Phylogenetic relationships of the fern genus Christiopteris shed new light onto the classification and biogeography of drynarioid ferns

Transcription

1 Botanical Journal of the Linnean Society, 2008, 157, With 2 figures Phylogenetic relationships of the fern genus Christiopteris shed new light onto the classification and biogeography of drynarioid ferns HARALD SCHNEIDER FLS 1,2 *, HANS-PETER KREIER 2, PETER HOVENKAMP 3 and THOMAS JANSSEN 2 1 Department of Botany, Natural History Museum, London SW7 5BD, UK 2 Albrecht-von-Haller Institut für Pflanzenwissenschaften, Georg-August University, Göttingen, Germany 3 Nationaal Herbarium Nederland, P.O. Box 9514, 2300 RA Leiden, the Netherlands Received 28 August 2007; accepted for publication 31 October 2007 Phylogenetic relationships of the SE Asiatic genus Christiopteris were explored by comparative analysis of sequence variation of four chloroplast genome regions that were successfully used in previous phylogenetic studies of Polypodiaceae. This small genus is nested within the drynarioid ferns, as recovered with good support by each of the methods applied to reconstruct the phylogeny, including maximum parsimony and maximum likelihood. The placement of Christiopteris within drynarioids enhances the need of a new generic classification for these ferns because Dynaria is paraphyletic in its current circumscription. Two alternative classifications are discussed. The results also support the hypotheses that leaf differentiation into litre collectors and trophosporophylls is an autapomorphy of drynarioid ferns and that SE Asia is the putative ancestral area for drynarioid and selligueoid ferns. Two clades of drynarioid ferns colonized Eastern Malesia independently, but only one of these colonization events created species endemic to New Guinea The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157, ADDITIONAL KEYWORDS: biogeography character evolution colonization Malesia phylogenetics plant diversity south-east Asia taxonomy. INTRODUCTION The recent years have witnessed considerable progress in our understanding of the phylogeny of recently derived ferns, especially Polypodiaceae (Schneider et al., 2002, 2004a, b, 2006; Smith et al., 2006). These studies have revealed many conflicts between existing classifications and inferred phylogenetic relationships. This is not surprising considering the instability of generic definitions proposed during the last century (Hennipman, Veldhoen & Kramer, 1990; Hovenkamp, 1996; Smith et al., 2006). Further progress in understanding phylogenies will rely largely on phylogenetic treatment of those taxa with *Corresponding author. Current address: Department of Botany, Natural History Museum, London SW7 5BD, UK. h.schneider@nhm.ac.uk contested relationships, for they can provide crucial clues to resolving the phylogeny of clades. The Malesian genus Christiopteris Copel. is one of these taxa with the potential to improve our current understanding of the phylogeny of drynarioid ferns (Hennipman et al., 1990; Janssen & Schneider, 2005). The genus includes two extant species, C. sagitta (H.Christ) Copel. occurring in the Philippines (Luzon and Mindanao) and C. tricuspis (Hook.) H.Christ found in continental southern Asia from India, Indochina and Hainan, to the Malay Peninsula (Hennipman & Hetterscheid, 1984; Hennipman, Hovenkamp & Hetterschied, 1998). The genus has not been included in any phylogenetic study using DNA sequences, despite its disputed affiliations. Hennipman et al. (1990) assigned Christiopteris to the tribe Microsoridae but suggested that it might be better placed in the tribe Selligueeae. Since then, phyloge- 645

2 646 H. SCHNEIDER ET AL. netic studies on the global relationships of Polypodiaceae have provided evidence for a separation between the drynarioid plus selligueoid and the microsoroid lineage including the tribes Microsoridae and Lepisoridae (Schneider et al., 2004b). The microsoroid lineage includes species with at least partially clathrate scales, whereas the drynarioids and their sister lineage the selligueoids have nonclathrate scales (Schneider et al., 2004a, b). The presence of isotoechous (non-clathrate scales) in Christiopteris suggests relationships to the drynarioid-selligueoid clade rather than the microsoroid clade. The findings of a study on the phylogeny of drynarioids (Janssen & Schneider, 2005) enhanced the interest in the putative relationships between Christiopteris and drynarioids. In this study, the authors suggested a scenario explaining the evolution of the drynarioid leaf dimorphisms with one leaf formed as litter collector and the other leaf formed as a trophosporophyll from a hypothetical ancestral leaf dimorphism as found in Christiopteris but also some selligueoid species, e.g. Selliguea triloba (Houtt.) M.G.Price (Janssen & Schneider, 2005). Christiopteris possesses two kinds of leaves: one has a long stalk, reduced lamina tissue and a pinnatifid lamina bearing sori; whereas the other has a short stalk, well-developed lamina tissue and a lobed lamina lacking sori. The second kind of leaves can be interpreted as a trophophyll, while the first kind has mainly the function of a sporophyll while maintaining the function of a trophophyll to some extent. Furthermore, the putative placement of Christiopteris within or closely to the drynarioids raises hopes to discover the natural genus classification of drynarioid ferns. By using sequences of the chloroplast genome (cpdna), convincing evidence was found that the current classification of drynarioid ferns as proposed by Roos (1985) requires substantial revision (Janssen & Schneider, 2005). The current classification recognizes two genera, Aglaomorpha Schott and Drynaria J. Sm., comprising together about 30 extant species. The broad genus concept of Aglaomorpha Schott was only introduced by Roos (1985) and includes an assemblage of species assigned previously to various monotypic or oligotypic genera, e.g. Dryostachyum J. Sm., Merinthosorus Copel., Photinopteris J. Sm., Pseudodrynaria C.Chr. and Thayeria Copel. Phylogenetic analyses based on morphological and cpdna (Janssen & Schneider, 2005) provided good support for the Aglaomorpha clade despite the remarkable disparity in leaf morphology among its members. Roos (1985) did not question the monophyly of the genus Drynaria J. Sm., which appeared clearly defined based on the putative apomorphy of leaf dimorphism. One type of leaf corresponds to a regular fern trophosporophyll, while the other type is a highly modified litter collector (Roos, 1985; Janssen & Schneider, 2005). The relatively low disparity in leaf morphology was a further argument to consider Drynaria a likely monophyletic genus. However, phylogenetic hypotheses based on cpdna rejected the monophyly of Drynaria. Instead, they provided evidence for a scenario in which the leaf dimorphism found in most species of Dyrnaria is a plesiomorphic character state of a paraphyletic Drynaria. Drynaria comprises three lineages, of which one forms a grade at the base of Aglaomorpha (Janssen & Schneider, 2005). Janssen & Schneider (2005) hesitated to devise a new generic classification of drynarioids because they were concerned about incomplete taxon sampling and the poorly supported deep relationships among the three lineages of drynarioid ferns (Janssen & Schneider, 2005). Thus, a phylogenetic study of the genus Christiopteris may help to address two major questions concerning drynarioids ferns: (1) the relationships of the two species currently accepted as members of the genus and (2) the historical biogeography of these ferns, especially the identification of the area of origin. To explore these issues, we generated DNA sequences of four cpdna markers identical to those used in previous studies (Schneider et al., 2004a, b; Janssen & Schneider, 2005) for three samples of Christiopteris, including both extant species, C. sagitta and C. tricuspis. MATERIAL AND METHODS PLANT MATERIAL AND DNA SEQUENCING Leaf material of Christiopteris sagitta was obtained from plants cultivated at the Hortus Botanicus at Leiden and the Botanischer Garten Berlin-Dahlem. Voucher specimens were deposited in the Nationaal Herbarium Nederland at Leiden (L.) and the herbarium of the Georg-August University Göttingen (GOET). We also obtained material of Christiopteris tricuspis from a herbarium voucher stored in the herbarium at Leiden. New sequences were also generated for Drynaria fortunei (specimen cultivated at the RBGE, originally collected by the Sino-British Cangshan Exp.) and a specimen of Aglaomorpha cultivated by Charles Alford (Vero Beach, Florida, USA), which does not to correspond with any known species of this genus, but lack of information about the area of origin hampers its description as a new species. Whole genomic DNA was extracted using the Invisorb Plant Mini Kit (Invitek, Berlin, Germany). Sequences of four chloroplast regions, rbcl, rps4, rps4-trns (GGA) intergenic spacer (IGS) (throughout the paper, the rps4 plus rps4-trns IGS are called rps4) andtrnl (CAA)

3 PHYLOGENY OF THE FERN GENUS CHRISTIOPTERIS 647 -trnf (GAA) IGS (called trnl-f IGS in the following) were generated using PCR primers and protocols used in previous studies (Haufler et al., 2003; Schneider et al., 2004a, b; Janssen & Schneider, 2005). Automated sequencing was carried out on a MegaBACE 1000 capillary sequencer using Dynamic ET Primer DNA sequencing reagent (Amersham Biosciences, UK). All sequences were assembled and edited using TreV (Staden Package; sourceforge/net/projects/staden) and finally deposited in GenBank (accession numbers given in Appendix 1). All other sequences have been used in previous studies (Schneider et al., 2004a, b; Janssen & Schneider, 2005). MacClade 4.08 (Maddison & Maddison, 2005) was used to generate manually the necessary alignments. Ambiguously aligned regions were excluded from all analyses. Gaps were treated as missing data and indel scoring was not performed. PHYLOGENETIC RECONSTRUCTIONS Phylogenetic analyses were carried out using a set of interchangeable phylogenetic software including FigTree v.1.0 (Rambaut, 2006), Garli (Zwickl, 2006), Geneious (Biomatters Ltd, 2007), Modeltest 3.7 (Posada & Crandall, 1998), MrBayes (Huelsenbeck & Ronquist, 2001), MrAIC (Nylander, 2007), PAUP* 4.0b (Swofford, 2002), PHYML (Guindon & Gascuel, 2003), Splitstree 4.0(Huson & Bryant, 2006) and Tracer 1.3 (Rambaut & Drummond, 2006). PAUP was used to perform maximum parsimony (MP) analyses as heuristic searches with TBR branch swapping and 100 randomly assembled replicates. All characters were treated as equally weighted and unordered. Maximum parsimony bootstrap analyses (MP-BS) were carried out as 1000 bootstrap replicates with heuristic searches, TBR and 10 randomly assembled replicates per bootstrap replicate. Splitgraphs were generated using the NeighbourNet algorithm (Bryant & Moulton, 2002) as implemented in Splitstree