Int. J. Plant Sci. 179(1):36–49. 2018. q 2017 by The University of Chicago. All rights reserved. 1058-5893/2018/17901-0003$15.00 DOI: 10.1086/694764 PHYLOGENY OF CAMPYLONEURUM (POLYPODIACEAE) Paulo H. Labiak1,* and Robbin C. Moran† *Universidade Federal do Paraná, Departamento de Botânica, C.P. 19031, 81531-980 Curitiba, Paraná, Brazil; and †New York Botanical Garden, 2900 Southern Boulevard, Bronx, New York 10458-5126, USA Editor: Maria von Balthazar Premise of research. Campyloneurum is entirely Neotropical with about 60 generally accepted species. Although a prominent, mostly epiphytic genus in Neotropical forests, no research has been done previously on its phylogeny or character evolution. Methodology. We obtained sequence data from four plastid markers (rbcL, trnG-trnR, trnL-trnF, and rps4trnS) of 44 species of Campyloneurum, or 73% of the species in the genus. The sequence data were analyzed using Bayesian and maximum likelihood methods. Herbarium specimens were examined to code morphological characters, and spores were imaged with the scanning electron microscope. Growth form and five morphological character states were optimized on the trees using parsimony. Pivotal results. We confirm most previous studies that Campyloneurum forms a clade with Microgramma and Niphidium. The three genera are generally characterized by simple entire leaves, areolate venation, and included veins within the areoles. Within Campyloneurum, seven main clades were recovered, some of which were defined by distinctive geography or morphological characters such as pruinose rhizomes, long-creeping rhizomes, or one-pinnate leaves. The spores were uniform and did not provide any grouping information. The genus has its highest endemism and diversity in the Andes, with the mountains of Costa Rica and Panama being a secondary center. A map is given showing the estimated species richness and percent endemism of Campyloneurum in different geographical regions of the Neotropics. Conclusions. This study presents the first phylogenetic analysis of Campyloneurum. The genus forms a clade with Microgramma and Niphidium. Within the genus, seven main clades were recognized, some of which are supported by distinctive morphological synapomorphies. A clade of five species is endemic to southeastern Brazil. Our tree suggests that many undescribed species occur in the genus, indicating the need for more taxonomic studies at the species level. Keywords: ferns, Microgramma, Neotropics, Niphidium, systematics. Online enhancement: appendix figure. Introduction found to be nested in Campyloneurum (Kreier et al. 2007). Thus, the total number of species generally recognized now stands at about 60. Campyloneurum is morphologically simple, characterized by entire leaves and prominent lateral veins running from the midrib to the margin and cross-connected by finer veins to create a series of areoles. The costal aereoles contain a single included veinlet, but the noncostular ones contain (depending on the species) two to five free veinlets (fig. 1A). When fertile, the sori are born at the apices of these veinlets. Exceptions to simple leaves and this general venation pattern exist. For instance, three species have one-pinnate laminae, not entire ones. These species form a clade and have been recognized formally as C. subgen. Decurrentia R. C. Moran and Labiak (Moran and Labiak 2016). In some species the laminae are narrowed to only 1–2 cm wide, and they present only one or two areoles between the costa and the margin, typically with a single included veinlet. Campyloneurum is a Neotropical fern genus distributed from southern Florida to southern Brazil and northern Argentina (Tryon and Tryon 1982; Lellinger 1988). It occurs in almost all terrestrial ecosystems, from sea level up to ca. 3500 m. Most of its species are epiphytic, but some are epipetric or hemipepiphytic (Labiak et al. 2017). Lellinger (1988) recognized 50 species of Campyloneurum. Since then, 10 additional species have been described (León 1995, 2004; Rojas 2005, 2017; León and Kessler 2013; Moran and Labiak 2016; Labiak et al. 2017). The monotypic Hyalotrichopteris, once recognized as distinct from Campyloneurum by Wagner and Farrar (1976) and Lellinger (1988), has now been 1 Author for correspondence; e-mail: plabiak@ufpr.br. Manuscript received June 2017; revised manuscript received September 2017; electronically published December 8, 2017. 36 LABIAK & MORAN—PHYLOGENY OF CAMPYLONEURUM 37 Fig. 1 Two characters and their states scored for Campyloneurum. A, Lateral veins prominent (C. atlanticum). B, Lateral veins not prominent (C. pittieri). C, Rhizomes pruinose (C. pittieri). D, Rhizomes not pruinose (C. brevifolium). Scale bars p 1 cm. Campyloneurum belongs to the Polypodiaceae (PPG I 2016), and most phylogenetic studies have revealed that it forms a clade with Microgramma C. Presl and Niphidium J. Sm. (Schneider et al. 2004; Kreier et al. 2007; Schuettpelz and Pryer 2007; Salino et al. 2008). Although the clade is strongly supported, relationships between the three genera are poorly supported. Schneider et al. (2004), Kreier et al. (2007), and Salino et al. (2008) recovered it as sister to Niphidium, whereas Schuettpelz and Pryer (2007) recovered it as sister to Microgramma. The close relationship of these three genera is also supported by morphology. They share simple entire leaves and anastomosing veins with included veinlets, although the patterns of the anastomosing veins differ for each genera (see “Discussion”). Sundue et al. (2015) recovered yet a third topology, with Campyloneurum as sister to a large clade composed of Microgramma, Niphidium, Pleopeltis, Pecluma, Polypodium, Phlebodium, and Pleurosoriopsis. Support values were not reported in this study, though. Although divergence time estimates for the origin of the crown Polypodiaceae are still controversial—with estimates varying from 55 Ma, during the Paleocene-Eocene (Schuettpelz and Pryer 2009), to 90 Ma, in the late Creataceous (Testo and Sundue 2016)—it is likely that most of the family’s lineages have diversified as epiphytes during the Oligocene and Miocene (33–5.3 Ma), a period where tropical forests were already dominated by angiosperms (Schneider et al. 2004; Schuettpelz and Pryer 2009). This seems to be the case for Campyloneurum, for which the estimates suggest an origin around 36.2 Ma (Schuettpelz and Pryer 2009) to 40.9 Ma (Testo and Sundue 2016). Campyloneurum is known from a Tertiary (lower Oligocene) fossil collected in northern Italy and described as “Polypodium (Campyloneurum) morellii Sqinabol” (Squinabol 1889–1891). The fossil is one-pinnate and has venation typical of the genus (see Squinabol’s illustration of the fossil reproduced in Moran and Labiak 2017). Because the fossil is one-pinnate, we assign it to subgen. Decurrentia. The fossil was collected with other primarily tropical fern genera, such as