American Journal of Botany 91(3): 474±489. 2004.

PATTERNS OF EVOLUTION IN WESTERN NORTH AMERICAN MIMULUS (PHRYMACEAE)1

PAUL M. BEARDSLEY,2,6 STEVE E. SCHOENIG,3 JUSTEN B. WHITTALL,4 AND RICHARD G. OLMSTEAD5

2Biology Department, Colorado College, 14 E. Cache La Poudre, Colorado Springs, Colorado 80903 USA; 3California Department of Food and Agriculture, 1220 N Street, Sacramento, California 95814 USA; 4Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, California 93106 USA; and 5Department of Biology, Box 355325, University of Washington, Seattle, Washington 98195 USA

A well-supported phylogeny is presented from both chloroplast DNA (the trnL/F region) and two regions of nuclear rDNA (ITS [internal transcribed spacer] and ETS [external transcribed spacer]) with nearly complete sampling for Mimulus (Phrymaceae) in western North America. Three separate genera are derived from within the clade that contains all the Mimulus species in western North America. The taxonomic status of the proposed sections of Mimulus and the relationships of many taxonomically dif®cult species are considered with observations on morphological evolution. Discordance between data sources provides support for the hypothesis that M. evanescens is a hybrid between M. latidens and M. brevi¯orus. In two major clades (Eunanus and Diplacus), patterns of genetic variation do not match the current taxonomy. The clustering of taxa in Eunanus is strongly associated with geographic distributions. Mimulus aurantiacus sensu Thompson, M. nanus, and M. ¯oribundus are found to be progenitor species to other species that appear to be derived from within them. Polyploidy and aneuploidy events are clustered near the tips of the phylogeny. Thus, these two mechanisms are concluded to have played a relatively small role in the evolution of persistent lineages in Mimulus. The phylo- genetic distribution of rare taxa is also examined.

Key words: chromosome evolution; cryptic diversity; ETS; ITS; Mimulus; Phrymaceae; polyploidy; trnL/F.

Systematists are charged with the dif®cult tasks of quanti- uncovered cryptic units of biodiversity and paraphyletic spe- fying and characterizing diversity in large groups of organisms cies (Avise, 2000; Omland et al., 2000; Schultheis, 2001). and of identifying units of biodiversity based on discontinu- Additional empirical studies of species boundaries may also ities in morphological or genetic diversity. The rapid devel- inform us regarding the relative importance of different modes opment of molecular systematics enables robust phylogenetic of species diversi®cation in different groups. It has been sug- estimates of species-rich groups, with complete or nearly com- gested that allopatric speciation via founder effect (Carson, plete sampling at the species level. The aim of this study is to 1968) or quantum speciation (Grant, 1981) may be more fre- construct a species-level phylogenetic hypothesis for a clade quent in plants than speciation by subdivision, especially for of Mimulus species that radiated primarily in western North annual taxa with small effective population sizes and low lev- America. These data enable opportunities to assess morpho- els of gene ¯ow (Levin, 1993; Rieseberg and Brouillet, 1994). logical (Scotland et al., 2003) and chromosome evolution This leads to the expectation that many plant species may be (Cunningham et al., 1998), better understand species delimi- paraphyletic (Rieseberg and Brouillet, 1994). If true, this poses tation and its relation to patterns of genetic diversity, and pro- pragmatic problems for species concepts requiring a strict ad- vide an evolutionary context for conservation (Soltis and Gitz- herence to monophyly (Mishler and Donoghue, 1982; de Quei- endanner, 1999; Baldwin, 2000). roz and Donoghue, 1988). Using the metaspecies concept, pos- With a rigorous phylogenetic hypothesis and relatively com- itively paraphyletic groups of populations would simply be plete sampling, changes in chromosome number may be op- rejected for species status, with no alternative offered for their timized and put in their proper historical context. Case studies taxonomic status (Olmstead, 1995). In general, we consider documenting these changes are necessary to help draw gen- current species delimitations as hypotheses and advocate for eralizations about the frequency of polyploid and aneuploid more empirical studies that analyze species boundaries through events in nature (Sang and Zhang, 1999; Baldwin and Wessa, the exploration of both genetic data (Beardsley et al., 2003) 2000; Schultheis, 2001), something about which very little is and factors associated with reproductive isolation (Ramsey et known (Ramsey and Schemske, 1998). Molecular systematic al., 2003). data also have led to a deeper understanding of the genetic Baldwin (2000) and Soltis and Gitzendanner (1999) both structure and evolutionary history of species in nature. Some emphasize the relevance of modern systematic data for con- studies demonstrate species delimitations that are concordant servation biologists. Within western North American Mimulus, with traditional systematic hypotheses, whereas others have molecular phylogenetic data are useful for circumscribing tax- onomically dif®cult rare species and identifying evolutionarily isolated taxa. Large-scale studies, such as the one reported 1 Manuscript received 8 April 2003; revision accepted 21 October 2003. here, are important to provide a foundation for future research The authors thank A. Angert, B. Baldwin, A. Colwell, K. Ritland, M. Tulig, focused on individual species and to identify lineages that re- and J. Willis for providing plant material or DNA and P. Reeves for helpful quire further attention. Studies that generalize about biological discussions. Funding was provided by grants to P. M. B. from ASPT, Sigma Xi, and the University of Washington Botany Dept. and NSF awards to R. G. properties associated with rarity (e.g., patterns of genetic var- O. # DEB-9509804 and # DEB-9727025. iation) would bene®t from knowledge of sister relationships, 6 E-mail: [email protected]. a valuable outcome of extensive phylogenetic sampling 474 March 2004] BEARDSLEY ET AL.ÐWESTERN NORTH AMERICAN MIMULUS 475

