Rhexia

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PHYLOGENETIC RELATIONSHIPS IN RHEXIA (MELASTOMATACEAE): EVIDENCEFROM DNA SEQUENCE DATA AND MORPHOLOGY

Gretchen M. Ionta,1,* Walter S. Judd,* Norris H. Williams,y and W. Mark Whitteny

*Department of Botany, University of Florida, Gainesville, Florida 32611, U.S.A.; and yFlorida Museum ofNatural History, University of Florida, Gainesville, Florida 32611-7800, U.S.A.

In the large, pantropical family Melastomataceae, Rhexia is unique in having a temperate distribution. Thespecies of Rhexia, the ‘‘meadow beauties,’’ are a common sight along roadsides and in open habitats in easternNorth America, ranging from Nova Scotia to Florida and the West Indies and west to eastern Texas. In order toelucidate interspecific phylogenetic relationships in Rhexia, we analyzed four independently evolving data sets:the internal transcribed spacer (ITS) regions of ribosomal nuclear DNA, the chloroplast region trnC-D, thenuclear gene that encodes chloroplast-expressed glutamine synthetase (ncpGS), and 37 morphological characters.The ITS and chloroplast topologies differed in the phylogenetic positions of several taxa (R. lutea, R. nashii, andR. salicifolia), and for R. cubensis, R. lutea, R. nashii, R. parviflora, and R. salicifolia, we recovered multiplealleles of ncpGS, which is single copy in most species of Rhexia, indicating that these individuals may be ofhybrid origin. Among the remaining taxa, we identified three major clades: R. petiolata, R. mariana, and R.virginica. We show evidence of a hybrid origin of R. nashii, with parentage from the R. mariana and R. virginicalineages.

Keywords: hybrid origin, Melastomataceae, ncpGS, phylogeny, Rhexia, trnC-D.

Introduction

In the large, pantropical family Melastomataceae (ca. 4500species in 150 genera), Rhexia L. is unique in having a tem-perate distribution (Renner 1993). The showy pink flowersof Rhexia, the ‘‘meadow beauties,’’ are a common sight alongroadsides and in various open habitats in eastern North Amer-ica, ranging from Nova Scotia to Florida and the West Indies(Cuba, Hispaniola, and Puerto Rico) and west to eastern Texas.The genus is centered in the southeastern coastal plain, with10 of the 11 currently recognized species (all but R. aristosa)occurring in Florida (Kral and Bostick 1969; W. S. Judd, G. M.Ionta, personal observations based on label data from herbar-ium specimens at FLAS, 2005). The species of Rhexia areplants of poorly drained, acidic, often sandy soils and are com-mon constituents of wet meadows, pine flat-woods, and road-side ditches. Most are early successional species that tend toinhabit areas that have been disturbed by processes such asburning or land clearing (Kral and Bostick 1969).Rhexia has been treated in the Generic Flora of the South-

eastern United States (Wurdack and Kral 1982), and taxo-nomic revisions of the genus have been published by James(1956) and Kral and Bostick (1969). Several apomorphiccharacters define the genus, including urceolate hypanthia,ovary locules opposite the petals (instead of alternating withthem), glabrous ovaries, anthers that are unilocular at matu-rity, and the loss of pedoconnectives (Renner 1993); thus,Rhexia has traditionally been treated as a monogeneric tribe,

Rhexieae. Recent higher-level DNA-based phylogenetic analy-ses, however, show with strong support that Rhexia is nestedwithin Melastomeae, a clade characterized by cochleate seeds(Clausing and Renner 2001). Rhexia is likely closely relatedto Arthrostemma Pav. ex D. Don, a relationship supported bymolecular data (Clausing and Renner 2001) and morphology:Rhexia and Arthrostemma share four-merous flowers, hypan-thia with glandular hairs, and strongly costate-tuberculateseeds (Whiffin and Tomb 1972; W. S. Judd, G. M. Ionta, per-sonal observation, 2005).Rhexia species are herbaceous to suffrutescent perennials,

easily recognized by their opposite leaves with acrodromousvenation (typical for Melastomataceae) and showy flowers withpink to magenta petals (in R. parviflora and some populationsof R. mariana var. mariana, white, and in R. lutea, yellow)borne at the apex of an urceolate hypanthium. The flowers ofRhexia are radially symmetrical except for the stamens: shortlybefore anthesis, the anthers twist counterclockwise on their fila-ments, coming to lie roughly parallel to one another and clus-tered toward the base of the flower. The unilocular anthers openby single apical pores. The seeds of most Rhexia are cochleate,a feature that supports the placement of the genus within thetribe Melastomeae. Rhexia alifanus is the exception, as it hasmuch larger, wedge-shaped, noncochleate seeds.James (1956) recognized two main groups within Rhexia,

based mainly on the size and shape of their anthers: thosewith straight, ascending anthers at most 2 mm long (R. petio-lata, R. nuttallii, and R. lutea) were placed in series A, and asecond group with descending, curved anthers 4–11 mm inlength (R. alifanus, R. mariana, R. virginica, R. salicifolia,R. cubensis, R. aristosa, and R. nashii) were placed in seriesB. Rhexia parviflora, which has slightly curved anthers of an

1 Author for correspondence; e-mail gionta@ufl.edu.

Manuscript received September 2006; revised manuscript received March

2007.

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intermediate length (3.5 mm), has been grouped with the long-anther species, but James (1956) suggested that this taxonmay share affinities with both groups. James (1956) and Kraland Bostick (1969) noted that those species with short an-thers tend to exhibit a uniform morphology throughout theirranges and are less weedy, more prone to self-fertilization,and less likely to hybridize. In contrast, they hypothesizedthat the long, curved anthers of the members of series B pro-mote outcrossing, resulting in species that are weedier andmore likely to hybridize and that exhibit greater intraspecificvariation. This variation is exemplified in R. mariana, thewidest-ranging species in the genus, which is highly variablein flower color (from white to deep lavender to rose laven-der), leaf width, and hypanthium size. Kral and Bostick(1969) noted that plants of R. mariana cloned from the sameparental stock and grown under different light conditionsvary greatly in leaf width, and they postulated that at leastsome of this variation may be environmentally induced. Intra-specific phenoplasticity has led to the description of severalvarieties of R. mariana, three of which are currently recog-nized. Rhexia alifanus is somewhat enigmatic: while its long,curved anthers clearly place it in series B, its noncochleateseeds and vertically oriented, flattened, isofacial leaves distin-guish it from all other species of Rhexia.Polyploidy and hybridization, both of which are known to

