Revisiting the Phylogeny of Bombacoideae (Malvaceae)

Accepted Manuscript

Revisiting the phylogeny of Bombacoideae (Malvaceae): Novel relationships, morphologically cohesive clades, and a new tribal classification based on multilocus 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

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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)

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

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,

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

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 resolve the

64 phylogeny of Bombacoideae. Specifically, we provide much-improved taxon sampling,

65 including around 70% of the known species and 100% of the genera of Bombacoideae.

66 In addition, we assess the evolution of selected morphological traits in order to find

67 synapomorphies that could aid in the identification of major clades.

69 2. Material and methods

70 2.1. Taxon sampling strategy

71 We sampled representatives of all genera of Bombacoideae and 108 of 160 known

72 species, including most species of Ceiba (15 of 18 species, four of which were

73 described in Chorisia: C. chodatii, C. insignis, C. speciosa, and C. ventricosa) and

74 Pseudobombax (23 of 25 species plus Pseudobombax sp. and P. aff. campestre [Mart.]

75 A.Robyns) (Appendix 1). Septotheca tessmannii Ulbr., the putative sister lineage to the

76 core Bombacoideae was also included, as were seven former Bombacaceae that are now

77 thought to fall outside Bombacoideae (Baum et al., 2004; Duarte et al., 2011):

78 Chiranthodendron pentadactylon Larreat., Fremontodendron californicum (Torr.)

79 Coville, Hampea appendiculata Standl., Ochroma pyramidale (Cav. ex Lam.) Urb.,

80 Patinoa sphaerocarpa Cuatrec., Pentaplaris dorotae L.O.Williams & Standl., and

81 Phragmotheca ecuadorensis W.S.Alverson. In all combined analyses, Sterculia

82 lanceolata Cav. and Sterculia nobilis Sm. were included as more distant outgroups to

83 root the tree. Sequences were obtained from 63 specimens collected in the field, 21

84 herbarium specimens, and 43 samples used in Duarte et al. (2011) (Appendix 1).

86 2.2. DNA Extraction, amplification, and sequencing

87 All sequences of matK and trnL-trnF plus 31 sequences of ITS from previous

88 phylogenetic studies (Alverson et al., 1999; Baum et al., 2004; Duarte et al., 2011) were

89 obtained from GenBank. For new sequences of ITS and the newly explored ETS and

90 trnS-trnG, laboratory work was performed at the Plant Molecular Systematics

91 Laboratory (LAMOL) of Feira de Santana State University (UEFS). Total DNA was

92 extracted from leaf tissue using DNeasy plant mini kits (Qiagen, Valencia, California).

93 The manufacturer’s protocol of the DNeasy plant mini kit was followed with the

94 exception of eluting with 50 µL, instead of 100 µL, of AE buffer in order to yield a

95 higher concentration of DNA.

96 Amplification was carried out in a 9700 GeneAmp Thermocycler (Applied

97 Biosystems, Singapore). Polymerase chain reactions (PCR) were performed using the

98 TopTaq Master Mix Kit (QIAGEN GmbH, Hilden, Germany) following the

99 manufacturer’s protocol and 5 pmol of each primer. Quantities of DNA for reactions 6

100 were not measured. In the approach used for ITS and ETS, the PCR reaction also

101 included 0.2 µL of BSA 0.3% (bovine serum albumin), 2 µL of betaine 5 M, and 0.2 µL

102 of DMSO 99.5% (dimethyl sulfoxide).

103 The ETS region was amplified using the primers 18S-IGS, developed by Baldwin

104 and Markos (1998) and AcR2, designed by J. Miller (CSIRO, Canberra) and used in

105 Acacia (Ariati et al., 2006) and Calliandra (Souza et al., 2014). This gene region was

106 amplified using the following PCR conditions: 1) an initial denaturing step of 97˚C for

107 1 minute; 2) 40 cycles of denaturing at 97˚C for 10 seconds, annealing 55˚C for 30

108 seconds, and extension at 72˚C for 20 seconds; and 3) a final extension step of 72˚C for

109 7 minutes.

110 The ITS region was amplified using the plant specific primers, ITS17 and ITS26

111 (Sun et al., 1994) and the following PCR conditions: 1) an initial denaturing step of

112 94˚C for 3 minutes; 2) 28 cycles of denaturing at 94˚C for 45 seconds, annealing 54˚C

113 for 60 seconds, and extension at 72˚C for 90 seconds; and 3) a final extension step of

114 72˚C for 7 minutes.

115 The trnS-trnG spacer region of chloroplast DNA was amplified using primers trnS

116 (GCU) and trnG (UCC), designed by Hamilton (1999). This chloroplast spacer region

117 was amplified using the following PCR conditions: 1) an initial denaturing step of 94˚C

118 for 1 minute, 2) 30 cycles of denaturing at 94˚C for 30 minute, annealing 55˚C for 40

119 seconds, and extension at 72˚C for 60 seconds; and 3) a final extension step of 72˚C for

120 5 minutes.

121 All PCR products were visualized using agarose gel electrophoresis and

122 successfully amplified products were cleaned using QIAquick PCR purification kits

123 (QIAGEN, Valencia, California) or by enzymatic treatment with Exonuclease I and

124 shrimp alkaline phosphatase (ExoSapIT, GE Healthcare, Buckinghamshire, U.K.) using 7

125 protocols recommended by manufacturers. The QIAquick protocol used a final elution

126 with 30 µL instead of 50 µL.

127 Cleaned PCR products were cycle-sequenced with the same primers as used for

128 amplifications using the Big Dye Terminator kit version 3.1 (Applied Biosystems,

129 Foster City, California, U.S.A.), except for ITS for which we used primers ITS4 (White

130 et al., 1990) and ITS92 (Desfeux and Lejeune, 1996). Complementary strands for each

131 region were sequenced using the automatic sequencers Spectrumedix SCE9624 and

132 ABI3130XL at LAMOL-UEFS. All newly generated sequences were uploaded to

133 GenBank (Benson et al., 2010) (see Appendix 1).

134

135 2.3. Data matrix and alignment

136 Complementary strands were combined and base-calling verified with the Staden

137 package (Staden, 1996). Alignments were performed in Muscle (Goujon et al., 2010)

138 and corrected by eye in Mesquite v3.02 (Maddison and Maddison, 2010), when

139 necessary. Six data matrices were constructed: 1) ETS only, 2) ITS only, 3) trnS-trnG

140 only, 4) ETS and ITS (nuclear matrix), 5) matK, trnS-trnG, trnL-trnF (plastid matrix),

141 6) ETS, ITS, matK, trnS-trnG, trnL-trnF (combined matrix).

142 were included in the combined matrices assuming that data sets differing considerably

143 in gene and taxon sampling can be gainfully combined (Cho et al., 2011) and frequently

144 increase the accuracy of phylogenies (Jiang et al., 2014). We ran SATé-II (Liu et al.,

145 2012) iterative alignment program with DendroPy 4.0.0 (Sukumaran and Holder, 2010)

146 using default settings on ITS dataset alone, ETS alone, and the two combined to check

147 the effect of alignments errors. All automatic alignment methods and manual

148 corrections produced similar results. The resulting matrices were submitted to

149 TreeBASE (study number S18447). 8

150

151 2.4. Phylogenetic analyses

152 Phylogenies were inferred from each data set using maximum parsimony,

153 maximum likelihood, and Bayesian analyses. A heuristic parsimony search with 10,000

154 random taxon-addition replicates was performed on each dataset in PAUP* 4.0b10

155 (Swofford, 2002) using tree bisection-reconnection (TBR) branch swapping and saving 156 15 trees per replicate. Trees saved in this first round were used as starting trees in a

157 second search using the same parameters and saving a maximum of 15,000 trees. A

158 total of 1,000 bootstrap pseudoreplicates (Felsenstein, 1985) were performed using

159 heuristic searches, TBR branch swapping, simple taxon addition, and saving 15 trees

160 per replicate. Gaps were treated as missing data.

161 MrModeltest version 2.3 was used to determine the model of evolution that best

162 fit the data for maximum likelihood and Bayesian inference analyses (Nylander, 2004).

163 Bayesian and maximum likelihood analyses were performed in MrBayes version 3.2.3

164 (Ronquist et al., 2012) and RAxML version 8.1 (Stamatakis, 2006, 2014), respectively,

165 using the Cyberinfrastructure for Phylogenetic Research (CIPRES) Portal 2.0 (Miller et

166 al., 2010). For Bayesian analyses, two independent MCMC runs were conducted, each

167 composed of four chains (one cold and three heated chains) of 50 million generations

168 with sampling every 10,000 generations. The convergence of the runs was assessed by

169 checking the standard deviation of split frequencies. program Tracer version 1.6

170 (Rambaut et al., 2014) was used to confirm stationarity of likelihood scores was

171 achieved early in the chain, and trees from the initial 25% of generations were discarded

172 as burn-in. Remaining trees were summarized in a consensus that included posterior

173 probabilities as branch support estimates. Maximum likelihood (ML) tree searches were

174 implemented with 1,000 fast bootstrap replicates under the GTRCAT model unlinked

175 for each partition.

176 Congruence among data partitions was assessed using the incongruence length

177 difference (ILD) test (Farris et al., 1995) as implemented in PAUP* v4.0b10 (Swofford,

178 2002) through a heuristic parsimony search with 500 random taxon addition replicates,

179 TBR branch swapping, and saving 10 trees per replicate. The possible presence of ITS

180 pseudogenes was investigated through comparison of length and substitution rates in

181 fast-evolving (ITS 1-2) and conserved (5.8S) regions and the presence of polymorphic

182 specimens, following the recommendation of Bailey et al. (2003).

183

184 2.5. Ancestral reconstruction of morphological traits in Bombacoideae

185 We analysed the evolution of five morphological characters often used to

186 recognize groups in the subfamily (see Supplemental data S1 – S2 and S5 – S7 with the

187 online version of this article). Although most traditional systematic and phylogenetic

188 studies in Bombacoideae have focused on floral characters (see Introduction), they have

189 been marked by several conflicts (Fig. 1), which seem to be common for taxonomic

190 classifications based on floral traits of Neotropical diverse groups (e.g., Cardoso et al.

