Phylogeny, Divergence Time Estimates, and Phylogeography of the Diploid Species of the Polypodium vulgare Complex (Polypodiaceae)

The Polypodium vulgare complex (Polypodiaceae) comprises a well-studied group of fern taxa whose members are cryptically differentiated morphologically and have generated a confusing and highly reticulate species cluster. Once considered a single species spanning much of northern Eurasia and North America, P. vulgare has been segregated into 17 diploid and polyploid taxa as a result of cytotaxonomic work, hybridization experiments, and isozyme studies conducted during the 20th century. Despite considerable effort, however, the evolutionary relationships among the diploid members of the P. vulgare complex remain poorly resolved. Here we infer a diploids-only phylogeny of the P. vulgare complex and related species to test previous hypotheses concerning relationships within Polypodium sensu stricto. Using sequence data from four plastid loci (atpA, rbcL, matK, and trnG-trnR), we recovered a monophyletic P. vulgare complex comprising four well-supported clades. The P. vulgare complex is resolved as sister to the Neotropical P. plesiosorum group and these, in turn, are sister to the Asian endemic Pleurosoriopsis makinoi. Using divergence time analyses incorporating previously derived age constraints and fossil data, we estimate an early Miocene origin for the P. vulgare complex and a late Miocene-Pliocene origin for the four major diploid lineages of the complex, with the majority of extant diploid species diversifying from the late Miocene through the Pleistocene. Finally, we use our node age estimates to reassess previous hypotheses, and to propose new hypotheses, about the historical events that shaped the diversity and current geographic distribution of the diploid species of the P. vulgare complex. Keywords—Divergence dating, Hawaiian Islands, Macaronesia, plastid sequence data, Pleistocene. Members of the Polypodium vulgare L. complex (Polypodiaceae) are among the best-studied examples of ferns that combine cryptic morphology with complex patterns of reticulate evolution. For much of the 19th and 20th centuries, taxonomists considered P. vulgare, the type species of Polypodium L., to be a single species spanning much of northern Eurasia and North America. Subtle variations in morphology, including differences in leaf shape, indument type, rhizome scale shape and coloration, spore size and ornamentation, and rhizome flavor, were either dismissed or treated as taxonomic varieties. As these cryptic characters (summarized by Shivas 1961a and Hennipman et al. 1990) were examined in greater detail and their strong correlations with geography were demonstrated, researchers began to split P. vulgare into segregate species. Using cytotaxonomic studies and hybridization experiments, Manton (1950) and Shivas (1961a, b) were the first to implicate a history of reticulation as one source of the taxonomic confusion. Based on these studies, they assigned specific status to each of the three sexually reproducing European cytotypes of P. vulgare (Manton 1947, 1950, 1951; Shivas 1961a, b). Subsequent investigations have led to the recognition of about ten diploid, six allotetraploid, and one allohexaploid species, as well as numerous sterile hybrids, that constitute the P. vulgare complex worldwide (Lloyd 1963; Taylor and Lang 1963; Valentine 1964; Lloyd and Lang 1964; Lang 1969, 1971; Whitmore and Smith 1991; Haufler and Zhongren 1991; Haufler and Windham 1991; Haufler et al. 1993; Haufler et al. 1995b; Schmakov 2001). Today, the name P. vulgare sensu stricto (s. s.) is applied only to the North European and Asian allotetraploid taxon derived from the diploid progenitors P. glycyrrhiza D.C. Eaton and P. sibiricum Sipliv. (Shivas 1961b; Haufler et al. 1995b). Despite progress in resolving the reticulate relationships and the origins of the polyploid members of the P. vulgare complex (Manton 1947, 1950; Shivas 1961a; Lloyd and Lang 1964; Lang 1971; Haufler et al. 1995a, b), the monophyly of the complex and phylogenetic relationships among its diploid taxa are not fully resolved. A synthesis of previous studies provides support for three major clades of diploid taxa, each united by a shared morphological character (Fig. 1; Manton 1950; Lloyd and Lang 1964; Lang 1971; Roberts 1980; Haufler and Ranker 1995; Haufler et al. 1995a, 1995b, 2000; Schneider et al. 2004; Otto et al. 2009). Two of these (clade A: P. appalachianum Haufler & Windham + P. amorphum Suksd. + P. sibiricum and clade G: P glycyrrhiza + P. californicum Kaulf. + P. fauriei Christ) are predominantly North American but extend to eastern Asia, and the third (clade C: P. cambricum L. + P. macaronesicum A.E. Bobrov) is found in Europe, primarily the Mediterranean region, and in Macaronesia. Members of the P. appalachianum clade (A) all have sporangiasters (see Fig. 1 i and ii; Martens 1943; Peterson andKott 1974), which are sterile, often glandular structures within sori that are homologous to sporangia. In contrast, the P. glycyrrhiza clade (G) is devoid of sporangiasters, but has hairs on the adaxial surface of the leaf midrib. The P. cambricum clade (C) has branching, hair-like paraphyses distributed among the sporangia (Fig. 1 iii and iv; Martens 1943, 1950; Wagner 1964). Relationships among and within these three clades, as well as the placement of the Hawaiian endemic P. pellucidum Kaulf., were not well resolved by previous studies (Fig. 1 a–f). Perhaps the most contentious relationship among diploids is that of P. scouleri Hook. & Grev., a western North American coast endemic; some molecular studies suggest it is sister to P. glycyrrhiza (Fig. 1 d, e; Haufler and Ranker 1995; Haufler et al. 2000), whereas others show it as sister to the Hawaiian endemic P. pellucidum (Fig. 1 f; Otto et al. 2009). On morphological grounds, the Polypodium vulgare complex has been considered closely related to the Neotropical P. plesiosorum Kunze group (Christensen 1928; Tryon and Tryon 1982; Haufler et al. 1995a, b). While recent phylogenetic work reveals the need for a recircumscription of the P. plesiosorum group (Otto et al. 2009), it comprises an estimated 20 diploid and polyploid species (A. R. Smith, pers. comm.; Tejero-Dı́ez and Pacheco 2004; Luna-Vega et al. 2012). Whereas some molecular analyses support a sister relationship between