New insights into the phylogeny of Pleopeltis and related Neotropical genera (Polypodiaceae, Polypodiopsida)

Molecular Phylogenetics and Evolution 53 (2009) 190–201

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Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev

New insights into the phylogeny of Pleopeltis and related Neotropical genera (Polypodiaceae, Polypodiopsida) Elisabeth M. Otto a,1, Thomas Janßen a,2, Hans-Peter Kreier a, Harald Schneider a,b,* a b

Albrecht-von-Haller Institute of Plant Sciences, Georg-August University Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany Department of Botany, The Natural History Museum, London SW7 5BD, UK

a r t i c l e

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Article history: Received 25 January 2009 Revised 16 March 2009 Accepted 4 May 2009 Available online 10 May 2009 Keywords: Biogeography Central America Chloroplast DNA Dicranoglossum Epiphytism Ferns Neurodium Phylogenetics Polypodium Pseudocolysis

a b s t r a c t The fern family Polypodiaceae plays an important role in Neotropical epiphyte diversity. Most of its American representatives are assembled in a monophyletic clade that, apart from the grammitids, nearly exclusively comprises species restricted to the New World. The phylogenetic relationships of these ferns are still insufficiently understood and many taxonomic problems, such as natural circumscriptions of the genera Polypodium and Pleopeltis, were unresolved. Here we address one of the two main lineages within New World Polypodiaceae including Pecluma, Phlebodium, Pleopeltis, and Polypodium. Our study is based on DNA sequence data from four plastid regions that were generated for 72 species representing all putative major taxonomic groups within this lineage. The analyses reveal three major clades: (1) Polypodium plus Pleurosoriopsis; (2) Pecluma plus Phlebodium, and some species of Polypodium; and (3) Pleopeltis and related genera. The last clade contains species of Pleopeltis and Polypodium as well as Microphlebodium, Neurodium, Dicranoglossum, and Pseudocolysis. All species included in the clade display conspicuous persistent peltate laminar scales that are not found in other species of this lineage. Our results suggest a reconsideration of the generic concept of Pleopeltis with peltate laminar scales being the genus’ key character. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction The Neotropics contribute about 30% of known plant species to worldwide plant diversity (Smith et al., 2004). Since epiphytes comprise about a third of tropical American plant species, they play an important role in Neotropical species richness (Benzing, 1990). Many epiphytes are ferns, with some lineages being almost exclusively adapted to the airy habitat. One example is the fern family Polypodiaceae, a very modern (Schneider et al., 2004b) and highly diverse group of mostly epiphytic ferns (Schneider et al., 2004c). During the last 15 years the application of molecular phylogenetics has revolutionized our understanding of relationships among ferns. This progress resulted in subsequent revisions of classification schemes at the familial and generic levels (Smith et al., 2006b), which can best be illustrated for some of the most extensively studied families such as Dryopteridaceae (Liu et al., * Corresponding author. Address: Department of Botany, The Natural History Museum – London, Cromwell Road, London SW7 5BD, UK. Fax: +44 20 7942 5529. E-mail addresses: [email protected], [email protected] (H. Schneider). 1 Present address: Institute of Applied Genetics, Free University Berlin, AlbrechtThaer-Weg 6, 14195 Berlin, Germany. 2 Present address: Department of Botany and Molecular Evolution, Research Institute Senckenberg, Senckenberganlage 25, 60325 Frankfurt, Germany. 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.05.001

2007; Li et al., 2008), Hymenophyllaceae (Ebihara et al., 2006), and Polypodiaceae (Schneider et al., 2004a). In these families, broad phylogenetic studies found several genera to be para- or polyphyletic and thus indicate the need for new classifications at the generic level (Ebihara et al., 2004, 2006; Schneider et al., 2004a,c, 2006a,b; Janßen and Schneider, 2005; Hennequin et al., 2006; Kreier and Schneider, 2006; Kreier et al., 2007). In an extensive phylogenetic study of Polypodiaceae using chloroplast markers, Schneider et al. (Schneider et al., 2004a, 2006b) gave strong evidence for a monophyletic Neotropical clade within the family. This clade comprises three lineages, with the genus Synammia sister to two species-rich lineages. Of those, one lineage comprises the grammitid ferns—corresponding to the former family Grammitidaceae—and related genera whereas the second lineage consists of most species of the genus Polypodium and some of its segregates. In Polypodiaceae, Polypodium is a prime example prevailing unnatural taxonomic concepts. Some authors (Tryon and Tryon, 1982; Moran and Riba, 1995; Mickel and Smith, 2004) expressed doubts that Polypodium in its broadest definition represented a natural unit. Several generic segregates from Polypodium s.l., e.g., Microgramma, Pecluma, and Pleopeltis, have been established in attempts to move towards a natural classification. Because of the lack of convincing information, other authors (Hennipman et al. in Kubitzki, 1990) have favoured a broadly defined genus Polypodium instead.

E.M. Otto et al. / Molecular Phylogenetics and Evolution 53 (2009) 190–201

On the contrary, Tryon and Tryon (1982) and Moran (in Moran and Riba, 1995) accepted not only most of the established segregates but discussed also several putative natural groups within the large generic concept of Polypodium. Schneider et al.’s (2004a) phylogenetic framework provided strong support for many accepted segregates such as the genera Campyloneurum, Goniophlebium, Microgramma, Niphidium, Pecluma, and Phlebodium. In addition, phylogenetic results suggested the establishment of a new Neotropical genus Serpocaulon (Smith et al., 2006a) and the recognition of a commonly forgotten segregate, the southern South America genus Synammia (Schneider et al., 2006b). However, some of the commonly recognized segregates were found to be nested within other genera. In particular the so-called Pleopeltis clade, containing the species closely related to Pleopeltis, seemed to be formed by species previously assigned to different genera including Pleopeltis, Dicranoglossum, Marginariopsis, Microphlebodium, Neurodium, Pseudocolysis, and some species of Polypodium. Other species of Polypodium showed close relationships to Pecluma and Phlebodium but not to Polypodium s.s. In summary, generic affiliations of many species assigned to Polypodium are still unclear and, although many segregates are now well established, the remaining putative groups are poorly circumscribed. As mentioned above the Pleopeltis clade recovered by Schneider et al. (2004a) includes some species of Polypodium. These species share persistent peltate scales on the leaf lamina or rachis and therefore were sometimes treated as the independent genus Marginaria or as Polypodium subg. Marginaria (de la Sota, 1965, 1966; Tryon and Tryon, 1982; Windham, 1993). Apart from monotypic Neurodium all the other genera in the Pleopeltis clade show persistent peltate laminar scales similar to those of Polypodium subg. Marginaria. This kind of peltate scale is also present in the sori of species belonging to Pleopeltis, the so-called peltate paraphyses that are the genus’ key character in its classic definition (Tryon and Tryon, 1982; Windham, 1993). Similarities between the peltate paraphyses and the peltate scales on the lamina led Windham (1993) to argue for the hypothesis that, as a result of sorus fusion, the peltate paraphyses are homologous with the laminar scales and are hence found now in sori of Pleopeltis. Since Schneider et al.’s (2004a) molecular results indicated that Pleopeltis might not be monophyletic and peltate paraphyses evolved at least twice independently within the Pleopeltis clade, this character provides limited evidence to support the monophyly of the genus. These findings lead to the hypothesis that all species of the clade could together represent the genus Pleopeltis, with the genus being based on persistent peltate laminar scales. If this turns out to be true, a substantial redefinition and expansion of the generic concept is necessary, with many smaller genera being subsumed. The acceptance of an expanded genus Pleopeltis comprising, among others, all species of Polypodium subg. Marginaria would resolve some conflicts between biological evidence and classification that have resulted in proposed intergeneric hybrids. Several hybrids between Pleopeltis and Polypodium subg. Marginaria are known. Wagner and Wagner (1975) described a hybrid between Polypodium (Pleopeltis) thyssanolepis and Pleopeltis macrocarpa. Anthony and Schelpe (1985) discovered a hybrid between Polypodium (Pleopeltis) polypodioides and Pleopeltis macrocarpa and established the hybrid genus xPleopodium. Mickel and Beitel (1987) added four additional taxa to this genus. Generic hybrids have often proven to be the result of unnatural generic units and usually are indicators of para- or polyphyletic taxonomic concepts (Pintér et al., 2002; Schneider et al., 2004d). The presence of hybrids thus gives a further hint towards the need of a taxonomic reconsideration. In this study, we inferred a detailed chloroplast DNA phylogeny not only for the Pleopeltis clade but also for related genera belonging to the Neotropical Polypodiaceae, lineage II. In addition, we collected morphological data on species belonging to the Pleopeltis