191 2012, 2013; Lohmann & Taylor, 2014). The characters and the states coded were as

192 follows: i) Prickles on trunk or branches: absent, present; ii) Calyx shape: lobed,

193 truncate, laciniate; iii) Endocarp type: undifferentiated, papyraceous, spongy, kapok; iv)

194 Seed shape: non-winged, winged; v) Seed number per fruit: 1 – 4, 10 – 30, 50 – 800.

195 The morphological data were compiled from herbarium, observations, and the literature

196 (Baum, 1995; Bentham, 1843, 1862; Cuatrecasas, 1950, 1953, 1954a,b; Ducke,

197 1935a,b, 1938; Dugand, 1943; Gibbs and Alverson, 2006; Gibbs and Semir, 2003;

198 Oliver, 1876; Paula, 1969; Pittier, 1914, 1916, 1921; Robyns, 1963; Ruiz and Pavon, 10

199 1797; Shepherd and Alverson, 1981; Steyermark, 1987; Ulbrich, 1914). We would note

200 that prickles in Bombax, Ceiba, and Spirotheca have a distinctive, sharp-pointed

201 morphology that differs from, for example, Cavanillesia “spines,” which are corky,

202 irregularly shaped protuberances (see Supplemental data S1, F).

203 Ancestral state reconstructions were performed using ML, implemented in

204 Mesquite v3.02 (Maddison and Maddison, 2010), using the Markov k-state 1 parameter

205 (Mk1) model of evolution (Pagel, 1999; Schluter et al., 1997), which assumes equal

206 rates of change between any two character states. We reconstructed the five characters’

207 evolution over a sample of 1,000 post burn-in trees derived from BI analysis of the

208 combined data. For these analyses, we pruned outgroup taxa in order to avoid ancestral

209 reconstruction being biased by the low diversity sampled for outgroups, taking care to

210 keep the branch lengths of the trees unchanged. The ancestral states supported by a

211 threshold log-likelihood ratio of 2.0 were summarized on the BI 50% majority rule

212 consensus tree. When ancestral state reconstruction was ambiguous (character state was

213 not definitely resolved), proportional likelihoods of the alternative states were provided.

214

215 3. Results

216 3.1. Analysis of individual data sets

217 We generated 83 new sequences of ETS, 52 of ITS, and 61 of trnS-trnG for a total

218 of 196 new sequences. The Akaike Information Criterion in MrModeltest found GTR +

219 Γ as the best fitting model for ETS and ITS, GTR + I + Γ for matK and trnS-trnG, and

220 HKY + Γ for trnL-trnF. Summary data for individual and combined data sets, and

221 parsimony results, are presented in Table 1 and Figs. S3 – S4. The trnS-trnG data set

222 had both the lowest number and percentage of informative characters. The ETS data set

223 had the highest percentage of parsimony informative characters whereas ITS had the

224 highest number of informative characters (Table 1).

225 The ILD test suggested a lack of significant conflict among the three cpDNA data

226 sets (p=0.052), despite matK yielding a distinct optimal tree. There was also no

227 evidence of discordance between the two nuclear markers (p=0.876). The ILD test

228 suggested, however, existence of significant conflict between the nuclear and plastid

229 data (p=0.002). A major cause of conflict related to the placement of the genus

230 Spirotheca. The matK data support Spirotheca as sister to Ceiba, ITS data set support it

231 as sister to Bombax, trnL-trnF placed it in a basal grade with Pochota fendleri and

232 Pseudobombax, the ETS data set resolved it as sister to a clade composed by Ceiba and

233 Pachira s.l., and trnS-trnG lacked relevant resolution. Since matK had the most

234 divergent signal of relationships for Spirotheca (S. rosea), this sequence was excluded

235 from combined analyses. After removing this sequence an ILD test no longer detected

236 significant discordance between the plastid and nuclear data sets.

237 The five genes supported somewhat different outgroup relationships and also

238 differed in the optimal branching order among the major bombacoid clades. However,

239 few of the deep relationships gained strong support from any one gene, leading us to

240 conclude that the combined data set should provide the best estimate of the phylogenetic

241 relationships of Bombacoideae. This tree is shown in Figure 2, with ML bootstrap

242 support (BS) and Bayesian posterior probabilities (PP) used to summarize clade

243 robustness.

244

245 3.2. Clades of Bombacoideae

246 The combined analysis supported the monophyly of the core Bombacoideae clade

247 (BI = 1.0; ML =94), identified previously (Baum et al., 2004; Duarte et al., 2011). The 12

248 following three major clades of the core Bombacoideae emerged from our combined

249 analysis of nrDNA and cpDNA, all with clade PP of 1.0: 1) the “winged seed clade”

250 containing Bernoullia, Gyranthera, and Huberodendron; 2) the “spongy endocarp

251 clade” comprising Adansonia, Aguiaria, Catostemma, Cavanillesia, and Scleronema;

252 and 3) the “Kapok clade” including Bombax, Ceiba, Eriotheca, Pachira,

253 Pseudobombax, Rhodognaphalon, and Spirotheca. Within the Kapok clade, three

254 subordinate clades with PP of 0.97-1.0 were recognized: i) the “Paleotropical Bombax

255 clade” including Bombax and Rhodognaphalon; ii) the “Pachira s.l. clade” composed of

256 Eriotheca and Pachira; iii) the “Striated bark clade” including Ceiba, Neobuchia Urb.,

257 Pochota, Pseudobombax, and Spirotheca (Fig. 2; see Figs. S5 – S7).

258

259 3.3. Ancestral state reconstruction of morphological traits

260 We aimed to reconstruct the evolution of five morphological characters (see

261 Appendix 2) in order to identify possible synapomorphies and infer the character state

262 of nine target nodes, as marked on Fig. 2. The optimizations were mostly unambiguous,

263 allowing confident assignment of ancestral state for most of the target nodes and the

264 identification of synapomorphies for the major clades (Table 2; Figs. 3 – 4, S5 – S7).

265 Only two nodes showed any ambiguity: the last common ancestor of the core

266 Bombacoideae was ambiguous for endocarp type (proportional likelihood of the

267 probable alternative states: papyraceous: 0.5, spongy: 0.3, kapok: 0.19) and seed

268 number per fruit (proportional likelihood of the probable alternative states: 50 to 800:

269 0.52, 10 to 30: 0.44); and the last common ancestor of the Kapok clade was unresolved

270 for the presence/absence of prickles (proportional likelihood of the probable alternative

271 states: present: 0.856, absent: 0.144).

272 13

273 4. Discussion

274 4.1. Monophyly and major clades of Bombacoideae

275 Our analyses confirmed the monophyly of the core Bombacoideae and of all

276 genera except for Pachira (see ‘The Pachira s.l. clade’ below). The monospecific

277 Septotheca was well supported as falling outside of the core Bombacoideae, as

278 suggested by previous molecular phylogenies (Baum et al., 2004; Duarte et al., 2011). 279 Septotheca inhabits seasonally flooded Amazonian forests and has simple leaves, very

280 distinct from the palmately compound (very rarely unifoliate) leaves of the core

281 Bombacoideae but similar to outgroups.

282 The three major bombacoid lineages inferred from the combined analysis -- the

283 Winged seed clade, the Spongy endocarp clade, and the Kapok clade -- were also

284 recovered in a previous analysis based on ITS, matK, and trnL-trnF data sets (Duarte et

285 al., 2011). These lineages are morphologically well characterized in relation to floral

286 and fruit traits, though fruit characters have previously been under-emphasized in

287 bombacoid systematics.

288 Our analyses of five morphological traits identified useful synapomorphies for

289 major clades, since there was little ambiguity in ancestral state reconstructions.

290 Ambiguity regarding prickles in the ancestral of the Kapok clade reflects homoplasy

291 (see Fig. S5) as will be more fully discussed.

292

293 4.1.1. The Winged seed clade

294 This clade is sister to the other two lineages of the core Bombacoideae and is

295 composed of about ten species of the genera Bernoullia, Gyranthera, and

296 Huberodendron. Representatives of this clade are huge, buttressed trees inhabiting wet

297 forests in the Neotropics. The fruits are typically dehiscent (contrary to Duarte et al.,

298 2011: 695) and large (to 30 cm long), enclosing 10 – 30 winged seeds (to ~20 cm long)

299 surrounded by a papyraceous endocarp (Fig. 2; Fig. S2, E – G; see also Cuatrecasas,

300 1950: 88; Gleason, 1934: 109; Oliver, 1876: plate 1170). These fruit and seed characters

301 along with their distinctive scorpioid inflorescences (Fig. S1, H; Ducke, 1935a;

302 Gleason, 1934; Oliver, 1873; Pittier, 1921) represent putative morphological

303 synapomorphies of the Winged seed clade and useful diagnostic characters for

304 identification purposes when combined with other traits.

305 Species in the Winged seed clade also share staminal filaments that are

306 completely connate bearing distal, sessile, “polythecate” anthers (i.e., with many sessile

307 thecae on an elongated staminal tube), though this may be plesiomorphic for the

308 Malvatheca clade (i.e., Malvoideae + Bombacoideae) (von Balthazar et al., 2006).

309 Representatives of the Winged seed clade also have subaggregate wood parenchyma,

310 with very narrow lines regularly alternating with 2 – 4 tangential rows of fibers that may

311 extend from ray to ray, in cross section, mostly small flowers (ca. 2.5 cm long), and

312 lobed calyces (Fig. S1, H; Cuatrecasas, 1950; Détienne et al., 1983; Gleason, 1934;

313 Oliver, 1876; Pittier, 1914, 1921).

314

315 4.1.2. The Spongy endocarp clade

316 This clade encompasses about 30 species of the mostly Amazonian genera

317 Aguiaria, Catostemma, Cavanillesia, and Scleronema, together with eight or nine

318 species of Adansonia, a genus native to the Paleotropics. The molecular data are

319 ambiguous as to whether Cavanillesia or Adansonia are sister to the rest of the clade

320 (Fig. S3). Due to this ambiguity, the polarity of seed number per fruit for the Spongy

321 endocarp clade was unresolved. The four Neotropical genera form a morphologically 15

322 cohesive group distinguished from other bombacoids by the relatively small flowers, 1 –

323 2-ovulate locules, and widely spaced bands in wood parenchyma (Détienne et al., 1983;

324 Metcalfe & Chalk, 1950; Paula, 1969, 1975; Record, 1949). Furthermore, whereas

325 Adansonia has many seeds per fruit and the likely ancestral condition of palmately

326 compound leaves, the Neotropical taxa have 1 – 4 seeds and simple (Cavanillesia) or 1-

327 foliolate leaves (with the sole exception of Catostemma digitatum which has 3 – 5-

328 foliolate leaves).

