnL^UAA- trnF^GAA</i> intergenic spacer, and <i>rpL16</i> intron).<br> <i>Key results:</i> The new phylogeny confirms the identity of several recently described species and proposed new taxa. Nine subclades are newly identified within the two major clades in Botrychium s.s.: Lanceolatum and Lunaria. Chloroplast DNA was variable enough to separate morphologically cryptic species in the Lunaria clade. On the contrary, much less variation is seen within the morphologically variable Lanceolatum clade despite sampling over the same broad geographic range. The chloroplast region <i>psbA-trnH^GUG</i> is identified as an efficient DNA barcode for the identification of cryptic taxa in <i>Botrychium</i> s.s.<br> <i>Conclusions:</i> The combined increase in species representation, samples from throughout the geographic range of each species, and sequencing of multiple plastid DNA regions supports morphologically cryptic species in <i>Botrychium</i> s.s.