Goniopteris, Hymenophyllum, Hypolepis, Lygodium, and Pteris (Bonci et al. 2011). A paleoclimatic study based on foliar physiognomy concluded that the fossil site was “an alluvial plain with flooded areas, meanders and small lakes, located within the tropical basal and premontane belt” (Bonci et al. 2011, p. 145). Because of its simple morphology, the species-level taxonomy of Campyloneurum has proved challenging. Similar species must often be distinguished primarily on the basis of rhizome scales, and the characters of these scales are often subtle and variable, such as color, habit, and shape of the cells. The genus lacks potentially informative characters, such as the presence or absence of indusia and paraphyses, different types of laminar scales, and erect rhizomes (all rhizomes in the genus are creeping). Only two species (C. aphanophlebium (Kunze) T. Moore and C. anetioides (Christ) R. M. Tryon and A. F. Tryon) have 38 INTERNATIONAL JOURNAL OF PLANT SCIENCES hairs; thus, characters pertaining to pubescence, such as color, density, distribution, and number of cells, are lacking for other species in the genus. No phylogenetic work at the species level has been done previously on Campylonerum. The purpose of this study is to produce a phylogenetic tree to understand the relationships between species and clades within the genus and to use this tree to examine character evolution and biogeography. Material and Methods Taxon Sampling Because of the conflicting results on the phylogenetic placement of Campyloneurum, we selected outgroup genera belonging to the Polypodiaceae that had been previously recovered as relatives of Campyloneurum (Schneider et al. 2004; Kreier et al. 2007; Schuettpelz and Pryer 2007; Salino et al. 2008; Sundue et al. 2015; Testo and Sundue 2016). These genera are Aglaomorpha Schott (1 sp.), Arthromeris (T. Moore) J. Sm. (1 sp.), Ascogrammitis Sundue (1 sp.), Ceradenia L.E. Bishop (1 sp.), Drynaria (Bory) J. Sm. (1 sp.), Leucotrichum Labiak (1 sp.), Microsorum Link (1 sp.), Melpomene A.R. Sm. & R.C. Moran (1 sp.), Microgramma C. Presl (5 spp.), Niphidium J. Sm. (4 spp.), Pecluma (2 spp.), Phlebodium J. Sm. (1 sp.), Platycerium Desv. (1 sp.), Pleopeltis Humb & Bonpl. ex Willd. (2 spp.), Pleurosoriopsis Fomin (1 sp.), Polypodium (1 sp.), Selliguea Bory (1 sp.), Serpocaulon A.R. Sm. (3 spp.), and Stenogrammitis Labiak (1 sp.). Besides these Polypodiaceae genera, we also included one species of Davallia. As for the ingroup, we obtained 124 specimens representing 44 species of the 60 generally accepted species of Campyloneurum (ca. 73% of the genus diversity). Fifteen samples are undetermined, and they are labeled as “sp.” and numbered in the tree (fig. 2). For most outgroups, we used sequences available on GenBank. We used only our own sequences of Campyloneurum for the analysis because of the time and expense involved in examining other vouchers, as well as to avoid missing data for some markers. DNA Extraction Total genomic DNA was extracted from silica-dried material using standard protocols of the Qiagen DNeasy Plant Mini Kit (Valencia, CA). For herbarium samples the protocols were basically the same, with an additional step incubating the samples at 427C for 12 h with the lysis buffer. Amplifications and Sequencing Our analysis includes four plastid markers: rbcL, trnG-trnR, trnL-trnF, and rps4-trnS. Amplifications were made by PCR in 15-mL reactions using 0.4 mL of nondiluted genomic DNA, 7.5 µL of GoTaq Green Master Mix (Promega), 0.8 mL of 5M betaine solution (Q-solution), 2.5 mL of 2.5 mg/mL BSA solution, and 2 mL of each primer at 3 mM. For rbcL we used the primers “ESRBCL1F” and “ESRBCL1361R” (Schuettpelz and Pryer 2007), for rps4-trnS the primers “rps4–3r.f” (Skog et al. 2004) and “trnSr” (Souza-Chies et al. 1997), for trnG-trnR the primers “TRNG1F” and “TRNR22R” (Nagalingum et al. 2007), and for trnL-trnF the primers “e” and “f,” designed by Taberlet et al. (1991). For rbcL and trnG-trnR we used a PCR program with an initial denaturation step of 5 min at 947C, followed by 35 cycles of 1 min at 947C, 1 min at 507C, 2.5 min at 727C, and a final extension period of 10 min at 727C. For rps4-trnS and trnL-trnF, we used one initial denaturation step of 5 min at 947C, followed by 35 cycles of 1 min at 947C, 30 s at 507C, 1 min at 727C, and a final extension period of 7 min at 727C. The PCR products were checked on a 1% agarose gel with ethidium bromide. We used the same amplification primers for the sequencing process, adding the internal primers “ESRBCL628F” and “ESRBCL654R” for rbcL (Schuettpelz and Pryer 2007) and “43F1” and “63R” for trnG-trnR (Nagalingum et al. 2007). All PCR products were sequenced by Macrogen-USA. The resulting sequences were assembled using Geneious 9 (Biomatters, Auckland, NZ). Newly obtained consensus sequences were submitted to GenBank. Voucher information and GenBank accession numbers are listed in appendix A. Alignment and Phylogenetic Analyses Consensus sequences were automatically aligned using MUSCLE (Edgar 2004), as implemented in Geneious 9R. The alignments were visually inspected, and regions of uncertain homology were identified and trimmed for the phylogenetic analysis. The best evolutionary model was calculated using jModeltest2 (Guindon and Gascuel 2003; Darriba et al. 2012), under the Bayesian information criterion. We used Mesquite v3.2 (Maddison and Maddison 2017) to construct the data matrices. Phylogenetic analyses were conducted under Bayesian inference (BI) and maximum likelihood (ML) methods. The BI analyses were performed using MrBayes 3.2.6 in CIPRES (Miller et al. 2010). Parameters were set to two runs and four chains each (three heated and one cold), with 20 million generations, sampling every one-thousandth generation. We applied the models suggested by jModeltest2 for each marker, with parameters unlinked. To test whether Markov chain Monte Carlo (MCMC) reached stationarity, we examined the log-likelihood (lnL) plots using Tracer 1.6 (Rambaut and Drummond 2013). Convergence was also examined using AWTY (Wilgenbusch et al. 2004). Branch support was obtained from the posterior probability (PP) values from the MCMC analysis, after discarding the first 20% of the samples as burn-in. The ML analyses were performed using RAxML v8.1.17 (Stamatakis 2014), applying the models of evolution to each marker. Branch support was estimated using 1000 bootstrap replicates (ML-BS). We conducted two analyses using a different number of species in each one. In the first, we included only one specimen per species, and in the second, we included all the samples sequenced, which included duplicates of the same species. The results were the same for the main relationships within Campyloneurum. For simplicity, we present in this article only the first (reduced) tree (fig. 2), and the second (enriched) tree in appendix B, available online. Morphological Data Six characters were scored for all taxa included in this study. Of these characters, five were morphological and one pertained to growth form (figs. 3, 4). All character data were generated Fig. 2 Cladogram from the Bayesian inference (BI) of the combined data set of the cpDNA markers rbcL, rps4-trnS, trnG-trnR, and trnL-trnF. Numbers on the branches represent the posterior probability (PP) values of the Bayesian inference and the bootstrap values of the maximum likelihood analysis (ML-BS). Thickened lines represent the clades with full support in both analyses. Support values bellow 0.7 of PP and 70% of ML-BS are not shown. An arrow indicates the clade corresponding to Campyloneurum, and the inset image represents the main clades that are discussed in the text. Fig. 3 Ancestral state reconstruction of growth form, rhizome habit, and rhizome epidermis for Campyloneurum and outgroups, using maximum parsimony. 