American Journal of 101(2): 287–299. 2014.

P HYLOGENY OF LAMIIDAE 1

N ANCY . EFULIO-RODRIGUEZ 2 AND R ICHARD . OLMSTEAD 2,3

2 Department of Biology, Box 355325, University of Washington, Seattle, Washington 98195 USA

• Premise of the study: The Lamiidae, a composed of approximately 15% of all fl owering , consists of fi ve orders: , , , , and ; and four families unplaced in an : , Metteniusi- aceae, Oncothecaceae, and Vahliaceae. Our understanding of the phylogenetic relationships of Lamiidae has improved signifi - cantly in recent years, however, relationships among the orders and unplaced families of the clade remain partly unresolved. Here, we present a phylogenetic analysis of the Lamiidae based on an expanded sampling, including all families together, for the fi rst time, in a single phylogenetic analyses. • Methods: Phylogenetic analyses were conducted using maximum parsimony, maximum likelihood, and Bayesian approaches. Analyses included nine plastid regions ( atpB , matK , ndhF , psbBTNH , rbcL , rps4 , rps16 , trnL - F , and trnV -atpE ) and the mitochondrial rps3 region, and 129 samples representing all orders and unplaced families of Lamiidae. • Key results: Maximum Likelihood (ML) and Bayesian provide good support for Boraginales sister to Lamiales, with successive outgroups (Solanales + ) and Gentianales, together comprising the core Lamiidae. Early branching patterns are less well supported, with Garryales only poorly supported as sister to the above ‘core’ and a weakly supported clade composed of Icacinaceae, , and Oncothecaceae sister to all other Lamiidae. • Conclusions: Our phylogeny of Lamiidae reveals increased resolution and support for internal relationships that have remained elusive. Within Lamiales, greater resolution also is obtained, but some interrelationships remain a challenge.

Key words: Boraginales; chloroplast; Garryales; Gentianales; Lamiales; Lamiidae; mitochondrial; Solanales.

Current understanding of Lamiidae phylogeny comes from been unable to establish precise sister group relationships two decades of molecular phylogenetic research aimed at un- among major within Lamiidae, beyond fi nding that Gar- derstanding the phylogeny of and angiosperms in ryales, Icacinaceae, Oncothecaceae, and Metteniusiaceae repre- general ( Olmstead et al., 1992 , 1993 , 2000 ; Chase et al., 1993 ; sent lineages, and that Lamiales, Gentianales, Solanales, Soltis et al., 2000, 2011 ; Albach et al., 2001; Kårehed, 2001; Boraginales, and Vahliaceae form a well-supported clade. Bremer et al., 2002 ; González et al., 2007 ). Through these stud- Lamiidae are one of two major clades, along with Campanu- ies, the of Lamiidae is well established, and in- lidae ( Cantino et al., 2007 ; Tank and Donoghue, 2010 ), com- cludes four large clades: (1) Boraginales (forget-me-nots, prising the core ( the Angiosperm Phylogeny heliotropes); (2) Gentianales (coffee, gentians, milkweeds); (3) Group (APG), 1998 ; APG II, 2003 ; APG III, 2009 ; Soltis et al., Lamiales (African violets, foxglove, mint, , , trum- 2011). Lamiidae (sensu Olmstead et al., 1992) are roughly pet creeper, ); and (4) Solanales (morning glory, petu- equivalent to the subclass Lamiidae of Takhtajan (1987, but not nia, potato, tomato), as well as a series of smaller clades of less 1997). In several early studies Lamiidae were referred to as as- well-known plants that mostly form a basal-grade relative to the terids ( Chase et al., 1993 ), euasterids I ( APG, 1998 ; APG III, large clades (Garryales, Icacinaceae, Metteniusiaceae, Oncoth- 2009 ), or lamiids ( Bremer et al., 2002 ; Judd and Olmstead, ecaceae, and Vahliaceae). Despite a progressive increment in 2004). Adding to this confusion, Cantino et al. (2007) used the number of genes and/or taxa sampled, these studies have Lamiidae for the smaller, less-inclusive clade (Lamiales, Genti- anales, Solanales, and Boraginales.), coining a new name, Gar- ryidae, for the clade recognized here as Lamiidae. This stemmed 1 Manuscript received 6 November 2013; revision accepted 7 January from the exclusion of Garryales from Lamiidae because of poor 2014. resolution in an early paper based on rbcL ( Olmstead et al., The authors thank Doug and Pam Soltis for coordinating the Angiosperm of Life Project; Mike Schoenborn for his help with the sequencing of the 1993 ). Priority, and common, informal recognition under the rps3 mitochondrial region; Bruce Godfrey (Director of the Comparative variety of names listed above, suggests that Lamiidae is best Genomics Center-University of Washington) for assistance in the sequencing used to refer to the more inclusive clade. facility; and Yin-Long Qiu and . Tank for sharing primers information. We Within Lamiidae, the largest and most intractable clade is La- are grateful to the following colleagues: Mark Chase, Laszlo Csiba, Claude miales (sensu Olmstead et al., 1993 , 2000 , 2001 ; Soltis et al., dePamphillis, Barry Hammel, Maribeth Latvis, Jeffrey Paul Benca, Jim Smith, 2000, 2011 ; Bremer et al., 2002; Schäferhoff et al., 2010). Sasa Stefanović, Lena Struwe, Eva Wallander, Alexandra Wortley, Gregory As currently delimited, Lamiales is composed of 25 families Young, Andrea D. Wolfe, and Dave Tank; as well as the Missouri Botanical (, , Byblidaceae, , Garden, Rancho Santa Ana Botanic Garden, Royal Botanic GardensUK), and Carlemanniaceae, , , Lentibulari- the Smithsonian Institution (US) for providing DNA or tissue for DNA aceae, , , , , extraction. This study was supported by a National Science Foundation (NSF) Assembling the Tree of Life grant EF-0431184 to RGO, and NSF Systematics , , , , Plan- grants DEB-0542493 and DEB 1020369 to RGO. taginaceae sensu APG, Plocospermataceae, Rehmanniaceae, 3 Author for correspondence (-mail: [email protected]) , , , , Thomandersiaceae, and ), and over 20 000 doi:10.3732/ajb.1300394 ( Olmstead et al., 2000 ; Olmstead, 2012, http://depts.washington.