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clade to find synapomorphies and reconstruct character evolution. Based on the data obtained, we suggest a redefinition of the genus Pleopeltis, to include all species of the Pleopeltis clade. We also attempt to clarify the phylogenetic situation of other genera closely related to the Pleopeltis clade, with special emphasis on Polypodium.

2. Materials and methods 2.1. Taxon sampling To infer the hypothesis of an expanded genus Pleopeltis, we sampled 48 specimens of 45 species belonging to the Pleopeltis clade. Of the included species 26 were assigned to Pleopeltis and 15 to Polypodium. One species each belonged to Dicranoglossum, Microphlebodium, Neurodium, and Pseudocolysis (Table 1). To reconstruct the phylogeny of the whole Neotropical polypod lineage II, we also included three species of the genus Pecluma, two species of Phlebodium, the monotypic genus Pleurosoriopsis, and 22 Polypodium species that lack the persistent scales on lamina and rachis. To polarize the character set, we assembled a multitaxon outgroup including four taxa belonging to the Neotropical polypod lineage I: Campyloneurum brevifolium, Campyloneurum phyllitidis, Microgramma percussa, and Serpocaulon triseriale. The selection of outgroup and ingroup taxa reflects the results presented in Schneider et al. (2004a). We took particular care to sample the type species of Polypodium (P. vulgare), Pleopeltis (P. angusta), and Marginaria (Pleopeltis/Polypodium polypodioides). 2.2. DNA extraction, amplification, sequencing, and alignment Sequences of two coding (rbcL, rps4) and two non-coding cpDNA regions (trnLUAA-trnFGAA intergenic spacer (trnL-F IGS), rps4-trnSGAA intergenic spacer (rps4-trnS IGS)) were generated for each species. Material for DNA extraction was provided from field collections by colleagues, or obtained from cultivated material of various botanical gardens, and from herbarium specimens (Table 1). Total DNA was extracted from silica-dried material with the Invisorb Spin Plant Mini Kit (Invitek). The extracts were used directly for PCR, using primers described in previous publications: rbcL (Haufler and Ranker, 1995; Haufler et al., 2003), rps4 + rps4trnS IGS (Nadot et al., 1994, 1995; Smith and Cranfill, 2002), and trnL-F IGS (Taberlet et al., 1991; Trewick et al., 2002). Reactions were carried out in a volume of 25 ll with 10 nM dNTP, 20 nM MgCl, 0.01 nM primer, 4% (v/v) DMSO, 0.25 U Taq (BiotaqTM Polymerase, Bioline), and 1 ll DNA. After initial denaturation at 94 °C, 30 cycles of 94 °C 1 min, 49 °C 1 min, 72 °C 2 min were run for rbcL and of 94 °C 15 s, 52 °C 30 s, 72 °C 1 min for trnL-F and rps4 + rps4-trnS. Fragments were purified using the GFXTM DNA and Gel Band Purification Kit (GE Healthcare). Sequencing was carried out on a Mega BACE 1000 Capillary Sequencer (Amersham Biosciences) using DYEnamic ET-Primer DNA Sequency Reagent (Amersham Biosciences). Sequences were edited and assembled with Pregap4 and Gap4 (Bonfield et al., 1995; Bonfield and Staden, 1996) and aligned manually in Se-Al v2.0a11 [http:// evolve.zoo.ox.ac.uk] and MacClade 4.0 (Maddison and Maddison, 2000). Ambiguously aligned regions and indels were excluded from phylogenetic analyses. Twenty-seven non-overlapping indels or deletions within the two spacer-regions were scored as binary characters and added to initial phylogenetic analyses. Their inclusion did not change the results and also did not improve the resolution of poorly resolved parts of the tree. Thus, we did not include the indel matrix in the final analyses. All newly generated sequence data were submitted to GenBank. Acces-

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Table 1 List of specimens used in this study. Information is provided in the sequence taxon name, origin of voucher specimen, collector of voucher specimen (herbarium deposited), GenBank accession number of trnL-F = trnL-F IGS/rbcL/rps4 = rps4+rps4-trnS IGS. A dash (–) indicates that the region was not sampled for the specimen. Collected material was obtained either form field collection or sometimes from material cultivated in one of the following Botanical Gardens: Botanical Garden Berlin Dahlem (BGBD) Old Botanical Garden of the Georg-August University Goettingen (ABGG), Botanical Garden of the University of Zurich (BGZ). Taxon

Voucher

trnL-F

rbcL

rps4

Campyloneurum brevifolium Lodd. Campyloneurum phyllitidis (L.) C. Presl Dicranoglossum panamense (C. Chr.) L.D. Gómez Neurodium lanceolatum (L.) Fée Pecluma alfredii (Rosenstl.) M.G. Pricce Pecluma eurybasis (C. Chr.) M.G. Price Pecluma ptilodon (Kunze) M.G. Price Phlebodium areolatum (Humb. & Bonpl. ex Willd.) J. Sm.