occur in Rhexia, complicate species delimitation and effortsto elucidate interspecific relationships. Kral and Bostick (1969)published chromosome counts of Rhexia that showed the fol-lowing: six species are consistently diploid (R. petiolata, R.nuttallii, R. alifanus, R. parviflora, R. salicifolia, and R. aris-tosa: n!11); four possess individuals that form part of a poly-ploid series (R. mariana: n!11, 22; R. cubensis: n!11, 22, 33;R. nashii: n!22, 33; R. virginica: n!11, 22); and two may con-sist entirely of polyploids (R. lutea: n!22; R. nashii, n!22, 33).In all cases they were unable to detect morphological differ-ences between diploid and polyploid individuals of the samespecies. In their field observations of Rhexia, Kral and Bostickobserved naturally occurring hybrid swarms involving R. mari-ana var.mariana plus R. nashii, R. virginica, or R. salicifolia.Inference of phylogenetic relationships among species uti-

lizing data from a single gene/region can produce misleadingresults due to processes such as the rapid concerted evolutionof the ribosomal DNA region (Sanderson and Doyle 1992)and interspecific hybridization followed by repeated back-crossing (Doyle 1992; Soltis and Kuzoff 1995; Wendel et al.1995; Yoo et al. 2002). In order to elucidate interspecificphylogenetic relationships in Rhexia, we analyzed four inde-pendently evolving data sets: the internal transcribed spacer(ITS) regions of ribosomal nuclear DNA, the chloroplast re-gion trnC-D (trnC-ycf6-psbM-trnD), the nuclear gene thatencodes chloroplast-expressed glutamine synthetase (ncpGS),and 37 morphological characters.

Material and Methods

Taxon Sampling

The 11 currently recognized species of Rhexia (as per Kraland Bostick 1969) and two outgroup taxa were included in

this study (table 1). Rhexia mariana is represented by R. ma-riana var. mariana. Outgroup choice was based on previouslypublished higher-level phylogenetic analyses (Clausing andRenner 2001; Renner and Meyer 2001) showing Arthrostemmaand Dissotis to be closely related to Rhexia. Multiple acces-sions (no. in parentheses) of R. alifanus (3), R. aristosa (2),R. cubensis (2), R. lutea (2), R. mariana (2), R. petiolata (2),R. salicifolia (2), and R. virginica (3) were sequenced for ITSin order to assess intraspecific variation. In all cases, differentaccessions of the same species had identical ITS sequences,and thus one exemplar of each species was included in ouranalyses.For most Rhexia species, we extracted genomic DNA from

silica-dried leaves field-collected for this study. Leaf materialof R. aristosa was obtained from plants grown from cryopre-served shoot tips at the Center for Research of EndangeredWildlife, Cincinnati Zoo and Botanical Garden. Additionalsamples of R. aristosa and R. cubensiswere obtained from her-barium specimens. Dissotis rotundifolia genomic DNA wasextracted from a horticultural specimen grown at the Univer-sity of Florida Department of Botany greenhouse. GenomicDNA was isolated using a modification of the CTAB extrac-tion protocol outlined by Doyle and Doyle (1987). ExtractedDNA was purified using Qiagen QIAQuick columns (Qiagen,Santa Clarita, CA), following the manufacturer’s protocol, inorder to remove secondary compounds that might inhibit am-plification.

DNA Amplification and Sequencing

Ribosomal DNA was amplified in 50-mL reactions withthe primers 17SE and 26SE of Sun et al. (1994), using atouchdown thermal cycling program with an initial annealingtemperature of 76"C, decreasing 1"C per cycle for 15 cycles,followed by 15 cycles at 61"C. With difficult samples (e.g.,herbarium specimens), ITS-1 and ITS-2 were amplified sepa-rately, using the primers and protocol of Blattner (1999). ThetrnC-D region of chloroplast DNA was amplified and se-quenced using the primers and protocol of Demesure et al.(1995). The nuclear gene region ncpGS was amplified using theprimers Rhex839 (ACCAAYGGAGTCATGC) and Rhex1056(CWTCTTCCCTCATGCTCTTYGT), which were modifiedfor this study from the primers GScp687F and GScp1056R ofYockteng and Nadot (2004). Amplification of ncpGS was per-formed in 50-mL reactions with 4.0 mmol/L of MgCl2. An ini-tial 10 min denaturation at 95"C was followed by 34 cycles of1 min at 95"C, 1 min at 50"C, 1 min at 72"C, and a final exten-sion of 7 min at 72"C.Products of polymerase chain reactions (PCRs) were puri-

fied using Qiagen QIAQuick columns following the manufac-turer’s protocol. Primers used for cycle sequencing were thesame as those used in PCR amplification with the addition offour internal primers for the trnC-D region: ycf6R, ycf6F,psbMR, and psbMF (see Shaw et al. 2005). Sequencing re-actions were performed using Applied Biosystems Big Dyereagents. All sequencing was done on Applied Biosystems(Foster City, CA) ABI 377 sequencers at the InterdisciplinaryCenter for Biotechnology Research, University of Florida. Se-quences were manually edited and assembled using Sequencher3.0 (Gene Codes Corporation, Ann Arbor, MI).

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Consensus sequences were visually aligned using SeAl 2.0(Rambaut 1996), and regions of ambiguous alignment wereidentified and excluded from analyses. Directly sequencedPCR products of ribosomal DNA for R. nashii and R. parvi-flora and ncpGS sequences of Arthrostemma ciliatum, D. ro-tundifolia, R. cubensis, R. parviflora, R. nashii, R. salicifolia,and R. lutea contained multiple polymorphic loci. In order toidentify allele sequences, we cloned the PCR products forthese taxa. Molecular cloning of amplification products wasperformed at the Interdisciplinary Center for BiotechnologyResearch using Invitrogen (Carlsbad, CA) TOPO-TA kits. Wethen sequenced 6–10 clones per individual.

ncpGS and PCR Recombinants

Directly sequenced PCR products of ncpGS for seven taxa(A. ciliatum, D. rotundifolia, R. cubensis, R. parviflora, R.nashii, R. salicifolia, and R. lutea) were heterozygous at sev-eral loci; we subsequently cloned PCR products of these taxabut failed to identify the allele sequences for all but R. nashiiand A. ciliatum (two alleles recovered for each) due to diffi-culties differentiating PCR artifacts (i.e., PCR recombinants)from ‘‘real’’ sequences. Given the documented ploidy level ofthese Rhexia species (diploid, tetraploid, and in one casehexaploid), one would expect at most six different alleles;however, in all but R. nashii and A. ciliatum, we recoveredeight to 10 different ncpGS alleles, leading us to conclude

that these might represent recombinant DNA artifacts. Wesubsequently analyzed a pruned data set including those taxathat are homozygous for ncpGS (clean sequences, no poly-morphic loci) that was comprised of six species of Rhexia(R. petiolata, R. nuttallii, R. alifanus, R. aristosa, R. virginica,and R. mariana var. mariana) and one outgroup taxon (A.ciliatum). The two alleles of ncpGS we identified for A. cilia-tum differ by only three base pairs. Preliminary analysesshowed that using either cloned sequence alone resulted inthe same topology as analyses including both clones. As a re-sult, we randomly chose one cloned A. ciliatum sequence foruse in subsequent analyses.

