Grana, 2016 http://dx.doi.org/10.1080/00173134.2016.1184307 Spores of Serpocaulon (Polypodiaceae): morphometric and phylogenetic analyses VALENTINA RAMÍREZ-VALENCIA1,2 & DAVID SANÍN 3 1Smithsonian Tropical Research Institute, Center of Tropical Paleocology and Arqueology, Grupo de Investigación en Agroecosistemas y Conservación de Bosques Amazonicos-GAIA, Ancón Panamá, Republic of Panama, 2Laboratorio de Palinología y Paleoecología Tropical, Departamento de Ciencias Biológicas, Universidad de los Andes, Bogotá, Colombia, 3Facultad de Ciencias Básicas, Universidad de la Amazonia, Florencia Caquetá, Colombia Abstract The morphometry and sculpture pattern of Serpocaulon spores was studied in a phylogenetic context. The species studied were those used in a published phylogenetic analysis based on chloroplast DNA regions. Four additional Polypodiaceae species were examined for comparative purposes. We used scanning electron microscopy to image 580 specimens of spores from 29 species of the 48 recognised taxa. Four discrete and ten continuous characters were scored for each species and optimised on to the previously published molecular tree. Canonical correspondence analysis (CCA) showed that verrucae width/verrucae length and verrucae width/spore length index and outline were the most important morphological characters. The first two axes explain, respectively, 56.3% and 20.5% of the total variance. Regular depressed and irregular prominent verrucae were present in derived species. However, the morphology does not support any molecular clades. According to our analyses, the evolutionary pathway of the ornamentation of the spores is represented by depressed irregularly verrucae to folded perispore to depressed regular verrucae to irregularly prominent verrucae. Keywords: character evolution, ferns, eupolypods I, canonical correspondence analysis useful in phylogenetic analyses of several other Serpocaulon is a fern genus restricted to the tropics groups of ferns (Wagner 1974; Pryer et al. 1995; and subtropics of the Americas that contain 48 Moran et al. 2007, 2010; Schneider et al. 2009). named taxa (Smith et al. 2006; Labiak & Prado Here, we use spore morphology as a phylogenetic 2008; Rojas-Alvarado & Chaves-Fallas 2013; Downloaded by [David Sanín] at 12:13 07 July 2016 tool to (a) explore the evolution of different spore Schwartsburd & Smith 2013; Sanín 2014, 2015, traits within different species of Serpocaulon,(b) personal observation, October 2015; Sanín & Torrez qualify the sculptural elements and the complexity 2014; Chaves-Fallas et al. 2015) and its monophyly of the surface ornamentation or patterns that they is strongly supported (Schneider et al. 2004b). How- form, and (c) provide a potential tool to classify ever, despite the strong support at the clade level, monolete verrucate spores and increase the taxo- interspecific relationships of the genus for some nomic resolution of spore records. clades are still not completely resolved using only molecular information (Kreier et al. 2008). In this context, the incorporation of new macro- and micro- Material and methods morphological characters can provide evidence for Spores were obtained from herbaria specimens that species delimitation in the genus and allow for the represent 25 of the species of Serpocaulon.Thissample reconstruction of phylogenetic character evolution. means 47% of the 48 described taxa (Table I). Those Spore morphology in Serpocaulon has not been exemplars are deposited in the following herbaria: studied in this context, although spores have been Correspondence: David Sanín, Facultad de Ciencias Básicas, Universidad de la Amazonia, Cr. 11 No. 5-69, Florencia Caquetá, Colombia. E-mail: [email protected] (Received 22 November 2015; accepted 31 March 2016) © 2016 Collegium Palynologicum Scandinavicum 2 V. Ramírez-Valencia and D. Sanín COL, FAUC, HUA, PSO and UPCB; abbreviations (Rasband 1997)(Figure 1E–G, Table II)andopti- follow Thiers (2016); see ‘Specimens examined’.Gen- misedonthesametopology(Figure 3)usingR. erally, one specimen per species and 20–50 spores per Morphological descriptions follow Tryon and stub were examined. We studied all species included in Lugardon (1991) and Punt et al. (2007). Verrucae the phylogeny of Kreier et al. (2008), with the excep- patterns were quantified from four drawings based tion of S. giganteum, S. gilliesii, S. latissimum, S. lorici- on pictures of a 5000× SEM image of each speci- forme and S. silvulae. Serpocaulon giganteum and S. men, using CorelDraw, Photoshop and Ilustrator loriciforme were excluded because we do not accept software packages for the creation of patterns their specific entity. The other three species were (Figure 1). excluded because we were not able to obtain any of In order to characterise