EVOLUTION OF MORPHOLOGICAL TRAITS IN VERBENACEAE

PREMISE OF THE STUDY
A new infrafamilial circumscription of the Verbenaceae with eight tribes: Casselieae, Citharexyleae, Duranteae, Lantaneae, Neospartoneae, Petreeae, Priveae, and Verbeneae, has been recently proposed, on the basis of molecular phylogenetic studies. Two genera, Dipyrena and Rhaphithamnus, remain unplaced. The aim of this work is to reconstruct the evolutionary history of morphological characters traditionally employed in the classification of the Verbenaceae, with special attention to tribes Verbeneae and Lantaneae.


METHODS
Twenty-one characters, related to habit and vegetative morphology, inflorescence and floral morphology, ovary and fruit morphology, as well as chromosome number, were optimized over a molecular phylogeny of Verbenaceae.


KEY RESULTS
All tribes are supported by at least one morphological trait except tribes Duranteae and Citharexyleae. Suffrutescent habit, sessile flowers, and four cluses are synapomorphies for tribe Verbeneae. Gynoecium with short style and entire stigma are synapomorphic traits for tribe Lantaneae. Sessile flowers and unicarpellate ovaries are morphological synapomorphies for the new tribe Neospartoneae. Suffrutescent habit is a synapomorphic trait for tribe Priveae. Homothetic pleiobotrya and absence of the adaxial staminode are synapomorphic traits for tribe Casselieae. Undivided fleshy fruits are probably a synapomorphic trait for tribe Petreeae. Putative plesiomorphies for the ancestor of the Verbenaceae are discussed as well as synapomorphic traits within other Verbenaceae clades.


CONCLUSIONS
Many of the characters traditionally employed in classification have proven to be very homoplastic, or have been shown not to support relationships within the family. Moreover, traditional assumptions concerning character polarity have in some cases been shown to be incorrect.

Verbenaceae, as recently circumscribed, comprises approximately 32 genera and 840 species ( Cantino et al., 1992 ;Atkins, 2004 ;Marx et al., 2010 ).They are an important element in the fl oras of North and South America, especially in warm temperate and tropical regions.The Verbenaceae includes shrubs or small trees, suffruticose plants, and herbs, and occurs in open and forested, xeric and mesic, habitats.
Recent molecular phylogenetic studies ( Marx et al., 2010 ;Yuan et al., 2010 ) identifi ed the major lineages in the family and resolved generic relationships throughout most of the group.This resulted in a revised classifi cation for Verbenaceae, dividing the family into eight tribes ( Marx et al., 2010 ).Within Verbenaceae, tribes Verbeneae and Lantaneae represent relatively recent radiations with historically uncertain generic boundaries.The fi ve genera and approximately 175 species of the tribe Verbeneae often have been combined under Verbena in earlier treatments ( Schauer, 1847 ;Briquet, 1895 ;Perry, 1933 ;Troncoso, 1974 ).Most species of Verbeneae are found in temperate North or South America.Several recent molecular phylogenetic studies of Verbeneae ( O'Leary et al., 2009 ;Yuan and Olmstead, 2008a , b ) have resulted in recircumscriptions of genera, and a robust phylogenetic backbone for this tribe.Similarly, the approximately 275 species of Lantaneae have been sorted traditionally into genera on the basis of a few easily observable traits (e.g., fruit type and infl orescence architecture) into seven to nine genera (e.g., Atkins, 2004 ).Most species from this tribe are found in tropical North or South America, with a secondary radiation in Africa.Ongoing work on Lantaneae also indicates that generic circumscriptions need to be reassessed ( Marx et al., 2010 ;Lu-Irving and Olmstead, in press ).
Molecular phylogenetic studies of Verbenaceae ( Marx et al., 2010 ;Yuan et al., 2010 ) found that traditional divisions of the family into tribes (e.g., Atkins, 2004 ), based on various combinations of morphological traits, were severely discordant with phylogenetic relationships.Similarly, at the generic level within Verbeneae and Lantaneae, morphology-based delimitations have been shown to be discordant with phylogenetic relationships, both with respect to which genera comprise each tribe and generic circumscriptions.Thus, the major objectives of this study are to identify morphological characters that support phylogenetic relationships within Verbenaceae as inferred on the basis of molecular data ( Marx et al., 2010 ;Yuan et al., 2010 ;O'Leary et al., 2009 ;Lu-Irving and Olmstead, in press ), including, where possible, those clades recognized as tribes, and to reconstruct the evolutionary history of those morphological characters traditionally used in the systematics of the family.
• Premise of the study: A new infrafamilial circumscription of the Verbenaceae with eight tribes: Casselieae, Citharexyleae, Duranteae, Lantaneae, Neospartoneae, Petreeae, Priveae, and Verbeneae, has been recently proposed, on the basis of molecular phylogenetic studies.Two genera, Dipyrena and Rhaphithamnus , remain unplaced.The aim of this work is to reconstruct the evolutionary history of morphological characters traditionally employed in the classifi cation of the Verbenaceae, with special attention to tribes Verbeneae and Lantaneae.• Methods: Twenty-one characters, related to habit and vegetative morphology, infl orescence and fl oral morphology, ovary and fruit morphology, as well as chromosome number, were optimized over a molecular phylogeny of Verbenaceae.• Key Results: All tribes are supported by at least one morphological trait except tribes Duranteae and Citharexyleae.Suffrutescent habit, sessile fl owers, and four cluses are synapomorphies for tribe Verbeneae.Gynoecium with short style and entire stigma are synapomorphic traits for tribe Lantaneae.Sessile fl owers and unicarpellate ovaries are morphological synapomorphies for the new tribe Neospartoneae.Suffrutescent habit is a synapomorphic trait for tribe Priveae.Homothetic pleiobotrya and absence of the adaxial staminode are synapomorphic traits for tribe Casselieae.Undivided fl eshy fruits are probably a synapomorphic trait for tribe Petreeae.Putative plesiomorphies for the ancestor of the Verbenaceae are discussed as well as synapomorphic traits within other Verbenaceae clades.• Conclusions: Many of the characters traditionally employed in classifi cation have proven to be very homoplastic, or have been shown not to support relationships within the family.Moreover, traditional assumptions concerning character polarity have in some cases been shown to be incorrect.November 2012] O'LEARY ET AL.-MORPHOLOGICAL TRAITS IN VERBENACEAE because the cpDNA tree for Verbeneae shows evidence for multiple chloroplast transfer events ( Yuan and Olmstead, 2008a , b ;O'Leary et al., 2009 ).