(Kruckeberg and Rabinowitz, 1985; Gitzendanner and Soltis, genetic distribution of rare taxa. The relevance of these results 2000). to our understanding of speciation (e.g., Barraclough and Nee, Species in the genus Mimulus have become model systems 2001) and the interpretation of other evolutionary events (e.g., for the study of evolutionary processes in nature due to the shifts in breeding system and changes in pollination syndrome) diversity of interesting traits and adaptations in the genus. As will be discussed in a future paper (P. M. Beardsley, unpub- currently described, Mimulus contains approximately 120 spe- lished data). cies, of which approximately 75% occur only in western North We report the results of analyses of DNA sequences from America (Grant, 1924). Mimulus species vary with respect to both the chloroplast and the nuclear genome. Data from the ploidy level (Vickery, 1978), breeding system (including fre- chloroplast genome comes from the leucine (trnL) intron and quent shifts among pollinators and to self-pollination), and ac- the intergenic spacer between trnL and trnF (trnL/F) (Gilley climation to extreme environments. Species within Mimulus and Taberlet, 1994). The nuclear genome is represented by are model systems for investigating the genetics of speciation sequences of both the internal transcribed spacer (ITS) and (Hiesey et al., 1971; Bradshaw et al., 1998), inbreeding de- external transcribed spacer (ETS) regions (Baldwin et al., pression (Darwin, 1876; Dudash and Carr, 1998), mating sys- 1995; Baldwin and Markos, 1998) of the nuclear rDNA. tem evolution (Leclerc-Potvin and Ritland, 1994), the evolu- tion of heavy metal tolerance (Macnair, 1983), and cytological Taxonomic historyÐRelationships among species of Mi- patterns of evolution (Vickery, 1978). Most of the intensely mulus have been controversial. In the last major systematic analyzed species of Mimulus are distributed in western North treatment of the genus, Grant (1924) divided the approximate- America. Despite extensive studies on evolutionary processes ly 120 species into two subgenera and 10 sections. Subgenus within Mimulus, our understanding of patterns of evolutionary Mimulus (Synplacus sensu Grant) is based on the character of change in the group remains poorly developed. the placenta being ®rmly united, forming a central column or Recent phylogenetic analyses (Beardsley and Olmstead, separating only at the apex. Taxa within subgenus Schizopla- 2002) identi®ed two geographically distinct clades that contain cus possess a placenta that is divided to the base. Prior mo- all the taxa traditionally placed in Mimulus. One clade contains lecular work (Beardsley and Olmstead, 2002) demonstrated all the Mimulus taxa from Australia, eastern North America, that the subgenus Schizoplacus is monophyletic and derived and others from scattered localities elsewhere in the world. from within subgenus Mimulus, which is not monophyletic. This clade includes section Mimulus (Eumimulus sensu Grant The 10 sections proposed by Grant (1924) were Eumimulus, [1924]) and Australian taxa in section Paradanthus, as well Erythranthe, Simiolus, Paradanthus, Eunanus, Oenoe, Dipla- as three small genera not previously allied with Mimulus. Spe- cus, Mimulastrum, Pseudoenoe, and Tropanthus (ϭ Berend- cies from the Australian clade have also become established tiella), the last three being monotypic. Differing views on re- in eastern North America, South Africa, Madagascar, and In- lationships among and within sections have been proposed and dia. A second clade contains all of the Mimulus species in some sections have been elevated to genera (McMinn, 1951; western North America, including sections Diplacus, Eunanus, Pennell, 1951). The taxonomic history of Mimulus or sections Oenoe, Simiolus (which subsequently became established in therein has been reviewed by Grant (1924), McMinn (1951), Chile), Erythranthe, and western North American taxa in sec- Vickery (1969), and Argue (1980). Thieret (1954, 1967) re- tion Paradanthus (which contains some species that are also viewed species within the genera Hemichaena and Berend- found in China and Japan) (Beardsley and Olmstead, 2002). tiella. Descriptions and taxonomic histories of species and sec- Also included in this clade of western North American Mi- tions within Mimulus that have proven to be the most dif®cult mulus are species previously classi®ed in the genera Leuco- taxonomically follow. carpus, Berendtiella, and Hemichaena. Confusion in the taxonomic status of rare species alters their DiplacusÐAuthors working on Diplacus have proposed a conservation priority and complicates protective measures number of taxonomic hypotheses. This section of Mimulus was (Skinner et al., 1995). The partitioning of morphological var- given generic status ®rst by Nuttall (1838) who emphasized iation into species has been particularly dif®cult in Mimulus the woody nature and dehiscence characters of the fruit. At (Skinner et al., 1995; Tibor, 2001), which is problematic, be- least 12 authors have granted Diplacus generic status (e.g., cause many of its species are rare (Table 1). In California, 29 Nuttall, 1838; Jepson, 1925; McMinn, 1951; Beeks, 1962; Mimulus taxa are being monitored by the California Native Grant, 1993), whereas at least 18 have treated it as a section Plant Society (CNPS), and 10 species have been identi®ed as of Mimulus (e.g., Grant, 1924; Munz, 1935; Pennell, 1947; species that are rare, threatened, or endangered throughout Thompson, 1993). Attempts to divide the variation in Diplacus their range (Tibor, 2001). In fact, in California, where many into discrete taxonomic units have been controversial. More genera contain taxa whose species boundaries are dif®cult to than 60 speci®c or subspeci®c taxa have been proposed delineate, Mimulus is comparable with Malacothamnus (As- (Beeks, 1962). In the most recent treatment (Thompson, 1993), teraceae) as the genus with the highest number of rare taxa the diversity within Diplacus is described as only two species, (12) with taxonomic problems (Skinner et al., 1995). As an M. clevelandii Brandegee and M. aurantiacus Curtis. No fur- example of the confusion surrounding the delimitation of spe- ther breakdown into varieties in M. aurantiacus was recog- cies in Mimulus, many previously recognized rare species were nized due to the high crossability of all taxa (McMinn, 1951) recently reclassi®ed as synonyms of more common species and the high frequency of hybrids between forms. This broadly (Thompson, 1993), thus altering priorities for conservation. de®ned M. aurantiacus will be referred to as M. aurantiacus The goals of this study are to (1) develop a rigorous phy- sensu Thompson. These taxonomic decisions remain contro- logenetic hypothesis for the relationships among Mimulus spe- versial (e.g., Waayers, 1996). For purposes of clarity, taxa in cies in the western North America clade, (2) analyze sectional- Diplacus described herein will follow the treatment of Munz and species-level relationships within Mimulus, (3) interpret (1974), which recognized seven species and four additional changes in chromosome number, and (4) examine the phylo- subspecies. 476 AMERICAN JOURNAL OF BOTANY [Vol. 91

TABLE 1. Listing of rare Mimulus taxa.