with LogDet distances (Lockhart et al., 1994) and four dimensions. Models for parameter-based analyses were selected using the software tools Modeltest and/or MrAIC with the implemented hierarchical likelihood ratio test (hlrt), Akaike information criterion (AIC) and the Bayesian information criterion (BIC), respectively (Posada & Crandall, 1998; Nylander, 2007). The GTR invgamma was found to be suitable for most maximum likelihood analyses and Bayesian inference of phylogeny. The GTR model is used as the standard model in several of the employed software packages such as GARLI (Zwickl, 2006). MrBayes (Huelsenbeck & Ronquist, 2001) was used to carry out the Bayesian inference of phylogeny (BY) using either a single model for the whole data set or two models of which one was applied to coding regions and the other one to non-coding regions. The results were investigated using Tracer and the Bayesian consensus trees were created using PAUP. Posterior probability support values (PP) were interpreted as significant if P Maximum likelihood (ML) analyses were performed in parallel using PHYML (Guindon & Gascuel, 2003) and GARLI (Zwickl, 2006) and compared with results of ML analyses using PAUP. Parameters were estimated during the analyses in PHYML and GARLI. These two programs were also used to generate bootstrap analyses using a non-parametric bootstrap procedure (ML-BS). Congruence among the four employed chloroplast genome markers was explored visually by comparing the bootstrap consensus trees obtained by MP-BS analyses of each individual region for putative different sister relationships with a BS support of at least 75%. To infer evidence for relationships among the recovered lineages of drynarioids, we employed various tests implemented in PAUP including the Templeton test (Templeton, 1983), the Kishino Hasegawa test (Kishino & Hasegawa, 1989) and the Shimodaira Hasegawa test (Shimodaira & Hasegawa, 1999). We used the morphological data set used in previous phylogenetic studies on drynarioids (Roos, 1985; Janssen & Schneider, 2005) to identify putative synapomorphic character states for the main lineages within the drynarioids. To do so, we included both species of Christiopteris by scoring characters from existing descriptions and careful examination of specimens stored in the herbaria of the Georg-August University Göttingen (GOET), the Nationaal Herbarium Nederland at Leiden (L.) and the Natural History Museum London (BM). MacClade (Maddison & Maddison, 2005) was used to handle morphological data and distribution ranges used to reconstruct ancestral morphological character states and ancestral distribution ranges, respectively, within an MP framework. RESULTS Sequences of the trnl-f IGS generated for two samples of C. sagitta were identical, whereas the pairwise similarity of trnl-f sequences for C. sagitta and C. tricuspis was 98%. The level of pairwise sequence similarities among all drynarioid ferns was about 89% for this cpdna marker. Maximum parsimony analyses based on trnl-f IGS sequences recovered C. tricuspis as sister to C. sagitta. Christioperis tricuspis was excluded from all other analyses because we were unable to obtain any other region than the trnl-f IGS from the herbarium material. The combined data set consisted of 2312 included characters, of which 653 (~ 28%) were parsimony-

4 648 H. SCHNEIDER ET AL core L N M D S P Aglaomorpha leporella Aglaomorpha heraclea Aglaomorpha drynarioides Aglaomorpha novoguineensis Aglaomorpha cornucopia ca Aglaomorpha acuminata Aglaomorpha pilosa Aglaomorpha parkinsoni Aglaomorpha Aglaomorpha hieronymi Aglaomorpha spec. Aglaomorpha splendens Aglaomorpha coronans Aglaomorpha meyeniana Drynaria mollis Drynaria fortunei northern Drynaria Christiopteris sagitta Drynaria sparsisora Christiopteris Drynaria descensa Drynaria quercifolia core Drynaria Drynaria bonii Drynaria rigidula Drynaria volkensii Drynaria willdenowii Drynaria laurentii Afro-Madagascan Drynaria Arthromeris wallichii Arthromeris lehmannii Selliguea lateritia Selliguea heterocarpa Selliguea hellwigii Selliguea enervis Selliguea lanceola Selliguea plantaginea Selliguea feei Gymnogrammitis dareiformis Selliguea laciniata Polypodiopteris brachypoda Platycerium coronarium Pyrrosia polydactyla Polypodium pelludicum Polypodium vulgare Pleopeltis polypodioides Pleopeltis angusta Pecluma ptilodon Serpocaulon ptilorhizon Microgramma percussa Campyloneurum chlorolepis Campyloneurum angustifolium Microsorum musifolium Microsorum cuspidatum Leptochilus macrophyllum Lemmaphyllum carnosum Goniophlebium subauriculatum Microsorum linguiforme Lecanopteris sinuosa Thylacopteris papillosa Loxogramme abyssinica Dictymia mackeei Figure 1. Phylogram retained in the maximum likelihood analyses performed using PHYML and GARLI. Bootstrap support is given using for 100% and for 90% for ML-BS above branches and for MP-BS below branches. Thick branches indicate a posterior value of P = Major clades are specified with bold letters below branches: core, core Polypodiaceae; ca, core Aglaomorpha; D, drynarioids; L, loxogrammoids; M, microsoroids; N, Neotropical clade; P, platycerioids; S, selligueoids. Major clades within drynarioids and selligueoids are annotated considering biogeographical distributions and/or taxonomy. informative and 411 (~ 18%) were variable but parsimony-uninformative. MP analyses resulted in 108 most parsimonious trees with a tree length of 2337 steps and the following characteristics: consistency index (excluding uninformative characters) of , retention index of and a rescaled consistency index of Base frequencies for the combined data set were as follows: A = 29.1%, C = 20%, G = 21.7% and T = 29.2% with a GC content of 41.7%. ML analyses with different software found the same topology with a - ln = (PHYML) with the corresponding parameters: gamma = 0.497, proportion of invariant = and the GTR model withatoc= 1.201, A to G = 3.622, A to T = 0.468, C to G = 0.784, C to T = and G to T = 1.0 (fixed). In all topologies obtained from the phylogenetic analyses, Christiopteris was found to be part of the drynarioid clade, but topologies obtained in different phylogenetic analyses recovered alternative sister relationships within the drynarioids (Fig. 1). Four clades were recovered within the drynarioids in all phylogenetic analyses, including analyses identifying support values: (1) a clade including the three Afro-Madagascan species of Drynaria (D. laurentii,

5 PHYLOGENY OF THE FERN GENUS CHRISTIOPTERIS 649 core Aglaomorpha Agl. mey. Christiopteris Agl. cor. Afro-Madagascan Drynaria Northern Drynaria Core Drynaria Drynarioids remaining Polypodiaceae 0.01 Selligueoids Figure 2. Splitsgraph obtained in a NeighbourNet analyses performed using SPLITSTREE. The analyses were performed with Logdet distances, equal angle splits transformation, least square weight modification and maximum dimension set to four. The analyses found 141 splits of a total weight and a fit of Branches cut by // are shown with less than half of their original length. D. volkensii and D. willdenowii); (2) a clade comprising the core of Drynaria consisting of D. rigidula and the members of the D. quercifolia complex (D. bonii, D. decensa, D. quercifolia and D. sparsisora); (3) a clade formed by the genus Aglaomorpha together with species of Drynaria occurring at the northern rim of the distribution range of drynarioid ferns (D. fortunei and D. mollis); and (4) Christiopteris. In model-based analyses, Christiopteris was found to be either the sister clade to the Afro-Madagascan clade (BY) or the sister to the Aglaomorpha clade plus northern Drynaria grade (ML). The strict consensus tree of the most parsimonious trees showed a polytomy comprising four lineages (Christiopteris clade, Aglaomorpha northern Drynaria grade, Afro- Madagascan Drynaria clade and core Drynaria clade) but 65% of the most parsimonious trees showed Christiopteris as sister to the Afro-Madagascan Drynaria clade. Other MPTs showed Christiopteris as sister to the Aglaomorpha plus northern Drynaria grade, sister to the core Drynaria clade or sister to all drynarioid ferns. Splitsgraph analyses support the evidence for insufficient resolution but give evidence for Christiopteris to be nested within the drynarioid clade (Fig. 2). Various tests were performed and the hypotheses recovered using ML analyses (Christiopteris sister to the Aglaomorpha plus northern Drynaria grade) were found to fit best with the data in both MP or ML frameworks, but only in the latter the differences among hypotheses were found to be significant (Tables 1, 2). DISCUSSION CHRISTIOPTERIS AND THE EVOLUTION OF DRYNARIOID FERNS All analyses provided unequivocal evidence for Christiopteris as belonging to the drynarioid ferns and rejected the alternative hypothesis of close relationships to the microsoroid ferns. The results obtained by the analyses of cpdna sequences are in concordance with morphological evidence, because, among old world clades of Polypodiaceae isotoechous (nonclathrate), scales are found only in the platycerioids, drynarioids and selligueoids (Schneider et al., 2004a, b). Other characters supporting the drynarioid relationships are the presence of blackish cells within the rhizome cortex and pith, the strong anastomosing venation with excurrent and recurrent free veinlets

6 650 H. SCHNEIDER ET AL. Table 1. Exploring evidence for relationships within drynarioid ferns by comparing five hypotheses recovered using maximum parsimony measures Topology TRM TL G-fit KH TE CH(AD(CD(ND, AG) MP (CH, AD) (CD(ND, AG) BY, MP ((CH, AD)CD)(ND, AG) MP (AD, CD)(CH(ND, AG) MP AD(CD)(CH(ND, AG) ML, MP CD(AD)(CH(ND, AG) MP The dataset was reduced to drynarioids and selligueoids. Clade abbreviations: AD, Afro-Madagascan Drynaria; AG, Aglaomorpha; CD, core Drynaria; CH, Christiopteris; ND, northern Drynaria. Other abbreviations: G-fit, Goloboff-fits; KH, P-values obtained in a two-tailed Kishino Hasegawa test using RELL bootstrap distribution (1000 replicates); TE, P-values obtained in a Templeton test; TL, tree length; TRM, topology was recovered in analyses using one of the following methods: Bayesian inference of phylogeny (BY), maximum likelihood (ML), maximum parsimony (MP). The hypothesis selected as the best fit with the data is printed in bold. P < 0.05 is interpreted as significant. Table 2. Exploring evidence for relationships within drynarioid ferns by comparing five hypotheses using maximum likelihood measures Topology TRM KH SH CH(AD(CD(ND, AG) MP (CH, AD) (CD(ND, AG) BY, MP ((CH, AD)CD)(ND, AG) MP (AD, CD)(CH(ND, AG) MP AD(CD)(CH(ND, AG) ML, MP CD(AD)(CH(ND, AG) MP Same data set as in Table 1. See Table 1 for abbreviations except: SH, P-values obtained in a one-tailed Shimodaira Hasegawa test using RELL bootstrap distribution (1000 replicates). The hypothesis selected as the best fit with the data is printed in bold. P < 0.05 is regarded as significant. and the dimorphic