American Journal of Botany 101(2): 287–299, 2014 ; http://www.amjbot.org/ © 2014 Botanical Society of America 287 288 AMERICAN JOURNAL OF BOTANY [Vol. 101 edu/phylo/Classifi cation.pdf). Lamiales phylogeny has proven similarity criterion (Simmons et al., 2004) using the program Se-Al (vers. diffi cult to resolve ( Wortley et al., 2005 ). Even though it is 2.0a11; Rambaut, 1996–2002). Gaps were treated as missing data. Ambigu- strongly supported as a monophyletic group, analyses of up to 17 ously aligned regions ( matK ~450 nt; psbBTNH ~175 nt; trnV ~525 nt; trnL-F ~430 nt; rps16 ~1100 nt; rps3 ~300 nt; ndhF ~30 nt) were excluded from genes ( Soltis et al., 2011 ) have failed to provide a fully resolved analyses. phylogeny. The recent study of Schäferhoff et al. (2010), with three chloroplast gene regions, included the broadest taxon sam- Maximum parsimony—Maximum parsimony (MP) analyses were per- pling to date, but also left many relationships among core fami- formed for each marker separately (results not shown) and combining all 10 lies poorly resolved. genes using PAUP* (version 4.0b10; Swofford, 2002 ). Heuristic searches were Here, we provide a Lamiidae phylogeny with the broadest performed with 100 random stepwise-addition replicates, using tree bisection- taxon sampling to date. Ten gene regions (~17 214 bp) were reconnection branch swapping (TBR), and with MULTREES on and 10 trees sequenced, including both chloroplast and mitochondrial ge- held at each step. This was followed by a second set of searches using the con- sensus of the trees obtained as a reverse constraint and 1000 replicates to search nomes. Within Lamiidae, we specifi cally focused on Lamiales, for other islands of equal or shorter trees (Catalan et al., 1997). Bootstrap values because of the lack of resolution in previous studies. (BP) for clades were obtained using similar settings described above, but sav- ing no more than fi ve trees and with MULTREES off (DeBry and Olmstead, 2000 ). Gaps were treated as missing data. MATERIALS AND METHODS Model selection—Nucleotide substitution models for each gene were deter- Taxon sampling— Taxon sampling consisted of 129 samples representing mined by the program jModelTest (Posada, 2008) using the Akaike information all four orders and the fi ve additional families unplaced to order in Lamiidae. criterion (AIC). The nucleotide substitution models obtained for each gene are Sampling was concentrated on resolving family relationships within Lamiales. as follows: atpB (TVM + I + G), matK (GTR + G), ndhF (GTR + I + G), Six taxa were chosen as the outgroups based on previous studies (e.g., psbBTNH (TVM + I + G), rbcL (TVM + I + G), rps3 (TVM + G), rps4 (TVM + G), Olmstead et al., 2000 ; Bremer et al., 2002 ; Soltis et al., 2011 ). Because it has rps16 (GTR + G), trnL - F (TVM + G), and trnV - atpE (TVM + G). been shown that Campanulidae are sister to Lamiidae (together forming the core Asteridae, Judd and Olmstead, 2004), four taxa were selected representing Bayesian analysis—Bayesian Markov Chain Monte Carlo (MCMC, Yang the main clades of campanulids, Helwingia (), Aralia (), and Rannala, 1997) posterior probabilities (PP) were calculated with MrBayes Lonicera (), and Pentaphragma (). Representatives of Eri- vers. 3.1.2 (Ronquist and Huelsenbeck, 2003). A data matrix combining all 10 cales (Rhododendron ) and (Cornus ) represent successive sister groups genes was employed. The sequence data were partitioned by gene regions; the to the core asterids. Voucher information and GenBank accession numbers of GTR + G DNA substitution model was selected because some of the models for the sequences used in this studied are provided in Appendix 1. the individual gene regions obtained using jModelTest are not implemented in MrBayes, thus, the more complex (parameter-rich) GTR + G model was used DNA isolation, amplifi cation, and sequencing — Total genomic DNA was (Ronquist et al., 2005). Three parallel runs with four MCMC chains were run extracted from collected preserved in silica gel and leaves removed from for 45 500 000 generations and sampled every 500 generations. Analyses were herbarium specimens using the modifi ed 2× CTAB method ( Doyle and Doyle, run until the value of the average standard deviation of split frequencies dropped 1987); the DNA was subsequently purifi ed with QIAquick spin columns ac- below 0.01. The fi rst 25% of samples were discarded as burn-in ( Huelsenbeck cording to the protocol of the manufacturer (Qiagen, Valencia, California, and Ronquist, 2001). Stationarity was verifi ed using the program Tracer version USA). We sequenced nine plastid regions, including six coding regions ( atpB , 1.4 (Rambaut and Drummond, 2003–2007). matK + 3′ trnK intron, ndhF , psbBTNH , rbcL, rps4 ), three noncoding regions ( rps16 intron, trnL - F intron and spacer, trnV - atpE spacer), and one mitochon- Maximum Likelihood— Maximum Likelihood (ML) analysis was carried drial coding region (rps3 ). The psbBTNH plastid region consists of four genes out using RAxML (version 7.0.4; Stamatakis, 2006) at the CIPRES PORTAL 2 ( psbB , psbT , psbN, and psbH); in this study, they were amplifi ed and sequenced (http://www.phylo.org/portal2/login!input.action). A data matrix combining all as one unit (see Appendix S1, see Supplemental Data with the online version of plastid and mitochondrial genes was employed. Each gene was treated as a this article). We included available DNA sequences of the abovementioned separate partition; the GTR + G model was used for all partitions and 1000 gene regions from GenBank. To build a complete database, in a few cases, rapid bootstrap (BS) replicates were performed. The ML and Bayesian trees composite terminals combining data from different species of the same were visualized and edited using Dendroscope software version 2.4 ( Huson were generated (in each such case, the species are much more closely related to et al., 2007). Topological differences between the optimal tree obtained in this each other than they are to other included taxa). study and that of Schäferhoff et al. (2010) were tested using the Shimodaira- Amplifi cation reactions for all chloroplast and the mitochondrial genes were Hasagawa () test (Shimodaira and Hasegawa, 1999) and the Approximately run according to the following steps: (1) 1 cycle was run with a denaturing step ° ° ° Unbiased (AU) test (Shimodaira, 2002) implemented in CONSEL (Shimodaira at 94 for 45 , an annealing step at 54 C for 45 s, and an extension at 72 C and Hasegawa, 2001 ). for 2 min; this was followed by (2) 38 cycles with a denaturing step at 94° C for 45 s, an annealing step at 52° C for 45 s, an extension step at 72° C for 90 s, and was fi nished with a fi nal extension at 72° C for 7 min. When genes were unable to amplify as a whole unit, amplifi cations were done in two or more overlap- RESULTS AND DISCUSSION ping pieces. Missing sequences are a consequence of amplifi cation failure or lack of DNA availability. PCR products were purifi ed using the Morgan and The analyses included 135 samples, 6 of which were out- Soltis (1993) polyethelene glycol (PEG ) protocol. The sequencing reaction vol- groups. The length of the combined data matrix consists of ume was 5 μ ; reactions were performed using 0.5 μ L of DNA, 1 μ L of primer 17 214 base pairs (including gaps and excluding ambiguously with a concentration of 1.6 ng/ μ L, 1.5 μ L of Big Dye buffer 5 ×, 0.25 μL of ABI Prism Big Dye Terminator vers. 3.1 (Applied Biosystems, Foster City, Califor- aligned regions). Of these, 8824 characters were constant and nia, USA), and 1.75 μ L of distilled water. Cycle sequencing products were 8390 were variable (6109 characters were parsimony informa- cleaned with sephadex reusing PERFORMA DTR V3 96-Well Short Plates tive). For most DNA regions. a few taxa were missing; the (EdgeBioSystems, Gaithersbuerg, Maryland, USA). Sequencing products were missing data per gene are: atpB (6.7%), matK (4.4%), ndhF visualized on an ABI 3100 or 3730xl Genetic Analyzer (Applied Biosystems, (7.4%), psbBTNH (10.4%), rbcL (2.2%), rps4 (6.7%), rps16 Foster City, California, USA). (3.7%), trnL - F (2.2%), trnV - atpE (11.9%), and rps3 (25.9%). Although tree topologies generated from MP, ML, and Sequence assembly, alignment and phylogenetic analysis — Sequences Bayesian approaches varied, the differences occurred only on were assembled and edited using Sequencher 4.5 (Gene Codes Corp., Ann Arbor, Michigan, USA). Initial automated alignments of the individual genes poorly supported nodes (Fig. 1, Appendices S2 to S5). The were made using the MAFFT (vers. 6.71; Katoh and Toh, 2008) with the E- maximum parsimony tree (Appendices S2 and S3) provided INS-i algorithm, and was subsequently polished carefully by eye based on the lower support values and less resolution than the ML (Appendix S4) February 2014] REFULIO-RODRIGUEZ AND OLMSTEAD — PHYLOGENY OF LAMIIDAE 289 and Bayesian trees (Fig. 1, Appendix S5). The Bayesian tree Our results are not consistent with those of González et al. provides strong support and the ML tree provides moderate (2007), despite the fact that we included in our analyses only support for the relationships (Garryales, (Gentianales, ((Solanales the atp and rbc L sequences obtained by González et al. (2007) , + Vahliaceae), (Boraginales + Lamiales)))). Other studies sug- because of the lack of available leaf material or DNA. gest that ML bootstrap values tend to be conservative ( Suzuki et al., 2002) and Bayesian posterior probabilities tend to over- Garryales—Two families and three genera are recognized in estimate support ( Douady et al., 2003 ; Simmons et al., 2004 ). Garryales (APG III, 2009): Eucommiaceae ( ) and Relationships found in our ML tree for the most part agree with ( and ). This clade was unanticipated the ML analyses of Soltis et al. (2011), which includes a subset by any traditional classifi cations, including that of Cronquist of these taxa and DNA regions, along with some additional, (1981) , which put the three genera in different subclasses, but more conservative, DNA regions appropriate to the deeper phy- was fi rst recognized by Olmstead et al. (1993) based on rbcL logenetic questions addressed in that study. sequences. Despite their disparate positions in traditional clas- With growing certainty, from the fi rst molecular phyloge- sifi cations, these three taxa share a suite of traits from sec- netic studies in asterids (Olmstead et al., 1992, 1993 ; Chase ondary chemistry, morphology, and ovular anatomy et al., 1993), through more recent asterid studies of 4 (Albach ( Olmstead et al., 1993 ). All of our analyses strongly support the et al., 2001 ) and 6 genes ( Bremer et al., 2002 ), and an angiosperm monophyly of the Garryales, which is consistent with prior re- study of 17 genes ( Soltis et al., 2011 ), the monophyly of the sults (e.g., Soltis et al., 2000 , 2011 ; Bremer et al., 2002 ; Lens major clades of Lamiidae has become generally accepted, but et al., 2008 ). In our ML and Bayesian analyses, the Garryales is relationships among them remain unresolved. Furthermore, the supported as sister to the remainder of Lamiidae with strong basal relationships remained obscure. Our more comprehensive support in the Bayesian analysis, but only weakly in the ML sampling of the Lamiidae yielded Bayesian (Fig. 1) and ML analysis, and not in the MP analysis, where they fall among trees (Appendix S4) with a considerable increment in resolu- Icacinaceae, Metteniusaceae, and Oncothecaceae with poor tion and support relative to previous studies. However, the in- support. This placement is consistent with the results of Bremer clusion of taxa representing more of the putative basal lineages et al. (2002) , but is at odds with several other studies, most of (e.g., Metteniusaceae) than included in any single previous which place Garryales either with representatives of Icacinaceae study could not resolve the basal Lamiidae relationships with (Soltis et al., 2000, 2011 ; Kårehed, 2001; González et al., 2007); confi dence. or Icacinaceae sister to other Lamiidae, exclusive of Garryales ( Lens et al., 2008 ); or Oncothecaceae sister to other Lamiidae, Major clades of Lamiidae— The following discussion sum- exclusive of Garryales (Hilu et al., 2003). Our study represents marizes results for the clades of Lamiidae. the greatest amount of data yet compiled to address this ques- tion with all of the relevant families included, but still leaves Icacinaceae, Metteniusaceae, and Oncothecaceae— The re- this portion of the tree poorly understood. sults of all three analyses placed Metteniusaceae sister to On- cothecaceae, and the ML and Bayesian analyses (Fig. 1B) found Core Lamiidae—The “core” Lamiidae is composed of four that combined clade to be sister to Icacinaceae (including large clades—Boraginales, Gentianales, Lamiales, and So- and ), with this inclusive clade sister to the lanales—and the small genus Vahlia , which is unassigned to rest of Lamiidae. However, this clade is recovered with low any of the larger clades. Lamiidae, composed of these main support in the ML and Bayesian analyses (BS = 56% and PP = component clades, were recognized in the earliest rbcL studies 0.86, respectively), and support for the included clades is also of Asteridae ( Olmstead et al., 1992 , 1993 ), and have been re- weak (Cassinopsis + Icacina: BS = 71%/PP = 0.98; covered with strong support in subsequent molecular phyloge- + : BS = 69%/PP = 0.86). In contrast, the MP analy- netic studies (Soltis et al., 1997, 2000 , 2007 , 2011 ; Olmstead sis found weak evidence for a clade consisting of these taxa et al., 2000; Albach et al., 2001; Bremer et al., 2002; Hilu et al., plus Garryales as sister to the rest of Lamiidae, and did not re- 2003). However, relationships among these fi ve groups have cover a monophyletic Icacinaceae (Cassinopsis and Icacina ). never been resolved with confi dence (defi ned here as MP/ML Icacinaceae have been found to be nonmonophyletic in pre- BS > 70%; Bayesian PP > 0.90), and, of fi ve prior studies that vious studies with more extensive sampling within the family obtained resolution among the four main groups (disregarding ( Kårehed, 2001 ; Bremer et al., 2002 ; Lens et al., 2008 ). In our Vahlia, which was not always included), no two obtained the ML and Bayesian trees, Icacina and Cassinopsis , both mem- same relationships ( Olmstead et al., 2000 ; Albach et al., 2001 ; bers of Icacinaceae s.s. ( Kårehed, 2001 ), form a clade with Hilu et al., 2003; Soltis et al., 2007, 2011 ). Our results are the moderate support (BS = 79%, PP = 0.98). This is consistent fi rst to provide resolution with moderate-to-strong support for with results of Kårehed (2001) and Bremer et al. (2002), who relationships among the major lineages of Lamiidae. In our found weak support for Icacinaceae s.s., including Cassinopsis analyses, Lamiales and Boraginales are sister groups (ML BS = and Icacina , but was contradicted by Lens et al. (2008) . Be- 65%; PP = 1.0). Solanales (and Vahlia) are sister to Lamiales cause we did not include members of Icacinaceae s.l. that are (inclusive group ML BS = 73%; PP = 1.0), and core Lamiidae outside of the narrowly defi ned Icacinaceae s.s., additional receive maximum bootstrap/posterior partition support in all work on Icacinaceae s.l. is needed to fully understand the base analyses. The placement of Vahlia as sister to Solanales was of Lamiidae. obtained in both Bayesian (PP = 1.0) and ML analyses (BS = The position of Metteniusaceae within Lamiidae was fi rst ex- 45%), though unresolved in the strict consensus of the MP plored by González et al. (2007) . They included Metteniusa in analysis. an analysis of the Soltis et al. (2000) data and found Metteniusa and Oncotheca to be successive sister groups to the core Lamii- Gentianales— Gentianales is composed of fi ve families, Apoc- dae, excluding Garryales, but they indicated that a sister group ynaceae, , , , and Rubi- relationship between the two taxa was nearly as parsimonious. aceae. In all of our analyses, this clade and the included families 290 AMERICAN JOURNAL OF BOTANY [Vol. 101