329 The Spongy endocarp clade was recovered in previous phylogenetic assessment

330 (Duarte et al., 2011) except for the inclusion of Aguiaria, which is here evaluated in

331 phylogenetic studies for the first time. The morphological similarities of Aguiaria to

332 Catostemma and Scleronema were first noted by Ducke (1937). Baum et al. (2004)

333 speculated that Aguiaria should be phylogenetically close to these two genera, a view

334 which is supported by the present study. Aguiaria, Catostemma, and Scleronema share

335 the unifoliolate leaves. The latter two genera, however, are distinguished by having the

336 3-locular ovaries as a possible synapomorphy. These three genera are endemic to

337 Amazonian wet forests whereas Cavanillesia, the other Neotropical genera, inhabits

338 seasonally dry forests.

339 Genera within the clade are rather variable in floral traits, and characterized by

340 flowers from a few centimeters (Aguiaria, Catostemma, Cavanillesia, and Scleronema)

341 to almost 30 cm long (Adansonia) and stamens ranging from 50 to more than one

342 thousand. All species have lobed calyces, but this is a plesiomorphic trait for the core

343 Bombacoideae inherited by the Winged seed clade rather than a synapomorphy for this

344 clade (Fig. S1, G – H). Additional support for the monophyly of the spongy endocarp

345 clade comes from fruit morphology (Fig. 3, Fig. S2).

346 At first glance, species in the Spongy endocarp clade have diverse fruit types (see

347 Fig. S2, A – D). Fruits are woody and indehiscent in Adansonia and Scleronema (Baum,

348 1995; Bentham, 1862), large samaras with five papyraceous wings in Cavanillesia

349 (Ruiz and Pavon, 1797), and woody, tardily dehiscent capsules in Catostemma

350 (Bentham, 1843; Shepherd and Alverson, 1981). In Aguiaria, the small fruits (less than

351 4 cm long) are unique among Angiosperms in having a coriaceous, dehiscent exocarp

352 that splits off into five valves, all of which remain attached to the base of an indehiscent

353 endocarp (see Fig. S2, C; see also line drawing in Ducke 1935b). In spite of these

354 differences, most taxa in this clade exhibit a spongy endocarp (Barroso et al., 1999,

355 2002; Baum, 1995; Ducke, 1935b; Kubitzki and Bayer, 2003; Paula, 1969; Schumann,

356 1886). Catostemma may be the exception, however: while fruit-derived fibers cover the

357 seeds, they have been described as mucilaginous rather than spongy (Shepherd and

358 Alverson, 1981). It may also be noteworthy that in Adansonia, Aguiaria, and

359 Catostemma, the endocarp tends to adhere to the seed surface (see Fig. S2, A – C).

360 We hypothesize that spongy endocarps constitute a morphological synapomorphy

361 for this clade, as strongly supported by ancestral state reconstruction (Fig. S3).

362 Developmental and anatomical studies of bombacoid fruits are needed to shed light on

363 homologies of the endocarps among Spongy endocarp clade genera as well as to

364 identify processes that may have generated the diversity of fruits in this group.

365

366 4.1.3. The Kapok clade

367 This clade constitutes the largest lineage of the core Bombacoideae, including ca.

368 120 Neotropical species, except for Bombax and Rhodognaphalon, which are

369 Paleotropical. Members of the clade have loculicidal capsules with endocarp modified

370 into woolly tissue (”kapok”) that surrounds non-winged, typically lightweight, and 17

371 numerous seeds (see Fig. S2, H – K), though the kapok is reduced and seeds larger in a

372 few, derived species. Fruits with kapok and truncate calyces (see Fig. 2, S1-I, S6) are

373 putative synapomorphies for this clade as supported by our ancestral character state

374 reconstructions (Figs. 3, S6).

375 The Kapok clade is composed of genera historically placed in Bombax s.l. (i.e.,

376 Bombax, Bombacopsis Pittier, Eriotheca, Pachira, Pseudobombax, and

377 Rhodognaphalon, Rhodognaphalopsis A.Robyns) plus Ceiba and Spirotheca. In

378 addition to kapok and truncate calyces, most species have seeds that are smooth (i.e.,

379 non-striate), maculate or dotted, and occasionally have a raised hilum. A possible

380 chemical synapomorphy for this clade is the presence of cyanidin-3,5-diglucoside

381 (Paula et al., 1997; Refaat et al., 2013), but studies on several genera are lacking. The

382 presence of prickles on trunks or branches is probably plesiomorphic for the Kapok

383 clade with two subsequent losses, one in the Pachira s.l. clade and one in

384 Pseudobombax (see Fig. S5).

385 The first cladogenesis within the Kapok clade separates the two Paleotropical

386 genera, Bombax and Rhodognaphalon from the much more diverse Neotropical taxa in

387 the Pachira s.l. and Striated bark subordinate clades.

388

389 4.1.3.1. The Paleotropical Bombax clade

390 This clade includes the Paleotropical genera Bombax and Rhodagnaphalon.

391 Hutchinson (1967) argued in favor of placing Rhodognaphalon in synonymy with

392 Pachira based on the shared presence of elongated flowers. Previous molecular analyses

393 rejected this view but the phylogenetic placement of the genus remained uncertain

394 (Duarte et al., 2011). Our combined analysis supports Rhodognaphalon as sister to

395 Bombax, a relationship not previously hypothesized. All other performed analyses of

396 individual or combined markers, however, were equivocal on the phylogenetic

397 placement of Rhodognaphalon.

398 The two genera in the Paleotropical Bombax clade share the presence of prickles

399 on the trunks and/or branches (Robyns, 1963: 15). Although adults of Rhodognaphalon

400 are often unarmed, prickled saplings have been reported (Voorhoeve, 1965: 72). The

401 presence of coriaceous (vs. woody) valves, five-winged columellae that are persistent in

402 fruit, and red petals are probable synapomorphies for this clade, though white or whitish

403 petals have been reported for R. brevicuspe (Sprague) Roberty (Robyns, 1963;

404 Voorhoeve, 1965: 71). According to Robyns (1963) and Voorhoeve (1965),

405 Rhodognaphalon can be distinguished from Bombax mainly based on the oblong-linear,

406 persistent calyces (vs. cupuliform and deciduous), the stamens organized in one whorl

407 (vs. two), the reddish brown (vs. white) kapok, and the larger seeds, which are also

408 fewer in number than in Bombax. Further work on the phylogeny and biogeography of

409 Bombax + Rhodognaphalon is warranted.

410

411 4.1.3.2. The Pachira s.l. clade

412 This clade was recovered in most of our analyses and encompasses about 70

413 Neotropical species of Eriotheca and Pachira. The Pachira s.l. clade corresponds to the

414 Pachira clade of Duarte et al. (2011) and includes various, previously recognized

415 segregates and synonyms of Pachira: Bombacopsis, Pochota Ram.-Goyena, and

416 Rhodognaphalopsis. Due to the lack of distinctive morphological characters,

417 Bombacopsis and Rhodognaphalopsis have often been merged into other genera by

418 recent workers. Steyermark and Stevens (1988) treated them as Pochota, distinguished

419 from the two species they retained in Pachira s. str. by the latter’s very long flowers. 19

420 However, more frequently Bombacopsis and Rhodognaphalopsis are treated synonyms

421 of Pachira (Alverson, 1994; Alverson and Mori, 2002; Alverson and Steyermark, 1997;

422 Fernández-Alonso, 1998, 2003), as supported by our analyses. Pochota was re-

423 established as a monotypic genus (Alverson and Duarte, 2015) based on previous

424 molecular evidence (Duarte et al., 2011) and its phylogenetic and morphological

425 affinities will be discussed below.

426 Striate seeds have previously been identified as a possible synapomorphy for the

427 Pachira s.l. clade (Duarte et al., 2011). Additionally, the alternate eophylls (i.e., first

428 leaves produced by seedlings; Robyns, 1963: 15), the lack of prickles on trunks and

429 branches, and leaflets with brochidodromous venation are possible synapomorphies for

430 the Pachira s.l. clade (though brochidodromous venation is also present in Spirotheca).

431 Taken together, the monophyly of the Pachira s.l. clade is well supported.

432 Whereas Duarte et al. (2011) found support for trees in which Eriotheca is

433 paraphyletic relative to Pachira, our data supported a monophyletic Eriotheca

434 embedded in a paraphyletic Pachira. The prior result might be an artifact of its less

435 dense taxon sampling, but further work is needed before concluding that either genus is

436 non-monophyletic. We identified two clades within Pachira, one composed of

437 Amazonian species and the other of extra-Amazonian species, with the latter appearing

438 more closely related to Eriotheca (see Fig. 2).

439 In the extra-Amazonian Pachira lineage, P. endecaphylla is sister to the other

440 species and is distinguished by numerous, small seeds surrounded by abundant kapok

441 like in Eriotheca. The other four extra-Amazonian species, in contrast, have few, large

442 seeds, and scarce kapok, similar to the Amazonian Pachira insignis.

443 Within the Amazonian Pachira lineage, two sublineages were supported. The first

444 (P. aquatica – P. mawarinumae) is composed of species inhabiting seasonally flooded 20

445 (“igapó”) forests with 7 – 11 leaflets (rarely 5), larger flowers and fruits (both to 30 cm

446 long), and finely hairy exocarps. The second (P. gracilis – P. flaviflora) is composed of

447 species from white-sand vegetation (“campinas” and “campinaranas,” Anderson, 1981),

448 characterized by a reduced number of leaflets (1 – 3, rarely 5), small, slender flowers (to

449 12 cm long), and smaller (to 10cm long), often glabrous fruits.

450 Eriotheca is a South American genus characterized by flowers that are smaller

451 than in Pachira (typically < 4 cm), staminal tubes that are surmounted by entirely free

452 filaments (vs. filaments organized in phalanges) (Robyns, 1963, Duarte et al., 2011),

453 multiflorous cymes (vs. 1 – 2-flowered cymes), and more or less reniform anthers when

454 dehisced (vs. oblong anthers).

