Accepted Manuscript Revisiting the phylogeny of Bombacoideae (Malvaceae): Novel relationships, morphologically cohesive clades, and a new tribal classification based on mul- tilocus phylogenetic analyses Jefferson G. Carvalho-Sobrinho, William S. Alverson, Suzana Alcantara, Luciano P. de Queiroz, Aline C. da Mota, David A. Baum PII: S1055-7903(16)30087-2 DOI: http://dx.doi.org/10.1016/j.ympev.2016.05.006 Reference: YMPEV 5515 To appear in: Molecular Phylogenetics and Evolution Received Date: 21 May 2015 Revised Date: 21 April 2016 Accepted Date: 2 May 2016 Please cite this article as: Carvalho-Sobrinho, J.G., Alverson, W.S., Alcantara, S., de Queiroz, L.P., da Mota, A.C., Baum, D.A., Revisiting the phylogeny of Bombacoideae (Malvaceae): Novel relationships, morphologically cohesive clades, and a new tribal classification based on multilocus phylogenetic analyses, Molecular Phylogenetics and Evolution (2016), doi: http://dx.doi.org/10.1016/j.ympev.2016.05.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Revisiting the phylogeny of Bombacoideae (Malvaceae): novel relationships, morphologically cohesive clades, and a new tribal classification based on multilocus phylogenetic analyses Jefferson G. Carvalho-Sobrinhoa,*, William S. Alversonb, Suzana Alcantarac, Luciano P. de Queirozd, Aline C. da Motad, David A. Baumb a Colegiado de Ciências Biológicas, Universidade Federal do Vale do São Francisco – UNIVASF, BR-407, Km 12, Vila CS-01, Petrolina, Pernambuco, 56300-990, Brazil. b Department of Botany, Birge Hall, University of Wisconsin-Madison, Madison, Wisconsin, 53706, U. S. A. c Departamento de Botânica-CCB, Universidade Federal de Santa Catarina – UFSC, Florianópolis, SC, 88040900, Brazil. d Programa de Pós-Graduação em Botânica, Universidade Estadual de Feira de Santana – UEFS, Av. Transnordestina, s/n, Novo Horizonte, Feira de Santana, Bahia, 44036900, Brazil. *Corresponding author: Colegiado de Ciências Biológicas, Universidade Federal do Vale do São Francisco – UNIVASF, BR-407, Km 12, Vila CS-01, Petrolina, Pernambuco, 56300-990, Brazil (J.G. Carvalho-Sobrinho). Email address: [email protected] (J.G. Carvalho-Sobrinho) 1 Abstract: Bombacoideae (Malvaceae) is a clade of deciduous trees with a marked dominance in many forests, especially in the Neotropics. The historical lack of a well- resolved phylogenetic framework for Bombacoideae hinders studies in this ecologically important group. We reexamined phylogenetic relationships in this clade based on a matrix of 6,465 nuclear (ETS, ITS) and plastid (matK, trnL-trnF, trnS-trnG) DNA characters. We used maximum parsimony, maximum likelihood, and Bayesian inference to infer relationships among 108 species (~70% of the total number of known species). We analysed the evolution of selected morphological traits: trunk or branches prickles, calyx shape, endocarp type, seed shape, and seed number per fruit, using ML reconstructions of their ancestral states to identify possible synapomorphies for major clades. Novel phylogenetic relationships emerged from our analyses, including three major lineages marked by fruit or seed traits: the winged-seed clade (Bernoullia, Gyranthera, and Huberodendron), the spongy endocarp clade (Adansonia, Aguiaria, Catostemma, Cavanillesia, and Scleronema), and the Kapok clade (Bombax, Ceiba, Eriotheca, Neobuchia, Pachira, Pseudobombax, Rhodognaphalon, and Spirotheca). The Kapok clade, the most diverse lineage of the subfamily, includes sister relationships (i) between Pseudobombax and “Pochota fendleri” a historically incertae sedis taxon, and (ii) between the Paleotropical genera Bombax and Rhodognaphalon, implying just two bombacoid dispersals to the Old World, the other one involving Adansonia. This new phylogenetic framework offers new insights and a promising avenue for further evolutionary studies. In view of this information, we present a new tribal classification of the subfamily, accompanied by an identification key. Keywords: baobab, fruit traits, kapok group, Neotropics, tribal classification 2 1 1. Introduction 2 Bombacoideae is a lineage of Malvaceae (Alverson et al., 1999; Bayer et al., 3 1999; Nyffeler et al., 2005) that includes trees with outstanding ecological importance in 4 the tropics. It encompasses about 17 genera and 160 species with ca. 90% of the species 5 distributed in the Neotropics. Bombacoideae comprises some of the most abundant and 6 dominant tree species in many Neotropical forests (Andel, 2001; Ferreira and Prance, 7 1998; Linares-Palomino and Alvarez, 2005; Pennington and Sarukhán, 1968; 8 Pennington et al., 2009; Prance et al., 1976). In the Paleotropics, it is represented by 9 fewer than 18 native species in three genera: Adansonia L. (eight or nine species), 10 Bombax L. (three or four species), and Rhodognaphalon (Ulbr.) Roberty (three species). 