To assess patterns of evolution of the 21 morphological and chromosome characters, we optimized each character under a parsimony criterion onto the summary phylogenetic tree using the program Mesquite version 2.72 ( Maddison and Maddison, 2005 ).To identify those morphological characters that support relationships based on molecular data, we recorded all morphological synapomorphies at each node.To further evaluate the utility of morphological characters in delimiting monophyletic groups and their degree of homoplasy, the molecularbased tree was used to infer the number of evolutionary steps of these characters using the program Mesquite version 2.72 ( Maddison and Maddison, 2005 ).
The phylogeny of the Verbenaceae is now well-resolved with respect to most generic relationships and the primary lineages within the family.The relationships among families in the portion of Lamiales where Verbenaceae is found, however, still remain unclear, or weakly supported ( Schaferhoff et al., 2010 ), with some support for the small African family, Thomandersiaceae, as sister to Verbenaceae ( Soltis et al., 2011 ;Refulio and Olmstead, unpublished data).Given the uncertainty of outgroup relationships, character optimizations are restricted to the ingroup, which will leave the ancestral node reconstructions unresolved in some instances.

MATERIALS AND METHODS
Accessions examined -Representatives of all tribes within Verbenaceae were examined.In all cases, species were selected to span the morphological variation within each genus (Appendix S1, see Supplemental Data with the online version of this article).All genera included in the family-wide study ( Marx et al., 2010 ) were sampled (including more than one species of most larger genera): Verbeneae -33 species, Lantaneae -29 species, Casselieae -4 species, Neospartoneae -5 species, Duranteae -8 species, Priveae -3 species, Citharexyleae -3 species, and Petreeae -1 species.The monotypic Dipyrena and one of the two species of Rhaphithamnus, genera not assigned to tribe, were examined.The only exceptions were the monotypic Xolocotzia (Petreeae), for which no material was available, and the monotypic Hierobotana , which was also missing in previous phylogenetic studies of tribe Verbeneae ( Yuan and Olmstead, 2008a , b ;O'Leary et al., 2009 ) and its phylogenetic position is still uncertain ( Marx et al., 2010 ).
Morphological data -Twenty discrete morphological characters and one cytological character were scored based on our experiences working with the group, observations of herbarium specimens and taxonomic literature, including all major treatments of the family and revisions of its genera ( Schauer, 1847 ;Briquet, 1895 ;Troncoso, 1974 ;Atkins, 2004 ;Botta, 1989 ;O'Leary et al., 2007aO'Leary et al., , 2008O'Leary et al., , 2010O'Leary et al., , 2011 ; ;O'Leary and Múlgura, 2010 ;Peralta, 2010 ).These 21 characters (Appendix 1) represent habitat and vegetative morphology (characters 1 -2), infl orescence and fl oral morphology (characters 3 -13), ovary and fruit morphology (characters 14 -20), and chromosome number (character 21).All characters were treated as unordered, 14 characters were binary, and 7 were multistate (Appendix S1, see Supplemental Data with the online version of this article).For a few taxa (e.g., the genus Chascanum ), some character states could not be assessed because of absence of existing material and literature information.Therefore, these states were scored as missing.Similarly, cytological data were missing for several taxa.Since the data are not being used for phylogenetic inference, some mutually dependent characters (chars.17 -19) were employed, to better illustrate the evolution of traits.
Evolution of morphological characters -A summary tree depicting relationships within the family was assembled from the family-wide studies ( Marx et al., 2010 ;Yuan et al., 2010 ), in combination with more focused studies of Verbeneae ( Yuan and Olmstead, 2008a , b ;O'Leary et al., 2009 ) and Lantaneae ( Lu-Irving and Olmstead, in press ).This tree was employed as a phylogenetic hypothesis on which to reconstruct character evolution.The portion of the tree below the node connecting Verbeneae and Lantaneae refl ects the congruence of chloroplast ( Marx et al., 2010 ) and nuclear ( Yuan et al., 2010 ) genome studies.The placement of Dipyrena in a trichotomy with Verbeneae and Lantaneae, refl ects the confl ict in its positions in those two studies.Verbena and Glandularia are constrained to monophyly based on phylogenetic inference from nuclear genes, the common evolutionary trend of unidirectional evolution from woody to herbaceous.The suffrutescent habit is a synapomorphy for Verbeneae and found in the basal grade formed by Mulguraea and the ancestor of Junellia .In the ancestor of Verbena plus Glandularia a shift to herbaceous habit occurred, constituting a synapomorphy for this clade.A similar shift occurred in Priveae, where the suffrutescent habit is a synapomorphy for the tribe and a shift to herbaceous habit is found in Pitraea .These results suggest that in Verbenaceae herbs have always evolved from suffruticose ancestors, and that suffruticose plants have always evolved from ancestors that were shrubs or small trees.The only case where this is not confi rmed is the reversion from a suffruticose ancestor to a shrubby habit within Junellia .This reversion was suggested by Sanders (2001) in that woody growth in Junellia might be a secondary development.
