Cardoso Et Al Molecular Phylogeny Vataireoid

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American Journal of Botany 100(2): 403421. 2013.

American Journal of Botany 100(2): 403421, 2013 ; http://www.amjbot.org/ 2013 Botanical Society of America

Taxonomic classifi cations often rely primarily on fl oral mor-phologies. Recent phylogenetic evidence, however, reveals that fl oral traits can be less reliable than vegetative and fruit-ing characters as predictors of phylogenetic relatedness (e.g., Pennington et al., 2001 ; Borba et al., 2001 , 2002 ; Cameron, 2005 ; Lohmann, 2006 ; Martin et al., 2008 ; Chase et al., 2009 ; Waterman et al., 2009 ; Salazar and Dressler, 2011 ; Cardoso et al., 2012a, b ).

Far from being an exception among the classifi cation of fl owering plant families, the classifi cation of the ecologically and economically important Papilionoideae (Leguminosae) has long been infl uenced by the great emphasis placed on fl oral traits (e.g., Bentham, 1865 ; Arroyo, 1981 ; Polhill and Raven, 1981 ; Lima and Vaz, 1984 ; Polhill, 1994 ; Tucker and Douglas, 1994 ; Tucker, 1997 ). The highly specialized papilionate fl ow-ers in papilionoid legumes are typically distinguished from the mostly radial mimosoid and the generally bilaterally sym-metrical, but nonpapilionate caesalpinioid fl owers by having standard, wing, and keel petals clearly differentiated, stamens enveloping the ovary, and a strong bilateral symmetry that of-ten involves a fusion of fl oral organs and limited access to the nectaries and pollen. This kind of fl ower organization repre-sents an ecological alternative to other fl oral types in that pollen and nectar become available after tripping or during succes-sive pollinator visits ( Arroyo, 1981 ; Westerkamp and Claen-Bockhoff, 2007 ). The papilionate fl ower is largely associated with bee pollination but was retained during the evolution of pollination systems involving birds and bats ( Arroyo, 1981 ; Bruneau, 1997 ). In Papilionoideae taxonomy, genera with more

1 Manuscript received 9 June 2012; revision accepted 28 November 2012.

We are grateful to the curators of the cited herbaria for loans of specimens for our morphological or molecular studies or for making their collections available during our visit, to S. Cardoso and V. Maia for sending some DNA samples, to Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renovveis (IBAMA) for issuing collecting permission (SISBIO 22753-1), to A. Popovkin for translating some German and Russian bibliographies about Luetzelburgia taxonomy, to A. Rapini, M. Simon, and two anonymous reviewers for constructive comments on the manuscript, to R. Aguilar for kindly providing a photograph of Vatairea lundellii , to A.M. Bastos, A.A. Cabaas-Fader, E.L. Cabral, M. Caceres, R. Camacho, D.S. Carneiro-Torres, J.G. Carvalho-Sobrinho, A.S.F. Castro, A.A. Conceio, E. Crdula, C. Correia, A.L. Crtes, E.R. Drechsler-Santos, A. Flores, A.P. Fortuna-Perez, R.M. Harley, J.G. Jardim, I.B. Lima, M.C. Machado, R. Machado, J. Marinho, W. Medina, M.O.T. Menezes, E. Mitch, P.L. Moraes, M.F. Moro, T.M. Moura, G. Parada, J.C. Prazeres-Neto, P.G. Ribeiro, N.P. Smith, E.R. Souza, R.M. Salas, R.M. Santos, D. Soto, N. Taylor, and F. Wartchow for assistance with fi eldwork or providing sample materials, and to the staff at HUEFS herbarium for their attention to the fi rst authors herbarium specimens and loans. Fieldwork and DNA sequencing were also partially sponsored by Programa de Pesquisa em Biodiversidade do Semi-rido (PPBIO), Projeto Biodiversidade do Bioma Mata Atlntica (PROBIO II/MCT/JBRJ), Instituto do Milnio do Semi-rido (IMSEAR), Sistema Nacional de Pesquisa em Biodiversidade (SISBIOTA, processes CNPq 563084/2010-3 and FAPESB PES0053/2011), Myndel Botanical Foundation, and FAPESB (PNX0014/2009). This paper is part of the fi rst authors Ph.D. thesis prepared in the Programa de Ps-graduao em Botnica (PPGBot-UEFS) and supported by SWE grant from CNPq (process 201621/2010-0) at Montana State University, Bozeman, USA.

5 Author for correspondence ([email protected])

doi:10.3732/ajb.1200276

A MOLECULAR PHYLOGENY OF THE VATAIREOID LEGUMES UNDERSCORES FLORAL EVOLVABILITY THAT IS GENERAL TO

MANY EARLY-BRANCHING PAPILIONOID LINEAGES 1

DOMINGOS CARDOSO 2,5 , LUCIANO PAGANUCCI DE QUEIROZ 2 , HAROLDO CAVALCANTE DE LIMA 3 , ELISA SUGANUMA 2 , CSSIO VAN DEN BERG 2 , AND MATT LAVIN 4

2 Departamento de Cincias Biolgicas, Universidade Estadual de Feira de Santana, Av. Transnordestina, s/n, Novo Horizonte, 44036-900, Feira de Santana, Bahia, BRAZIL; 3 Instituto de Pesquisas Jardim Botnico do Rio de Janeiro, Rua Pacheco Leo, 915 22460-030, Rio de

Janeiro, BRAZIL; and 4 Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman 59717 USA

Premise of study: Flowering traits can sometimes be overemphasized in taxonomic classifi cations. The fused and completely differentiated papilionate fl oral organs in the neotropical legume trees Vatairea and Vataireopsis were traditionally used in part to ascribe these genera to the tribe Dalbergieae. In contrast, the free and mostly undifferentiated fl oral parts of Luetzelburgia and Sweetia fi t the circumscription of the primitive Sophoreae. Such divergent fl oral morphologies thought to divide deep phylogenetic lineages indeed may be prone to episodic transformation among close papilionoid relatives.

Methods: We sampled 26 of 27 known species of Luetzelburgia , Sweetia , Vatairea , and Vataireopsis in parsimony and Bayes-ian phylogenetic analyses of nuclear ribosomal ITS/5.8S and six plastid ( matK , 3 -trnK , psbA-trnH , trnL intron, rps16 intron, and trnD-T ) DNA sequence loci.

Key results: The analyses of individual and combined data sets strongly resolved the monophyly of each of Luetzelburgia , Sweetia , Vatairea , and Vataireopsis . Vataireopsis was resolved as sister to the rest and the morphologically divergent Luetzelburgia and Vatairea were strongly resolved as sister clades. Floral morphology was generally not a good predictor of phylogenetic relatedness.

