Accepted Manuscript Molecular phylogeny of the Brazilian endemic genus Orthophytum (Bromelioideae, Bromeliaceae) and its implications on morphological character evolution Rafael B. Louzada, Katharina Schulte, Maria das Graças L. Wanderley, Daniele Silvestro, Georg Zizka, Michael H.J. Barfuss, Clarisse Palma-Silva PII: DOI: Reference: S1055-7903(14)00099-2 http://dx.doi.org/10.1016/j.ympev.2014.03.007 YMPEV 4844 To appear in: Molecular Phylogenetics and Evolution Received Date: Revised Date: Accepted Date: 26 January 2013 27 February 2014 10 March 2014 Please cite this article as: Louzada, R.B., Schulte, K., Wanderley, M.L., Silvestro, D., Zizka, G., Barfuss, M.H.J., Palma-Silva, C., Molecular phylogeny of the Brazilian endemic genus Orthophytum (Bromelioideae, Bromeliaceae) and its implications on morphological character evolution, Molecular Phylogenetics and Evolution (2014), doi: http://dx.doi.org/10.1016/j.ympev.2014.03.007 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. 1 Molecular phylogeny of the Brazilian endemic genus Orthophytum (Bromelioideae, 2 Bromeliaceae) and its implications on morphological character evolution 3 Rafael B. Louzada *, Katharina Schulte , Maria das Graças L. Wanderley , Daniele Silvestro , 4 Georg Zizkae, Michael H.J. Barfussf, Clarisse Palma-Silva 5 a 6 b 7 c 8 4814, Australia. 9 d a, b,c d e b Universidade Federal de Pernambuco, Departamento de Botânica, Recife 50670–901, Pernambuco, Brazil. Australian Tropical Herbarium, James Cook University, PO Box 6811, Cairns QLD 4878, Australia. Centre for Tropical Biodiversity and Climate Change, James Cook University, Discovery Drive, Townsville, QLD Instituto de Botânica, Secretaria do Meio Ambiente, Avenida Miguel Stéfano 3687, São Paulo 01061-970, São Paulo, 10 Brazil. 11 e 12 Frankfurt am Main D–60325, Germany. 13 f 14 1030 Vienna, Austria. 15 *Corresponding author: Telephone +55 81 2126-8864. 16 E-mail address: rb.louzada@gmail.com (R.B. Louzada). Department of Botany and Molecular Evolution, Research Institute Senckenberg and J.W. Goethe University, Department of Systematic and Evolutionary Botany, Faculty of Life Sciences, University of Vienna, Rennweg 14, 17 18 19 20 21 22 23 1 24 Abstract 25 The saxicolous genus Orthophytum (~ 60 species, Bromeliaceae) is endemic to eastern Brazil and 26 diversified in xeric habitats of the Caatinga and campos rupestres. Within the genus, two main 27 groups are discerned based on the presence or absence of a pedunculate inflorescence, which are 28 further subdivided into several morphological subgroups. However, these systematic hypotheses 29 have not yet been tested in a molecular phylogenetic framework. Here we present the first 30 phylogenetic analysis of Orthophytum using nuclear and plastid markers (phytochrome C, and 31 trnH-psbA and trnL-trnF spacers). Forty species representing the two main groups and all 32 subgroups of Orthophytum, and the related genera Cryptanthus (8 spp.) and Lapanthus (2 spp.) 33 were analyzed. The phylogenetic reconstruction revealed a well-supported clade termed Eu- 34 Orthophytum, containing species with pedunculate inflorescences only. The Orthophytum species 35 with sessile inflorescence formed two clades: 1) the amoenum group, and 2) the vagans group plus 36 O. foliosum, the only pedunculate Orthophytum species found outside Eu-Orthophytum. The 37 vagans clade is in sister group position to Eu-Orthophytum. Within the latter, the subgroup mello- 38 barretoi was sister to the most diversified clade, termed Core Orthophytum. Morphological 39 character state reconstructions of floral characters used in previous taxonomic treatments as key 40 diagnostic characters (penduncle presence, corolla form, and petal appendage form) showed 41 different levels of homoplasy. 42 43 Keywords: Bromelioideae; bromeliads; Espinhaço Range; phytochrome C; PHYC; trnH-psbA 44 spacer; trnL-trnF spacer. 45 46 1. Introduction 47 Bromeliaceae (ca. 4300 spp.; Butcher and Gouda, cont. updated) is an almost exclusively 48 Neotropical family, with only one species (Pitcairnia feliciana (A. Chev.) Harms & Mildbraed) 2 49 occurring in West Africa. The family has traditionally been divided in three subfamilies: 50 Pitcairnioideae, Bromelioideae and Tillandsioideae (Smith and Downs, 1974, 1977, 1979; Smith 51 and Till, 1998). The monophyly of Pitcairnioideae has been questioned in several molecular 52 phylogenetic studies (e.g. Barfuss et al., 2005; Crayn et al., 2004; Givnish et al. 2007; Horres et al., 53 2000; Terry et al., 1997), and recently a new classification for Bromeliaceae based on molecular 54 phylogenetic evidence from the gene ndhF was proposed by Givnish et al. (2007, 2011) dividing 55 Bromeliaceae into eight subfamilies: Brocchinioideae, Lindmanioideae, Tillandsioideae, 56 Hechtioideae, Navioideae, Pitcairnioideae, Puyoideae and Bromelioideae. 57 Subfamily Bromelioideae comprises 33 genera and approximately 950 species distributed in 58 tropical and subtropical America with a center of diversity in southeastern Brazil (Butcher and 59 Gouda, cont. updated; Smith and Downs, 1979). The monophyly of the subfamily is supported by 60 both morphological and molecular evidence, with Puya as sister group (Barfuss et al., 2005; Crayn 61 et al., 2004; Givnish et al., 2004, 2007, 2011; Horres et al., 2000; 2007; Schulte et al., 2005, 2009; 62 Schulte and Zizka, 2008; Terry et al., 1997). Nevertheless the inter- and infrageneric relationships 63 within the subfamily are poorly understood (Brown and Leme, 2000; Schulte et al., 2009). Recent 64 molecular studies based on plastid and nuclear data identified several basal lineages within the 65 subfamily (Greigia Regel, Ochagavia Phil., Fascicularia Mez, Deinacanthon Mez, Bromelia Juss.) 66 (Schulte et al., 2005, 2009; Schulte and Zizka, 2008). Fernseea Baker was reported as sister to a 67 clade comprising the remainder of the subfamily, termed Eu-Bromelioideae (Schulte et al., 2009; 68 Schulte and Zizka, 2008). Among the latter, the genera Orthophytum Beer, Cryptanthus Otto & A. 69 Dietr., Ananas Mill., Neoglaziovia Mez, and Acanthostachys Klotzsch were identified as early 70 divergent lineages (basal eu-bromelioids) whereas the more advanced bromelioids, characterized by 71 the tank habit (a central water collecting tank formed by the leaf sheaths), formed a moderately- 72 supported clade, termed the core bromelioids (Givnish et al., 2011; Schulte et al., 2009; Schulte and 73 Zizka, 2008; Sass and Specht, 2010). Whereas core bromelioids comprise the majority of species 3 74 and epiphytes, the more basal lineages lack a central external water reservoir and are mainly 75 terrestrial or lithophytes. 