leaves. Closer relationships to drynarioids than to selligueoids are supported by the spore ornamentation. Spherical bodies (also called globules) on the spore surface are found in most of the members of the Aglaomorpha clade, as well as in various species of the three Drynaria clades (Tryon & Lugardon, 1990; van Uffelen, 1993). The absence of specialized litter collectors is unusual for drynarioid ferns in general but can be found in selected species of both Drynaria (e.g. D. parishii) and Aglaomorpha (e.g. A. acuminata). Chlorophyllous spores as found in Christiopteris are a unique character within the drynarioids, but this character has evolved several times independently within the Polypodiaceae (Schneider, unpublished results). The inclusion of Christiopteris within the drynarioids did not provide new insights in the evolution of litter-collecting leaves in drynarioids because phylogenetic reconstructions found no or only weak evidence for this taxon to be sister to all other drynarioids. Instead, the leaf dimorphism found in Christiopteris is likely the result of an evolutionary reversal. The rather short branches at the base of the extant drynarioids and the long branch leading to the extant drynarioid crown may suggest a long isolation of the lineage with a rather recent adaptive radiation in which the extant diversity of these ferns was established. Strong modifications of leaf morphology, especially the differentiation of leaf types, are very likely connected to the proposed adaptive radiation. This hypothesis requires further study based on estimates of divergence times and ecological niche reconstructions. CLASSIFICATION OF DRYNARIOID FERNS The currently accepted classification of the Drynaridae with three genera (Aglaomorpha, Christiopteris and Drynaria) is not in concordance with the recovered phylogenetic relationships (Roos, 1985; Janssen & Schneider, 2005). The first two genera, Aglaomorpha and Christiopteris appear to be natural units, whereas the genus Drynaria is paraphyletic as long as the other two genera are accepted. Rejection of paraphyletic taxonomic units is a generally accepted imperative and two putative solutions are discussed here (Ebach, Williams & Morrone, 2006). One solution includes the acceptance of a single genus including all species of drynarioid ferns. Based on the rule of priority, the genus name Aglaomorpha Schott (published in 1836) would have to be preferred over the names Drynaria J.Sm. (published in 1841) and Christiopteris Copel. (published in 1905). This concept has the major disadvantage of circumscribing a morphologically highly disparate assemblage. Especially the two species of Christiopteris do not look like anything in this clade. However, support for this classification comes from reports on successfully

7 PHYLOGENY OF THE FERN GENUS CHRISTIOPTERIS 651 hybridization of species belonging to the Aglaomorpha clade (A. coronans) and core Drynaria clade (D. rigidula) (Hoshizaki, 1991). The alternative classification results in the acceptance of up to four genera. Within this scheme, the first genus corresponds to Christiopteris as currently defined including two species (C. sagitta and C. tricuspis), whereas the second genus Aglaomorpha would have to be extended to include not just the currently accepted member species A. acuminata, A. brooksii, A. cornucopia, A. coronans, A. drynarioides, A. heraclea, A. hieronymi, A. latipinna, A. leporella, A. nectarifera, A. meyeniana, A. novoguineensis, A. parkinsoni, A. pilosa and A. splendens, but also four species currently assigned to Drynaria: D. delavayi, D. fortunei, D. mollis and D. sinica (Janssen & Schneider, 2005). The third genus would correspond to the core component of Drynaria comprising D. bonii, D. descensa, D. involuta, D. quercifolia, D. rigidula and D. sparsisora (Janssen & Schneider, 2005). The fourth genus lacks an existing taxon name but would likely correspond with the Afro-Madagascan clade comprising D. laurentii, D. volkensii and D. willdenowii. Unfortunately, the current sampling for DNA evidence does not include eight species assigned either to Aglaomorpha or Drynaria. In summary, 22% of the 32 known species of drynarioids are not included in the molecular systematic studies and thus taxonomic conclusions appear premature. Five out of the eight missing taxa can be easily assigned to a clade based on their morphological similarities, e.g. A. brooksii and A. nectarifera to the Aglaomorpha clade, D. delavayi and D. sinica to the northern Drynaria grade and D. involuta to the core Drynaria clade (Janssen & Schneider, 2005). However, the relationships of D. parishii, D. pleuridioides and D. propinqua require investigation before any assignment can be made with confidence. Morphological similarities suggest that D. pleuridioides has close relationships to the Afro-Madagascan clade (Roos, 1985; Janssen & Schneider, 2005), but this is in conflict with the distribution data because this species does not occur in the range of the Afro-Madagascan clade but in the Malesian region (Sumatra, Java, Lesser Sunda Islands, Sulawesi, Ambon), which is more likely within the range of the core Drynaria clade. The other two species of unclear relationships, D. parishii and D. propinqua, may be members of the core Drynaria clade (Janssen & Schneider, 2005) but this requires support by genomic data. BIOGEOGRAPHICAL IMPLICATIONS The placement of Christiopteris in the drynarioid ferns enhances existing