Species Section NHP statusa Rank or status Comments California, CNPS listb M. acutidens Paradanthus G2Q/S2? 3 Synonym of M. inconspicuus in Thompson (1993) M. aridus Diplacus G4?/S3.3 4 Synonym of M. aurantiacus in Thompson (1993) M. brandegei Oenoe GX/SX 1A Synonym of M. latifolius in Thompson (1993) M. clevelandii Diplacus G3G4/S3.2 4 Threatened by recreation M. cusickii Diplacus G4G5/S1.3 2 Known in California in Warner Mountains M. diffusus Paradanthus G4Q/S3.3 4 Synonym of M. palmeri in Thompson (1993) M. evanescens Paradanthus G2/S1.2 1B Six occurrences in California, one in Oregon M. exiguus Paradanthus G2/S2.2 1B Threatened by development and vehicles M. ®licaulis Paradanthus G2/S2.2 1B Threats are logging and reforestation with herbi- cides (ϭ M. bioletti) M. ¯emingii Diplacus G3/S3.3 4 Synonym of M. aurantiacus in Thompson (1993) M. glabratus ssp. utah- Simiolus G5T5/S1.1 2 Less than 10 occurrences; synonym of M. gutta- ensis tus in Thompson (1993) M. glaucescens Simiolus G3/S3.3 4 Serpentine soils M. gracilipes Paradanthus G3/S3.2 1B Range extension? M. grayi Paradanthus G3/S3.3 4 Synonym of M. inconspicuus in Thompson (1993) M. inconspicuus Paradanthus G3/S3.3 4 Does not include M. grayi, M. acutidens M. johnstonii Eunanus G3/S3.3 4 Only in San Gabriel and San Bernardino moun- tains M. laciniatus Simiolus G3/S3.3 4 M. microphyllus Simiolus G3/S3.3 4 Synonym M. guttatus in Thompson (1993) M. mohavensis Eunanus G2/S2.2 1B Threats are development, mining, non-native plants, and vehicles M. norrisii Paradanthus G2/S2.3 1B Less than 10 occurrences M. nudatus Simiolus G3/S3.3 4 Serpentine soils M. parryi Eunanus G3G4/S2.3 2 M. pictus Mimulastrum G2/S2.2 1B M. pulchellus Paradanthus G3/S3.2 1B M. purpureus Paradanthus G2/S2.2 1B Less than 20 occurrences, threats are develop- ment and vehicles M. pygmaeus Oenoe G4/S3.2 4 Candidate for Oregon listing M. rattanii spp. decur- Eunanus G4T3/S3.2 4 Synonym of M. rattanii in Thompson (1993); atus threat is sand mining M. rupicola Oenoe G3/S3.3 4 M. shevockii Paradanthus G1/S1.2 1B Seven occurrences, residential develop M. subsecundus Eunanus G3/S3.3 4 Synonym of M. fremontii in Thompson (1993) M. traskiae Oenoe GX/SX 1A Only known from type M. whipplei Simiolus GX/SX 1A Only known from type; synonym of M. guttatus in Thompson (1993) Oregon, ORNHP listc M. bolanderi Eunanus G4/S1 2 M. clivicola Paradanthus G4/S2 4 M. douglasii Oenoe G4G5/S2 4 M. evanescens Paradanthus G2/S2 1 Species of concern (Federal) M. hymenophyllus Paradanthus G1/S1 1 Species of concern (Federal) M. jepsonii Eunanus G4/S3 4 M. jungermannioides Paradanthus G2/S2 1 M. kelloggii Oenoe G4/S3 4 M. latidens Paradanthus G4/S1 2 M. patulus Paradanthus G3/S3 4 M. tricolor Oenoe G4/S2 2 Utah, State listd M. parryi Eunanus G3G4/S2 Watch Mimetanthe pilosa (ϭ Own genus or Mi- G5/S3 Peripheral Mimulus pilosus) muluoides M. bigelovii var. cuspi- Eunanus G5/S1 Peripheral Synonym of M. spissus (Grant, 1924) datus M. brewerii Paradanthus G5/S1 Peripheral M. cardinalis Erythranthe G5/S1 Peripheral; probably mislabeled speci- men of M. verben- aceus M. galbratus ssp. fre- Simiolus G5T5/S? Peripheral montii M. primuloides Paradanthus G4/S1 Peripheral M. glabratus ssp. utah- Simiolus G5T5/S3? Need data, question- ensis able taxa March 2004] BEARDSLEY ET AL.ÐWESTERN NORTH AMERICAN MIMULUS 477

TABLE 1. Continued.

Species Section NHP statusa Rank or status Comments Idaho, State liste M. alsinoides Paradanthus G5/S1 1 M. ampliatus Paradanthus G1Q/S1 GP1 M. clivicola Eunanus G4/S3 M M. hymenophyllus Paradanthus G1/S1 GP1 Species of concern (Federal) M. patulus Paradanthus G2Q/S1 GP3 Species of concern (Federal) Montana, State list M. brevi¯orus Paradanthus G4/S1 Species of concern One occurrence M. nanus Eunanus G5/S1 Species of concern Six occurrences M. patulus Paradanthus G2Q/S1 Species of concern Four occurrences M. primuloides Paradanthus G4/S2 Species of concern Thirteen occurrences New Mexico, State status M. guttatus Simiolus G5/S3? Dropped M. nasutus Simiolus G4?Q/S4? Dropped M. nasutus var. nasutus Simiolus G4?QT4?/S4? Dropped M. primuloides Paradanthus G4/S1? Dropped Colorado M. eastwoodiae Erythranthe G3?/S1 M. gemmiparus Simiolus G2/S2 M. ringens Mimulus G5/SH Washington M. pulsiferae Paradanthus G4?/S2 M. suksdor®i Paradanthus G4/S2 M. jungermannioides Paradanthus G2/SH Nevada M. ovatus Eunanus G1G3Q/S1S3 Synonym of M. bigelovii var. ovatus; Thompson (1997) says this is a hybrid between M. cusi- ckii and M. nanus var. mephiticus M. angustifolius Eunanus G1Q Unknown; M. ovatus? Wyoming, State priority M. nanus Eunanus G5/S1 Low On geysers of Yellowstone M. rubellus Paradanthus G5/S1 Low Note: Data collected from Stone (1998), Spackman et al. (1999), Rush et al. (2000), Kagan et al. (2001), More®eld (2001), Tibor (2001), Beauvais (2002), Crispin (2002), Johnson and Tonne (2002), and Mancuso and Cooke (2002). a Natural Heritage Program status rank abbreviations codes: G, global; S, state; 1, extreme rarity, typically less than ®ve occurrences or very few individuals; 2, rarity, typically 6±20 occurrences or few individuals; 3, either very rare and local in range or found locally but in a restricted range, 21±100 occurrences; 4, widespread, but rare in parts of the range at the periphery, more than 100 occurrences; 5, widespread, but rare in parts of the range; X, extinct or extirpated; ?, rank is inexact; Q, taxonomic question. b California Native Plant Society (CNPS) rank codes: 1A, presumed extinct in California (CA); 1B, rare, threatened, or endangered in California; 2, rare, threatened, or endangered in California but more common elsewhere; 3, more information needed; 4, plants of limited distribution. c Oregon Natural Heritage Program List (ORNHP): 1, threatened or extinct throughout range; 2, threatened or extinct in Oregon; 3, more information needed; 4, taxa of concern but not currently endangered. d Utah state status: Watch, plants regionally endemic but without rangewide viability concern; Peripheral, plants rare or uncommon in Utah, more common and widespread outside the state. e Idaho Native Plant Society rank: 1, state priority, taxa in danger of becoming extinct in Idaho; M, monitor; GP, global priority.

