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Systematics |
Received for publication August 3, 2001. Accepted for publication February 19, 2002.
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 INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: ETS • ITS • Mimuleae •Mimulus •Phryma • Phrymaceae • Scrophulariaceae •trnL/F
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| MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Research onMimulus has continued withstudies of inbreeding depression (Carr and Dudash, 1996; Carr,Fenster, and Dudash, 1997; Karron et al., 1997; Dudash and Carr,1998; Willis, 1999) the genetics of speciation (Bradshaw etal., 1995, 1998), mating system evolution (Leclerc-Potvin andRitland, 1994), and the ecological effects of hybridization(Beeks, 1962; Waayers, 1996).As currently described,Mimulus contains approximately 120 species,of which some 75% occur only in western North America. However,Mimulus is worldwide in distribution. Four species exist inAustralia (Grant, 1924; Barker, 1982) (one of them also occursin South Africa and India), 10 in Chile (von Bohlen, 1995a),approximately 19 in Mexico (Grant, 1924; Vickery, 1997), 4 inthe Himalayas (Yamazaki, 1993), 1 in Madagascar, and 2 in easternNorth America. The relationships among western North AmericanMimulus,Mimulus species distributed outside western North America,and several genera putatively closely related toMimulus remainuncertain.
Dumortier (1829) first proposed tribe Mimuleae (Scrophulariaceae).Von Wettstein (1891) placedMimulus withMazus,Dodartia,Monttea,Melosperma, andLancea in subtribe Mimulinae of tribe Gratioleae.Leucocarpus,Hemichaena, andBerendtiella were placed in tribeScrophularieae. Pennell (1920) redefined tribe Mimuleae to containLeucocarpus andMimulus, though in subsequent works, Pennellplaced Mimuleae in tribe Gratioleae (Pennell, 1935, 1947). Pennell(1935) also transferredHemichaena andBerendtiella to tribeGratioleae, noting a similarity in floral structure toMimulus.Burtt (1965) reestablished Mimuleae as a tribe, diagnosed onthe presence of two characters: (1) tubular, toothed calycesand (2) bilamellate stigmas that are receptive only on the innersurface and that close together with contact. Thieret (1954,1967) suggested the removal ofMelosperma andMonttea to Melospermeae.This left tribe Mimuleae sensu Argue (1984) composed ofMimulus(125 species),Berendtiella (5 species),Hemichaena (1 species),Leucocarpus (1 species),Dodartia (1 species),Lancea (2 species),andMazus (25 species). The relationships amongMimulus andother members of Mimuleae are poorly understood. In his analysisof pollen, Argue (1980, 1984) found no character or combinationof characters that separatesDodartia,Lancea,Leucocarpus,Mazus, orMimulus.
Barker (1982) considered the Australian generaGlossostigma,Peplidium, andElacholoma (subtribe Limoselleae of tribe Gratioleae,sensu Bentham [1876]) to be members of Mimuleae. Similar tothe traditional members of Mimuleae, these Australian generapossess tubular, toothed calyces, though inGlossostigma thecalyx has three or four lobes instead of the typical five lobes.The stigmas of these Australian genera are structurally differentfrom those of species traditionally assigned to Mimuleae. Inthe monotypicElacholoma, the stigma lobes are relatively longand are receptive over most of their length (Barker, 1982).The stigmas ofGlossostigma andPeplidium contain one largeand one vestigial stigma lobe. The large lobe covers the mouthof the corolla and is receptive only on the outer surface (Barker,1982). Upon being touched, this large stigma lobe moves fromcovering the mouth to pressing against the upper corolla lip,exposing the anthers. In order to assess the homology of thesestigmatic movements to the touch-sensitive stigmatic movementsin traditional Mimuleae, a sound phylogenetic hypothesis isneeded.
Though much has been learned about processes of evolution inMimulus, the systematic placement of the genus and the relationshipsamong species within it remain unresolved. Molecular studieshave demonstrated that the traditionally recognized Scrophulariaceaeare not monophyletic (Olmstead and Reeves, 1995; Olmstead etal., 2001). In their analysis of Scrophulariaceae using threechloroplast genes, Olmstead et al. (2001) found that the singlerepresentative ofMimulus formed the sister group to OrobanchaceaeandPaulownia in some trees and to Lamiaceae in others. Additionaltaxa were not sampled withinMimulus or tribe Mimuleae. Olmsteadet al. (2001) dismembered the traditional Scrophulariaceae butdid not reassignMimulus to any clade of family rank.