76 Orthophytum is a saxicolous (rarely terrestrial) genus endemic to eastern Brazil where it 77 underwent considerable diversification (Fig. 1). The species generally inhabit the top of granitic- 78 gneiss inselbergs in the regions of the Atlantic Rainforest and the Caatinga, and quartzitic- 79 sandstone outcrops in the Brazilian campos rupestres („rocky fields‟) along the Espinhaço Range. 80 Two centers of diversity can be recognized, one in the Espinhaço Range and the other in the 81 Atlantic Rain Forest area in the Brazilian states of Minas Gerais and Espírito Santo (Louzada and 82 Wanderley, 2010). 83 The genus was described by Beer (1854) based on one unnamed collection of a pedunculate 84 species known today as Orthophytum glabrum (Mez) Mez (Louzada and Wanderley, 2010). Ule 85 (1908) described two new genera from Brazil (Sincoraea Ule and Cryptanthopsis Ule), both with 86 sessile inflorescences, which were subsequently regarded as synonyms of Orthophytum (Smith, 87 1955; Smith and Downs, 1979). In the taxonomic treatment for Bromeliaceae in Flora Neotropica 88 (Smith and Downs, 1979), 17 species of Orthophytum were recognized. Today the genus comprises 89 about 60 species (Louzada and Wanderley, 2011), the majority described in the last two decades, 90 and a taxonomic revision of the group is urgently needed to assess the conservation status of the 91 species. 92 Within Orthophytum two main morphological groups are traditionally recognized based on 93 the presence or absence of a peduncle (or stalk, sometimes in bromeliads also called a scape; Leme 94 2004; Louzada and Wanderley, 2010; Wanderley, 1990; Wanderley and Conceição, 2006). These 95 groups of species were termed “complexes” in Leme (2004) and each one was subdivided into 96 subgroups also called “subcomplexes”. However in this study we adopted the terms groups and 97 subgroups instead of complexes and subcomplexes because we understand these morphological 98 groups of species are not species complexes according to its idea. The first group comprises the 4 99 majority of species and is termed the “group with scapose inflorescence” which is divided into three 100 subgroups: disjunctum, leprosum, and mello-barretoi (Leme, 2004). The other is the “group with 101 sessile inflorescence” which comprises three subgroups: amoenum, vagans, and supthutii (Leme, 102 2004). Recently, a new genus was established (Lapanthus Louzada & Versieux) to better 103 accommodate the species of the supthutii subgroup (Louzada and Versieux, 2010). Nevertheless, 104 the validity of these taxonomic hypotheses has not yet been tested in a molecular phylogenetic 105 framework. 106 In previous phylogenetic studies on Bromelioideae, Orthophytum has usually been 107 represented by only a few taxa (Ramírez-Morillo, 1996; Schulte et al., 2005, 2009; Schulte and 108 Zizka, 2008) and the genus was found to be the sister group of Cryptanthus. However, due to the 109 low taxon sampling the hypotheses outlined above and the monophyly of the genus could not be 110 properly tested yet. Therefore, a more comprehensive phylogenetic study is needed to clarify inter- 111 and intrageneric relationships of Orthophytum. 112 Here we present a molecular phylogeny of Orthophytum and related genera based on the 113 plastid intergenic spacer regions trnL-trnF and trnH-psbA, and the low-copy nuclear gene 114 phytochrome C (PHYC). The objectives were (1) to assess the phylogenetic relationships between 115 Orthophytum, Cryptanthus, and Lapanthus, and the monophyly of the genera, (2) to elucidate 116 intrageneric relationships in Orthophytum, (3) to investigate the evolution and taxonomic 117 significance of morphological characters previously used in the taxonomy of Orthophytum. 118 119 2. Material and methods 120 2.1. Taxon sampling 121 In the present study a molecular data set of 54 species from six genera (Table 1) was 122 analyzed. In Orthophytum, 40 of the about 60 recognized species (i.e. 67 % of known diversity) 123 were included to investigate all of the morphological groups and subgroups described by Leme 5 124 (2004) including the two species of the supthutii subgroup today recognized as the genus Lapanthus 125 (Louzada and Versieux, 2010). In addition, eight species of the genus Cryptanthus comprising 126 representatives from the two subgenera and six of eight sections described by Ramírez-Morillo 127 (1996) were included in the data set. Outgroup species were included from the early diverging 128 Bromelioideae: Bromelia (2), Ochagavia (1) and from the mono-generic subfamily Puyoideae (1) 129 based on Givnish et al. (2011) and Schulte et al. (2009). 130 2.2. DNA extraction, amplification and sequencing 131 Total genomic DNA was extracted from leaf material using a cetyltrimethylammonium 132 bromide (CTAB) procedure (Doyle and Doyle, 1987) modified by Horres et al. (2000). The 133 phytochrome C (PHYC) gene was amplified using primers phyc515f-br AAG CCC TTY TAC GCT 134 ATC CTG CAC CG and phyc1699r-br ATW GCA TCC ATT TCA ACA TCT TCC CA. Internal 135 primers were used for sequencing (phyc974f-br GCT CCT CAC GGC TGC CAC GCT CA and 136 phyc1145r-mo CCT GMA RCA RGA ACT CAC AAG CAT ATC). The trnL-trnF and trnH-psbA 137 regions were amplified using universal primers described in Shaw et al. (2005) and Sang et al. 138 (1997), respectively. The two plastid regions and the nuclear gene were chosen because they have 139 proven to be most informative markers for Bromeliaceae (e.g., Barfuss et al., 2005: trnL-trnF; 140 Givnish et al., 2011: trnL-trnF, trnH-psbA; Jabaily & Sytsma, 2010: PHYC). Amplifications were 141 carried out in a Veriti Thermal Cycler (Applied Biosystem Corp., Foster City, California). Plastid 142 regions were amplified with 10 μL reactions following Palma-Silva et al. (2009). The nuclear 143 region PHYC was amplified with 10 μL as follows: 1x Taq buffer (Fermentas), 1.5 mM MgCl2 144 (Fermentas), l00 μmol deoxynucleotide triphosphate, 10 pmol of each primer , 1 U Taq DNA 145 polymerase (Fermentas) and 10-20 ng of DNA template, using a standard cycling program: 2 min 146 denaturation at 95o C followed by 35 cycles of 95o C denaturation for 30 s, 30 s annealing at 59o C, 147 and 2 min extension at 70o C and a final elongation step at 70o C for 7 min. The PCR products were 148 cleaned using ExoSAP-IT (USB Corp., Cleveland, Ohio) following the manufacturer‟s protocol. 