Morphological Characters

Herbarium specimens from the Florida Museum of Natu-ral History (FLAS) and the University of North Carolina(UNC) were examined. Thirty-seven phenotypic characters(table 2) were measured on 68 specimens (including thoseused for molecular analyses; see table 1), representing the11 species of Rhexia, A. ciliatum, and D. rotundifolia. Char-acter scorings for phenotypic characters, i.e., morphologicaland anatomical (hereafter referred to as ‘‘morphological char-acters’’), are based on the authors’ observations of herbariumand living material, supplemented by information obtainedfrom published revisionary and monographic studies (James1956; Kral and Bostick 1969).

Table 1

Voucher Information for Specimens Included in DNA-Based Cladistic Analysis of Rhexia

GenBank accession nos.a

Taxon Voucher ITS trnC-D ncpGS

Arthrostemma ciliatum Pav. ex D. Don Oaxaca, Mexico; Penneys 1436 (FLAS) DQ985619 DQ985644 DQ985657,DQ985658

Dissotis rotundifolia (Sm.) Triana Alachua Co., FL; cult. Ionta 391 (FLAS) DQ985620 DQ985645R. alifanus Walter Nassau Co., FL; Ionta 227 (FLAS) DQ985621R. alifanus Walter Alachua Co., FL; Ionta 413 (FLAS) DQ985622 DQ985646 DQ985659R. alifanus Walter St. Johns Co., FL; J. R. Abbott 14892 (FLAS) DQ985623R. aristosa Britton Bladen Co., NC; Bostick 337-1 (NCU) DQ985624R. aristosa Britton No voucher; tissue culture at Cincinnati Zoo DQ985625 DQ985647 DQ985660R. cubensis Griseb. Gulf Co., FL; Ionta 400 (FLAS) DQ985626 DQ985648R. cubensis Griseb. Franklin Co., FL; Bostick 388-1 (NCU) DQ985627R. lutea Walter St. Johns Co., FL; J. R. Abbott 14882 (FLAS) DQ985628 DQ985649R. lutea Walter Liberty Co., FL; Zomlefer 802 (FLAS) DQ985629R. mariana L. Alachua Co., FL; Ionta 148 (FLAS) DQ985630R. mariana L. Liberty Co., FL; Ionta 397 (FLAS) DQ985631 DQ985650 DQ985661R. nashii Small Franklin Co., FL; Ionta 398 (FLAS) DQ985632,

DQ985633DQ985651 DQ985662,

DQ985663R. nuttallii C. W. James St. Johns Co., FL; J. R. Abbott 14883 (FLAS) DQ985634 DQ985652 DQ985664R. parviflora Chapm. Liberty Co., FL; Ionta 395 (FLAS) DQ985635,

DQ985636DQ985653

R. petiolata Walter Alachua Co., FL; Ionta 147 (FLAS) DQ985637 DQ985654 DQ985665R. petiolata Walter Bay Co., FL; Ionta 402 (FLAS) DQ985638R. salicifolia Kral and Bostick Leon Co., FL; Ionta 407 (FLAS) DQ985639 DQ985655R. salicifolia Kral and Bostick Bay Co., FL; J. R. Abbott 14995 (FLAS) DQ985640R. virginica L. Madison Co., FL; Ionta 202 (FLAS) DQ985641R. virginica L. Liberty Co., FL; Ionta 396 (FLAS) DQ985642 DQ985656 DQ985666R. virginica L. Pulaski Co., KY; J. R. Abbott 17408 (FLAS) DQ985643

a Cloned specimens are represented by more than one GenBank number.

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Morphological characters used in this study were readilydivisible into discrete states, thus arbitrary decisions relatingto state delimitation (Stevens 1991) were avoided. Characterswith abundant infrataxon variation and those that could notbe delimited into discrete states were excluded, e.g., leaf lengthand width, inflorescence architecture, hypanthium length, ca-lyx length, petal length and width, ovary position, and cap-sule length. Multistate characters were treated as unordered(table 2). Species varying in particular characters were scoredas ‘‘?’’; characters coded as both 0/1 within a terminal taxonare denoted with a ‘‘v,’’ and those coded as both 1/2 are de-noted with a ‘‘b’’ in table 3.

Phylogenetic Analyses

Phylogenies were estimated using maximum parsimony;branch and bound searches were conducted using PAUP* 4.0b10(Swofford 1999). All characters were unordered and equallyweighted, gaps were treated as missing data, and the MulTreesoption was in effect. Relative support for clades was evaluatedusing 1000 bootstrap replicates (Felsenstein 1985) with 10 random-addition replicates and tree bisection and reconnection branchswapping.Congruence of separate data sets was assessed using a con-

ditional combination approach (Huelsenbeck et al. 1996), as

Table 2

Morphological and Anatomical Characters Used in Cladistic Analysis of Rhexia (Melastomataceae: Melastomeae)

1. Stolons present (0), absent (1).2. Rootstock nontuberous (0), tuberous (1).3. Stems (in cross section) more or less quadrangular (0); stems with two broad convex faces alternating with two narrower concave faces (1).4. Stems with scattered elongate multicellular hairs (0); stems with such hairs restricted to nodal line (1); stems lacking such hairs (2). Note: Allspecies have minute multicellular hairs at the nodal line.5. Stems lacking longitudinal wings or with short wings (0); stems with four well-developed wings (1).6. Leaves ovate to broadly ovate or broadly elliptic, or narrowly ovate/elliptic (0); leaves more or less linear (1).7. Leaves conspicuously petiolate (0); leaves sessile to short, inconspicuously petiolate (1).8. Leaves with indumentum of elongate multicellular hairs (0); leaves glabrous (1).9. Hairs of leaves glandular headed (0); hairs of leaves nonglandular (1).10. Leaves held parallel with the ground (0); leaves held perpendicular to the ground (1).11. Leaves bifacial (0), unifacial (1).12. Stomata mainly on abaxial lamina surface (0); stomata more or less equally distributed (1).13. Stomatal density high, ca. 529 to over 882/mm2 (0), low, ca. 235/mm2 (1).14. Leaf with palisade chlorenchyma only adaxially (0); leaf with palisade chlorenchyma both adaxially and abaxially (1).15. Epidermal cells of adaxial leaf surface more or less flat (0); epidermal cells bullate (1).16. Leaves flat (0); leaves slightly to strongly V shaped (1).17. Inflorescence few flowered, the bracts similar to the leaves (0); inflorescence variably cymose, the bracts narrow, distinctly different fromthe leaves (1).