EunanusÐSpecies delimitation has been dif®cult in Mimu- (1993) suggests that hybridization amongst taxa is common lus sect. Eunanus (Thompson, 1991), and generations of sys- and zones of clinal intermediacy exist. Speci®cally, Thompson tematists have interpreted the same morphological data differ- (1993) reports hybrids between the following species in Cal- ently. Grant (1924) described 23 species in Eunanus, whereas ifornia: M. constrictus (Grant) Pennell with M. whitneyi Gray Pennell (1951) recognized 25 and Thompson (1993) just 17. and M. johnstonii Grant, M. layneae (Greene) Jepson with M. Numerous species or subspecies in Eunanus are being moni- nanus Hook. & Arn. and M. mephiticus Greene, M. nanus with tored by National Heritage Programs in western states: four in M. mephiticus, and M. leptaleus Gray with M. whitneyii. In- California (Tibor, 2001), two each in Oregon (Kagan et al., vestigations of Eunanus taxa in the Paci®c Northwest by Ezell 2001) and Utah (Stone, 1998), and one each in Idaho (Man- (1971) and Meinke (1992a) indicate further species delimita- cuso and Cooke, 2002), Montana (Crispin, 2002), Nevada tion problems (e.g., M. jepsonii Grant). (More®eld, 2001), and Wyoming (Beauvais et al., 2002) (Ta- ble 1). Thompson (1991) analyzed species delimitation in ®ve SimiolusÐThis section is clearly circumscribed morpholog- Eunanus taxa using ®eld, herbarium, laboratory, and test gar- ically (Grant, 1924; Vickery, 1978), yet the species making up den data and noted that ``these ®ve taxa thus stretch any spe- this section display a high degree of environmental plasticity. cies de®nition to its practical limit'' (p. 224). Thompson The complexity of this group has resulted in much taxonomic 478 AMERICAN JOURNAL OF BOTANY [Vol. 91 confusion, which Grant (1924) lamented ``has become rather Mimulus brewerii (Greene) Cov.ÐThis species possesses burdensome'' (p. 109). tardily dehiscent corollas like members of Schizoplacus, a ped- icel length that was thought to be midway between both sub- ParadanthusÐThe section Paradanthus is most likely not genera, and an apically separating placenta like subgenus Mi- a natural group (Grant, 1924), a result supported by pollen mulus. Greene (1885) placed M. brewerii in Eunanus, Pennell data (Argue, 1980, 1986). Within this section are several al- (1951) and Vickery (1969) placed it in its own section (Mon- liances that have attracted taxonomic attention. In particular, imanthe), whereas Grant (1924) and Thompson (1993) placed an alliance of 13 species centering around the widespread M. it in Paradanthus. moschatus Lindley are noted for a high number of endemic species, large variation in breeding system (Meinke, 1992b; Mimulus pygmaeus GrantÐThis species possesses a differ- M. Carlson, Oregon State University, unpublished data), and ent capsule symmetry and anther development from typical problems in species delimitation (Hitchcock and Cronquist, members of Oenoe (Argue, 1980). Grant (1924) and Thomp- 1969; Argue, 1986; Thompson, 1993; Meinke, 1995b; Whit- son (1993) retained this species in Oenoe, whereas Pennell tall, 1999; Tibor, 2001). (1947, 1951) and Vickery (1969) placed it in its own section Microphyton. ErythrantheÐExtensive cytogenetic, hybridization, and transplant studies by Vickery (1978) and Hiesey et al. (1971) MATERIALS AND METHODS have con®rmed the close relationships of members of this sec- Taxon samplingÐAn attempt was made to sample all western North Amer- tion, which includes M. lewisii Pursh and M. eastwoodiae ican species of Mimulus, which proved dif®cult due to problems with syn- Rydb., two species that Grant (1924) had placed in section onymy. Overall, 115 accessions were sampled. In choosing species to sample, Paradanthus. The desert species M. parishii Greene was not we mostly followed the species designations of Thompson (1993) for Cali- included in these analyses, though both Greene (1885) and fornian taxa, with a few exceptions, and the descriptions of Hitchcock and Grant (1924) suggested a possible close relationship with M. Cronquist (1969), Meinke (1995a, b) and Ezell (1971) for taxa in the Paci®c lewisii. There is some confusion about the identity of popu- Northwest. Multiple accessions from distinct portions of the range in wide- lations in southwest Utah and northwest and central Arizona spread species are included. Fewer species occur in the Rocky Mountain with red corollas. Kearney and Peebles (1960) consider these states, and their speci®c designations are less controversial. Also sampled specimens as a variety of M. cardinalis whereas other workers were representative species from Chile (M. depressus Phil., M. cupreus Dom- (Vickery and Wullstein, 1987) maintain that they are a distinct brain, and M. luteus L.), and from Asia (M. nepalensis Benth., M. sessilifolius species (M. verbenaceus Greene). Estimates of relationships Maxim., M. bodinieri Vaniot, and M. tenellus Bunge). Three species from the among species and populations in Erythranthe were recently Australian and eastern North American clade of Mimulus (M. ringens L., M. made using polymorphic DNA markers (Beardsley et al., gracilis R. Br., and M. uvedaliae Benth) were identi®ed in previous studies 2003), which indicate that M. verbenaceus is distinct from M. as appropriate outgroups (Beardsley and Olmstead, 2002). For species used cardinalis and should retain its species status. in this study, voucher specimens, and GenBank accession numbers, see the Supplemental Data accompanying the online version of this article. Mimulus mohavensis LemmonÐMimulus mohavensis has a ϭ Molecular methodsÐThe modi®ed cetyltrimethyl ammonium bromide distinctive chromosome number (n 7; Thompson, 1993) and (CTAB) method of Doyle and Doyle (1987) was used to extract total genomic has been dif®cult to place taxonomically due its distinct co- DNA, which was further puri®ed using Qiagen Qiaquick spin-columns (Qia- rolla and tricolpate pollen (other members of Eunanus have gen, Valencia, California, USA). The trnL/F region was ampli®ed using the 5±7 pollen apertures) (Argue, 1980). Similar to M. pictus trn-c and trn-f primers (Taberlet et al., 1991). For taxa in which trnL/F did (Greene) Gray, this species has a distinctive white, rotate co- not amplify, the intron and spacer were ampli®ed separately, using the trn-c rolla with purple vein patterns that resemble a web. Grant and trn-d primers to amplify the trnL/F intron and trn-e and trn-f to amplify (1924) and Vickery (1969) placed this species in its own sec- the spacer. The entire ITS region was ampli®ed using the its4 and its5 primers tion (Mimulastrum), whereas Pennell (1947) placed it in Eu- (Baldwin, 1992). For taxa in which the entire region did not amplify, ITS 1 nanus, and both von Wettstein (1891) and Thompson (1993) and ITS 2 were ampli®ed separately, using the its5 and its2 primers for ITS placed it in section Mimulastrum with M. pictus, due to the 1 and the its3 and its4 primers for ITS 2. To amplify a portion of the ETS similarity of their corollas. region, we used the 3Ј 18S-IGS primer of Baldwin and Markos (1998) and the Mimulus speci®c 5Ј ETS-B primer of Beardsley and Olmstead (2002). Mimulus pictusÐGrant (1924) described this species as pos- Sequences of both strands of the PCR product were generated on an ABI 377 sessing a calyx similar to those in Diplacus; a style, stigma, (Applied Biosystems, Foster City, California, USA). To sequence the trnL/F and capsule similar to those of species within Oenoe; and a region, we used the internal primers of Taberlet et al. (1991), trn-d and trn- corolla like M. mohavensis. Grant (1924) and Vickery (1969) e, and the external trnL-2C and trnL-2F (Beardsley and Olmstead, 2002). The placed M. pictus in its own section (Pseudoenoe), Pennell ITS was sequenced using the external PCR primers, its4 and its5, and the two (1951) placed it in Oenoe, whereas von Wettstein (1891) and internal primers, its2 and its3. The ETS was sequenced using the 18S-E primer Thompson (1993) placed it in Mimulastrum. of Baldwin and Markos (1998), which is slightly internal to 18S-IGS primer, and the ETS-B primer. Ampli®cation and sequencing of the ITS region in the M. moschatus alliance was as described in Whittall (1999). Mimulus pilosus (Benth.) WatsonÐThe placement of this species has been complicated by its unique calyx, which is AnalysesÐConsensus sequences for trnL/F, ITS, and ETS were aligned irregular and has weak angles and partially separated sepals manually using the program Se-Al version 1 (A. Rambaut, University of Ox- (Argue, 1980). Greene (1886), von Wettstein (1891), and ford, Oxford, UK) (see Supplemental Data accompanying the online version Grant (1924) elevated this species to the monogeneric Mime- of this article). tanthe whereas other workers (Pennell, 1947, 1951; Vickery, The three DNA regions were analyzed both individually and in combination 1969; Thompson, 1993) placed it in its own section within using the program PAUP* 4.0b8a (Swofford, 1998). Trees were constructed Mimulus (sect. Mimuloides). for each DNA region separately and examined for hard topological incon- March 2004] BEARDSLEY ET AL.