cult. ABGG, Kreier s.n. (GOET) cult. BGBD, Schuettpelz s.n. (GOET) cult. BGBD, Schuettpelz s.n. (GOET) Costa Rica, Monro 3217 (BM), Mexico, Kimnach 95 (UC) Bolivia, Kessler s.n. (Goet) Mexico, Leferbe 3085 (UC) Mexico, Krömer 2706 (Goet) cult, ABGG, Schuettpelz 623 (GOET) Mexico, Krömer 2664 (GOET) Mexico, Krömer 2122 (GOET) Bolivia, Jimenez 2356 (LPD) Mexico, Krömer 2775 (GOET) Bolivia, Jimenez 2462 (LPD) Bolivia, Jimenez 1938 (LPD) cult. BGZ, Schneller s.n. (Z) Bolivia, Jimenez 1113 (LPD) Mexico, Tejero-Diez 4937 (GOET) Mexico, Tejero-Diez 4909 (GOET) Bolivia, Jimenez 664 (LPD) Mexico, Tejero-Diez 4908 (GOET) Bolivia, Jimenez 817 (LPD) Costa Rica, Krieger 2328 (COLO) Mexico, Tejero-Diez 4951 (GOET) Bolivia, Jimenez 2508 (LPD) Grand Comores, Rakotondrainibe 6803 (P) Mexico, Tejero-Diez 4929 (GOET) Mexico, Tejero-Diez 4933 (GOET) Paraguay, Bohs 3185 (COLO) Mexico, Tejero-Diez 4947 (GOET) Mexico, Tejero-Diez 4922 (GOET) Mexico, Tejero-Diez 4942 (GOET) Bolivia, Sundue 815 (NY) Bolivia, Jimenez 1450 (LPD) Mexico, Tejero-Diez 4938 (GOET) Bolivia, Jimenez 1318 (GOET) Costa Rica, Sundue 913 (NY) Japan, Kato s.n. (T) USA, Haufler & Soltis s.n. (KANU) USA, Haufler s.n. (KANU) Mexico, Lautner 98-83 (GOET) cult. ABGG, Schwertfeger s.n. (GOET) Brasil, Smith. S.n. (UC) Mexico, Krömer 2705 (GOET) Dominica, Hill 28995 (UC) Costa Rica, Ranker 1830 (COLO) Mexico, Krömer 2707 (GOET) Japan, Yahara s.n. (TI) Costa Rica, Krieger 2332 (COLO) Mexico, Tejero-Diez 4941 (GOET) Barrington & Haufler 922 (KANU) cult. ABGG, Schwertfeger s.n.(GOET) Guattemala, Lautner 91-7 (GOET) cult. BGBD, Schuettpelz 622 (GOET) Mexico, Tejero-Diez 4905 (GOET) Mexico, Kessler 13494 (GOET) cult., ABGG, Schwertfeger s.n. (GOET) Costa Rica, Sundue 960 (NY) Mexico, Tejero-Diez 4940 (GOET) Mexico, Krömer 2635 (GOET) Ecuador, Moran 6768 (NY) Costa Rica, Sundue 961 (NY) Costa Rica, Sundue 920 (NY) Costa Rica, Krieger 2327 (COLO) Hawai’i, Li s.n. (KANU) Mexico, Tejero-Diez 4943 (GOET) Mexico, Tejero-Diez 4930 (GOET) Mexico, Lautner 01-39 (GOET) Mexico, Lautner 02-44 (GOET) Mexico, Krömer 2718 (GOET) Mexico, Krömer 2702 (GOET) Mexico, Krömer 2646 (GOET) Mexico, Tejero-Diez 4946 (GOET)

EF104513 EF104514 – EU650061 – FJ825691 AF159193 FJ825690 DQ642258 EU650083 EU650083 DQ642259 EU650067 DQ642260 DQ642261 EU650097 DQ642262 EU650064 EU650066 EU650088 EU650069 DQ642263 EU650062 EU650072 EU650068 DQ642266 EU650063 EU650074 EU650082 EU650064 EU650065 EU650085 EU650081 DQ642268 EU650073 DQ642270 EU650070 – AF159182 AF159181 – FJ825689 FJ825688 EU650091

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Pleopeltis angusta Humb. & Bonpl. ex Willd. var. angusta Pleopeltis angusta var. stenoloma (Fée) Farw. Pleopeltis appressa M. Kessler & A.R. Sm. Pleopeltis astrolepis (Liebm.) E. Fourn. Pleopletis ballivanii (Rosenst.) A.R. Sm. Pleopeltis bombycina (Maxon) A.R. Sm. Pleopeltis bombycina (Maxon) A.R. Sm. Pleopeltis buchtienii (H. Christ & Rosenst.) A.R. Sm. Pleopeltis conzattii (Weath.) R.M. Tryon and A.F. Tryon Pleopeltis crassinervata (Fée) T. Moore Pleopeltis disjuncta M. Kessler & A.R. Sm. Pleopeltis fallax (Schltdl. & Cham.) Mickel & Beitel Pleopeltis fraseri (Mett. Ex Kuhn) A.R. Sm. Pleopeltis fructuosa (Maxon & Weath. ex Weath.) Lellinger Pleopeltis guttata (Maxon) E.G. Andrews & Windham Pleopeltis intermedia M. Kessler & A.R. Sm. Pleopeltis macrocarpa (Bory ex Willd.) Kaulf. Pleopelits mexicana (Fée) Mickel & Beitel Pleopeltis munechii (H. Christ) A.R. Sm. Pleopeltis pleopeltifolia (Raddi) Alston Pleopeltis polylepis (A. Roem. ex Kunze) T. Moore Pleopeltis polypodioides (L.) E.G. Andrews & Windham var. polypodioides Pleopeltis polypodioides var. acicularis (Weath.) E.G. Andrews & Windham Pleopeltis pycnocarpa (C. Chr.) A.R. Sm. Pleopeltis remota (Desv.) A.R. Sm. Pleopeltis thyssanolepis (A. Braun ex Klotzsch (E.G. Andrews & Windham Pleopeltis tweediana (Hook.) A.R. Sm. Pleopeltis wiesbaurii (Sodiro) Lellinger Pleurosoriopsis makinoi (Maxim.) Fomin. Polypodium amorphum Suksd. Polypodium appalachianum Haufler & Windham Polypodium arcanum Maxon Polypodium cambricum L. Polypodium chnoophorum Spreng. Polypodium collinsii Maxon Polypodium dulce Poir. Polypoodium echinolepis Fée Polypodium fauriei Nakai Polypodium friedrichsthalianum Kunze Polypodium furfuraceum Schltdl. & Cham. Polypodium glaberulum Mickel & Beitel Polypodium glycyrrhiza D.C. Eaton Polypodium hartwegianum Hook. Polypodium hirsutissimum Raddi Polypodium lepidotrichum (Fée) Maxon Polypodium longipinnulatum E. Fourn. Polypodium macaronesicum A.E. Bobrov Polypodium macrolepis Maxon Polypodium madrense J. Sm. Polypodium martenzii Mett. Polypodium monosorum Desv. Polypodium montigenum Maxon Polypodium murorum Hook. Polypodium myriolepis H. Christ Polypodium pellucidum Kaulf. Polypodium platylepis Mett. ex Kuhn Polypodium plebeium Schltl. & Cham. Polypodium plesiosorum Kunze Polypodium pyrrholepis (Fée) Maxon Polypodium rhachypterygium Liebm. Polypodium rhodopleuron Kunze Polypodium rzedowskianum Mickel Polypodiium sanctae-rosae (Maxon) C. Chr.