Conclusions: Luetzelburgia , Sweetia , Vatairea , and Vataireopsis are unequivocally resolved as the vataireoid clade. Fruit and vegetative traits are found to be more phylogenetically conserved than many fl oral traits. This explains why the identity of the vataireoids has been overlooked or confused. The evolvability of fl oral traits may also be a general condition among many of the early-branching papilionoid lineages.

Key words: convergence; fl oral evolution; Leguminosae; morphology; Papilionoideae; phylogeny.

404 AMERICAN JOURNAL OF BOTANY [Vol. 100

Rizzini, 1971 ). The understanding of the evolutionary history of the vataireoid clade is also particularly important given that the clade includes ecologically confi ned genera that are taxo-nomically diverse in South America and there mostly in Brazil, a country notable for its biological diversity and endemism. A phylogenetic perspective of the vataireoids could provide in-sights into how ecology and geography interact to shape phy-logeny ( Lavin, 2006 ; Schrire et al., 2009 ; Pennington et al., 2006 , 2009 , 2010 ; Srkinen et al., 2012 ). This study also sets the stage for a taxonomic revision of the genus Luetzelburgia , which re-mains the only vataireoid genus not yet monographed (D. Cardoso et al., unpublished manuscript).

Pennington et al. (2000a , 2001 ) postulated at least nine rever-sions from the papilionate fl ower had occurred during early papilionoid evolution. The vataireoid clade provides an excel-lent group with which to detail this issue because it includes genera distinguished from each other by fl oral parts with vary-ing degrees of differentiation and fusion. Phylogenetic analysis of this group provides an opportunity to test contrasting hypoth-eses about conservative vs. labile fl oral organization. We thus identify the most phylogenetically conserved morphologies that can then be used to apomorphically diagnose the vataireoid clade.

MATERIALS AND METHODS

Taxon sampling The species of the vataireoid clade were comprehen-sively sampled for DNA sequence and morphological variation. The sampling included 26 of the 27 known species in the genera Luetzelburgia , Sweetia , Vatairea , and Vataireopsis . Among these is one new Luetzelburgia species, which is here provisionally called L. guianensis . Only one undescribed species of Vatairea that is morphologically most similar to V. erythrocarpa (collected from the rain forest at Reserva Natural Caon del Ro Claro in northwestern Colombia) was not included in the current study. Sampling multiple conspecifi c accessions was guided by an effort to capture the full extent of morphological variation and geographic range distribution for each species (Appendix 1).

Outgroup sampling was guided by the matK phylogenies of Wojciechowski et al. (2004) and Lavin et al. (2005) , which suggest a sister relationship of the vataireoid and the lecointeoid clades (sensu Herendeen, 1995 and Mansano et al., 2004a ): Exostyles , Harleyodendron , Holocalyx , Lecointea , and Zollernia . This putative sister group relationship is corroborated by the leaves or leafl ets of these two groups that often have nonentire margins (e.g., crenate, serrate, or spinescent). Such leaf and leafl et margins were hypothesized as synapomorphic for the lecointeoid clade ( Mansano et al., 2004a ), but they have also been re-ported as common in the vataireoids ( Lima, 1982a ; Cardoso et al., 2008 ).

Morphological data Binary and multistate characters were scored for le-cointeoid and vataireoid species and included three vegetative, 22 fl oral, and fi ve fruit and seed characters (Appendix 2). All characters were treated as unor-dered and unweighted ( Fitch, 1971 ). Morphological data for the ingroup spe-cies were scored from extensive fi eld collections and about 1500 herbarium specimens, including types, deposited in the herbaria: ALCB, ASE, BHCB, CEN, CEPEC, CTES, EAC, F, GUA, HBR, HRB, HUEFS, IAN, IPA, INPA, JPB, K, LPB, MIRR, MBM, MBML, MG, MO, NY, PEUFR, R, RB, SI, SP, SPF, SPSF, U, UB, UEC, UESC, UFMT, USZ, VIC, and XAL (acronyms after Thiers, 2011 ). Morphological character scorings derived from the fi eld and her-barium sources were validated against the taxonomic literature to ensure that all potentially informative morphologies were being considered for analysis. Scor-ing the morphological variation for the outgroup species relied mostly on her-barium specimens and literature sources (e.g., Cowan, 1979 ; Barneby, 1989 , 1992 ; Mansano and Lewis, 2004 ; Mansano et al., 2004b ; Mansano and Vianna-Filho, 2010 ). Evolution of morphological characters was investigated in the total combined parsimony analysis of molecular and morphology data sets using accelerated transformation in the program PAUP* version 4.0b10 ( Swofford, 2002 ). Reconstructing characters as such allowed the identifi ca-tion of potential diagnostic apomorphies for each of the principal vataireoid subclades.

radial than bilateral symmetry or with incompletely differen-tiated petals and free stamens have been classifi ed into the primitive tribes Swartzieae and Sophoreae (e.g., Cowan, 1981 ; Polhill, 1981a , b , 1994 ). The evolutionary history of the early-branching papilionoids, therefore, provides many opportunities to investigate whether traditional taxonomies emphasizing fl oral morphology accurately refl ect phylogenetic relationships. If fl oral morphologies are found to have evolved in a noncon-servative manner, then the question of why this is so can begin to be addressed by studying the specifi c instances of indepen-dent fl oral evolution.

The four early-branching papilionoid genera that are the fo-cus of this study are Luetzelburgia Harms, Sweetia Spreng., Vatairea Aubl., and Vataireopsis Ducke. Because they have contrasting fl oral morphologies, they have been neglected as a potentially phylogenetic cohesive group and classifi ed into dis-parate papilionoid groups. Sweetia is the most distinctive genus with very small fl owers (less than 10 mm long) in combination with subequal calyx lobes, fl abellate standard petals, nearly free stamens, and undifferentiated lateral petals. Because Luetzel-burgia and Sweetia have a syndrome of weakly papilionate fl o-ral features, they have been traditionally classifi ed into the primitive papilionoid tribe Sophoreae ( Polhill, 1981b , 1994 ). Vatairea and Vataireopsis share a completely differentiated papilionate fl ower, including stamens with fi laments fused into a tube and lateral petals differentiated into wing and keel petals ( Lima, 1980 , 1982a ). Although Vataireopsis and Luetzelburgia share crimped petals and a standard lacking an emarginate apex, the truly papilionate fl oral morphology of Vatairea and Vatair-eopsis weighed heavily in classifying these genera into the tribe Dalbergieae ( Polhill, 1981c ).