455

456 4.1.3.3. The Striated bark clade

457 The genera Spirotheca, Neobuchia, Ceiba, Pochota fendleri, and Pseudobombax

458 together form a well supported clade in the combined analysis. Spirotheca appears as

459 sister to Bombax in the nuclear analysis, though this result is not strongly supported

460 (Fig. S3). With exception of Pseudobombax, members of this clade have prickles on the

461 trunk and/or branches. Besides the prickled trunks and/or branches, Spirotheca,

462 Neobuchia, and Ceiba share a similar floral morphology in having just five or fifteen

463 “filaments” (i.e., lobes at the apex of their staminal columns), stamens with appendages,

464 and 2 – 4-thecate anthers (Gibbs and Alverson, 2006; Gibbs and Semir, 2003).

465 However, it is not yet clear whether these shared floral traits are homologous. The

466 plastid tree, which includes a Spirotheca-Neobuchia-Ceiba clade, supports the

467 homology of these flowers, but the nuclear tree does not. We suspect that the plastid

468 DNA, which has less homoplasy, reflects the true relationships, but analysis of further

469 nuclear markers is needed. 21

470 Except for Spirotheca, a genus of hemiepiphytes mostly found in wet upper-

471 elevation forests, species in the Striated bark clade are predominantly deciduous while

472 flowering, living in areas with seasonal climates. Trunks are characteristically swollen

473 above or below the ground and display longitudinal striations that result from

474 chlorophyll-rich underbark being exposed between vertical bands of the periderm,

475 leaving the trunk with a marbled, grey/green aspect (see Fig. S1, A – E). While

476 conspicuous striations can be absent in adults of Pochota fendleri and Spirotheca, they

477 occur in juvenile plants. The eucamptodromous venation of Ceiba, Pochota fendleri,

478 and Pseudobombax is likely apomorphic for these three genera and differs from the

479 putatively plesiomorphic brochidodromous venation seen in Spirotheca and the Pachira

480 s.l. clade.

481 Our data support the merging of Ceiba and Chorisia Kunth as adopted in the last

482 revision of Ceiba (Gibbs & Semir, 2003). The monospecifc genus Neobuchia was

483 supported as sister to Ceiba, as previously hypothesized by Duarte et al. (2011).

484 Neobuchia paullinae Urb. is endemic to Haiti, and like many Ceiba species, has

485 crenulate to serrate leaflets, deciduous calyces, and a similar androecium.

486 Pochota fendleri has a distinct wood anatomy (Détienne et al. 1983) and our data

487 strongly supported it as sister to Pseudobombax. This relationship has not previously

488 been hypothesized but is supported by the putative synampomorphy of hippocrepiform

489 anthers. Furthermore, members of the Pseudobombax-Pochota fendleri clade differ

490 from its likely sister clade (Spirotheca-Neobuchia-Ceiba) by having calyces persistent

491 in fruit, petals densely covered abaxially by stellate or tufted trichomes, appearing

492 brownish to nigrescent (never white or red), 100 – 1,500 fertile stamens with

493 monothecate anthers, kapok brown (vs. white), fruit columella entire (vs. 5-divided, see

494 Fig. S2, K), and nectariferous glands on the receptacle (absent in Ceiba).

495 Besides the presence of woody prickles on the trunks, Pochota fendleri can be

496 distinguished from all species of Pseudobombax by its heavy wood and the coriaceous

497 valves (vs. woody) that are persistent (vs. caducous) in dehisced fruits. The absence of

498 prickles in Pseudobombax (Carvalho-Sobrinho et al., 2013) represents an unambiguous

499 reversion of this character in the core Bombacoideae. Furthermore, Pseudobombax is

500 characterized by leaflets with entire margins and manifest a distinctive synapomorphy

501 of petioles that are distally dilated or expanded with leaflets having inarticulate

502 petiolules (Carvalho-Sobrinho and Queiroz, 2011; Carvalho-Sobrinho et al., 2014;

503 Robyns, 1963).

504

505 4.2. A new tribal classification of the Bombacoideae

506 Our data do not support the monophyly of Adansonieae sensu Hutchinson (1967),

507 even including Ceiba as proposed by Bakhuizen van den Brink (1924), leaving most

508 species without an obvious tribal affiliation. Therefore, we propose here a new tribal

509 classification of Bombacoideae based on the combined tree derived from the five

510 nuclear and chloroplast markers (Fig. 2) and supplemented by morphological data.

511 It seems logical to recognize the three major clades of core Bombacoideae at the

512 tribal rank. The Winged seed clade does not match any previously-named tribe; we

513 name it Bernoullieae as typified on the genus first described in the clade. The Spongy

514 endocarp clade corresponds to a recircumscribed Adansonieae because it contains the

515 type species of two previously defined tribes, Adansonieae and ‘Catostemmateae’, but

516 the former was the only validly published. The Kapok clade, on the other hand,

517 corresponds to the Takhtajan (1997) tribes ‘Ceibeae’ and Bombaceae (excluding

518 Adansonia and Gyranthera, Fig. 1), but the latter is the sole name validly published at

519 the tribal rank that includes Kapok clade genera. 23

520

521 4.3. Taxonomic treatment

522

523 Tribe Adansonieae Horan., Char. Ess. Fam.: 192. 17 Jun 1847 (as ‘Adansoniae’).–T:

524 Adansonia L. (1759).

525 Important treatments of included taxa: Alverson (1994), Alverson and Mori (2002),

526 Alverson and Steyermark (1997), Baum (1995), Ducke (1935a,b, 1937, 1938), Paula

527 (1969), Robyns (1964), Steyermark (1987)

528

529 Small to large unarmed trees, often with swollen trunks. Leaves simple and 3 – 5-

530 nerved at base, or palmately compound, with pinnately-veined leaflets, sometimes

531 reduced to one leaflet. Inflorescences axillary, terminal, or ramiflorous. Flowers

532 usually small (c. 2.5 cm long), reaching c. 30 cm in length (Adansonia). Calyx

533 lobed or laciniate, sometimes completely enclosing the corolla in mature buds,

534 with lobes reflexed or coiled at the base of the flower at anthesis (Adansonia).

535 Stamens usually 15 – 120 or numerous (Adansonia), filaments partially fused into

536 a cylindrical staminal tube, often apically dilated. Ovary usually 2 – 9-carpellate,

537 ovules per locule few (1 – 2) to numerous (Adansonia). Anthers monothecate.

538 Fruits samaras, woody berries, or tardily dehiscent capsules, endocarp spongy,

539 rarely undifferentiated (Scleronema). Seeds usually few (1 – 4), or numerous

540 (Adansonia), not winged.

541 Genera included: Adansonia, Aguiaria, Catostemma, Cavanillesia, Scleronema.

542 Recommended phylogenetic clade definition: The most inclusive clade containing

543 Adansonia digitata L. but not Bernoullia flammea Oliv., Bombax ceiba L., Ceiba

544 pentandra (L.) Gaertn., or Pachira aquatica Aubl.

545

546 Tribe Bernoullieae Carv.-Sobr., tr. nov.–T: Bernoullia Oliv. (1876).

547 Important treatments of included taxa: Alverson and Mori (2002), Cascante-Marin

548 (1997), Pennington et al. (2004), Robyns (1964)

549 Diagnosis: Inflorescences scorpioid, fruits woody loculicidal capsules with

550 papyraceous endocarps enclosing numerous winged seeds.

551 Large unarmed trees, often buttressed. Leaves palmately compound, 3 – 5-

552 foliolate, pinnately-veined, or 1-foliolate (appearing as simple leaves) and 3-

553 veined at base. Inflorescences terminal, scorpioid. Flowers usually small (ca. 2.5

554 cm in length), but to ca. 15 cm long in Gyranthera. Calyx lobed. Stamens 5 – 20,

555 filaments completely fused in a staminal tube except for short lobes, often

556 laterally cleft on one side. Anthers “polythecate” (i.e., with many sessile thecae on

557 an elongated staminal tube), sometimes spirally twisted. Ovary 5-carpellate,

558 ovules ca. 10 per locule. Fruits large (to 30 cm long), woody loculicidal capsules

559 with papyraceous endocarps. Seeds 10 – 30, large (ca. 6 – 9 cm long), winged.

560 Genera included: Bernoullia, Gyranthera, Huberodendron.

561 Recommended phylogenetic clade definition: The most inclusive clade containing

562 Bernoullia flammea Oliv. but not Adansonia digitata L., Bombax ceiba L., Ceiba

563 pentandra (L.) Gaertn., or Pachira aquatica Aubl.

564

565 Tribe Bombaceae Kunth, Syn. Pl. 3: 258. 28 Feb 1824. –T: Bombax L., nom. et typ.

566 cons. (1753).

567 Important recent treatments: Alverson and Mori (2002), Alverson and Steyermark

568 (1997), Fernández-Alonso (1998, 2001), Gibbs and Alverson (2006), Gibbs and

569 Semir (2003), Robyns (1963, 1964)

570 Trees, rarely shrubs or stranglers (Spirotheca), trunks often swollen and prickled.

571 Leaves palmately compound, 3 – 11-foliolate, rarely 1-foliolate, leaflets

572 pinnately-veined. Inflorescences axillary or terminal, often reduced to solitary

573 flowers. Flowers from 2.5 cm to 30 cm long. Calyx truncate, rarely lobed (Ceiba

574 and Neobuchia). Stamens 5 – 1500, fused in a staminal tube, often organized in

575 groups (phalanges). Anthers monothecate, bithecate (Ceiba and Neobuchia), or 4-

576 thecate and spirally twisted (Spirotheca). Ovary 5 – 8-carpellate, ovules numerous

577 per locule. Fruits loculicidal capsules with kapok. Columella persistent, entire or

578 5-fid. Seeds numerous, not winged, often maculate or striate, small, rarely 2 – 3

579 cm in length.