Leaf blades in Verbenaceae are typically entire.However, divided leaf blades may have evolved from entire leaf blades independently in a species of Bouchea (Duranteae) and in Burroughsia (Lantaneae) and several times within Verbeneae (char.2) (Appendix S3, see Supplemental Data with the online version of this article).Reversions to entire leaf blades appear to have occurred only within Glandularia .Sanders (2001) suggested Verbena and Glandularia could be distinguished from the rest of the Verbenaceae by their more divided leaf blades.However, leaf incision shows a high level of homoplasy particularly DISCUSSION Habit and vegetative morphology -Most species of Verbenaceae are woody, with Glandularia, Verbena, Phyla , and Pitraea the only herbaceous genera (char.1) (Appendix S2, see Supplemental Data with the online version of this article).Woodiness in Verbenaceae is represented by those plants that are small trees, shrubs, lianas, or suffruticose plants.Shrubs or small trees are plesiomorphic in most Verbenaceae tribes.The suffrutescent habit has apparently evolved from shrubs or small trees at least fi ve times within the family, constituting a synapomorphy for the clade that includes Bouchea, Chascanum , and Stachytarpheta (within Duranteae), the clade of Tamonea plus Casselia (within Casselieae), Priveae, and Verbeneae.Within Lantaneae the suffrutescent habit appears in certain taxa, such as Lippia dulcis Trevir.and Burroughsia .The herbaceous habit has probably evolved independently at least three times in Pitraea (Priveae), Phyla (Lantaneae), and within the tribe Verbeneae.No evidence of a reversion from herbaceous to woody habit has been found in Verbenaceae, but the suffrutescent habit appears to have reverted to shrubby habit in Junellia .Although examples of woodiness evolving from herbaceous ancestors are found in other families such as Apiaceae ( Calviño et al., 2006( Calviño et al., , 2008 ) ), Pericallis in the Asteraceae ( Panero et al., 1999 ), or Echium in the Boraginaceae ( Böhle et al., 1996 ), Verbenaceae exemplifi es Fig. 2. Evolution of the infl orescence, presence or absence of terminal fl orescence (character 3).This character is reconstructed as having four steps within Verbenaceae.
Moldenke.Loss of the terminal fl orescence, constitutes a synapomorphy for all the remaining Lantaneae, i.e., the Lippia + Lantana and Aloysia + Xeroaloysia clade.Within the tribe Verbeneae all members share the plesiomorphic state, e.g., infl orescences with a terminal fl orescence.
Presence of axillary fl orescences (char.4, Fig. 3 ) is the plesiomorphic and most frequent state for Verbenaceae.The character is ambiguously reconstructed and is the most homoplastic of all characters, with at least 14 steps.Loss of axillary fl orescences, (giving rise to monobotrya from heterothetic pleiobotrya), likely occurred unambiguously and independently several times within the family, e.g., in Recordia (Duranteae), Pitraea (Priveae), and Acantholippia (Lantaneae).All other shifts are ambiguously reconstructed and therefore reversions to presence of axillary fl orescences cannot be ruled out.
Classical views of infl orescence evolution ( Fig. 4A ) suggest that compound infl orescences with axillary and terminal fl orescences (heterothetic pleiobotrya) gave rise, on the one hand, to compound truncate infl orescences with only axillary fl orescences (homothetic pleiobotrya) by loss of the terminal fl orescence, and, on the other hand, simple forms reduced to one terminal fl orescence (monobotrya), by loss of axillary fl orescences ( Sell, 1976( Sell, , 1980 ; ;Troll, 1964Troll, , 1969 ) ). Martínez et al. (1996) and Múlgura within Verbeneae (at least 10 changes).Nevertheless, the character of divided leaf blades is a synapomorphy for Glandularia , whereas that of entire leaf blades constitutes a plesiomorphy for Verbena , where divided leaves only appear in the North American Verbena clade.

Infl orescence and fl oral morphology -
The presence of a terminal fl orescence is the plesiomorphic state for the family (char.3, Fig. 2 ).It can take the form of a spike or raceme, found either with or without axillary fl orescences in Verbenaceae, and referred to as heterothetic pleiobotrya or monobotrya, respectively.The shift to the absence of a terminal fl orescence, referred to here as a homothetic pleiobotrya, seems to have occurred at least four times independently within Verbenaceae, once in the ancestor of tribe Casselieae, once in the ancestor of the genus Rhaphithamnus, and twice within Lantaneae.Although reconstruction is ambiguous for the ancestor of Lantaneae, excluding Coelocarpum , the loss of the terminal fl orescence and its regain is less probable than two parallel losses, so we consider this last option to be more likely.Within Lantaneae, presence of a terminal fl orescence is the plesiomorphic state, found only in Coelocarpum and the clade Aloysia + Acantholippia , with the exception of Aloysia catamarcensis Fig. 3. Evolution of the infl orescence, presence or absence of axillary fl orescences (character 4).This character is reconstructed as having a minimum of 14 steps within Verbenaceae.
ing a nonhomoplasious synapomorphy for this clade (Appendices S5, S6C, see Supplementary Data with the online version of this article), whereas the Aloysia pro parte + Xeroaloysia and the Aloysia pro parte + Acantholippia clades both have 4-toothed calyces (Appendix S6B, see Supplementary Data with the online version of this article).Since Coelocarpum is the only member of this tribe with a 5-toothed calyx and is sister to the rest of the clade, a 5-toothed calyx is plesiomorphic for Lantaneae, within which a shift to a 4-toothed calyx occurred once and a subsequent shift to a bilobed calyx resulted in a synapomorphy for the Lippia + Lantana clade.This suggests an apparent progressive reduction in calyx teeth from fi ve to four to two.