6 149 Cycle sequencing was carried out with the Big Dye Terminator kit v.3.1 (Applied Biosystem Corp., 150 Foster City, California) with an initial 60 s denaturation at 95o C, followed by 30 cycles at 96o C 151 denaturation for 10 s, 10 s annealing at 50o C, and 2 min extension at 60o C. The sequences were 152 generated on an ABI 3730 DNA Analyzer sequencer. 153 2.3. Alignment of sequences and data congruence 154 The sequences were assembled and edited with the software Geneious 5.1.7 (Drummond et 155 al., 2011) and initially aligned with MAFFT (Kazutaka et al., 2002) followed by manual 156 adjustments in Geneious. Congruence among data partitions of the two plastid and one nuclear 157 marker was assessed a) by visual inspection of the tree topologies based on the plastid versus the 158 nuclear data set and b) with the incongruence length difference (ILD) test (Farris et al., 1994) 159 implemented in PAUP*4.0b10 (Swofford, 2002) employing 100 replicates (heuristic search, 10 160 random addition replicates, tree-bisection-reconnection (TBR) branch swapping), saving a 161 maximum of 1,000 most parsimonious trees per replicate. 162 2.4. Phylogenetic analysis 163 A maximum parsimony (MP) analysis was performed in PAUP*4.0b10. Heuristic searches 164 were conducted with 10,000 random taxon addition replicates and TBR branch swapping. The 165 statistical support was estimated by bootstrap analysis with 1,000 pseudoreplicates, each with 10 166 random taxon addition replicates and TBR branch swapping. The degree of homoplasy was 167 estimated using consistency (CI) and retention (RI) indices. 168 Bayesian inference analyses (BI) were run in MrBayes 3.2 (Ronquist et al. 2012). The best-fit 169 model (GTR+I+G) for the combined dataset was determined using the Akaike Information 170 Criterion (Akaike, 1973) as implemented in MrModeltest 2.2 (Nylander, 2004). Four simultaneous 171 Markov chains Monte Carlo (MCMC) were run for 10,000,000 generations sampling every 1,000 172 generations. After examining the MCMC convergence using Tracer (Rambaut and Drummond, 173 2007), the initial 2,000,000 generations from each run were discarded from the analysis as burn-in 7 174 while the remaining trees were used to construct a consensus tree with posterior probabilities (PP) 175 assessing the statistical nodal support. Two partitioning schemes were tested, one unlinking the 176 model parameters between nuclear and plastid regions, the other unlinking all markers (i.e. three 177 partitions). The best-fit model was chosen by Bayes factor test based on the harmonic mean of the 178 respective log likelihoods (Kass and Raftery, 1995). 179 2.5. State character reconstruction 180 To examine the evolution of key morphological characters used in previous taxonomic 181 treatments (Smith and Downs, 1979; Leme, 2004) and to assess their taxonomic value, ancestral 182 character state reconstructions were performed with maximum parsimony using Mesquite 2.75 183 (Maddison and Maddison, 2011) and a Bayesian framework in RASP (Yu et al. 2013). The analyses 184 were run on the Bayesian consensus tree based on the combined plastid and nuclear data set, 185 exluding the outgroup. Three characters were examined: Peduncle presence: [0] absent, [1] present; 186 corolla form: [1] clavate, [2] tubular; and petal appendages form: [1] fimbriate, [2] sacciform 187 lacerate, [3] acute, [4] cuppuliform lacerate, [5] absent). Character states were scored from fresh 188 material, herbarium sample and literature (Louzada and Wanderley, 2010; Smith and Downs 1979). 189 190 3. Results 191 3.1. Phylogenetic relationship 192 Sequences for the two plastid and one nuclear loci were generated for 54 accessions of 193 Bromelia, Cryptanthus, Lapanthus, Ochagavia, Orthophytum and Puya (Table 1). The final 194 alignment comprised 605 positions for trnH-psbA, 851 for trnL-trnF intergenic spacer regions, and 195 1,124 for the nuclear gene PHYC. The combined dataset yielded an alignment of 2,580 characters in 196 length with 273 variable characters. The number of parsimony informative characters was 105 (4%) 197 for the ingroup (Orthophytum, Cryptanthus, and Lapanthus), and 78 (3 %) for Orthophytum. PHYC 198 alignment presented few double peaks, inferred as allelic variation, which were treated as 8 199 ambiguous data. The partition homogeneity test indicated that the different data partitions of the 200 combined matrix (PHYC vs. two plastid regions) are not significantly incongruent (P-value = 201 0.067). The phylogenetic consensus trees based on the plastid versus the nuclear data set did not 202 yield any statistically supported incongruent topologies. Thus, in the following we discuss the 203 phylogenetic relationships among Orthophytum and related genera based on the combined data set. 204 A comparison of the two partitioning schemes in the Bayesian analyses showed that the model with 205 three unlinked partitions outperforms the model with two partitions (log Bayes factor: 22.7, 206 harmonic means of the log-likelihood: -6430.35 and -6441.70 respectively). The results of the 207 Bayesian inference (BI) based on the unlinked partition scheme are therefore presented below 208 unless noted otherwise. 209 In the MP analysis of the combined data matrix 130,093 most parsimonious trees of 392 steps 210 in length were found (CI = 0.75; RI = 0.89). The MP (not shown) and the BI consensus trees of the 211 combined data set show a moderate to highly-supported clade containing the genera Orthophytum, 212 Cryptanthus and Lapanthus (BS 71, PP 1). This group comprises four main clades (1–4) in the BI 213 (Fig. 2). 214 The first main clade receives moderate to high statistical support (BS 66, PP 1) and unifies the 215 species of the amoenum subgroup sensu Leme (2004). The clade comprises seven of the ten 216 investigated Orthophytum species with sessile inflorescences and short caulescent habit (Fig. 2, 3). 217 Within the first main clade, O. burle-marxii, O. heleniceae, O. ophiuroides and O. ulei form a 218 moderately to highly-supported clade (BS 69, PP 1) in which the sister group relationship between 219 O. burle-marxii and O. ophiuroides receives a moderately to high statistical support (BS 66, PP 220 0.99). 