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E.M. Otto et al. / Molecular Phylogenetics and Evolution 53 (2009) 190–201 Table 1 (continued) Taxon

Voucher

trnL-F

rbcL

rps4

Polypodium scouleri Hook. & Grev. Polypodium subpetiolatum Maxon Polypodium vulgare L. Pseudocolysis bradeorum L.D. Gómez Serpocaulon triseriale (Sw.) A.R. Sm.

cult. ABGG, Schwertfeger s.n. (GOET) Mexico, Krömer 2662 (GOET) cult., ABGG, Schwertfeger s.n. (GOET) Mexico, Palacios-Rios 2960 (UC) Bolivia, Jimenez 1994 (LPD)

FJ825678 FJ825677 EF551119 – DQ151980

FJ825693 FJ825692 EF55105 AY362614 DQ151926

FJ825664 FJ825663 EF551081 AY36268 DQ151953

sion numbers and voucher information are given in Table 1. All matrices were submitted to TreeBase (SN3923). 2.3. Phylogenetic analyses Maximum parsimony analyses (MP) were conducted with PAUP4.0b10 (Swofford, 2003). The program was set to 1000 random additions and TBR branch swapping. If more than one tree was found, results were summarized as strict consensus as implemented in PAUP. Tree statistics, tree length, consistency index (CI), homoplasy index (HI), retention index (RI), and rescaled consistency index (RC) were also calculated using PAUP. Constant characters were ignored when calculating these indices. Branch support was estimated by 100 bootstrap replicates (MP-BS), each conducted with 10 random additions and TBR branch swapping. MP analyses were performed with the sequence data with and without the indel score matrix. Considering the Akaike information criterion the program Modeltest (Posada and Crandall, 1998) selected the GTR+G+I model of substitution. This model was used in maximum likelihood analyses (ML) that were performed with PHYML (Guindon and Gascuel, 2003). Branch support was estimated by 1000 bootstrap replicates (ML-BS). Bayesian inference of phylogeny (BY) was carried out with MRBAYES 3.0 (Huelsenbeck and Ronquist, 2001). Four MCMC chains were run for 10 million generations under the GTR+G+I model, sampling every 100th generation and excluding the burn-in period. Alternative runs were performed without partitioning the data set, with the data set partitioned into coding versus non-coding regions, or split into four different partitions with one for each of the four included regions. The program TRACER (http://tree.bio.ed.ac.uk/software/tracer/) was used to calculate the statistics for each Bayesian analysis. Trees sampled during the burn-in phase were excluded and posterior probabilities (PP) were estimated using the majority rule consensus tree of PAUP. 2.4. Reconstruction of ancestral character states and distribution ranges To explore the evolution of morphological disparity of Pleopeltis, we assembled a morphological character matrix including 41 characters comprising characters of the rhizome, leaf, and indument (available by request for the authors). This matrix was assembled based on careful evaluation of herbarium vouchers (usually several per species) deposited at the herbaria of the Universities of Goettingen and Zurich. Information concerning the distribution ranges was obtained from online databases (http://tropicos.org, http:// www.gbif.org), the Goettingen herbarium, and printed floras. Two approaches, maximum parsimony and maximum likelihood, were applied to reconstruct ancestral character state changes and putative ancestral distribution ranges. The reconstructions were carried out with the phylogram recovered in the ML analyses of the chloroplast DNA data set. Both reconstructions were carried out with the Ancestral State Reconstruction Packages included in the software Mesquite 1.12 (Maddison and Maddison, 2006). The MP reconstructions were preformed with both ACCTRAN and DELTRAN optimizations whereas the ML reconstructions were pursued with the Mk1 model.

3. Results The four markers together yielded 2454 included positions (1785 coding, 669 non-coding). Twenty-seven deletions and/or insertions [indels] were coded as binary characters and added to the maximum parsimony data set. Five hundred and fifty-five positions of the included 2481 positions were parsimony-informative. The MP analysis of the molecular data set resulted in 216 most parsimonious trees with a tree length of 1645 steps, CI = 0.560, and RI = 0.852 (Fig. 1). The best tree found in the maximum likelihood analysis had a log-likelihood of ln = 15429.55 with the parameters for the GTR+G+I model estimated as f(A) = 0.2934, f(C) = 0.1990, f(G) = 0.2197, f(T) = 0.2879, A–C = 1.2323, A–G = 3.0253, A–T = 0.4657, C–G = 0.6563, C–T = 3.6416, G–T = 1.0 (fixed), gamma shape (G) = 0.360 with four categories, and proportion of invariant (I) = 0.000 (Fig. 2). All analyses of the molecular data recovered the same global phylogeny that only differed in the hypothetical relationships within some clades (Figs. 1–3). The ingroup comprised three strongly supported major clades: (1) Pleopeltis clade (MPBS = 100%, ML-BS = 100%, PP p = 1.00), (2) Pecluma clade (MPBS = 96%, ML-BS = 91%, PP p = 1.00), and (3) Polypodium clade (MP-BS = 96%, ML-BS = 90%, PP p = 1.00). The three analyses recovered slightly different relationships within the Pleopeltis clade (Figs. 1–3). Pseudocolysis bradeorum was found to be sister to all other members of the clade in ML and MP results but to be nested among the other clade species in all the Bayesian analyses (one, two, or four model partitions). The remaining Pleopeltis clade comprised three subclades that were found in all analyses. Subclade I (MP-BS = 84%, ML-BS = 74%, PP p = 1.0) comprised 18 species including the representatives of Dicranoglossum and Neurodium as well as the type species of Pleopeltis (P. angusta) and Marginaria (P. polypodioides). In the results obtained in the Bayesian inference (Fig. 3), this subclade also included Pseudocolysis bradeorum. Subclade II (MP-BS = 67%, MLBS = 85%, PP p = 1.00) contained 18 species with Microphlebodium muenchii being sister to the remaining strongly supported subclade. Subclade III (MP-BS = 94%, ML-BS = 91%, PP p = 1.0) corresponded to the Polypodium squamatum group and included 9 species. The results of the MP and ML analyses suggested the P. squamatum subclade to be the sister to a monophylum comprising the other two subclades, but this hypothesis lacked support. The Bayesian analyses recovered a polytomy instead. For the Pecluma clade identical topologies were found in all three analyses. Monophyletic Phlebodium (MP-BS = 100%, ML-BS = 100%, PP p = 1.00) was the sister to the remaining species. The next split in the clade included a well-supported branch (MP-BS = 100%, MLBS = 100%, PP p = 1.00) including Polypodium hartwegianum and P. longipinnulatum. The other taxa were nested within two clades, one of which corresponds to the genus Pecluma, the other to three species of Polypodium (P. chnoophorum, P. dulce, P. rhachypterygium). The latter clade was strongly supported (MP-BS = 100%, MLBS = 96%, PP p = 1.00), but its sister relationship to Pecluma did not get as much support (MP-BS = 75%, ML-BS = 84%, PP p = 0.98). Partly conflicting relationships were inferred for the Polypodium clade (Figs. 1–3). The sister relationship of the monotypic Asian

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Fig. 1. Strict consensus tree of 216 most parsimonious trees recovered in the maximum parsimony analyses of the molecular data set. Bootstrap values are given above branches. The arrow points toward the position of Pseudocolysis bradeorum. Subclades I, II, III of the Pleopeltis clade follow the description given in the text.

genus Pleurosoriopsis to the remaining clade got only low support (MP-BS = 59%, ML-BS = 63%). The Bayesian topology showed this species to be nested in a polytomy. The remaining species of the clade either formed a poorly supported grade (ML) or split into two subclades. One of the two subclades (Polypodium s.s.) found in MP and BY (MP-BS = 90%, PP p = 1) comprised the northern temperate species of Polypodium, whereas the other (P. plesiosorum group) included species occurring mainly in Central America (MP-BS = 100%, ML-BS = 99%, PP p = 1.00).