Fig. 1. Bayesian majority-rule consensus tree resulting from an analysis of combining all 10 genes. Numbers above branches are Bayesian posterior probability/ML bootstrap/MP bootstrap. An asterisk indicates posterior probability of 1.0 or bootstrap of 100%; a hyphen (-) indicates the branch was not obtained in the ML bootstrap consensus, or received <5% bootstrap support in the MP analysis. February 2014] REFULIO-RODRIGUEZ AND OLMSTEAD — PHYLOGENY OF LAMIIDAE 291

Fig. 1. Continued. are strongly supported as monophyletic groups (BS = 100%, PP = 2009 ). Our study agrees with all prior studies in fi nding 1.00), an outcome that is in accordance with previous works in sister to the remaining families. However, a succession of previous which suffi cient sampling permits such a conclusion (Olmstead studies with increasing amounts of data (one locus: Olmstead et al., et al., 1993; Backlund et al., 2000; Jiao and Li, 2007; Frasier, 1993 ; 2000 ; two loci: Backlund et al., 2000 ; three loci: Jiao and Li, 292 AMERICAN JOURNAL OF BOTANY [Vol. 101

2007 ; four loci: Frasier, 2009 ) have not agreed upon relationships sister to Lamiales. This clade has been recognized in all broad mo- among the other four families, nor has any study provided strong lecular studies of asterids and angiosperms, in which both Borag- support for one set of relationships. With data from 10 DNA re- inaceae and Hydrophyllaceae have been sampled (e.g., Olmstead gions, our Bayesian results (Fig. 1) provide strong support for et al., 1992, 1993 , 2000 ; Soltis et al., 1997, 2000 , 2011 ; Albach and Gentianaceae as sisters (PP = 1.0), and for that et al., 2001 ), and all studies that have included suffi cient sampling of clade as sister to a clade composed of Loganiaceae and Gelsemi- both families have found that the two families, as traditionally de- aceae (PP = 0.98). Bremer et al. (2002) , with a 6-gene data set, but fi ned, are not both monophyletic ( Olmstead et al., 1993 ; Ferguson, only one taxon per family, obtained the same relationships. Our 1999; Gottschling et al., 2001; Soltis et al., 2011; Nazaire and parsimony analysis (Appendixes S2, S3) is congruent with respect Hufford, 2012 ; Cohen, 2013 ; Weigend et al., 2013 ). In addition, to familial relationships, while our ML analysis (Appendix S4) the parasitic Lennoaceae have been found to belong within the places Gentianaceae as sister to Gelsemiaceae + Loganiaceae with Boraginales ( Gottschling et al., 2001 ; Nazaire and Hufford, 2012 ), weak support. as suggested by Yatskievych and Mason (1986) . Our results provide strong support that Saccifolium belongs in The Angiosperm Phylogeny Group (1998, 2003 , 2009 ) recog- Gentianaceae as suggested by Judd and Olmstead (2004) and indi- nized a broadly defi ned (including Hydrophyllaceae cated in Frasier (2009), and that belongs with Gelsemi- and Lennoaceae). This Boraginaceae s.l. has been followed in re- aceae (Struwe and Olmstead, unpublished). cent textbooks (e.g., Judd et al., 2008 ), angiosperm-wide phyloge- netic studies (e.g., Soltis et al., 2011 ), and some comprehensive Vahliaceae— This small clade consists of one genus with fi ve phylogenetic studies of the group ( Ferguson, 1999 ; Nazaire and African species. Vahlia was once thought to belong in Saxifra- Hufford, 2012). However, given that this clade represents a more gaceae (e.g., Cronquist, 1981 ), but early rbcL evidence placed it in or less equally divergent of similar age to the other three Lamiidae ( Morgan and Soltis, 1993 ). Subsequent studies placed it major clades of core Lamiidae (Gentianales, Lamiales, Solanales), in various isolated positions in Lamiidae ( Chase et al., 1993 ; Soltis an alternative classifi cation that would elevate the rank of this clade et al., 1997, 2000 ; Albach et al., 2001; Bremer et al., 2002), but to an order has been adopted in some studies ( Gottschling and with little support. In Soltis et al. (2011), Vahliaceae is sister to Hilger, 2001; Gottschling et al., 2001, 2005 ; Diane et al., 2002; core Lamiidae (Gentianales, Solanales, Boraginales, Lamiales). Hilger and Diane, 2003 ; Luebert and Wen, 2008 ; Weigend Weigend et al. (2013) , in a study of Boraginales, with limited out- and Hilger, 2010 ; Luebert et al., 2011 ; Cohen, 2013 ; Weigend group sampling among other lamiid groups, placed Vahlia sister to et al., 2013). This classifi cation would elevate former subfamilies Lamiales, with minimal support. In this study, Vahliaceae is sister of Boraginaceae to the rank of family (Boraginaceae, Cordiaceae, to Solanales with high support (PP = 1.00) in the Bayesian tree Ehretiaceae, Heliotropiaceae); keep Hydrophyllaceae at the rank (Fig. 1B), but with low support (BS = 45%) in the ML tree (see of family (although it will likely need to be split into two families); Appendix S4) and MP bootstrap analysis (BS = 29%; unresolved and recognize two small families, Wellstediaceae (formerly Bor- in the strict consensus tree). aginaceae) and Codonaceae (formerly Hydrophyllaceae). Our re- sults place the latter two families, each composed of one small Solanales—Solanales include fi ve families (APG III, 2009), So- African genus, as successive sister groups to Boraginaceae s.s., lanaceae, , , Hydroleaceae, and consistent with other studies ( Cohen, 2013 ; Weigend et al., 2013 ). Sphenocleaceae. Our results strongly support the monophyly of There are still a number of issues to resolve in the phylogeny of the clade and its constituent families. Relationships among fami- Boraginales that will affect the fi nal classifi cation of the clade. lies are strongly supported with the three small families Montini- Whereas ITS sequences support a monophyletic Hydrophyllaceae aceae, Hydroleaceae, and Sphenocleaceae forming a clade sister to ( Gottschling et al., 2001 ; Nazaire and Hufford, 2012 ), most another clade composed of the large families Convolvulaceae and chloroplast DNA studies ( Luebert and Wen, 2008 ; Nazaire and . These relationships are consistent with most prior Hufford, 2012; Weigend et al., 2013) instead identify two clades, studies (e.g., Olmstead et al., 1993 , 2000 ; Soltis et al., 2000 , 2011 ; with one corresponding to the mostly woody Nameae be- Albach et al., 2001 ; Bremer et al., 2002 ). ing more closely related to the woody tropical (Cor- Whereas circumscriptions of Solanaceae and Convolvulaceae diaceae, Ehretiaceae, Heliotropiaceae). Our results are consistent and their close relationship to each other, have long been accepted with other chloroplast DNA studies, albeit with limited taxon (e.g., Stebbins, 1974 ; Cronquist, 1981 ; Takhtajan, 1997 ), the three sampling, in fi nding that Nameae is a distinct clade from other small families comprising the remainder of Solanales had not been Hydrophyllaceae with relatively strong support in all analyses allied in traditional classifi cations. The pantropical was ( Fig. 1 ). Given the uncertainty concerning monophyly of tradi- traditionally placed in the otherwise mostly temperate, North tional Hydrophyllaceae, and the fact that the two clades are American Hydrophyllaceae in Solanales by Cronquist (1981) , each distinct in both chloroplast and nuclear gene trees, it may but in Boraginales by Takhtajan (1997) . Sphenocleaceae (one be desirable to recognize two families—Hydrophyllaceae s.s., African and one pantropical species) was placed in Campanulales and a new family based on tribe Nameae. Similarly, while cp- ( Cronquist, 1981 ; Takhtajan, 1997 ). Montiniaceae (South DNA studies place Lennoaceae within Ehretiaceae ( Nazaire and ) is represented here by and Kali- and Hufford, 2012; Weigend et al., 2013), ITS sequences sug- phora, which were assigned to Grossulariaceae and Cornaceae, gest a position for Lennoaceae sister to Ehretiaceae (Gottschling respectively, by Cronquist (1981) , but were each monogeneric et al., 2001 ), or sister to Cordiaceae, with weak support families assigned to Hydrangeales by Takhtajan (1987) . Solanales (Nazaire and Hufford, 2012). As with other parasitic groups have been suggested to be of Gondwanan origin, with the three (e.g., Cuscuta — Stefanović and Olmstead, 2004), Lennoaceae main clades each tracing its origin to one of the primary continental are diffi cult to place, due to the inability to amplify some chlo- fragments following the split up of Gondwana ( Olmstead, 2013 ). roplast DNA regions (e.g., ndhF , Weigend et al., 2013; Olm- stead, unpublished), and accelerated substitution rates resulting Boraginales— Our results obtain a strongly supported Boragina- in long branches in both chloroplast and nuclear gene trees les (Boraginaceae s.l.), including Hydrophyllaceae, in a position ( Nazaire and Hufford, 2012 ; Weigend et al., 2013 ). February 2014] REFULIO-RODRIGUEZ AND OLMSTEAD — PHYLOGENY OF LAMIIDAE 293

