TAXON 60 (4) • August 2011: 1123–1137 Hirai & al. • New Neotropical fern genus Moranopteris Moranopteris: A new Neotropical genus of grammitid ferns (Polypodiaceae) segregated from Asian Micropolypodium Regina Y. Hirai,1 Germinal Rouhan,2 Paulo H. Labiak,3 Tom A. Ranker4 & Jefferson Prado1 1 Instituto de Botânica, Caixa Postal 68041, CEP 04045-972, São Paulo-SP, Brazil 2 Muséum national d’Histoire naturelle, UMR CNRS 7205 ‘Origine, Structure et Evolution de la Biodiversité’, Botanique, 16 rue Buffon CP 39, 75005 Paris, France 3 Universidade Federal do Paraná, Departamento de Botânica, Caixa Postal 19031, CEP 81531-980, Curitiba-PR, Brazil 4 Department of Botany, University of Hawaii, 3190 Maile Way, Honolulu, Hawaii 96822, U.S.A. Author for correspondence: Regina Y. Hirai, [email protected] Abstract Moranopteris, a new genus of grammitid ferns here described, is segregated from Micropolypodium on the basis of molecular phylogenetic analyses (plastid sequences including atpB, rbcL, and trnL-trnF) and morphological characters. Bayesian and maximum parsimony analyses support Moranopteris as sister clade to a group that includes Micropolypodium s.str. and 11 or 12 other genera, mostly from the Old World. A morphological synapomorphy for the species of Moranopteris is the presence of minute and slightly catenate branched hairs (with or without a setiform branch) and sometimes unbranched hairs occurring together on the stipes, costae, and laminar tissues abaxially. In contrast, Micropolypodium s.str. has only minute, catenate, and unbranched hairs borne on the same parts of the leaves. The new genus Moranopteris comprises 28 spe- cies—including two new species here described, M. rupicola and M. simplex—and one hybrid, occurring in the Neotropics. The 27 necessary new combinations are here proposed. Our results suggest that Micropolypodium is confined to eastern Asia. Keywords cpDNA; Grammitidaceae; molecular phylogeny; Neotropics; Paleotropics Supplementary Material The alignment is available in the Supplementary Data section of the online version of this article (http://www.ingentaconntect.com/content/iapt/tax). INTRODUCTION netic analyses (using rbcL, atpB, and trnL-F) on a grammitid fern genus. The first phylogenetic study focusing on the gram- Hasebe & al. (1994, 1995) and Pryer & al. (1995) first mitid ferns worldwide, however, was conducted by Ranker investigated fern phylogeny based on rbcL, and morphology & al. (2004), who included 73 species of grammitid ferns (rep- plus rbcL, respectively. Hasebe & al. (1994) showed that Poly- resenting 16 genera) and using molecular (plastid sequences podiaceae Bercht. & J. Presl plus Grammitidaceae Newman atpB and rbcL) as well as morphological characters. Results form a monophyletic group. These studies provided the first of this latter study highlighted that many of the traditionally insight that Polypodiaceae were not monophyletic, because recognized genera were not monophyletic. For example, none of Grammitidaceae appear nested within it. However, in these the classically accepted genera, e.g., Ctenopteris Blume, Gram- studies, the family Grammitidaceae was represented by a single mitis Sw., Prosaptia C. Presl, and Xiphopteris Kaulf. were species, Micropolypodium okuboi (Yatabe) Hayata, the type of supported as monophyletic. Furthermore, some more recently Micropolypodium Hayata. described genera, e.g., Lellingeria A.R. Sm. & R.C. Moran, Using three plastid regions (the two genes rbcL and rps4, and Terpsichore A.R. Sm., were also not supported as mono- and the intergenic spacer rps4-trnS) and expanding the sam- phyletic. pling of so-called polygrammoid ferns (Polypodiaceae s.str. In the last few years, phylogenetic studies have been per- and grammitids) to 98 selected species, Schneider & al. (2004) formed to resolve relationships within predominantly New obtained similar results, and further recovered Grammitida- World grammitids, i.e., Melpomene A.R. Sm. & R.C. Moran ceae as monophyletic but still nested within a larger clade cor- (Lehnert & al., 2009), Lellingeria (Labiak & al., 2010a, b), responding to Polypodiaceae s.l. Grammitidaceae, therefore, Terpsichore (Sundue & al., 2010), and the “Lellingeria myo- are now usually referred to informally as the “grammitid ferns”. suroides clade” (Ranker & al., 2010). Based on these findings, Smith & al. (2006, 2008), in their Ranker & al. (2004) showed that Micropolypodium was classification for the leptosporangiate ferns, included all genera not monophyletic, with species of the genus Terpsichore nested formerly treated as Grammitidaceae (e.g., Parris, 1990; Smith, within it. In their analyses, out of 24 known Micropolypodium 1995) in Polypodiaceae, which, according to this circumscrip- species (Smith, 1992: 22 in the New