ÐWESTERN NORTH AMERICAN MIMULUS 479 gruencies. The phylogenetic position of only one accession, M. evanescens that this group possesses a shared mutation in one of the prim- Meinke, showed a well-supported difference among the three regions. Thus, er binding regions for ETS. M. evanescens was removed from the large-scale analysis. To further explore Analyses of 114 taxa, excluding M. evanescens, using max- the placement of M. evanescens, a smaller ingroup of 17 taxa, strongly sup- imum parsimony (MP) as the optimality criterion, recovered ported as being monophyletic in the large scale analysis, was constructed with over 25 000 most parsimonious trees, but only 25 000 were two outgroups and the data combinations from each DNA fragment were saved. These trees had a length of 3517 steps (consistency explored using the Incongruence Length Difference (ILD) test (Farris et al. index [CI] ϭ 0.485; retention index [RI] ϭ 0.876). When the [1994] as implemented in PAUP*). The following comparisons were made: consensus tree was used as a reverse constraint for 50 000 ITS vs. ETS, trnL/F vs. ITS, and ETS vs. trnL/F. For each test, 100 replicates random sequence additions, no trees were recovered that were were analyzed with a heuristic search, each with 10 random sequence addition either shorter or the same length. One of the 25 000 MP trees replicates. Comparisons were made both including and excluding M. evanes- is depicted in Figs. 1 and 2. The analysis provided robust cens. All subsequent searches were completed using the combined data from all support for many of the relationships among species of the three DNA regions. Parsimony searches were conducted using heuristic western North American Mimulus. The major clades recovered searches. Gaps were scored as missing data. In cases where greater than are discussed in the following section. 25 000 most parsimonious trees existed, 25 000 trees were saved and a strict Results of the ILD test for the smaller analysis of 17 taxa, consensus tree was generated. The consensus tree was used as a reverse con- including M. evanescens, were as follows: ITS vs. trnL/F, P straint in additional searches made using 50 000 random sequence addition ϭ 0.23; ITS vs. ETS, P ϭ 0.01; and ETS vs. trnL/F, P ϭ replicates, tree bisection and reconnection (TBR) swapping and MULTREES 0.01. The results when M. evanescens was excluded were: ITS off (Catalan et al., 1997; Rice et al., 1997). Relative support for individual vs. ETS, P ϭ 0.11; and ETS vs. trnL/F, P ϭ 0.22, indicating branches was estimated using bootstrap values (Felsenstein, 1985). Bootstrap that M. evanescens was the source of the incongruence. Data values were calculated using 100 replicate full heuristic searches with MUL- from the different DNA regions were combined, with the ex- TREES on and TBR branch swapping. ception of M. evanescens, for which the ITS and trnL/F data were combined and considered separately from the ETS data. Chromosome countsÐPublished chromosome counts (Chuang and Heck- Analysis of this data set using MP resulted in two trees of ard, 1992; Meinke, 1992b; Thompson, 1993; Vickery, 1995, 1997) were length 621 steps, CI ϭ 0.850, RI ϭ 0.739, and rescaled con- mapped onto inferred trees and parsimony was used to optimize changes in sistency index (RC) ϭ 0.628. One of the two MP trees is chromosome number. It was assumed that a two-fold difference in the chro- shown in Fig. 3. mosome number between taxa implied a polyploid event that could only in- Figures 4 and 5 show the parsimony reconstructed chro- crease the total number of chromosomes and not the reverse. mosome numbers for many of the Mimulus taxa and the in- ferred chromosomal events that have occurred in the evolution RESULTS of western North American Mimulus. From this analysis, 13 polyploid and 15 aneuploid events are inferred and the inferred The manual alignment of the trnL/F sequences required nu- base chromosome number for the clade is n ϭ 8. merous short gaps to account for putative insertions and de- letions. The total aligned length of the trnL/F region was 1099 DISCUSSION bases. The trnL/F alignment had 398 variable and 227 parsi- mony informative sites. We were unable to amplify trnL/F in Recovered clades and morphological evolutionÐMolecu- 14 accessions: M. torreyi Gray 2, both accessions of M. pi- lar data support the monophyly of Mimulus in western North losus, M. pygmaeus, M. aurantiacus ssp. australis (McMinn) America, if the genera Leucocarpus, Hemichaena, and Ber- Munz, M. longi¯orus (Nutt.) Grant, M. montiodes (Gray) 1, endtiella are included. The major clades recovered in this anal- M. nelsonii Grant, M. eastwoodiae, M. verbenaceus, M. bod- ysis (Figs. 1 and 2) are described below. Percentages refer to inieri, M. tenellus, M. dentatus Benth., and M. sessilfolius.We bootstrap values (Felsenstein, 1985). were only able to amplify the intron for Berendtiella. The total aligned length of the ITS region was 654 bases. Clade AÐThis clade (100%) was recovered by Beardsley The ITS alignment had 331 variable and 252 parsimony in- and Olmstead (2002) and includes Hemichaena, Berendtiella, formative sites. We were not able to obtain ITS sequences for and members of Mimulus subgenus Schizoplacus. A morpho- ®ve accessions: both accessions of M. pilosus, M. pygmaeus, logical synapomorphy for the group is a placenta that is di- M. brewerii, and M. sessilfolius. We were only able to obtain vided to the base. Clade A contains Clades B, C, D, E, and F. data for ITS 2 for M. viscidus Congdon., M. rattani Gray, M. tricolor Lindley, M. angustatus Gray, M. congdonii Robinson, Clade BÐThis group (100%) consists of the genera Hemi- and M. rupicola Cov. & Grant. The dif®culty in amplifying chaena and Berendtiella. Thieret (1972) suggested that Hem- ITS 1 in many species is due to a unique stem-loop structure ichaena and Berendtiella could be distinguished from other in this region (Whittall, 1999), a problem that was sometimes closely related genera by their bracteolate cymose in¯ores- overcome by using 5% dimethyl sulfoxide (DMSO) in both cence. ampli®cation and sequencing. The aligned ETS sequences were 480 bp in length. The ETS Clade CÐThe Schizoplacus group (sensu Beardsley and alignment had 373 variable and 293 parsimony informative Olmstead, 2002) (100%) contains sections Oenoe, Eunanus, sites. We were not able to amplify ETS in M. androsaceus and Diplacus. Species in this group generally have a persistent Greene, M. purpureus Grant, M. shevockii Heckard & Baci- corolla and a pedicel that is longer than the calyx. The taxo- gal., and M. brewerii. Though one should interpret negative nomically dif®cult M. pilosus was found to be sister to all PCR results with great caution, data from the other two DNA other Mimulus in this clade. Mimulus rupicola and M. parryi regions indicate that M. androsaceus, M. purpureus, and M. Gray are sister species (100%) and are sister to the remainder shevockii form a clade (100% bootstrap), which may indicate of this clade. A close relationship between these taxa has not 480 AMERICAN JOURNAL OF BOTANY [Vol. 91