221 In the second main clade, the two species of Lapanthus form a strongly supported clade (BS 222 97, PP 1), which is found as the sister group to a clade including Cryptanthus tiradentensis in the 223 first diverging lineage plus the highly-supported group (five species) of Cryptanthus subg. 9 224 Cryptanthus sensu Ramírez-Morillo (1996) (BS 100, PP 1). Nevertheless, the sister group 225 relationship receives no statistical support in the BI analysis and the node collapses in the strict 226 consensus of the MP analysis. Within the Cryptanthus subg. Cryptanthus clade, the first divergent 227 lineage is C. bahianus (sect. Bahianae), sister group to a moderately to well-supported clade (BS 228 84, PP 0.99) with C. colnagoi (sect. Cryptanthus), C. diamantinesis (sect. Bahianae), C. warren- 229 loosei, (sect. Bahianae), and C. zonatus (sect. Zonatae). 230 The third main clade shows a highly supported lineage with two long caulescent species with 231 sessile inflorescences of Cryptanthus subg. Hoplocryptanthus (C. odoratissimus, C. microglaziovii; 232 Fig. 2 (BS 95, PP 1) as sister group to a large, highly-supported clade comprising the remaining 233 species of Orthophytum (BS 96, PP 1). Nevertheless, the sister group relationship between the latter 234 and the Cryptanthus subg. Hoplocryptanthus clade does not receive statistical support. 235 The first diverging clade (A) within the large Orthophytum clade is well supported (BS 91, PP 236 1) and consists of three species with sessile inflorescences and long caulescent habit, which 237 constitute the vagans subgroup (O. zanonii, O. vagans, O. pseudovagans) sensu Leme (2004), plus 238 a pedunculate species (O. foliosum) nested within the vagans subgroup. Thus, the clade includes 239 members of the two main morphological groups (sessile and pedunculate inflorescences; Fig. 2). 240 Within the vagans clade, O. zanonii is sister to a well-supported clade with O. vagans, O. 241 pseudovagans and O. foliosum (BS 86, PP 0.98). 242 Next diverging is a well-supported clade (BS 91, PP 1), termed Eu-Orthophytum clade in the 243 following, which comprises all species with pedunculate inflorescences. Its sister group relationship 244 to the vagans clade is well supported (BS 96, PP 1). The Eu-Orthophytum clade splits into two 245 highly supported clades (B, C) and relationships between the two clades receive high statistical 246 support (Fig. 2). Clade B consists of the species of the mello-barretoi subgroup (BS 96, PP 1), 247 represented in our sampling with four out of six species and covering almost the complete 248 geographic distribution of the subgroup. The BI tree shows Orthophytum mello-barretoi as sister 10 249 species of O. schulzianum (BS 75, PP 0.96), both forming a sister group to O. diamantinense and O. 250 graomogolense. In the MP strict consensus tree the phylogenetic relationships within the mello- 251 barretoi subcomplex remain unresolved. 252 Clade C, in the following termed the Core Orthophytum clade, is strongly supported (BS 100, 253 PP 1) and comprises the majority of Orthophytum species, all possessing pedunculate 254 inflorescences in lax spikes of spikes or spikes densely arranged and petal apices obtuse to 255 subacute. Several subclades receive moderate to high support but relationships between these 256 subclades remain largely unclear due to a lack of resolution or statistical support. Noteworthy 257 groups of the Core Orthophytum clade are the glabrum clade (PP 0.97), the fosterianum clade, the 258 saxicola clade (BS 93, PP 1) and the sucrei clade (Fig. 2). 259 3.2. State character reconstruction 260 Both Bayesian and maximum parsimony analyzes reconstructed sessile inflorescences as 261 ancestral character state for the group, with two independent shifts to pedunculate inflorescences: 1) 262 in Orthophytum foliosum and 2) in the Eu-Orthophytum clade (Fig. 2, 3). The character state 263 reconstructions of the corolla form indicates that a tubular corolla represents the ancestral character 264 state in the Cryptanthus-Orthophytum clade with at least two shifts to a clavate corolla in the 265 vagans clade (excluding O. foliosum) and the mello-barretoi clade (Fig. 4A). The ancestral 266 character state reconstructions of petal appendages form indicate that sacciform lacerate petal 267 appendages represent the ancestral state character in the group and that it can be considered a 268 synapomorphy of the amoenum clade. The acute petal appendages are present in Lapanthus, and the 269 absence of petal appendages is shared by at least two clades including Cryptanthus species (Fig. 270 4B). In the next diverging lineage (vagans clade) cupuliform lacerate petal appendages were 271 reconstructed as ancestral state with one shift for sacciform lacerate appendages in Orthophytum 272 foliosum. Fimbriate appendages appear to be a synapomorphy of the Core Orthophytum clade. 273 11 274 275 4. Discussion In Bromelioideae, the reconstruction of infrageneric relationships based on DNA sequence 276 data has proven difficult due to low sequence divergence of molecular markers used so far, yielding 277 phylogenetic reconstructions with generally low resolution and support values (Sass and Specht 278 2010; Schulte et al. 2009; Sousa et al., 2007; Sousa, 2011). In Bromelioideae, those studies were 279 conducted within the more advanced Eu-Bromelioideae, the core bromelioids, which are 280 characterized by the presence of a central water collecting tank and which apparently underwent a 281 rapid radiation starting around 5.5 Ma mainly in a largely continuous habitat, the Brazilian Atlantic 282 rainforest (Givnish et al., 2011; Schulte et al., 2005, 2009). In contrast, Orthophytum represents an 283 early diverging lineage within the Eu-Bromelioideae that diversified extensively in xeric vegetation 284 types in Southeast and Northeast Brazil, the campos rupestres and Caatinga, with their vast 285 diversity of naturally isolated microhabitats (the inselbergs), which promoted genetic isolation and 286 thus fostered the evolution of numerous microendemics in Orthophytum (Smith and Downs, 1979). 287 Here we present and discuss the first molecular phylogeny of the genus based on a comprehensive 288 sampling of its known diversity. This allowed us to identify several highly supported lineages 289 within the genus, to elucidate infrageneric phylogenetic relationships, and to highlight critical issues 290 for future research. 