40 Fig. 4 Ancestral state reconstruction of lamina division, lateral veins, and laminar hairs for Campyloneurum and outgroups, using maximum parsimony. 41 42 INTERNATIONAL JOURNAL OF PLANT SCIENCES from original observation of living material or herbarium specimens and those for spores from images taken with the scanning electron microscope (SEM). These spore images were generated using a JEOL JSM-5410LV SEM equipped with a JEOL Orion 5410 software interface, at the New York Botanical Garden. The spore images shown in this article, and others not shown, are available publicly at http://www.plantsystem atics.org. Morphological and spore character states were optimized onto the ML tree under a maximum parsimony criterion using Mesquite (Maddison and Maddison 2017). Geographical Data A map showing the number of species of Campyloneurum in different regions of high species richness and endemism was compiled from studies of herbarium specimens at MO, NY, and UPCB. The numbers represent our estimates and do not include the 15 undetermined species listed as “sp.” and numbered in figure 2. The shaded areas on the map are the regions of high species richness and endemism delineated by Tryon (1972); however, we modified Tryon’s Central America region. We recognize one region from Mexico to northern Nicaragua and another region consisting of Costa Rica and western Panama, the mountains of which are geographically isolated and known to contain high endemism and diversity (Moran 2008). Results In our results, Campyloneurum formed a clade with Microgramma and Niphidium (ML-BS p 51%; PP p 0.78; fig. 2). The first divergent lineage in our analysis is an Old World clade composed of Arthromeris, Selliguea, Drynaria, and Aglaomorpha (ML-BS p 81%; PP p 0.53). Microsorum is sister to Platycerium (ML-BS p 87%; PP p 0.98), Pecluma is sister to Pleopeltis (ML-BS p 80%; PP p 0.99), and Serpocaulon is sister to the grammitid clade (Ascogrammitis, Ceradenia, Leucotrichum, and Stenogrammitis; ML-BS p 70%; PP p 0.99). The next divergent lineage is Pleurosoriopsis, the relationships of which were not resolved by any method of analysis. Phlebodium and Polypodium formed a clade (ML-BS p 56%; PP p 0.96), which was recovered as sister to Niphidium, Microgramma, and Campyloneurum, but with very low support values in all analyses (ML-BS p 51%; PP p 0.78). Campyloneurum was resolved as monophyletic (fig. 2; MLBS p 74%; PP p 1.0) and sister to Microgramma (ML-BS p 74; PP p 0.99; fig. 2). Within Campyloneurum, seven main clades were recovered (fig. 2), most of them with high support. These clades were C. subgen. Decurrentia (ML-BS p 100%; PP p 1), C. rigidum (ML-BS p 100%; PP p 1), the southeastern Brazilian clade (ML-BS p 69%; PP p 0.98), the Phyllitidis clade (ML-BS p 99%; PP p 0.98), the Costatum clade (ML-BS p 90%; PP p 0.98), the Repens clade (ML-BS p 94%; PP p 1), and the Pruinose clade (ML-BS p 97%; PP p 1). The results of the morphological character optimizations (figs. 3, 4) revealed that most characters were homoplastic within Campyloneurum, such as rhizome habit, growth form, shape of cells in the rhizome scales, and prominence of lateral veins. Some characters defined clades, such as one-pinnate laminae, rhizome pruinosity, and laminar hairs. Discussion Outgroup genera. In our analysis, Campyloneurum formed a clade with Niphidium and Microgramma. Although this corroborates most previous results based on molecular data (Schneider et al. 2004; Kreier et al. 2007; Schuettpelz and Pryer 2007; Salino et al. 2008; Testo and Sundue 2016), support values were still low. Our results do not agree with those of Sundue and Testo (2015). They recovered Campyloneurum as sister to a clade consisting of Microgramma, Niphidium, Pecluma, Phlebodium, Pleopeltis, Pleurosoriopsis, and Polypodium. Because they did not cite support values for this clade and its internal branches, it is difficult to assess the finding. Morphologically, Campyloneurum, Microgramma, and Niphidium are alike by having simple entire leaves and anastomosing veins. What distinguishes them are patterns of venation and cell shape of the rhizome scales. Regarding venation, Microgramma differs most conspicuously from the others by lacking prominent main lateral veins between the costa and the margin (de la Sota and Pérez-García 1982). Niphidium has prominent main lateral veins as in Campyloneurum but differs by the pattern of fine reticulate veinlets between them, consisting of smaller areoles containing both excurrent and recurrent veinlets (Lellinger 1972). The sori are often supplied by two or more veinlets (Lellinger 1972). In contrast, Campyloneurum typically has prominent lateral veins that are cross-connected by veinlets forming a series of well-defined areoles (fig. 1A). In most species, the areoles contain two excurrent veinlets, but in a few species, the areoles contain three or four (e.g., C. magnificum C. Presl, C. pascoense R. M. Tryon, and C. tucumanense (Hieron.) Ching). In Campyloneurum, the costal areoles differ from the others by containing only a single veinlet. When present, the sori are born at the apices of the included veinlets. On our tree (fig. 4), this venation pattern optimizes at the base of the Campyloneurum clade and is therefore considered a possible synapomorphy for the genus. It is the most common pattern in the genus. In the Pruinose clade, however, the venation complexity is often reduced in association with narrow (1–2-cmwide) laminae. In this clade, the main lateral veins are similar in width to the other veins. The shape of the cells in the rhizome scales is also a possible synapomorphy for Campyloneurum (figure not shown). They are isodiametric in the basal groups of the genus but elongated in the outgroup genera Microgramma and Niphidium. Spores. The spores are relatively uniform throughout the genus and do not provide characters to distinguish the main clades or species (fig. 5). All are monolete, fabiform, and (at least when fresh) yellow, as is typical for the