The presence of an anther connective tissue surpassing the theca is the single morphological trait that supports the monophyly of Mulguraea (char.7) (Appendix S7, see Supplementary Data with the online version of this article), a genus recently segregated from Junellia ( O' Leary et al., 2009 ).This character state also occurs independently in Bouchea (Duranteae), but could not be confi rmed in its sister genus Chascanum .
A glandular anther connective appendix (char.8) (Appendix S8, see Supplementary Data with the online version of this article) seems to have evolved independently at least fi ve times in Verbenaceae.Within the tribe Verbeneae the presence of a glandular appendix in the anthers is a synapomorphy for Glandularia, but this trait has been lost in Glandularia incisa (Hook.)Tronc.but also appeared independently in Junellia crithmifolia (Gillies & Hook.)N. O'Leary & P. Peralta and related species once assigned to Glandularia ( O' Leary et al., 2009 ).This character has been used traditionally to differentiate between Verbena and Glandularia ( Schnack, 1964 ) and it constitutes a synapomorphy for Glandularia, whereas Verbena bears the plesiomorphic state, e.g., absence of an anther connective appendix.Outside the Verbeneae, presence of a glandular anther connective appendix also appears at least 3 times independently in species from tribe Lantaneae, but where it is not useful to defi ne groups.
Plants of Verbenaceae generally have four fertile stamens.A fi fth structure in the staminal whorl is sometimes present in the adaxial (or ventral) position and is always sterile.This staminode (char.9) (Appendix S9, see Supplementary Data with the online version of this article), is found in at least one genus from each of the fi rst diverging tribes of Verbenaceae from Petreeae to Neospartoneae, and is present also in the family Thomandersiaceae ( Wortley et al., 2007 ;Refulio and Olmstead, unpublished data).The staminode character is highly homoplastic, with at least 7 changes, and its reconstruction is ambiguous.The probable ancestors of tribes Duranteae, Citharexyleae, Casselieae, Priveae, and Neospartoneae could either have had a staminode or not.However, in Lantaneae, Verbeneae, and the genus Dipyrena , the staminode is always absent, leaving only the four staminal structures in the androecium.The absence of the staminode is plesiomorphic for Verbeneae, despite the suggestion by Sanders (2001) that absence of the staminode is a synapomorphy for Verbeneae.
In the possible ancestor of Stachytarpheta , within Duranteae, the fi fth staminode is absent (plesiomorphic state, char.9), and a unique condition from which the two abaxial lateral stamens are sterile probably arose.The monotypic genus Hierobotana (not included here), which has only two stamens and no staminodes, and Stachytarpheta are the only genera of Verbenaceae with two fertile stamens.
The length of the style relative to the length of the ovary (char.11) (Appendix S10, see Supplementary Data with the online version of this article) has been a diagnostic trait used to distinguish Verbena from Glandularia ( Schnack andCovas, et al. (1998 , 2002 ) suggested that this evolutionary trend exists in Verbenaceae, as well.By looking at the reconstructions of characters 3 and 4 together ( Figs. 2, 3 ) it is possible to confi rm that homothetic pleiobotrya found in Casselieae, Rhaphithamnus, Aloysia catamarcensis (Lantaneae) and a large clade within Lantaneae, are always derived from heterothetic pleiobotrya.Homothetic pleiobotrya can therefore be considered a fi nal step, although reversion to heterothetic pleiobotrya cannot be ruled out within Lantaneae.This analysis also confi rms the derivation of monobotrya from heterothetic pleiobotrya by loss of axillary fl orescences.However, unexpectedly, according to classical assumptions about the evolution of infl orescences, reversions from monobotrya to heterothetic pleiobotrya also occur ( Fig. 4B ).The frequency with which these character transformations have occurred in Verbenaceae renders infl orescence architecture to be of limited value for defi ning tribal groups.
Pedicellate fl owers (char.5) (Appendix S4, see Supplementary Data with the online version of this article) are the plesiomorphic state for the family, found in all members of the fi rst diverging tribes from Petreeae to Rhaphithamnus , with the exception of a clade within Duranteae where sessile fl owers are found ( Stachytarpheta + Bouchea + Chascanum ).Reconstruction of this trait is ambiguous along the stem of the tree above the divergence of Rhaphithamnus , with pedicellate fl owers found only in Coelocarpum and Dipyrena , and the fl owers being sessile in the rest of the taxa.For the tribe Verbeneae, presence of sessile fl owers constitutes either a plesiomorphy or a synapomorphy, the latter having been suggested by Sanders (2001) .
Calyx dentition (char.6) (Appendix S5, see Supplementary Data with the online version of this article) has been used traditionally as a diagnostic trait within the family Verbenaceae ( Schauer, 1847 ;Troncoso, 1974 ;Botta, 1979 ;Atkins, 2004 ).A fi ve-toothed calyx (Appendix S6A, see Supplementary Data with the online version of this article) is the plesiomorphic state, and found in most members of the family with the exception of Lantaneae.In this tribe, the clade of Lippia + Lantana has a bilobed calyx, constitut-Fig.4. Schematic representation of infl orescence evolution in Verbenaceae according to (A) classical hypothesis ( Troll, 1964( Troll, , 1969 ; ;Sell, 1976Sell, , 1980 ) ) and (B) maximum parsimony reconstruction of characters 3 and 4. Within tribe Lantaneae reversion to heterothetic pleiobotrya cannot be ruled out.Within tribes Citharexyleae, Neospartoneae, Lantaneae and Verbeneae a shift from monobotrya to heterothetic pleiobotrya are equally likely.
synapomorphy.All members of Verbeneae have bilobed stigmas, a trait that several authors have employed as a diagnostic trait for the tribe ( Sanders, 2001 ;Atkins, 2004 ).However, it constitutes a plesiomorphic trait for Verbeneae.