580 Genera included: Bombax, Ceiba, Eriotheca, Neobuchia, Pachira, Pochota,

581 Pseudobombax, Rhodognaphalon, Spirotheca.

582 Recommended phylogenetic clade definition: The most inclusive clade containing

583 Bombax ceiba L., Ceiba pentandra (L.) Gaertn., and Pachira aquatica Aubl. but not

584 Adansonia digitata L., Bernoullia flammea Oliv., Bombax ceiba L., or Catostemma

585 fragrans Benth.

586

587 KEY TO THE TRIBES OF BOMBACOIDEAE (MALVACEAE)

588

589 1. Inflorescences scorpioid cymes. Filaments fused into a staminal tube that is often

590 laterally cleft on one side; anthers “polythecate” (i.e., with many sessile theceae on

591 an elongated staminal tube), sometimes spirally twisted. Fruits loculicidal capsules

592 with papyraceous endocarp. Seeds winged...... Bernoullieae Carv.-Sobr., tr. nov.

593 1. Inflorescences cymose but not scorpioid. Filaments fused for one-third of their length

594 or less, or if completely fused then never cleft. Anthers appearing 1–2-thecate, or if

595 twisted then 4-thecate. Fruits woody berries, samaras, or loculicide capsules with

596 endocarp spongy or modified into kapok. Seeds not winged.

597 2. Trees, never stranglers. Calyces lobed or laciniate, sometimes completely

598 enclosing the corolla in mature buds. Fruits samaras, woody berries, or loculicidal

599 capsules, with endocarps spongy, rarely undifferentiated, without kapok......

600 ...... Adansonieae Horan. (1847)

601 2. Trees, rarely shrubs or stranglers. Calyces truncate, if lobed then stamens 5(–

602 15), bithecate. Fruits loculicidal capsules with endocarp modified as kapok......

603 ...... Bombaceae Kunth (1824)

604

605 Acknowledgments

606 Thanks are due to Charles Zartman, Christine Bacon, Domingos Cardoso, Marlon

607 Machado, and Paulo Kaminski who provided plant material for this study, and Daiane

608 Trabuco, Elvia Souza, and Luane do Carmo for assistance in the molecular laboratory.

609 We thank the directors and staff of the molecular laboratory in UEFS (LAMOL) and the

610 directors, curators, and staff of the herbaria who loaned specimens, especially Cláudia

611 Gonçalves, Marc Pignal, and Jacques Florence (P), Hans-Joachim Esser (M), Martin

612 Cheek and Cátia Canteiro (K), Lorenzo Ramella and Nicolas Fumeaux (G). We also

613 thank the Fundação de Amparo à Pesquisa do Estado da Bahia–FAPESB (process

614 APP0006/2011), the Conselho Nacional de Desenvolvimento Científico e Tecnológico–

615 CNPq (processes 300811/2010-1 and 563546/2010-7-REFLORA), the Programa de

616 Apoio a Núcleos de Excelência (PRONEX, process PNX0014/2009), and the Sistema

617 Nacional de Pesquisa em Biodiversidade (SISBIOTA CNPq 563084/2010-3/FAPESB

618 PES0053/2011) for financial support. This paper is part of the PhD thesis of JGCS

619 prepared in the Programa de Pós-Graduação em Botânica (PPGBot–UEFS) and

620 supported by a grant from the Coordenação de Aperfeiçoamento de Pessoal de Nível

621 Superior–CAPES. JGCS also thanks CNPq (process 158916/2014-0), the Reflora

622 programme (process BEX 5415/13-6) for a sandwich fellowship, and the Department of

623 Botany of the Smithsonian Institution, Washington, D.C., for a José Cuatrecasas

624 Fellowship. Partial funding for DAB was provided by NSF grant DEB-1354793.

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Table 1. Matrix and tree statistics for the individual and combined data sets.

nrETS nrITS matK trnL-F trnS-G nuclear plastid Combined (nuclear) (nuclear) (plastid) (plastid) (plastid) No. taxa included in each matrix 83 91 58 59 61 107 95 118

Length of aligned matrices (bp) 532 907 2629 1356 1041 1439 5026 6465

540 201 185 884 798 1686 No. variable characters 344 (64.7%) 412 (15.7%) (59.8%) (14.8%) (17.8%) (61.6%) (15.9%) (26.1%) 391 166 104 64 655 334 994 No. parsimony informative characters 264 (49.6%) (43.3%) (6.3%) (7.7%) (6.1%) (45.6%) (6.6%) (15.4%)

102 12255 3105 4587 > 15,000 821 5193 315

1065 2053 590 273 249 3143 1131 4161

0.5352 0.4584 0.7678 0.8278 0.8193 0.4808 0.7807 0.5703

0.4902 0.4066 0.5798 0.7235 0.6281 0.4314 0.6101 0.4727

0.8370 0.7709 0.7405 0.8813 0.8490 0.7932 0.7971 0.7946

Notes: MP = Maximum parsimony.

Table 2. Results of the ancestral state reconstruction of selected morphological traits of Bombacoideae (Malvaceae) using a maximum likelihood approach. Ancestral character state inferred using a log-likelihood ratio threshold of 2.0.When ancestral state reconstruction for a node is ambiguous, proportional likelihoods of the alternative states are provided. Rate of transition: Mesquite's estimated rate of change parameter under the Markov k-state 1 parameter (Mk1) model of evolution, in which any particular change between characters' states is equally probable.

Node number & reference clade Rate of 1-Ancestor of core 2-Winged 3-Spongy 4- 5- 6- 7- 8-Striated 9- Trait transitio Bombacoideae seed clade endocarp ‘Catostem Kapok- Paleotropical Pachira bark clade Pochota+Pseu n clade matae’ Clade Bombax s.l. clade dobombax clade Prickles present: on trunk 0.856 4,74 absent absent absent absent present absent present present or absent: branches 0.144 Calyx 3,85 lobed lobed lobed lobed truncate truncate truncate truncate truncate shape papyraceous: 0.5 Endocarp 1,48 spongy: 0.3 papyraceous spongy spongy kapok kapok kapok kapok kapok type kapok: 0.19 Seed non- non- non- non- 1,48 winged winged non-winged non-winged non-winged shape winged winged winged winged 50 – 800: Seed 50 – 800: 0.52 0.84 number 3,94 10 – 30 1 – 4 50 – 800 50 – 800 50 – 800 50 – 800 50 – 800 10 – 30: 0.44 1 – 4: per fruit 0.14

FIGURE CAPTIONS

Fig.1. Taxonomic placement of genera of Bombacoideae (Malvaceae) according to different classification systems. Dotted lines indicate changes in genus circumscription.

Genera described after the previous treatment are indicated by an asterisk. Quotation marks indicate tribes not validly published.

Fig. 2. Bayesian inference (BI) consensus cladogram based on the combined (plastid and nuclear) data set showing the three major lineages of Bombacoideae (Malvaceae) recovered: the Winged seed clade (green), the Spongy endocarp clade (orange), and the

Kapok clade (blue). Numbers above the branches represent Bayesian posterior probabilities ( ≥ 0.75) as revelead with MrBayes, numbers below the branches indicate maximum likelihood (ML) bootstrap support values ( ≥ 50%) as obtained with RAxML; only values for nodes discussed on the text are shown. Numbers within circles indicate target nodes for reconstruction of ancestral states of selected morphological traits using

ML. 2A. Relationships in the Spongy endocarp clade. Photos on the right show the fruits of (a) Huberodendron swietenioides, (b) Adansonia digitata, and (c) Ceiba jasminodora. Photo credits: (a) University of Michigan Herbarium, (b) J. Pierre, (c)

Marlon Machado. 2B. Relationships in the Kapok clade. Species names in the

Amazonian and extra-Amazonian Pachira clades refer to basionyms, except by names described in Bombax, to illustrate historical generic concepts.

Fig. 3. Ancestral character states reconstruction of endocarp type as shown on the BI majority rule consensus cladogram. Ancestral states reconstructions were performed with ML optimization as implemented in Mesquite v 3.02. Branch lengths are illustrated here without scale, for aesthetical purposes only. Colors represented in the nodes 43

indicate the proportional likelihood of the states reconstructed for each node (see text).

Numbers within circles indicate target nodes. Character transitions discussed in the text are indicated by arrows.

Fig. 4. Ancestral character states reconstruction of seed shape as shown on the BI majority rule consensus cladogram. Ancestral states reconstructions were performed with ML optimization as implemented in Mesquite v 3.02. Branch lengths are illustrated here without scale, for aesthetical purposes only. Colors represented in the nodes indicate the proportional likelihood of the states reconstructed for each node (see text).

Numbers within circles indicate target nodes. The character transition to winged seeds is indicated by an arrow.

SUPPLEMENTAL DATA (S1 – S7)

S1. Bark and floral characters in Bombacoideae (Malvaceae). A. Unarmed striated bark of Pseudobombax calcicola. B. Prickled striated bark of Ceiba erianthos. C–D. Striated bark of Spirotheca elegans: prickled bark of an adult individual (C) and unarmed seedling (D). E. Prickled striated bark of Pochota fendleri. F. Unstriated bark of

Cavanillesia umbellata with corky irregularly shaped protuberances. G. Lobed calyces of Cavanillesia umbellata. H. Lobed calyces of Huberodendron allenii. I. Truncate calyx of Pseudobombax munguba. Photo credits: A–D and F: M. Machado; E, G, and I:

J. Carvalho-Sobrinho; H: R. Aguilar.

S2. Fruit and seed characters of Bombacoideae (Malvaceae). A. Woody berry (cracked to expose the inner part) of Adansonia. B. Woody capsule with colored endocarp of

Catostemma. C. Coriaceous 1-seeded-capsule of Aguiaria. D. Samara and seed of

Cavanillesia. E–G. Woody capsule with winged seeds of Huberodendron. H–K. Woody capsule with kapok of Pseudobombax (H), Eriotheca (I), and Ceiba (J–K). Photo credits: A: J. Marie; B: C. Zartman; C: D. Cardoso; D: Steve (Newton’s Apple); E, G:

Cuatrecasas (1950, Fig. 9 and 11); F: University of Michigan Herbarium; H: M

Machado; I: J. Carvalho-Sobrinho; J: Martius & Zuccarini (1824, tab. 97); K: P.

Hendrigo.