nongrammitid Polypodiaceae (Tryon and Lugardon 1991). The spore surfaces vary from smooth to shallowly or prominently verrucate. The intergradations between these are continuous, and for that reason we did not optimize the characters on the tree. Irregularly overlying the spore surface are varying amounts of spherical deposits, or globules (Tryon and Tryon 1982; Tryon and Lugardon 1991). Besides the spore images in figure 5, other ones of subgen. Decurrentia and the southeastern Brazilian clade can be found, respectively, in Moran and Labiak (2017) and Labiak et al. (2017). LABIAK & MORAN—PHYLOGENY OF CAMPYLONEURUM 43 Fig. 5 Spores of some main clades of Campyloneurum. A, C. magnificum, subgen. Decurrentia clade (Colombia, Sanín et al. 5153, NY). B, C. atlanticum, southeastern Brazilian clade (Brazil, Vanni et al. 281, MO). C, C. ensifolium, Pruinose clade (Mexico, Gutierrez et al. 547, NY). D, C. xalapense, Costatum clade (Mexico, McVaugh 18998, NY). E, C. sphenodes, Repens clade (Ecuador, Clark et al. 5401, MO). F, C. angustifolium, Pruinose clade (Costa Rica, Mickel 3199, NY). Scale bars p 10 mm. When compared to the spores of Microgramma and Niphidium, those of Campyloneurum do not exhibit any distinctive characteristics. They are basically the same as those of Niphidium as depicted by Tryon and Tryon (1982) and Tryon and Lugardon (1991). Niphidium spores are smooth and bear irregular spherical deposits. The spores of some species of Microgramma, such as M. lycopodioides (Tryon and Lugardon 1991), are verrucate and resemble the spores found in Campyloneurum (e.g., fig. 5A). Microgramma, however, has some species with papillate spores and others with rugate ones (see M. reptans (Cav.) A. R. Sm. and M. megaphylla (Desv.) de la Sota; Tryon and Lugardon 1991). These two character states are not present in Campyloneurum. Main subclades of Campyloneurum. Campyloneurum subgen. Decurrentia is sister to the rest of the genus (fig. 2). It contains three species that were monographed by Moran and Labiak (2017). These species have one-pinnate leaves, a character state that represents a synapomorphy for the clade. It is the only evolution of one-pinnate leaves in the clade formed by Campyloneurum, Microgramma, and Niphidium (fig. 4). Campyloneurum rigidum clade. This species alone forms a clade sister to the southeastern Brazilian and Phyllitidis clades (fig. 2). It is endemic to southeastern Brazil and characterized by laminae so thick that the veins are hidden or obscured (Vasques and Prado 2011). Southeastern Brazilian clade. This is sister to the Phyllitidis clade (fig. 2). It contains five species, all endemic to southeastern Brazil, and all were included in our analysis. The species were described and illustrated for a treatment of Campyloneurum for the state of São Paulo, Brazil, by Vasques and Prado (2011) and Labiak et al. (2017). We know of no morphological synapomorphies that characterize the clade. Growth habit within the southeastern Brazilian clade varies (fig. 3). Two species, C. atlanticum R. C. Moran and Labiak and C. herbaceum (Christ) Ching, are hemiepiphytes, but C. crispum Fée is holoepiphytic (Labiak et al. 2017). As for the other species in the clade, C. fallax Fée is epiphytic and C. nitidum (Kaulf.) C. Presl may be epiphytic, saxicolous, or rarely terrestrial (P. H. Labiak, personal observation) Phyllitidis clade. This is sister to the southeastern Brazilian clade (figs. 2, 3). It contains about seven described species. The two most widespread ones (C. phyllitidis (L.) C. Presl and C. brevifolium (Lodd. ex Link) Link) are similar morphologically and in need of biosystematic revision. Tetraploids have been documented for C. phyllitidis (L.) C. Presl in Florida, Jamaica, Trinidad and Tobago, and the Galapagos (see review by Walker 44 INTERNATIONAL JOURNAL OF PLANT SCIENCES 1985). Although its species generally have short-creeping rhizomes and rhizome scales with isodiametric cells, we know of no morphological synapomorphies that characterize this clade. Two species of the Phyllitidis clade are slightly atypical within the clade. Campyloneurum nitidissimum (Mett.) Ching has hard black scales with acute apices and elongated cells. In contrast, the other species in the clade have brown rhizome scales with obtuse apices and isodiametric cells. Another species, C. cubense Fée, is atypical by long-creeping rhizomes (unlike the compact or short-creeping rhizomes found in other members of the clade), only two rows of areoles between the costa and the margin (as opposed to generally five to 12 in the others), and small laminae up to 35 cm long (generally 0.5– 1.5 m long in the other species). Costatum clade. This is sister to the Repens and Pruinose clades (fig. 2). In our analysis it contains three species, and these are similar morphologically. We do not know of a morphological synapomorphy for the clade. All three species have longdecurrent lamina bases and cuspidate apices. The group is primarily Central American. It is named for C. costatum (Kunze) C. Presl, the most widespread species in the clade, occurring in the Antilles and Central America (León 1992). Repens clade. This is sister to the Pruinose clade (fig. 2). It contains 22 species in our analysis, and we estimate that the clade contains (if undescribed species are included) a total of 30 species. The clade is characterized by long-creeping rhizomes (fig. 3). Typically, the internodes are 10–20 times the width of the rhizomes. The clade is named for Campyloneurum repens (L.) C. Presl, the type of the genus. Within the clade is a subclade consisting of four species endemic to the mountains of Costa Rica and Panama (fig. 2, C. falcoideum to C. talamancanum). Besides long-creeping rhizomes, many species in the Repens clade are probably hemiepiphytic. In Costa Rica, one of us (R.C. Moran) has observed the gametophytes of C. serpentinum (Christ) Ching growing at the base of trunks. The sporeling produces a long root that grows to the soil, where it branches profusely. After contact with the soil, the shoot grows upward, adhering to the trunk by short lateral roots. This growth habit is like that found in Elaphoglossum amygdalifolium (Mett. ex Kuhn) Christ (Lagomarsino et al. 2012), Colysis ampla (F. Muell. ex Benth.) Copel. (Testo and Sundue 2014), and two species of Campyloneurum belonging to the southeastern Brazilian clade (Labiak et al. 2017). Field observations pertaining to growth habit are needed for more species of the Repens clade. Pruinose clade. This is sister to the Repens clade (fig. 2). It contains 21 species in our analysis, and we estimate that it might contain more than 30 species if undescribed ones are included. A possible synapomorphy for the group is pruinose rhizomes, a character easily seen in the field and on herbarium specimens (figs. 1C, 3). Among the species in the clade, the pruinosity may be variable. In certain specimens, it may