Patterns of Diversification in the Xeric-adapted Fern Genus Myriopteris (Pteridaceae)

Strong selective pressures imposed by drought-prone habitats have contributed to extensive morphological convergence among the 400 + species of cheilanthoid ferns (Pteridaceae). As a result, generic circumscriptions based exclusively on macromorphology often prove to be non-monophyletic. Ongoing molecular phylogenetic analyses are providing the foundation for a revised classification of this challenging group and have begun to clarify its complex evolutionary history. As part of this effort, we generated and analyzed DNA sequence data for three plastid loci (rbcL, atpA, and the intergenic spacer trnG–trnR) for the myriopterid clade, one of the largest monophyletic groups of cheilanthoid ferns. This lineage encompasses 47 primarily North and Central American taxa previously included in Cheilanthes but now placed in the recircumscribed genus Myriopteris. Here, we infer a phylogeny for the group and examine key morphological characters across this phylogeny. We also include a brief discussion of the three well-supported Myriopteris subclades, along with a review of reproductive mode and known ploidy levels for members of this early diverging lineage of cheilanthoid ferns. Keywords—Apomixis, Cheilanthes, convergence, molecular phylogeny, myriopterid. Cheilanthoid ferns have been called “the most contentious group of ferns with respect to practical and natural generic classification” (Tryon and Tryon 1982: 248). Members of this clade are best known for their ability to thrive in habitats too dry for most other ferns, and the taxonomic confusion plaguing the group has often been attributed to extensive morphological convergence resulting from selection imposed by arid environments (Tryon and Tryon 1973, 1982; Kramer et al. 1990; Rothfels et al. 2008). A recent series of molecular systematic studies (Gastony and Rollo 1998; Kirkpatrick 2007; Prado et al. 2007; Schuettpelz et al. 2007; Zhang et al. 2007; Rothfels et al. 2008; Windham et al. 2009; Beck et al. 2010; Eiserhardt et al. 2011; Link-Perez et al. 2011; Sigel et al. 2011; Li et al. 2012) has begun to clarify relationships among the 400 + species of cheilanthoid ferns and provides the foundation for a new, phylogenetically-based classification of the group. These studies indicate that the most significant barrier to recognizing monophyletic genera within the cheilanthoid clade is the current circumscription of the genus Cheilanthes Sw. Every molecular phylogenetic analysis with broad sampling across cheilanthoids has shown that Cheilanthes is polyphyletic; species currently assigned to the genus reside in five of the six major cheilanthoid clades identified by Rothfels et al. (2008), Windham et al. (2009), and Eiserhardt et al. (2011). For this reason, taxonomists are working to redefine the genus by segregating out monophyletic groups that are not closely related to the generitype, Cheilanthes micropteris Sw. One such clade that is phylogenetically distant from Cheilanthes s. s. has recently been transferred to the genus Myriopteris (Fig. 1; see Grusz 2013; Grusz and Windham 2013). Aside from a single disjunct species endemic to southern Africa and a few widespread species that extend to South America and certain Caribbean islands, members of this group are limited to North and Central America whereas Cheilanthes s. s. is largely confined to the Southern Hemisphere. Previously referred to as the myriopterid ferns, this clade contains roughly 10% of all cheilanthoid species diversity (Fig. 1; Windham et al. 2009) and thus constitutes a critical group for phylogenetic analysis. Previous studies have shown that the myriopterids constitute a well-supported clade (e.g. Windham et al. 2009; Eiserhardt et al. 2011), yet phylogenetic relationships among the species of this group are poorly known. To better understand the evolutionary history of the newly recircumscribed genus Myriopteris, we estimate a phylogeny for the clade and map key morphological characters across this phylogeny. Because polyploidy and apomixis are important evolutionary processes among myriopterid ferns, we also summarize the available data on reproductive mode and ploidy level for all species included in our analyses, and examine their distribution across the myriopterid tree. Materials and Methods Taxon Sampling—A total of 68 accessions representing 40 (of 47 total) myriopterid taxa were included in our molecular phylogenetic analyses (Table 1). Four outgroup taxa (Argyrochosma microphylla, Astrolepis windhamii, Paragymnopteris marantae, and Pellaea atropurpurea) were selected from the pellaeid clade, which was resolved as sister to Myriopteris in all previous molecular studies with sufficient sampling (Gastony and Rollo 1998; Kirkpatrick 2007; Rothfels et al. 2008; Windham et al. 2009; Eiserhardt et al. 2011). We included multiple accessions of wide ranging taxa withinMyriopteris, attempting to sample across their geographic distribution. DNA Extraction, Amplification, and Sequencing—For each individual sampled (see Appendix 1), genomic DNA was extracted from silica-dried leaf fragments or air-dried herbarium specimens using the DNeasy plant mini kit (Qiagen, Valencia, California) following the protocol described in Schuettpelz and Pryer (2007). Three plastid loci, rbcL (1,343 bp), atpA (1,872 bp), and the intergenic spacer, trnG–trnR (1,293 bp), were amplified for all accessions. The PCR reactions were conducted using 1 + PCR buffer IV containing MgCl2 (ABgene, Epsom, U. K.), combined with 200 mM each dNTP, 100 mg/ml BSA, 50 U/ml Taq polymerase, 0.5 mM of each locus-specific primer pair (Table 2), and 1 ml template DNA for a 25 ml reaction. The PCR amplifications entailed an initial denaturation step (94 C for 5 min) followed by 35 denaturation, annealing, and elongation cycles (94 C for 1 min, 45 C for 2 min, and 72 C for 2 min) and a final elongation step (72 C for 10 min). Amplicons were visualized on a 1% agarose gel. The PCR purification and sequencing followed the protocol of Grusz et al. (2009). All 178 newly obtained sequences were subsequently deposited in GenBank (Appendix 1). Sequence Alignment and Data Sets—Sequence fragments were assembled and edited using Sequencher 4.8 (Gene Codes Corporation, Michigan). Manual alignments of the resulting consensus sequences were