Fig. 1. One of 25 000 most parsimonious (MP) trees resulting from the analysis of combined trnL/F, ITS, and ETS data with M. evanescens excluded. Numbers on the branches indicate bootstrap values estimated using parsimony. Capital letters denote groups mentioned in the text. Rare species or species that are reported to contain rare subspecies that have a global Natural Heritage designation of G1, G2, or G3 are noted to the right of the taxon name. Question marks indicate groups in which species designation is not clear and may contain rare species or subspecies. The reconstructed phylogeny is continued in Fig. 2. been previously suggested. The base chromosome number of 1). Mimulus mohavensis is derived from within the Eunanus M. rupicola (n ϭ 8) is different than the base chromosome group. Bentham (1876) originally proposed that species in Eu- number of all the species assigned to sect. Oenoe (n ϭ 9) nanus be recognized at the generic level based on their having (Thompson, 1993). Mimulus mohavensis and M. pictus both a divided placenta, pubescent styles, and small, nearly regular, belong in this clade, but are not closely related. Their radial, corollas. Grant (1924) pointed out, however, that these char- salverform corollas, which are white with a purple weblike acters are of dubious quality. All of the species in Schizoplacus pattern, thus represent a case of convergent ¯oral evolution. have a divided placenta, making this character a pleisiomorphy Clade C contains Clades D, E, and F. for Eunanus. Some species in Eunanus have relatively large, strongly zygomorphic corollas (e.g., M. brevipes). Finally, Clade DÐSection Eunanus was found to be monophyletic many other species in Schizoplacus have pubescent styles. (100%) with the exclusion of M. parryi and M. torreyi (Fig. The results further suggest that patterns of genetic diversi- March 2004] BEARDSLEY ET AL.ÐWESTERN NORTH AMERICAN MIMULUS 481