An additional intriguing result of molecular analyses of Lamialesis the close relationship betweenMimulus and the taxonomicallyisolatedPhryma (Phrymaceae) (R. G. Olmstead, unpublished manuscript).Phryma is monotypic with a disjunct distribution in easternAsia and eastern North America. The taxonomic position ofPhrymahas been unsettled for years: some authors place it in Verbenaceae(e.g., Cronquist, 1981) and others in its own family Phrymaceae(e.g., Schauer, 1847; Moldenke, 1971; Lu, 1990).Phryma hasa distinctive pseudomonomerous gynoecium, which develops intoa one-seeded fruit (Chadwell, Wagstaff, and Cantino, 1992).A close relationship betweenPhryma andMimulus has never beensuggested.
Relationships withinMimulus have also been a source of taxonomiccontroversy. Grant (1924) divided the approximately 120 speciesinto two subgenera and ten sections. SubgenusMimulus (Synplacussensu Grant) is based on the character of the placentae beingfirmly united to form a central column or separating only atthe apex. Taxa within subgenusSchizoplacus possess placentaethat are divided to the base. The subgeneric assignments madeby Grant have not been challenged, although differing viewson relationships among and within sections have been proposedand some sections have been elevated to generic rank (McMinn,1951; Pennell, 1951). Vickery (1969) recognized seven majorsections inMimulus (Mimulus,Erythranthe,Simiolus,Paradanthus,Eunanus,Oenoe andDiplacus) and five monotypic sections. Thompson's(1993) treatment ofMimulus in California follows Vickery, butreassignsM. mohavensis (monotypic sect.Mimulastrum) andM.pictus (monotypic sect. Pseudoenoe) to sect.Mimulastrum andplacesM. pygmaeus (monotypic sect.Microphyton) in sect.Oenoe.The monophyly of some of the sections is suspect, because othersections have been suggested to be derived from within them(Grant, 1924; D. Thompson, personal communication).
The goals of this study are to: (1) develop a rigorous phylogenetichypothesis for the higher level relationships ofMimulus andother members of the tribe Mimuleae, (2) examine the relationshipofMimulus to putatively closely related genera, (3) test thehypothesis of monophyly of the genusMimulus, (4) analyze subgenericand sectional relationships withinMimulus, and (5) assess biogeographicalrelationships and morphological character changes in a phylogeneticcontext.
We report the results of analyses of DNA sequences from boththe chloroplast and nuclear genome. Data from the chloroplastgenome come from the leucine (trnL) intron and the intergenicspacer betweentrnL andtrnF (trnL/F) (Gilley and Taberlet,1994). This region has been shown in previous studies to providegood phylogenetic resolution within families and genera in theLamiales (McDade and Moody, 1999). The nuclear genome is representedby sequences of both the internal transcribed spacer (ITS) andexternal transcribed spacer (ETS) regions (Baldwin et al., 1995;Baldwin and Markos, 1998) of nuclear rDNA. We collected nucleargenome data for two reasons: (1) to compare phylogenetic hypothesesfrom different genomes and (2) to help resolve more distal portionsof the phylogeny. Our expectation in starting this project wasthat the substitution rate oftrnL/F, ETS, and ITS would overlap,but overall thetrnL/F region would have more slowly evolvingsites. This information would be useful for resolving the deepernodes in the phylogeny. The ETS and ITS would show higher overallsubstitution rates that would aid in resolving the distal portionsof the phylogeny.
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| RESULTSDISCUSSIONLITERATURE CITED |
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as most closely related toMimulus, includingPaulowniaceae, Lamiaceae, Orobanchaceae, Pedaliaceae, and Acanthaceae.Some of thetrnL/F and ITS sequences for the preceding familieswere obtained from GenBank. Species used in this study, voucherspecimens, and GenBank accession numbers are listed on the BotanicalSociety of America website (http://ajbsupp.botany.org/v89/).A representative of Veronicaceae,Mohavea breviflora, was usedas an outgroup in all analyses, a choice that is supported byprevious molecular analyses of Lamiales (Olmstead et al., 2001).