Although the four vataireoid genera harbor disparate fl oral morphologies, preliminary molecular phylogenetic analyses fo-cusing on the early-branching lineages of Papilionoideae sug-gested they collectively might form a clade along with the radially symmetrical-fl owered genera Exostyles Schott and Harleyodendron R.S.Cowan ( Ireland et al., 2000 ; Pennington et al., 2001 ) . However, a recent analysis of the lecointeoid le-gumes (sensu Herendeen, 1995 ) involving molecular and mor-phological data confi rmed a closer relationship of Exostyles and Harleyodendron with Holocalyx Micheli, Lecointea Ducke, Uribea Dugand & Romero, and Zollernia Wied-Neuw. & Nees ( Mansano et al., 2004a ). The close relationship of Luetzel-burgia , Sweetia , Vatairea , and Vataireopsis , the vataireoid clade, was foreshadowed by Pennington et al. (2000a , 2001 ) and Wojciechowski et al. (2004) . These preliminary molecular results also agreed with earlier but neglected studies of Lima (1980 , 1982a ), who postulated the identity of this group after detailed morphological analysis stemming from the infl uential works of Ducke (1932) and Yakovlev (1976) . Regardless, the vataireoid clade was represented in previous phylogenetic studies by at most one accession per genus and not necessar-ily by all four genera. This clade was resolved as one of many early branches within the well-supported 50-kb inversion clade, which comprises most species and genera of papilionoid legumes ( Pennington et al., 2000a , 2001 , 2005 ; Wojciechowski et al., 2004 ; Lavin et al., 2005; Cardoso et al., 2012c ). The monophyly of the vataireoid clade was thus suspect, as was the circumscription of the constituent genera. Luetzelburgia spe-cies, for example, had been variously misplaced within Vatairea or Vataireopsis and vice versa because several concerned spe-cies shared a distinctive samara fruit morphology involving a large distal wing and small lateral ones (e.g., Ducke, 1930 ;

405February 2013] CARDOSO ET AL.PHYLOGENY OF THE VATAIREOID LEGUMES

[TBR] branch swapping, steepest descent, bootstrap resampling). All charac-ter state transformations were weighted equally and unordered ( Fitch, 1971 ) and maxtrees was set to 10 000. Clade support was estimated with nonpara-metric bootstrap resampling ( Felsenstein, 1985 ) as implemented in PAUP*, where 10 000 bootstrap replicates were each analyzed using the heuristic search parameters mentioned. TreeRot.v3 ( Sorenson and Franzosa, 2007 ) and PAUP* were used to calculate Bremer supports ( Bremer, 1994 ) and par-titioned Bremer supports (PBS), the latter of which provides a relative mea-sure of how different data partitions contribute to the Bremer support for each node in a combined data analysis ( Baker and DeSalle, 1997 ; Baker et al., 1998 ).

Phylogenetically informative insertions and deletions included 17 from the matK/trnK sequence data, 1 from the psbA-trnH data set, 54 from the rps16 intron data set, 17 from the trnL intron data set, and 68 from the trnD-T data set. Gaps were not coded for the ITS data set because they were generally auta-pomorphic and from ambiguously aligned regions. The psbA-trnH inter-genic spacer, which is known for inversions associated with palindromic sequences ( Simpson et al., 2006 ; Whitlock et al., 2010 ), had one unambigu-ous indel, which was a large deletion of 377 bp in all outgroup species. In-dels were coded as binary (presence/absence) following the simple gap coding method of Simmons and Ochoterena (2000) as implemented in the program GapCoder ( Young and Healy, 2003 ). Aligned data sets are acces-sioned in TreeBASE (http://treebase.org, study no. S12754) and also at website http://www.montana.edu/mlavin/data/vataireoids.txt. Voucher spec-imens and GenBank accession numbers for the DNA sequences generated during this study are presented in Appendix 1.

Bayesian analyses ( Yang and Rannala, 1997 ; Lewis, 2001 ) of the indi-vidual ITS data set and the combined data sets were performed with the pro-gram MrBayes version 3.1 ( Ronquist and Huelsenbeck, 2003 ) using the Cyberinfrastructure for Phylogenetic Research (CIPRES) Portal 2.0 ( Miller et al., 2010 ). Two separate runs of a Metropolis-coupled MCMC permutation of parameters were each initiated with a random tree and four chains set at default temperatures ( Huelsenbeck et al., 2001 ). The best-fi tting nucleotide substitution model for each partition was selected via the Akaike information criterion (AIC) ( Akaike, 1974 ) as implemented in the program ModelTest version 3.7 ( Posada and Crandall, 1998 ). The substitution model GTR+I+G was selected for the ITS region, the model K81uf+G for both matK/trnK and rps16 , TVM+G for psbA-trnH and trnL intron, and K81uf+I+G for trnD-T . In the total combined Bayesian analysis, the morphological and indel data were each analyzed with a gamma rates model. All data partitions were unlinked and parameter estimates were made separately for each partition. Markov chains were run for 10 7 generations and sampled every 10 5 generation such that sampling yielded 100 nonautocorrelated Bayesian trees from each run. The program Tracer version 1.3 ( Rambaut and Drummond, 2004 ) was used to identify likelihood stationarity, and trees sampled from these generations were summarized in a consensus that included posterior probabilities as branch support estimates.

We assessed the combinability of DNA markers by comparing clade sup-ports between individual data partitions ( Wiens, 1998 ). Because Bayesian pos-terior probability values are often biased high (e.g., Suzuki et al., 2002 ; Alfaro et al., 2003 ; Erixon et al., 2003 ), we used the more conservative parsimony bootstrap supports to identify clade confl ict between the molecular partitions. Incongruent clades with bootstrap supports >80% were taken as evidence for not combining data sets.

To examine whether outgroup choice affected the topology of the in-group (e.g., because of a potential long branch leading to the outgroup), we followed Holland et al. (2003) and Bergsten (2005) and performed all par-simony analyses using only the vataireoids and evaluated ingroup interrelations in the unrooted network. This issue was also addressed by sampling exten-sively, taxonomically and genetically, among the early-branching papilionoid lineages and using them as outgroups. This effort is part of another study ( Cardoso et al., 2012c ).

Missing data Despite repeated efforts to optimize PCR conditions, we were unable to obtain amplifi able DNA from some herbarium specimens for certain markers (Appendix 1). Sequence coverage in the combined analysis of 71 accessions was ca. 99%. Only Vataireopsis iglesiasii was not sequenced for matK and trnD-T . The only sequences not generated for the outgroups were trnD-T for Exostyles godoyensis , E. aff. venusta , and Lecointea hatschbachii (Appendix 1). Wiens (2003 , 2006 ) showed no negative impact of including in-completely sampled taxa in a concatenated analysis. When accessions sampled for 50% of the data are included they can break up long branches and improve phylogenetic accuracy ( Wiens, 2005 ).

