(Verbenaceae) Using Multiple Loci

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Botanical Journal of the Linnean Society, 2013, 171, 103–119. With 5 ﬁgures

Investigating the evolution of Lantaneae (Verbenaceae) using multiple loci

PATRICIA LU-IRVING* and RICHARD G. OLMSTEAD

Department of Biology, University of Washington, Seattle, Washington 98195, USA

Received 3 February 2012; revised 29 June 2012; accepted for publication 23 August 2012

Lantaneae are an example of a taxonomically problematic, widespread and recently radiated Neotropical lineage. Taxonomy in Lantaneae is difficult because of complex, overlapping patterns of shifts in morphological traits among members; monophyly of the traditional genera cannot be assumed without additional information from molecular data. We took a multi-locus approach to infer phylogenetic relationships in Lantaneae, resolving major clades among a broad representative sample that covers the morphological, taxonomic and geographical diversity of this group. Data from multiple, independent loci reveal individual gene trees that are incongruent with one another, with varying degrees of support. Without reliable, applicable methods to determine the sources of such incongruence and to resolve it, we present the consensus between well-supported topologies among our data sets as the best estimate of Lantaneae phylogeny to date. According to this consensus tree, fleshy fruits in Lantaneae have been derived from dry fruits at least five times; taxonomic schemes separating genera based on fruit characteristics are artificial. Lantaneae have shifted into the Neotropics from the southern temperate subtropics and have colonized Africa in at least two separate long-distance dispersal events. This study provides a first pass at a broad Lantaneae phylogeny, but two important areas remain unresolved: the position of Acantholippia relative to Aloysia; and species-level relationships in the Lantana–Lippia clade. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 103–119.

ADDITIONAL KEYWORDS: Lantana –Lippia – long-distance dispersal – Neotropics – pentatricopeptide repeat (PPR) loci – recent radiation.

INTRODUCTION With the increasing range of modern tools available to systematists, great progress has been made in the The Neotropics are globally renowned as a region of last several years in untangling the evolutionary remarkable floristic diversity. Much of this species histories of difficult taxa in important Neotropical richness is concentrated in large, endemic (or nearly families. Recent examples, in addition to those cited endemic) lineages, such as Cactaceae, Bromeliaceae above, can be found in cycads (González, Vovides & and Bignoniaceae (Gentry, 1982). In these and other Bárcenas, 2008), palms (Eiserhardt et al., 2011; characteristic Neotropical lineages, ‘problematic’ taxa Ludeña et al., 2011), Fabaceae (Torke & Schaal, 2008), are common: plant groups in which traditional clas- Annonaceae (Chatrou et al., 2012) and Podostemaceae sifications are at odds with newly obtained molecular (Tippery et al., 2011). Each of the lineages studied in evidence. Examples include Mammillaria Haw. in these examples has in common particular characteris- Cactaceae (Butterworth & Wallace, 2004), subfamily tics that make it problematic: it is species-rich and Bromelioideae in Bromeliaceae (Schulte, Barfuss & geographically widespread, classifications within the Zizka, 2008; Sass & Specht, 2010), Tabebuia Gomes group are historically difficult and previous broad, ex DC. (Grose & Olmstead, 2007) and tribe Bignon- molecular phylogenetic studies fail to resolve relation- ieae (Lohmann, 2006) in Bignoniaceae. ships within it. Here we present an additional example from our work in Lantaneae: a morphologically diverse group of several hundred species forming the most *Corresponding author. E-mail: [email protected] species-rich tribe of Verbenaceae.

BACKGROUND INFORMATION branch lengths in Lantaneae found in the molecular study of Marx et al. (2010). After recent recircumscription (Marx et al., 2010), Adding to the problems associated with describing tribe Lantaneae is monophyletic, containing two the wide range of morphologies in Lantaneae, the major genera (Lantana L. and Lippia L.) and seven tribe is also geographically wide-ranging. The origin smaller genera. It is sister to tribe Verbeneae (Yuan of Lantaneae is in subtropical South America, and et al., 2009b; Marx et al., 2010). The two principal the centre of diversity is in the Neotropics (Atkins, genera of Lantaneae comprise c. 75% of the species 2004; Marx et al., 2010; Olmstead, 2012). The native (Lippia with c. 200 species and Lantana with c. 150 distribution spans the southern states of the USA, species; Atkins, 2004). However, some taxonomists Mexico and Central America, the Caribbean and consider that there are too many names in Lantana South America; a few species also occur on the (López-Palacios, 1991; Verdcourt, 1992; Santos, 2002), other side of a trans-Atlantic disjunction, in Africa preferring to recognize as few as 55 species (Sanders, and Madagascar. Some members, most notably the 2001). Other genera are smaller: Aloysia Palau (30 Lantana camara L. species group, have been globally species); Phyla Lour. (five species; O’Leary & introduced as ornamentals and spread as weeds, Múlgura, 2012); Nashia Millsp. (seven species); Acan- apparently hybridizing with native species in some tholippia Griseb. (six species); Coelocarpum Balf. f. parts of the Neotropics (Sanders, 1987), further con- (five species); Burroughsia Moldenke (two species; fusing taxonomic efforts. Native African species are Moldenke, 1940); and the monotypic Xeroaloysia assigned to both Lantana and Lippia, suggesting at Tronc. (numbers of species from Atkins, 2004, unless least two distinct colonization events. otherwise attributed). Many members of Lantaneae There is a growing effort to address the troublesome are of ecological and ethnobotanical significance in classification schemes in Lantaneae and to produce their natural settings; for example, Acantholippia sal- generic revisions (e.g. Silva, 1999; Salimena, 2002; soloides Griseb., a community dominant in the alti- Silva & Salimena, 2002; Santos, 2002; Sanders, 2001, plano used locally as a culinary herb. Others are of 2006; Siedo, 2008; O’Leary et al., 2012; O’Leary & global economic and/or ecological importance; for Múlgura, 2012). However, because Lantaneae are example, Aloysia citriodora Palau (lemon verbena), species-rich, geographically widespread and recently commonly cultivated for its medicinal and culinary radiated, these taxonomic efforts are hindered by the uses, and Lantana camara (lantana), a popular orna- common problems that such a group presents. Their mental and weed of global significance. focus is often on specific geographical regions, usually The evolutionary history of Lantaneae presents a defined by political boundaries, which may or may not difficult problem. The large numbers of species in be of biogeographic significance. Additionally, many Lantaneae encompass a great deal of morphological taxonomic revisions focus on single genera, tradition- variation, ranging from herbs to shrubs and small ally circumscribed, under the implicit assumption that trees, with a diverse spectrum of leaf morphologies generic boundaries are of evolutionary significance. and inflorescence architectures. Members of Lanta- There is a clear need for a broad, well-resolved phy- neae are found in many different habitats, including logenetic hypothesis for Lantaneae, which has yet to moist lowland forests, the fire-prone cerrado and the be addressed in detail in a molecular phylogenetic dry altiplano, each with accompanying morphological study. adaptations. Attempts to partition this wide range of variation according to generic and infrageneric boundaries traditionally rely heavily on fruit mor- PHYLOGENY RECONSTRUCTION USING MULTIPLE phology (Chamisso, 1832; Schauer, 1847; Briquet, INDEPENDENT LOCI 1895, 1904; Moldenke, 1959; Troncoso, 1974). Accord- Phylogenetic systematic studies in plants over the ing to one scheme, species with schizocarpous fruit last three decades have made great use of sequence are assigned to Lippia and species with fleshy drupes data from plastid DNA, and recent studies that are placed in Lantana (Schauer, 1847; Troncoso, sample very broadly across large Neotropical groups 1974). Alternatively, the number of mericarps or continue to rely on it (e.g. Lohmann, 2006; Olmstead pyrenes per fruit has also been used to separate et al., 2008, 2009; Marx et al., 2010; Bárcenas, Yesson Lantana from Lippia (Chamisso, 1832; Silva, 1999). & Hawkins, 2011; Givnish et al., 2011). However, the However, generic boundaries in Lantaneae are plastid genome has a lower rate of molecular change blurred by species that are difficult to assign unam- than the nuclear genome, and individual plastid biguously to genus, presumably attributable to con- loci are often insufficiently variable to provide reso- vergence in these (and other) important diagnostic lution between species in recently diversified groups traits. These confounding morphological patterns are (Small, Cronn & Wendel, 2004). The nuclear genome consistent with recent radiation, as are the short is an extensive source of variable DNA regions, and

© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 103–119 PHYLOGENY OF LANTANEAE 105 variable nuclear loci are often much richer sources of MATERIAL AND METHODS information for molecular phylogenetic studies in SAMPLING such groups (Small et al., 2004; Whittall et al., 2006; Taxa were chosen to broadly represent the morphologi- Steele et al., 2008). Additionally, hybridization and/or cal and geographical variation found in Lantaneae. All incomplete lineage sorting may be common among genera belonging to the tribe were sampled (Acanthol- recently diverged species; their effects can only be ippia, Aloysia, Burroughsia, Coelocarpum, Lantana, exposed by multi-locus approaches. For example, Lippia, Nashia, Phyla, Xeroaloysia). Forty-seven tribe Verbeneae have a complicated evolutionary species of Lantaneae were chosen as the ingroup, and history of plastid transfer, incomplete lineage sorting seven species from related lineages were chosen as and convergent character evolution, which was only outgroups. Voucher information and GenBank acces- revealed by molecular phylogenetic studies using sion numbers for all taxa sampled are listed in the multiple loci (Yuan & Olmstead, 2008a, b; O’Leary Supporting Information (Table S1). et al., 2009; Yuan et al., 2009b). As genomic resources and sequencing technologies continue to be developed, the information content of the nuclear genome has become increasingly accessible to and drawn upon by DNA EXTRACTION, AMPLIFICATION AND SEQUENCING phylogenetic studies; the COSII genes in Solanaceae DNA was extracted from dried leaf tissue that was are one example of this (Levin, Whelan & Miller, collected in the field and preserved in silica gel or 2009). sampled from herbarium specimens. Extractions were Yuan et al. (2009a) developed approaches to carried out following a standard 2 ¥ cetyl trimethyl- utilize the pentatricopeptide repeat (PPR) gene family ammonium bromide (CTAB) method (modified from as a source of multiple nuclear loci suitable for use Doyle & Doyle, 1987); DNA was purified by isopropa- in phylogenetic studies and optimized primers to nol precipitation and some extractions were further amplify and sequence several of these loci in Verben- purified using a DNA cleanup kit (Promega Corp.). aceae (Yuan et al., 2009b). PPR genes encode peptides PCRs were performed in a Perkin-Elmer thermocy- with unusually high substitution rates. There are a cler, under the following general reaction conditions: large number of PPR loci, and these are highly diver- 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, gent from one another. The shared presence of many 50 °C for 30 s, 72 °C for 1.5–2.5 min, followed by 72 °C of these loci in such distantly related groups as for 10 min. Universal primers were used to amplify the Brassicaceae (Arabidopsis thaliana (L.) Heynh.) and trnT-L (Taberlet et al., 1991), rpl32-trnL (Shaw et al., Poaceae (rice, maize) suggests that the present 2007) and trnQ-rps16 (Shaw et al., 2007) regions from diversity of PPR genes is attributable to ancient the plastid genome. The ETS region of the nuclear duplications (Yuan et al., 2009a, b). Yuan et al. 18S/26S rDNA was amplified using the 18S-IGS (2009a) screened the genomes of A. thaliana and rice primer of Baldwin & Markos (1998) with a custom for intron-less PPR genes with a single orthologue in primer designed to amplify ETS in Lamiales (ETS-B: each, and published a list of > 100 of these. The loci on 5′-ATAGAGCGCGTGAGTGGTG-3′). The AT1G09680 this list are valuable as phylogenetic tools because and AT5G39980 PPR genes (hereafter referred to as they can be directly sequenced and easily and unam- PPR11 and PPR123, from the order in which they are biguously aligned, problems caused by doubtful listed by Yuan et al., 2009a) were amplified using orthology are avoided, and they can potentially be primers optimized for use in Verbenaceae by Yuan developed for use in any plant group. et al. (2009b). Primers specific to the AT3G25970 We took a multi-locus approach to reconstruct a region (hereafter referred to as PPR81; Yuan et al., phylogeny for Lantaneae, in order to test monophyly 2009a) in Verbenaceae were designed following the of the genera, investigate the extent to which fruit procedure outlined by Yuan et al. (2009a); the following characters are homoplasious, and seek evolutionary primers were successfully used to amplify a fragment patterns in geographical distribution in the tribe. We of the coding sequence of approximately 1.2 kb in collected DNA sequences across a broad sample of the length: PPR81-400f (5′-AGTGCRCTTTTWGATATGTA tribe, from three PPR genes along with the nuclear YGCAAAGTG-3′) and PPR81-1630r (5′-TCRACTGC external transcribed spacer (ETS) region and three ACATGCRTAATKTTCCAT-3′). All PCR products were plastid loci (trnT-L, rpl32-trnL and trnQ-rps16). Two purified by polyethylene glycol (PEG) precipitation. of the PPR loci used in this study were amplified Cycle sequencing reactions were carried out in using primers designed by Yuan et al. (AT1G09680 a Perkin-Elmer thermocycler using BigDye ver. 3.1 and AT5G39980; 2009b); a third (AT3G25970) was (Applied Biosystems Inc.), following a standard selected from the original list of those with a single Applied Biosystems sequencing protocol. For all loci orthologue in A. thaliana and rice (Yuan et al., 2009a) except ETS, internal sequencing primers were used and new primers were designed to amplify it. in addition to PCR primers to obtain overlapping

© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 103–119 106 P. LU-IRVING and R. OLMSTEAD reads across fragments (see also Supporting Informa- 50 million generations). Longer MrBayes analyses tion, Table S2). Products of sequencing reactions were carried out using the NSF TeraGrid via the were purified by precipitation in sodium acetate CIPRES portal (Miller, Pfeiffer & Schwartz, 2010). and ethanol or by passing through Sephadex G-50 When summarizing consensus trees over all runs, the columns. An Applied Biosystems genetic analyser was first 25% of sampled trees were considered burn-in used to generate raw sequence data; the reads were and were discarded. then edited and assembled using Sequencher (Gene Codes Corp.). FRUIT EVOLUTION AND BIOGEOGRAPHY A semi-strict (combinable component) consensus tree PHYLOGENETIC ANALYSES between trees inferred from different loci was con- Sequences were aligned using MAFFT ver. 6 online structed using PAUP* (Swofford, 2000); relationships (Katoh et al., 2002); alignments were then inspected that were not well supported in individual trees (boot- and manually adjusted where necessary. Sequence strap value > 80% and posterior probability > 0.9) alignments for the three plastid loci (trnT-L, trnL- were considered unresolved and collapsed before rpl32 and trnQ-rps16) were concatenated and ana- creating the consensus. We used Mesquite ver. 2.75 lysed as a single data set. Alignments for nuclear (Maddison & Maddison, 2011) to score taxonomically loci were treated as separate data sets. Phylogenetic important fruit characters and geographical distribu- reconstructions were performed individually for each tions, to map them onto the consensus tree and to data set, and for a supermatrix consisting of data infer the most parsimonious character states and from all loci (plastid and nuclear) in concatenation. distributions at ancestral nodes. The supermatrix was treated as consisting of a single partition. RESULTS The suitability of different models of evolution to the data was assessed using jModeltest 0.1 (Posada, 2008). DATA COLLECTION The GTR + I +Gmodel was selected and applied to all Complete or nearly complete sequences of each target analyses. Phylogenetic reconstructions for individual locus were obtained for the majority of taxa included data sets and supermatrices were carried out using in this study. Only the sequences of the PPR81 locus maximum likelihood and Bayesian approaches, as for three taxa (Lippia rehmannii H. Pearson, Lantana implemented in GARLI (ver. 2.0; Zwickl, 2006) and rugosa Thunb., Burroughsia fastigiata (Brandegee) MrBayes (ver. 3.1.2; Ronquist & Huelsenbeck, 2003). Moldenke) were not available; sequences for these Shimodaira–Hasegawa (SH) tests (Shimodaira & taxa were treated as missing data in the phylogenetic Hasegawa, 1999) were carried out to gauge the com- analyses from all concatenated sequences, and not patibility of the results of analyses of individual loci included in the individual analyses of the PPR81 with one another. Tree likelihood scores were calcu- locus. A few other sequences were partial for some lated and SH tests performed using PAUP* ver. 4b10 taxa, or included short regions of missing data (DNA (Swofford, 2000), with RELL optimization and 5000 from herbarium specimens was occasionally of poor replicates under the GTR + I +Gmodel. quality, making amplification difficult). The ETS Maximum likelihood analyses used two replicate region for Lippia origanoides Kunth. was amplified runs, which were run with the generation threshold and sequenced from a different DNA accession for termination at 20 000 generations, and termina- (individual) from that which provided sequences for tion score threshold 0.05. Bootstrapping was carried other loci; ETS could not be sequenced directly from out with 100 replicates, with the generation threshold the original accession because of a length polymor- for termination lowered to 10 000 to facilitate faster phism. The sequences from ETS and from the PPR analysis (as recommended in the GARLI manual, ver. loci contained some single nucleotide allelic differ- 0.96). ences within individuals, which were scored as poly- Bayesian analyses used two replicate runs, each morphisms in alignments. consisting of four chains, which were run for at least The total aligned sequence data gathered were one million generations, and sampled every 1000 gen- 400 bp of ETS (all taxa), 1180 bp of PPR11 [except erations. Convergence between runs was assessed by Lippia lupulina Cham.: 761 bp; Lippia diamantinensis examining standard deviations of split frequencies, Glaz.: 854 bp; Lantana trifolia L.: 753 bp; Citharexy- and by using AWTY (Wilgenbusch, Warren & Swof- lum montevidense (Spreng) Moldenke: regions of ford, 2004) to plot split frequencies over different missing sequence totalling 253 bp], 1059 bp of PPR81 runs. Analyses that had not converged after one [except Dipyrena glaberrima (Gillies & Hook.) Hook.: million generations were run until convergence diag- 914 bp, Lippia dulcis Trevir.: 913 bp, Lippia javanica nostics indicated they had reached stationarity (up to (Burm.f.) Spreng: 923 bp; sequences from Lippia

© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 103–119 PHYLOGENY OF LANTANEAE 107 rehmannii, Lantana rugosa and Burroughsia fastig- FRUIT EVOLUTION AND BIOGEOGRAPHY iata were excluded], 1047 bp of PPR123 (except Lippia The consensus between well-supported topologies of lupulina: 773 bp). Plastid loci were completely ampli- individual data sets is shown in Figures 4 and 5. Fruit fied and sequenced for all taxa [except trnQ-rps16 of characters important in separating Lantana from Phyla nodiflora (L.) Greene, for which approximately Lippia are mapped in Figure 4, with parsimony recon- ′ 250 bp were missing from the 3 end]. Plastid loci structions of ancestral states. Geographical ranges varied in length, from 626–698 bp for trnT-L frag- of members of Lantaneae sampled in this study are ments, 738–1010 bp for trnL-rpl32 fragments and mapped in Figure 5, with putative ancestral distribu- 1065–1652 bp for trnQ-rps16 fragments and, in com- tions inferred by parsimony. bination, provided 4335 bp of aligned sequence data. After alignment and concatenation, the supermatrix of all sequence data consisted of 8734 aligned positions. DISCUSSION These results provide the first phylogenetic hypoth- eses for Lantaneae, which are broadly sampled and PHYLOGENETIC ANALYSES sufficiently resolved to reveal the major groups within The results of phylogenetic reconstructions from the tribe. These major clades are consistent between individual data sets are depicted in Figures 1–3A; gene trees, despite some points of incongruence in Figure 3B shows the results of phylogenetic analysis their relationships to one another and the relation- of the supermatrix consisting of all data in concate- ships among taxa within them. The monophyly of nation. In SH tests, individual data sets all rejected Lantaneae sensu Marx et al. (2010) is confirmed. The the best likelihood trees from the other data sets with short branch lengths in the tribe, particularly in the < P 0.000 (see also Supporting Information, Table S3). Lantana–Lippia clade, are consistent with a recent < The combined tree was rejected with P 0.05 by the radiation. We find strong evidence for the non- plastid data, PPR81 and PPR123, but not by ETS monophyly of the major genera of Lantaneae. Species (P = 0.118) and PPR11 (P = 0.09). of Lantana and Lippia are interspersed throughout Well-supported clades are consistent between the the Lantana–Lippia clade, and Nashia, Burroughsia maximum likelihood and Bayesian analyses for each and Phyla are nested in it, as is a lineage of Aloysia data set; relationships that are resolved differently by spp. The remaining Aloysia spp. sampled here are maximum likelihood and Bayesian analyses receive allied with Acantholippia spp. and Xeroaloysia in a low support. Three out of five gene trees place Coe- grade leading to the Lantana–Lippia clade. Major locarpum in a sister relationship with the rest of taxonomic revisions are required in Lantaneae; in Lantaneae, with good support; conflicting topologies order to achieve monophyletic genera, Lantana and receive poor support in the other two gene trees. Two Lippia must either be fragmented into many smaller well-supported clades of Aloysia spp. are present in genera, or lumped into a single genus. Our phyloge- all gene trees: the Aloysia citriodora clade, and the netic analysis reveals multiple independent shifts in Aloysia gratissima (Gillies & Hook.) L. D.Benson the fruit characteristics historically used to diagnose clade, which includes Xeroaloysia ovatifolia (Mold- genera (fleshiness and number of pyrenes); we also enke) Tronc. However, there is conflict between gene show that the African members of Lantaneae repre- trees about whether these two clades together form sent at least two independent colonization events. a clade (ETS and plastid trees do not feature this The finding that the Lantana camara species complex clade; all three PPR genes do). The tree inferred from is not immediately related to most other Lantana plastid data places Acantholippia salsoloides as sister spp. is of note to tropical conservationists investigat- to the A. citriodora clade, with good support, but trees ing biological means to control invasive L. camara from the four nuclear loci place this species in various populations. other relationships, with varying levels of support. The tree inferred from all loci in concatenation is consistent with the plastid gene tree with regard to ANALYSES OF INDIVIDUAL DATA SETS the placement of Coelocarpum, the two Aloysia clades Areas of each individual tree that did not receive good mentioned above, and A. salsoloides. Acantholippia support were sometimes reconstructed differently by seriphioides (A.Gray) Moldenke is consistently the different methods of phylogenetic inference used reconstructed in a well-supported sister relationship here (indicated by dashed lines in Figs 1–3). This is with a large clade comprising all sampled species of probably indicative of a lack of phylogenetic signal in Lantana and Lippia. This large Lantana–Lippia the data in these areas. clade also contains the sampled members of Nashia, The contrast between the relatively slow rate Burroughsia and Phyla and one Aloysia sp. [Aloysia of change of the plastid genome and the higher sub- barbata (Brandegee) Moldenke]. stitution rates of the nuclear genome is evident in the

Figure 1. See caption on next page.