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was used to extract total genomic DNA,which was further purified using Qiaquick spin-columns (Qiagen,Valencia, California, USA).ThetrnL/F region was amplified using thetrn-c andtrn-f primers(Taberlet et al., 1991). We had difficulty amplifying both theintron and spacer as one fragment for three species (Mimulusuvedaliae,Lancea tibetica, andBerendtiella rugosa). For thesetaxa, the intron and spacer were amplified separately, usingthetrn-c andtrn-d primers to amplify thetrnL/F intron andtrn-e andtrn-f to amplify the spacer. The entire ITS regionwas amplified using the its4 and its5 primers (Baldwin, 1992).InHemichaena fruiticosa andMimulus gracilis, we had difficultyamplifying the entire fragment so the ITS1 and ITS2 were amplifiedseparately, using the its5 and its2 primers for ITS1 and theits3 and its4 primers for ITS2. To amplify a portion of the3' end of the ETS, we used the 3' 18S-IGS primer of Baldwinand Markos (1998). The 5' primer, named ETS-B (5'-ATAGAGCGCGTGAGTGGTG-3')(A. Yen, University of Washington, unpublished manuscript) wasdesigned usingMimulus sequences as a reference. The polymerasechain reaction (PCR) conditions for all three DNA regions wereas follows: 35 cycles of 94°C for 1 min, 50°C for 1min, and 72°C for 1 min. The PCR products were purifiedusing Qiagen Qiaquick spin-columns according to the manufacturer'sprotocol.
Sequences of both strands of the PCR product were generatedon an ABI 377 (Applied Biosystems, Foster City, California,USA). To improve sequence quality for thetrnL/F region of allsampled taxa, the internal primers of Taberlet et al. (1991),trn-d andtrn-e, were used for sequencing as were two primersthat we designed usingMimulus sequences as a reference,trnL-2C(5'-ATCGGTAGACGCTACGGACT-3') and trnL-2F (5'-CGGGATAGCTCAGCTGGTAG-3')that are just internal totrn-c andtrn-f, respectively. TheITS was sequenced using the external PCR primers, its4 and its5,and the two internal primers, its2 and its3. The ETS was sequencedusing the 18S-E primer of Baldwin and Markos (1998), which isslightly internal to 18S-IGS primer, and theMimulus specificETS-B primer. Electropherograms for each region were compiledand compared using the program Sequencher version 3.0 (GeneCodes Corporation, Ann Arbor, Michigan, USA), from which a consensussequence was generated.
Analyses
Consensus sequences fortrnL/F, ITS, and ETS of all taxa werealigned manually using the program Se-Al version 1 (A. Rambaut,University of Oxford, Oxford, United Kingdom). Some regionswere too different to be confidently aligned for ITS and ETSsequences, but would be valuable for analyses within subgroups.Regions that were difficult to align with confidence were excluded.Alignments are available at the Botanical Society of Americawebsite (http://ajbsupp.botany.org/v89/).
The three DNA regions were analyzed both individually and incombination using the program PAUP* 4.0b3a (Swofford, 1998).The default PAUP settings were used except as noted. Parsimonysearches were conducted using heuristic searches with 1000 randomsequence addition replicates, to find multiple islands of trees,if present (Maddison, 1991). Gaps were scored as missing data.Support for individual branches was estimated using bootstrapvalues (Felsenstein, 1985). Bootstrap values were calculatedusing 1000 full heuristic search replicates.
Maximum likelihood estimates of the phylogeny were also madeusing PAUP* for the nrDNA,trnL/F, and combined data sets. Ineach case, one of the most parsimonious trees was selected andPAUP* was used to estimate the transition : transversion ratio(ti : tv) (with two rate categories) and the shape parameterfor the gamma distribution. Base frequencies were determinedusing the empirical values. The estimated values were enteredas fixed data and were used in a heuristic search. Using theseparameters corresponds to the HKY85 model of evolution.
The incongruence length difference (ILD) test (Farris et al.,1994, as implemented in PAUP*) was used to assess potentialconflicts between the phylogenetic signal from different DNAfragments. The following comparisons were made: ITS vs. ETSandtrnL/F vs. combined ITS and ETS. For each test, 100 replicateswere analyzed with an heuristic search, each with ten randomsequence addition replicates.
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| DISCUSSIONLITERATURE CITED |
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Compared to ITS, the approximately 450 bp of the 3' end of theETS that we analyzed was relatively easy to amplify and sequence.Complete sequences for both strands required only two sequencingreactions. The aligned ETS sequences were 491 bp in length.Pairwise distances among all sampled taxa ranged from 0.7% to40.1% for ETS compared to a range of 0% to 27.9% for ITS. TheGC content of ETS (56.2%) was similar to that of ITS (59.2%).Seven regions totaling 123 bp could not be aligned confidentlyand were excluded from the analysis (bases 93–104, 159–200,263–284, 368–373, 390–398, 439–451,and 470–488 in the alignment).