The distinction between the traditional Boraginaceae and Hy- studies, with some families better represented (i.e., more dis- drophyllaceae pertains to the number of ovules per carpel, with persed phylogenetic distribution) in one or the other study. two ovules per carpel in the former, and four-to-many ovules per Within Lamiales, all family-level clades with two or more rep- carpel in the latter. The placement of (formerly Hydro- resentatives were supported by a Bayesian PP and/or ML boot- phyllaceae) as sister to the clade composed of and Bor- strap values of greater than 0.90, consistent with previous aginoideae (Boraginaceae s.s.), and the placement of the two studies that led to currently recognized families ( APG III, clades of Hydrophyllaceae at the base of the other lineage leading 2009); all nodes representing interfamilial relationships, except to the primarily woody, tropical borages (Cordiaceae, Ehretiaceae, for two, have similarly strong support. Our results provide an Heliotropiaceae), suggests that the ancestral carpel morphology incremental improvement over those of Schäferhoff et al. included multiple ovules. It also suggests that a reduction to two (2010), with 11 nodes (Fig. 1) receiving strong support (Bayes- ovules per carpel occurred independently in Boraginoideae, along ian PP and/or ML BS > 0.90 or 90%, respectively) that were with the gynobasic style characteristic of that group, and in the weakly supported, or not obtained in that study (Appendixes S6 woody borages, where the gynobasic style is not found (Olmstead and S7). Most confl ict between the two studies are for clades and Ferguson, 2001 ; Weigend et al., 2013 ). weakly supported in that study, except for one clade composed of Acanthaceae, Bignoniaceae, , Martyniaceae, Lamiales—Most 20th century classifi cations recognized a Pedaliaceae, Schlegeliaceae, Thomandersiaceae, and Verben- small order Lamiales, which was centered on Lamiaceae and aceae, which received strong support in the Bayesian analysis Verbenaceae (e.g., Stebbins, 1974; Cronquist, 1981; Takhta- of Schäferhoff et al. (2010), but which was contradicted strongly jan, 1987 ), but the earliest rbcL phylogenies of Asteridae in the Bayesian analysis here (less strongly in our ML analysis). (Olmstead et al., 1992, 1993 ) identifi ed a large clade com- A likelihood ratio test comparing our tree with the optimal tree posed of all or parts of several orders in those earlier classifi ca- obtained from a search in which the taxa forming the confl ict- tions (e.g., Callitrichales, Gentianales, Lamiales, Plantaginales, ing clade in Schäferhoff et al. (2010) was constrained to mono- and Scrophulariales sensu Cronquist, 1981 ). This clade was phyly, using both the Shimodaira-Hasegawa (Shimodaira and named Lamiales by Olmstead et al. (1993), and has been re- Hasegawa, 1999 ) and Approximately Unbiased ( Shimodaira, covered consistently in subsequent molecular phylogenetic 2002) methods, found that the results of Schäferhoff et al. studies with strong support (Olmstead et al., 2000; Soltis et al., (2010) were signifi cantly less likely. This appears, therefore, to 2000 , 2011 ; Bremer et al., 2002 ; Hilu et al., 2003 ; Schäferhoff be one place where the much greater DNA sequence data in this et al., 2010). It is supported here with 100% bootstrap sup- study resulted in a signifi cant difference relative to that study. port in the ML and MP analyses, and a posterior probability A series of increasingly inclusive clades were obtained with of 1.0 in the Bayesian analysis. Despite being a universally high support, which have been identifi ed in previous studies recognized group today, fully resolving phylogenetic rela- with fewer loci and often with weaker support ( Olmstead et al., tionships within Lamiales, even with multilocus data sets, has 2001 ; Oxelman et al., 2005 ; Schäferhoff et al., 2010 ). These proven diffi cult, with incremental improvements obtained as include: more data are applied to the problem (Olmstead et al., 2001; 1. Lamiaceae/Orobanchaceae clade, including the small fam- Bremer et al., 2002; Schäferhoff et al., 2010; Soltis et al., ilies Mazaceae, Paulowniaceae, Phrymaceae, and Rehmanni- 2011). aceae—Mazaceae (Dodartia, , ), excluded from In contrast with the diffi culty in resolving backbone rela- Phrymaceae, where it was initially placed by Beardsley and tionships, well-supported clades within Lamiales have been Olmstead (2002), and Rehmanniaceae ( , Trianaeo- identifi ed and assigned the rank of family in recent studies phora – Albach et al., 2009 ; Xia et al., 2009 ), are the most (Acanthaceae— et al., 1995; McDade et al., 2000, recently described families of Lamiales (Reveal, 2011). Lami- 2013 ; Bignoniaceae— Spangler and Olmstead, 1999 ; Olmstead aceae are sister to a clade that is composed of the other et al., 2009; Gesneriaceae—Smith et al., 1997; Perret et al., families. 2013 ; Lamiaceae— Wagstaff et al., 1998 ; Bendiksby et al., 2. The Lamiaceae/Orobanchaceae clade plus the eight fami- 2011 ; Lentibulariaceae— Jobson et al., 2003 ; Müller et al., lies mentioned previously—the eight families, which form a 2004; Linderniaceae—Rahmanzadeh et al., 2005; Oleaceae— grade leading to the Lamiaceae/Orobanchaceae clade in this Wallander and Albert, 2000; Orobanchaceae— Young et al., study, together formed a clade in the study of Schäferhoff et al. 1999; Bennett and Mathews, 2006; Phrymaceae—Beardsley (2010) . Both studies obtained this inclusive clade, called the and Olmstead, 2002; —Albach et al., 2005; Scro- “higher core Lamiales” by Schäferhoff et al. (2010) , with strong phulariaceae—Korn hall et al., 2001; Kornhall and Bremer, support. However, some of the weakest branch support values at 2004 ; Oxelman et al., 2005 ; Thomandersiaceae— Wortley et al., the suprafamilial level are found within this clade. Within this 2007; Verbenaceae—Marx et al., 2010). The most signifi cant clade, Martyniaceae and Pedaliaceae occur as successive sister changes from premolecular classifi cations have come in the re- groups to Acanthaceae, with modest support for Martyniaceae + circumscription of Lamiaceae/Verbenaceae (Cantino, 1992a, Acanthaceae (also obtained in the MP analysis), but very weak 1992b ; Wagstaff and Olmstead, 1997 ; Wagstaff et al., 1998 ), support for the more inclusive group, which was not obtained in and the splitting of Scrophulariaceae in multiple segregate fami- optimal parsimony or likelihood trees. This clade also includes a lies ( Olmstead et al., 2001 ; reviewed in Tank et al., 2006 ). well-supported clade that is composed of Thomandersiaceae as The study of Schäferhoff et al. (2010) was the fi rst to include sister to Verbenaceae. Thomandersiaceae were fi rst recognized all 25 currently recognized families ( APG III, 2009 ; Reveal, as distinct from Acanthaceae by Wortley et al., (2007) and de- 2011 ; Olmstead, 2012 ) in a three-gene analysis (matK, rps16, fi ned as its own family. This relationship was not resolved in trnLF ). Our study also includes all 25 families and adds 7 ad- Schäferhoff et al. (2010), but was obtained by Soltis et al. (2011). ditional gene regions to the 3 included in that study, for a total Schlegeliaceae are sister to Lentibulariaceae, but with minimal of 10 DNA regions (9 chloroplast, 1 mitochondrial) and a total sampling of both groups and a long branch leading to aligned length of over 17 000 bases. Sampling is similar in both (Lentibulariaceae). Thus, this pairing may be questionable. 294 AMERICAN JOURNAL OF BOTANY [Vol. 101