Molecular data DNA isolations, polymerase chain reaction (PCR) ampli-fi cations, and template purifi cations were performed with Qiagen kits (i.e., Qia-gen, Santa Clara, California, USA). Some samples were isolated using a modifi ed version of the 2 CTAB procedure ( Doyle and Doyle, 1987 ) and purifi ed with sepharose CL-6B (Sigma, St. Louis, Missouri, USA). PCR purifi cation through enzymatic treatment with exonuclease I (EXO) and shrimp alkaline phos-phatase (SAP) and the DNA sequencing were performed at the High-Throughput Genomics Unit at the University of Washington, Seattle, Washington, USA.

Our molecular data sets were derived from one nuclear and six plastid DNA regions. The nuclear ribosomal 5.8S and fl anking internal transcribed spacers (ITS region; Baldwin et al., 1995 ) were analyzed because this region has been phylogenetically informative at and above the species level for papilionoid le-gumes (e.g., Lavin et al., 2003 ; Saslis-Lagoudakis et al., 2008 ; Torke and Schaal, 2008 ; Schrire et al., 2009 ; Ireland et al., 2010 ; Pennington et al., 2010 ; Queiroz and Lavin, 2011 ; Delgado-Salinas et al., 2006 , 2011 ). The complete plastid matK protein-coding region and its fl anking 3 - trnK intron ( Hilu and Liang, 1997 ) were included because they have been phylogenetically informa-tive at many taxonomic levels ( Hu et al., 2000 ; Miller and Bayer, 2001 ; Lavin et al., 2003 ; Wojciechowski et al., 2004 ; Pennington et al., 2010 ; Delgado-Salinas et al., 2011 ; Cardoso et al., 2012a , c ). The plastid psbA-trnH intergenic spacer was informative in the caesalpinioid genus Pomaria ( Simpson et al., 2006 ) and the mimosoid tribes Acacieae and Ingeae ( Miller et al., 2003 ). The plastid rps16 intron has been phylogenetically informative in tribe Glycininae ( Lee and Hymowitz, 2001 ) and the caesalpinioid genus Senna ( Marazzi et al., 2006 ). The plastid trnD-T intergenic spacer includes trnY GUA - and trnE UUC -coding genes and is as potentially variable as ITS ( Shaw et al., 2005 ) and was informative in the tribes Robineae ( Pennington et al., 2011 ; Queiroz and Lavin, 2011 ) and Psoraleeae ( Egan and Crandall, 2008 ), and the species-rich genus Mimosa ( Simon et al., 2011 ). The plastid trnL intron has been shown to be rela-tively informative in many groups of Papilionoideae (e.g., Ireland et al., 2000 ; Lavin et al., 2001 , 2003 ; Torke and Schaal, 2008 ; Saslis-Lagoudakis et al., 2008 ). The trnL-F intergenic spacer was not used in the phylogenetic analysis because a preliminary sequencing of all species of Luetzelburgia and the sam-pled species of Sweetia , Vatairea , and Vataireopsis (Appendix 1) revealed a large deletion of ca. 400 bp at the 5 end of this region. Sampling many genetic loci potentially improves phylogenetic accuracy (e.g., Soltis et al., 1998 ; Rokas and Carroll, 2005 ), as can sampling more species or accessions (e.g., Graybeal, 1998 ; Pollock et al., 2002 ; Zwickl and Hillis, 2002 ; Heath et al., 2008 ) because of the increased probability of subdividing long branches.

Amplifi cation and sequencing primers and reaction conditions for the ITS region were described in Delgado-Salinas et al. (1999) and Lavin et al. (2003) and for matK / trnK in Wojciechowski et al. (2004) . The four universal primers (C, D, E, and F) described by Taberlet et al. (1991) were used for the trnL intron and trnL-F . Primers used for psbA-trnH were described by Kress et al. (2005) . The primers used for rps16 and trnD-T were those described by Shaw et al. (2005) . Reaction conditions for psbA-trnH , rps16 , trnD-T , trnL intron, and trnL-F consisted of 40 cycles of denaturation at 94 C for 1 min, annealing at 50 C for 30 s, and extension at 72 C for 1 min.

The phylogenetic utility of nuclear ribosomal repeat sequences can be com-promised by paralagous evolution (e.g., Bailey et al., 2003 ). Pseudogenes are recognized, however, by the numerous small insertion-deletion regions that oc-cur even in the 5.8S region and by not being GC-rich (e.g., Bailey et al., 2003 ; Hughes et al., 2006 ). To detect when and where intraindividual ITS/5.8S se-quence variation might arise, we used an annealing temperature of 50 C and direct sequencing of PCR products. All PCR products from vataireoids and outgroups sequenced cleanly in both the forward and reverse directions. Ad-ditionally, the 5.8S sequences included no indels or divergent sequences. We also subjected ITS/5.8S sequences to a Bayesian Markov chain Monte Carlo (MCMC) analysis ( Yang and Rannala, 1997 ) in which base frequencies and among-site substitution rates were estimated separately for each of the ITS1, 5.8S, and ITS2 regions. The similar estimates of relative frequencies of nucle-otide bases and substitution classes among these three regions suggested no evi-dence of pseudogenes.

Alignment and phylogenetic analysis Forward and reverse reads were assembled with Sequencher 4.1 software (Gene Codes, Ann Arbor, Michigan, USA). Sequences were aligned manually in Se-Al software ( Rambaut, 1996 ) using the similarity criterion of Kelchner (2000) and Simmons (2004) to avoid inconsistencies derived from automated multiple alignments. Parsimony anal-yses of individual and combined data sets were performed with PAUP* version 4.0b10 ( Swofford, 2002 ) and involved the standard approaches that maximized the detection of global optima and clade stability (e.g., retention of all most par-simonious trees, random addition replicates with tree-bisection-reconnection


The partitioned Bremer support scores were nearly uniformly positive, with no confl ict for all nodes defi ning the genera but with some confl ict at the nodes defi ning intergeneric relation-ships ( Fig. 2 ).

The well-supported monophyly and intergeneric relation-ships among each of the vataireoid genera were consistently resolved during an unrooted analysis and after performing sep-arate and combined analyses of matK and trnL intron data, which were comprehensively sampled for early-branching lin-eages of Papilionoideae.

Morphological evolution in the vataireoid clade Although the intergeneric relationships were poorly supported in the total combined analysis ( Table 1 ; Fig. 3 ), the strict consensus gen-erally resolved the same relationships as did the individual and combined molecular analyses. Our analyses rejected any hypothesis of monophyly involving a group marked by nonpap-ilionate fl owers (i.e., a Sweetia - Luetzelburgia clade) or by pap-ilionate fl owers (i.e., a Vatairea - Vataireopsis clade). No matter the combination of data or method of analysis, variation in de-gree of fl oral differentiation was as likely the result of indepen-dent evolution as homology ( Figs. 2, 3 ).