Flowers in the Verbenaceae have a slender style base (char.13) (Appendices S12, S13, see Supplementary Data with the online version of this article), with the exception of several Junellia species, which have enlarged style bases.Within Junellia this character has been used to distinguish the subgenera Junellia and Thryothamnus ( Botta, 1989 ;Peralta et al., 2008 ).Its reconstruction on the phylogeny shows that the presence of an enlarged style base is a nonhomoplasious synapomorphy for the clade of J. tridactylites (Lag.)Moldenke to Junellia seriphioides (Gillies & Hook.ex Hook.)Moldenke, traditionally treated in Junellia subgenus Junellia .In contrast, both Junellia spathulata (Gillies & Hook.ex Hook.)Moldenke, traditionally treated under Junellia subgenus Thryothamnus ( Botta, 1989 ), and J. crithmifolia (formerly in Glandularia , but recently combined under Junellia by O' Leary et al., 2009 ) have slender style bases.Thus, the enlargement of the style base appears to be a diagnostic trait for subgenus Junellia . 1944Junellia . , 1946 ; ;Schnack, 1964 ;Troncoso, 1974 ;Botta, 1993 ).Most of the early diverging tribes of Verbenaceae (Petreeae, Citharexyleae, Casselieae, and Priveae), have short styles (reconstruction of the character in Duranteae is ambiguous), and this character is inferred to be ancestral in the family.Long styles appeared prior to the divergence of Rhaphithamnus , but short styles are likely present in both the ancestors of Lantaneae and the genus Verbena .The character of short styles constitutes a synapomorphy for Lantaneae and for Verbena .In spite of its homoplastic nature within Verbenaceae, it is a good diagnostic trait to distinguish Verbena from other members of Verbeneae.
An entire, unlobed stigma is the plesiomorphic state in Verbenaceae, and present, with few exceptions, in tribes Petreeae, Duranteae, Citharexyleae, and Casselieae.A bilobed stigma has appeared independently at least four times in Verbenaceae from apparent ancestors with an entire stigma (char.12) (Appendix S11, see Supplementary Data with the online version of this article), e.g., in Recordia and Bouchea (Duranteae, where it is uncertain in Chascanum ), Parodianthus (Casselieae), and in the probable ancestor of tribes Priveae to Verbeneae.Reversion from a bilobed to entire stigma supports the monophyly of Neosparton and of Lantaneae, where an entire stigma constitutes a In Verbenaceae the most frequent type of fruit (char.16, Fig. 6 ) is a dry schizocarp, but some members of the family have fl eshy drupaceous fruits with internal pyrenes.This character shows a high level of homoplasy with the fl eshy fruit having evolved independently at least 8-10 times within Verbenaceae from dryfruited ancestors.Within Lantaneae, the lack of resolution inhibits an unambiguous reconstruction of this character, but multiple origins of fl eshy fruits are evident ( Lu-Irving and Olmstead, in press ).Schauer (1847) reunited several genera under an enlarged tribe Verbeneae (including Lippia, Aloysia , and Priva ) on the basis of their dry fruits, however, this trait is plesiomorphic for all these taxa.Without outgroups in our analysis, it is impossible to unambiguously reconstruct the morphology of the ancestor of Verbenaceae.Yet, by examining fruit variation of families inferred to be close to Verbenaceae ( Schaferhoff et al., 2010 ;Soltis et al., 2011 ; Refulio and Olmstead, unpublished data), we found that most of them have dry fruits, and therefore, a dry fruit is more likely for the ancestor of Verbenaceae.Fleshy fruits are Ovary and fruit morphology -The calyx completely covering the fruit (char.14) (Appendix S14, see Supplementary Data with the online version of this article), is a plesiomorphic character for all Verbenaceae tribes, except Citharexyleae and Casselieae, where it is ambiguously resolved.The transformation to a calyx not fully enclosing the fruit has occurred at least 6 times independently, e.g., in Recordia and Bouchea (Duranteae), Phyla (Lantaneae), Dipyrena , and possibly in the ancestor of the clade comprising Citharexyleae + Casselieae.In Rehdera (Citharexyleae), the calyx does not persist in the fruit.Despite its homoplastic nature, this character is useful to distinguish the tribe Verbeneae, with the plesiomorphic state, from its likely sister taxon Dipyrena glaberrima (Gillies & Hook.ex Hook.)Hook., where the calyx only partially covers the fruit at maturity.
The number of carpels (char.15, Fig. 5 ) in Verbenaceae is one or two, except in Duranta , which typically has four carpels, and/or occasionally fi ve.Bicarpellate ovaries predominate in Lamiales, including Thomandersiaceae ( Wortley et al., 2007 ;Soltis et al., 2011 ;Refulio and Olmstead, unpublished data).When observed in Verbenaceae, anatomical evidence suggests that the occurrence of a single carpel arose through the abortion of one carpel early in development ( Junell, 1934 ;Atkins, 2004 ).Thus, bicarpellate ovaries are likely to be ancestral in Verbenaceae, despite the fact that two early diverging lineages (Petreeae and Duranteae) have unicarpellate ovaries at maturity.The presence of two developed carpels is inferred throughout Fig. 6.Evolution of the type of fruit (character 16).This character is reconstructed as having 10 steps within Verbenaceae.and the innovation consists of the splitting of fruits along a second longitudinal "transverse plane" segregating carpels ( Fig. 8A , "t.p.") as codifi ed in character 19 ( Fig. 10 ).This leads to an increase in the number of dispersal units from 2 to 4 (char.17).Presence of 4 cluses has been used traditionally to distinguish Verbeneae ( Troncoso, 1974 ), and our results show the character constitutes a nonhomoplasious synapomorphy for this tribe.