3. The preceding clade plus Byblidaceae, Linderniaceae, diverging clades, even at deep phylogenetic nodes ( Oliver, Stilbaceae, and Scrophulariaceae s.s.—our results provide sup- 2013). Individual nuclear gene regions are unlikely to provide port for a clade composed of Byblidaceae and Linderniaceae, more resolution than a multilocus chloroplast tree, but a hope which was unresolved in the Bayesian analysis depicted in Fig. for the future, with the availability of whole genomes and im- 1 of Schäferhoff et al. (2010) , but which was obtained in their proved coalescent methods that can handle both large numbers ML analysis. The correct placement of Byblidaceae and Len- of taxa and loci, is a tree of angiosperms that can accommo- tibulariaceae has been problematic because of the accelerated date variation in individual gene trees. With progress on these substitution rates in the chloroplast genomes of both of these fronts, studies of divergence timing and rates, biogeographic carnivorous groups (Müller et al., 2004), and is most interpretations, and a more detailed understanding of pheno- likely responsible for the parsimony analysis in this study and typic evolution of this large and important clade are now more that of Schäferhoff et al. (2010) to obtain a weakly supported feasible. clade composed of these two families, because parsimony is more susceptible to long branch attraction. The Bayesian analy- sis (using BEAST; Drummond and Rambaut, 2007 ), of the data LITERATURE CITED of Schäferhoff et al. in Perret et al. (2013 ; suppl. Figure 1), also obtains a clade composed of Byblidaceae and Lentibulariaceae. A LBACH , D . C . , . UN , . S ØREN ROSENDAL , AND L . ONG-QING. 2009 . Phy- Therefore, uncertainty still exists regarding the correct place- logenetic placement of Triaenophora (formerly Scrophulariaceae) with ment of these two groups. Scrophulariaceae are sister to all some implications for the phylogeny of Lamiales. Taxon 58 : 749 – 756 . other members of this clade. A LBACH , D. C. , H. . MEUDT , AND B . XELMAN . 2005 . Piecing together the “new” Plantaginaceae. American Journal of Botany 92 : 297 – 315 . 4. The preceding clade plus Plantaginaceae—our results, as A LBACH , D. C. , P. S. SOLTIS , D. E. S OLTIS , AND R. G. OLMSTEAD . 2001 . well as other recent studies (Albach et al., 2005; Oxelman et al., Phylogenetic analysis of asterids based on sequences of four genes. 2005 ; Schäferhoff et al., 2010 ), do not support recognition of a Annals of the Missouri Botanical Garden 88 : 163 – 212 . segregate Gratiolaceae (sensu Rahmanzadeh et al., 2005 ). A NGIOSPERM PHYLOGENY GROUP . 1998 . An ordinal classifi cation of the families 5. A clade composed of all families, except Plocospermata- of fl owering plants. Annals of the Missouri Botanical Garden 85 : 531 – 553 . ceae, Oleaceae, Carlemanniaceae, and Tetrachondraceae—this A NGIOSPERM PHYLOGENY GROUP. 2003 . An update of the Angiosperm clade is characterized by bilateral symmetry and two pairs of Phylogeny Group classifi cation for the orders and families of fl owering and has been called the “Core Lamiales” (Schäferhoff plants: APG II . Botanical Journal of the Linnean Society 141 : 399 – 436 . et al., 2010). We included , a diffi cult to place genus A NGIOSPERM PHYLOGENY GROUP. 2009 . An update of the Angiosperm Phylogeny Group classifi cation for the orders and families of fl owering sometimes allied with Gesneriaceae ( Oxelman et al., 1999 ; plants: APG III . Botanical Journal of the Linnean Society 161 : 105 – 121 . Bremer et al., 2002; Perret et al., 2013), which was not included B ACKLUND , M., B . OXELMAN , AND B . B REMER . 2000 . Phylogenetic relation- in Schäferhoff et al. (2010) . Peltanthera is supported in a posi- ships within the Gentianales based on ndhF and rbcL sequences, with tion as sister to a clade composed of Gesneriaceae and Calceo- particular reference to the Loganiaceae. American Journal of Botany lariaceae in our study with strong support in all three analyses. 87 : 1029 – 1043 . This is in contrast to its placement in Perret et al. (2013, suppl. B EARDSLEY , P. M . , AND R. G. OLMSTEAD . 2002 . Redefi ning Phrymaceae: The Figure 1), in which it is placed as sister to Gesneriaceae, but placement of , tribe Mimuleae, and Phryma , character evolu- with weak support (the non-Gesneriaceae data for their study tion and biogeography. American Journal of Botany 89 : 1093 – 1102 . were drawn from those of Schäferhoff et al., 2010 ). They con- B ENDIKSBY , M . , L . HORBEK , A.-C. S CHEEN , C . L INDQVIST , AND O . R YDING . sidered Peltanthera to be included in Gesneriaceae, but our re- 2011 . 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A phylogenetic analysis of morphological and molecu- order, however, noting that this tree represents a maternal phy- lar characters of Boraginaceae: evolutionary relationships, , logeny based primarily on cpDNA, and discordance between and patterns of character evolution. (early access): http:// individual gene trees and the species tree is expected in rapidly onlinelibrary.wiley.com/doi/10.1111/cla.12036/full February 2014] REFULIO-RODRIGUEZ AND OLMSTEAD — PHYLOGENY OF LAMIIDAE 295

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A PPENDIX 1. Species included in this study, with assignment to order (where appropriate), family, authorities, voucher information (herbarium acronym), and GenBank accession number (atpB, matK, ndhF, psbBTNH, rbcL, rps4, rps16, trnLF, trnV, rps3). GenBank numbers in bold are new for this study; “na” indicates no sequence available for this region for this accession.