evidence for a diversity centre and putative area of origin of drynarioid ferns and selligueoid ferns in the south-eastern continental Asia plus the most western parts of the Malesian region (Table 3). Both lineages, drynarioids and selligueoids, show a similar distribution range throughout the subtropical to tropical continental Asia, Malesia and Australasia, with the exception of the Afro- Madagascan clade of Drynaria which is the only representative of these ferns outside the Asian Australasian regions (Roos, 1985; Rödl-Linder, 1994; Hovenkamp, 1998; Janssen & Schneider, 2005; Janssen, Kreier & Schneider, 2007). The shared range suggests a similar biogeographical history of both lineages. This hypothesis is supported by the position of Eastern Malesian and Pacific island endemics in the derived clades of drynarioids. This pattern is particularly pronounced in the drynarioid ferns, with the New Guinea endemics nested exclusively within the core Aglaomorpha clade (Table 3; Janssen & Schneider, 2005). In other clades of drynarioids, species with relatively restricted ranges are endemic to continental Asia, Western Malesia or the Philippines. As an example, the core Drynaria clade comprises several species, e.g. D. quercifolia, D. rigidula and D. sparsisora, with large distribution ranges, but other species, e.g. D. bonii, D. descensa, are endemic to Indochina and areas of Western Malesia such as Borneo and the Philippines (Table 3; Roos, 1985; Janssen & Schneider, 2005). The ranges of the widespread species of the core Drynaria clade may be the result of secondary range expansion, but this requires confirmation by conducting phylogeographical studies of these species. The large ranges of some species in the core Drynaria clade contrast with the tendency of other clades to comprise species with smaller more distinct ranges. A remarkable differentiation is found in the Aglaomorpha plus northern Drynaria grade. Species of the northern Drynaria grade and the clades of Aglaomorpha, which do not belong to core Aglaomorpha, occur in continental Asia or the Philippines, whereas species belonging to the core Aglaomorpha clade occur mainly or exclusively in areas of the Malay Archipelago, preferably in Central and Eastern Malesia. The phylogenetic pattern suggests a range expansion for the Aglaomorpha clade from continental Asia via the Philippines to Eastern Malesia. Future research may be focused on the hypothesis that these putative range expansions in the Aglaomorpha and core Drynaria clade are correlated with the formation of the Malay Archipelago as the result of the collision between the Australian craton and SE Asia in the Miocene (Ridder-Numan, 1996; Hall, 1998). ACKNOWLEDGEMENTS This investigation was supported by the German Science Foundation (DFG) grant to HS (SCHN758/2)

8 652 H. SCHNEIDER ET AL. Table 3. Distribution ranges of drynarioid ferns sorted according to recovered lineages (Fig. 1) Taxa TA MA HM CH IN WM PH CM NG AP Afro-Mada. Drynaria D. laurentii E D. volkensii E D. wildenowii E Core Drynaria D. bonii (E) () D. descensa D. quercifolia () D. rigidula D. sparsisora Christiopteris C. sagitta E C. tricuspis (E) () Northern Drynaria D. fortunei E D. mollis E Basal Aglaomorpha A. coronans A. meyeniana () (E) Core Aglaomorpha A. acuminata A. cornucopia E A. drynarioides A. heraclea A. hieronymi E A. latipinna E A. novoguineensis E A. parkinsoni E A. pilosa A. splendens E Information obtained from Roos (1985), Hennipman et al. (1998), Hovenkamp & Roos (1998a, b). Abbreviations: AP, Australia and Pacific islands to the South of Solomon islands; CH, China (including Taiwan); CM, Sulawesi Moluccas; HM, Himalaya; IN, Indochina; MA, Madagascar; NG, New Guinea (including New Hebrides, Solomon Islands); PH, Philippines; PI, Pacific Islands (including Australia); TA, Tropical Africa; WM, Western Malesia. Distribution attributes:, widespread occurrence in this area; (), only one of few locations at the fringe of this area; E, endemic to this area; (E), nearly endemic to this area. A. leporella was excluded because its natural occurrence is unknown (Roos, 1985). as part of the Schwerpunkt programme (SPP 1127) Radiations origin of biological diversity. We thank Tom Ranker and an anonymous reviewer for comments on this study. REFERENCES Bryant D, Moulton V NeighborNet: an agglomerative method for the construction of planar phylogenetic networks. In: Guig PR, Gisfield D, eds. Algorithms in bioinformatics, WABI 2002 volume LNCS Heidelberg: Springer Verlag, Ebach MC, Williams DM, Morrone JJ Paraphyly is bad taxonomy. Taxon 55: Guindon GJ, Gascuel P A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52: Hall R The plate tectonics of Cenozoic SE Asia and the distribution of land and sea. In: Hall R, Holloway JD, eds. Biogeography and geological evolution of SE Asia. Leiden: Backhuys Publishers, Haufler CH, Grammer WA, Hennipman E, Ranker TA, Smith AR, Schneider H Systematics of the ant-fern genus Lecanopteris (Polypodiaceae): testing phylogenetic hypothesis with DNA sequences. Systematic Botany 28: Hennipman E, Hetterscheid WLA The emendation of the fern genus Christiopteris, including the transference of two taxa to micosoroid Polypodiaceae. Botanische