S3. Bayesian inference (BI) consensus cladogram of combined nuclear data (ETS and

ITS) showing relationships in Bombacoideae (Malvaceae). Numbers above the branches represent Bayesian posterior probabilities ( ≥ 0.75) as revelead with MrBayes, numbers

below the branches indicate maximum likelihood bootstrap support values ( ≥ 50%) as obtained with RAxML. Numbers within circles indicate target nodes.

S4. Bayesian inference (BI) consensus cladogram of combined plastid data (matK, trnL-

F, and trnS-trnG) showing relationships in Bombacoideae (Malvaceae). Numbers above the branches represent Bayesian posterior probabilities ( ≥ 0.75) as revelead with

MrBayes, numbers below the branches indicate maximum likelihood bootstrap support values ( ≥ 50%) as obtained with RAxML. Numbers within circles indicate target nodes.

S5. Ancestral character states reconstruction of prickles on trunk or branches as shown on the BI majority rule consensus cladogram. Ancestral states reconstructions were performed with ML optimization as implemented in Mesquite v 3.02. Branch lengths are illustrated here without scale, for aesthetical purposes only. Colors represented in the nodes indicate the proportional likelihood of the states reconstructed for each node

(see text). Numbers within circles indicate target nodes. Character transitions discussed in the text are indicated by arrows.

S6. Ancestral character states reconstruction of the calyx shape as shown on the BI majority rule consensus cladogram. Ancestral states reconstructions were performed with ML optimization as implemented in Mesquite v 3.02. Branch lengths are illustrated here without scale, for aesthetical purposes only. Colors represented in the nodes indicate the proportional likelihood of the states reconstructed for each node (see text). Numbers within circles indicate target nodes. Character transitions discussed in the text are indicated by arrows.

S7. Ancestral character states reconstruction of seed number per fruit as shown on the

BI majority rule consensus cladogram. Ancestral states reconstructions were performed with ML optimization as implemented in Mesquite v 3.02. Branch lengths are illustrated here without scale, for aesthetical purposes only. Colors represented in the nodes indicate the proportional likelihood of the states reconstructed for each node (see text).

Numbers within circles indicate target nodes. Character transitions discussed in the text are indicated by arrows.

Appendix 1. Origin and voucher specimens for DNA sequences used in the study.

Numbers preceded by KM or KR refer to newly generated sequences. “....” = missing sequences.

Taxon, collection locality, voucher collector and number, (Herbarium acronyms

[following the Index Herbariorum (http://sweetgum.nybg.org/ih/]), GenBank accession numbers: nrETS, nrITS, matK, trnL-trnF, or trnS-trnG.

Adansonia digitata L., Pacific Tropical Garden acc. no. 770032002, KM453023,

HQ658372*, AY321168*, HQ696738*, ....; Adansonia gregorii F.Muell., Wendel s.n.

(ISC), ...., HQ658374*, HQ696688*, HQ696740*, KM453112; Adansonia grandidieri

Baill., D.A.Baum 345 (MO), ...., HQ658373*, HQ696687*, HQ696739*, ....; Adansonia

“kilima” Pettigrew, K.L. Bell, Bhagw., Grinan, Jillani, Jean Mey., Wabuyele & C.E.

Vickers, J. Pettigrew 402, …., JN400327*, …., JN400300*, ….; Adansonia madagascariensis Baill., accession A, …., AF028532*, …., JN300407*, ….;

Adansonia perrieri Capuron, isolate 208, …., AF028538*, …., JN400292*, ….;

Adansonia rubrostipa Jum. & H.Perrier, …., AF028531*, …., …., ….; Adansonia suazerensis H.Perrier ...., AF028529*, …., …., ….; Adansonia za Baill., D.A.Baum 357

(MO), ...., AF028536*, HQ696689*, HQ696741*, ....; Aguiaria excelsa Ducke,

D.Cardoso 3343 (HUEFS), KM453024, KM453161, ...., ...., KM453109; Bernoullia flammea Oliv., T.S.Cochrane s.n. (WIS), KM453027, HQ658366*, HQ696685*,

HQ696732*, ....; Bombax anceps Pierre, KYUM<174>, …., …., AB924835*, …., ….;

Bombax buonopozense P.Beauv., Pac. Trop. Bot. Gard. acc. no. 770474001,

KM453025, HQ658376*, AY321171*, HQ696742*, ....; Bombax ceiba L.,

J.G.Carvalho-Sobrinho 3073 (HUEFS), KM453026, KM453163, ...., ...., KM453109,

W.S.Alverson s.n. (WIS), ...., ...., HQ696690*, HQ696743*, ....; Catostemma albuquerquei Paula, J.G.Carvalho-Sobrinho 3117 (HUEFS), KM453028, KM453165,

...., ...., KM453111; Catostemma fragrans Benth., W.S.Alverson 4030 (WIS),

KM453029, HQ658370*, AY589069*, HQ696736*, KM453121; Catostemma milanezii Paula, J.G.Carvalho-Sobrinho 3116 (HUEFS), KM453030, KM453166, ....,

...., ....; Cavanillesia chicamochae Fern.Alonso, D. Castellanos 83, KM245242, ...., ....,

KM488630, ....; Cavanillesia platanifolia (Bonpl.) Kunth., Fairchild Botanical Gardens acc. no. FG83343A, KM453031, HQ658371*, HQ696686*, HQ696737*, ....;

Cavanillesia umbellata Ruiz & Pav., J.G.Carvalho-Sobrinho 2987 (HUEFS),

KM453032, ...., ...., ...., ....; Ceiba acuminata (S.Watson) Rose, Fairchild Botanical

Gardens acc. no. X-2–206, ...., HQ658385*, HQ696700*, HQ696752*, ....; Ceiba aesculifolia (Kunth) Britten & Baker.f., Fairchild Botanical Gardens acc. no. 83301,

KM453033, HQ658384*, HQ696699*, HQ696751*, ….; Ceiba chodatii (Hassl.)

Ravenna, G.Schmeda 1170 (US), KM453034, ...., ...., ...., ....; Ceiba crispiflora (Kunth)

Ravenna, Pacific Tropical Garden acc. no. 750726001, KM453035, HQ658387*,

AY321169*, HQ696754*, ....; Ceiba erianthos (Cav.) K.Schum., E.R.Souza 710

(HUEFS), KM453036, KM453167, ...., ...., KM453113; Ceiba glaziovii (Kuntze)

K.Schum., J.G.Carvalho-Sobrinho 2967 (HUEFS), KM453037, ...., ...., ...., KM453116;

Ceiba insignis (Kunth) P.E.Gibbs & Semir, J.Campos & P.López 4953 (US),

KM453038, KM488629, ...., ...., ....; Ceiba jasminodora (A.St.-Hil.) K.Schum.,

J.G.Carvalho-Sobrinho 3070 (HUEFS), KM453039, KM453168, ...., ...., KM453131;

Ceiba pentandra (L.) Gaertn., J.G.Carvalho-Sobrinho s.n. (HUEFS), KM453040,

KM453169, ...., ...., KM453117, W.S.Alverson s.n. (WIS), ...., HQ658386*,

HQ696701*, HQ696753*, ....; Ceiba pubiflora (A.St.-Hil.) K.Schum., J.G.Carvalho-

Sobrinho 3066 (HUEFS), KM453041, KM453170, ...., ...., KM453118; Ceiba rubriflora Carv.-Sobr. & L.P.Queiroz, J.G.Carvalho-Sobrinho 574 (HUEFS),

KM453042, KM453171, ...., ...., KM453119; Ceiba samauma (Mart.) K.Schum.,

J.G.Carvalho-Sobrinho s.n. (HUEFS), KM453043, ...., ...., ...., ....; Ceiba schottii Britten

& Baker.f., Fairchild Botanical Gardens acc. no. 83302, ...., HQ658389*, HQ696703*,

HQ696756*, ....; Ceiba speciosa (A.St.-Hil.) Ravenna, W.S.Alverson s.n. (WIS),

KM453044, HQ658388*, HQ696702*, HQ696755*, ....; Ceiba ventricosa (Nees &

Mart.) Ravenna, J.G.Carvalho-Sobrinho 3124 (HUEFS), KM453045, KM453172, ....,

...., ....; Chiranthodendron pentadactylon Larreat., Wendt s.n. (WIS), KM453046,

HQ658356*, AY321164*, HQ696722*, ....; Eriotheca bahiensis M.C. Duarte & G.L.

Esteves, M.C.Duarte 89 (SPF), ...., HQ658398*, HQ696712*, HQ696766*, ....;

Eriotheca candolleana (K. Schum.) A.Robyns, M.C.Duarte 99 (CVRD), ....,

HQ658394*, HQ696718*, HQ696772*, ....; J.G.Carvalho-Sobrinho 3137 (HUEFS), ....,

...., ...., ...., KM453120; Eriotheca crenulaticalyx A.Robyns, L.P.Queiroz s.n. (HUEFS),

KM453047, KM453173, ...., ...., KM453122; Eriotheca discolor (Kunth) A.Robyns,

Campo 6110 (MO), ...., …., HQ696775, HQ696720, ….; Eriotheca dolichopoda

A.Robyns, M.C.Duarte 92 (CEPEC), ...., HQ658402*, HQ696719*, HQ696773*, ....;

J.G.Carvalho-Sobrinho 3123 (HUEFS), KM453048, KM453174, ...., ...., KM453123;

Eriotheca estevesiae Carv.-Sobr., G.Pereira-Silva 5392 (HUEFS), KM488628,

KM283224, ...., ...., KM453160; Eriotheca globosa (Aubl.) A.Robyns, R.O.Perdiz 745

(CEPEC), KM453049, ...., ...., ...., ....; Eriotheca gracilipes (K.Schum.) A.Robyns,

J.G.Carvalho-Sobrinho 3037 (HUEFS), KM453050, ...., ...., ...., KM453124;

M.C.Duarte 120 (SP), ...., ...., HQ696708*, HQ696762*, ....; Eriotheca longipedicellata

(Ducke) A.Robyns, M.C.Duarte 93 (IAN, SP), ...., ...., HQ696716*, HQ696770*, ....;

Eriotheca longitubulosa A.Robyns, M.C.Duarte 96 (SP), ...., ...., HQ696717*,

HQ696771*, ....; Eriotheca macrophylla (K. Schum.) A.Robyns, M.C.Duarte 106 (SP),

...., ...., HQ696713*, HQ696767*, KM453108; J.G.Carvalho-Sobrinho 2949 (HUEFS),

KM453051, KM453175, ...., ...., KM453125; Eriotheca obcordata A.Robyns, B.M.Silva

107 (HUEFS), ...., HQ658403*, ...., HQ696774*, ....; Eriotheca parvifolia (Mart.)