GARRYALES. AUCUBACEAE , Thunb., R. G. Olmstead na, na, na, na, na, na, na. RUBIACEAE , Cephalanthus occidentalis L., R. 2008-2 (WTU); M. J. Moore 327 (FLAS) for trnLF, trnV, GQ997051, G. Olmstead 90-007 (WTU);, AJ236190, AY538377, AJ236288, na, na, GQ997060, GQ997066, GQ997084/GQ997096/GQ997095/GQ997089, HQ385114, AF004033, AJ346955, na, na. Cephalanthus salicifolius GQ997098, GQ997121, GQ997116, KJ161976, KJ161969, HQ385030. Humb. & Bonpl., na, na, na, na, AJ346975, na, na, na, na, na. Coffea EUCOMMIACEAE , Oliv ., R. G. Olmstead 97-11 arabica L., R. G. Olmstead 2007-105 (WTU); EF044213, EF044213, (WTU), HQ384789, AJ429317, AJ429113, HQ384680, L01917, EF044213, EF044213, EF044213, EF044213, EF044213, EF044213, HQ385138, AJ431028, AJ430905, na, HQ385028. GARRYACEAE , EF044213, HQ385005. Galium aparine L ., N. Fraga et al. 1282 (RSA); na, Dougl. ex Lindl., R. G. Olmstead s.n. (WTU), AJ235479, HQ384560, na, HQ384655, X81091, HQ385112, HQ385189, HQ412968, AJ429319, HQ384864, HQ384681, L01919, HQ385139, AJ431030, HQ412907, HQ385003. gratissima (Wall.) Sweet , R. Shefferson HQ412986, HQ412924, HQ385029. ICACINACEAE , Cassinopsis s.n. (UC);, HQ384771, AJ429325, AJ011987, HQ384657, HQ384914, ilicifolia (Hochst.) Sleumer, voucher unknown, na, AJ429312, AJ429110, HQ385115, AJ431036, AJ430911, AJ429678, HQ385006. Psychotria na, AJ428896, na, AJ431023, AJ430900, AJ429667, na. Cassinopsis hispidula Standl. ex Steyerm ., R. Loredo 3881 (MO); HQ384770, madagascariensis Baill., B. Lewis & Razafi mandimbison 594 (MO), HQ384561, HQ384851, HQ384656, HQ384913, HQ385113, HQ385190, HQ384787, na, na, HQ384678, na, HQ385137, HQ385205, na, na, HQ412969, HQ412908, HQ385004. LAMIALES. ACANTHACEAE, HQ385026. Icacina mannii Oliv., H. P. Bourobou Bourobou et al. 666 montanus (Nees) T. Anders ., D. J. Harris 1931 (K), HQ384715, (MO), HQ384788, HQ384577, AJ400888, HQ384679, HQ384929, na, HQ384511, AJ429115, HQ384599, L12592, HQ385052, DQ059146, HQ385206, HQ412985, na, HQ385027. METTENIUSACEAE , AF061823, HQ412860, HQ384951. americana Vahl, R. G. Metteniusa tessmanniana (Sleumer) Sleumer, Betancur 10386 (COL, K), Olmstead 90-011 (WTU), AJ236178, HQ384510, HQ384825, HQ384598, AM421129, na, na, na, AM421128, na, na, na, na, na. ONCOTHECACEAE , L14401, HQ385051, HQ385154, HQ412935, HQ412859, HQ384950. Oncotheca balansae Baill ., Jaffre 3238 (NOU), AJ235549, AJ429320, R. Br. sp. , D. J. Harris 5722 (FHO), HQ384717, HQ384513, AJ429114, na, AJ131950, na, AJ431031, AJ430530, AJ429673, na. HQ384827, HQ384601, HQ384879, HQ385054, HQ385155, HQ412936, VAHLIACEAE , Vahlia capensis Thunb ., J. H. J. Vlok 2546 (MO), HQ412862, HQ384952. alata Bojer, R. G. Olmstead 93-45 AJ236217, AJ429316, AJ429112, HQ384677, L11208, HQ385136, (WTU), HQ384716, HQ384512, HQ384826, HQ384600, HQ384878, AJ431027, HQ412984, AJ429671, HQ385025. GENTIANALES. HQ385053, AJ609131, AJ608564, HQ412861, na. BIGNONIACEAE , APOCYNACEAE, oblongifolia (Hochst.) Codd , Matthaei capreolata L., J. R. Abbott et al. 24872 (FLAS), HQ384723, Bot. Gard.; no voucher;, HQ384763, HQ384553, HQ384847, HQ384649, HQ384518, DQ222566, HQ384607, HQ384884, HQ385060, HQ385160, X91758, HQ385104, HQ385185, HQ412962, HQ412903, HQ384995. FJ870021, HQ412867, HQ384958. aff. speciosa Warder, R. G. Alstonia scholaris (L.) R. Br., na, AJ429321, na, na, X91760, na, Olmstead 88-003 (WTU), HQ384724, HQ384519, L36397, HQ384608, DQ660558, AJ430907, AJ429674, na. Alstonia vitiensis Seem ., G. Mc HQ384885, HQ385061, HQ385161, HQ412940, HQ412868, HQ384959. Pherson et al. 19471 (MO);, na, na, na, na, na, HQ385107, na, na, na, na. scaber Ruiz & Pav., R. G. Olmstead 2007-155 (WTU), Apocynum cannabinum L ., R. G. Olmstead 88-002 (WTU);, HQ384762, HQ384728, HQ384523, AF102630, HQ384612, AF102646, HQ385065, HQ384552, HQ384846, HQ384648, HQ384907, HQ385103, DQ660563, HQ385165, HQ412943, HQ412872, HQ384963. mimosifolia HQ412961,, HQ384993. Asclepias syriaca L ., Michigan, USA; no voucher;, D. Don, L. Lohmann 369 (MO), HQ384729, AJ429328, EF105012, HQ384761, HQ384551, na, HQ384647, HQ384906, HQ385102, HQ384613, HQ384888, HQ385066, AJ431039, AJ430914, AJ429681, DQ660564, HQ412960, HQ412902, HQ384992. Kopsia fruticosa (Roxb.) HQ384964. Vent. , Waimea Bot. Gard. #79S51, no A. DC .,, na, na, AJ235824, na, X91763, na, DQ660588, AM295091, na, na. voucher, HQ384725, HQ384520, AF102635, HQ384609, HQ384886, Kopsia paucifl ora . f., K. Larsen et al. 43442 (MO);, HQ384765, HQ385062, HQ385162, HQ412941, HQ412869, HQ384960. HQ384555, na, HQ384651, na, HQ385106, na, na, na, HQ384997. Nerium heterophylla (DC.) Britton , S. O. Grose 056 (WTU), HQ384726, oleander L ., E. Kempton s.n. (RSA), Judd 8076 (FLAS) for trnLF, trnV, HQ384521, AY500455, HQ384610, HQ384887, HQ385063, HQ385163, GQ997632, GQ997641, GQ997647, GQ997665/GQ997677/GQ997676/ HQ412942, HQ412870, HQ384961. stans Juss., R. G. Olmstead GQ997670, GQ997679, GQ997702, GQ997697, KJ161977, KJ161970, 2003-4 (WTU), HQ384727, HQ384522, AF102639, HQ384611, AF102655, HQ384994. Vinca minor L., F. M. Roberts & R. L. Allen 6607 (RSA);, HQ385064, HQ385164, AY008826, HQ412871, HQ384962. HQ384764, HQ384554, HQ384848, HQ384650, HQ384908, HQ385105, BYBLIDACEAE , linifl ora Salisb., R. G. Olmstead 2008-36 DQ660623, HQ412963, HQ412904, HQ384996. GELSEMIACEAE, (WTU), HQ384703, HQ384500, HQ384816, HQ384586, HQ384870, sempervirens (L.) St. Hil., J. R. Abbott et al. 24871 (FLAS);, HQ385039, AJ431070, AJ430941, AJ429533, HQ384936. AJ236193, HQ384556, AF130170, HQ384652, HQ384909, HQ385108, CALCEOLARIACEAE , L. sp., J. L. Clark 6747 (US), AJ431033, AJ430908, AJ429675, HQ384998. brunonis Didr ., HQ384746, HQ384541, AF123679, na, HQ384899, na, na , na, HQ412891, Razafimandimbison 477 (UPS);, HQ384766, HQ384557, AJ235828, na. Calceolaria mexicana Benth , T. B. Croat et al. 93268 (MO), na, na, na, HQ384653, HQ384910, HQ385109, HQ385186, HQ412964, na, HQ384632, na, HQ385086, AJ609202, AJ608611, na, na. Ruiz HQ384999. Pteleocarpa lamponga Bakh. ex K.Heyne , N. S. Mat Asri, FRI & Pav. sp., U.C. Berkeley Bot. Gard., no voucher, na, na, AF123684, na, 26732 (KEP);, HQ384767, HQ384558, HQ384849, HQ384654, HQ384911, AF123666, na, na, na, na, na. G. Don , R. G. Olmstead HQ385110, HQ385187, HQ412965, na, HQ385000. GENTIANACEAE, 2007-152 (WTU), HQ384747, HQ384542, na, HQ384633, na, HQ385087, affi ne Balf. f. , AJ236195, na, na, na, L11684, na, na, na, na, na. HQ385178, HQ412953, HQ412892, HQ384980. CARLEMANNIACEAE , Exacum quinquenervium Griseb ., D. Rabehevitra et al. 1028 (MO);, na, Carlemannia tetragona Hook.f ., Grierson & Long 3027 (K), HQ384757, na, na, na, na, HQ385100, na, HQ412959, na, na. procera Holm, HQ384548, DQ673290, HQ384643, DQ673316, HQ385097, HQ385182, Michigan, USA; no voucher;, HQ384758, na, HQ384845, HQ384644, HQ412957, HQ412899, HQ384989. GESNERIACEAE , L14398, HQ385098, na, na, na, HQ384990. Gentiana purpurea L. , na, hirsutus (Mart.) Wiehler, Marie Selby Bot. Gard., no voucher, HQ384743, AJ429323, na, na, na, na, AJ431034, AJ430909, AJ429676, na. Sabatia HQ384540, HQ384840, HQ384629, HQ384897, HQ385083, HQ385176, angularis (L.) Pursh , R. G. Olmstead 90-008 (WTU);, HQ384759, HQ412951, HQ412889, HQ384979. Peltanthera fl oribunda Benth ., B. HQ384549, na, HQ384645, HQ384905, HQ385099, HQ385183, Hammell 24638 (INB, MO), HQ384748, AJ429330, HQ384842, HQ412958, HQ412900, HQ384991. Saccifolium bandeirae Maguire & HQ384634, HQ384900, HQ385088, AJ431041, AJ430916, AJ429682, Pires , Piliackas et al. s.n. (MJG);, HQ384760, HQ384550, na, HQ384646, HQ384981. Saintpaulia ionantha H. Wendl. , J. Smith 3702 (SRP), AJ242609, HQ385101, HQ385184, na, HQ412901, na. LOGANIACEAE, HQ384744, na, HQ384841, HQ384630, HQ384898, HQ385084, Spigelia humboldtiana Cham. & Schltdl., S. Suazo 347 (MO);, HQ384769, HQ385177, HQ412952, HQ412890, na. racemosum (Ruiz & HQ384559, HQ384850, na, HQ384912, HQ385111, HQ385188, Pavon) Barringer , Bremer 5350 (USP), HQ384745, na, AF027283, HQ412967, HQ412906, HQ385002. Strychnos nux-vomica L ., H. Forbes HQ384631, AJ001763, HQ385085, AJ609142, AJ430917, na, na. s.n. (UC);, HQ384768, Z70193, na, KJ161982, L14410, na, AF004094, caulescens Vatke, voucher unknown, na, na, na, na, na, na, HQ412966, HQ412905, HQ385001. Strychnos spinosa Lam ., na, na, na, AJ431043, na, AJ429684, na. Streptocarpus holstii Engl ., Matthaei Bot. na, na, JF268464, na, na, na, na. Strychnos potatorum L.f., na, na, AJ235841, Gard., no voucher, HQ384742, HQ384539, L36415, HQ384628, HQ384896, 298 AMERICAN JOURNAL OF BOTANY [Vol. 101