Several groups were found within each of the Pleopeltis subclades. In subclade I, Neurodium lanceolatum and Dicranoglossum panamense were found to be sister to each other. The MP results indicated this group as the sister to the remaining subclade but this hypothesis lacked support in the bootstrap analyses (MP-BS < 50%). The other molecular methods suggested these two taxa to be part of the unresolved subclade. All analyses supported a group (Polypodium murorum group: MP-BS = 99%, ML-BS = 100%, PP p = 1.00) comprising four South American species: Pleopeltis buchtienii, P.

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Fig. 2. Phylogram obtained in a maximum likelihood analyses of the molecular data set. ML bootstrap values are given above or below branches. The arrow points toward the position of Pseudocolysis bradeorum. Subclades I, II, III of the Pleopeltis clade follow the description given in the text.

pycnocarpa, Polypodium monosorum, and P. murorum. Another South American species, Pleopeltis appressa, was found to be the sister of this clade in all three analyses but support for this relationship was low. A second group (MP-BS = 95, ML-BS = 90, PP p = 1.00) found in all analyses included five species of South American or South to Central American distributional ranges: Pleopeltis pleopeltifolia, P. tweediana, P. fraseri, P. remota, and P. ballivanii (Pleopeltis remota group). Pleopeltis disjuncta, from Peru and Bolivia, Polypodium friedrichsthalianum and P. furfuraceum, from Mexico

and Central America, formed another assemblage within subclade I (P. furfuraceum group: MP-BS = 100%, ML-BS = 100%, PP p = 1.00). The relationships of P. polypodioides were unclear. The two sampled varieties showed distinctive sequences and were never found to be sister in any of the performed analyses. Due to contradicting and poorly supported topologies the positions of the mentioned lineages remained ambiguous. Apart from Microphlebodium muenchii, subclade II included two taxa of ambiguous relationships (Pleopeltis thyssanolepis and

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Fig. 3. Majority role consensus tree calculated for all sampled trees obtained in the plateau phase of the Bayesian inference of phylogeny analyses using a single model for the molecular matrix. p-Values are given above branches. The arrow points toward the position of Pseudocolysis bradeorum. Subclades I, II, III of the Pleopeltis clade follow the description given in the text.

Polypodium platylepis) plus two lineages that were found in all analyses. One of them (MP-BS = 91%, ML-BS = 94%, PP p = 1.00) contained the simple-bladed Pleopeltis species and Pleopeltis fallax. They formed two groups. The Pleopeltis macrocarpa group comprised P. macrocarpa and its Mexican relatives, P. conzattii, P. crassinervata, P. mexicana, and P. polylepis, whereas the P. wiesbaurii group consisted of Pleopeltis (Marginariopsis) wiesbaurii, Pleopeltis

astrolepis, P. fallax, P. fructuosa, and P. intermedia. The other supported lineage (MP-BS = 85%, ML-BS = 81%, PP p = 0.99) in subclade II (Polypodium plebeium group) comprised Central American species with pinnatifid leaves: Pleopeltis guttata, Polypodium madrense, P. montigenum, P. plebeium, and P. rzedowskianum. Subclade III consisted of species that have been included in the Polypodium squamatum group. In the subclade two distinct lineages

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were found. One (MP-BS = 100%, ML-BS = 100%, PP p = 1.00) consisted of three South American samples (Polypodium bombycina (Zurich specimen), P. hirsutissimum, and P. macrolepis) whereas the other lineage (MP-BS = 75%, ML-BS = 88%, PP p = 0.98) included six Central American species (Polypodium collinsii, P. lepidotrichum, P. macrolepis, P. myriolepis, P. pyrrholepis, and P. sanctae-rosae). The two samples of P. bombycina showed distinct sequences and fell into different parts of the subclade. The Bolivian sample was sister to the remaining taxa, but the other sample (‘‘Zurich”) nested within the South American lineage.

4. Discussion This study presents a phylogeny of one of the two main Neotropical lineages of Polypodiaceae (lineage II, see Section 1) with special emphasis on the Pleopeltis clade. The studied lineage, including Pecluma, Phlebodium, Pleopeltis, Polypodium, and a few smaller genera, was shown to be monophyletic in a previous study by Schneider et al. (2004a). Its roughly 150 species comprise three major entities: the Polypodium, Pecluma, and Pleopeltis clades. Because our data do not give convincing support for a sister relationship of the Polypodium clade to the other two clades, the relationships among the three remain ambiguous. Nevertheless all three clades were found to be strongly supported in our analyses. The expanded sampling provides additional insights into the phylogenetic relationships within each clade and the natural classification of Polypodium. 4.1. Polypodium clade Apart from the monotypic East Asian genus Pleurosoriopsis, this clade comprises mostly north-temperate species of Polypodium related to Polypodium vulgare, the type of Polypodium, and P. plesiosorum and allies in Mexico and Central America. Pleurosoriopsis might be the sister to the remaining clade, which would support its classification as a segregate genus. The species of Polypodium probably belong to two independent groups but further evidence is required to confirm this hypothesis. The first group includes all northern temperate species of the genus Polypodium. Our sampling comprises representatives occurring in Europe (P. cambricum, P. vulgare), Macaronesia (P. macaronesicum), northern North America (P. amorphum, P. appalachianum, P. glycyrrhiza, P. scouleri), northeastern Asia (P. fauriei), and Hawai’i (P. pellucidum). Both taxonomic sampling strategy and a low level of variation restricted the resolution within and support of this putative group. The second group is supported in all analyses. It includes nearly exclusively taxa occurring from Mexico to Panama. These species were assigned previously to either the Polypodium plesiosorum group or to the P. dulce or P. subpetiolatum groups (Moran and Riba, 1995; Smith et al., 2006a), or they were treated as part of an assemblage that contained the members of Polypodium s.s., the P. dulce and the P. plesiosorum group (Tryon and Tryon, 1982). Compared to these concepts our results indicate a monophyletic group that contains all members assigned to P. plesiosorum but only part of the P. dulce group which was found to be polyphyletic with some species being nested within the Pecluma clade (see below). 4.2. Pecluma clade This clade comprises two well-established segregates of Polypodium. In addition it contains some members of the polyphyletic Polypodium dulce group (Moran and Riba, 1995; Smith et al., 2006a). These taxa form two strongly supported groups that are closely related to Pecluma. The low support values for the sister relationships of Pecluma and the two Polypodium splin-