18. Sepals acute to acuminate (0); sepals obtuse to rounded (1).19. Hypanthium not constricted to only moderately constricted, not as following (0); strongly and shortly constricted, with neck strongly flaringdistally (1).

20. Hypanthium with elongate multicellular hairs (0); hypanthium glabrous (1).21. Hairs of hypanthium highly, 6stellate branched (0); hairs glandular headed, more or less scattered, or hairs lacking (1); hairs nonglandular,clustered toward apex of hypanthium (2).

22. Hypanthium neck shorter than or more or less equaling the body (0); hypanthium neck longer than the body (1).23. Petals 5 (0); petals 4 (1).24. Petals glabrous but with gland-headed hairs along margin (0); petals with multicellular, often gland-headed hairs (1); petals glabrous except forapical hair (2).

25. Petals at anthesis horizontally spreading, plane (0); petals at anthesis ascending, the adaxial surface concave (1).26. Petals deep to pale pink/purple or white (0); petals yellow (1).27. Stamens 6 dimorphic (0); stamens monomorphic (1).28. Anthers 4–11 mm long, usually curved (0); anthers 6 2 mm long, straight (1). Note: R. parviflora is coded as ‘‘v’’ as it has the intermediatevalue of 3–3.5 mm anthers.

29. Anthers more or less smooth (0); anthers minutely papillose (1).30. Mature anthers bilocular (0), unilocular (1).31. Dorsal connective appendages lacking or very short (0); dorsal connective appendage elongated, narrowly conical filiform, to .4 mm long (1).32. Pedoconnective present, i.e., stamen with anther connective basally 6 prolonged, with a conspicuously bifid to elongate-flattened ventralappendage (0); pedoconnective absent (1).

33. Ovary with multicellular hairs (0); ovary glabrous (1).34. Seeds cochleate (0); seeds noncochleate (1).35. Testa surface smooth (0); testa surface costate tuberculate (1).36. Testa surface of regular to irregular costae (0), papillae in lines (1), uniformly papillose (2).37. Capsule nonurceolate (0); capsule urceolate (1).

Note. Presumed plesiomorphic states are listed first (0), followed by apomorphic states (1, 2, etc.). Polarization assumes rooting with Disso-tis and Arthostema; multistate character states are considered unordered. (For characters varying between Dissotis and Arthrostemma, the statein Dissotis is coded as 0, and for characters in which Dissotis is polymorphic or unknown, the 0/1 states are arbitrarily coded.)

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described by Soltis et al. (2001). Each data set was initiallyanalyzed separately; we then compared the resultant topolo-gies and searched for instances of strongly supported contra-dictory clades. Following the approach of Mason-Gamer andKellogg (1996), we established a bootstrap value cutoff of 70%for assessing strong conflict between data sets.The following analyses were performed: (1) ITS alone, in-

cluding the 11 species of Rhexia (two cloned sequences eachof R. parviflora and R. nashii) and two outgroup taxa; (2)chloroplast data alone, including the 11 species of Rhexiaand two outgroup taxa; (3) a pruned ncpGS data set (see ex-planation above); (4) morphological data, including all 11species of Rhexia and two outgroups; (5) analyses of pruneddata sets (ITS, trnC-D and morphology, and molecular andmorphological data combined); and (6) separate analyses ofpruned data sets (ITS, trnC-D, and ncpGS), with the additionof R. nashii sequences.Morphological character-state transformations were mapped

onto one of the most parsimonious trees (MPTs) from the un-pruned morphological analysis using the output of PAUP*and MacClade 4.05 (Maddison and Maddison 2001). Mor-phological characters were also traced onto the MPT result-ing from the pruned total evidence analysis.

Results

ITS Data Set

The aligned ITS data set, comprising the 11 species of Rhexia(two clones of R. parviflora and R. nashii were included),Arthrostemma ciliatum and Dissotis rotundifolia, is 768 bplong with 188 variable and 70 parsimony-informative char-acters. A branch-and-bound search resulted in one MPT(fig. 1A) of 256 steps (Consistency Index "CI# ! 0:887; Reten-tion Index "RI# ! 0:788; Rescaled Consistency Index "RC# !0:699). The monophyly of Rhexia is supported with 100% BS(bootstrap support). Within Rhexia, there is moderate supportfor two major clades: R: alifanus$ R: nuttallii$ R: petiolata

(R. petiolata clade; BS 72%), and R: lutea$ R: cubensis$ R:mariana var. mariana $ R: parviflora (R. mariana clade; BS80%). There is weak support (BS 57%) for a clade comprisedof R: virginica$ R: nashii$ R: aristosa$ R: salicifolia (R.virginica clade). The R. petiolata clade contains two of thethree short-anther species, R. petiolata and R. nuttallii, whichare included in series A of James (1956). Sister to these twospecies is R. alifanus, which is included in series B of James(1956) because of its long anthers, but which also has manyfeatures unique among species of Rhexia. Sister to the R. petio-lata clade is a clade containing two subclades: the R. marianaclade and the R. virginica clade. Rhexia lutea, the other short-anther species, is in the R. mariana clade.

Chloroplast Data Set

The aligned plastid data set, comprising the 11 species ofRhexia, A. ciliatum and D. rotundifolia, is 2245 characterslong with 308 variable characters, 86 of which are parsimonyinformative. A branch and bound search resulted in threeMPTs (fig. 1B, strict consensus) of 351 steps (CI ! 0:926;RI ! 0:836; RC ! 0:775). The monophyly of Rhexia is againsupported (BS 100%). Rhexia lutea, a short-anther species(series A of James 1956), is sister to the rest of the genus, withstrong support. Among the remaining Rhexia species, there isgood support for three major clades. A R. petiolata clade con-tains R. nuttallii, R. petiolata, and R. alifanus (BS 89%), mir-roring the results of the ITS analysis. As in the ITS analysis,sister to this R. petiolata clade is a clade containing two well-supported subclades: a clade containing R. mariana var.mariana(BS 87%) and a clade containing R. virginica (BS 96%). TheR. mariana and R. virginica clades recovered in the trnC-D to-pology differ from those of the ITS topology in three key ways.In the plastid analysis, R. lutea is sister to the rest of the genus,while in the ITS analysis, this species is part of the clade con-taining R. mariana var. mariana. Rhexia nashii and R. salicifo-lia, both part of the R. virginica clade in the ITS analysis, are inthe clade containing R. mariana var. mariana in the trnC-D

Table 3

Morphological Character-State Values for Taxa Used inCladistic Analysis of Rhexia

1 2 3Species 1234567890123456789012345678901234567

Dissotis rotundifolia 00000000v000?0000000000000000000000?0Arthrostemma ciliatum 1100000000000001100v100b0000000000100R. petiolata 1012001010000001001110121011010110121R. nuttallii 1012001000000001011010111011010110101R. lutea 1010011000000011101010121111010110111R. salicifolia 1100011001010101100010110010110110101R. parviflora 00v000000001000110001012001v111110101R. aristosa 1101111000010101100020110010110110101R. virginica 11001010000000011000101v0010110110101R. mariana 00100v1000000001100010120010111110101R. cubensis 01100110000100011000111v0010111110101R. nashii 0v10001000000001100v11110010111110101R. alifanus 10120011?01111001000101100100111110?1

Note. D. rotundifolia and A. ciliatum used as outgroups. ? ! unknown ornot applicable; v ! 0=1; b ! 1=2.