Figure 1. A, maximum likelihood phylogeny inferred from DNA sequences from nuclear ETS (400 bp), for 47 species of Lantaneae and seven outgroup species. Branches in bold are supported by > 80% of bootstrap replicates and posterior probability values > 0.9 in Bayesian analyses of the same data. Dashed lines indicate branches not present in the phylogeny inferred by Bayesian analysis. B, maximum likelihood phylogeny inferred from DNA sequences from nuclear locus PPR 11 (1180 bp), for 47 species of Lantaneae and seven outgroup species. Branches in bold are supported by > 80% of bootstrap replicates and posterior probability values > 0.9 in Bayesian analyses of the same data. Dashed lines indicate branches not present in the phylogeny inferred by Bayesian analysis. ᭣ branch lengths and resolution of the trees shown scope of this study, the question of which topology in Figures 1–3A (note that the ETS tree is drawn to best reflects the evolutionary history of this species half the scale of the other trees). The concatenated remains open. The position of Acantholippia salsolo- plastid matrix was several times the length (aligned ides relative to the two Aloysia clades will affect how positions) of any other locus sequenced, but did Acantholippia and Aloysia are recircumscribed and not provide enough information to resolve relation- should be resolved before revision can take place. ships in the Lantana–Lippia clade (although deeper Given the generally poor resolution of the backbone nodes in Lantaneae were resolved with confidence). of the Lantana–Lippia clade, a future study using This is consistent with our expectations and with denser sampling and additional loci would be findings in the sister group to Lantaneae, Verbeneae required to study the evolution of this group in detail, (Yuan & Olmstead, 2008a, b). Plastid sequence and the placement of Lippia rhodocnemis and Lippia would be needed in great quantities compared with aristata would be best addressed therein. The situa- nuclear sequence in order to provide enough information in which the position of a few lineages are in tion to resolve relationships at the species level in strongly supported conflict between gene trees was Lantaneae. also found in Verbeneae, the sister tribe of Lantaneae Plastid data could not resolve relationships (Yuan & Olmstead, 2008a, b; O’Leary et al., 2009; between closely related species and the rapidly evolv- Yuan et al., 2009b), and in the problematic Neotropi- ing nuclear ETS region failed to resolve many of the cal palm tribe Bactridinae (Eiserhardt et al., 2011; deeper nodes with confidence. In contrast, sequences Ludeña et al., 2011). In these examples, the question from PPR genes provided the greatest resolution of how the conflicting lineages are related to one over the whole tree. All nuclear loci sequenced for this another, and to other lineages within their respective study had polymorphic sites in some individuals, tribes, also has yet to be resolved. which did not affect direct sequencing (they were When phylogenetic signals between gene trees are coded as polymorphisms in alignments). Some allelic in conflict, the pattern of species divergence is some- variation is to be expected of nuclear loci, but would times best represented by the combined phylogenetic require the isolation of individual alleles via cloning signals; i.e. the best estimate of the species tree is in order to be studied in more detail. provided by analysing the conflicting loci in concatenation (the total evidence approach; Kluge, 1989). This approach provides a good approximation of the INCONGRUENCE BETWEEN LOCI species tree under circumstances when stochastic Trees reconstructed from different individual data error in the finite data partitions is the cause of sets differ in their topologies, and are not compatible incongruence (Olmstead & Sweere, 1994; Gadakgar, with one another according to SH topology tests (see Rosenberg & Kumar, 2005) and is an attractive pros- also Supporting Information, Table S3). However, pect when individual data sets do not provide enough most of the differences are in relationships that information to resolve a tree. Combined analyses are not well supported, and are thus probably best have been commonly performed in phylogenetic explained by insufficent information and/or noise studies over the last 10–20 years (reviewed briefly by (‘soft incongruence’; Seelanan, Schnabel & Wendel, Edwards, 2009; recent examples in Neotropical plants 1997). Our results also include a few instances include studies by Sass & Specht, 2010; Eiserhardt of well-supported incongruence between loci with et al., 2011). However, analysis of combined data respect to the placement of (1) Dipyrena glaberrima does not reliably reflect the species tree under other among the outgroups, (2) Acantholippia salsoloides, circumstances, such as when conflicting evolutionary (3) Lippia rhodocnemis Mart. & Schauer/Lippia her- histories underlie individual genes as a result of mannioides Cham. and (4) Lippia aristata Schauer. incomplete lineage sorting, hybridization or gene Conflict between different loci over the placement of duplication and extinction (Maddison, 1997; Slowin- Dipyrena glaberrima has been previously reported ski & Page, 1999; Kubatko & Degnan, 2007). Alter- (Marx et al., 2010) and, whereas it lies outside the native approaches, most commonly assuming that

Figure 2. See caption on next page.

Figure 2. A, maximum likelihood phylogeny inferred from DNA sequences from nuclear locus PPR 81 (1059 bp), for 44 species of Lantaneae and seven outgroup species. Branches in bold are supported by > 80% of bootstrap replicates and posterior probability values > 0.9 in Bayesian analyses of the same data. Dashed lines indicate branches not present in the phylogeny inferred by Bayesian analysis. B, maximum likelihood phylogeny inferred from DNA sequences from nuclear locus PPR 123 (1047 bp), for 47 species of Lantaneae and seven outgroup species. Branches in bold are supported by > 80% of bootstrap replicates and posterior probability values > 0.9 in Bayesian analyses of the same data. Dashed lines indicate branches not present in the phylogeny inferred by Bayesian analysis. ᭣ incomplete lineage sorting is the cause of incongru- is monophyletic. Acantholippia contains two distinct ence, rely on coalescent theory to infer the most likely lineages, Aloysia contains at least two (the relation- species tree from a number of individual gene trees ship of the A. citriodora clade to the A. gratissima (e.g. Liu, 2008; Kubatko, Carstens & Knowles, 2009; clade should be considered equivocal, pending further Heled & Drummond, 2010; for review, see Knowles, investigation, and denser sampling, of these groups 2009; Degnan & Rosenberg, 2009). and of Acantholippia). Lantana spp. form two distinct Unfortunately, no widely accessible method yet clades, a Lantana trifolia clade and a Lantana exists to tease apart the effects of incomplete lineage camara clade. Lippia spp. are distributed throughout sorting from hybridization and gene duplication/ the Lantana–Lippia clade, and form the background extinction (but see Than & Nakhleh, 2009; Choi & Hey, from which Nashia inaguensis Millsp., Burroughsia 2011). Any of these mechanisms could be the cause fastigiata, Phyla nodiflora, Aloysia barbata and the of the incongruence seen among our data sets for two Lantana clades are derived. Lantaneae. It might even be the case that there is no Our results show that assuming correspondence single bifurcating tree that adequately describes the between traditional taxa and evolutionary lineages is pattern of descent of the species of Lantaneae from not valid in Lantaneae and should not be accepted their common ancestor; polytomy and reticulation may uncritically in other, difficult Neotropical groups. be characteristic of evolutionary history in difficult, Generic revisions in Lantaneae should proceed care- recently diversified groups such as Lantaneae. fully, contingent on thorough re-evaluation of the Although the tree inferred from our combined data morphological characters that correspond with evolu- is fully resolved with reasonable support (Fig. 3B), we tionary lineages. Based on our results, Lantana and do not assume that it necessarily corresponds with Lippia will need to be fragmented or lumped together the Lantaneae species tree. Relationships that are in with the smaller genera which nest in the Lantana– conflict between loci are often resolved in favour of Lippia clade. In either scenario, genera will not be the larger data sets, or of the majority of data sets; i.e. easy to define morphologically. We can identify no minority conflicting signals from individual loci are morphological characteristics that have not under- masked in the combined analysis, although they may gone multiple, parallel shifts among the major clades provide equally valid alternative estimates of phylog- of Lantaneae. Taxonomic revisions in the tribe will eny. We feel that it is more conservative and more probably involve recircumscribing genera based on representative of our current understanding to leave combinations of traits, rather than on one to a few unresolved any nodes where well-supported conflict diagnostic characters. Densely sampled molecular exists. We thus consider the semi-strict consensus phylogenetic studies are needed to investigate each between well-supported topologies of individual gene clade of Lantaneae, guided by the broad phylogenetic trees to be the best current estimate of Lantaneae results published here, before reliable revisions can phylogeny. be made.