291 292 293 4.1. Phylogenetic relationship within Orthophytum and related genera The phylogenetic reconstructions depict a well-supported clade unifying the Orthophytum 294 species with pedunculate inflorescence plus the vagans clade, which comprises three species with 295 sessile inflorescences and short stem, and one species with a pedunculate inflorescence (O. 296 foliosum) (Fig. 2, 3). 297 The species belonging to the amoenum subgroup sensu Leme (2004) formed a well-supported 298 clade (Fig. 2), thus supporting Leme‟s taxonomic concept. The subgroup is characterized by sessile 12 299 inflorescences (Fig. 3), short and inconspicuous stems, inner leaves and primary bracts contrasting 300 with the outer leaves in color, and white petals with a pair of sacciform lacerate appendages inserted 301 laterally to the antepetalous stamens (Leme, 2004; Louzada and Wanderley, 2010). Leme (2007) 302 also noticed a morphological affinity between Cryptanthus subgen. Hoplocryptanthus sect. 303 Schwackeanae and Orthophytum, stating that species of this section are morphologically more 304 similar to the species of Orthophytum than to any other Cryptanthus species. This morphological 305 similarity is especially pronounced among species of the amoenum subgroup and Cryptanthus 306 subgen. Hoplocryptanthus sect. Schwackeanae (here represented by C. tiradentenis), which mainly 307 differ in the presence or absence of petal appendages. 308 Lapanthus, a genus recently erected to accommodate two aberrant species (L. itambensis and 309 L. duartei) formerly placed within the sessile inflorescence group of Orthophytum (Louzada and 310 Versieux, 2010), forms a well-supported monophyletic clade in sister group position to Cryptanthus 311 tiradentensis (subg. Hoplocryptanthus), and a highly supported clade uniting the species of 312 Cryptanthus subgen. Cryptanthus, however, this sister group relationship is not supported. The 313 species of Cryptanthus subg. Cryptanthus are andromonoecious, usually short caulescent with 314 sessile inflorescences, never pseudopedunculate, and usually without petal appendages or calli 315 (Ramírez-Morillo, 1996). In Bromeliaceae, andromonoecy is only reported for Cryptanthus subg. 316 Cryptanthus (Ramírez-Morillo, 1996), therefore this feature represents a potential synapomorphy 317 for the clade. Lapanthus is morphologically more similar to Cryptanthus subg. Hoplocryptanthus 318 sect. Schwackeanae because both groups possess scented and hermaphroditic flowers (Louzada and 319 Versieux, 2010; Ramírez-Morillo, 1996). Moreover, the species of Lapanthus and Cryptanthus 320 subg. Hoplocryptanthus sect. Schwackeanae share a similar habitat in the southern portion of the 321 Espinhaço Range, inhabiting quartzite-sandstone and iron rocky outcrops. 322 323 In clade 3, the first divergent lineage and sister group of the remaining Orthophytum species comprises Cryptanthus odoratissimus and C. microglaziovii, which belong to the subg. 13 324 Hoplocryptanthus, sections Mesophyticae and Hoplocryptanthus, respectively. This subgenus is 325 characterized by a usually long caulescent habit (sometimes short caulescent), sessile 326 inflorescences, and hermaphroditic flowers. 327 The next diverging clade unifies the three species of the vagans subgroup sensu Leme (2004) 328 plus Orthophytum foliosum (Fig. 2). The species of the vagans subgroup possess an interesting 329 morphological affinity to Cryptanthus odoratissimus and C. microglaziovii. They have a long 330 caulescent habit with a sessile inflorescence (Fig. 3), which is rare in Orthophytum but not in 331 Cryptanthus subg. Hoplocryptanthus sect. Hoplocryptanthus, including C. microglaziovii. The 332 molecular phylogeny indicates that the vagans subgroup in its original circumscription may 333 constitute a paraphyletic lineage, and that Orthophytum foliosum may need to be included. The 334 latter species has a pedunculate inflorescence and was placed in the disjunctum subgroup by Leme 335 (2004). The position of the three species under the subgroup vagans (Leme, 2004; sessile 336 inflorescence and long caulescent habit), which is closely related to the pedunculate species of 337 Orthophytum, raises a few questions. Are the plants really long caulescent? Is the structure found in 338 Orthophytum foliosum really homologous to the peduncle of the other Orthophytum species? 339 Developmental and anatomical studies of the peduncles in Orthophytum are required to address 340 these questions. 341 The outlined morphological affinities between the well-supported clades outside the Eu- 342 Orthophytum clade revealed in the molecular phylogeny lead us to the conclusion that the generic 343 boundaries of Orthophytum, Cryptanthus, and Lapanthus need to be carefully revised as in their 344 current circumscription they may not constitute monophyletic lineages. To this aim, a broader 345 sampling of the genus Cryptanthus appears vital as well as the inclusion of further molecular 346 markers. Traditionally, the genera Orthophytum and Cryptanthus have been separated based on the 347 presence or absence of petal appendages (Orthophytum: present, Cryptanthus: absent). The 348 taxonomic utility of this character for the delimitation of genera in Bromeliaceae has been 14 349 questioned repeatedly (Brown and Terry, 1992; Zizka et al., 1999), and a recent molecular 350 phylogenetic study demonstrated that this character is homoplastic in subfamily Bromelioideae 351 (Schulte and Zizka, 2008). 352 Although the chromosome number 2n=50 prevails in Bromeliaceae (Brown et al., 1997; 353 Brown and Gilmartin, 1989; Cotias-de-Oliveira et al., 2004; Gitai et al., 2005; Louzada et al., 2010; 354 Palma-Silva et al., 2004; Ramírez-Morillo and Brown, 2001, among other), Cryptanthus species 355 studied so far exhibited lower chromosome counts 2n=34, 36 or 54 (e.g. Ceita et al., 2008; 356 Ramírez-Morillo, 1996; Ramírez-Morillo and Brown, 2001) whereas in Orthophytum a polyploidy 357 series of 2n=50, 100, and 150 has been reported (Cotias-de-Oliveira et al., 2000; Louzada et al., 358 2010). In Lapanthus the only report is for L. duartei (= O. supthutii) with 2n=50 (Louzada et al., 359 2010). Therefore, a potentially useful character to delimit Cryptanthus from 360 Orthophytum/Lapanthus may be their chromosome numbers if the reduction of chromosome 361 numbers was the initial isolating event that preceded the evolution of Cryptanthus. Nevertheless, 362 this hypothesis remains to be tested in a phylogenetic framework. So far, chromosome counts for 363 Cryptanthus are available only for a handful of species (9 spp.; Ramírez-Morillo and Brown, 2001). 