1059IONTA ET AL.—PHYLOGENETIC RELATIONSHIPS IN RHEXIA

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analysis. Finally, R. cubensis appears in the R. mariana cladein both analyses; however, its sequence is identical to thatof R. mariana var. mariana in the ITS analysis, while in thetrnC-D analysis it is closely allied with R. salicifolia (BS 100%).

ncpGS

The aligned ncpGS data set (pruned, comprising six Rhexiaspecies$ A: ciliatum) is 463 characters long, with 63 variable

and nine parsimony-informative characters. A branch andbound search resulted in three MPTs (see fig. 1C, strict consen-sus) of 71 steps (CI ! 0:944; RI ! 0:692; RC ! 0:653). As inall other analyses, Rhexia is monophyletic with 100% BS,and three main clades are supported: a R. petiolata clade(R: petiolata$ R: nuttallii$ R: alifanus; BS 79%), a R. virgin-ica clade (R: virginica$ R: aristosa) with little support (BS54%), and a clade containing only R. mariana. Relationshipsamong these clades are not resolved.

Fig. 1 Cladograms based on parsimony analyses of independent data sets. Numbers above branches indicate bootstrap values. Vertical barsindicate R. petiolata, R. mariana, and R. virginica clades for each analysis. A, MPT from the ITS analysis. B, Strict consensus tree from trnC-Danalysis. C, Strict consensus tree from pruned ncpGS analysis. D, One of two MPTs from analysis of morphological data set; arrows indicatenodes that collapse in strict consensus. Letters indicate putative synapomorphies: a ! 1, 16, 17, 21, 23, 24, 35; b ! 7, 27, 30, 32, 33, 37; c ! 3;d ! 19, 24(2), 25, 28; e ! 4%2&, 17(0); f ! 12, 31; g ! 1%0&, 29; h ! 24%2&; i ! 2, 22; j ! 2, 5, 29; k ! 6, 12, 14 (see table 1).

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Morphology

The morphological data set, comprising the 11 species ofRhexia, A. ciliatum, and D. rotundifolia, includes 37 variablecharacters, 24 of which are parsimony informative. A branchand bound search resulted in two MPTs of 59 steps (CI !0:595; RI ! 0:667; RC ! 0:463), which differ only in rela-tionships within the clade containing R. parviflora, R. marianavar. mariana, R. cubensis, and R. nashii. One tree supportsthe sister-group relationship of R. cubensis and R. parviflora,with R. mariana var. mariana sister to that pair followedby R. nashii, which is sister to the three above-mentionedspecies. The other tree (see fig. 1D) supports a R. marianavar. mariana$ R: parviflora clade, which is sister to R:cubensis$ R: nashii. DELTRAN optimizations are employedin figure 1D and in the following discussion, unless otherwisenoted.The monophyly of Rhexia (BS 96%) is supported by the fol-

lowing putative synapomorphies: sessile to shortly petiolateleaves (char. 7; lost in R. parviflora), monomorphic stamens(char. 27), unilocular anthers (char. 30), a lack of pedoconnec-tives (char. 32), glabrous ovaries (char. 33), and urceolate cap-sules (char. 37). The genus is morphologically distinctive; allof these features except the first are uniquely derived.Within Rhexia, only a few clades receive bootstrap sup-

port. The clade comprising R. petiolata, R. nuttallii, and R.lutea (BS 90%) is supported by the putative synapomorphiesof strongly and shortly constricted hypanthia (char. 19),petals concave and ascending at anthesis (char. 25), anthersstraight and ca. 2 mm long (char. 28), and petals glabrous ex-cept for an apical hair (char. 24-2). The first three synapo-morphies are unique for this clade. Glabrous petals arehomoplasious, having been lost in R. nuttallii and evolved in-dependently in R. mariana var. mariana and R. parviflora.Within this clade, the sister-group relationship between R.petiolata and R. nuttallii is moderately supported (BS 71%);these species have stems lacking multicellular hairs (char. 4-2)and reduced inflorescences with inflorescence bracts similar tothe leaves (char. 17-0). Glabrous stems are also present in R.alifanus. Rhexia aristosa is sister to R. salicifolia (BS 70%), aplacement supported by their more or less linear leaves (char.6) with stomata distributed on both surfaces (char. 12) andpalisade chlorenchyma present both adaxially and abaxially(char. 14); all three characters are homoplasious. These twospecies are consistently placed as sister to R. virginica by theirtuberous rootstocks (char. 2), stems with four well-developedlongitudinal wings (char. 5), and minutely papillate anthers(char. 29). Stem winging is a uniquely derived feature (lost inR. salicifolia), but the other two characters evolve in paral-lel elsewhere on the tree: tubers in R. cubensis and R. nashiiand papillose anthers in the R: parviflora$ R: mariana var.mariana$ R: cubensis$ R: nashii clade.Rhexia alifanus is highly autapomorphic yet nested within

the phylogenetic structure of the genus. Particularly distinc-tive apomorphies include glabrous leaves (char. 8), flat andunifacial leaves (chars. 16-0 and 11) with low stomatal densi-ties (char. 13), and noncochleate seeds (char. 34) with a smoothtesta (char. 35-0). In the morphological analysis, R. alifanusconsistently appears as sister to the R: parviflora $ R:mariana var. mariana$ R: cubensis$ R: nashii clade, but

this position is not well supported. Putative synapomorphieslinking R. alifanus with this clade include leaf stomatal dis-tribution (char. 12) and anthers with elongated, narrowlyconical-filiform dorsal connective appendages (char. 31).Rhexia parviflora, R. mariana var. mariana, R. cubensis,

and R. nashii form a clade on the basis of their nontuberousrootstock (char. 2-0) and minutely papillose anthers (char.29). Papillose anthers also characterize R. salicifolia, R. aris-tosa, and R. virginica, an apparent parallelism based on thetopologies of the two morphology-based trees.

