Infraspecific diversification of the star cloak fern (Notholaena standleyi) in the deserts of the United States and Mexico.

PREMISE: Not all ferns grow in moist and shaded habitats. One well-known example is Notholaena standleyi, a species that thrives in deserts of the southwestern United States and Mexico. This species exhibits several "chemotypes" that differ in farina (flavonoid exudates) color and chemistry. By integrating data from molecular phylogenetics, cytology, biochemistry, and biogeography, we circumscribed the major evolutionary lineages within N. standleyi and reconstructed their diversification histories. METHODS: Forty-eight samples were selected from across the geographic distribution of N. standleyi. Phylogenetic relationships were inferred using four plastid and five nuclear markers. Ploidy levels were inferred using spore sizes calibrated by chromosome counts, and farina chemistry was compared using thin-layer chromatography. RESULTS: Four clades are recognized, three of which roughly correspond to previously recognized chemotypes. The diploid clades G and Y are found in the Sonoran and Chihuahuan deserts, respectively; they are estimated to have diverged in the Pleistocene, congruent with the postulated timing of climatological events separating these two deserts. Clade P/YG is tetraploid and partially overlaps the distribution of clade Y in the eastern Chihuahuan Desert. It is apparently confined to limestone, a geologic substrate rarely occupied by members of the other clades. The cryptic (C) clade, a diploid group known only from southern Mexico and highly disjunct from the other three clades, is newly recognized here. CONCLUSIONS: Our results reveal a complex intraspecific diversification history of N. standleyi, traceable to a variety of evolutionary drivers including classic allopatry, parapatry with or without changes in geologic substrate, and sympatric divergence through polyploidization.

Low-copy nuclear sequence data confirm complex patterns of farina evolution in notholaenid ferns (Pteridaceae).

Notholaenids are an unusual group of ferns that have adapted to, and diversified within, the deserts of Mexico and the southwestern United States. With approximately 40 species, this group is noted for being desiccation-tolerant and having "farina"-powdery exudates of lipophilic flavonoid aglycones-that occur on both the gametophytic and sporophytic phases of their life cycle. The most recent circumscription of notholaenids based on plastid markers surprisingly suggests that several morphological characters, including the expression of farina, are homoplasious. In a striking case of convergence, Notholaena standleyi appears to be distantly related to core Notholaena, with several taxa not before associated with Notholaena nested between them. Such conflicts can be due to morphological homoplasy resulting from adaptive convergence or, alternatively, the plastid phylogeny itself might be misleading, diverging from the true species tree due to incomplete lineage sorting, hybridization, or other factors. In this study, we present a species phylogeny for notholaenid ferns, using four low-copy nuclear loci and concatenated data from three plastid loci. A total of 61 individuals (49 notholaenids and 12 outgroup taxa) were sampled, including 31 out of 37 recognized notholaenid species. The homeologous/allelic nuclear sequences were retrieved using PacBio sequencing and the PURC bioinformatics pipeline. Each dataset was first analyzed individually using maximum likelihood and Bayesian inference, and the species phylogeny was inferred using *BEAST. Although we observed several incongruences between the nuclear and plastid phylogenies, our principal results are broadly congruent with previous inferences based on plastid data. By mapping the presence of farina and their biochemical constitutions on our consensus phylogenetic tree, we confirmed that the characters are indeed homoplastic and have complex evolutionary histories. Hybridization among recognized species of the notholaenid clade appears to be relatively rare compared to that observed in other well-studied fern genera.