Although the number of seeds per dispersal unit has been used traditionally in Verbenaceae systematics (e.g., Troncoso, 1974 ;Briquet, 1895 ), we did not code this character, because it results from the correlation between the number of carpels (char.15, Fig. 5 ), which are biovulate, and the separation of the ovary (chars.18 and 19, Figs. 9, 10 ) along one or two longitudinal planes ( Fig. 8 ).Furthermore, the repeated parallel abortion of one carpel (char.15, Fig. 5 ) does not always result in a net change in the number of dispersal units, because this last depends also on the separation of the ovary (chars.18 and 19, Figs. 9, 10 ).In schizocarpic fruits, two-seeded mericarps come from a bicarpellate ovary that separates only along the medial plane of the ovary; whereas one-seeded units (cluses) originate from an unicarpellate ovary that also separates along the medial plane of the ovary, or from a bicarpellate ovary that separates along both the medial and transverse plane.Taxa with fl eshy fruit may produce pyrenes, and these can be two-seeded (e.g., likely to be derived in Petreeae, as it is elsewhere in the family, and no reversals back to dry fruits are inferred. Another important fruit trait is whether the fruit is divided into one of more units at maturity (char.17, Fig. 7 ).The most frequent condition within Verbenaceae is a divided fruit with 2 dispersal units (either cluses or mericarps).Fruits in the most closely related families, including Thomandersiaceae ( Wortley et al., 2007 ;Soltis et al., 2011 ; Refulio and Olmstead, unpublished data) are bicarpellate and multiovulate, and most often are capsules, with seeds as the dispersal unit.Thus, there is no direct inference from outgroups to bear on the question of ancestral fruit in Verbenaceae.The presence of 2 dispersal units is the result of the separation of the fruit along a longitudinal plane orientated on the median of the carpels (or carpel), the "medial plane" ( Fig. 8A, B , "m.p.") that splits them into halves, as codifi ed in character 18 ( Fig. 9 ).Each dispersal unit consists of two adjacent half-carpels (tribes Citharexyleae, Casselieae (except Casselia ), and genera Rhaphithamnus and Dipyrena ), or half of one carpel (tribes Duranteae, Lantaneae, except Coelocarpum , Neospartoneae, and Casselia ).The transformation to an undivided fruit has likely occurred independently numerous times from ancestors with 2 dispersal units, almost always correlated with a shift to fl eshy fruits.A shift to 4-parted fruits has occurred only once in the possible ancestor of the tribe Verbeneae, dently in both the ancestor of Mulguraea and in that of Junellia ( Fig. 11 , asterisk), rendering an enlarged cluse base a plesiomorphic state for Verbena + Glandularia .However, a unique origin of this trait in the likely ancestor of Verbeneae, with a possible later reversal in Verbena + Glandularia , cannot be ruled out with these data.Elsewhere in the family Verbenaceae, this trait can be expressed as a broad base, except where the fruits are undivided.
The evolution of the basic chromosome number (char.21, not shown) is impossible to reconstruct in the family because of lack of information for many members.The common ancestor of tribe Verbeneae, is unambiguously reconstructed as x = 10, as inferred by Yuan and Olmstead (2008a) , although we cannot be certain if this is a synapomorphy or a plesiomorphy.The sister relationship of Verbena ( x = 7) and Glandularia ( x = 5) makes it impossible to know whether there was a sequential reduction from x = 10, to x = 7, and to x = 5, or if both of those lineages are derived independently from ancestors with x = 10.As Yuan and Olmstead (2008a) indicate, these results contradict the hypothesis of an origin of Junellia by polyploidy from ancestors with x = 5 ( Poggio et al., 1988 ;Botta and Brandham, 1993 ).
All previous classifi cations of Verbenaceae relied on some combination of carpel anatomy, fruit morphology, and infl orescence structure to circumscribe tribes ( Schauer, 1847 ;Briquet, 1895 ;Troncoso, 1974 ;Sanders, 2001 ;Atkins, 2004 ).Despite differences among these classifi cations (depending on which traits were emphasized), all included two or more tribes that were in confl ict with the molecular phylogenetic relationships as reconstructed by Marx et al. (2010) .Infl orescence structure was reconstructed as two characters (characters 3 and 4), one of which is highly homoplastic.The loss of a terminal fl orescence (char.3), and subsequent development of indeterminate growth in the infl orescence, is inferred to have occurred four times ( Fig. 2 ), but the loss of axillary fl orescences (char.4), resulting in a monobotrya (e.g., a unique terminal spike), may have occurred as many as 12-15 times ( Fig. 3 ).Similarly, the characters related to carpel anatomy and fruit morphology (chars.15-17) also are highly homoplastic ( Figs. 1, 5-7 ).The diffi culty with which carpel number is interpreted has misled nearly all previous workers who constructed classifi cations of Verbenaceae. Only Junell (1934) , whose recommendations for restructuring the classifi cations of Lamiaceae and Verbenaceae were ignored for nearly 60 years ( Cantino et al., 1992 ), correctly interpreted the carpel anatomy of Verbenaceae.

Conclusions-Synapomorphic morphological characters in
Verbenaceae-The examined morphological and chromosome characters supporting the monophyly of groups inferred by phylogenetic analyses of molecular data are summarized in Fig. 11 .Different combinations of characters states of the four kinds examined (e.g., habit and vegetative morphology, infl orescence and fl oral morphology, ovary and fruit morphology, and chromosome number) support tribal relationships within Verbenaceae.