9 PHYLOGENY OF THE FERN GENUS CHRISTIOPTERIS 653 Jahrbücher für Systematik, Pflanzengeschichte und Pflanzengeographie 105: Hennipman E, Hovenkamp PH, Hetterschied WLA Christiopteris. Flora Malesiana Series II 3: Hennipman E, Veldhoen P, Kramer KU Polypodiaceae. In: Kubitzki K, ed. Families and genera of vascular plants vol. I: pteridophytes and gymnosperms (Kramer KU, Green PS, vol. eds). Heidelberg: Springer Verlag, Hoshizaki BJ An intergenic hybrid: Aglaomorpha Drynaria. American Fern Journal 81: Hovenkamp P The inevitable instability of generic circumscriptions in Old World Polypodiaceae. In: Camus JM, Gibby M, Johns RJ, eds. Pteridology in perspective. Kew: Royal Botanic Gardens Kew, Hovenkamp P An account of the Malay-Pacific species of Selliguea. Blumea 43: Hovenkamp P, Roos MC. 1998a. Aglaomorpha. Flora Malesiana Series II 3: Hovenkamp P, Roos MC. 1998b. Drynaria. Flora Malesiana Series II 3: Huelsenbeck JP, Ronquist F MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17: Huson DH, Bryant D Application of phylogenetic networks in evolutionary studies. Molecular Biology and Evolution 23: Janssen T, Kreier H-P, Schneider H Origin and diversification of African ferns with special emphasis on Polypodiaceae. Brittonia 59: Janssen T, Schneider H Exploring the evolution of humus collecting leaves in drynarioid ferns (Polypodiacae, Polypodiidae). Plant Systematics and Evolution 252: Kishino H, Hasegawa M Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Honionoidea. Journal of Molecular Evolution 29: Lockhart PJ, Steel MA, Hendy MD, Penny D Recovering evolutionary trees under a more realistic model of sequence evolution. Molecular Biology and Evolution 11: Maddison DR, Maddison WP Macclade Sunderland, MA: Sinauer Associates. Nylander JA MrAIC. Pl Program distributed by the author. Uppsala, Sweden: Evolutionary Biology Centre, Uppsala University. Available at ~nylander/mraic/mraic.html/ Posada D, Crandall KA Modeltest: testing the model of DNA substitution. Bioinformatics 14: Rambaut A FigTree version 1.0. Computer program distributed by the author. Oxford, UK: Department of Zoology, University of Oxford. Available at evolve.zoo.ox.ac.uk/software.html?id=figtree Rambaut A, Drummond A Tracer, version 1.3. Computer program distributed by the authors. Oxford, UK: Department of Zoology, University of Oxford. Available at Ridder-Numan JWA The historical biogeography of Southeast Asian genus Spatholobus (Leguminosae, Papilionideae) and its allies. Blumea Supplement 10: Rödl-Linder G Revision of the fern genus Polypodiopteris (Polypodiaceae). Blumea 39: Roos MC Phylogenetic systematics of the Drynrioideae (Polypodiaceae). Verhandelingen der Koninklijke Akademie van Wetenschappen. Afdeeling Natuurkunde, Tweede Sectie 85: Schneider H, Janssen T, Hovenkamp P, Smith AR, Cranfill R, Haufler CH, Ranker TA. 2004a. Phylogenetic relationships of the enigmatic Malesian fern Thylacopteris (Polypodiaceae, Polypodiidae). International Journal of Plant Sciences 165: Schneider H, Kreier H-P, Wilson R, Smith AR The Synammia enigma: evidence for a temperate lineage of polygrammoid ferns (Polypodiaceaem Polypodiidae) in southern South America. Systematic Botany 31: Schneider H, Smith AR, Cranfill R, Haufler CH, Ranker T, Hildebrand T Gymnogrammitis dareiformis is a polygrammoid fern (Polypodiaceae) resolving an apparent conflict between morphological and molecular data. Plant Systematics and Evolution 234: Schneider H, Smith AR, Cranfill R, Hilfebrand TJ, Haufler CH, Ranker TA. 2004b. Unraveling the phylogeny of polygrammoid ferns (Polypodiaceae & Grammitidaceae): exploring aspects of the diversification of epiphytic plants. Molecular Phylogenetics and Evolution 31: Shimodaira H, Hasegawa M Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Molecular Biology and Evolution 16: Smith AR, Pryer KM, Schuettpelz E, Korall P, Schneider H, Wolf PG A classification for extant ferns. Taxon 55: Swofford DL PAUP* phylogenetic analysis using parsimony (*and other methods). Sunderland, MA: Sinauer Associates. Templeton AR Phylogenetic inference from restriction endonuclease cleavage site maps with particular reference to humans and apes. Evolution 37: Tryon AF, Lugardon B Spores of pteridophytes. Heidelberg: Springer Verlag. van Uffelen GA Sporogenesis in Polypodiacae (Filicales) III. Species of several genera, spore characters and their value in phylogenetic analyses. Blumea 37: Zwickl DJ Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion. PhD Thesis, Austin, TX: University of Texas at Austin.

10 654 H. SCHNEIDER ET AL. APPENDIX I Information vouchers and GenBank accession numbers of taxa included in this study. Acquired gene regions are given in the sequence rbcl/rps4 REG/trnL-F IGS with rps4 REG corresponding to rps4 pls rps4-trns (GGA) IGS and trnl-f IGS corresponding to trnl (CAA)-trnF (GAA) IGS. Cultivated material was obtained either from various Botanical Gardens [Botanic Garden Berlin Dahlem (B), Botanic Garden Munich (M), Botanic Garden of the University Göttingen (GOET), Botanic Garden of the University Heidelberg (HEID), Botanic Garden of the University Tübingen (TUEB), Hortus Botanicus Leiden (HBL), New York Botanic Garden (NY), Royal Botanic Gardens Edinburgh (RBGE), SELBY Botanic Garden (SEL), University of California Botanic Gardens (UCBG), University of Utrecht Botanic Garden (UTR)] or from Charles Alford (CAG) who is a commercial fern grower in Florida (USA). If possible, the accession number of the garden is given. Abbreviations of herbaria used to deposit vouchers are given as on Index Herbariorum ( Taxon Origin Voucher GenBank accession number Drynarioids Aglaomorpha acuminata Cult. HEID Igersheim s.n. (HEID) AY529147/AY529172/AY (Willd.) Hovenkamp