Parsimony reconstruction of ancestral states identifi ed the potential morphological synapomorphies for the vataireoid clade and for each of its constituent genera ( Fig. 3 ). The vatair-eoid clade is defi ned by 10 synapomorphies of which the most unequivocal are the highly congested leaves at the distal ends of fascicled branches (character 1: state 1), corolla bilaterally sym-metrical (9: 0), the standard petal clearly differentiated from the other petals (13: 1) and with a distinct macula (15: 1), keel pet-als free but with overlapping distal ends (22: 1), and samara with a long distal wing arising from a basal seed chamber (26: 1) ( Figs. 4, 5 ).

Vataireopsis , the earliest-branching genus of the vataireoid clade, has eight synapomorphies ( Fig. 3 ), three of which are unique to this genus: the curved hypanthium (6: 1), gynoecium laterally attached on hypanthium wall (25: 1), and the mesocarp concentrated only in the ventral margin of the seed chamber (29: 1). Sweetia is marked by nine macromorphological autapo-morphies, which readily distinguish it from other vataireoid genera. These include small fl owers, less than 10 mm long

RESULTS

Analysis of nuclear ITS data Parsimony analysis produced the maximum number of trees (564 steps, CI = 0.63, and RI = 0.92; Table 1 ). ITS had the highest number and percentage of phylogenetically informative characters ( Table 1 ). The mono-phyly of the vataireoid clade and of its four main lineages cor-responding to the genera Luetzelburgia , Sweetia , Vatairea , and Vataireopsis were each well supported ( Fig. 1 ; Table 1 ). Rela-tionships among the vataireoid genera in the strict consensus are weakly resolved at best. Within Luetzelburgia and Vatairea , species relationship are often weakly resolved. Among the 20 species represented by multiple DNA accessions in the vataireoid genera, well-supported species monophyly was ob-served only in Luetzelburgia andrade-limae , Sweetia fruticosa , Vatairea fusca , V. guianensis , V. lundellii , and Vataireopsis speciosa ( Fig. 1 ).

Analyses of plastid data All individual and combined cp-DNA parsimony analyses strongly supported the monophyly of the vataireoid clade and the four genera ( Table 1 ; Appendices S1S6, see Supplemental Data with the online version of this article). In contrast to the weakly resolved intergeneric relation-ships recovered with ITS, the placements of Vataireopsis as the earliest-branch and Sweetia as sister to Luetzelburgia and Vatairea were better resolved with varying support among most individual cpDNA sequence data sets and with strong support in the combined cpDNA analysis ( Table 1 ; Appendices S1S6).

Analyses of combined nuclear and plastid data Signifi -cant incongruence and decreased resolution were not observed after combining molecular data sets in this study. The combined analysis of molecular data involved a matrix of 71 accessions for the seven nuclear and plastid DNA markers in which eight species were represented by single accessions and the remain-ing species by 26 accessions. Both parsimony and Bayesian analyses resolved the same relationships as did the individual analyses with respect to the monophyly of the vataireoid clade and the four genera ( Fig. 2 ). Interrelationships among the four genera were strongly resolved and were similar to those from the combined cpDNA analysis (Appendix S6).

TABLE 1. Summary of the phylogenetic analyses of the vataireoid legumes, including the characteristics of the sequence data and resulting trees for the different data sets analyzed.

DNA marker N Aligned length No./% PI MPT L CI RI Luetzelburgia Sweetia Vatairea Vataireopsis (Lue,Vat) (Swe (Lue,Vat))ITS 71 844 220/26.1 10 4 564 0.63 0.92 100/1.0* 100/1.0* 100/1.0* 100/1.0* / / matK/trnK 74 1871 198/10.5 720 276 0.93 0.98 100 100 97 100 91 79 psbA-trnH 92 641 98/15.2 10 4 140 0.86 0.97 90 100 56 96 rps16 intron 71 1010 135/12.7 10 4 239 0.86 0.96 100 100 78 100 71 trnL intron 94 631 69/10.7 17 95 0.93 0.99 82 100 84 100 62 87 trnD-T 80 1782 223/12.1 8562 317 0.88 0.98 100 100 90 100 84Plastid combined 71 5935 700/11.5 10 4 1064 0.87 0.97 100/1.0* 100/1.0* 100/1.0* 100/1.0* 96/1.0* 97/1.0*ITS+plastid combined 71 6779 920/13.3 139 1675 0.77 0.95 100/1.0* 100/1.0* 100/1.0* 100/1.0* 80/1.0* 76/1.0*Morphology+combined 71 6779 950/13.6 171 1775 0.75 0.94 100/1.0* 100/1.0* 100/1.0* 100/1.0* 57/1.0* sc/1.0*

Notes: N = number of accessions; Aligned length = length of the aligned molecular matrix; No./% PI = number of parsimony informative characters and percentage of total characters that are parsimony informative; MPT = number of most parsimonious trees; L = length of MPT; CI = consistency index; RI = retention index. The number of parsimony informative characters, MPT, L, CI, and RI reported for the plastid markers were derived from the analysis that included the unambiguous gaps coded as additional characters. Parsimony bootstrap values ( 50%) are provided for each vataireoid genus as well as for the subclades Luetzelburgia + Vatairea (Lue,Vat) and Sweetia + Luetzelburgia + Vatairea (Swe (Lue,Vat)). Posterior probability (*) from the Bayesian analysis is provided only for the most comprehensive analyses of individual ITS region and combined data sets. A node with bootstrap support less than 50% but resolved in the strict consensus is represented by sc. Nodes neither resolved in the strict consensus nor in the Bayesian analysis are represented by .


Fig. 1. Majority-rule consensus tree and respective phylogram derived from the Bayesian analysis of the ITS region showing the relationships among the vataireoid legume genera Luetzelburgia , Sweetia , Vatairea , Vataireopsis , and outgroups. Bayesian posterior probabilities and parsimony bootstrap sup-port values are reported above and below branches, respectively; branches in bold are those supported by 100% posterior probability. All GenBank acces-sion numbers after taxon names refer to sequences newly reported in this study (Appendix 1).


Fig. 2. Majority-rule consensus tree and respective phylogram derived from the combined nuclear (ITS) and plastid ( matK/trnK , psbA-trnH , rps 16, trnL , and trnD-T ) Bayesian analysis showing the relationships among the vataireoid legume genera Luetzelburgia , Sweetia , Vatairea , and Vataireopsis . Bayesian posterior probabilities and parsimony bootstrap support values are reported above and below branches, respectively; branches in bold are those supported by 100% posterior probability. Numbers above bars represent Bremer support ( Bremer, 1994 ). Partitioned Bremer support (PBS) scores ( Baker and DeSalle, 1997 ; Baker et al., 1998 ) for the six DNA partitions are represented by green bars above internodes (positive) and red bars below internodes (negative). The shortest bars represent PBS scores of 0.5 steps. PBS scores that are 20 steps are scaled by tallest bars. Bremer support and PBS scores are not displayed for internal branches in the vataireoid genera.