HQ385082, HQ385175, HQ412950, HQ412888, na. LAMIACEAE , HQ384517, HQ384831, HQ384606, HQ384883, HQ385059, AF482539, mollis Siebold & Zucc ., Missouri Bot. Gard. 897684, no HQ412939, HQ412866, HQ384957. PHRYMACEAE , Mimulus voucher, HQ384701, HQ384498, AY310134, HQ384584, HQ384868, aurantiacus Curtis, A. Colwell 97-CA-JTNP-2 (WTU), na, na, na, na, na, HQ385037, HQ385145, HQ412928, HQ412847, HQ384934. na, na, na, na, HQ384945. Mimulus guttatus Fisch. ex DC ., voucher trichotomum Thunb ., R. G. Olmstead 2002-134 (WTU), HQ384695, unknown, KJ161980, KJ161979, KJ161984, KJ161983, KJ161981, HQ384492, AF130146, HQ384578, HQ384865, HQ385031, HQ385140, KJ161985, KJ161978, KJ161975, KJ161968, na. L., FJ952051, HQ412842, na. tomentosa Roxb ., Fairchild Bot. Gard. P. D. Cantino 1376 (BHO), HQ384710, AJ429341, AJ429118, HQ384593, #59259, no voucher, HQ384702, HQ384499, HQ384815, HQ384585, U28881, HQ385046, AJ609150, AJ430928, AJ429696, HQ384944. HQ384869, HQ385038, AJ505411, HQ412929, HQ412848, HQ384935. PLANTAGINACEAE , angustifolia Benth ., R. G. Olmstead purpureum L. , S. Wagstaff 88-031 (BHO), HQ384696, HQ384493, 2007-106 (WTU), HQ384740, HQ384537, na, HQ384626, na, HQ385080, na, HQ384579, Z37403, HQ385032, HQ385141, AJ608588, HQ412843, na, na, HQ412886, HQ384978. Angelonia pubescens Benth ., voucher HQ384930. rotundifolia Huds., voucher unknown, na, na, na, na, unknown, na, na, AF123675, na, AF123672, na, AJ609214, AJ608618, na, Z37417, na, na, na, na, na. Mentha spicata L ., R. G. Olmstead 92-191 na. majus L ., GRIN PI 420370, grown from seed, no voucher; (WTU), HQ384698, HQ384495, HQ384813, HQ384581, na, HQ385034, M. J. Moore 314 (FLAS) for trnLF, trnV, GQ996968, GQ996977, na, na, na, na. Ehrh ., voucher unknown, na, na, na, na, GQ996983, GQ997001/GQ997013/GQ997012/GQ997006, GQ997015, na, na, AJ505418, AY618526, na, na. odorata Blanco , W. Gillis GQ997038, GQ997033, KJ161974, KJ161967, HQ384977. glabra 9430 (FTG), HQ384697, HQ384494, HQ384812, HQ384580, HQ384866, L ., R. G. Olmstead 2007-154 (WTU), HQ384738, HQ384535, HQ384837, HQ385033, HQ385142, HQ412925, HQ412844, HQ384931. HQ384624, HQ384895, HQ385078, HQ385172, HQ412949, HQ412884, calycina F. Muell. ex Benth ., RBGK 386.86.08142, no voucher, HQ384700, HQ384975. fl oridana Nutt ., V. E. McNeilus 92-225 (RSA), HQ384497, HQ384814, HQ384583, HQ384867, HQ385036, HQ385144, HQ384741, HQ384538, HQ384839, HQ384627, na, HQ385081, HQ412927, HQ412846, HQ384933. agnus-castus L. , K.-J. Kim HQ385174, na, HQ412887, na. Gratiola pilosa Michx ., voucher unknown, 2804 (TEX), HQ384699, HQ384496, AF130149, HQ384582, U78716, na, na, na, na, AF026827, na, na, AJ608591, na, na. persica Poir., HQ385035, HQ385143, HQ412926, HQ412845, HQ384932. R. G. Olmstead 92-144 (WTU), HQ384739, HQ384536, HQ384838, LENTIBULARIACEAE , Kunth, R. G. Olmstead HQ384625, na, HQ385079, HQ385173, AF513336, HQ412885, 97-5 (WTU), HQ384704, HQ384501, na, HQ384587, HQ384871, HQ384976. PLOCOSPERMATACEAE , buxifolium HQ385040, HQ385146, AF482626, HQ412849, HQ384937. Benth., P. Tenorio L. 20446 (MO), HQ384756, AJ429315, HQ384844, LINDERNIACEAE , plantagineum Hochst ., voucher HQ384642, HQ384904, HQ385096, AJ431026, HQ412956, AJ429670, unknown, na, AY667461, na, na, na, na, AY492201, AY492174, na, na. HQ384988. REHMANNIACEAE , N.E.Br., B. Oxelman baillonii Godefroy ex André, R. G. Olmstead 98-52 (WTU), na, 2336 (WTU), HQ384709, HQ384505, HQ384820, HQ384591, HQ384874, AY492166, AJ617583, na, AB259805, na, AJ609130, AJ608563, na, na. HQ385044, DQ856490, AJ608572, HQ412853, HQ384942. MARTYNIACEAE , lutea (Lindl.) Van Eselt ., Porter & Machen SCHLEGELIACEAE , fuscata A.H.Gentry, J. L. Clark 8578 12121 (RSA), HQ384731, HQ384525, HQ384832, HQ384615, HQ384890, (US), HQ384718, HQ384514, HQ384828, HQ384602, HQ384880, HQ385068, HQ385167, HQ412945, HQ412874, HQ384966. Martinia HQ385055, HQ385156, na,, HQ384953. Schlegelia parvifl ora (Oerst.) annua L ., P. Jenkins 97-149 (ARIZ), HQ384730, HQ384524, AF190906, Monach ., voucher unknown, na, na, na, na, na, na, na, AJ430932, AJ429696. HQ384614, HQ384889, HQ385067, HQ385166, HQ412944, HQ412873, SCROPHULARIACEAE , meridionalis (L. f.) Kuntze, R. G. HQ384965. MAZACEAE , N.E.Br., J. Aagard 2004-1 Olmstead 2009-17 (WTU), HQ384737, HQ384534, na, HQ384623, na, (WTU), HQ384705, HQ384502, HQ384817, HQ384588, HQ384872, HQ385077, na, HQ412948, HQ412883, HQ384974. Alonsoa unilabiata HQ385041, HQ385147, AF479004, HQ412850, HQ384938. OLEACEAE , (L.f.) Steud., voucher unknown, na, na, AF188184, na, AF026821, na, phillyreoides Labill., E. Wallender 20 (GB), HQ384753, AJ609217, na, na, na. Burch. ex Benth sp., Hort. Wor. Bot. HQ384545, DQ673263, HQ384639, HQ384902, HQ385093, AF225226, Gard, no voucher, na, na, AF123676, na, AF123671, na, AJ609210, AF231818, HQ412896, HQ384985. Vahl sp., P. A. Reeves 11 AJ608607, na, na. davidii Franch ., R. G. Olmstead 88-007 (WTU), HQ384754, HQ384546, HQ384843, HQ384640, HQ384903, (WTU), HQ384734, HQ384530, HQ384835, HQ384619, L14392, HQ385094, HQ385181, HQ412955, HQ412897, HQ384986. Jasminum HQ385073, HQ385169, AF380861, HQ412879, HQ384970. nudifl orum Lindl., voucher unknown, DQ673255, DQ673255, DQ673255, mauritianum A. DC., RBGKew #1984-4220, no voucher, AJ236166, DQ673255, DQ673255, DQ673255, DQ673255, DQ673255, DQ673255, HQ384532, HQ384836, HQ384621, HQ384894, HQ385075, AJ609161, na. L., R. G. Olmstead 89-007 (WTU), HQ384750, HQ412947, HQ412881, HQ384972. fruticans Benth ., R. G. HQ384543, AF130164, HQ384636, HQ384901, HQ385090, HQ385180, Olmstead 2007-107 (WTU), HQ384736, HQ384533, na, HQ384622, na, AF380876, HQ412894, na. arbor-tristis L., D. Spooner 6910 HQ385076, HQ385171, na, HQ412882, HQ384973. Nemesia strumosa (WIS), HQ384755, HQ384547, AF130161, HQ384641, U28877, Benth ., voucher unknown, na, na, AF123688, na, AF123663, na, na, HQ385095, AF225272, AF231863, HQ412898, HQ384987. europaea AJ608631, na, na. capensis E.Mey. ex Benth., R. G. Olmstead L., Y. L. Qiu 95043 (IND), HQ384752, AJ429335, AF130163, HQ384638, 2007-153 (WTU), HQ384735, HQ384531, AJ617608, HQ384620, AJ001766, HQ385092, AJ431047, AJ430922, AJ429687, HQ384984. HQ384893, HQ385074, HQ385170, AJ608575, HQ412880, HQ384971. vulgaris L ., Cultivated, no voucher, HQ384751, HQ384544, californica Cham. & Schltdl ., R. G. Olmstead 93-1 (WTU), DQ673277, HQ384637, DQ673303, HQ385091, AF225292, AF231882, HQ384733, HQ384529, HQ384834, HQ384618, HQ384892, HQ385072, HQ412895, HQ384983. OROBANCHACEAE , linariifolia AJ609224, HQ412946, HQ412878, HQ384969. STILBACEAE , Benth. , D. Tank 2001-49 (WTU), HQ384707, HQ384504, HQ384819, na, Campylostachys cernua Kunth, R. G. Olmstead 99-09 (WTU), na, AF026823, HQ385043, EF103788, EF103866, HQ412852, HQ384940. HQ384527, HQ384833, HQ384616, HQ384891, HQ385070, HQ385168, philippensis (Cham. & Schltdl.) Benth ., J. Armstrong 1163 AJ608621, HQ412876, HQ384967. Euthystachys abbreviata A. DC ., J. (PSU), HQ384708, na, AF123686, HQ384590, AF123664, na, AJ609169, Rourke & D. J. McDonald, no voucher, AJ236173, HQ384526, AF147715, na, na, HQ384941. Lindenbergia Lehm. sp ., voucher unknown, na, na, Z29671, HQ385069, AJ609185, AJ608626, HQ412875, na. AJ429337, na, na, na, na, AJ431049, AJ430924, AJ429689, na. lucida L., L. Festo & M. A. Mwangoka 1862 (MO), HQ384732, HQ384528, groenlandica Retz ., J. Aagard s.n. (WTU), HQ384706, HQ384503, AF188185, HQ384617, AF026828, HQ385071, AJ609180, AJ621109, HQ384818, HQ384589, HQ384873, HQ385042, HQ385148, HQ412930, HQ412877, HQ384968. TETRACHONDRACEAE , HQ412851, HQ384939. PAULOWNIACEAE , tomentosa procumbens L ., , USA, no voucher, HQ384749, AJ429351, Steud ., R. G. Olmstead 88-008 (WTU), AJ236174, AJ429339, L36406, AJ011986, HQ384635, AJ011989, HQ385089, HQ385179, HQ412954, HQ384592, L36447, HQ385045, HQ385149, AJ608578, HQ412854, HQ412893, HQ384982. patagonica Skottsb ., voucher HQ384943. PEDALIACEAE , lugardii N.E.Br. ex Stapf , unknown, na, AJ429352, AF027272, na, AF254787, na, AJ431064, cultivated, UW Greenhouse, no voucher, HQ384720, HQ384516, AJ430939, AJ430389, na. THOMANDERSIACEAE , Thomandersia HQ384830, HQ384604, HQ384881, HQ385057, HQ385158, HQ412938, laurifolia Baill ., Cable 4012 (RBGE), HQ384719, HQ384515, HQ384829, HQ412864, HQ384955. indicum L. , cultivated, no voucher, HQ384603, AY919280, HQ385056, HQ385157, HQ412937, HQ412863, HQ384721, AJ429340, L36413, HQ384605, HQ384882, HQ385058, HQ384954. VERBENACEAE , berlandieri B.L. Rob ., L. HQ385159, AJ430927, HQ412865, HQ384956. grandidieri Woodbury 803 (FTG), HQ384712, HQ384507, HQ384822, HQ384595, (Baill.) Ihlenf. & Straka , R. G. Olmstead 99-01 (WTU), HQ384722, HQ384876, HQ385048, HQ385151, HQ412932, HQ412856, HQ384947. February 2014] REFULIO-RODRIGUEZ AND OLMSTEAD — PHYLOGENY OF LAMIIDAE 299