364 4.2. Phylogenetic relationships in Eu-Orthophytum 365 The first diverging lineage within the Eu-Orthophytum clade represents the mello-barretoi 366 subgroup sensu Leme (2004, 2008), thus supporting the monophyly of the subcomplex. These 367 species are characterized by pedunculate inflorescences with basal primary bracts narrowly 368 triangular and elongate, the usually red inflorescences in congested spikes of glomerules, and green 369 petals with obtuse-cucullate apices. It remains to be evaluated if the subgroup should be recognized 370 with a formal status within the genus (Leme and Paula, 2008) but this should await the inclusion of 371 O. eddie-estevesii, the northernmost species of the group, in the phylogenetic analysis. 372 373 Core Orthophytum (Fig. 2) arises as sister group to the subgroup mello-barretoi and comprises the majority of Orthophytum species, morphologically characterized by lax 15 374 inflorescences in spikes of spikes or spikes, greenish-white petals with white lobes, and obtuse to 375 subacute apices. Within Core Orthophytum four lineages were found with species that, according to 376 their morphological features, may be included in the leprosum and the disjunctum subgroup sensu 377 Leme (2004). The leprosum subgroup is mainly characterized by leaves that do not form a distinct 378 rosette before and at anthesis, and the disjunctum subgroup by possessing a distinct rosette before 379 and after anthesis. 380 The fosterianum clade comprises six pedunculate species (O. alvimii, O. fosterianum, O. 381 grossiorum, O. gurkenii, O. lanuginosum, O. magalhaesii) with inflorescences in lax spikes of 382 spikes, and leaves and primary bracts with lepidote-lanate indumentum, the latter representing the 383 morphological synapomorphy of the group (Hutchinson, 1983; Leme and Paula, 2003, 2005). The 384 second lineage in Core Orthophytum, named sucrei clade, comprises O. boudetianum sister to O. 385 sucrei (BS 84, PP 1), and O. estevesii sister to O. pseudostoloniferum (BS 82, PP 1). These are 386 small sized species with inflorescences that are spikes, inhabiting granitic-gneiss rocky outcrops in 387 the central region of the Brazilian state of Espírito Santo. 388 A highly-supported clade unifies two other groups termed glabrum and saxicola clades. The 389 glabrum clade shelters four species including Orthophytum glabrum, the generic type (Smith, 390 1955). Orthophytum leprosum occurs as sister to a clade with O. glabrum, O. horridum and O. 391 lucidum, and according Leme (2004) the first two species are placed in the leprosum subgroup, and 392 the remaining two other species are placed in the disjunctum subgroup. Based on the present 393 analysis we conclude that the leprosum subgroup is not monophyletic, firstly due to O. glabrum 394 being more closely related to O. lucidum and O. horridum than to O. leprosum, and secondly by the 395 placement of O. falconii (leprosum subcomplex) in the saxicola clade. The combination of features 396 such as stiff coriaceous leaves being glabrous or lepidote with adpressed scales, can characterize the 397 species included in the glabrum clade. However, more morphological studies are necessary in order 398 to better define the group. 16 399 The final diverging clade is a highly supported group termed the saxicola clade which 400 includes the terrestrial species Orthophytum falconii in sister group position to the remaining 401 species of the saxicola clade. Orthophytum falconii is known only from the type collection and 402 resembles morphologically O. benzingii (= O. leprosum) due the absence of a rosette at anthesis 403 (Leme, 2003). For that reason, it was placed in the leprosum subgroup (Leme, 2004). 404 The saxicola clade certainly represents the most diversified group within Core Orthophytum 405 with the dwarf O. saxicola and the robust O. riocontense. As with the glabrum clade, to define one 406 or more morphological features, which characterize the saxicola clade is a challenging task. In 407 contrast to the other subclades within Core Orthophytum, in the saxicola clade both inflorescences 408 types (spikes or lax spikes of spikes) can be found in different species. For example spikes are 409 observed in O. saxicola and O. harleyi whereas lax spikes of spikes are present in O. argentum and 410 O. toscanoi or even both inflorescence types can be found in the same species (e.g. O. 411 conquistense). 412 4.3. Character state reconstructions of key morphological characters 413 In the past, the genus Orthophytum was informally subdivided into two major groups 414 according to the inflorescence type, the sessile and the pendunculate inflorescence group (Leme, 415 2004; Smith and Downs, 1979; Wanderley, 1990). Mapping this character onto the obtained 416 phylogeny revealed that pedunculate inflorescences evolved twice within Orthophytum, once within 417 the vagans clade (O. foliosum) and in a clade that entirely consists of representatives with 418 pedunculate inflorescences, termed Eu-Orthophytum clade and comprising the majority of species. 419 In contrast, the Orthophytum species with sessile inflorescences were found in two clades: the 420 amoenum and the vagans clade, which both received moderate to high support values (Fig. 2). 421 Relationships between these two clades remain unclear due to a lack of resolution in the deeper 422 nodes of the phylogeny. Besides these two clades, the genera Cryptanthus and Lapanthus both 423 consist of species with sessile inflorescences. Mapping the inflorescence type onto the phylogeny 17 424 showed that sessile inflorescences could be regarded as the plesiomorphic condition in 425 Orthophytum, and pedunculate inflorescences as the derived one (Fig. 3). Although pedunculate 426 inflorescences apparently arose twice within the genus, the character state possesses a valuable 427 phylogenetic signal as it characterizes a major lineage, the Eu-Orthophytum clade. 