Fig. 3. Strict consensus of 171 most parsimonious trees derived from the total combined analysis of morphological and molecular (ITS, matK/trnK , psbA-trnH , rps16 , trnL , and trnD-T ) data showing the relationships among the vataireoid genera Luetzelburgia , Sweetia , Vatairea , Vataireopsis , and out-groups. Bayesian posterior probabilities and parsimony bootstrap support values are reported above and below branches, respectively; branches in bold are those supported by 90100% of parsimony bootstrap. Ancestral morphological character states were reconstructed through an acctran optimization. Char-acter state numbers are listed in Appendix 2. , unambiguous synapomorphies; =, parallelisms; , reversals.


(4: 0), calyx valvate (7: 1) and with fi ve more or less equally spaced lobes (8: 1), standard petal fl abelliform (14: 6) and with-out a macula (15: 0) ( Fig. 3 ). Vatairea is defi ned by six synapo-morphies, most of which evolved independently in the genus Vataireopsis ( Fig. 3 ) except for the standard petal emarginate (17: 1) and seed testa fused with endocarp (30: 1). These non-homologies were detected also in the total combined analysis. Eight characters were reconstructed as synapomorphies of Lu-etzelburgia ( Fig. 3 ), six exclusive to this genus including petals covered by sericeous indumentum in the middle portion of the outer surface (12: 1), standard petal with auricles (16: 1), and the undifferentiated wing-like and keel-like petals biauriculate (19: 2).

DISCUSSION

The monophyly of the vataireoid clade The vataireoids comprise lineages that are morphologically very different. Polhill (1981b) weighted the presence of weakly papilionate fl oral characters in Luetzelburgia ( Fig. 4 ) and Sweetia to classify these genera in his Myroxylon group of the papilionoid tribe Sophoreae. The strongly papilionate fl owers of Vatairea and Vataireopsis together with indehiscent fruits ( Fig. 5 ) led to their placement among the genera of tribe Dalbergieae ( Polhill, 1981c ). This clade, unequivocally resolved here, represents but one of the many cases with a complex taxonomic history whose resolution in part changed the classifi cation of many of the early-branching papilionoid legumes ( Pennington et al., 2001 ; Wojciechowski et al., 2004 ; Lewis et al., 2005 ; Cardoso et al., 2012a, c ).

The close affi nity of the vataireoid genera was indeed hy-pothesized earlier in the morphological studies of Ducke (1932) , Yakovlev (1976) , and Lima (1980 , 1982a) . The last study em-phasized samaroid fruit morphology ( Figs. 4, 5 ) to suggest that the tribe Dalbergieae should be expanded to include both Luet-zelburgia and Sweetia in the same subgroup as Vatairea and Vataireopsis . Yakovlev (1976) placed little taxonomic value on the degree of connation of staminal fi laments such that the nearly free stamens of Luetzelburgia did not necessarily signify a distant relationship to Vatairea and Vataireopsis , which have fused staminal fi laments. The proposed taxonomy of these au-thors concerning the vataireoids was overshadowed by Polhills (1981a , 1994 ) classifi cation, however. Our results are generally consistent with those of Ducke (1932) , Yakovlev (1976) , and Lima (1980 , 1982a) . In addition to the several morphological synapomorphies enumerated ( Fig. 3 ), the vataireoid clade is also marked by a large deletion of ca. 400 bp in the trnL-F in-tergenic spacer, a putative molecular synapomorphy not de-tected in any other early-branching papilionoid legumes (e.g., Ireland et al., 2000; D. Cardoso, personal observation). Our analyses provide, therefore, additional support for the exclusion of Exostyles and Harleyodendron from the vataireoid clade, in agreement with Mansano et al. (2002 , 2004a ).

Monophyly and relationships among the vataireoid gen-era The four principal vataireoid lineages all correspond to currently recognized genera. The rain forest genus Vataireopsis is unexpectedly resolved as the earliest branch of the vataireoid clade. The similarity of the papilionate fl owers of Vataireopsis and Vatairea ( Lima, 1980 , 1982a ), which was used to suggest they should be amalgamated ( Polhill, 1981c ), is shown here not to indicate a sister relationship of these two genera.

The monospecifi c genus Sweetia is resolved as sister to a Luetzelburgia + Vatairea clade. This clade of three genera is also apomorphically defi ned by fruits with an indistinct meso-carp (28: 0), which reverts back to a distinct mesocarp in Vatairea . Mainly because of its small fl owers much less than 10 mm long, each with subequal calyx lobes and undifferentiated and nonoverlapping wing and keel petals, Sweetia was once thought to belong to the genus Acosmium Schott s.l. ( Bentham, 1865 ; Mohlenbrock, 1963 ). Yakovlev (1969) reinstated the ge-nus comprising two species: S. fruticosa and S. atrata Mohlenb., the latter of which is a synonym of Machaerium acutifolium Vogel. The taxonomic identity of Sweetia as distantly related to Acosmium s.l. was later confi rmed in phylogenetic studies of trnL intron and matK sequences ( Pennington et al., 2001 ; Wojciechowski et al., 2004 ), and recently in a phylogeny that sampled all lineages ever referred to Acosmium s.l. ( Cardoso et al., 2012a ).

Sweetia holds the greatest number of plesiomorphic fl oral traits of the vataireoids (see state assignments in characters 8, 18, 19, 20, 22, and 23 in Appendix 2) yet was not resolved as the sister to the rest of the vataireoid clade ( Figs. 2, 3 ), a fi nding further underscoring the lability of fl oral morphology in this group. Sweetia also has the smallest samara of the vataireoid clade and does not bear the additional lateral wings on seed chamber, which is common to many Luetzelburgia , some Vatairea , and all Vataireopsis species ( Figs. 4, 5 ; Lima, 1980 , 1982a ; Cardoso et al., 2008 ). It is widespread in the South American seasonally dry tropical forests and woodlands (SDTF) from the Caatinga region in northeastern Brazil, southward to the southeastern Brazilian dry forest remnants, Paraguay, and Missiones, Ar-gentina, and from there northward into the Chiquitana and inter-Andean valleys of Bolivia. Because of its scattered distri-bution in the SDTF biome, S. fruticosa may be shown to have an intraspecifi c phylogeny that is strongly geographically struc-tured to the degree resolved for Cyathostegia (Benth.) Schery, which is endemic to the inter-Andean dry valleys of Ecuador and Peru ( Pennington et al., 2010 ).