It is diffi cult to infer synapomorphic traits for the tribe Petreeae, since it is the fi rst diverging tribe within Verbenaceae and no sister families of Verbenaceae were sampled for comparison.However, three morphological synapomorphies from the fruit probably support this tribe, i.e., the entire, undivided (char.17, state 0 and char.18, state 0), and fl eshy fruit (char.16, state 1).Also, reduction to one carpel, through abortion, is another likely synapomorphy.In a separate analysis, Rueda (1994) identifi ed bulbous-based hairs on the leaves and rounded lobes of the calyx (vs.dentate calyx lobes) as synapomorphies of Petreeae.However, he surveyed these traits only for a small number of putatively related genera.
Lantana , Neosparton , and Petrea ), four-seeded (Citharexyleae, except for Rehdera ), or up to 8-seeded ( Duranta ), depending on the number of carpels.In Rhaphithamnus , the fruit is not divided but there are two pyrenes, and each is two-seeded, because both carpels are separated along the medial plane.
The current circumscription of the tribe Verbeneae is supported by the separation of the fruit longitudinally along the transverse plane of the ovary (char.19, state 1), a nonhomoplasious synapomorphy.This innovation, coupled with the plesiomorphic condition that separates the fruit longitudinally along the medial plane of the ovary (char.18), results in the separation of the fruit into 4 units, called cluses (char.17, state 2), a characteristic only found in this tribe, and present in all its members.The Verbeneae is also supported by the suffrutescent habit (char.1, state 1) No synapomorphic traits could be found for tribe Duranteae.However the clade of Stachytarpheta + Bouchea + Chascanum is supported by three synapomorphies, i.e., suffrutescent habit (char.1, state 1), pedicellate fl owers (char.5, state 1), and probably the absence of an adaxial staminode (char.9, state 0).
The tribe Citharexyleae also has no apparent synapomorphies.The absence of evident morphological synapomorphies for Citharexyleae and Duranteae, and their placement as consecutive branches on the phylogenetic tree of Verbenaceae, helps explain why the genera Citharexylum and Duranta were united in nearly all previous classifi cations ( Marx et al., 2010 ).
The clade that groups all Verbenaceae tribes except Duranteae and Petreeae is supported by the presence of bicarpellate ovaries (char.15, state 1), although this is likely to be the ancestral state in the family.The clade from tribe Priveae to tribe Verbeneae is supported by the presence of a bilobed stigma (char.12, state 1), and the clade from Rhaphithamnus to Verbeneae is supported by long styles (char.11, state 1).Furthermore, the clade of Lantaneae + Dipyrena + Verbeneae is supported by the absence of an adaxial staminode (char.9, state 0), although there is some ambiguity in the reconstruction of this trait.
In this study we carried out an exhaustive search for morphological characters that might be synapomorphies within Verbenaceae with a special focus on tribes Verbeneae, based on our knowledge of the group ( O'Leary, 2007 ), and Lantaneae ( Lu-Irving and Olmstead, in press ).Characters traditionally employed in the classifi cation of Verbenaceae were taken into consideration, since one main objective was to analyze their evolution within the family and principally in relation to each tribe.Many of the characters traditionally employed in classifi cation have proven to be very homoplastic, or have not been shown to support relationships within the family.Moreover, traditional assumptions concerning character polarity have been shown to be incorrect in some cases.More detailed studies within other tribes, working with larger samples, and cryptic (and historically unused) characters, may yet identify morphological traits diagnostic for additional clades.
Online Supplemental Materials -The online version of this publication has links to fi gures which are not essential for a basic understanding of the main paper but may help in its comprehension.
Woodiness distributed throughout the above-ground parts of the plant has been coded as shrub or small tree.Presence of persistent woody stems only on the base of the plant has been coded as suffrutescent, and complete absence of woody structures has been coded as herb.
Verbenaceae leaf blades are always simple with differences in the division of the blade (e.g., lobed, dissected).Leaf blades were coded either as entire when there is no incision, or divided, irrespective of the degree of its incision.
A fl orescence is the minimum expression of fl ower arrangement.In Verbenaceae fl orescences are either racemes or spikes varying in arrangement, spacing, number of fl owers, and development of its rachis, this last character sometimes results in capitulate fl orescences.Florescences are organized into simple or compound infl orescences, and in terminal or axillary position.
Recent studies ( Martínez et al., 1996 ;Martínez and Múlgura, 1997 ;Múlgura et al., 1998 ;2002 ;Drewes and Martínez, 1999 )  (3) homothetic pleiobotrya (compound infl orescence, many fl orescences only axillary, see Múlgura et al., 2002 : 5, Fig. 2; O'Leary and Múlgura, 2012 : 580, Fig. 1A ), and considered these cases to be evolutionary stages of one character.In this study, we coded these characteristics as two different characters (chars.3 and 4) because we understand that the presence of a terminal fl orescence is independent of the presence of axillary fl orescences.Therefore for character 3, plants with monobotrya and heterothetic pleiobotrya are here coded as terminal fl orescence present, and plants with homothetic pleiobotrya are coded as terminal fl orescence absent.
Plants with heterothetic and homothetic pleiobotrya are coded as axillary fl orescences present, and monobotrya or absence of axillary fl orescences are coded as absent.See observation under character 3.
Flowers can be arranged in racemes or spikes, in the fi rst case fl owers are briefl y pedicellate, in the second they are sessile.
In Verbenaceae, the calyx is a short or long tube, and its apex can be 2-, 4-, or 5-toothed (Appendix S6, see Supplemental Data with the online version of this article).