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ters indicate the need for further evidence. Pecluma is separated from the other groups by its apomorphic character state of basally attached rhizome scales, while its relatives possess peltate or pseudopeltate scales. The new related but previously unrecognised lineages may be treated either as part of Pecluma or described as two new genera. The first alternative has the disadvantage of ignoring the scale apomorphy of Pecluma, whereas the second option introduces two rather small genera that will be difficult to define. The long branches in the ML phylogram (Fig. 2) are notable and may indicate longer separation and/or extinction events. However, they could also be the result of altered mutation rates. Additional studies considering molecular and morphological data are needed to resolve the relationships among these ferns. This would provide the required fundament for a natural classification. 4.3. Pleopeltis clade This strongly supported clade comprises all the species of Pleopeltis and Polypodium subg. Marginaria included in this study, as well as Dicranoglossum panamense, Marginariopsis (Pleopeltis) wiesbaurii, Microphlebodium (Pleopeltis) muenchii, Neurodium lanceolatum, and Pseudocolysis (Polypodium) bradeorum. Schneider et al. (2004a) also found Polypodium ensiforme from southern Africa to be part of this clade, but we currently lack material suitable to DNA studies for this species and so this species was excluded from this study. Pseudocolysis might be the sister to the remaining clade but with bootstrap values of 66% (ML) and 78% (MP) this hypothesis is not very well supported. Additionally, the Bayesian analysis provides a different result, with Pseudocolysis being part of a fully supported subclade I. It is unclear if the Bayesian results were caused by inconsistencies of the analytical procedure or they reflect the true relationships, in which case the maximum likelihood and maximum parsimony results are misleading. The sequences of the intergenic spacers trnL-F and rps4-trnS that are still missing for Pseudocolysis are needed to answer this question. The main apomorphy of the Pleopeltis clade members are persistent peltate scales on the mature leaf, with Neurodium lanceolatum being the only exception. Nectaries occur on the leaf blades of many but apparently not all species in the clade and could provide additional phylogenetic support for the clade (Koptur et al., 1982, 1998). By careful examination of herbarium specimens belonging to 35 species, it was possible to confirm the presence of these structures for 31 species. Since they usually are very inconspicuous and can only be seen on well-preserved specimens, it is possible that other species share this trait. Our study found evidence to support an expanded generic concept of Pleopeltis that includes all species of the Pleopeltis clade. This means that Dicranoglossum, Marginariopsis, Microphlebodium, Neurodium, and Pseudocolysis are part of the redefined genus. In addition, the species previously assigned to Polypodium subg. Marginaria and the members of Pleopeltis are part of this clade. The type of Presl’s genus Marginaria, which is Marginaria (Pleopeltis) polypodioides, nests within the same clade as Pleopeltis angusta, the type of Pleopeltis. Persistent peltate laminar scales are the main character of the redefined genus. The presence of these scales is a taxonomically distinctive character because it does not occur in the closely related Pecluma or Polypodium clades. The presence of leaf nectaries as a second generic character needs confirmation. As mentioned before, Pleopeltis has been defined by the presence of peltate scales in the sorus. Our results found taxa with this kind of paraphysis to belong to two different subclades within the Pleopeltis clade. The type species Pleopeltis angusta belongs to subclade I, whereas Pleopeltis macrocarpa and allies are nested in a lineage of subclade II. Soral scales can also be observed in Microphlebodium muenchii, but they are pseudopeltate and similar to laminar scales in this species.

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Several options for a taxonomic treatment may be considered. As discussed above, all species nested within the Pleopeltis clade may be treated as members of the genus Pleopeltis. With one exception in the Neotropics, the genus is determined by the presence of persistent scales on the leaf. In the Neotropics, only species of Microgramma and Serpocaulon have this character. The vast majority of species of Pleopeltis occur in the Neotropics, but a few species occur also in the Afro-Madagascan region and one is also found in India. The second taxonomic option is to accept four genera in the Pleopeltis clade, and these four genera would correspond to subclades I to III plus Pseudocolysis or three genera respectively if Pseudocolysis is part of subclade I. Most of these putative genera would be difficult to define morphologically, making this taxonomic treatment problematic. Finally, subclades I to III may be included in Pleopeltis with Pseudocolysis constituting a separate genus. This solution would fit best with the leaf morphology but in the end our results are not strong enough to fully support this hypothesis. This last classification can be a temporary solution until further evidence on the actual position of Pseudocolysis is found. Apart from the ambiguous position of Pseudocolysis, the placement of previously segregated small genera is rejected. Marginariopsis wiesbaurii nests within the Pleopeltis macrocarpa group and shares with that the simple leaves (except for P. fallax) and peltate paraphyses. Marginariopsis was described as a monotypic genus because of its strong leaf dimorphism and conspicuous coenosori. The unusual chromosome number of n = 35 seems to support this separation, but Hooper (1995) and Krieger (2007) showed that these characters were taxonomically uninformative, since all the simple-leaved members of the Pleopeltis macrocarpa group have to some extent fused sori, dimorphic leaves and chromosome numbers of n = 34 or n = 35. The recent interpretation of leaf dimorphism in the P. macrocarpa group by Krieger (2007) required, however, some confirmation. The pseudopeltate laminar and soral scales of monotypic Microphlebodium muenchii are exceptional within the Pleopeltis clade. Although a sister relationship of this species to subclade II is not fully supported, M. muenchii nests well within the Pleopeltis clade. Dicranoglossum is undoubtedly a member of subclade I and closely related to Pleopeltis angusta, whose leaves are similar to those of Dicranoglossum panamense. Tryon and Tryon (1982) suspected a close relationship to Pleopeltis. Because of its glabrous leaves and distal, marginal sori, monotypic Neurodium lanceolatum might still be the most unusual fern in the Pleopeltis clade. Our results found it to be very closely related to Dicranoglossum panamense, with which it shares the arrangement of the coenosori and the short creepingrhizome. However, the sorus structure is a highly variable character in Dicranoglossum and additional studies including other species of Dicranoglossum are required to explore the evolution of this character. Sampling Pleopeltis repanda may provide an improved understanding of the relationships of this clade because this species may be closely related to Neurodium lanceolatum (Smith, personal communication). The Pleopeltis clade and the three subclades therein are well supported but still some taxa are problematic. Microphlebodium muenchii seems to be distantly related to subclade II but has distinct differences in the cpDNA sequences, as illustrated by the length of the branch separating M. muenchii and clade II in Fig. 2, as well as low support values, suggests a long separation. Thus the relationship between the two could be an artifact and should be investigated further. Sampling the second species of Microphlebodium (Mickel and Smith, 2004) may improve these results. As mentioned above, the position of Pseudocolysis bradeorum also requires additional research. A further notable concern is the position of the Bolivian sample of Pleopeltis (Polypodium) bombycina as sister to the remaining Polypodium squamatum group (subclade III) that includes a second sample of Pleopeltis bombycina of un-