be poorly developed or apparently absent (e.g., C. amphostenon (Klotzsch) Fée, C. asplundii (C. Chr.) Ching, and C. chlorolepis Alston). We suspect that this is largely caused by the use of high heat during specimen drying and perhaps to a lesser degree by abrasion. Nevertheless, all species of this clade in our analysis (fig. 2) typically have specimens exhibiting pruinose rhizomes. Some species of the Pruinose clade have tristichous rhizomes instead of the normal distichous ones. This was noted by Hovenkamp (1992) for C. angustifolium (Sw.) Fée. Distichous versus tristichous rhizomes are difficult to discern on herbarium specimens, and for that reason we did not include them in our optimizations. More field observations of fresh rhizomes are needed to determine whether tristichous rhizomes constitute a possible synapomorphy for a group of species in the Pruinose clade. Besides pruinose rhizomes, many species in the Pruinose clade are characterized by narrow laminae (!3 cm) and lateral veins that are not prominent, being approximately the same thickness as the cross veins. Geography. Campyloneurum is entirely Neotropical and occurs primarily in the mountains at middle elevations (León 1992). This is reflected by the fact that the genus is most diverse in the Andes, where we estimate that 36 species occur (some undescribed), 12 of which are endemic (fig. 6). The mountains of Costa Rica and western Panama have been an important secondary center, with 18 species, five of which are endemic. The mountains of southeastern Brazil harbor nine species, eight of which are endemic. Five of these endemics form what is called here the southeastern Brazilian clade (fig. 2). Endemic clades in southeastern Brazil have been documented in other fern genera belonging to the Dryopteridaceae, such as Megalastrum Holttum (unpublished results) Polybotrya Willd. (Moran and Labiak 2015) and Stigmatopteris C. Chr. (Moran and Labiak 2016). Within the Polypodiaceae, an endemic clade has been reported for Moranopteris R.Y. Hirai & J. Prado (Hirai et al. 2011). Thus, for the Polypodiaceae, Campyloneurum provides a second example of species forming a clade endemic to the Atlantic Rain Forest. The least diverse region for the genus is the Guyanan Shield, with only seven species, none of which are endemic. We attribute this to the fact the region contains no extensive mountain ranges providing continuous midelevation habitats. Much work remains to be done on the species-level taxonomy of Campyloneurum. For instance, the monophyly of some species was not confirmed by our analysis that included multiple samples (see app. B). This can usually be seen in those species that have been traditionally treated in a broad sense, such as C. repens (Aubl.) C. Presl, C. amphostenon (Kunze ex Klotzsch) Fée, and C. angustifolium (Sw.) Fée. Although it is possible that the placement of some specimens (numbered as spp. 1–15) is due to the low resolution within some clades, a morphological analysis of these specimens suggests that, in many cases, they represent undescribed species. Recognizing new species can be challenging because many times they are best distinguished by subtle features of the rhizome scales that need to be observed under a microscope. The scales are sometimes caducous or (if present) abraded, thus making interpretation and comparison difficult. The genus awaits monographic treatment, which will undoubtedly find species to be added to the phylogenetic tree presented here. Hybridization and polyploidy have received little study in Campyloneurum. No interspecific hybrids have been proposed within the genus, and only 10 of the genus’s estimated 60 species have chromosome counts (see León 1992 for a summary). So far, only two ploidy levels have been found: diploids (n p 37) and tetraploids (n p 74). Alloploidy has been suggested to occur by Walker (1966) in tetraploid C. phyllitidis (L.) C. Presl from Jamaica based on the bimodal distribution of chromosome lengths; however, he did not suggest possible parentage. Because most species of Campyloneurum have not been assessed for hybridization or documented for ploidy level, it is LABIAK & MORAN—PHYLOGENY OF CAMPYLONEURUM 45 Fig. 6 Number of species and endemics (in parentheses) of Campyloneurum in various regions (shaded) of the Neotropics with high endemism and species richness. unknown how these phenomena might influence the results in our tree (fig. 2). Acknowledgments We thank Judith Garrison Hanks for help in taking the spore images shown in figure 7, Weston Testo and Michael Sundue for sharing silica-dried material, and Blanca León for helpful discussions about the taxonomy of the genus. Michael Sundue and an anonymous reviewer provided many helpful comments on the manuscript. Fabian Michelangeli graciously hosted one of us (P.H. Labiak) during a three-week research visit to New York. This work was partially funded by a grant from CNPq (307514/2016-1) to P.H. Labiak. Appendix A List of the species and specimens used in this study and their respective GenBank accession numbers. The information is given in the following order: species, voucher (herbarium), locality, and GenBank accession numbers for rbcL, rps4-trnS, trnG-trnR, and trnL-trnF. Missing sequences are indicated by a dash. Full information is provided only for newly generated sequences, which are indicated by an asterisk. Aglaomorpha pilosa Copel., AY529156.1, AY529180.1, —, —; Arthromeris himalayensis (Hook.) Ching, JQ685378.1, JQ685442.1, —, —; Ascogrammitis anfractuosa (Kunze ex Klotzsch) Sundue, GU476853.1, KM106108.1, —, —; 46 INTERNATIONAL JOURNAL OF PLANT SCIENCES *Campyloneurum abruptum (Lindm.) B. León, Prance 19105 (NY), Brazil, MF318030, MF318065, MF318195, MF318319; *Campyloneurum abruptum (Lindm.) B. León, Prance 19113 (NY), Brazil, MF318031, MF318066, MF318196, MF318320; *Campyloneurum aglaolepis (Alston) de la Sota, Jiménez 2452 (NY), Bolivia, MF317973, MF318067, MF318197, MF318321; *Campyloneurum aglaolepis (Alston) de la Sota, Villaroel 1636 (NY), Bolivia, MF318056, MF318068, MF318198, MF318322; *Campyloneurum aglaolepis (Alston) de la Sota, Zarate 1128 (NY), Bolivia, MF318060, MF318081, MF318212, MF318332; *Campyloneurum amazonense B. León, Wurdack 844 (NY), Peru, —, —, MF318199, —; *Campyloneurum amphostenon (Kunze ex Klotzsch) Fée, Liogier 12791 (NY), Dominican Republic, —, MF318069, MF318200, MF318323; *Campyloneurum amphostenon (Kunze ex Klotzsch) Fée, Testo 697 (VT), Costa Rica, MF318050, MF318070, MF318201, MF318324; *Campyloneurum anetioides (Christ) R.M. Tryon & A.F. Tryon, Mickel 2362 (NY), Costa Rica, MF317991, MF318071, MF318202, MF318325; *Campyloneurum angustifolium (Sw.) Fée, Crosby 704 (NY), Jamaica, —, MF318073, MF318204, —; *Campyloneurum angustifolium (Sw.) Fée, Crosby 739 (NY), Jamaica, —, MF318074, MF318205, —; *Campyloneurum angustifolium (Sw.) Fée, Helwig 460 (NY), Mexico, —, MF318075, MF318206, MF318326; *Campyloneurum