428 The vagans clade and the Cryptanthus subg. Hoplocryptanthus clade possess an interesting 429 long caulescent habit due to the elongation of the vegetative stem bearing the sessile inflorescence, 430 characterizing probably an intermediate stage in the evolution of Orthophytum. Although three of 431 four species included in the vagans clade have sessile inflorescences and one pedunculate, all are 432 composed of congested spikes of glomerules. The same inflorescence branching type is found in the 433 next diverging mello-barretoi clade. Moreover, O. pseudovagans, O. vagans and O. zanoni have 434 petals with obtuse-cucullate apices, only present in these two clades, reinforcing the hypothesis of a 435 clade with intermediate features. 436 Flower characters such as petal appendages and corolla form have been widely used for 437 classification in Bromeliaceae (Smith and Downs, 1974, 1977, 1979) and are considered valuable 438 for inter- and intraspecific classification in Cryptanthus, Lapanthus and Orthophytum (Leme, 2004; 439 Louzada and Wanderley 2010; Louzada and Versieux, 2010). Our ancestral state reconstructions of 440 the form of the corolla and the petal appendages indicated different levels of homoplasy (Fig. 4), 441 which implies a limited taxonomic value of these characters in the group. 442 Corolla form mapped in our phylogeny (Fig. 4A) demonstrates that clavate and tubular 443 corolla arose at least twice in the evolution of the three genera. The type of corolla can be 444 associated with specific pollinators, however reproductive biology studies in these genera are still 445 incipient. 446 As mentioned above, petal appendages have been used as a diagnostic character at genus 447 level. This feature has been also highlighted by Schulte and Zizka (2008) where taxonomic 448 significance of the presence or absence of this character in higher taxonomic levels was evaluated in 18 449 a phylogenetic framework. Furthermore, the authors also emphasized that this character is 450 inappropriate for generic delimitation in Bromelioideae. Taking into account the presence or 451 absence of petal appendages, our data confirm the results published by Schulte and Zizka (2008) 452 (Fig. 4B), however the form of these appendages have not been evaluated at the infra-generic level 453 before. 454 The presence of petal appendages seems to be an ancestral character in the group with the 455 appearance of sacciform lacerate appendages in the amoenum clade (Fig. 2 and 4B). In the next two 456 diverging lineages, which comprise two clades of Cryptanthus genus, the petal appendages are 457 absent and a second independent origin of petal appendages is inferred in the clade including Eu- 458 Orthophytum and vagans clades. 459 Our character state reconstruction showed that the sacciform lacerate petal appendages arose 460 two times independently and therefore are homoplastic. Interestingly, the species of the amoenum 461 and mello-barretoi clades (Figs. 2 and 4B) that present sacciform lacerate appendages are 462 distributed in two extremes of the same mountain chain (Espinhaço Range), indicating the 463 convergent evolution of petal appendages form in the same environment. 464 Fimbriate, acute and cuppuliform lacerate petal appendages are exclusive characters of the 465 Core Orthophytum, Lapanthus and vagans clades respectively, and may be putative 466 synapomorphies of these clades. 467 468 469 5. Conclusions The study presents the first phylogeny of the Brazilian endemic genus Orthophytum. The 470 presented results show the first molecular phylogenetic evidence that Orthophytum as well as its 471 two traditional morphological groups (sessile inflorescence and pedunculate groups) may not be 472 monophyletic. However, with the weak internal node support in trees from the different 473 phylogenetic analyzes, we cannot confirm the non-monophyletic status of Orthophytum. 19 474 Additionally, Cryptanthus species appear at least in the two lineages. However, the two traditional 475 groups amoenum and mello-barretoi were confirmed as natural groups with high statistical support. 476 Mapping the inflorescence type onto the phylogeny showed the pedunculate condition as 477 derived in the evolution of Orthophytum and related. Bayesian and parsimony ancestral state 478 character reconstructions indicated varying level homoplasy in the form of the corolla and the petal 479 appendages, two characters which are frequently used as diagnostic in Cryptanthus, Lapanthus and 480 Orthophytum. 481 Finally, for future studies in Orthophytum we suggest the inclusion of additional variable 482 molecular markers to assess the monophyly of the genus as well as to include a wider sampling for 483 Cryptanthus. Furthermore, phylogeographic studies in closely related Orthophytum species would 484 be desirable to increase our understanding of interspecific relationships. 485 486 6. Acknowledgements 487 The authors thank São Paulo Research Foundation/FAPESP (graduate research fellowship RBL 488 08/52912-5 and CP-S 2009/52725-3) and CNPq for financial support; IBAMA, IEF for collection 489 permits; Lisa Campbell for language editing; Maria Cláudia Medeiros, Gisele Silva, Marlon 490 Machado, Geyner Alves, Diego Pinangé, Ana Paula Prata, Daniel Melo, Rodrigo Oliveira for 491 assistance during field work; Ana Maria Benko-Iseppon, Fábio Pinheiro, Diego Pinangé, Geyner 492 Alves, Rodrigo Pegorin and Cesar Redivo for lab assistance; Leonardo M. Versieux, Rafaela C. 493 Forzza, Tânia Wendt, Tarciso Filgueiras and two anonymous reviewers and editor for their much 494 valuable comments on the paper. 495 496 6. References 20 497 Akaike, H., 1973. 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Examination of subfamilial phylogeny in 632 Bromeliaceae using comparative sequencing of the plastid locus ndhF. Am. J. Bot. 84, 664– 633 670. 634 635 636 637 Ule, E., 0 Beitra฀ge zur Flora von Bahia. Bot. Jahrb. Syst. 42, 191–238. anderley, .G.L., 0. Diversidade e distribuic฀ o geogr fica das esp cies de Orthophytum (Bromeliaceae). Acta Bot. Bras. 4, 169–175. anderley, .G.L., Conceic฀ o, . ., 200 . otas taxono฀micas e uma nova esp cie do 638 ge฀nero Orthophytum Beer (Bromeliaceae) da Chapada Diamantina, Bahia, Brasil. 639 Sitientibus ser. Ci. Biol. 6, 3–8. 640 641 Yu, Y., Harris, A.J., He, X-J. 2013. RASP (Reconstruct Ancestral States in Phylogenies) 2.1 beta. Available at http://mnh.scu.edu.cn/soft/blog/RASP. 26 642 643 Zizka, G., Horres, R., Nelson, E.C., Weising, K. 1999. Revision of the genus Fascicularia Mez (Bromeliaceae). Bot. J. Linn. Soc. 129, 315–332. 