Pruned Data Sets

Analyses of pruned ITS and trnC-D data sets (not shown)resulted in topologies congruent to those recovered in analysesof the pruned ncpGS data set (fig. 1C); the three clades recov-ered in the ncpGS data set are again present. Analysis of thepruned morphological data set resulted in three MPTs (notshown) with excellent support (BS 96%) for R: nuttallii$ R:petiolata and moderate support for R: aristosa$ R: virginica(BS 76%) but <50% BS for other relationships within Rhexia.Visual inspection of the topologies resulting from separateanalyses of the pruned data sets revealed no strongly sup-ported incongruence, so we then combined these pruned datasets (the three molecular regions plus morphology) in a total-evidence analysis. The combined analysis of the four pruneddata sets (ITS$ trnC-D$ ncpGS$morphology) resulted in asingle MPT (fig. 2) with strong support for the following clades:(1) R. petiolata clade (R: nuttallii$ R: petiolata$ R: alifanus;

Fig. 2 Most parsimonious tree from combined pruned data set ofthree molecular data sets plus morphology. Numbers above branchesindicate bootstrap values. Vertical bars indicate R. petiolata, R. mari-ana, and R. virginica clades.

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BS 99%); (2) R. virginica clade (R: virginica$ R: aristosa; BS99%); and (3) R. mariana clade (an isolated species, sister tothe R: virginica clade; BS 96%). As in the separate ITS andtrnC-D analyses (complete and pruned data sets), the R. petio-lata clade is sister to the R. mariana clade and the R. virginicaclade.The monophyly of Rhexia is supported by the morpho-

logical synapomorphies identified in the morphology-basedanalysis (see above). The R: nuttallii$ R: petiolata clade issupported by the following putative synapomorphies: reducedinflorescences with bracts similar to leaves (char. 17-0), hy-panthia strongly and shortly constricted with necks stronglyflaring distally (char. 19), petals ascending at anthesis (char.25-0), and anthers straight and ca. 2 mm long (char. 28). Thehighly autapomorphic R. alifanus is supported as sister tothe R: nuttallii$ R: petiolata clade by the synapomorphy ofstems lacking multicellular hairs (char. 4-2). The presence ofstems with two broad convex faces alternating with two nar-rower concave faces is an additional synapomorphy underDELTRAN optimization. The R: virginica clade is supportedby tuberous rootstocks (char. 2), quadrangular stems (char.3-0, if ACCTRAN optimization is employed), and stems withfour well-developed longitudinal wings (char. 5). The positionof R. mariana var. mariana as sister to the R: virginica clade issupported by the presence of minutely papillose anthers (char.29). It is noteworthy that this character was mapped as homo-plasious on the morphology-based cladograms, but here, in thecombined DNA$morphology tree, it is resolved as a uniqueand nonhomoplasious synapomorphy. A morphological char-acter unique to R. mariana var. mariana (in this pruned taxonset) is the presence of stolons (char. 1-0).

Addition of Rhexia nashii to Pruned Molecular Data Sets

The topologies resulting from separate analyses of thepruned data sets$ R: nashii were congruent with those thatdid not include R. nashii sequences, with the exception of theITS tree, in which R. virginica and R. aristosa no longer forma clade. Analysis of the pruned ITS data set including the twoR. nashii sequences resulted in three MPTs (fig. 3A, strict con-sensus). One R. nashii ITS sequence type is strongly supportedas sister to R. virginica (BS 94%), while the other is sister toR. mariana var. mariana, but with weak support (BS 57%).Analysis of the pruned trnC-D data set$ R: nashii resulted inthree MPTs (fig. 3B, strict consensus). In this case, R. nashii isstrongly allied with R. mariana var. mariana (BS 100%). Ad-dition of the two ncpGS alleles of R. nashii identified for thisstudy to the ncpGS pruned data set resulted in three MPTs(fig. 3C, strict consensus). One allele of R. nashii is sister to R.virginica (BS 71%), and the other is sister to R. mariana var.mariana (BS 100%). One ncpGS allele of R. nashii is identicalto the ncpGS sequence of R. mariana var. mariana.

Discussion

Major Clades

We identified three major clades within Rhexia: the R. pe-tiolata clade (R: petiolata$ R: nuttallii$ R: alifanus), the R.mariana clade (R. mariana var. mariana), and the R. virginica

clade (R: virginica$ R: aristosa) (figs. 1A–1C, 2). In the ITS,trnC-D, and combined, pruned analyses, the R. petiolataclade is sister to the R. mariana clade and R. virginica clade.Circumscription of these clades differs in the topologies re-sulting from analyses of the complete chloroplast and ribo-somal DNA data sets, and these clades are not present in thetrees recovered from analysis of the complete morphologicaldata set. Removal from subsequent analyses of those taxa (R.nashii, R. cubensis, R. salicifolia, R. lutea, and R. parviflora)that appear in different clades in the chloroplast and ITSdata sets, or that are heterozygous for ncpGS, led to congru-ent topologies for ITS, trnC-D, and ncpGS. The pruned mor-phological data set supports the alliance of R. petiolata andR. nuttallii (BS 96%), but the relationships among the remain-ing taxa are unresolved. The total-evidence analysis (threemolecular regions$morphology) of the pruned data sets re-sulted in one MPT with excellent support for these threeclades (fig. 2).

The Rhexia petiolata Clade

The topologies resulting from the analyses of the threemolecular data sets and the pruned, combined data sets (mo-lecular alone and molecular$morphology) reveal a well-supported clade containing R. petiolata, R. nuttallii, and R.alifanus (see figs. 1A–1C, 2). Rhexia petiolata and R. nuttalliiare well supported as sister taxa by molecular and morpholog-ical data with the exception of the chloroplast marker: in theanalyses of the trnC-D data sets, R. nuttallii, R. petiolata, andR. alifanus form a well-supported clade, but relationshipsamong the three taxa are unresolved. The morphological datasupport R. petiolata and R. nuttallii as sister taxa but do notsupport a close relationship between these two taxa and R.alifanus (fig. 1D).Rhexia petiolata and R. nuttallii share features, including

short anthers and cupped petals; their well-supported rela-tionship as sister taxa in the majority of our analyses con-firms the hypotheses of James (1956) and Kral and Bostick(1969) that these two taxa are closely related. Analyses ofmorphological data (complete and pruned data sets), in keep-ing with evidence from the three independently evolving mo-lecular data sets and the total-evidence analysis, support thehypothesis that R. petiolata and R. nuttallii are sister taxa(fig. 1D). The two taxa are united by four morphologicalsynapomorphies (see results of morphology-based analysis).Rhexia alifanus is well supported as sister to R: petiolata$