Fig. 2. A continuation of the phylogeny presented in Fig. 1.

®cation in Eunanus are not congruent with patterns suggested the north-central Sierra Nevada groups with the northern by the current classi®cation (Fig. 1). Mimulus nanus is not clade). Little resolution exists amongst Eunanus species in the monophyletic in two distinct ways. First, a cluster of individ- Sierra Nevada and Transverse mountain ranges. uals from the Sierra Nevada (100%) is distinct from another cluster from the Cascade Mountains in Oregon and Washing- Clade EÐTaxa within Oenoe fall into two well-supported ton and the Rocky Mountains in Montana (78%). Second, M. clades. One clade (99%) contains M. pygmaeus, M. angusta- nanus in the Northwest is paraphyletic, because three currently tus, and M. pulchellus (Greene) Grant. A second clade (99%) recognized species, M. cusickii (Greene) Rattan, M. jepsonii, of taxa from Oenoe contains M. douglasii (Benth.) Gray, M. and M. clivicola Greenm. have been derived from within it. congdonii, and M. kelloggii (Greene) Gray. Sister to this clade Only one individual from both M. jepsonii and M. clivicola is the taxonomically dif®cult taxon M. torreyi. This larger and two individuals from M. cusickii were sampled, however, clade is sister to Diplacus (Clade F) and, thus, Oenoe is weak- so the monophyly of each of the derivative species cannot ly supported (48%) as paraphyletic. Relationships among Oen- currently be addressed. The results also indicate that all Eu- oe species are strongly supported. Mimulus rupicola was not nanus species from the Paci®c Northwest and Rocky Moun- included in either of the two Oenoe clades. tains are differentiated from species that occur throughout the Sierra Nevada and Transverse mountain ranges in California Clade FÐSection Diplacus is found to be monophyletic (53%), with only one exception (M. jepsonii, sampled from (100%) and derived from within western North American Mi- 482 AMERICAN JOURNAL OF BOTANY [Vol. 91

phological synapomorphy of calyces with strongly developed rib angles, sometimes called corky (Grant, 1924; Pennell, 1951; Heckard and Shevock, 1985). Clade H contains a rela- tively large number of rare taxa. Four of the 10 Mimulus spe- cies identi®ed by the California Native Plant Society (CNPS) (Tibor, 2001) as being rare, threatened, or endangered through- out their range are in this clade as are M. rupestris Greene (known from one population, Vickery and Wullstein, 1987) and M. eastwoodiae (rare in Colorado; Spackman et al., 1999).

Clade IÐSection Erythranthe is supported as monophyletic (81%), a result con®rmed in other studies (Beardsley and Olm- stead, 2002; Beardsley et al., 2003). In her monograph, Grant (1924) based sect. Erythranthe on an extremely bilabiate co- rolla, exserted style and stamens, and hairy stamens. Her choice of these characters led her to exclude M. lewisii and M. eastwoodiae from the section. Mimulus lewisii and M. eas- twoodiae have since been transferred back to Erythranthe (Pennell, 1951) and extensive cytogenetic, hybridization, and transplant studies by Vickery (1978) and Hiesey et al. (1971) have con®rmed the close relationship of these species, but have failed to provide synapomorphies for the section. The results presented here suggest that Erythranthe is monophy- letic if M. parishii is included. They also suggest that M. lew- Fig. 3. One of two MP trees resulting from the analysis of combined trnL/ isii and M. cardinalis are very closely related and have di- F, ITS, and ETS data for clade K, except for M. evanescens in which the verged recently. Phylogenetic relationships among taxa in Er- ETS data are considered separately from the combined ITS/trnL/F data. An asterisk indicates the node that collapses in the strict consensus of MP trees. ythranthe are unresolved on the basis of ITS, ETS, and trnL/ Numbers above the branches indicate bootstrap values estimated using par- F sequences; however, analysis of ampli®ed fragment length simony. polymorphisms (AFLPs) provided well-supported estimates of relationships (Beardsley et al., 2003). mulus (Fig. 1). Patterns of genetic diversi®cation are not con- Clade JÐSection Simiolus is monophyletic (100%) includ- gruent with those suggested by the current classi®cation (see ing a clade (93%) comprising the three Chilean Simiolus spe- below). cies. Species in the Simiolus clade possess a mature calyx that is much in¯ated with lower teeth that turn up and fold over Clade GÐThis well-supported group (92%) contains the the lateral teeth, nearly closing the ori®ce, and a personate segregate genus Leucocarpus, plus Mimulus sections Ery- corolla with two prominent hairy ridges down the lower lip thranthe, Simiolus, and non-Australian members of Paradan- (Grant, 1924). Yellow corolla color is not a consistent char- thus. Leucocarpus is sister to the rest of the Mimulus taxa in acter, because some Chilean Simiolus species have cream- or clade G. The one species in Leucocarpus has a baccate, in- pink-colored corollas. The relatively low genetic distances dehiscent fruit that is derived from the dehiscent capsules amongst taxa indicate that this clade is relatively young and found in most species in Phrymaceae sensu Beardsley and that its constituent species are very closely related. Olmstead (2002). Burtt (1965) describes the fruit as being a white berry, with thin skin and with most of the substance of Clade KÐThis strongly supported group (100%) consists of the fruit derived from the ¯eshy placenta. Within the Mimulus two major clades. The ®rst (54%) contains M. latidens (Gray) portion of this clade, M. inconspicuus (Gray) is sister to the Greene and a well-supported clade (89%) previously recog- clade comprising the rest of the Mimulus species (99%). Mi- nized as the M. moschatus alliance (Whittall, 1999). This clade mulus dentatus (Paci®c Northwest) and M. sessilifolius (Japan) contains taxa primarily endemic to the Paci®c Northwest, a are sister species (97%), but not otherwise closely associated large percentage of rare or threatened taxa, and substantial with any other group. Some species (e.g., M. gemmiparus We- variation in breeding systems (Meinke, 1995b; Whittall, 1999). ber and M. alsinoides Benth.), without evident morphological If Thompson's (1993) synonymy of M. dudleyi Grant with M. synapomorphies linking them to any other group, also are not ¯oribundus Lindley is accepted, M. ¯oribundus would be par- strongly united with any of the well-supported clades on the aphyletic because M. norrisii Heckard & Shevock has been basis of the molecular data. Clade G contains Clades H, I, J, derived from within it. However, M. dudleyi is differentiated and K. both morphologically and geographically from the polymor- phic and widespread M. ¯oribundus. Sister to the M. moscha- Clade HÐThis clade (100%) contains taxa in Paradanthus tus alliance is a well-supported clade (80%) that is native to mostly from the Sierra Nevada and southern California Asia and Japan. [though M. suksdor®i (Gray), M. brewerii, M. rubellus (Gray), and M. primuloides Benth. have wider distributions] and Er- Systematic conclusionsÐ(1) Mimulus in western North ythranthe (Clade I), which is derived from within Paradan- America is not monophyletic because Leucocarpus, Berend- thus. Mimulus ®licaulis Watson and M. bicolor Benth. are re- tiella, and Hemichaena are derived from within this clade. solved as sister species (61%), a result supported by the mor- Given the data presented here and previously (Beardsley and March 2004] BEARDSLEY ET AL.ÐWESTERN NORTH AMERICAN MIMULUS 483