A.Robyns, J.G.Carvalho-Sobrinho 2870 (HUEFS), KM453053, ...., ...., ...., KM453126;

M.C.Duarte 109 (SP), ...., HQ658401*, HQ696710*, HQ696764*, ....; Eriotheca pentaphylla (Vell. emend. K. Schum.) A. Robyns, M.C.Duarte 75 (SP), …., ….,

HQ696768*, HQ696714*, ….; Eriotheca pubescens (Mart.) A.Robyns, J.G.Carvalho-

Sobrinho 2873 (HUEFS), ...., ...., ...., ...., KM453127; J.G.Carvalho-Sobrinho 2878

(HUEFS), KM453054, ...., ...., ...., KM488631; M.C.Duarte 115 (SP), ...., HQ658397*,

HQ696709*, HQ696763*, ....; Eriotheca roseorum (Cuatrec.) A.Robyns, Fuentes 1167

(MO), …., …., HQ696765*, HQ696711*, ….; Eriotheca ruizii (K.Schum.) A.Robyns,

P.M.Peterson 9487 (US), …., …., HQ696777*, HQ696721*, ….; Eriotheca saxicola

Carv.-Sobr., J.G.Carvalho-Sobrinho 3165 (HUEFS), KM453055, ...., ...., ...., ....;

J.G.Carvalho-Sobrinho 3167 (HUEFS), ...., KM453176, ...., ...., ....; J.G.Carvalho-

Sobrinho 3146 (HUEFS), ...., ...., ...., ...., KM453129; Eriotheca squamigera (Cuatrec.)

Fern.Alonso., Neill 12522,…., …., …., HQ696776*, ....; Eriotheca surinamensis

(Uittien) A.Robyns, M.C.Duarte 97 (SP), ...., HQ658400*, HQ696715*, HQ696769*,

....; Eriotheca sp., J.G.Carvalho-Sobrinho 3125 (HUEFS), KM453052, KR076544, ….,

…., ….; Fremontodendron californicum (Torr.) Coville, Ex. Rancho Santo Ana Bot.

Garden, Prop. no. 5996, Herb. no. 12343, KM453056, HQ658357*, AY321165*,

HQ696723*, KM453114; Gyranthera caribensis Pittier, Iltis et al. s.n (WIS), ....,

HQ658368*, AY589071*, HQ696734*, KM453110; Hampea appendiculata Standl.,

W.S.Alverson 2179 (WIS)…., U56781, AY589062, …., ….; Huberodendron patinoi

Cuatrec., W.S.Alverson 2201 (WIS), KM453057, HQ658367*, AY589072*,

HQ696733*, ....; Huberodendron swietenioides (Gleason) Ducke, J.G.Carvalho-

Sobrinho 3292 (HUEFS), ...., KM453162, ...., ...., KM453129; Neobuchia paulinae

Urb., Cult. Jardin Botanico, Santo Domingo, Dominican Republic, …., ….,

HQ696707*, HQ696760*, ….; Ochroma pyramidale (Cav. ex Lam.) Urb.,

J.G.Carvalho-Sobrinho 3077 (HUEFS), KM453058, ...., ...., ...., KM453132; W.S.

Alverson s.n. (WIS), ...., HQ658363*, AY321172*, HQ696729*, ....; Pachira aquatica

Aubl., J.G.Carvalho-Sobrinho s.n. (HUEFS), KM453059, ...., ...., ...., KM453133; W.S.

Alverson s.n. (WIS), ...., ...., AY321170*, HQ696759*, ....; Pachira brevipes

(A.Robyns) W.S.Alverson, J.G.Carvalho-Sobrinho 3097 (HUEFS), KM453060, ...., ....,

...., KM453139; P.Fine 1060 (UC), ...., HQ658391*, HQ696694*, ...., ....; Pachira endecaphylla (Vell.) A.Robyns, J.G.Carvalho-Sobrinho 3130 (HUEFS), KM453061,

KM453182, ...., ...., KM453134; Pachira faroensis (Ducke) W.S.Alverson, D.Cardoso

3418 (HUEFS), KM453068, KM453179, ...., ...., ....; Pachira flaviflora (Pulle)

Fern.Alonso, P.Fine 1062 (UC), ...., HQ658379*, HQ696693*, HQ696746*, ....;

Pachira glabra Pasq., M.C.Duarte 70 (SP), ...., HQ658393*, HQ696706*, HQ696761*,

....; J.G.Carvalho-Sobrinho 2863 (HUEFS), KM453062, KM453177, ...., ....,

KM453135; Pachira gracilis (A.Robyns) W.S.Alverson, T.D.M.Barbosa 1296 (INPA),

KM453063, ...., ...., ...., ....; Pachira humilis Spruce ex Benth., D.Cardoso 3413

(HUEFS), KM453067, KM453178, ...., ...., ....; Pachira insignis (Sw.) Sw. ex Savigny,

J.G.Carvalho-Sobrinho 3106 (HUEFS), KM453064, ...., ...., ...., KM453136; P.Fine

1061 (UC), ...., HQ658390*, HQ696704*, HQ696757*, ....; Pachira mawarinumae

(Steyerm.) W.S.Alverson, D.Cardoso 3419 (HUEFS), KM453069, KM453180, ...., ....,

....; Pachira minor (Sims) Hemsl., G. Davidse 4901...., ...., HQ696705*, HQ696758*,

....; Pachira moreirae Carv.-Sobr. & W.S.Alverson, J.G.Carvalho-Sobrinho 2963

(HUEFS), KM453065, KF477294, ...., ...., KM453138; Pochota fendleri (Seem.) W.S.

Alverson & M.C.Duarte [≡ Pachira quinata], W.S.Alverson 2174 (WIS), ...., ....,

HQ696692*, HQ696745*, ....; P.E.Kaminski s.n. (HUEFS), KM453074, KM453184,

...., ...., KM453141; Pachira retusa (Mart.) Fern.Alonso, M.V.Moraes 532 (HUEFS),

KM453066, KF477293, ...., ...., KM453140; Pachira aff. mawarinumae (Steyerm.)

W.S.Alverson, D.Cardoso 3425 (HUEFS), KM453070, KM453181, ...., ...., ....; Pachira sp., J.G.Carvalho-Sobrinho 3176 (HUEFS), KM453071, ...., ...., ...., ....; Patinoa sphaerocarpa Cuatrec., W.S.Alverson s.n. (WIS), ...., HQ658364*, AY589074*,

HQ696730*, ....; Pentaplaris doroteae L.O.Williams & Standl., B.E.Hammel 18736

(MO), ...., HQ658358*, AY321163*, HQ696724*, KM453115; Phragmotheca ecuadorensis W.S.Alverson, W.S.Alverson 2223 (WIS), ...., ...., AY589068*, ...., ....;

Pseudobombax amapaense A.Robyns, J.G.Carvalho-Sobrinho 3105 (HUEFS),

KM453092, KM453200, ...., ...., KM453154; Pseudobombax andicola A.Robyns,

W.J.Eyerdam 25320 (F), KM453077, ...., ...., ...., ....; C.Antezana 1122 (NY), ....,

KM453186, ...., ...., KM453142; Pseudobombax argentinum (R.E.Fries) A.Robyns,

C.Saravia-Toledo 11476 (LPB), KM453078, ...., ...., ...., ....; Pseudobombax cajamarcanus Fern.Alonso, C.Díaz 2189 (MO), KM453079, ...., ...., ...., ....;

Pseudobombax calcicola Carv.-Sobr. & L.P.Queiroz, J.G.Carvalho-Sobrinho 2993

(HUEFS), KM453080, KM453187, ...., ...., KM453144; Pseudobombax campestre

(Mart.) A.Robyns, J.G.Carvalho-Sobrinho 2872 (HUEFS), KM453081, KM453188, ....,

...., KM453145; Pseudobombax croizatii A.Robyns, Oldham s.n. (WIS), ....,

HQ658382*, HQ696697*, HQ696749*, ....; Pseudobombax ellipticoideum A.Robyns,

M.J.Balick 3704 (MO), KM453084, ...., ...., ...., ....; M.G.Aguilar 3621 (MO), ...., ...., ....,

...., ....; Pseudobombax ellipticum (Kunth) Dugand, J.G.Carvalho-Sobrinho 3131

(HUEFS), KM453083, KM453190, ...., ...., KM488632; Fairchild Botanical Gardens acc. no. FG X.1-101, ...., KM453189, ...., ...., KM453146; Pseudobombax grandiflorum

(Cav.) A.Robyns, J.G.Carvalho-Sobrinho 3198 (HUEFS), KM453085, ...., ...., ....,

KM453148, Fairchild Botanical Gardens acc. no. FG-65-35, ...., ...., HQ696698*,

HQ696750*, ...., J.G.Carvalho-Sobrinho 2946 (HUEFS), ...., KM453191, ...., ...., ....;

Pseudobombax grandiflorum var. majus A.Robyns, J.G.Carvalho-Sobrinho 3069

(HUEFS), KM453088, KM453195, ...., ...., KM453150; Pseudobombax guayasense

A.Robyns, T.D.Pennington 14519 (K), KM453086, …, …., …., ….; Pseudobombax longiflorum (Mart.) A.Robyns, J.G.Carvalho-Sobrinho 2880 (HUEFS), ...., ...., ...., ....,

...., J.G.Carvalho-Sobrinho 2875 (HUEFS), KM453087, KM453194, ...., ...., ....,

J.G.Carvalho-Sobrinho 2882 (HUEFS), ...., ...., ...., KM453149; Pseudobombax marginatum (A.St.-Hil., Juss. & Cambess.) A.Robyns, R.Small s.n. (ISC), ...., ....,

HQ696696*, HQ696748*, KM453147, L.P.Queiroz 14753 (HUEFS), KM453089,

KM453197, ...., ...., KM453151; Pseudobombax millei A.Robyns, J.G.Carvalho-

Sobrinho s.n. (HUEFS), KM453090, KM453198, ...., ...., ....; Pseudobombax mininum

Carv.-Sobr. & L.P.Queiroz, J.G.Carvalho-Sobrinho 2887 (HUEFS), KM453091,

KM453199, ...., ...., KM453152; Pseudobombax munguba (Mart.) Dugand,

J.G.Carvalho-Sobrinho 3105 (HUEFS), KM453092, KM453200, ...., ...., KM453153;

Pseudobombax parvifolium Carv.-Sobr. & L.P.Queiroz, J.G.Carvalho-Sobrinho 3029

(HUEFS), KM453094, KM453201, ...., ...., KM453154; Pseudobombax petropolitanum

A.Robyns, J.G.Carvalho-Sobrinho 3071 (HUEFS), KM453096, ...., ...., ...., KM453155,

J.G.Carvalho-Sobrinho 3171 (HUEFS), ...., ...., ...., ...., ...., J.G.Carvalho-Sobrinho

3173 (HUEFS), ...., KM453203, ...., ...., ....; Pseudobombax pulchellum Carv.-Sobr.,

A.H.Gentry 75227 (MO), KM453097, ...., ...., ...., ....; Pseudobombax septenatum (Jacq.)