Aglaomorpha cornucopia Cult. M Janssen 2255 (GOET) AY529148/AY529173/AY (Copel.) M.C.Roos Aglaomorpha coronans Cult. HEID Igersheim s.n. (HEID) AF470349/AY459184/AY (Mett.) Copel. Aglaomorpha drynarioides Cult. B Janssen 2256 (GOET) AY529149/AY529174/AY (Hook.) M.C.Roos Aglaomorpha heraclea Cult. GOET Janssen 2249 (GOET) AY529150/AY529175/AY (Kunze) Copel. Aglaomorpha hieronymi Cult. HEID Hagemann 2601 (HEID) AY529151/AY529176/AY (Brause) Copel. Aglaomorpha latipinna Irian Jaya Mangen 2230 (L.) / /AY (C.Chr.) M.C.Roos Aglaomorpha leporella Cult. HEID Janssen 2253 (GOET) AY529152/AY529177/AY (Goebel) C.Chr. Aglaomorpha meyeniana Cutl. GOET Janssen 2260 (GOET) AY529153/AY459185/AY52940 Schott Aglaomorpha Cult. B Janssen 2254 (GOET) AY529154/AY529178/AY novoguineensis (Brause) C.Chr. Aglaomorpha parkinsoni Cult. GOET Janssen 2259 (GOET) AY529155/AY529179/AY (Baker) Parris & M.C. Roos Aglaomorpha pilosa Cult. B Janssen 2258 (GOET) AY529156/AY529180/AY (Hook. & Bauer) Copel. Aglaomorpha splendens Cult. CAG Smith s.n. (UC) AY529157/AY529181/AY (Hook. & Bauer) Copel. Aglaomorpha spec. Cult. CAG Smith s.n. (UC) EU128497/EU128504/EU Christiopteris sagitta Cult. HBL Hovenkamp s.n. (L.) EU128498/EU128505/EU (H.Christ) Copel. Christiopteris sagitta Cult. B Schuettpelz 613 (B, GOET) / /EU (H.Christ) Copel. Christiopteris tricuspis Malay Peninsula Mahmud Sider s.n (L.) / /EU (Hook.) H.Christ Drynaria bonii H.Christ Cult. B Janssen 2248 (GOET) AY529158/AY529182/AY Drynaria descensa Copel. Cult. HEID (GOET) AY529159/AY529183/AY Drynaria fortunei (Kunze Cult. RBGE Sino-British Cangshan Exp. EU128499/EU128506/EU ex Mett.) J.Sm. 715 (E) Drynaria laurentii (de Wild. & T. Durand) Hieron. Cult. SEL Smith s.n. (UC) AY529161/AY529185/AY529478

11 PHYLOGENY OF THE FERN GENUS CHRISTIOPTERIS 655 APPENDIX I Continued Taxon Origin Voucher GenBank accession number Drynaria mollis Bedd. Cult. GOET Janssen 2257 (GOET) AY529162/AY529186/AY Drynaria quercifolia (L.) Cult. GOET Janssen 2247 (GOET) AY529165/AY529187/AY J. Sm. Drynaria rigidula Bedd. Cult. B Janssen 2251 (GOET) AF470339/AY096221/AY Drynaria sparsisora Cult. TUEB Janssen 2246 (GOET) AY529167/AY529189/AY (Desv.) T. Moore Drynaria volkensii Hieron. Tanzania Hemp 3635 (UTB) AY529168/AY529190/ Drynaria willdenowii Cult. B Janssen 2250 (GOET) AY529169/AY529191/AY (Bory.) T. Moore Selligueoids Arthromeris lehmannii Taiwán Cranfill 696 (UC) AY096198/AY096216/AY (Mett.) Ching Arthromeris wallichiana Cult. RBGE Kirkpatrick 90/283 (E) EU128500/EU128507/EU (Spreng.) Ching Gymnogrammitis Nepal, India KEKE 1158 (K) AY096201/AY096219/ dareiformis (Hook.) Ching Tsutsumi 1043 (T) / /EU Polypodiopteris Borneo Kessler (UC) AY362557/AY362621/ brachypoda (Blume) Parris Selliguea enervis (Cav.) Java Wilson 2893 (UC) AY096200/AY096218/AY Ching Selliguea feei Bory Java Wilson 2862 (UC) AY096199/AY096217/AY Selliguea hellwigii (Diels) Cult. HBL Hovenkamp s.n. (L.) EU128501/EU128508/EU Hovenkamp Selliguea heterocarpa Malay Peninsula Jaman 5897 (UC) AY459172/AY362619/AY (Blume) Blume Selliguea laciniata (Bedd.) Malay Peninsula Jaman 5894 (UC) AY529271/AY529193/AY Hovenkamp Selliguea lanceola (Mett.) New Caledonia Munzinger 1253 (P) AY459173/AY459186/AY E.Fourn. Selliguea lateritia (Baker) Cult. HBL Hovenkamp s.n. (L.) EU128502/EU128509/EU Hovenkamp Selliguea plantaginea Tahiti Ranker 1897 (COLO) EU128503/EU128510/EU Brackenr. Loxogrammoids Dictymia mackeei Tindale Cult. RBGE Page (L.) DQ227292/DQ227295/ DQ Loxogramme abyssinica (Baker) M.G. Price Tanzania Hemp 3638 (DSM) DQ164443/DQ164474/ DQ Microsoroids Goniophlebium Cult. UCBG Smith s.n. (UC) AF470342/ /AY subauriculatum (Blume) C.Presl Cult. GOET Kreier s.n. (GOET) /DQ168812/ Lecanopteris sinuosa Cult. UTR Hennipman 7821 (L.) AF470321/AY362634/AY (Hook.) Copel. Lemmaphyllum carnosum Cult. UCBG Smith s.n. (UC) AF470332/AY362631/AY (Hook.) C. Presl Leptochilus macrophyllus (Blume) Noot. Cult. UCBG Craig s.n. (UC) AF470340/AY362639/AY083643

12 656 H. SCHNEIDER ET AL. APPENDIX I Continued Taxon Origin Voucher GenBank accession number Microsorum cuspidatum (D. Don) Tagawa Microsorum linguiforme (Mett.) Copel. Microsorum musifolium (Blume) Copel. Thylacopteris papillosa (Blume) J.Sm. Neotropical Clade Campyloneurum angustifolium (Sw.) Fée Campyloneurum chlorolepis Alston Microgramma percussa (Cav.) de la Sota Pleopeltis angusta Humb. & Bonpl. ex Willd. Pleopeltis polypodioides (L.) E.G.Andrews & Windham Polypodium pellucidum Kaulf. Cult. HBL no voucher AF470335/ /AY Cult. NY 470/77 Smith (UC) /AY096230/ New Guinea Ranker 1176 (UC) AF470334/ /AY Cult. HEID Hagemann s.n. (HEID) /AY362635/ Cult. UCBG U Smith s.n. (UC) AF470335/ /AY Cult. HEID Hagemann s.n. (UC) /AY362636/ Java Gravendel et al. 559 (L.) AY459174/AY459188/AY Cult. UCBG S1 Chisaki & Carter AF470344/AY362645/AY (UC) Cult. UCBG Smith 1159 (UC) AF470345/AY362646/AY Cult. GOET Schwertfeger s.n. (GOET) DQ642149/DQ642187/ DQ Cult. UCBG Wagner s.n. (UC) AY362590/AY362664/AF Guatemala León & Morales 3762 (UC) AY362592/AY362665/AF Hawai i Li et al. s.n. (KANU) U21149/ /AF Cult. UCBG Smith s.n. (UC) /AY096234/ Polypodium vulgare L. Cult. GOET Schwertfeger s.n. (GOET) EF551065/EF551081/EF Serpocaulon ptilorhizon (H. Christ) A. R. Sm. Bolivia Jimenez 1102 (UC) DQ151920/DQ151945/ DQ Platycerioids Platycerium coronarium (König ex Müller) Desv. Cult. GOET Kreier CG0404 (GOET) DQ164448/DQ164479/ DQ Pyrrosia polydactyla (Hance) Ching Cult. GOET Schneider s.n. (GOET) DQ164470/DQ164502/DQ164534