Petrea kohautiana C. Presl, J. L. Clark 6554 (US), HQ384714, HQ384509, L18798, HQ385125, AJ431069, AJ430947, AJ429534, na. BORAGINALES. HQ384824, HQ384597, HQ384877, HQ385050, HQ385153, HQ412934, BORAGINACEAE, offi cinalis L ., R. G. Olmstead 96-62 (WTU), HQ412858, HQ384949. dichotoma Vahl., R. G. HQ384785, AJ429308, L36393, HQ384676, HQ384927, HQ385133, Olmstead 951 (WTU), HQ384713, HQ384508, HQ384823, HQ384596, AJ431019, AJ430896, AJ429664, HQ385024. CODONACEAE , Codon U32161, HQ385049, HQ385152, HQ412933, HQ412857, HQ384948. royenii L ., voucher unknown, na, na, na, na, AM234925, na, na, na, na, na. Lag. & Rodr ., R. G. Olmstead 92-131 (WTU), Codon schenckii Schinz , R. Seydel 4315 (MO);, na, HQ384576, HQ384711, HQ384506, HQ384821, HQ384594, HQ384875, HQ385047, HQ384863, na, na, HQ385135, HQ385204, na, na, na. CORDIACEAE , HQ385150, HQ412931, HQ412855, HQ384946. SOLANALES. Cordia nevillii Alston , M. Thulin et al. 8293 (UPS), HQ384781, CONVOLVULACEAE , Convolvulus cneorum L., R. Olmstead 2002-13 HQ384571, HQ384860, HQ384672, HQ384923, HQ385129, HQ385199, (WTU), HQ384774, HQ384565, HQ384852, HQ384661, HQ384916, HQ412979, na, HQ385021. EHRETIACEAE , Ehretia acuminata HQ385119, HQ385194, HQ412972, HQ412912, HQ385011. Dinetus R. Br ., R. G. Olmstead 93-63 (WTU), M. J. Moore 317 (FLAS) for racemosus Sweet, Staples et al. 427 (A), voucher unknown, na, HQ384856, trnV, GQ997217, GQ997226, GQ997232, GQ997250, GQ997264, HQ384665, HQ384920, HQ385123, HQ385197, HQ412975, HQ412916, GQ997287, GQ997282, EU600008, KJ161971, HQ385020. NAMEAE , HQ385016. Erycibe coccinea (F. M. Bailey) Hoogland , Forster 27670 (A), californicum (Hook. & Arn.) Torr., R. G. Olmstead 95-21 HQ384776, HQ384568, HQ384855, HQ384664, HQ384919, HQ385122, (WTU), HQ384783, HQ384573, AF047820, HQ384674, HQ384925, HQ385196, HQ412974, HQ412915, HQ385015. Evolvulus pilosus Roxb ., HQ385131, HQ385201, HQ412981, HQ412921, HQ385022. Nama Cultivated, no voucher, HQ384775, HQ384566, HQ384853, HQ384662, demissum A. Gray var. demissum , Le Roy Gross et al. 882a (RSA), HQ384917, HQ385120, na, HQ412973, HQ412913, HQ385013. Humbertia HQ384782, HQ384572, HQ384861, HQ384673, HQ384924, HQ385130, madagascariensis Lam., G. McPherson 14267 (MO), HQ384777, HQ385200, HQ412980, HQ412920, na. HELIOTROPIACEAE , EU330288, HQ384857, HQ384666, HQ384921, na, na, HQ412976, Heliotropium arborescens L., R. G. Olmstead 96-64 (WTU), HQ384780, HQ412917, HQ385017. Ipomoea coccinea L., R. G. Olmstead 88-015 HQ384570, EF688911, HQ384671, L14399, HQ385128, EF688962, (WTU), na, na, na, na, na, na, na, na, na, HQ385012. Ipomoea purpurea EF688806, HQ412919, na. HYDROPHYLLACEAE , Hydrophyllum (L.) Roth, voucher unknown, EU118126, EU118126, EU118126, capitatum Dougl. ex Benth., R. G. Olmstead 2008-1 (WTU), HQ384784, EU118126, EU118126, EU118126, EU118126, EU118126, EU118126, na. HQ384574, AF047785, HQ384675, HQ384926, HQ385132, HQ385202, reclinata House ex Small , S. Stefanović 01-120 (TRTE), HQ412982, HQ412922, HQ385023. WELLSTEDIACEAE , Wellstedia AY100829, HQ384567, HQ384854, HQ384663, HQ384918, HQ385121, dinteri Pilg ., P. Goldblatt J. Manning 8763 (MO), HQ384786, HQ384575, HQ385195, AY101149, HQ412914, HQ385014. HYDROLEACEAE , HQ384862, na, HQ384928, HQ385134, HQ385203, HQ412983, Hydrolea ovata Nutt., R. G. Olmstead 89-09 (WTU), AJ236184, AJ429356, HQ412923, na. HQ384858, HQ384667, na, HQ385124, na, HQ412977, AJ429703, HQ385018. MONTINIACEAE , madagascariensis Hook.f., C. OUTGROUPS. HELWINGIACEAE, Helwingia japonica (Thunb.) F. Dietr., Birkinshaw et al. 565 (MO), HQ384779, AJ429358, HQ384859, HQ384669, no voucher, AF209596, AJ430195, AF130207, na, L11226, na, AJ431089, HQ384922, HQ385126, HQ385198, AJ430945, HQ412918, na. Montinia AJ430963, AJ429723, na. Helwingia Willd. sp. , Peng et al. 17408 (S), na, caryophyllacea Thunb., R. G. Olmstead 94-01 (WTU), AY100852, na, na, GQ983706, na, GQ983964, na, na, na, na. ARALIACEAE , Aralia HQ384569, AF130178, HQ384670, L11194, HQ385127, AJ431068, spinosa L., Plunkett 1371 (WS), GQ983605, AJ429371, AJ429125, HQ412978, AJ429706, HQ385019. SOLANACEAE , elegans GQ983696, L11166, GQ983963, GQ983999, AJ430957, GQ984102, na. Wydler, R. G. Olmstead 92-220 (WTU), HQ384772, HQ384563, PENTAPHRAGMATACEAE , Pentaphragma ellipticum Poulsen , no AY206746, HQ384659, AF035738, HQ385117, HQ385192, HQ412971, voucher, AJ318980, AJ429387, AF130183, na, L18794, na, AJ431099, HQ412910, HQ385009. Nicotiana tabacum L ., Matthaei Bot. Gard., no AJ430975, AJ429732, na. Pentaphragma (Roxb. ex Jack) Wall. ex G. voucher, Z00044, Z00044, Z00044, Z00044, Z00044, Z00044, Z00044, Don sp., Duangjai 49 (BRUN), na, na, na, GQ983761, na, GQ983969, na, Z00044, Z00044, BA000042. Petunia axillaris Britton, Sterns & na, na, na. CORNACEAE, Cornus fl orida L., M. J. Moore 328 (FLAS), Poggenb ., R. G. Olmstead S-60 (WTU), AJ236182, HQ384562, U08926, EU002157, EU002175, EU002215, EU002377, EU002276, EU002311, HQ384658, HQ384915, HQ385116, HQ385191, HQ412970, HQ412909, GQ998132, AY254238, KJ161972 , na. CAPRIFOLIACEAE, Lonicera HQ385008. Schizanthus pinnatus Ruiz & Pav ., R. G. Olmstead S-72 orientalis Lam ., no voucher, na, AJ430196, na, na, X87389, na, AJ431104, (WTU), HQ384773, HQ384564, U08929, HQ384660, U08619, HQ385118, AJ430980, AJ429737, na. Lonicera japonicaa Thunb., Smith s. n. (YU), HQ385193, AY101172, HQ412911, HQ385010. Solanum lycopersicum GQ983602, na, AF130194, GQ983785, na, GQ983915, na, na, na, na. L., Cultivated, no voucher, AM087200, AM087200, AM087200, ERICACEAE , Rhododendron simsii Planch., M. J. Moore 324 (FLAS), AM087200, AM087200, AM087200, AM087200, AM087200, AM087200, GQ997783, GQ997791.1, GQ997797, GQ99778215/GQ99778227/ HQ385007. SPHENOCLEACEAE , Sphenoclea zeylanica Gaertn ., N. GQ99778226/GQ99778220, GQ997829, GQ997851, GQ997846, De la Barra 943 (MO), HQ384778, AJ429360, AJ429119, HQ384668, GQ184592, KJ161973, na.