Although some Luetzelburgia species have been misplaced in different vataireoid genera often because of the small lateral wings born from the seed-chamber region of the fruit ( Ducke, 1930 ; Rizzini, 1971 ), our analyses strongly confi rm the mono-phyly of this genus ( Figs. 13 ). Luetzelburgia is readily identi-fi ed by several morphological synapomorphies, including the unique standard petal that is oblong to spathulate and all petals sericeous in the outer surface.

The sister relationship of Luetzelburgia and Vatairea was never postulated before because these genera differ greatly in fl oral morphology. This relationship is well supported with mo-lecular data only. Indeed, a tendency of leafl ets to have serrate, crenate or short-toothed margins is the only macromorphologi-cal character that is detected however inconsistently in most Luetzelburgia and Vatairea species but never in Sweetia and Vataireopsis . Rodrigues and Tozzi (2007) analyzed seedlings of all vataireoid genera and described 13-foliolate second eo-phylls only in Luetzelburgia and Vatairea , whereas Sweetia and Vataireopsis have 68-foliolate second eophylls. This character is in need of study among outgroups to say anything about the direction of change between these character states in the vatair-eoid clade. Regardless, more vegetative than fl oral traits have been phylogenetically conserved during the evolution of the vataireoid clade.

Relationships within Vataireopsis The genus Vataireopsis comprises four species of large trees confi ned to the South


Fig. 4. Representative morphology of an outgroup lecointeoid genus and the vataireoids. (A) Harleyodendron unifoliolatum . This Brazilian endemic monospecifi c genus is an example of the predominantly radially symmetrically fl owered lecointeoid legumes. (B) Luetzelburgia andrade-limae . A sterile individual showing fascicled leaves on terminal branches, a common feature in all vataireoid genera. (C) Luetzelburgia bahiensis . Flowering individual when leafl ess, a phenological trait also common to all vataireoids. (D) Luetzelburgia auriculata . Detail of a terminal branch with the fascicled leaves and the sparsely serrate leafl ets. (E) Luetzelburgia andrade-limae . A leafl ess branch holding only the terminal paniculate infl orescence. (F, G) Luetzelburgia bahiensis . (F) Detail of the fl owers to show the crimped petals and the free stamens; (G) different views of the samara to show the small wings on each side of the seed chamber, a unique trait of several vataireoids. (H) Luetzelburgia andrade-limae . An example of a Luetzelburgia samara without small wings on the seed chamber. All photos: Domingos Cardoso.


Fig. 5. Representative morphology of the vataireoids. (AD) Vataireopsis araroba . (A) Fascicled branches that lead to (B) terminal fascicled leaves; (C) detail of bilaterally symmetrical and truly papilionate fl ower with crimped petals; and (D) distally winged samaras showing also small wings on the seed chamber. (E) Vatairea guianensis . Flowering terminal branch still holding the fascicled leaves. (F) Vatairea lundellii . Detail of its strongly differenti-ated papilionate fl ower. (G) Vatairea macrocarpa . Samaras without small wings on seed chamber. Photos AE and G: Domingos Cardoso; F: Reinaldo Aguilar.


2010 ; De-Nova et al., 2011 ; Govindarajulu et al., 2011 ; Pennington et al., 2010 , 2011 ; Queiroz and Lavin, 2011 ; Srkinen et al., 2011 , 2012 ; Simon et al., 2011 ).

Because of the nonmonophyly of individual species and the poorly supported interspecies relationships in Luetzelburgia ( Figs. 13 ; D. Cardoso et al., unpublished manuscript), infer-ring morphological evolution in this genus is diffi cult. The par-simony optimization of the morphology shows a very complex pattern including high levels of homoplasy. For example, petals predominantly dark pinkish to red (10: 2) evolved indepen-dently in L. purpurea , L. sotoi , in the most recent common an-cestor (MRCA) of L. andina and L. guianensis , and in the MRCA of L. andrade-limae and L. guaissara . The absence of small lateral wings emanating from the seed-chamber portion of the mature samara also evolved independently in L. neuro-carpa and in the MRCA of L. andrade-limae and L. guaissara , but this state reversed back to the presence of these small wings in L. trialata . Similarly, leaves 1125-foliolate (2: 2) evolved in the MRCA of L. andrade-limae and L. guaissara , but this state reversed back to leaves with less than 10 leafl ets in L. trialata .

Relationships within Vatairea Vatairea with nine species of large trees (one of which is yet to be described) inhabits mostly neotropical rain forests ( Lima, 1982a ). Most Vatairea species have widespread distributions. Vatairea lundellii is common throughout Central America; V. heteroptera occurs throughout the Brazilian coastal Atlantic Forest; and V. guian-ensis , V. paraensis , V. sericea , V. erythrocarpa , and V. fusca are found throughout the Amazon region. Vatairea macrocarpa is the only species occurring in savanna vegetation, where it has been listed among the most dominant of the woody species ( Ratter et al., 2006 ). The shallow depth of the V. macrocarpa crown clade, which is distally nested within the Vatairea lin-eage ( Fig. 2 ), suggests a recent evolution and thus corroborates the hypothesis of a recently assembled Cerrado fl ora derived by niche evolution of rain forest ancestors ( Simon et al., 2009 ; Simon and Pennington, 2012 ).

As in the rain forest genus Vataireopsis , species relationships were fairly well supported and revealed low geographic phylo-genetic structure, as expected given the widespread distribution of all species. This is consistent with the phylogenies of other neotropical rain forest genera such as Clusia L. ( Gustafsson and Bittrich, 2003 ), Guatteria Ruiz & Pav. ( Erkens et al., 2007 ), Renealmia L. ( Srkinen et al., 2007 ), Ruellia ( Tripp, 2008 ), and the species-rich legumes Inga Mill. ( Lavin, 2006 ) and Swartzia Schreb. ( Torke and Schaal, 2008 ).

Character evolution in Vatairea is also primarily marked by convergence: small lateral wings on the fruit seed chamber (27: 1) evolved independently not only as a synapomorphy for the genera Luetzelburgia and Vataireopsis , but also in V. fusca and as a synapomorphy for V. erythrocarpa and V. paraensis . Leaves 1125-foliolate (2: 2) evolved independently as an autapomorphy for V. heteroptera and as a synapomorphy for V. fusca and V. lundellii . Large fl owers (>20 mm long) (4: 2) are also convergent in V. heteroptera , V. erythrocarpa , and V. guianensis ( Fig. 3 ).

