Zuluaga, Cameron, Croat and Medecilo, 2015 Testing the monophyly of Spathiphyllum... Testing the monophyly of Spathiphyllum, and the relationship between Asian and tropical American species. Alejandro Zuluaga and Kenneth Cameron Department of Botany University of Wisconsin-Madison, Madison, WI 53706 Thomas Croat P. A. Schulze Curator of Botany Missouri Botanical Garden P.O. Box 299, St. Louis, MO 63166 Melanie Medecilo Biological Sciences Department, De La Salle University-Dasmarinas Cavite, Phillipines ABSTRACT the only disjunct aroid genus between the As part of a broader phylogenetic study of Old and New World Tropics. the subfamily Monsteroideae we used three plastid and one nuclear markers to test the INTRODUCTION monophyly of the genus Spathiphyllum. Our results support the tribe Spathiphylleae as With the increased application of sister of the remaining Monsteroideae, and molecular data for phylogenetic analysis, the the monophyly of Spathiphyllum. The understanding of relationships within monotypic genus Holochlamys appears nested Araceae has been improved considerably within Spathiphyllum in a clade with the (Cabrera, 2008, Cusimano, 2011, Henríquez, Asian species and S. cannifolium. However, 2014). Additionally, new fossils and support for this clade is low. The section biogeographical studies have helped to Spathiphyllum shows a high support and is elucidate the historical and current sister to the remaining species in the genus. geographic distribution patterns in the As a consequence Spathiphyllum remains as family (Nauheimer, 2012a). As a result, aroid classification has changed Aroideana VOL 38E NO 1, 2015 107 Zuluaga, Cameron, Croat and Medecilo, 2015 Testing the monophyly of Spathiphyllum... dramatically; several genera have been America (Boyce & Croat, 2013). Unlike resurrected; and new genera have been Schismatoglottis, molecular evidence for the created (Cabrera, 2008, Cusimano, 2011). separation of Neotropical Homalomena is not Nevertheless, many critical issues are still in conclusive (Barabé, 2002, Gauthier, 2008, need of more data. Wong et al., 2013). The most plausible options for the assignment of the Before this molecular era there were Neotropical Homalomena would be the thought to be three geographically disjunct resurrection of the genus Adelomena, or the genera between the old world and new combination of these species of world tropics. Species in the genera Homalomena with Philodendron subgenus Homalomena, Schismatoglottis and Spathiphyllum Pterosmischum in the genus Elopium (Wong et were found in both tropical America and al., 2013). Thus, more extensive sampling tropical Asia, particularly within the Malay and more molecular data are necessary to Archipelago and Melanesia (van Steenis, fully understand the relationships among 1962, Grayum, 1990, Mayo et al., 1997). Homalomena, and the subgenera of However, recent studies (Barabé, 2002, Philodendron. Gauthier, 2008, Wong et al., 2013) have shown evidence challenging the monophyly Finally, we have the genus Spathiphyllum, of disjunct Schismatoglottis and Homalomena which has a different distribution pattern. (they are no longer disjunct in their revised The genus is more diverse in Tropical circumscriptions), leaving Spathiphyllum as America with about 50 species (Cardona, the only remaining aroid genus with a 2004), and only three species in Southeast tropical disjunction. Asia. Spathiphyllum commutatum Schott is widespread in Malesia and reaches The genus Schismatoglottis, with only three Micronesia; S. solomonense Nicolson is species in South America, and about 150 in restricted to the Solomon Islands; and S. tropical Asia, belongs to the tribe schlechteri (Engl. & K.Krause) Nicolson is Schismatoglottidae that in its current restricted to New Guinea. Neotropical circumscription comprises 11 genera. species are concentrated in the northern Several of these genera are small groups Andes, with only a few species reaching recognized thanks to molecular data (Wong Central America and Mexico (Cardona, et al., 2010a, 2010b, Low, 2014). The three 2004), and one species, S. leave reaches Neotropical species previously recognized Cocos Island in the Pacific Ocean west of in Schismattoglotis are now in the resurrected Costa Rica. No comprehensive phylogenetic genus Philonotion (Wong, 2010c) that belong studies, until now, have included more than to its own tribe Philonotieae. Equally, the three Spathiphyllum from Tropical America, genus Homalomena is considerable more or any of the Asian species. diverse in Tropical Asia with about 500 species, versus only 10 species in Tropical Aroideana VOL 38E NO 1, 2015 108 Zuluaga, Cameron, Croat and Medecilo, 2015 Testing the monophyly of Spathiphyllum... Spathiphyllum together with Holochlamys As