The ETS and ITS region are closely linked in the rDNA, therefore,we will only present results for analyses of combined rDNA data.Results from the partition homogeneity test (see below) justifyour decision. The combined nrDNA data set was 1187 bases inaligned length. After excluding ambiguously aligned sites, 874bases were used in the analysis, of which 540 were variableand 365 were parsimony informative. Parsimony analyses resultedin one most-parsimonious tree of length 1586 (CI = 0.541, RI= 0.656, RC = 0.355). For the ML analysis of nrDNA sequencedata (Fig. 1), the following parameters were estimated on oneof the most-parsimonious trees: ti : tv ratio = 2.037, gamma= 0.634. The MP tree differed from the ML tree in the resolutionof the deeper nodes and the ML tree resolvesM. floribundusas sister toM. nepalensis while the MP tree resolvesM. nepalensisas sister to a clade containingM. floribundus,M. guttatus,M. tilingii, andM. depressus.
Results of the partition homogeneity test for ITS vs. ETS andITS/ETS vs.trnL/F showed that none of the data sets were significantlydifferent from random pairwise partitions of the data (P = 0.14,P = 0.67, respectively). Therefore, we combined all three datasets in subsequent analyses.
The combined chloroplast and nrDNA data set was 2211 bases inaligned length. A total of 313 sites were difficult to alignand were excluded, resulting in 1898 included characters, ofwhich 879 were variable and 546 were parsimony informative.Parsimony analyses of the combined data resulted in three most-parsimonioustrees of length 2117 (CI = 0.599, RI = 0.691, RC = 0.413). Forthe ML analysis of the combined data, the following parameterswere estimated on one of the most parsimonious trees: ti : tvratio = 1.164, gamma = 0.471. The ML tree is congruent withthe strict consensus of the three MP trees and has a parsimonylength of 2118.
Figure 1 compares the ML trees resulting from the analysis ofchloroplast DNA and nrDNA. The phylogeny estimated usingtrnL/Fshows good resolution of deep nodes among genera of Mimuleaebut poor resolution among the families in the Lamiales. Thephylogeny estimated using nrDNA resolves some of the deepernodes in the phylogeny differently, though none are well supported.Analyses of the combined data (Fig. 2) resulted in a topologythat is congruent with the phylogeny estimated using only chloroplastdata, though more resolved, for the relationships of speciesofMimulus,Phryma,Leucocarpus,Hemichaena,Berendtiella, andPeplidium with the exception of the placement ofM. pulchellus.Relationships among and within these genera are identicallyresolved in the combined ML tree and one of the combined MPtrees. The relationships of families in the Lamiales to eachother are generally unresolved in all analyses. The major cladesidentified in our analyses are detailed below.
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(Mimulus,Leucocarpus,Hemichaena, andBerendtiella) andGlossostigma,Peplidium, andPhryma. We refer to this clade as the subfamilyPhrymoideae. The relationships among Phrymoideae, Mazoideae,andPaulownia are unresolved in the strict consensus of thethree MP trees.Within Phrymoideae, relationships among all sampled taxa arefully resolved in the combined analysis, with the exceptionof one node in the MP analyses, and most clades are well supported.Of the 20 resolved nodes in the MP analyses, only one has abootstrap value <76%, and only three have bootstrap values<92%. Clade D (BS = 100% all analyses) contains members ofMimulus sect.Mimulus (sect.Eumimulus sensu Grant [1924]),including the type species,M. ringens, and the Australian generaGlossostigma andPeplidium. Derived from within clade D is cladeE (BS = 96%), containingGlossostigma,Peplidium, andM. uvedaliae,which also is Australian in distribution. A sister relationshipbetween sampled members ofGlossostigma (which was not sampledfortrnL/F) andPeplidium was recovered in analysis of nrDNA(BS = 90%).
AllMimulus in western North American plus the generaBerendtiella,Hemichaena, andLeucocarpus form clade F (BS = 63%).Phrymafrom eastern North America and from eastern China form a clade(BS = 100%).Phryma is sister to clade F in the ML analysisof combined data and in two of the three MP trees. In one MPtree,Phryma is sister to the entire Phrymoideae. Clade G (BS= 93%) containsLeucocarpus and its sister clade H (BS = 100%),which containsMimulus sects.Simiolus,Erythranthe, andParadanthus.Within clade H are clades that correspond to sect.Erythranthe(M. cardinalis andM. lewisii [BS = 93%]) and sect.Simiolus(M. guttatus,M. tillingii, andM. depressus [BS = 100%]). WithinSimiolus, the two species from western North America form aclade (BS = 92%) that is sister to the species from Chile (M.depressus). SectionParadanthus is paraphyletic.