TAXONOMIC IMPLICATIONS FRUIT EVOLUTION Marx et al. (2010) considered the assignment of Coe- Classifications in Lantaneae have relied largely on locarpum to Lantaneae to be discordant, given the fruit characteristics to separate its principal genera, major morphological differences between this genus Lantana and Lippia. Schauer (1847), followed by and the other members of the tribe, but could not Troncoso (1974), assigned species with fleshy drupes place it with confidence as a lineage separate from the to Lantana and species with dry schizocarps to rest of Lantaneae. Our results open the possibility of Lippia. Under this scheme, Lippia brasiliensis (Link) excluding Coelocarpum from Lantaneae and confirm- T. R. S.Silva and Lippia macrophylla Cham. are ing the monophyly of the tribe, whether Coelocarpum placed in Lantana section Sarcolippia. More recent is included or not. However, none of the genera of revisions (Silva, 1999) follow Chamisso (1832) by Lantaneae represented here by more than one species defining Lippia as species with divided fruits, thus

Figure 3. See caption on next page.

Figure 3. A, maximum likelihood phylogeny inferred from DNA sequences from three plastid loci in combination (4335 aligned positions) for 47 species of Lantaneae and seven outgroup species. Branches in bold are supported by > 80% of bootstrap replicates and posterior probability values > 0.9 in Bayesian analyses of the same data. Dashed lines indicate branches not present in the phylogeny inferred by Bayesian analysis. B, maximum likelihood phylogeny inferred from all DNA sequences in combination (8734 aligned positions), for 47 species of Lantaneae and seven outgroup species. Branches in bold are supported by > 80% of bootstrap replicates and posterior probability values > 0.9 in Bayesian analyses of the same data. Dashed lines indicate branches not present in the phylogeny inferred by Bayesian analysis. ᭣

Figure 4. Semi-strict consensus between well-supported topologies of individual phylogenies for Lantaneae, with fruit characters mapped as indicated (left: fruit type; right: number of pyrenes/mericarps). Character states at ancestral nodes are parsimony reconstructions.

Figure 5. Semi-strict consensus between well-supported topologies of individual phylogenies for Lantaneae, with geographical distributions mapped as indicated; species occurring in more than one coded region are denoted with an additional circle. Distributions at ancestral nodes are parsimony reconstructions. Inset A, distribution of occurrence records for the species of Lantaneae included in this study (data from GBIF; records of globally invasive species and species with no georeferenced records omitted). grouping dry schizocarps together with dipyrenous cation schemes are artificial, confounded by charac- drupes under Lippia, and limiting Lantana to include ters that have undergone multiple independent shifts only species with monopyrenous drupes. This more in different lineages. recent scheme reassigns dipyrenous fleshy-fruited There have been at least five origins of a fleshy or species such as L. brasiliensis and L. macrophylla to leathery outer layer on the fruit in Lantaneae, four Lippia. Our results show that both of these classifi- of them in the Lantana–Lippia clade (Fig. 4). Fleshy

© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 103–119 PHYLOGENY OF LANTANEAE 115 fruited lineages identified in our results are: (1) here by L. rehmannii and L. javanica); and a lineage the Lantana trifolia clade; (2) the Lantana camara in a Lantana clade [represented here by L. vibur- clade; (3) Nashia; (4) the clade corresponding to the noides (Forssk.) Vahl and L. rugosa]. This frequency traditional Lantana section Sarcolippia (represented of colonization of Africa seems high, given that Lan- here by L. brasiliensis and L. macrophylla); and (5) taneae are a young lineage and that long-distance Xeroaloysia. Whether or not the common ancestor dispersal between Africa and South America has been of Coelocarpum + Lantaneae had fleshy fruits is found to be relatively infrequent in other lineages difficult to infer, attributable to the difficulty in (Crisp et al., 2009). placing fleshy-fruited Dipyrena relative to dry-fruited In the Americas, a geographical shift from Verbeneae and Lantaneae. If Dipyrena is sister to temperate/subtropical regions into the tropics can be Verbeneae + Lantaneae, it is most parsimonious to seen in Lantaneae (Fig. 5). Aloysia and Acantholip- reconstruct a dry-fruited ancestor for Lantaneae and pia, which form a grade at the base of the tribe, are hypothesize that Coelocarpum represents another distributed primarily in arid temperate regions of independent derivation of fleshy fruits (as shown in South America, extending north into the Andes. Fig. 4). If, however, Dipyrena is sister to Verbeneae Aloysia has an amphitropical distribution with a sec- (rather than to Verbeneae + Lantaneae), a fleshy- ondary radiation in Mexico and the south-western fruited ancestor for Lantaneae is the more parsimo- USA, which may be the result of long-distance dis- nious hypothesis. persal (P. Lu-Irving & R. G. Olmstead, unpubl. data). In the Lantana–Lippia clade, the independent Members of the Lantana–Lippia clade, derived from derivation of fleshy drupes from dry schizocarps the grade of Aloysia and Acantholippia, are found has resulted in dipyrenous fruits in two lineages (the throughout the tropics. This suggests a general Sarcolippia clade and Nashia) and monopyrenous pattern of movement into the tropics from the arid fruits in two lineages (the L. camara clade, and temperate or subtropical regions of South America the L. trifolia clade). In the L. trifolia clade, during the evolution of Lantaneae. Lippia aristata represents a subsequent shift Members of Lantaneae mainly occur in dry to semi- from monopyrenous fruits to dipyrenous fruits. The arid habitats, and rarely in wet forest environments. pattern of shifts in fruit type (dry to fleshy) and For example, Acantholippia seriphioides, which is subdivision (two mericarps to two pyrenes or to one sister to the rest of the Lantana–Lippia clade, inhab- pyrene; one pyrene to two pyrenes) in the Lantana– its arid uplands in Argentina, whereas the next Lippia clade reveals a complex history of fruit evolu- lineage to diverge consists of low or creeping suf- tion, which has had the consequence of misleading frutescent herbs found in dry scrub and dry to mesic taxonomic efforts based on fruit characteristics. disturbed habitats. Most of the rest of the clade are woody shrubs of open and disturbed habitats, forest edges, dry hills and cerrado. Occurrence records for BIOGEOGRAPHIC PATTERNS the species of Lantaneae sampled here (Fig. 5, inset Major clades in Lantaneae are geographically hetero- A) reveal geographical distributions that mostly geneous, suggesting that migration has been an exclude the Amazon or wet coastal forests, and important and common element in the evolution correspond with the distribution of seasonally dry of Lantaneae (Fig. 5). Old World representatives of tropical forest and chaco biomes as outlined by Pen- Lantaneae can be accounted for by at least three nington, Lavin & Oliveira-Filho (2009). The lineage intercontinental colonization events. Coelocarpum, corresponding to Lantana section Sarcolippia repre- endemic to Madagascar and Socotra, is sister to the sents a shift to wetter and more closed forest envi- rest of Lantaneae, and represents one lineage that ronments, but, this shift notwithstanding, the overall has dispersed to the Old World (Marx et al., 2010; biogeographical pattern in Lantaneae is one of niche Olmstead, 2012). Similar patterns of disjunction conservatism. Verbeneae, the sister clade to Lanta- between sister lineages (with distributions in the neae, generally occur in dry to semi-arid habitats in New World and in Madagascar) are found in other temperate zones, and are not diverse in the tropics. families; for example, Tsoala Bosser & D’Arcy in Aloysia spp. echo this pattern and, in the colonization Solanaceae (Olmstead et al., 2008) and groups of of the tropics represented by the Lantana–Lippia Fabaceae (Lavin et al., 2000; 2004). The legumes are clade, the environmental preferences of most of these particularly well-studied examples, in which large species reflect those of their ancestors. This is con- shifts in geographical range belie a high degree of sistent with findings that biome shifts are uncommon niche conservatism (Lavin et al., 2004). In addition to among plant lineages (Crisp et al., 2009; but see also Coelocarpum, two long-distance dispersals from the Simon et al., 2009). Neotropics to Africa are inferred in the Lantana– There is no discernible correlation between fruit Lippia clade: a lineage in a Lippia clade (represented type (whether fleshy or dry) and biogeographic

© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 103–119 116 P. LU-IRVING and R. OLMSTEAD patterns. A more densely sampled and fully resolved require large quantities of sequence data from rapidly phylogenetic hypothesis might reveal such a correla- evolving DNA regions in problematic, species-rich lin- tion, but, to date, if there is any consistent dispersal eages. Individual plastid loci are unlikely to provide advantage possessed by fleshy-fruited species in Lan- sufficient phylogenetic information in such groups. taneae, it is not apparent. In many dry-fruited species, If a molecular systematic study is to be undertaken segments of the hairy calyx persistently enclose the in a difficult group, such as Lantaneae, a period of mericarp, facilitating ectozoochory, just as the fleshy extensive preliminary work should first be carried out fruits are adapted to endozoochory. The different dis- in order to develop, evaluate and select the loci to persal strategies employed among members of Lanta- provide data for it. We expect that the potential of the neae have not been broadly studied, and are likely to nuclear genome as a resource for phylogenetic infor- be diverse in such a large and varied tribe. mation will be largely realized over the next decade. Growing access to complete genome sequences across a range of plant species will enable a variety of FUTURE PROSPECTS multi-locus approaches to be developed and applied in With a broadly representative sample of Lantaneae, divergent groups of flowering plants. With continuing we have identified major clades in the tribe and advances in sequencing technologies, we predict that revealed the extent to which they do or do not corre- large-scale sequencing approaches such as restriction spond with accepted genera. With the evolutionary site-associated DNA (RAD) tagging (Miller et al., history of lineages of Lantaneae outlined here, future 2007; Baird et al., 2008) and large-scale alignment of systematic studies can target specific groups for the entire linkage groups will replace the use of sets of dense sampling that will probably be necessary to well-characterized loci for phylogenetic studies. elucidate relationships at the species level. Particu- Most taxonomic and phylogenetic studies in large, larly important areas that have yet to be resolved geographically widespread plant groups are subject are: (1) the relationship of Acantholippia salsoloides to trade-offs between geographical and taxonomic and its (unsampled) affiliates with Aloysia spp. (this comprehensiveness and between breadth and depth will determine how these genera are redefined); and in treating the taxa in question. Broad molecular (2) species-level relationships in the Lantana–Lippia systematic studies across large groups guide the sam- clade (these will reveal the patterns of trait and pling of subsequent work focused on particular line- biogeographic evolution among these many species). ages in those groups. Our phylogenetic estimate for In Lantaneae, as in other problematic Neotropical Lantaneae was guided by a previous, broader study groups (e.g. Bactridinae, Bromelioideae and other of Verbenaceae (Marx et al., 2010) and, in turn, will examples cited above), a phylogenetic estimate using provide a foundation for further efforts to revise molecular data is essential as a basis for reliable genera, elucidate patterns of trait evolution at the taxonomic revisions and speculation on evolutionary species level and understand patterns of migration history. The difficult taxonomy of such groups hints at and colonization among the Neotropical flora better. the complex pattern of homoplasy that may exist in As phylogenetic data become more easily obtainable morphological characters used to define taxa. Shared in larger quantities, the trade-off between breadth ancestry among lineages cannot be unambiguously and depth should become less limiting, and we expect inferred from morphology alone. Molecular phyloge- that large, data-rich studies that are broadly and netic studies of difficult Neotropical groups should densely sampled will become more common. Moving consider evidence from multiple, independent loci. If forward, collaborative efforts will be needed to thor- major points of departure between gene histories oughly represent species-rich and geographically exist among the species under investigation, they widespread groups in molecular phylogenetic studies can be discovered by taking a multi-locus approach. at a range of taxonomic levels. The development of It is important to evaluate possible incongruence collaborative networks across international bounda- between gene trees, to avoid providing an inappropri- ries will be important in coming years, as we pool our ate interpretation of the species tree. Lineages that efforts and expertise to advance our understanding of are species-rich and recently radiated may be particu- evolution in problematic Neotropical plant groups. larly prone to the incongruence among phylogenetic signal from different loci that is attributable to incom- ACKNOWLEDGEMENTS plete lineage sorting and hybridization. In recently radiated lineages, nucleotide variability This study was supported by the National Science between taxa is an important criterion when selecting Foundation (NSF) (grants DEB 0542493 and DEB loci from which to infer phylogeny. Resolving mater- 1020369). The first author (P.L.I.) was able to partici- nal relationships at the level of species is a valuable pate in this Symposium thanks to travel funds pro- component of phylogenetic studies, but is likely to vided by the NSF to students of the American Society

© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 103–119 PHYLOGENY OF LANTANEAE 117 of Plant Taxonomists and the Botanical Society of Linder P. 2009. Phylogenetic biome conservatism on a America (DEB 1120802). Additional support, in the global scale. Nature 458: 754–756. form of Graduate Student Research Awards, was Degnan JH, Rosenberg NA. 2009. Gene tree discordance, provided by the Society of Systematic Biologists, phylogenetic inference, and the multispecies coalescent. the American Society of Plant Taxonomists and the Trends in Ecology and Evolution 24: 332–340. Botanical Society of America. We are grateful to Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure Segundo Leiva, Fátima Salimena, Lyderson Viccini, for small quantities of fresh leaf tissue. Phytochemical Bul- Loreta Freitas and Verônica Thode for assistance in letin, Botanical Society of America 19: 11–15. Edwards SV. 2009. Is a new and general theory of molecular the field and for providing plant material for this systematics emerging? Evolution 63: 1–19. study. Additional plant material was obtained with Eiserhardt WL, Pintaud J-C, Asmussen-Lange C, the help of the Plant Resources Center at the Uni- Hahn WJ, Bernal R, Balslev H, Borchsenius F. 2011. versity of Texas (TEX-LL), the Missouri Botanical Phylogeny and divergence times of Bactridinae (Arecaceae, Garden (MO) and the US National Herbarium (US). Palmae) based on plastid and nuclear DNA sequences. Taxon Valuable discussion and comments on the manuscript 60: 485–498. were provided by Yuan Yaowu, Ryan Miller, the Gadakgar SR, Rosenberg MS, Kumar S. 2005. Inferring Leaché laboratory at the University of Washington species phylogenies from multiple genes: concatenated and three anonymous reviewers. sequence tree versus consensus gene tree. Journal of Experi- mental Zoology 304B: 64–74. REFERENCES Gentry AH. 1982. Neotropical floristic diversity: phytogeo- graphical connections between Central and South America, Atkins S. 2004. Verbenaceae. In: Kadereit JW, ed. The fami- Pleistocene climatic fluctuations, or an accident of the lies and genera of flowering plants, Vol. 7. Berlin: Springer- Andean orogeny? Annals of the Missouri Botanical Garden Verlag, 449–468. 69: 557–593. Baird NA, Etter PD, Atwood TS, Currey MC, Shiver AL, Givnish TJ, Barfuss MHJ, Van Ee B, Riina R, Schulte K, Lewis ZA, Selker EU, Cresko WA, Johnson EA. 2008. Horres R, Gonsiska PA, Jabaily RS, Crayn DM, Smith Rapid SNP discovery and genetic mapping using sequenced JAC, Winter K, Brown GK, Evans TM, Holst BK, RAD markers. PLoS ONE 3: e3376. Luther H, Till W, Zizka G, Berry PE, Sytsma K. 2011. Baldwin B, Markos S. 1998. Phylogenetic utility of the Phylogeny, adaptive radiation, and historical biogeography external transcribed spacer (ETS) of 18S-26S rDNA: in Bromeliaceae: insights from an eight-locus plastid phyl- congruence of ETS and ITS trees of Calycadenia (Composi- ogeny. American Journal of Botany 98: 872–895. tae). Molecular Phylogenetics and Evolution 10: 449– González D, Vovides AP, Bárcenas C. 2008. Phylogenetic 463. relationships of the Neotropical genus Dioon (Cycadales, Bárcenas RT, Yesson C, Hawkins JA. 2011. Molecular Zamiaceae) based on nuclear and chloroplast DNA sequence systematics of the Cactaceae. Cladistics 27: 470–489. data. Systematic Botany 33: 229–236. Briquet I. 1895. Verbenaceae. In: Engler A, Prantl K, eds. Grose SO, Olmstead RG. 2007. Evolution of a charismatic Die natürlichen Pflanzenfamilien, Vol. 4. Leipzig: W. Engel- Neotropical clade: molecular phylogeny of Tabebuia s.l., mann, 132–182. Crescentieae, and allied genera (Bignoniaceae). Systematic Briquet I. 1904. Verbenaceae. Plantae Hassleriane. Bulletin Botany 32: 650–659. de l’Herbier Boissier ser. 2, 4: 1062–1066. Heled J, Drummond AJ. 2010. Bayesian inference of Butterworth CA, Wallace RS. 2004. Phylogenetic studies species trees from multilocus data. Molecular Biology and of Mammillaria (Cactaceae) – insights from chloroplast Evolution 27: 570–580. sequence variation and hypothesis testing using the para- Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a metric bootstrap. American Journal of Botany 91: 1086– novel method for rapid multiple sequence alignment based 1098. on fast Fourier transform. Nucleic Acids Research 30: 3059– Chamisso A. 1832. De plantis in Expeditione Romanzoffiana 3066. observatis dicunt. Verbenaceae. Linnaea 7: 105–128, 213– Kluge AG. 1989. A concern for evidence and a phylogenetic 272. hypothesis of relationships among Epicrates (Boidae, Ser- Chatrou LW, Pirie MD, Erkens RHJ, Couvreur TLP, pentes). Systematic Zoology 38: 7–25. Neubig KM, Abbott JR, Mols JB, Maas JW, Saunders Knowles LL. 2009. Estimating species trees: methods of RMK, Chase MW. 2012. A new subfamilial and tribal phylogenetic analysis when there is incongruence across classification of the pantropical flowering plant family genes. Systematic Biology 58: 463–467. Annonaceae informed by molecular phylogenetics. Botanical Kubatko LS, Carstens BC, Knowles LL. 2009. STEM: Journal of the Linnean Society 169: 5–40. species tree estimation using maximum likelihood for gene Choi SC, Hey J. 2011. Joint inference of population assign- trees under coalescence. Bioinformatics 25: 971–973. ment and demographic history. Genetics 189: 561–577. Kubatko LS, Degnan JH. 2007. Inconsistency of phyloge- Crisp MD, Arroyo MTK, Cook LG, Gandolfo MA, Jordan netic estimates from concatenated data under coalescence. GJ, McGlone MS, Weston PH, Westoby M, Wilf P, Systematic Biology 56: 17–24.

Lavin M, Schrire BP, Lewis G, Pennington RT, Delgado- benaceae) using the phylogenetic species concept. Botanical Salinas A, Thulin M, Hughes CE, Matos AB, Wojcie- Journal of the Linnean Society 170: 197–219. chowski MF. 2004. Metacommunity process rather than O’Leary N, Yuan YW, Chemisquy A, Olmstead RG. 2009. continental tectonic history better explains geographically Reassignment of species of paraphyletic Junellia s.l. to the structured phylogenies in legumes. Philosophical Transac- new genus Mulguraea (Verbenaceae) and new circumscrip- tions of the Royal Society B: Biological Sciences 359: 1509– tion of genus Junellia: molecular and morphological congru- 1522. ence. Systematic Botany 34: 777–786. Lavin M, Thulin M, Labat J-N, Pennington RT. 2000. Olmstead RG. 2012. Phylogeny and biogeography in Africa, the odd man out: molecular biogeography of dalber- Solanaceae, Verbenaceae, and Bignoniaceae: a comparison gioid legumes (Fabaceae) suggests otherwise. Systematic of continental and intercontinental diversification patterns. Botany 25: 449–467. Botanical Journal of the Linnean Society 171: doi: 10.1111/ Levin RA, Whelan AP, Miller JS. 2009. The utility of nuclear j.1095-8339.2012.01306.x. conserved ortholog set II (COSII) genomic regions for species- Olmstead RG, Bohs L, Abdel Migid H, Santiago-Valentin level phylogenetic inference in Lycium (Solanaceae). Molecu- E, Garcia VF, Collier SM. 2008. A molecular phylogeny of lar Phylogenetics and Evolution 53: 881–890. the Solanaceae. Taxon 57: 1159–1181. Liu L. 2008. BEST: Bayesian estimation of species trees Olmstead RG, Sweere JA. 1994. Combining data in phylo- under the coalescent model. Bioinformatics 24: 2542–2543. genetic systematics: an empirical approach using three Lohmann LG. 2006. Untangling the phylogeny of Neotropi- molecular data sets in the Solanaceae. Systematic Biology cal lianas (Bignonieae, Bignoniaceae). American Journal of 43: 467–481. Botany 93: 304–318. Olmstead RG, Zjhra ML, Lohmann LG, Grose SO, López-Palacios S. 1991. Aspectos críticos de la sistemática Eckert AJ. 2009. A molecular phylogeny and classification de Verbenaceae de Venezuela. Addenda delenda et corri- of Bignoniaceae. American Journal of Botany 96: 1731– genda a Verbenaceae Flora de Venezuela (1977). Pittieria 1743. 19: 40–52. Pennington RT, Lavin M, Oliveira-Filho A. 2009. Ludeña B, Chabrillange N, Aberlenc-Bertossi F, Woody plant diversity, evolution, and ecology in the tropics: Adam H, Tregear JW, Pintaud J-C. 2011. Phylogenetic perspectives from seasonally dry tropical forests. Annual utility of the nuclear genes AGAMOUS 1 and phytochrome Review of Ecology, Evolution, and Systematics 40: 437–457. B in palms (Arecaceae): an example within Bactridinae. Posada D. 2008. jModelTest: phylogenetic model averaging. Annals of Botany 108: 1433–1444. Molecular Biology and Evolution 25: 1253–1256. Maddison WP. 1997. Gene trees in species trees. Systematic Ronquist F, Huelsenbeck JP. 2003. MRBAYES 3: Bayesian Biology 46: 523–536. phylogenetic inference under mixed models. Bioinformatics Maddison WP, Maddison DR. 2011. Mesquite: a modular 19: 1572–1574. system for evolutionary analysis. Version 2.75. Available at: Salimena FRG. 2002. New synonyms and typifications in http://mesquiteproject.org Lippia sect. Rhodolippia (Verbenaceae). Darwiniana 40: Marx HE, O’Leary N, Yuan YW, Lu-Irving P, Tank DC, 121–125. Mulgura ME, Olmstead RG. 2010. A molecular phylogeny Sanders RW. 1987. Taxonomic significance of chromosome and classification of Verbenaceae. American Journal of observations in Caribbean species of Lantana (Verben- Botany 97: 1647–1663. aceae). American Journal of Botany 74: 914–920. Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the Sanders RW. 2001. The genera of Verbenaceae in the CIPRES gateway for inference of large phylogenetic trees. southeastern United States. Harvard Papers in Botany 5: In: Proceedings of the Gateway Computing Environments 303–358. Workshop (GCE 2010). New Orleans, LA: Institute of Elec- Sanders RW. 2006. Taxonomy of Lantana sect. Lantana trical and Electronics Engineers (IEEE), 1-8. (Verbenaceae): I. Correct application of Lantana camara Miller MR, Dunham JP, Amores A, Cresko WA, and associated names. Sida 22: 381–421. Johnson EA. 2007. Rapid and cost-effective polymorphism Santos IEM. 2002. A taxonomic revision of Lantana sect. identification and genotyping using restriction site associ- Lantana (Verbenaceae) in the Greater Antilles. Willdenowia ated DNA (RAD) markers. Genome Research 17: 240–248. 32: 285–301. Moldenke HN. 1940. Novelties in the Avicenniaceae and Sass C, Specht CD. 2010. Phylogenetic estimation of the Verbenaceae. Phytologia 1: 411–412. core bromelioids with an emphasis on the genus Aechmea Moldenke HN. 1959. A resume of the Verbenaceae, Avicen- (Bromeliaceae). Molecular Phylogenetics and Evolution 55: niaceae, Stilbaceae, Symphoremaceae, and Eriocaulaceae 559–571. of the world as to valid taxa, geographic distribution and Schauer JC. 1847. Verbenaceae. In: De Candolle AP, ed. synonymy. Yonkers: published by the author. Prodromus, Vol. 11. Lehre: Cramer. 522–700. O’Leary N, Múlgura ME. 2012. A taxonomic revision of the Schulte K, Barfuss MHJ, Zizka G. 2008. Phylogeny of genus Phyla (Verbenaceae). Annals of the Missouri Botani- Bromelioideae (Bromeliaceae) inferred from nuclear and cal Garden 98: 578–596. plastid DNA loci reveals the evolution of the tank habit O’Leary N, Denham SS, Salimena F, Múlgura ME. 2012. within the subfamily. Molecular Phylogenetics and Evolu- Species delimitation in Lippia section Goniostachyum (Ver- tion 51: 327–339.