R: nuttallii by the molecular data; however, this relationshipis difficult to correlate with the morphology of these taxa,and analyses of the morphological data sets do not supportthis grouping. Both MPTs recovered from the analysis of thecomplete morphological data set (one MPT shown in fig. 1D)place R. alifanus in a clade with other long-anther taxa, suchas R. mariana var. mariana and R. nashii, but without signifi-cant BS. Topologies resulting from the analysis of the prunedmorphological data set (removing taxa of putative hybrid origin)were largely unresolved; however, a R: alifanus$ R: petiolata$R: nuttallii clade was present in two of three MPTs. Rhexia ali-fanus has long anthers and belongs to series B of James (1956),whereas R. petiolata and R. nuttallii have short anthers and be-long to series A. In addition, R. alifanus has features that set it

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apart from R. petiolata, R. nuttallii, and the other members ofthe genus, including large, wedge-shaped noncochleate seeds(most members of the tribe Melastomeae have cochleate seeds),a conspicuously glandular-hairy hypanthium, and vertically ori-ented isofacial leaves with two double-palisade layers and awell-developed waxy cuticle. The uniqueness of R. alifanus is re-flected in the statement by Kral and Bostick (1969, p. 414) thatthis ‘‘most clearly marked species in the genus may actuallyhave closer affinities with some tropical-American genus.’’ Ouranalyses of both molecular and morphological data clearly

show R. alifanus to be a member of the genus Rhexia, and themolecular and total-evidence analyses overwhelmingly support aclose relationship of this enigmatic species to R. petiolata andR. nuttallii. It is evident (figs. 1D, 2) that the unique morphol-ogy of R. alifanus is likely the result of an accumulation of anumber of autapomorphies subsequent to its split from the R:petiolata$ R: nuttallii clade. Rhexia alifanus, with its long tap-root, tends to grow on drier sites than other species in the genus.Kral and Bostick (1969) suggest that the unique vegetative fea-tures of R. alifanus may represent adaptations to a more xeric

Fig. 3 A–C, Strict consensus trees based on parsimony analyses of pruned independent molecular data sets with the addition of R. nashiisequences. Numbers above branches indicate bootstrap values for clades containing R. nashii. A, ITS. B, trnC-D. C, ncpGS. D, Hypothesizedorigin of R. nashii superimposed on the MPT from the analysis of pruned molecular and morphological data sets combined.

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habitat. The R. petiolata clade, as a whole, is supported by arather cryptic character: these taxa lack the elongate, multicellu-lar hairs that are present on the stems of other species of Rhexia,either scattered in internodal regions or restricted to nodal lines.

The Rhexia virginica Clade

Rhexia virginica and R. aristosa form a clade in most anal-yses; exceptions are the analyses of the pruned ITS data set $R: nashii and the pruned morphological data set. In somecases, other taxa are present in this clade; topologies from thefollowing analyses show an expanded R. virginica clade: theunpruned ITS data set (R: virginica$ R: aristosa$ R: nashii $R: salicifolia; fig. 1A), the pruned ncpGS data set$ R: nashii(R: virginica$ R: aristosa$ one ncpGS allele of R. nashii; fig.3C), and the unpruned morphological data set (R: virginica$R: aristosa$ R: salicifolia; fig. 1D). Removal of taxa poten-tially of hybrid origin (pruned data sets) results in congruencebetween the three molecular data sets; combined analyses(molecular$morphology) of pruned data sets result in strongsupport (BS 99%; fig. 2) for a R: virginica$ R: aristosa clade.The R: virginica$ R: aristosa clade is supported by the

presence of tuberous rootstocks, strongly winged stems, and(if ACCTRAN optimization is employed) quadrangularstems. Kral and Bostick (1969) noted that R. aristosa andR. virginica also typically possess ‘‘spongy-thickened’’ root-stocks. We excluded this character from our analyses due todifficulties assessing relative sponginess of roots in herbariummaterial and because this character seems to be of variabledevelopment; i.e., many specimens of these species lacked no-ticeable spongy root tissue. Though not included in our analy-ses, the ability to develop such spongy tissue toward the baseof the stems is another potential synapomorphy for this clade.

Rhexia mariana

Rhexia mariana, the ‘‘most abundant, wide-ranging andweediest’’ species in the genus (Kral and Bostick 1969, p.409), has a range that overlaps with the ranges of all otherspecies of Rhexia. This species is known to hybridize withseveral other species of Rhexia both in the field and in green-house experiments (Kral and Bostick 1969). In our analysesof the complete molecular data sets, R. mariana (representedin this study by R. mariana var. mariana) appeared within aclade distinct from the R. petiolata and R. virginica cladesdescribed above. Circumscription of the R. mariana cladediffered between the ITS and trnC-D topologies: in the ITStree (fig. 1A), Rhexia mariana var. mariana shows an affinitywith R. parviflora and R. cubensis, while in the trnC-D trees(fig. 1B), it is allied with these two species along with R.nashii and R. salicifolia.Our subsequent removal of those taxa (R. lutea, R. parvi-

flora, R. cubensis, and R. salicifolia; pruned data sets) withpolymorphic ncpGS sequences was an attempt to clearly un-derstand the relationships between the ‘‘core’’ species ofRhexia, i.e., those taxa that were stable among the completemolecular data sets and were not heterozygous for ncpGS.Multiple copies of this nuclear gene, which is biparentally in-herited and occurs as a single copy in most plants (Emshwillerand Doyle 1998), can suggest a possible hybrid origin in taxa

with more than one copy. In the pruned data sets, R. marianavar. mariana appeared alone, either as sister to the R. virginicaclade (in the ITS and trnC-D analyses) or in a polytomy withthe R. virginica and R. petiolata clades (ncpGS). The positionof R. mariana var. mariana in the pruned morphological analy-sis is unresolved. Total-evidence analysis of the pruned data sets(molecular$morphology; fig. 2) strongly supports R. marianavar. mariana as sister to the R: virginica$ R: aristosa clade.This phylogenetic placement is supported morphologically bythe minutely papillose anthers characteristic of these threespecies.

Evidence That Rhexia nashii Is of Hybrid Origin

Rhexia nashii appeared in incongruent but well-supportedpositions in the separate complete ITS and trnC-D topologies(fig. 1A, 1B). Nuclear ribosomal DNA is known to be subjectto concerted evolution, which can mask the full parentallineage of a hybrid taxon, and the chloroplast is maternallyinherited; thus, markers from this genome reflect only a por-tion of the lineage of a taxon. Single-copy nuclear genes arebiparentally inherited and represent an additional line of evi-dence for elucidating relationships in groups with compli-cated evolutionary histories. The ncpGS gene region is singlecopy in most taxa, and in contrast to ITS, it is not subject toprocesses such as concerted evolution.In an attempt to identify the parental lineages of the puta-