Berendtiella andHemichaena, together with all members ofMimulussubgenusSchizoplacus form clade I (BS = 92%). All sampled speciesinMimulus subg.Schizoplacus are monophyletic (BS = 100%) andtogether comprise clade J that is sister to a clade comprisedofHemichaena andBerendtiella (BS = 99%). Within clade J areclades that correspond to sect.Eunanus (M. whitneyi andM.brevipes [BS = 100%]) and sect.Diplacus (M. aurantiacus andM. clevelandii [BS = 99%]). The latter plus two species ofMimulussect.Oenoe form a clade (BS = 92%).Diplacus appears to bederived from within a paraphyleticOenoe.
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). Neither source ofdata provided resolution of the relationships of Phrymaceaeto other families in the Lamiales. The chloroplast data wereless homoplastic than the nuclear data (trnL/F CI = 0.776 vs.nrDNA CI = 0.518) for analyses of all taxa. The combined analysisreflects the strongly supported components of the individualdata sets and results in a well-supported estimate of phylogenyinMimulus and related genera in tribe Mimuleae from which significantsystematic and evolutionary conclusions can be drawn.Systematic conclusions
(1)Mimulus and the genera derived from within it form a stronglysupported clade referred to as subfamily Phrymoideae (Fig. 2).This is not nested in any clade traditionally considered tohave the rank of family in this study. Similarly, the recentstudy of Olmstead et al. (2001) found thatMimulus was not associatedwith any of the named clades of Lamiales. Phrymoideae has awide geographic range and contains approximately 160 speciesthat are divergent morphologically and ecologically.
(2) There is weak support for a more inclusive clade, Phrymaceae,in which Phrymoideae and Mazoideae are sister groups. This relationshipis recovered in the ML analysis of the combined data, in twoof the three MP trees, and in 39% of the parsimony bootstrapdata sets. Given thatMazus andLancea share a set of morphologicalcharacters with species in Phrymoideae, we tentatively assignthese genera to Phrymaceae with the caveat that further researchis needed to confirm their sister relationship to Phrymoideae.Therefore, we propose that the clade recovered in this analysisthat contains all the representatives ofMimulus, as well asthe generaPhryma,Glossostigma,Peplidium,Leucocarpus,Berendtiella,andHemichaena,Mazus, andLancea be recognized at the rankof family; Phrymaceae (Schauer, 1847) has priority. A distinctadvantage in recognizingLancea andMazus within the Phrymaceaeis that synapomorphies can be diagnosed for Phrymaceae, as willbe discussed below in the section on morphological evolution.The scope of this newly defined Phrymaceae is very differentfrom its historical antecedents. Previously, the family wasmonotypic and limited in geographic range to eastern North Americaand eastern China. In our circumscription, Phrymaceae encompassesapproximately 190 species that are distributed worldwide.
(3) Relationships of Phrymaceae to other families in the Lamialesare generally unresolved. A similar lack of resolution has beenencountered in other molecular analyses of the Lamiales (Olmsteadet al., 2001; B. Oxelman, Uppsala University, unpublished manuscript).Sampling in this study was designed to include groups putativelyclosely related toMimulus and tribe Mimuleae, based on othermolecular studies of Lamiales. Given a limited sampling of thesegroups,Paulownia has the strongest support (albeit weak; BS= 37%) as sister to Phrymaceae. This relationship is recoveredin the ML analysis and in two of the three MP trees. The thirdMP tree resolvesPaulownia as sister to Phrymoideae and they,in turn, are sister to Mazoideae. If this latter relationshipturns out to be correct,Paulownia would simply be includedwithin Phrymaceae. At present, we assignPaulownia to the monogenericfamily Paulowniaceae.
(4) The genusMimulus is not monophyletic.Glossostigma,Peplidium,Phryma,Leucocarpus,Hemichaena, andBerendtiella are derivedfrom withinMimulus. Though it was not sampled, morphologicalevidence strongly implies that the Australian genusElacholomais also derived from withinMimulus (Barker, 1982). The monophylyofMimulus was not found in analyses of any of the individualdata sets or in any combination of the data sets. In the combineddata set, trees that are constrained to makeMimulus monophyleticare 94 steps longer. The nonmonophyly ofMimulus is not surprising,given that no clear synapomorphy for the group has been identified.In her monograph ofMimulus, Grant (1924) suggested that calyxcharacters best diagnosed the genus. Calyx characters withinMimulus, however, cannot be described in any way that wouldconsistently differentiateMimulus from other genera in tribeMimuleae.