Independent fl oral evolution in the early-branching Papil-ionoideae The vataireoid example that refutes the idea that fl oral characters are phylogenetically conserved ( Tucker and Douglas, 1994 ; Tucker, 1997 ) is exemplifi ed elsewhere, most notably in pollinator-driven convergent fl oral evolution of orchids (e.g., Borba et al., 2001 , 2002 ; Cameron, 2005 ; Chase et al.,

American tropical rain forests ( Lima, 1980 ). Vataireopsis araroba has the largest fl owers (>20 mm long) in the genus, whereas the other Vataireopsis species have medium-sized fl owers, 1120 mm long ( Lima, 1980 ). Vataireopsis iglesiasii is the only species to have the small lateral wings of the seed cham-ber ending next to the stipe, as well as a straight hypanthium ( Figs. 2, 3 ). This last fl oral trait is uncommon in Vataireopsis but common in the other vataireoid genera. The Amazonian V. iglesiasii is more closely related to the Brazilian Atlantic Forest endemic V. araroba than it is to other Amazonian en-demics ( Fig. 2 ). This relationship reveals an idiosyncratic phylogenetic pattern observed in many rainforest-inhabiting legumes in which phylogenies are more strongly ecologically than geographically structured ( Lavin et al., 2004 ; Schrire et al., 2005 ; Lavin, 2006 ).

Relationships within Luetzelburgia Luetzelburgia is the most diverse genus (13 spp., one not yet described) of the vataireoid clade ( Fig. 4 ). Clarifi cation of the taxonomy of Luet-zelburgia was attempted by Yakovlev (1976) . Extensive fi eld efforts in recent years, however, revealed that the genus is still in need of a more precise species-level circumscription ( Cardoso et al., 2008 ). This phylogenetic study of the vataireoid clade sets the stage for a forthcoming taxonomic revision of Luetzel-burgia together with a biogeographical and phylogenetic account for the genus (D. Cardoso et al., unpublished manuscript).

Luetzelburgia is ecologically similar to Sweetia in having a predilection to South American seasonally dry tropical forests and woodlands (SDTF; Cardoso et al., 2008 ). Seven species of Luetzelburgia are found in the Brazilian Caatinga, the largest and most isolated nucleus of SDTF ( Pennington et al., 2000b ; Queiroz, 2006 ). Luetzelburgia auriculata is widespread in the savannas and dry forests of the Cerrado and Caatinga in central and northeastern Brazil. Luetzelburgia praecox is widespread in the savannas of central Brazil. All remaining species are each narrowly distributed in disjunct dry forest patches in the Atlan-tic domain of southeastern Brazil, in southern and northern Amazonia, and the Chiquitano and inter-Andean dry forests of Bolivia.

The Luetzelburgia phylogeny is geographically structured as might be expected for lineages largely associated with the SDTF biome ( Pennington et al., 2006 , 2009 ). The morphologi-cally distinct Caatinga specialists, L. andrade-limae , L. bahien-sis , L. harleyi , L. neurocarpa , and L. purpurea , were resolved as closely related in the molecular combined analysis ( Fig. 2 ). The Atlantic dry forest sister endemics L. guaissara and L. tri-alata provide another example. However, both total combined and individual phylogenies did not reveal strongly supported clades within the genus. This result contrasts to the general pat-tern observed in the predominantly dry-forest genus Coursetia , in which well-supported geographical phylogenetic structure arises in both individual phylogenies of nuclear ITS and plastid trnD-T sequences ( Queiroz and Lavin, 2011 ; Srkinen et al., 2012 ). The geographical structuring in the phylogeny of Luet-zelburgia , but with weak clade support and a high level of in-complete lineage sorting was also revealed by analyses of a broad ITS data set of more than 200 accessions spanning the known geographical distribution and morphological variation of the genus (D. Cardoso et al., unpublished manuscript). Our results suggest that Luetzelburgia has recently evolved within the SDTF biome, which may explain why its phylogeny is less geographically structured compared to other SDTF lineages (e.g., Lavin et al., 2004 ; Lavin, 2006 ; Duno-de-Stefano et al.,


2009 ; Waterman et al., 2009 ) and of the radially symmetrically fl owered Gloxinieae lineages of Gesneriaceae ( Clark et al., 2011 ). It also has been widely detected in the early-branching papilionoids, including the tribe Amorpheae, which is marked by both nonpapilionate and papilionate-fl owered subclades ( McMahon and Hufford, 2004 ); the baphioid legumes with weakly papilionate fl owers sister to the non-protein-amino-acid-accumulating clade that includes the large majority of Papilionoideae ( Pennington et al., 2000a ; Wojciechowski et al., 2004; Cardoso et al., 2012c ); the dalbergioid genera Acosmium , Etaballia Benth., Inocarpus J.R.Forst. & G.Forst., and Riede-liella Harms, each marked by radial fl owers and each sister to different strongly papilionate lineages ( Lavin et al., 2001 ; Cardoso et al., 2012a ); and the nonpapilionate-fl owered genis-toid genera Cadia Forssk., Dicraeopetalum Harms, Guiano-dendron Sch.Rodr. & A.M.G.Azevedo, and Leptolobium Vogel where each is sister to disparate clades marked by bilateral, nearly to strongly papilionate fl owers ( Pennington et al., 2001 ; Lavin et al., 2005 ; Boatwright et al., 2008 ; Cardoso et al., 2012a, b, c ). Even the hierarchical signifi cance hypothesis ( Tucker, 1997 ), which posits that early developing fl oral traits will be more phylogenetically conserved than later developing traits, has been called into question in studies of Fabales, le-gumes, and closest relatives ( Bello et al., 2012 ). Indeed, the long held conventional view that a well-differentiated, strongly bilateral papilionate fl ower with fused fl oral parts has been de-rived from an open, radially symmetrical, nonpapilionate fl ower (e.g., Arroyo, 1981 ; Polhill, 1981a , 1994 ) has been challenged by many molecular systematic studies over the last decade (e.g., Pennington et al., 2000a ; Wojciechowski et al., 2004 ; Cardoso et al., 2012a , c ).

The vataireoid clade provides a vignette of how fl oral evolu-tion unfolds in the many diverse papilionoid lineages that once formed the tribes Swartzieae and Sophoreae. Papilionoid le-gumes with radial to weakly papilionate fl owers with free parts were traditionally classifi ed into one of these two tribes. These two tribes have been continuously recircumscribed ( Ireland, 2005 ; Pennington et al., 2005 ; Cardoso et al., 2012c ) because they harbor disparate and often distantly related lineages that are each often more closely related to ones marked by strongly papilionate fl owers. Given the pace at which recent molecular legume phylogenetic studies have been focusing on legume taxa once classifi ed into Swartzieae and Sophoreae ( Ireland et al., 2000 , 2010 ; Lavin et al., 2001 ; Pennington et al., 2001 ; Mansano et al., 2004a ; Wojciechowski et al., 2004 ; Cardoso et al., 2012a , c ), it is expected that more vataireoid-like examples will be forthcoming. The profusion of such examples will then prompt the question of what ecological conditions (e.g., perhaps the lack of specialist pollinators; Cronk and Mller, 1997 ) favor the rapid evolution of radial or undifferentiated fl oral morphologies from a papilionate ancestral condition.

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