known origin (‘‘Zurich”). The pattern raises questions concerning the monophyly of Pleopeltis bombycina, which is the species with the largest distributional range of any member of the Polypodium squamatum group, and also in respect of the origin of this clade (see below). In his monography de la Sota (1966) established two segregates of Pleopeltis bombycina, Polypodium insularum and P. apagolepis, but these taxa should be restudied taking into account DNA variation. The current evidence is insufficient to draw any conclusions. The low support for the relationships among the major subclades is notable. It could be rather difficult to resolve this problem because these lineages might have arisen from a rapid initial radiation of the Pleopeltis clade. This hypothesis is supported by the pattern of branch length distribution in Fig. 2. Similar patterns were also found with respect to the diversification of each of the three subclades. Short basal branches and lack of support for their nodes contrast with longer branches leading to extant species. This pattern suggests short intervals of rapid diversification after the departure from a uniform origin followed by longer intervals of rather constant evolution. Deciphering radiation events is now widely recognized as a common problem in our attempts to reconstruct the tree of life (Shavit et al., 2007; Whitfield and Lockhart, 2007). It will be interesting to find out which events might have triggered the rapid initial radiations within the Pleopeltis clade. The first step in future studies should address the low level of sequence variation within each of the three subclades by either exploring additional cpDNA markers or by employing sequences of nuclear markers. An expansion of the taxonomic sampling will help to overcome some ambiguities in our current understanding of the relationships within Pleopeltis. Particularly it may improve the resolution within each of the major subclades. As an example, none of the four applied methods found the two included specimens of Pleopeltis polypodioides to be monophyletic. In our results three different putative positions were recovered for the two subspecies but none gets sufficient bootstrap support or posterior confidence values. Pleopeltis polypodioides, the resurrection fern, has a large distribution range stretching from the southeastern United States down to southern South America. Up to seven subspecies are currently recognized for this highly variable species, with one also occurring in Africa. The African member of the group is sometimes accepted as a segregate species P. ecklonii. An increased sampling is required to confirm or disprove the monophyly of this taxon. The monophyly of P. polypodioides is currently studied in an independent study comprising a sampling of all putative segregates of this species (Sprunt and Schneider, personal communication). The results of this study will likely shed new light on the monophyly of this species. The results of this study confirmed several groups of closely related species. As an example, we discovered a very low genetic variation between the sister taxa of Pleopeltis macrocarpa, which indicates a very recent speciation. These results are in agreement with previous studies of isozyme variation suggesting a rapid ecological diversification of this group as an adaptive response to niche differentiation in tropical epiphytic habitats (Hooper, 1995; Hooper and Haufler, 1997; Haufler et al., 2000). The Pleopeltis clade also includes other species complexes that are potential candidates to study speciation patterns and the underlying processes such as the Cental American–Mexican Polypodium plebeium group, the already mentioned Pleopeltis macrocarpa group, and two mainly South American complexes, the Polypodium murorum and Pleopeltis remota groups. The importance of ecological differentiation is underlined by the results of studies documenting differentiations between species of Pleopeltis and/or related genera (Wolf and Flamenco-S, 2006). Currently accessible evidence is insufficient to reconstruct the evolution of putative adaptive traits such as the ability to withstand extreme desiccation, leaf surface reduction,

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or survival in different forests types and in different niches in the epiphytic community. Required studies on niche differentiation in epiphytic habitats are confronted with the complexity of host tree specific traits such as water-holding capacity of the bark, structuring within the tree, and the community structure of the vegetation that surrounds and covers it (Callaway et al., 2002). 4.4. Biogeography and the evolution of Pleopeltis and its relatives With 47 species more than half of the 72 ingroup species (Pecluma, Polypodium, and Pleopeltis) occur in Central America (Panama to Mexico, see Davies and Heywood, 1997). Approximately 50% of all included species have ranges restricted to this region. This dominance of Central American species is unlikely to be broken by including currently unsampled relatives of this lineage. The area including southern Mexico and Central America is not only the center of biodiversity of these ferns but it is also the putative center of origin for all major clades within Pleopeltis because species occurring exclusively outside of the area are clustered within few derived clades. The Polypodium clade is the only one including a group of northern temperate species besides a group with a Central American distribution range. Exclusively South American species are part of derived lineages of the Pleopeltis clade or Pecluma clade. Reconstruction of ancestral distribution ranges using either a maximum parsimony or maximum likelihood approach suggests southern Mexico and Central America as the putative areas of origin for the Pleopeltis clade. The same region was also found as the more probable alternative for the Polypodium clade. Within the Pleopeltis clade, the range of certain species expanded several times. Several species, e.g., Pleopeltis macrocarpa, P. polypodioides, P. thyssanolepis, and P. remota show large distributions including Central America and at least parts of South America. Two of these widely distributed species, Pleopeltis macrocarpa and P. polypodioides, have even successfully colonized southern Africa. Evidence was found for at least two to three radiations within the Pleopeltis clade after colonization of South America – one in southern Brazil and one or two in the Andes. One or two mainly Andean radiations are indicated by the clustering of Andean endemics in the Pleopeltis remota and Polypodium murorum clades. In the maximum likelihood analyses, these two clades are sister to each other, whereas independent origins are suggested in the Bayesian and MP results. The poor support of the relationships within subclade I of the Pleopeltis clade does not provide sufficient evidence to explore the hypothesis of one or more colonisations of the Andes from Central America or any further exploration of the biogeographic history of these lineages. Complex patterns of lineage exchange between Central and South America were already documented for several lineages of angiosperms. In some cases, Central America was the initial source area but was recolonised by offspring of originally Central American lineages that successfully colonized South America (Erkens et al., 2007). Several radiations appear to be limited to Central America, e.g., the Polypodium macrolepis and P. plebeium groups. The interpretation of other groups appears less unequivocal because some of the associated species have either also South American occurrences or exclusively South American distributions, e.g., Pleopeltis macrocarpa in the P. macrocarpa group and P. intermedia in the P. wiesbaurii group. Additional evidence is required to untangle the biogeographic history of these lineages. A well studied example (Hooper, 1995; Hooper and Haufler, 1997; Haufler et al., 2000) for speciation in Central America is the already mentioned group of Pleopeltis conzattii, P. crassinervata, P. mexicana, and P. polylepis. Pleopeltis macrocarpa, the sister to this quartet, is very widespread, but sequence comparison of chloroplast trnL-F IGS DNA of P. macrocarpa specimens collected in Chile, the Comoros, and Tanzania found not a single mutation (Janßen et al., 2007). This is congruent with the very