part of a broader systematic study of becarii form the tribe Spathiphylleae in the the subfamily Monsteroideae (Zuluaga, subfamily Monsteroideae (Cabrera, 2008, unpublished data) we tested the monophyly Cusimano, 2011). The relationships of the of Spathiphyllum and Holochlamys, and tribe Spathiphylleae within Monsteroideae investigated the relationships of the Asian have been controversial, but recent studies and American species of Spathiphyllum. strongly support Spathiphylleae as sister to the remaining Monsteroideae (Chartier, 2014, Henriquez, 2014). These two genera METHODS are very similar morphologically and Taxon Sampling and DNA sequencing. ecologically, differing mainly in a few During the sampling for the study of characteristics of the flowers. Whereas systematics of the subfamily Monsteroideae Spathiphyllum species have two to three we sequenced a total of seven accessions of locules and ovules with axillar placentation, the genus Spathiphyllum from Tropical Holochamys has one locule and ovules with America, S. commutatum and S. solomonense basal placentation (Bunting, 1960, Mayoet from Tropical Asia, and one accession of al.,1997). Nevertheless, anatomical studies Holochlamys becarii. Additional sequences for suggest that the Holochlamys ovary could S. phryniifolium were obtained from actually have two locules, and the GENBANK. For this study we used placentation may not be basal (Eyde, 1967, representatives from all ten additional Carvell,1989). Monsteroideae genera, plus six other aroids as outgroups. Currently, Spathiphyllum is divided into four sections based the fusion of the perianth DNA was extracted from silica-dried plant segments, the form and length of the pistils material using the DNeasy Plant Mini Kit relative to the perianth, and the attachment (Qiagen, Valencia, CA, USA) according to the spathe to the peduncle (Bunting, 1960). the manufacturer's specifications. Some Asian species together with S. cannifolium samples were extracted using a CTAB and S. leave form the section Massowia, method adapted from Doyle & Doyle which is recognized by having the perianth (1987). We sequenced four plastid markers, segments completely connate forming a cup matK, rbcL, trnC-petN spacer, and partial ycf1. around the pistil. This trait led Bunting PCR products were purified using ExoSap- (1960) to propose the monophyly of this It (Cleveland, OH). Sequencing reactions group. However, Williams & Dressler (1967) used the BigDye Terminator Cycle and Grayum (1984, 1990) suggested this Sequencing Kit, and cleaned using could be an artificial group with Holochlamys Agencourt CleanSeq (Beverly, MA) nested within it. Sections Amomophyllum, magnetic beads. BigDye Terminator Cycle Dysspathiphyllum, and Spathiphyllum have free Sequencing Ready Reaction Kit (Applied perianth segments. Biosystems, Foster City, CA, USA). Samples Aroideana VOL 38E NO 1, 2015 109 Zuluaga, Cameron, Croat and Medecilo, 2015 Testing the monophyly of Spathiphyllum... were sequenced using PE-Biosystems of the subfamily Monsteroideae version3.7 of Sequencing Analysis at the (Unpublished data). University Wisconsin–Madison Biotechnology Center. Trees derived from individual gene analyses are congruent, however the Molecular analysis. All sequences were resolution is low except for a clade manually edited in Geneious6.0 (Biomatters, comprising the species of the section http://www.geneious.com). Sequences were Spathiphyllum. Thus, a combined gene aligned in Geneious6.0 using a combination analysis is presented (Figure 1). All of Muscle (Edgar 2004) and MAFFT individual and combined analyses show the (Katoh 2002). Finally, all alignments were tribe Spathiphylleae as monophyletic and checked manually. We performed individual sister to the remaining Monsteroideae, thus and combined phylogenetic analyses. supporting the topology presented by Analyses for each marker/dataset were Chartier (2014) and Henríquez (2014). performed under Maximum Likelihood Section Spathiphyllum represented by S. using RaxML 7.0 (Statamakis, 2006), and cochlearispathum, S. phryniifolium, and S. wallisii MrBayes 3.1.2 on XSEDE, as implemented shows strong support, and is sister to the in the CIPRES portal. For RaxML, we used remaining Spathiphylleae. The genus the GTRGAMMA model in all cases. For Holochlamys appears nested within Bayesian analysis models were calculated Spathiphyllum in a clade with the Asian using Jmodeltest2 (Darriba, 2012). species and one accession of S. cannifolium (section Massowia). Section Amomophyllum (S. RESULTS. minor, S. floribundum, S. patulinervum) and one accession of S. cannifolium are sister to the So far at least one of the four molecular clade containing Holochlamys. However markers has been successfully sequenced for support for the last