644 645 Table 1. Studied material. Abbreviations: B, Herbarium of Botanical Garden of Berlin; FR, 646 Herbarium Senckenbergianum; HB, Herbarium Bradeanum; IBt, Instituto de Botânica, São Paulo; 647 K, Herbarium of Royal Botanical Gardens, Kew; MBML, Herbarium of Museu de Biologia Melo 648 Leitão; SP, Herbarium of Instituto de Botânica, São Paulo; WU, Herbarium of Vienna University. 649 650 Fig. 1. A-U. Diversity of Orthophytum. A. O. argentum. B. O. leprosum. C. O. boudetianum. D. O. 651 braunii. E. O. maracasense. F. O. albopictum. G. O. conquistense. H. O. humile. I. O. disjunctum. 652 J. O. eddie-estevesii. K. O. foliosum. L. O. magalhaesii. M. O. glabrum. N. O. hatschbachii. O. O. 653 ophiuroides. P. O. ulei. Q. O. graomogolense. R. O. grossiorum. S. O. horridum. T. O. sucrei. U. 654 O. lanuginosum (Fotos R.B. Louzada). 655 Fig. 2. Phylogram of Bayesian analysis of the combined nuclear and plastid data (PHYC, trnL-trnF 656 and trnH-psbA ). Numbers above the branches represent posterior probabilities (PP) and below 657 these branches are bootstrap values (BS). Numbers after terminal names indicate the chromosome 658 counts. 659 Fig. 3. Cladogram of Bayesian analysis of the combined nuclear and plastid data set (PHYC, trnL- 660 trnF and trnH-psbA) mapping the presence and absence of a peduncle. 661 Fig. 4. Ancestral character reconstructions for floral characters based on the Bayesian tree inference 662 of the combined nuclear and plastid data set (PHYC, trnL-trnF and trnH-psbA). (A) Corolla form. 663 (B) Petal appendages form. Branch colors indicate the ancestral reconstruction under maximum 664 parsimony. Pie diagrams at nodes indicate ancestral character reconstructions under Bayesian 665 framework. 666 27 667 Taxa Locality Voucher Bromelia pinguin L. ex cult. Schulte 300508-10 (FR) Bromelia serra Griseb. ex cult. Horres 029 (FR) Cryptanthus bahianus L.B. Sm. ex cult. Gartenherbar 11060a (B) Cryptanthus colnagoi Rauh & Leme ex cult. HBV 7103 (WU) Cryptanthus diamantinense Leme ex cult. Leme 3813 (HB) Cryptanthus microgaziovii I. Ramírez Brazil, ES, Santa Leopoldina Louzada et al. 12 (SP) Cryptanthus odoratissimus Leme ex cult. Kautsky et al. s.n. (HB) Cryptanthus tiradentensis Leme Brazil, MG, Tiradentes Louzada et al. 158 (SP) Cryptanthus warren-loosei Leme ex cult. 0013741 (WU) Cryptanthus zonatus (Vis.) Beer Brazil, PE, Recife IBT living collection Lapanthus duartei (L.B. Sm.) Louzada & Versieux Lapanthus itambensis (Versieux & Leme) Brazil, MG, Conceição do Mato Dentro Louzada et al. 28 (SP) Brazil, MG, Santo Antonio do Itambé Louzada et al. 30 (SP) ex cult. Horres 015a (FR) Orthophytum alvimii W. Weber Brazil, MG, Teófilo Otoni Louzada et al. 90 (SP) Orthophytum argentum Louzada & Wand. Brazil, BA, Rio de Contas Louzada et al. 110 (SP) Orthophytum boudetianum Leme & L. Kollmann Brazil, ES, Afonso Cláudio Louzada 135 (SP) Orthophytum braunii Leme Brazil, BA, Seabra Machado 50 (SP) Orthophytum burle-marxii L.B. Sm. & Read Brazil, BA, Lençóis Louzada & Moreira 45 (SP) Orthophytum conquistense Leme & M. Machado Brazil, BA, Vitória da Conquista Machado 277 (SP) Orthophytum diamantinense Leme Brazil, BA, Diamantina Louzada & Ribeiro 146 (SP) Orthophytum estevesii (Rauh) Leme Brazil, ES, Santa Teresa Fontana et al. 2959 (MBML) Orthophytum falconii Leme Brazil, BA, Candido Sales Reis & Falcon s.n. (HB 89876) Orthophytum foliosum L.B. Sm. Brazil, ES, Santa Teresa Louzada et al. 13 (SP) Orthophytum fosterianum L.B. Sm. Brazil, ES, Colatina Louzada et al. 17 (SP) Orthophytum glabrum (Mez) Mez Brazil, MG, Itaobim Louzada & Medeiros 139 (SP) Orthophytum graomogolense Leme & C.C. Paula Brazil, MG, Grão Mogol Louzada & Moreira 42 (SP) Orthophytum grossiorum Leme & C.C. Paula Brazil, MG, Carlos Chagas Leme et al. 5584 (HB) Orthophytum gurkenii Hutchison Brazil, ES, Baixo Guandu Louzada 133 (SP) Orthophytum harleyi Leme & M. Machado Brazil, BA, Érico Cardoso Louzada et al. 108 (SP) Orthophytum hatschbachii Leme Brazil, BA, Rio de Contas Louzada et al. 104 (SP) Orthophytum heleniceae Leme Brazil, BA, Andaraí Wanderley et al. 2544 (SP) Orthophytum horridum Leme Brazil, MG, Pedra Azul Louzada & Medeiros 138 (SP) Orthophytum humile L.B. Sm. Brazil, MG, Grão Mogol Louzada & Moreira 41 (SP) Orthophytum lanuginosum Leme & C.C. Paula Brazil, MG, Teófilo Otoni Louzada & Medeiros 143 (SP) Orthophytum lemei E. Pereira & I.A. Penna Brazil, BA, Morro do Chapéu Louzada et al. 186 Orthophytum leprosum (Mez) Mez Brazil, MG, Jacinto Louzada & Medeiros 141 (SP) Orthophytum lucidum Leme & H. Luther Brazil, BA, Jequitinhonha Louzada & Medeiros 142 (SP) Orthophytum macroflorum Leme & M. Machado Brazil, BA, Licínio de Almeida Machado s.n. (SP 441733) Louzada & Versieux Ochagavia litoralis (Phil.) Zizka, Trumpler & Zoellner 28 Orthophytum magalhaesii L.B. Sm. Brazil, ES, Vila Pavão Louzada 131 (SP) Orthophytum mello-barretoi L.B. Sm. Brazil, MG, Santana do Riacho Louzada & Medeiros 84 (SP) Orthophytum mucugense Wand. & Conc. Brazil, BA, Mucugê Louzada & Moreira 58 (SP) Orthophytum ophiuroides Louzada & Wand. Brazil, BA, Lençóis Louzada & Wanderley 88 (SP) Brazil, ES, Santa Teresa Leme et al. 6915 (MBML) Orthophytum pseudovagans Leme & L. Kollmann Brazil, ES, Águia Branca Demuner et al. 3464 (MBML) Orthophytum riocontense Leme Brasil, BA, Abaíra Machado 1206 (SP) Orthophytum saxicola (Ule) L.B. Sm. Brazil, BA, Itaberaba Louzada et al. 122 (SP) Orthophytum schulzianum Leme & M. Machado Brazil, MG, Diamantina Machado 1218 (SP) Orthophytum sucrei H. Luther Brazil, ES, Afonso Cláudio Louzada 136 (SP) Orthophytum toscanoi Leme Brazil, BA, Machado 1213 (SP) Orthophytum ulei Louzada & Wand. Brazil, BA, Mucugê Louzada & Wanderley 91 (SP) Orthophytum vagans M.B. Foster ex cult. Louzada s.n. (SP 442925) Orthophytum zanonii Leme Brazil, ES, Pancas Louzada et al. 18 (SP) Orthophytum sp. Brazil, BA, Jacaraci Machado 1207 (SP) Puya chilensis Molina ex cult. Chase 23824 (K) Orthophytum pseudostoloniferum Leme & L. Kollmann 668 29 Highlights The first phylogeny of the bromeliad genus Orthophytum is here presented. In the present study was sampled 40 of the about 60 recognized species of Orthophytum. The two main morphological groups of Orthophytum arise as non-monophyletic. The pedunculate inflorescence in the genus can be regarded as derived condition. View publication stats