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low genetic diversity among the other species. The biodiversity center of the four Central American taxa lies in southern Mexico. Hooper and colleagues (Hooper, 1995; Hooper and Haufler, 1997; Haufler et al., 2000) found evidence for an ecological differentiation instead of a biogeographical separation in their extensive study of Mexican populations. Thus, this group may be one of the best examples to infer evidence for sympatric speciation in ferns although phylogeographic studies of other Pleopeltis clade groups are likely to reveal similar patterns. To our knowledge, this study is the first phylogenetic study providing evidence for Central America as a species diversity hotspot and putative cradle for a particular lineage of ferns. Further conclusions concerning the origin of Neotropical Polypodiaceae are hindered by the lack of similar studies in the Neotropical sister lineage of Pleopeltis, a group that includes Campyloneurum, Microgramma, Niphidium, Serpocaulon, and the grammitids. Only the phylogeny of Serpocaulon has been explored sufficiently to allow some conclusions. Southern Brazil and Bolivia were found to be the possible areas of origin for this genus wherefrom it then colonized the Andes (Kreier et al., 2008). This hypothesis is in contrast to the Andean diversity of Pleopeltis that probably is the result of colonization from Central America. The pattern becomes more ambiguous if the exclusively southern South American genus Synammia, the putative sister to the two mentioned Neotropical lineages, is considered (Schneider et al., 2006b). Further examination of the early history of Neotropical Polypodiaceae should include two clades that were not sufficiently explored so far. The first one comprises the genera Campyloneurum, Microgramma, and Niphidium. Existing evidence (Janßen et al., 2007; Kreier et al., 2007; Salino et al., 2008) suggests a rather confusing geographic pattern in this clade because early separating species have distribution ranges covering both Central and South America. The second clade, the grammitids, is also a challenge. Ranker et al. (2004) discovered a group of 12 species assigned to the genus Terpsichore as sister to the about 700 species of this pantropical group. The mentioned 12 species occur either in both or at least one of the two regions of Central America and South America. Altogether the geographic situation of the grammitids and the Microgramma clade is very ambiguous so that without further investigations it is impossible to draw any final conclusions concerning the historical processes that caused the described distribution patterns. The diversification of Neotropical Polypodiaceae may have happened in the later Cenozoic as indicated by occurrences of ferns belonging to the Polypodium clade in the Californian Miocene (Kvacek et al., 2004) and divergence time estimates indicating the Polypodiaceae lineage as not older than the Eocene (Schneider et al., 2004b). Thus, the geographic distribution of clades likely reflects abiotic events during this period including the formation of a permanent land bridge between North and South America, the formation of the Andes, and dramatic climatic changes such as the Eocene–Oligocene cooling (Zanazzi et al., 2007). The latter event may be of particular interest for the establishment of a northern temperate clade. Future studies should be pursued to test the hypothesis that these events shaped the current diversity of these ferns. Also similarities to the diversification of other plants may be taken into account. 4.5. Ecological and morphological disparity of Pleopeltis The Pleopeltis clade shows a remarkable morphological disparity, especially concerning leaf shape and scale characters. Despite this great variety there are only a few apomorphies characterizing the different groups within the clade. Due to their morphological heterogeneity two of the three subclades cannot be distinguished by specific traits. Only subclade III that corresponds to the well established Polypodium squamatum group (Maxon, 1916; Weatherby, 1947; de la Sota, 1966) can be separated morphologically. It is

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characterized by netted venation combined with nectaries sitting on conspicuous lobes at the acroscopic pinna bases. Another well-defined lineage is the group of Pleopeltis macrocarpa and P. wiesbaurii, characterized by the combination of simple leaves and peltate paraphyses (except for P. fallax). Neurodium lanceolatum is simple leaved as well but lacks paraphyses whereas Pleopeltis angusta shows paraphyses but divided leaves. Scale density is a rather uninformative character with many unrelated species being very densely scaly, e.g., Pleopeltis polypodioides, P. thyssanolepis, and some species of the Polypodium squamatum group. Another inconsistent character is the presence of green spores, reported from Pleopeltis angusta, P. polypodioides, P. remota, and P. wiesbaurii (personal observation HS; communications Alan Smith, Susan Sprunt). The extreme desiccation tolerance shown by Pleopeltis polypodioides appears to be unusual but other species, e.g., P. crassinervata and Polypodium furfuraceum show poikilohydrous behavior (sensu Kessler and Siorak, 2007) as well. Poikilohydrous behavior is also observed in cultivated plants of the P. squamatum group (personal communication Alan Smith). The mentioned features probably are responses to non-biological stresses, such as exposure to sunlight or limited access to water. Some of them have been discussed as adaptive traits for ferns (Watkins et al., 2006; Kessler and Siorak, 2007). Since ecological studies demonstrated that species of the Pleopeltis clade differ greatly in their ecological preferences (Wolf and Flamenco-S, 2006), the lineage appears to be well suited to explore the evolution of adaptive characters in epiphytic habitats. Unfortunately, the available data do not allow us to infer if adaptation to different ecological niches in epiphytic habitats was the major force triggering the species richness and unique morphological disparity of this lineage. Acknowledgments The authors thank L. Bohs, M. Gibby, I. Jimenez, M. Kessler, J. Krieger, T. Krömer, R. Moran, E. Schuettpelz, M. Sundue, and D. Tejero-Diez for providing plant samples. We are grateful to T. Krömer and D. Tejero-Díez for supporting EO during her fieldwork in Mexico. The project was supported financially by the German Science Foundation (DFG Grant SCHN 785/2-2) under the Schwerpunkt Programm SPP 1127 ‘‘Radiations–Origin of Biological Diversity.” References Anthony, N.C., Schelpe, E.A.C.mL.E., 1985. X Pleopodium – a putative intergeneric fern hybrid from Africa. Bothalia 15, 555–559. Benzing, D.H., 1990. Vascular Epiphytes. General Biology and Related Biota. Cambridge University Press, Cambridge. Bonfield, J.K., Staden, R., 1996. Experiment files and their application during large scale sequencing projects. DNA Sequence 6, 109–117. Bonfield, J.K., Smith, K.F., Staden, R., 1995. A new DNA sequence assembly program. Nucleic Acids Res. 24, 4992–4999. Callaway, R.M., Reinhart, K.O., Moore, G.W., Moore, D.J., Pennings, S.C., 2002. Epiphyte host preferences and host traits: mechanisms for species-specific interactions. Oecologia 132, 221–230. Davies, S.D., Heywood, V.H., 1997. Centres of Plant Diversity: A Guide and Strategy for their Conservation. Union Internationale pour la Conservation de la Nature et de ses Ressources, Switzerland. de la Sota, E.R., 1965. Las especies escamosas del genero Polypodium L. (s.str.) en Brasil. Rev. Museo de la Plata Sección Botánica 9, 243–271. de la Sota, E.R., 1966. Revisión de las expecies americanas del grupo ‘‘Polypodium squamatum” L. Polypodiaceae (s. str.). Rev. Museo de la Plata Sección Botánica 10, 69–186. Ebihara, A., Dubuisson, J.-Y., Iwatsuki, K., Hennequin, S., Ito, M., 2006. A taxonomic revision of Hymenophyllaceae. Blumea 51, 221–280. Ebihara, A., Hennequin, S., Iwatsuki, K., Bostock, P.D., Matsumoto, S., Jaman, R., Dubuisson, J.-Y., Ito, M., 2004. Polyphyletic origin of Microtrichomanes (Prantl) Cope. (Hymenophyllaceae), with a revision of the species. Taxon 53, 935– 948. Erkens, R.H.J., Chatrou, L.W., Maas, J.W., van der Niet, T., Savolainen, V., 2007. A rapid diversification of rainforest trees (Guatteria; Annonaceae) following dispersal from Central into South America. Mol. Phylogenet. Evol. 44, 399–411.

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