(5) It is clear that generic boundaries within Phrymaceae requireextensive redefinition in order for genera to be monophyletic.Options for generic revisions include: (i) retain the traditionalMimulus sensu lato (s.l.) as one genus and reassign speciesof the six other genera toMimulus or (ii) break up the traditionalgenusMimulus. The advantages of option i are that fewer speciesin Phrymaceae would require a name change and all of the intenselystudied species in western North America would retain the namewith which they are currently identified. In this scenario,Phrymoideae would be synonymous withMimulus. The primary disadvantagesof option i are that at least six genera would require namechanges and that all the diversity contained in Phrymoideaewould be assigned to one genus. The latter situation is onlya problem, however, if one assumes that different ranks withina hierarchical classification have implications with regardsto amounts of diversity. Option ii would result in more generain Phrymaceae than option i but would require 105 to 185 namechanges, depending on where generic boundaries are drawn. Redrawinggeneric boundaries within Phrymaceae and in-depth discussionsof the implications of using traditional and phylogenetic nomenclaturalsystems (deQueiroz and Gauthier, 1992; Cantino et al., 1999)are beyond the scope of this paper, but will be the subjectof a future publication.
(6) Some subgeneric and sectional relationships are apparent.SubgenusSchizoplacus is shown to be monophyletic (BS = 100%)whereas subgenusMimulus is not. Preliminary evidence existsfor the monophyly of sects.Simiolus (BS = 100%),Erythranthe(BS = 93%),Diplacus (BS = 99%), andEunanus (BS = 100%). SectionMimulus is not monophyletic, becauseGlossostigma andPeplidiumare derived from within it, nor is sectionParadanthus. In definingsect.Paradanthus, Grant (1924) admitted that the species withinit were probably not closely related. The nonmonophyly of thissection was also postulated from pollen data (Argue, 1980).Mimulus bicolor (sect.Paradanthus) is closely related to speciesinErythranthe, whereas other species inParadanthus are moreclosely related to sect.Simiolus. SectionOenoe may also notbe monophyletic. More thorough analyses of relationships withinMimulus in western North America and Australia are in preparation.
Morphological evolution
Species within the redefined Phrymaceae are diverse in bothlife history and morphological traits. This clade includes specieswith habits ranging from annuals, attaining a final plant heightof a few centimeters, to perennials, some of which are woodyand attain a height of 4 m. Variation for reproductive mechanismsalso exists, including species that have outcrossing, mixed-mating,selfing, and asexual breeding systems. At least five speciesin Phrymaceae,M. congdonii,M. douglasii (Thompson, 1993),M. nasutus (Diaz and Macnair, 1998),M. pictus (Thompson, 1997),andGlossostigma cleistanthum (Barker, 1982), have some populationsthat are cleistogamous. Within Phrymaceae, insects pollinateflowers of most of the species, but hummingbird pollinationalso exists (M. cardinalis [Bradshaw et al., 1998] andM. flemingii[Stebbins, 1989]). Many species have corollas with bilateralsymmetry, but flowers of other species are radially symmetric.Barker (1982) notes that radially symmetrical corollas seemto be associated with a prostrate habit in Australian speciesofMimulus andPeplidium. The most common fruit type in Phrymaceaeis a readily dehiscent capsule containing numerous seeds, butexceptions exist.Phryma leptostachya has an achene (Whipple,1972).Leucocarpus perfoliatus has a baccate, indehiscent fruit(Burtt, 1965) described as a white berry, with thin skin andwith most of the substance of the fruit derived from the fleshyplacenta. Many species ofPeplidium possess capsules that arethick-walled and open only after the plant has senesced. Grant(1924) described a similar capsule in some species ofMimulussect.Oenoe. The phylogenetic distance betweenMimulus sect.Oenoe andPeplidium (Fig. 2) demonstrates that this characterevolved convergently in taxa from two different continents,possibly in response to common environmental conditions. Speciesin Phrymaceae also show variation in habitats, inhabiting sitesas varied as desert, riparian, and alpine environments. Somespecies extend over a large geographic range and have developedmany different races (e.g.,M. glabratus) whereas others arelocal endemics and several are rare (e.g.,M. exiguus).
It is of great practical importance to provide morphologicalsynapomorphies for described clades. Synapomorphies for someof the recovered clades are described below, along with evidencefor or against previous systematic and morphological hypotheses.A summary of the major morphological shifts in the family ispresented inFig. 3.