angustifolium (Sw.) Fée, Martínez 28464 (NY), Mexico, MF317986, MF318076, MF318207, MF318327; *Campyloneurum angustipaleatum (Alston) M. Mey. ex Lellinger, Nee 49544 (NY), Bolivia, MF318017, MF318078, MF318209, MF318329; *Campyloneurum angustipaleatum (Alston) M. Mey. ex Lellinger, Nee 50463 (NY), Bolivia, MF318018, MF318079, MF318210, MF318330; *Campyloneurum aphanophlebium (Kunze) T. Moore, Croat 11634 (NY), Panama, MF317961, MF318082, MF318213, MF318333; *Campyloneurum asplundii (C. Chr.) Ching, Núñez 360 (NY), Bolivia, MF318019, MF318083, MF318214, MF318334; *Campyloneurum asplundii (C. Chr.) Ching, Núñez 422 (NY), Bolivia, MF318020, MF318084, MF318215, MF318335; *Campyloneurum asplundii (C. Chr.) Ching, Sundue 824 (NY), Bolivia, MF318044, MF318129, MF318255, MF318375; *Campyloneurum atlanticum R.C. Moran & Labiak, Dittrich 561 (NY), Brazil, MF317962, MF318085, MF318216, MF318336; *Campyloneurum atlanticum R.C. Moran & Labiak, Labiak 4229 (NY), Brazil, MF318027, MF318086, MF318217, MF318337; *Campyloneurum atlanticum R.C. Moran & Labiak, Mazziero 982 (UPCB), Brazil, MF318014, MF318088, MF318219, MF318339; *Campyloneurum austrobrasilianum (Alston) de la Sota, Rosa 135 (NY), Brazil, MF318034, MF318089, —, MF318340; *Campyloneurum austrobrasilianum (Alston) de la Sota, Salvador 3991 (NY), Brazil, MF318036, MF318090, —, MF318341; *Campyloneurum brevifolium (Lodd. ex Link) Link, Wiggins 157 (NY), Costa Rica, MF318057, —, MF318223, MF318345; *Campyloneurum brevifolium (Lodd. ex Link) Link, Mickel 3515 (NY), Costa Rica, MF317994, MF318092, MF318221, MF318343; *Campyloneurum brevifolium (Lodd. ex Link) Link, Zak 2199 (NY), Ecuador, MF318061, MF318148, MF318273, MF318393; *Campyloneurum centrobrasilianum Lellinger, Alvarenga 799 (NY), Brazil, —, —, MF318224, —; *Campyloneurum centrobrasilianum Lellinger, Irwin 13057 (NY), Brazil, MF317968, MF318094, MF318225, MF318346; *Campyloneurum chlorolepis Alston, Betancourt 936 (NY), Colombia, —, MF318097, —, —; *Campyloneurum chlorolepis Alston, Zabalaga 33 (NY), Bolivia, MF318062, MF318098, MF318227, MF318349; *Campyloneurum chrysopodum Fée, Navarrete 1829 (NY), Ecuador, MF318016, MF318099, MF318228, MF318350; *Campyloneurum coarctatum (Kunze) Fée, Jiménez 563 (NY), Bolivia, MF317974, MF318100, MF318229, MF318351; *Campyloneurum coarctatum (Kunze) Fée, Sundue 899 (NY), Bolivia, MF318008, MF318102, MF318231, —; *Campyloneurum cochense (Hieron.) Ching, Jaramillo 4671 (NY), Ecuador, MF317969, MF318103, MF318232, MF318353; *Campyloneurum cochense (Hieron.) Ching, Sparre 18935 (NY), Ecuador, MF318041, MF318106, MF318235, MF318356; *Campyloneurum cochense (Hieron.) Ching, Lewis 2386 (NY), Ecuador, MF317978, MF318107, MF318236, MF318357; *Campyloneurum cochense (Hieron.) Ching, Grubb 1341 (NY), Ecuador, MF317963, MF318131, MF318257, —; *Campyloneurum costatum (Kunze) C. Presl, Testo 547 (VT), Costa Rica, —, MF318108, —, —; *Campyloneurum costatum (Kunze) C. Presl, Zanoni 35445 (NY), Dominican Republic, —, MF318109, —, —; *Campyloneurum crispum Fée, Labiak 4357 (NY), Brazil, —, MF318110, MF318237, MF318358; *Campyloneurum crispum Fée, Jardim 848 (NY), Brazil, —, MF318135, MF318286, MF318380; *Campyloneurum crispum Fée, Labiak 5392 (NY), Brazil, MF318028, MF318180, MF318303, MF318425; *Campyloneurum cubense Fée, Shafer 7977 (NY), Cuba, —, MF318111, MF318238, MF318359; *Campyloneurum cubense Fée, Underwood 660 (NY), Cuba, —, MF318112, —, —; *Campyloneurum cubense Fée, Mickel 9264 (NY), Haiti, MF317996, MF318177, MF318301, MF318421; *Campyloneurum decurrens C. Presl, Matos 1269 (NY), Brazil, MF317982, MF318113, MF318239, MF318360; *Campyloneurum decurrens C. Presl, Prado 2027 (NY), Brazil, MF318033, MF318114, MF318240, MF318361; *Campyloneurum densifolium (Hieron.) Lellinger, Saguástegui 15992 (NY), Peru, MF318035, MF318115, MF318241, MF318362; *Campyloneurum densifolium (Hieron.) Lellinger, Zogg and Gassner 13769 (NY), Ecuador, MF318064, MF318116, MF318242, MF318363; *Campyloneurum ensifolium (Willd.) J. Sm., Munn-Estrada 854 (NY), Mexico, MF318010, MF318194, MF318318, MF318440; *Campyloneurum falcoideum (Kuhn ex Hieron.) M. Mey. ex Lellinger, Moran 7975 (NY), Costa Rica, —, MF318117, MF318243, MF318364; *Campyloneurum falcoideum (Kuhn ex Hieron.) M. Mey. ex Lellinger, Testo 583 (VT), Costa Rica, MF318049, MF318118, MF318244, MF318365; *Campyloneurum fallax Fée, Labiak 3916 (NY), Brazil, MF318024, MF318119, MF318245, MF318366; *Campyloneurum fallax Fée, Pereira 106 (NY), Brazil, MF318023, MF318120, MF318246, MF318367; *Campyloneurum filiforme R.C. Moran & Labiak sp. nov. ined., Moran 6919 (NY), Ecuador, MF318000, MF318121, MF318247, MF318368; *Campyloneurum fuscosquamatum Lellinger, Arroyo 26 (NY), Bolivia, MF317951, MF318123, MF318249, MF318370; *Campyloneurum fuscosquamatum Lellinger, Paniagua 4102 (NY), Bolivia, MF318022, MF318125, MF318251, MF318372; *Campyloneurum fuscosquamatum Lellinger, Zak 3848 (NY), Ecuador, —, MF318156, MF318280, MF318400; *Campyloneurum herbaceum (Christ in Schwacke) Ching, Matos 2521 (NY), Brazil, MF317985, MF318087, MF318218, MF318338; *Campyloneurum LABIAK & MORAN—PHYLOGENY OF CAMPYLONEURUM 47 herbaceum (Christ in Schwacke) Ching, Mynssen 1237 (NY), Brazil, MF318011, MF318126, MF318252, MF318373; *Campyloneurum jamaicense R.C. Moran & Labiak sp. nov. ined, Christenhusz 3116 (NY), Jamaica, MF317959, MF318142, MF318267, MF318388; *Campyloneurum jamaicense R.C. Moran & Labiak sp. nov. ined, Maxon 235 (NY), Jamaica, MF317984, MF318157, MF318281, MF318401; *Campyloneurum lorentzii (Hieron.) Ching, Jiménez 2415 (NY), Bolivia, MF317971, MF318127, MF318253, MF318374; *Campyloneurum lorentzii (Hieron.) Ching, Jordan 534 (NY), Bolivia, —, MF318128, MF318254, —; *Campyloneurum macrosorum Fée, Kennedy 1878 (NY), Panama, MF317976, MF318132, MF318258, MF318377; *Campyloneurum magnificum T. Moore, Campos 4043 (NY), Peru, —, MF318133, —, MF318378; *Campyloneurum magnificum T. Moore, Smith 2824 (NY), Ecuador, MF318039, MF318134, MF318259, MF318379; *Campyloneurum nitidissimum (Mett.) Ching, Bennet 3404 (NY), Ecuador, MF317954, MF318136, MF318260, MF318381; *Campyloneurum nitidissimum (Mett.) Ching, Huayla 103 (NY), Bolivia, MF317967, MF318174, MF318298, MF318418; *Campyloneurum nitidissimum (Mett.) Ching, Jiménez 2445 (NY), Bolivia, MF317972, MF318175, MF318299, MF318419; *Campyloneurum nitidissimum (Mett.) Ching, Sundue 762 (NY), Bolivia, MF318007, MF318176, MF318300, MF318420; *Campyloneurum nitidum (Kaulf.) C. Presl, Zardini 6055 (NY), Paraguay, MF318063, MF318130, MF318256, MF318376; *Campyloneurum nitidum (Kaulf.) C. Presl, Labiak 4163 (NY), Brazil, MF318025, MF318137, MF318261, MF318382; *Campyloneurum nitidum (Kaulf.) C. Presl, Labiak 4182 (NY), Brazil, MF318026, MF318138, MF318262, MF318383; *Campyloneurum nitidum (Kaulf.) C. Presl, Mazziero 976 (UPCB), Brazil, MF318012, MF318139, MF318263, MF318384; *Campyloneurum ophiocaulon (Klotzsch) Fée, Stolze 