the relationships between them. We used four genera as outgroup taxa, including species morphology on a quantitative basis, a cano- Campyloneurum, Pleopletis, Polypodium and Micro- nical correspondence analysis (CCA) was performed gramma (see ‘Specimens examined’) (Schneider et al. (Figure 4). A Hellinger transformation of species 2004b; Smith et al. 2006; Kreier et al. 2008) measurements was made in order to increase the Spores were processed without treatment from statistical weight. The statistical analyses were per- herbarium specimens to aluminium scanning elec- formed using R. tron microscope (SEM) stubs with double-sided bonding tapes, covered with gold/palladium in a sputter-coater for five minutes, and then, imaged Results digitally using a Zeiss mod EVO 40 vp SEM at Spore morphology and ornamentation 15 kV (acceleration voltage) at magnifications of 5000× and 15000× in the SEM laboratory at the SEM images revealed that the surface ornamenta- Smithsonian Tropical Research Institute (STRI), tion of the 25 Serpocaulon species are considerably Panama City. Spore images, detailed morphological diverse despite them being grouped in the same description and measurements of all species exam- ornamentation category. All species are charac- ined for this study can be found at STRI Pollen terised by verrucate ornamentation consisting of Database http://biogeodb.stri.si.edu/jaramillo/palyno an element more than 1 µm wide that is wider morph/. than long and not constricted at the base. How- Four discrete morphological spore characters ever, the spores of Serpocaulon species show dif- (perispore, prominent verrucae, depressed regular ferent patterns of morphology: irregularly verrucae and depressed irregularly verrucae) and depressed verrucae, depressed regular verrucae, one vegetative character (blade dissection) irregularly prominent verrucae and with the pre- describedforthetreeprincipal morphologies (pin- sence of folded perine (Figure 1A–D). natisect, pinnate and simple (Lellinger 2002); All studied species of Serpocaulon and four out- Figure 1A–D, Table I) were observed, studied group species are assigned to five classification and optimised using Mesquite 2.75 (Maddison & patterns of ornamentation (Figure 2). Group Downloaded by [David Sanín] at 12:13 07 July 2016 Maddison 2011) onto the cpDNA strict consensus one includes 16 species that show spores with tree of Kreier et al. (2008, Figure 2); using the irregularly depressed verrucae, including three principle of parsimony. We have used binary char- members of the outgroup (Campyloneuron brevifo- acter states for all qualitative characters, except the lium, Microgramma lycopodioides, Polypodium vul- blade dissection that was multistate. The final trees gare, S. attenuatum, S. crystalloneuron, S. falcaria, and figures were made in Mesquite and R (R S. fraxinifolium, S. intricatum, S. lasiopus, S. levi- Development Core Team 2014). gatum, S. loriceum, S. menisciifolium, S. polystichum, Ten continuous characters, including four S. ptilorhizon, S. subandium and S. triseriale). indexes (verrucae width/verrucae length [VW/VL], Group two contains five species whose spores verrucae length/spore length [VL/SL], verrucae exhibitedfoldedperine(S. catharinae, S. dissimile, width/spore length [VW/SL], verrucae width/spore S. latipes, S. sessilifolium and S. wagneri). Group width [VW/SW], perimeter verruca [PV], area ver- three contains two species with spores with rucae [AV], diameter verrucae [DIV], density ver- depressed regular verrucae (S. eleutherophlebium rucae [DEV], degree of sphericity [RV] and and S. patentissimum), and group four contains Outline [O]; obtained from the average of three six species with spores with prominent verrucae measurements from a central oval) were measured (S. appressum, S. caceresii, S. dasypleuron, S. mar- (Figure 1E–G) following the suggestions of Ragha- itimum, S. polystichum and S. richardii)(Table I). van et al. (2005), Mander et al. (2013)andHan This last group represents the principal polytomy et al. (2014). They were analysed using Image J in the phylogenetic analysis. Additionally, Spores of Serpocaulon (Polypodiaceae) 3 Figure 1. Ornamentation type verrucae: A. Spores with irregularly depressed verrucae – (Serpocaulon triseriale). B. Spores with perine Downloaded by [David Sanín] at 12:13 07 July 2016 (Serpocaulon sessilifolium). C. Spores with depressed regular verrucae (Serpocaulon patentissimum). D. Spores with prominent verrucae (Serpocaulon caseresii). E. Spore outline/verrucae index measure. F. Graphic representation of some measures. G. Graphic representation of the verrucae density measure. H. Pinnatisect dissection; incised all the way down to the axis, the segments not contracted at their base. I. Pinnate dissection;