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Grant (1924) diagnosed subgenusSchizoplacus primarily on thebasis of having a placenta that is divided to the base. Othertraits associated withSchizoplacus were relatively short pedicels(usually shorter than the calyx) and glandular pubescent styles.Sister taxaHemichaena andBerendtiella have united placentaethus making divided placentae a synapomorphy forSchizoplacus.Berendtiella andHemichaena have pedicels that are shorter thanthe calyx and glandular pubescent styles, making these characterssynapomorphies for the more inclusive clade.
The characters that Grant (1924) used to diagnose sect.Mimulus(Eumimulus sensu Grant [1924]) were tubular calyces with equalteeth and a distinctly bilabiate, blue corolla with glabrouspalatine ridges. Presumably, the description of tubular calyceswas meant to distinguish the calyces in sect.Mimulus from thosein sect.Simiolus, which are strongly inflated and sagitallycompressed, and sects.Paradanthus andErythranthe, which havesharp, definite angles and flat sides. However, according toour results, these characters are not consistent within theclade containing sect.Mimulus. The Australian generaGlossostigmaandPeplidium are clearly derived from within sect.Mimulusand do not have equal calyx teeth, many have radially symmetricflowers, many others have flowers that are not blue, and somehave sharply angled calyx lobes. Overall, the distribution ofmorphological characters in this clade needs to be reexaminedin light of the phylogenetic evidence. At present, no clearsynapomorphies exist for the clade containingMimulus sect.Mimulus.
Barker (1982) described two derived morphological charactersfor these genera: one-celled anthers and the presence of onelarge and one vestigial stigmatic lobe. InGlossostigma andPeplidium, the large stigmatic flap covers the corolla mouthand, upon being touched, is triggered back against the uppercorolla lip. This stigmatic movement is apparently homologousto and derived from the stigmatic condition in the rest of thePhrymaceae in which two equal or nearly equal stigma flaps,receptive on the inner surface only, close upon touch (Fetscherand Kohn, 1999).
The monotypic genusPhryma has been difficult to place taxonomicallydue to its having a pseudomonomerous gynoecium that developsinto an achene. The results presented here indicate thatPhrymais derived from within the traditionally recognized genusMimulus.In comparison to other species in Phrymaceae,Phryma sharessome floral characteristics, but many of its reproductive charactersare derived. All other taxa in the Phrymaceae are bicarpellateand have numerous seeds, thus the enigmatic gynoecium and fruitofPhryma is derived. Whipple (1972) described the calyx ofPhryma as persistent, zygomorphic, and as having five ridgeswith three hooked adaxial lobes and two short subulate abaxiallobes. The three elongated and hooked adaxial corolla lobescan be interpreted as being derived from the ancestral conditionof relatively short, uncurved adaxial lobes. It is possiblethat this morphological switch is an adaptation to epizoochorousdispersal, in that these hooked upper lobes allow the fruitto become attached to animal fur (Holm, 1913). The flowers ofPhryma have two stigmatic surfaces at the tip of the style,but, in general, flowers are much reduced, which makes determinationof stigmatic characters difficult.Phryma is also characterizedby the reflexed movement of the mature calyx and fruit (Whipple,1972). While in flower, the calyx is perpendicular to the stem,while in fruit the calyx points abaxially and is parallel tothe stem. Its common name "lopseed" reflects this fruit orientation.
The following characters have been used to diagnoseDiplacus:(1) plants are shrubs, semi-shrubs, or perennial from a woodycaudex; (2) glutinous exudation from the leaves; (3) prismaticcalyx; and (4) investing, linear, oblong capsule (Grant, 1924).This combination of characters separatesDiplacus from othergroups in Phrymaceae.
In an analysis of morphological characteristics ofHemichaena,Berendtiella, andLeucocarpus, Thieret (1972) suggested thatHemichaena andBerendtiella could be distinguished from otherclosely related genera by their inflorescences of bracteolatecymes. Species in this clade are woody, as are members of theDiplacus clade, suggesting the convergent evolution of woodin these two groups (Fig. 3). Additionally, species in bothDiplacus and theHemichaena-Berendtiella clade have leaves thatare revolute at their margins, a character that is possiblya convergent adaptation to the dry habitats in which plantsof both groups live. Thieret (1972) postulated thatLeucocarpusandHemichaena were closely related due to their possessionof reticulate seeds and distinctive calyces and stigmas. Theestimated phylogeny (Fig. 2), however, shows thatLeucocarpusandHemichaena are not closely related. The calyx and stigmacharacters are sympleisiomorphies.