tive hybrid species R. nashii, we added the two ncpGS allelesthat we had identified for this taxon to an analysis of thepruned ncpGS data set. In the resulting topology (fig. 3C),one allele of R. nashii is sister to R. mariana var. mariana(BS 100%), and the other is sister to R. virginica (BS 71%).Addition of R. nashii sequences to the pruned ITS data set(see fig. 3A) resulted in one cloned sequence as sister to R.virginica (BS 94%) and the other as sister to R. mariana var.mariana, but with only 57% BS . In contrast, addition of R.nashii sequences to the pruned trnC-D data set (see fig. 3B) re-sulted in strong support (BS 100%) for R. nashii as sister to R.mariana var. mariana. These results indicate a possible hybridorigin for R. nashii, with the maternal parent derived from theR. mariana lineage and the paternal parent from the R. virginicalineage. One ncpGS allele sequence of R. nashii is identical toR. mariana var. mariana; the other differs from R. virginicaby only one base pair. It cannot, however, be assumed thatR. nashii is a direct hybrid of these two taxa, since the lackof divergence in ncpGS sequence data between these taxa isin keeping with the low number of parsimony-informativecharacters (only nine) in this data set. Rhexia nashii se-quences of ITS and trnC-D regions are quite different fromthose of R. virginica and R. mariana var. mariana, a reflectionof an overall greater variability in these gene regions in Rhexia.Therefore, we hypothesize that R. nashii is of ancient hybrid or-igin, with parents from the R. mariana var. mariana lineage andthe R. virginica lineage (fig. 3D).Rhexia nashii shares apomorphies with both R. mariana

var. mariana (the presence of stolons, stomata mainly on ab-axial leaf surface, and elongate dorsal anther appendages; seetables 2 and 3) and R. virginica (tuberous rootstocks andpetals with multicellular hairs; the latter condition is variablein R. virginica). The conjunction of stolons and tubers in the

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rootstock of R. nashii supports a hybrid origin of this species.In their current distribution, R. mariana var. mariana andR. virginica are sympatric. Kral and Bostick (1969) reportednaturally occurring hybrid swarms involving R. mariana var.mariana and R: virginica, but the hybrids they observed did notresemble R. nashii, and very few viable seeds were formed.This supports our hypothesis that R. nashii is of ancient hybridorigin from the R. mariana and R. virginica lineages ratherthan a recent hybrid of these two taxa.

Other Taxa with Ambiguous Placementsand PCR Recombination

Rhexia lutea, R. parviflora, R. cubensis, and R. salicifoliawere also excluded from the pruned data sets. As notedabove, these taxa appeared in different places in the overallITS and trnC-D topologies and/or had more than one copyof ncpGS (R. parviflora also had two copies of ITS, as did R.nashii, as discussed above). We were unsuccessful in separat-ing ‘‘real’’ ncpGS sequences from artifacts for these taxa dueto problems with Taq polymerase error and/or PCR recombi-nation. In PCR recombination, artifact sequences that com-bine the features of heterogeneous sequences are formed invitro when uncompleted PCR products act as primers in sub-sequent PCR cycles and anneal to different templates (Jansenand Ledley 1990; Bradley and Hillis 1997; Cronn et al.2002). Recombinants can account for a large proportion ofthe sequences in a PCR product: Jansen and Ledley (1990)found 25% of sequences to be recombinants, and Cronnet al. (2002) documented up to 89% recombinants, depend-ing on the gene system. Differentiating ‘‘real’’ sequences fromrecombinants can be difficult if not impossible, particularly ifthe diploid progenitors of hybrid taxa are not known a pri-ori. Emshwiller and Doyle (2002) were able to identify arti-fact sequences in Oxalis by comparing diagnostic nucleotidesand from phylogenetic context. We were unable to identifywith confidence the actual ncpGS sequences from among themany artifact sequences; thus, we can only speculate as tothe reasons for the incongruence between the ITS and trnC-Dtopologies. Here, we briefly discuss possible evolutionary his-tories of R. lutea and R. salicifolia.James (1956) placed R. lutea in series A due to its short an-

thers and cupped petals; this relationship is supported by ourmorphological analysis (fig. 1D). Like R. alifanus, R. luteahas features that set it apart from other species in the genus,the most distinctive being corolla color: R. lutea has yellowpetals, and all other species have purple, pink, or white petals.Kral and Bostick (1969, p. 388), citing additional differencesin this species based on chromatographic and anatomical

analyses, suggested that ‘‘it would appear that R. lutea in se-ries ‘A’ [of James] . . . may actually represent [a] distinct sec-tion.’’ Rhexia lutea came out in very different places in theITS and trnC-D analyses: as sister to the rest of the genus inthe trnC-D analysis (fig. 1B), and as sister to the R. marianaclade in the ITS analysis (fig. 1A). This incongruence, coupledwith multiple heterozygous ncpGS loci, suggests a possiblehybrid origin of this species. Rhexia lutea clearly shares fea-tures of the R. petiolata clade but does not appear in thisclade in either of the DNA-based analyses. This indicates thatthe features used by James to define series A (cupped petals,short anthers), while synapomorphies for R: petiolata$ R:nuttallii, may either be homoplasious (evolving in parallel inR. lutea) or were passed to R. lutea as a result of ancient hy-bridization with a member (either extinct or extant) of the R.petiolata clade. The placement of R. lutea as sister to the R.mariana clade in the ITS topology is intriguing: R. parviflora,with anthers of intermediate length, appears in this clade.Without an additional independently evolving line of evidence(i.e., correctly identified ncpGS alleles or another indepen-dently evolving marker), it will be difficult to ascertain the re-lationship of R. lutea to the rest of the genus.Rhexia salicifolia is sister to R. aristosa (as part of the R.

virginica clade) in the ITS topology (fig. 1A; BS 99%). In thetrnC-D analysis, it is supported as sister to R. cubensis aspart of the R. mariana clade (fig. 1B; BS 100%). Kral andBostick (1969, p. 404) noted similarities of this species toboth R. virginica and R. cubensis and reported that James‘‘was of the opinion that this taxon could perhaps have beenof hybrid origin between R. cubensis and R. virginica.’’ Ourevidence does not point to R. salicifolia as a direct hybrid ofthese taxa but suggests instead that the species may be of hy-brid origin, from parents of the R. aristosa lineage (R. virgin-ica clade) and the R. cubensis lineage (R. mariana clade). Asis the case with R. lutea (as well as R. parviflora and R. cu-bensis), elucidating the evolutionary history of R. salicifoliawill require either additional sequencing of ncpGS (after re-solving the problems involving PCR recombination) or theuse of another, independently evolving marker.

Acknowledgments

We thank Richard Abbott, Bernadette Plair, Darin Penneys,and Wendy Zomlefer for providing samples of Rhexia andArthrostemma; Louise Kirn of the U.S. National Forest Ser-vice for assistance with locating populations of R. parvifloraand R. salicifolia; Kent Perkins of FLAS for assistance withherbarium materials; and the staff and curator of NCU forherbarium loans.

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