1668 (NY), Ecuador, MF318042, MF318091, MF318220, MF318342; *Campyloneurum ophiocaulon (Klotzsch) Fée, Holm-Nielsen 5775 (NY), Ecuador, MF317966, MF318140, MF318264, MF318385; *Campyloneurum ophiocaulon (Klotzsch) Fée, Grijalva 520 (NY), Ecuador, MF317964, MF318143, MF318268, MF318389; *Campyloneurum parvisquama R.C. Moran & Labiak sp. nov. ined, Rodriguez 5051 (NY), Colombia, —, MF318144, MF318269, —; *Campyloneurum pascoense R.M. Tryon & A.F. Tryon, Serrano 6057 (NY), Bolivia, MF318037, MF318146, MF318271, MF318391; *Campyloneurum pascoense R.M. Tryon & A.F. Tryon, Solomon 11919 (NY), Bolivia, MF318040, MF318147, MF318272, MF318392; *Campyloneurum pentaphyllum (Willd.) Pic. Serm., Smith 2456 (NY), Colombia, —, MF318149, —, —; *Campyloneurum phyllitidis (L.) C. Presl, Boom 11148 (NY), Curacao, MF317955, MF318150, MF318274, MF318394; *Campyloneurum phyllitidis (L.) C. Presl, Jiménez 2661 (NY), Bolivia, MF317975, MF318151, MF318275, MF318395; *Campyloneurum phyllitidis (L.) C. Presl, Lavestre 1890 (NY), Dominican Republic, MF317977, MF318152, MF318276, MF318396; *Campyloneurum phyllitidis (L.) C. Presl, Liogier 10249 (NY), Puerto Rico, MF317979, MF318153, MF318277, MF318397; *Campyloneurum repens (Aubl.) C. Presl, Hollowell 522 (NY), Guyana, MF317965, MF318154, MF318278, MF318398; *Campyloneurum repens (Aubl.) C. Presl, Lleras 17287 (NY), Brazil, MF317980, MF318155, MF318279, MF318399; *Campyloneurum rigidum J. Sm., Mazziero 981 (UPCB), Brazil, MF318013, MF318159, MF318283, MF318403; *Campyloneurum rigidum J. Sm., Prado 1969 (NY), Brazil, MF318032, MF318160, MF318284, MF318404; *Campyloneurum serpentinum (Christ) Ching, Testo 225 (VT), Costa Rica, MF318046, MF318158, MF318282, MF318402; *Campyloneurum serpentinum (Christ) Ching, Mickel 3287 (NY), Costa Rica, MF317992, MF318161, MF318285, MF318405; *Campyloneurum solutum (Klotzsch) Fée, Jaramillo 9608 (NY), Ecuador, MF317970, MF318162, MF318286, MF318406; *Campyloneurum solutum (Klotzsch) Fée, Navarrete 1409 (NY), Ecuador, MF318015, MF318163, MF318287, MF318407; *Campyloneurum solutum (Klotzsch) Fée, Sigel 36 (NY), Ecuador, MF318038, MF318164, MF318288, MF318408; *Campyloneurum sp. 1, Thompson 7313 (NY), Dominican Republic, MF318054, MF318080, MF318211, MF318331; *Campyloneurum sp. 2, Luteyn 13362 (NY), Ecuador, MF317981, MF318122, MF318248, MF318369; *Campyloneurum sp. 3, Porter 4918A (NY), Panama, MF318029, MF318077, MF318208, MF318328; *Campyloneurum sp. 4, Moran 6806 (NY), Ecuador, MF317998, MF318168, MF318292, MF318412; *Campyloneurum sp. 5, Moran 6233 (NY), Ecuador, MF317997, MF318124, MF318250, MF318371; *Campyloneurum sp. 6, Boeke 3208 (NY), Peru, MF317956, MF318095, —, MF318347; *Campyloneurum sp. 7, Mickel 2178 (NY), Costa Rica, MF317990, MF318104, MF318233, MF318354; *Campyloneurum sp. 8, Mickel 3330 (NY), Costa Rica, MF317993, MF318105, MF318234, MF318355; *Campyloneurum sp. 9, Testo 901 (VT), Mexico, MF318053, MF318165, MF318289, MF318409; *Campyloneurum sp. 10, Asplund 19660 (NY), Ecuador, MF317953, MF318096, MF318226, MF318348; *Campyloneurum sp. 11, Taylor 11528 (NY), Costa Rica, MF318045, MF318093, MF318222, MF318344; *Campyloneurum sp. 12, McCarthy 195 (NY), Ecuador, MF317989, MF318101, MF318230, MF318352; *Campyloneurum sp. 12, Moran 6248 (NY), Ecuador, —, MF318169, MF318293, MF318413; *Campyloneurum sp. 13, Callejas 4692 (NY), Colombia, MF317958, MF318072, MF318203, —; *Campyloneurum sp. 14, Zabalaga 1112 (NY), Bolivia, MF318059, —, MF318266, MF318387; *Campyloneurum sp. 14, Núñez 733 (NY), Bolivia, MF318021, MF318141, MF318265, MF318386; *Campyloneurum sp. 15, Testo 532 (NY), Costa Rica, MF318047, MF318166, MF318290, MF318410; *Campyloneurum sp. 15, Testo 792 (NY), Costa Rica, MF318051, MF318167, MF318291, MF318411; *Campyloneurum talamancanum R.C. Moran & Labiak sp. nov. ined, Testo 582 (VT), Costa Rica, MF318048, MF318170, MF318294, MF318414; *Campyloneurum talamancanum R.C. Moran & Labiak sp. nov. ined, Testo 852 (VT), Costa Rica, MF318052, MF318171, MF318295, MF318415; *Campyloneurum talamancanum R.C. Moran & Labiak sp. nov. ined, Sundue 1678 (NY), Costa Rica, MF318006, MF318172, MF318296, MF318416; *Campyloneurum tenuipes Maxon, Maya 3827 (NY), Mexico, MF317987, MF318173, MF318297, MF318417; *Campyloneurum tucumanense (Hieron.) Ching, Martinez 1979 (NY), Argentina, MF317983, MF318145, MF318270, MF318390; *Campyloneurum vulpinum (Lindm.) Ching, Stolze 1743 (NY), Ecuador, MF318043, —, MF318302, MF318424; *Campyloneurum vulpinum (Lindm.) Ching, Mickel 8157 (NY), Dominican Republic, MF317995, MF318178, —, MF318422; *Campyloneurum vulpinum 48 INTERNATIONAL JOURNAL OF PLANT SCIENCES (Lindm.) Ching, Mickel 8920 (NY), Dominican Republic, —, MF318179, —, MF318423; *Campyloneurum wurdackii B. León, Wurdack 41303 (NY), Venezuela, MF318058, MF318181, MF318304, MF318426; *Campyloneurum xalapense Fée, Munn-Estrada 1128 (NY), Mexico, MF318009, MF318193, MF318317, MF318439; Ceradenia kalbreyeri (Baker) L.E. Bishop, GU476905.1, KM106128.1, —, —; Davallia mariesii T. Moore ex Baker, JX103717.1, JX103759.1, —, —; Drynaria mollis Bedd., AY529162.1, AY529186.1, —, —; Leucotrichum organense (Gardner) Labiak, GU376492.1, JN654946.1, —, —; Melpomene flabelliformis (Poir.) A.R. Sm. & R.C. Moran, Labiak 4432 (NY), Brazil, GU387028.1, GU387114.1, GU387197.1, GU387278.1; *Microgramma dictyophylla (Kunze ex Mett.) de la Sota, Moran 7632 (NY), Ecuador, MF318005, MF318183, MF318306, MF318428; *Microgramma lindbergii (Mett. ex Kuhn) de la Sota, Bolli A74 (NY), Paraguay, MF317957, MF318184, MF318307, MF318429; *Microgramma lycopodioides (L.) Copel., Acevedo-Rodriguez 14064 (NY), Dominican Republic, MF317950, MF318185, MF318308, MF318430; *Microgramma tobagensis (C. Chr.) C.D. Adams & Baksh.-Com. ex Rudd, Moran 6927 (NY), Ecuador, MF318001, MF318182, MF318305, MF318427; *Microgramma vacciniifolia (Langsd. & Fisch.) Copel., Christenhusz 4985 (NY), Brazil, MF317960, MF318186, MF318309, MF318431; Microsorum fortunei (T. Moore) Ching, EU482955.1, EU483006.1, —, —; *Niphidium albopunctatissimum Lellinger, Tupayachi 6329 (NY), Peru, MF318055, —, MF318311, MF318433; *Niphidium albopunctatissimum Lellinger, Arroyo 2779 (NY), Bolivia, MF317952, MF318187, MF318310, MF318432; Niphidium crassifolium (L.) Lellinger, EU250351.1, EU250358.1, —, —; *Niphidium longifolium (Cav.) C.V. Morton & Lellinger, Moran 6844 (NY), Ecuador, MF317999, MF318188, MF318312, MF318434; *Niphidium nidulare (Rosenst.) Lellinger, Mickel 1913 (NY), Costa Rica, MF317988, MF318189, MF318313, MF318435; Pecluma eurybasis (C. Chr.) M.G. Price, EF463255.1, FJ825676.1, —, —; Pecluma ptilotos (Kunze) M.G. Price, AY362588.1, AY362661.1, —, —; Phlebodium areolatum (Humb. & Bonpl. ex Willd.) J. Sm., FJ825704.1, FJ825675.1, —, —; Platycerium grande J. Sm., DQ164451.1, DQ164482.1, —, —; Pleopeltis bombycina (Maxon) A.R. Sm., AY362612.1, EU650175.1, —, —; *Pleopeltis macrocarpa (Bory ex Willd.) Kaulf., Moran 7531 (NY), Ecuador, MF318002, MF318192, MF318316, MF318438; Pleurosoriopsis makinoi (Maxim. ex Makino) Fomin, AY362613.1, AY362685.1, —, —; Selliguea hellwigii (Diels) Hovenkamp, EU128501.1, EU128508.1, —, —; Serpocaulon fraxiniifolium (Jacq.) A.R. Sm., Jiménez 696 (UC), Bolivia, DQ151909.1, DQ151934.1, —, DQ151961.1; Serpocaulon levigatum (Cav.) A.R. Sm., EF551073.1, EF551103.1, —, —; Serpocaulon loriceum (L.) A.R. Sm., EF551074.1, EF551104.1, —, —; Stenogrammitis wittigiana (Fée & Glaz. ex Fée) Labiak, Labiak 4441 (UPCB), Brazil, GU387029.1, GU387106.1, GU387189.1, GU387270.1. Literature Cited Bonci MC, V Grazia, S Tacchino, M Piazza 2011 Oligocene fossil leaves of the Perrando Collection: history, preservation, and paleoclimatic meaning. Boll Soc Paleontol Ital 50:145–164. Darriba D, GL Taboada, R Doallo, D Posada 2012 jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772. de la Sota ER, B Pérez-García 1982 Nerviación y dimorfismo foliar en Microgramma Presl (Polypodiaceae) s. str. Biotica 7:45–64. Edgar RC 2004 MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. Guindon S, O Gascuel 2003 A simple, fast and accurate method to estimate large phylogenies by maximum-likelihood. Syst Biol 52: 696–704. Hirai RY, G Rouhan, PH Labiak, T Ranker, J Prado 2011 Moranopteris: a new Neotropical genus of grammitid ferns (Polypodiaceae) segregated from Asian Micropolypodium. Taxon 60:1123– 1137. Hovenkamp P 1992 The significance of rhizome morphology in the systematics of the polypodiaceous ferns (sensu stricto). Am Fern J 80:33–43. Kreier H-P, A Rojas-Alvarado, AR Smith, H Schneider 2007 Hyalotrichopteris is indeed a Campyloneurum (Polypodiaceae). Am Fern J 97:127–135. Labiak P, F Matos, G Rouhan, J Garrison Hanks, RC Moran 2017 Notes on the taxonomy and growth habits of three species of Campyloneurum (Polypodiaceae) from southeastern Brazil. Am Fern J 107:1–20. Lagomarsino LP, AL Grusz, RC Moran 2012 Primary hemiepiphytism and gametophyte morphology in Elaphoglossum amygdalifolium (Dryopteridaceae). Brittonia 64:226–235. Lellinger DB 1972 A revision of the fern genus Niphidium. Am Fern J 62:101–120. ——— 1988 Some new species of Campyloneurum and a provisional key to the genus. Am Fern J 78:14 –35. León B 1992 A taxonomic revision of the fern genus Campyloneurum (Polypodiaceae). PhD diss. Aarhus University, Aarhus, Denmark. ——— 1995 A new species of Campyloneurum (Polypodiaceae) from northwestern Ecuador. Novon 5:42–44. ——— 2004 A new species of Campyloneurum (Polypodiaceae) from northern Peru. Rev Peru Biol 11:135–137. León B, M Kessler 2013 Campyloneurum poloense (Polypodiaceae), a new combination and lectotypification for a Bolivian fern. Phytotaxa 119:59–60. Maddison WP, DR Maddison 2017 Mesquite: a modular system for evolutionary analysis. Version 3.2. http://mesquiteproject.org. Miller MA, W Pfeiffer, T Schwartz 2010 Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Pages 1–8 in Proceedings of the Gateway Computing Environments Workshop, 14 Nov. 2010, New Orleans, LA Moran RC 2008 Biogeography of ferns and lycophytes. Pages 369– 396 in C Haufler, TA Ranker, eds. The biology and evolution of ferns and lycophytes. Cambridge University Press, Cambridge. Moran RC, P Labiak 2015 Phylogeny of the polybotryoid fern clade (Dryopteridaceae). Int J Plant Sci 176:880–891. ——— 2016 Phylogeny and character evolution in the Neotropical fern genus Stigmatopteris (Dryopteridaceae). Brittonia 68:476–488. ——— 2017 The 1-pinnate species of Campyloneurum (Polypodiaceae). Brittonia 68:186–196. Nagalingum NS, H Schneider, KM Pryer 2007 Molecular phylogenetic relationships and morphological evolution in the heterosporous fern genus Marsilea. Syst Bot 32:16–25. PPG (Pteridophyte Phylogeny Group) I 2016 A community-derived classification for extant lycophytes and ferns. J Syst Evol 54:563 – 603. LABIAK & MORAN—PHYLOGENY OF CAMPYLONEURUM Rambaut A, AJ Drummond 2013 Tracer. Version 1.6. http://tree.bio .ed.ac.uk/software/tracer/. Rojas AF 2005 El complejo de Campyloneurum angustifolium (Sw.) Fée (Polypodiaceae) in Costa Rica. Lankesteriana 5:41–48. ——— 2017 Novelties in Campyloneurum (Polypodiaceae) from Mesoamerica. Am J Plant Sci 8:921–940. Salino A, TE Almeida, AR Smith, AN Gómez, HP Kreier, H Schneider 2008 A new species of Microgramma (Polypodiaceae) from Brazil and recircumscription of the genus based on phylogenetic evidence. Syst Bot 33:630–635. Schneider H, AR Smith, R Cranfill, TJ Hildebrand, CH Haufler, TA Ranker 2004 Unraveling the phylogeny of polygrammoid ferns (Polypodiaceae and Grammitidaceae): exploring aspects of the diversification of epiphytic plants. Mol Phylogenet Evol 31:1041–1063. Schuettpelz EH, KM Pryer 2007 Phylogeny of ferns inferred from 400 leptosporangiate species and three plastid genes. Taxon 56: 1037–1050. ——— 2009 Evidence for a Cenozoic radiation of ferns in an angiospermdominated canopy. Proc Natl Acad Sci USA 106:11200–11205. Skog JE, JT Mickel, RC Moran, M Volovsek, EA Zimmer 2004 Molecular studies of representative species in the fern genus Elaphoglossum (Dryopteridaceae) based on cpDNA sequences rbcL, trnL-F, and rps4-trnS. Int J Plant Sci 165:1063–1075. Souza-Chies TT, G Bittar, S Nadot, L Carter, E Besin, B Lejeune 1997 Phylogenetic analysis of Iridaceae with parsimony and distance methods using the plastid gene rps4. Plant Syst Evol 204:109–123. Stamatakis A 2014 RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312– 1313. 49 Sundue MA, WL Testo, TA Ranker 2015 Morphological innovation, ecological opportunity, and the radiation of a major vascular epiphyte lineage. Evolution 69:2482–2495. Taberlet P, L Gielly, G Pautou, J Bouvert 1991 Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol Biol 17:1105–1109. Testo WL, MA Sundue 2014 Primary hemiepiphytism in Colysis ampla (Polypodiaceae) provides new insight into the evolution of growth habit in ferns. Int J Plant Sci 175:526–536. ——— 2016 A 4000-species dataset provides new insight into the evolution of ferns. Mol Phylogenet Evol 105:200–211. Tryon AF, B Lugardon 1991 Spores of the Pteridophyta. Springer, New York. Tryon RM 1972 Endemic areas and geographic speciation in tropical American ferns. Biotropica 4:121–131. Tryon RM, AF Tryon 1982 Ferns and allied plants, with special reference to tropical America. Springer, New York. Vasques DT, J Prado 2011 Campyloneurum C. Presl (Polypodiaceae) no estado de São Paulo, Brasil. Hoehnea 38:147–163. Wagner WH Jr, DR Farrar 1976 The Central American fern genus Hyalotricha and its family relationships. Syst Bot 1:348–362. Walker TG 1966 A cytotaxonomic survey of the ferns of Jamaica. Trans R Soc Edinb 66:169–237. ——— 1985 Cytotaxonomic studies of the ferns of Trinidad. 2. The cytology and taxonomic implications. Bull Br Mus (Nat Hist) Bot 13:149–249. Wilgenbusch JC, DL Warren, DL Swofford 2004 AWTY: a system for graphical exploration of MCMC convergence in Bayesian phylogenetic inference. http://ceb.csit.fsu.edu/awty.