Dugand, E.Villanueva 835 (LPB), KM453098, ...., ...., ...., ....; Pseudobombax simplicifolium A.Robyns, J.G.Carvalho-Sobrinho 3027 (HUEFS), KM453099, ...., ....,

...., KM453156, J.G.Carvalho-Sobrinho 3062 (HUEFS), KM453100, KM453204, ....,

...., ....; Pseudobombax aff. campestre (Mart.) A.Robyns, J.G.Carvalho-Sobrinho 3080

(HUEFS), KM453076, KM453205, ...., ...., KM453143; Pseudobombax tomentosum

(Mart.) A.Robyns, J.G.Carvalho-Sobrinho 2874 (HUEFS), KM453101, KM453206, ....,

...., ....; J.G.Carvalho-Sobrinho 2879 (HUEFS), KM488627, KM453207, ...., ....,

KM453157; Rhodognaphalon schumannianum A.Robyns, M.W.Chase 5973 (K), ....,

HQ658380*, HQ696695*, HQ696747*, ....; Scleronema micranthum (Ducke) Ducke,

J.G.Carvalho-Sobrinho 3098 (HUEFS), KM453102, ...., ...., ...., KM453158;

W.S.Alverson s.n. (WIS), ...., HQ658369*, AY589070*, HQ696735*, ....; Scleronema praecox (Ducke) Ducke, J.G.Carvalho-Sobrinho 3276 (HUEFS), KM453103, ...., ....,

...., KM453159; Septotheca tessmannii Ulbr., J.Rios 1917 (MO), KM453104,

HQ658365*, AY589073*, HQ696731*, ....; Spirotheca elegans Carv.-Sobr.,

M.C.Machado & L.P.Queiroz, J.G.Carvalho-Sobrinho 2964 (HUEFS), KM453105,

KM453208, ...., ...., ....; Spirotheca rivierii (Decne.) Ulbr., J.G.Carvalho-Sobrinho s.n.

(HUEFS), KM453106, KM453209, ...., ...., ....; Spirotheca rosea (Seem.) P.E.Gibbs &

W.S.Alverson, W.S.Alverson 2185 (WIS), KM453107, HQ658378*, HQ696691*,

HQ696744*, .....; Sterculia lanceolata Cav., YCC635, ...., AF460184*, HQ415311*,

AY328151*, ....; Sterculia nobilis Sm., YCC632, ...., AF460183*, ...., JN676078*, ....

Appendix 2. Matrix used in the reconstruction of ancestral states of morphological characters of Bombacoideae (Malvaceae) using a maximum likelihood approach.

Prickles on Seed number per trunk or Calyx shape Endocarp type Seed shape fruit branches Adansonia digitata 0 2 1 0 2 Adansonia grandidieri 0 2 1 0 2 Adansonia gregorii 0 2 1 0 2 Adansonia kilima 0 2 1 0 2 Adansonia madagascariensis 0 2 1 0 2 Adansonia perrieri 0 2 1 0 2 Adansonia rubrostipa 0 2 1 0 2 Adansonia suazerensis 0 2 1 0 2 Adansonia za 0 2 1 0 2 Aguiaria excelsa 0 0 1 0 0 Bombax anceps 1 1 2 0 2 Bombax buonopozense 1 1 2 0 2 Bombax ceiba 1 1 2 0 2 Bernoullia flammea 0 0 0 1 1 Catostemma albuquerquei 0 0 1 0 0 Catostemma fragrans 0 0 1 0 0 Catostemma milanezii 0 0 1 0 0 Cavanillesia chicamochae 0 0 1 0 0 Cavanillesia platanifolia 0 0 1 0 0 Cavanillesia umbellata 0 0 1 0 0 Ceiba acuminata 1 0 2 0 2 Ceiba aesculifolia 1 0 2 0 2 Ceiba chodatii 1 0 2 0 2

Prickles on Seed number per trunk or Calyx shape Endocarp type Seed shape fruit branches Ceiba crispiflora 1 0 2 0 2 Ceiba erianthos 1 0 2 0 2 Ceiba glaziovii 1 0 2 0 2 Ceiba insignis 1 0 2 0 2 Ceiba jasminodora 1 0 2 0 2 Ceiba pentandra 1 0 2 0 2 Ceiba pubiflora 1 0 2 0 2 Ceiba rubriflora 1 0 2 0 2 Ceiba samauma 1 0 2 0 2 Ceiba schottii 1 0 2 0 2 Ceiba speciosa 1 0 2 0 2 Ceiba ventricosa 1 0 2 0 2 Eriotheca bahiensis 0 1 2 0 2 Eriotheca candolleana 0 0 2 0 2 Eriotheca crenulaticalyx 0 1 2 0 2 Eriotheca discolor 0 1 2 0 2 Eriotheca dolichopoda 0 1 2 0 2 Eriotheca estevesiae 0 1 2 0 2 Eriotheca globosa 0 1 2 0 2 Eriotheca gracilipes 0 1 2 0 2 Eriotheca longipedicellata 0 1 2 0 2 Eriotheca longitubulosa 0 1 2 0 2 Eriotheca macrophylla 0 1 2 0 2 Eriotheca obcordata 0 1 2 0 2 Eriotheca parvifolia 0 1 2 0 2 Eriotheca pentaphylla 0 0 2 0 2 Eriotheca pubescens 0 1 2 0 2

Prickles on Seed number per trunk or Calyx shape Endocarp type Seed shape fruit branches Eriotheca roseorum 0 1 2 0 2 Eriotheca ruizii 0 1 2 0 2 Eriotheca saxicola 0 1 2 0 2 Eriotheca sp.CS3125 0 1 2 0 2 Eriotheca squamigera 0 1 2 0 2 Eriotheca surinamensis 0 1 2 0 2 Gyranthera caribensis 0 0 0 1 1 Huberodendron patinoi 0 0 0 1 1 Huberodendron swietenioides 0 0 0 1 1 Neobuchia paullinae 1 1 2 0 2 Pachira aff. mawarinumae 0 1 2 0 2 Pachira aquatica 0 1 2 0 1 Pachira brevipes 0 1 2 0 2 Pachira endecaphylla 0 1 2 0 2 Pachira faroensis 0 1 2 0 2 Pachira flaviflora 0 1 2 0 2 Pachira glabra 0 1 2 0 1 Pachira gracilis 0 1 2 0 2 Pachira humilis 0 1 2 0 2 Pachira insignis 0 1 2 0 1 Pachira mawarinumae 0 1 2 0 2 Pachira minor 0 1 2 0 2 Pachira moreirae 0 1 2 0 1 Pachira retusa 0 1 2 0 1 Pachira sp.CA85 0 1 2 0 2 Pachira sp.CS3176 0 1 2 0 1 Pochota fendleri 1 1 2 0 2

Prickles on Seed number per trunk or Calyx shape Endocarp type Seed shape fruit branches Pseudobombax aff. campestre 0 1 2 0 2 Pseudobombax amapaense 0 1 2 0 2 Pseudobombax andicola 0 1 2 0 2 Pseudobombax argentinum 0 1 2 0 2 Pseudobombax cajamarcanus 0 1 2 0 2 Pseudobombax calcicola 0 1 2 0 2 Pseudobombax campestre 0 1 2 0 2 Pseudobombax sp.CB305 0 1 2 0 2 Pseudobombax croizatii 0 1 2 0 2 Pseudobombax ellipticum 0 1 2 0 2 Pseudobombax ellpiticoideum 0 1 2 0 2 Pseudobombax grandiflorum 0 1 2 0 2 Pseudobombax guayasense 0 1 2 0 2 Pseudobombax longiflorum 0 1 2 0 2 Pseudobombax grandiflorum var. majus 0 1 2 0 2 Pseudobombax marginatum 0 1 2 0 2 Pseudobombax millei 0 1 2 0 2 Pseudobombax minimum 0 1 2 0 2 Pseudobombax munguba 0 1 2 0 2 Pseudobombax parvifolium 0 1 2 0 2 Pseudobombax petropolitanum 0 1 2 0 2 Pseudobombax pulchellum 0 1 2 0 2 Pseudobombax septenatum 0 1 2 0 2 Pseudobombax simplicifolium 0 1 2 0 2 Pseudobombax tomentosum 0 1 2 0 2 Rhodognaphalon schumannianum 1 1 2 0 2 Scleronema micranthum 0 0 3 0 0

Prickles on Seed number per trunk or Calyx shape Endocarp type Seed shape fruit branches Scleronema praecox 0 0 3 0 0 Spirotheca elegans 1 1 2 0 2 Spirotheca rivieri 1 1 2 0 2 Spirotheca rosea 1 1 2 0 2

Graphical abstract

Highlights

 We reconstructed a phylogeny of Bombacoideae (Malvaceae) based on plastid and

nuclear ribosomal DNA.

 Novel phylogenetic relationships emerged including clades that are

morphologically cohesive based on fruit and seed traits.

 The phylogenetic placement of Pochota fendleri, a historically incertae sedis taxon,

is resolved.

 We present a new tribal classification and an identification key to the tribes.