World and two in the Pa- tion, would comprise ca. 1200 species and 56 genera. leotropics; none shared by these two areas), they sampled only In their study of the Hawaiian Adenophorus Gaudich., three Neotropical species (Micropolypodium taenifolium (Jen- Ranker & al. (2003) were the first to focus molecular phyloge- man) A.R. Sm., M. zurquinum (Copel.) A.R. Sm., M. hyalinum 1123 Hirai & al. • New Neotropical fern genus Moranopteris TAXON 60 (4) • August 2011: 1123–1137 (Maxon) A.R. Sm.), and no Asian taxa. Mickel & Smith (2004) to 62 species representing 22 grammitid genera. Voucher in- mentioned some preliminary, unpublished molecular results on formation and GenBank accession numbers are listed in the Neotropical species of Micropolypodium indicating that the Appendix. Asian species (including the type of the generic name, M. oku- DNA extraction. — Genomic DNA was isolated from ei- boi) of Micropolypodium s.str. were not most closely related to ther field-collected, silica gel–dried leaves or from herbarium the Neotropical species. specimens when fresh material was unavailable. Total genomic According to Ranker & al. (2004), the Neotropical spe- DNA was extracted using the Qiagen DNeasy Plant Mini Kit cies of Micropolypodium formed a well-supported clade that (Valencia, California, U.S.A.) following the manufacturer’s included two, and possibly three, species of the Terpsichore protocol, but with an additional proteinase K digestion during achilleifolia group as defined by Smith (1993): T. achilleifolia the lysis step for the herbarium material: 30 μL of proteinase (Kaulf.) A.R. Sm., T. longisetosa (Hook.) A.R. Sm., and, pos- K (20 mg/mL), and 30 μL of β-mercaptoethanol (98%) were sibly, T. gradata (Baker) A.R. Sm. Although the last taxon was added per tube, and the tubes were incubated on a tipping plate not included in analyses by Ranker & al. (2004), it appeared at 42°C for 12 hours. to be related to the two other species of Terpsichore, and all Amplifications and sequencing. — Three plastid DNA three species are morphologically similar to Micropolypodium sequences were amplified by PCR, the two coding regions atpB because of their orangish to golden rhizome scales, leaves with and rbcL, and the non-coding spacer trnL-trnF. For atpB, we determinate growth, conspicuous hydathodes on the adaxial used the primers ESATPB172F and ESATPE45R (Schuettpelz laminar surfaces, and dark red-brown unbranched setae on & Pryer, 2007), for rbcL the primers ESRBCL1F and ESRB- laminar tissues. Based on these results of Ranker & al. (2004), CL1361R (Schuettpelz & Pryer, 2007), and for trnL-trnF the Labiak & Matos (2007) described and assigned a new hybrid primers “e” and “f ” (Taberlet & al., 1991). The two genes were from Rio de Janeiro (Brazil) to Micropolypodium (M. ×bra- typically amplified using a program beginning with one initial dei Labiak & F.B. Matos), and also combined T. achilleifolia denaturation step of 5 min at 94°C, followed by 35 cycles of and T. gradata in the genus (= M. achilleifolium (Kaulf.) Labiak 1 min at 94°C, 1 min at 50°C, 2.5 min at 72°C, and a final & F.B. Matos and M. gradatum (Baker) Labiak & F.B. Matos). extension period of 10 min at 72°C. For trnL-F, we used the Because Ranker & al. (2004) did not include any Paleotrop- following protocol: 5 min at 94°C/35× (1 min, 94°C; 30 s, 50°C; ical Micropolypodium in their sampling, and because levels of 1 min, 72°C)/7 min at 72°C. The PCR products were sequenced morphological homoplasy are high among the Neotropical and by the High-Throughput Genomics Unit at the University of Paleotropical species of Micropolypodium (and more generally Washington, using the amplification primers plus the internal among all grammitid ferns; Ranker & al. 2004), we conducted primers 493F and 910R for atpB (Schuettpelz & Pryer, 2007), molecular phylogenetic studies focused on Micropolypodium and ESRBCL628F and ESRBCL654R for rbcL (Schuettpelz with a much broader sampling of the species named in the & Pryer, 2007). genus. The three major aims of this study were (1) to test the Alignment and phylogenetic analyses. — Forward and monophyly of Micropolypodium s.l.; (2) to confirm the mono- reverse sequences obtained for all species were edited and as- phyly of the Neotropical species of Micropolypodium, as sug- sembled using Sequencher v.4 (Gene Codes Corporation, Ann gested by previous studies (Ranker & al., 2004); and (3) to Arbor, Michigan, U.S.A.). Consensus sequences were then au- investigate the evolutionary relationships between Micropoly- tomatically aligned using Muscle v.3.6 using the default param- podium and other grammitid genera; the here presented results eters (Edgar, 2004), and the resulting alignments were manu- will lay the foundation for a taxonomic revision