A final observation made by Thieret (1972) on theHemichaena-Berendtiellaclade is the curious presence of two flowers in each axil thathe called superposed inflorescences. The presence of two supernumerarybuds in the axil of each leaf has also been reported inM. guttatusandM. gemmiparus (Moody, Diggle, and Steingraeber, 1999). Ineach of these species, the distal bud ultimately becomes a floweror a lateral branch. InM. guttatus, the proximal bud remainsinactive for the entire life of the plant. InM. gemmiparus,the proximal bud becomes a brood bulbil that functions as anasexual propagule, which is the primary means of propagationof this species (Beardsley, 1997). Therefore, among North AmericanPhrymaceae, supernumerary buds can be seen to take on at leastthree different forms: a flower inHemichaena-Berendtiella,a brood bulbil inM. gemmiparus, and dormant inM. guttatus.
Biogeography
Within Phrymoideae are two clades that represent distinct radiationsand document two centers of diversity. The first center of diversityis in western North America and is well recognized and oftenstudied. From within this radiation of species in western NorthAmerica, several species have colonized other continents, possiblythrough long-distance dispersal.Mimulusdepressus (Chile) isa member of sect.Simiolus, indicating a relatively recent establishmentofMimulus in South America. The presence of a suite of morphologicaltraits makes it highly probable that most species ofMimulusin Chile are closely related and reflect one colonization event.Pollen (Argue, 1981) and other morphological data (von Bohlen,1995b) indicate thatM. crinitus andM. bridgesii are not closelyrelated to other ChileanMimulus and reflect at least one additionalcolonization event.Mimulus nepalensis (Tibet and China) isderived from within a clade of taxa that are found primarilyin California and the Pacific Northwest. In addition, morphologicalevidence (Grant, 1924) and preliminary sequence data (P. M.Beardsley, unpublished data) suggest thatM. sessilifolius representsa second, distinct, relatively recent establishment ofMimulusin Asia.
The greatest number of species in North America occurs in California,with declining numbers of species to the north, extending tosouthern Alaska. Fewer species occur in the Great Basin, RockyMountains, and the desert Southwest. Some species not sampledin this study in sectionsErythranthe,Simiolus, andParadanthusare also found in Mexico. Our data suggest that these specieswere derived relatively recently from ancestors in California.
TheHemichaena-Berendtiella clade is found primarily in centraland southern Mexico, and one species,Hemichaena fruiticosa,is native to southern Mexico, Guatemala, and Costa Rica (oneof the two species in the family with a tropical distribution).Hemichaena fruiticosa is found in diverse habitats, both disturbedand undisturbed, in the mountains from 900 to 3500 m (Thieret,1972). The second species in Phrymaceae found in Central AmericaisLeucocarpus perfoliatus, which grows along stream banks inforests from 1350 to 2000 m (Pennell, 1920).Leucocarpus issister to a large clade (approximately 60 species) that radiatedextensively in western North America, indicating that some combinationof higher rates of speciation or lower rates of extinction intemperate habitats than in tropical habitats exists in thisclade.
Phrymaceae have undergone a second radiation in Australia. AllAustralian taxa withinMimulus,Glossostigma, andPeplidium,together comprising approximately 30 species, fall within oneclade. The geographic division between the Australian speciesand the rest of the taxa in Phrymoideae, which are primarilyNorth American, is certainly of ancient origin. The reconstructedancestral distribution of the common ancestor of Phrymoideaeis unresolved.
Populations ofPhryma leptostachya exist in eastern North America(var.leptostachya) and eastern China and Japan (var.asiatica).Similar to other studies (Lee et al., 1996; Xiang et al., 2000),we recovered substantial divergence in the sampled DNA sequencesbetween the two varieties. Using a molecular clock based ondivergence inrbcL sequences, Xiang et al. (2000) estimatedthe time of divergence between the two varieties at 5.45 ±2.47 million years. Hara (1962) demonstrated limited morphologicaldivergence between the two varieties. Differences were limitedto the shape, size, and pubescence of leaves. This lack of differentiationis perhaps explained byPhryma's greatly reduced floral displaythat offers limited opportunities for morphological change.Closely related groups of species in the newly defined Phrymaceaeoften differ morphologically in floral characters, while retainingpleisiomorphic vegetative characters.
The results of this study begin to clarify patterns of relationshipinMimulus, tribe Mimuleae, and other closely related genera.In light of this molecular evidence, we propose the recognitionof a dramatically expanded Phrymaceae containing many speciesthat have become model systems for the study of evolutionaryprocesses. Future phylogenetic studies in Phrymaceae will continueto inform the systematics of the group and will serve as a usefulstarting point for further comparative analyses.
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2 Author for reprint requests (pbeard{at}u.washington.edu)
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