11 Whether the disjunct distribution of Ceiba pentandra (L.) Gaertn. and Pachira glabra 12 Pasq. in the African and American continents is natural or anthropogenic has long been 13 controversial (Dick et al., 2007; Robyns, 1963). Ignoring these two widely cultivated 14 species, three independent migrations from the Neotropics to the Paleotropics are 15 usually invoked to explain the worldwide distribution of Bombacoideae (Duarte et al., 16 2011). 17 Most traditional systematic and phylogenetic studies in Bombacoideae have 18 focused on floral characters, especially the androecium (Bentham, 1843, 1862; 19 Carvalho-Sobrinho et al., 2009; Duarte et al., 2011; Gibbs and Alverson, 2006; Gibbs 20 and Semir, 2003; Nyffeler et al., 2005; Robyns, 1963). Genera possessing flowers with 21 many filaments partially connate in a tube and monothecate anthers were historically 22 placed in the tribe Adansonieae (Hutchinson, 1967; Schumann, 1886, 1895; see Fig. 1), 23 which comprised most species of Bombacoideae. According to some classification 24 systems, Adansonieae was also characterized by possession of palmately compound 25 leaves, resulting in the inclusion of Ceiba Mill., Bernoullia Oliv., Gyranthera Pittier, 3 26 and Spirotheca Ulbr. (e.g., Bakhuizen van den Brink, 1924; Barroso et al., 2002). Such 27 a broadened circumscription of Adansonieae makes the tribe rather variable in flower 28 traits: Ceiba and Spirotheca have five stamens with two or four thecae each (Gibbs and 29 Alverson, 2006; Gibbs and Semir, 2003); whereas Bernoullia and Gyranthera have 30 anthers with many distal, sessile thecae on an elongated staminal tube (von Balthazar et 31 al., 2006). Furthermore, Bernoullia and Gyranthera exhibit distinct capsular fruits 32 enclosing winged seeds that differ from the woody berries of Adansonia and from the 33 capsules with kapok and non-winged seeds of Ceiba and most other Adansonieae 34 genera. 35 Recent studies based on morphological (Carvalho-Sobrinho and Queiroz, 2011) 36 and molecular data (Duarte et al., 2011), however, indicate that Adansonieae as 37 conceived historically is not monophyletic without inclusion of genera with simple or 1- 38 foliolate leaves traditionally placed in the tribes Durioneae, Hampeae, 39 ‘Catostemmateae’, and Matiseae (Fig. 1): Catostemma Benth., Cavanillesia Ruiz & 40 Pav., Huberodendron Ducke, and Scleronema Benth (Edlin, 1935; Hutchinson, 1967; 41 Schumann, 1895; Takhtajan, 1997). However, all the aforementioned genera compose a 42 molecularly well-supported clade named the core Bombacoideae (Baum et al., 2004; 43 Duarte et al., 2011), which may be defined as corresponding to the smallest 44 monophyletic group containing Gyranthera and Bombax (Baum et al., 2004). 45 At the generic level, previous molecular work has supported monophyly of most 46 currently accepted genera. However, Bombacopsis and Rhodognaphalopsis, erected by 47 Pittier (1916) and Robyns (1963), respectively, have been shown to be embedded within 48 Pachira s. lat. (Duarte et al., 2011). Likewise, a recent molecular study suggested that 49 Eriotheca Schott & Endl. forms a paraphyletic grade relative to Pachira Aubl. (Duarte 50 et al., 2011). This same study also found that one species of Pachira, P. quinata 4 51 (=Bombacopsis quinata), is not related to the remainder of the genus and was recently 52 transferred to Pochota (Alverson and Duarte, 2015). Finally, the wisdom of fusing 53 Ceiba and Chorisia Kunth into Ceiba has long been controversial (Gibbs & Semir, 54 2003; Gibbs et al., 1988; Ravenna, 1998), but has not yet been thoroughly assessed in a 55 phylogenetic framework. 56 Despite recent efforts to clarify the phylogeny of Bombacoideae, uncertainty 57 remains, especially in regards to intergeneric relationships. The lack of a well resolved 58 phylogenetic framework hampers development of a coherent tribal classification and 59 investigation of the tempo and mode of evolution of Bombacoideae, which is crucial for 60 understanding the diversification of the group and of the Neotropical flora. Here, we use 61 newly generated DNA sequence data of the trnS-trnG spacer region of plastid DNA 62 (cpDNA) and the External Transcribed Spacer (ETS) of the nuclear ribosomal DNA 63 (nrDNA), in combination with previously studied markers, to better