The fruiting calyx has been coded as completely covering the fruit whenever no part of the fruit is visible, as in some Glandularia where the calyx teeth are fused and contorted, enclosing the fruit (e.g., see O' Leary et al., 2007b : 504, Fig. 1B,G).If any of the apical part of the fruit is visible, then coding is as partially covering the fruit (e.g., see O'Leary and Múlgura, 2010 : 167, Fig. 1b,f).If the calyx falls during development of the fruit, then this has been coded as not persistent.
In Verbenaceae a schizocarpic fruit can be divided into two or four units, depending on the number of partitions of the ovary.The partition along the "medial plane" ( Figure 8 , "m.p.") separates the fruit longitudinally into two units.A unicarpellate ovary ( Figure 8B ) partitions into two 1-seeded units, called cluses, each cluse representing half a carpel; a bicarpellate ovary ( Figure 8A ) partitions into two 2-seeded units, called mericarps, each one representing two adjacent half-carpels.In some taxa, a second partition segregating carpels along the "transverse plane" ( Figure 8A , "t.p."), perpendicular to the "medial plane", also occurs, deriving four one-seeded units, again called cluses, because each one represents half a carpel.Whenever the fruit is fl eshy (drupaceous) this character was coded as not divided.However, Tamonea is the only example of a dry fruit which does not split (not schizocarpic), and here also coded as not divided.
18. Separation of the fruit longitudinally along the medial plane of the ovary, splitting carpels (or carpel) into halves: 0, absent; 1, present.
See observations under character 17 and Figure 8 "m.p.".Even if the fruit is drupaceous, there can be an internal separation which delimits pyrenes, as in Rhaphithamnus, but the number of dispersion units remains one (see Troncoso, 1974 : 381, Fig. 29k, l).
The most frequent condition in Verbenaceae is an enlarged mericarp or cluse base with an internal fold of the commissural wall, here coded as enlarged (e.g., see Botta, 1989 : 381, Fig. 2D,E).In a group of taxa within tribe Verbeneae, where cluse bases are narrowed and lack an internal fold of the commissural wall, this was coded as narrowed (e.g., see Botta, 1989 : 381, Fig. 2B,C).But, whenever the fruit is dry but not divided (e.g., Tamonea ), or is a fl eshy drupaceous fruit, this character was coded as not divided fruit.
This character was coded into seven states, representing all the different basic chromosome numbers that have been reported in Verbenaceae.
Verbenaceae fl owers have anthers with two thecae, which are joined to each other and to the fi lament by the connective tissue.Sometimes this tissue can be prominently developed with an apiculate apex that surpasses the length of the theca.O' Leary et al. (2011 : 22, Fig. 7E) illustrate stamens with anther connective tissue not surpassing thecae.Botta (1999 : 191, Fig. 141f,f') shows stamens with anther connective tissue surpassing thecae.
Anther connective tissue in Verbenaceae is enlarged sometimes, as explained in character 7, and other times it appears as an apical and very obvious glandular anther appendix.In several cases the appendix can even protrude from the corolla limb (see O'Leary et al. 2011 : 20, Fig. 6G).These cases are coded as present.In other cases the tissue does not enlarge to form a noticeable gland, but just a little glandular tissue is visible (e.g., Dipyrena , Pitraea, see Troncoso, 1974 : 363, Fig. 20g), which in this study this has been coded as absent, as no real appendix is developed.9. Adaxial staminode: 0, absent; 1, present.
Staminodes are generally reduced to a brief antherless fi lament.Verbenaceae fl owers normally have 4 fertile stamens, inserted on the corolla and alternating with the corolla lobes.In some genera 1 staminode is present, and always in the ventral adaxial position, representing a fi fth sterile stamen (see Troncoso, 1974 : 363, Fig. 20f).Whenever a plant has at least one fl ower bearing a staminode, even if staminodes are absent in other fl owers from the same individual, the character is coded as present.The lack of any staminodes is coded as absent.
In Stachytarpheta only 2 stamens are fertile, and the other two in adaxial lateral position are reduced to sterile staminodes (see Atkins, 2005 : 192, Fig. 5D,J).
In most Verbenaceae fl owers, the style base is somewhat inserted at the apex of the ovary, and here coded as a slender style base.However, in some species of Junellia , the style base is enlarged and expands in fruit to partially or even completely cover the fruit, and here coded as an enlarged style base (Appendix S13, see Supplemental Data with the online version of this article).

Fig. 1 .
Fig. 1.Number of evolutionary steps of each of the 21 characters.Bars represent the following classes of characters shown in Appendix 1: life history and vegetative morphology (white bars); infl orescence and fl oral morphology (light-gray bars); ovary and fruit morphology (dark-gray bars); and chromosome number (black bar).Character numbers refer to those presented in Appendix 1.

Fig. 5 .
Fig.5.Evolution of number of carpels (character 15).This character is reconstructed as having seven steps within Verbenaceae (comparison with related families and developmental studies, cited in the text, suggests that both tribes Petreeae and Duranteae represent independent reduction in carpel number).

Fig. 7 .
Fig. 7. Evolution of the number of units resulting from the of division of the fruit (character 17).This character is reconstructed as having 12 steps within Verbenaceae.

Fig. 10 .
Fig. 10.Evolution of the separation of the fruit longitudinally along the transverse plane of the ovary (character 19).This character is reconstructed as having one step within Verbenaceae.

Fig. 11 .
Fig. 11.Examined synapomorphic characters mapped onto a simplifi ed version of the tree depicting relationships of Verbenaceae used elsewhere in this paper.Numbers correspond to those characters listed and described in Appendix 1, with states in parentheses.Probable plesiomorphies of the ancestor of Verbenaceae are provided in the box.