Seelanan T, Schnabel A, Wendel JF. 1997. Congruence Tippery NP, Philbrick CT, Bove CP, Les DH. 2011. Sys- and consensus in the cotton tribe (Malvaceae). Systematic tematics and phylogeny of Neotropical riverweeds Botany 22: 259–290. (Podostemaceae: Podostemoideae). Systematic Botany 36: Shaw J, Lickey EB, Schilling EE, Small RL. 2007. Com- 105–118. parison of whole chloroplast genome sequences to choose Torke BM, Schaal BA. 2008. Molecular phylogenetics of noncoding regions for phylogenetic studies in Angiosperms: the species-rich Neotropical genus Swartzia (Leguminosae– the Tortoise and the Hare III. American Journal of Botany Papilionoideae) and related genera of the Swartzioid clade. 94: 275–288. American Journal of Botany 95: 215–228. Shimodaira H, Hasegawa M. 1999. Multiple comparisons of Troncoso NS. 1974. Los géneros de Verbenaceas de Suda- log-likelihoods with applications to phylogenetic inference. merica extra-tropical (Argentina, Chile, Bolivia, Paraguai, Molecular Biology and Evolution 16: 1114–1116. Uruguay y sur de Brasil). Darwiniana 18: 295–412. Siedo SJ. 2008. Systematics of Aloysia (Verbenaceae). Verdcourt B. 1992. Verbenaceae. In: Polhill RM, ed. Flora of Doctoral thesis, University of Texas at Austin. Tropical East Africa. Rotterdam: A.A. Balkema, 1–155. Silva TRS. 1999. Redelimitação e revisão taxonômica do Whittall JB, Medina-Marino A, Zimmer EA, Hodges SA. genero Lantana L. (Verbenaceae) no Brasil. Doctoral thesis, 2006. Generating single-copy nuclear gene data for a recent Universidade de São Paulo. adaptive radiation. Molecular Phylogenetics and Evolution Silva TRS, Salimena FRG. 2002. Novas combinações 39: 124–134. e novos sinônimos em Lippia e Lantana (Verbenaceae). Wilgenbusch JC, Warren DL, Swofford DL. 2004. AWTY: Darwiniana 40: 57–59. a system for graphical exploration of MCMC convergence Simon MF, Grether G, de Queiroz LP, Skema C, in Bayesian phylogenetic inference. Available at: http://ceb. Pennington RT, Hughes CE. 2009. Recent assembly of csit.fsu.edu/awty the cerrado, a Neotropical plant diversity hotspot, by in situ Yuan YW, Liu C, Marx HE, Olmstead RG. 2009a. The evolution of adaptations to ﬁre. Proceedings of the National pentatricopeptide repeat (PPR) gene family, a tremendous Academy of Sciences of the United States of America 106: resource for plant phylogenetic studies. New Phytologist 20359–20364. 182: 272–283. Slowinski JB, Page RDM. 1999. How should species phyl- Yuan YW, Liu C, Marx HE, Olmstead RG. 2009b. An ogenies be inferred from sequence data? Systematic Biology empirical demonstration of using pentatricopeptide repeat 48: 814–825. (PPR) genes as plant phylogenetic tools: phylogeny of Ver- Small RL, Cronn RC, Wendel JF. 2004. Use of nuclear benaceae and the Verbena complex. Molecular Phylogenetics genes for phylogeny reconstruction in plants. Australian and Evolution 54: 23–35. Systematic Botany 17: 145–170. Yuan YW, Olmstead RG. 2008a. A species-level phylogenetic Steele PR, Guisinger-Bellian M, Linder R, Jansen RK. study of the Verbena complex (Verbenaceae) indicates two 2008. Phylogenetic utility of 141 low-copy nuclear regions in independent intergeneric chloroplast transfers. Molecular taxa at different taxonomic levels in two distantly related Phylogenetics and Evolution 48: 23–33. families of rosids. Molecular Phylogenetics and Evolution Yuan YW, Olmstead RG. 2008b. Evolution and phylogenetic 48: 1013–1026. utility of the PHOT gene duplicates in the Verbena complex Swofford DL. 2000. PAUP*: phylogenetic analysis using (Verbenaceae): dramatic intron size variation and footprint parsimony (*and other methods). Version 4b.10. Sunderland: of ancestral recombination. American Journal of Botany 95: Sinauer Associates. 1166–1176. Taberlet P, Gielly L, Pautou G, Bouvet J. 1991. Universal Zwickl DJ. 2006. Genetic algorithm approaches for the primers for ampliﬁcation of three non-coding regions of phylogenetic analysis of large biological sequence datasets chloroplast DNA. Plant Molecular Biology 17: 1105–1109. under the maximum likelihood criterion. PhD dissertation, Than C, Nakhleh L. 2009. Species tree inference by mini- The University of Texas at Austin. Available at: http:// mizing deep coalescences. PLoS Computational Biology 5: garli.googlecode.com e1000501.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Table S1. Voucher information for the specimens from which DNA was obtained, and GenBank identiﬁcation numbers for the sequence data generated in this study. Table S2. Sequences of primers used in this study. Table S3. Results of SH tests (P-values) on trees inferred from different data sets.