Fig. 4. Mapped chromosome numbers for many of the Mimulus taxa and the inferred chromosomal events using MP that have occurred in the evolution of western North American Mimulus. Shaded boxes indicate inferred polyploid events. Open boxes indicate inferred aneuploid events. Lowercase letters indicate the reference for the chromosome numbers. (a) Vickery (1995); (b) Thompson (1993); (c) Meinke (1992b); (d) Chuang and Heckard (1992); (e) Vickery (1997). The reconstruction of chromosomal events is continued in Fig. 5.

Olmstead, 2002), it is clear that generic assignments need to in Mimulus, which supports its recognition as a section within be reconsidered in Mimulus. This comprehensive work will be Mimulus and argues against its recognition as a segregate ge- presented in a future publication and will include the clade of nus. Oenoe (excluding M. rupicola and including M. torreyi) mostly Australian Mimulus, which contains the eastern North is paraphyletic, with Diplacus being derived from within it American type species M. ringens. (although a monophyletic Oenoe is only slightly less parsi- (2) The following sections are monophyletic: Diplacus, Er- monious). Paradanthus is not monophyletic and needs exten- ythranthe (including M. parishii), Simiolus (excluding M. gem- sive revision. These results provide no support for Mimulas- miparus), and Eunanus (excluding M. parryi and M. torreyi trum, because M. mohavensis and M. pictus are not closely and including M. mohavensis). Diplacus is derived from with- related. 484 AMERICAN JOURNAL OF BOTANY [Vol. 91

Fig. 5. A continuation of the reconstruction of chromosomal events presented in Fig. 4. Uppercase letters represent equally parsimonious changes. The change indicated by (A) could have taken place on either of the two branches. A change from n ϭ 15 to n ϭ 14 could have occurred at either (B) or (C). A change from n ϭ 30 to n ϭ 31 could have occurred at (D) or (E). The change at (F) occurred on the branch leading to M. brewerii, but no branch length is represented; therefore, the bar is drawn next to the species name.

(3) Mimulus mohavensis is derived from within Eunanus. within the North American Mimulus clade, accompanied by This species possesses a large number of morphological au- 15 aneuploid events. Clade A, which contains species in sub- tapomorphies, including a unique base chromosome number genus Schizoplacus plus Hemichaena and Berendtiella, differs (n ϭ 7) (Vickery, 1995). Mimulus pictus does not cluster in from Clade G, which contains Mimulus species in Erythranthe, any of the larger recognized clades. It is weakly supported as Simiolus, and Paradanthus plus Leucocarpus, in the frequency sister to the clade containing Oenoe and Diplacus. Mimulus of chromosomal events. In Clade A, only one polyploid event brewerii is closely related to other taxa in the nonmonophy- and three aneuploid events are inferred. At least 12 polyploid letic Paradanthus, including M. rubellus, as suggested by and 12 aneuploid events are inferred to have occurred in Clade Grant (1924). Mimulus pygmaeus is closely related to other G. Most of the chromosomal rearrangements have taken place taxa in Oenoe, making its unusual capsule symmetry and an- in Simiolus (Vickery, 1995). The documentation of a strong ther development (Argue, 1980) autapomorphic. phylogenetic component for the nonrandom clustering of chro- mosomal changes has also been made in Downingia (Schulth- Chromosomal evolutionÐInferred patterns of chromosom- eis, 2001). In this case, one main clade of Downingia remained al evolution in western North American Mimulus are shown uniformly n ϭ 11 whereas the second main clade was highly in Figs. 4 and 5. The ancestral chromosome number was in- variable for chromosome number. ferred to be n ϭ 8. Though the presence of multiple chro- Polyploidy is often cited as a major mechanism for plant mosome numbers in an individual species can make the in- speciation (Stebbins, 1971; Grant, 1981). Based on an exam- ference of the total number of changes dif®cult, we estimate ination of the chromosome numbers in different sections, that at least 13 independent polyploid events have occurred Vickery (1995) suggested that speciation by aneuploidy and March 2004] BEARDSLEY ET AL.ÐWESTERN NORTH AMERICAN MIMULUS 485 polyploidy was extensive in Mimulus. The relative importance been an area of intense debate for well over a century (Steb- of these two processes can be estimated in light of the given bins, 1971; Grant, 1981). Within the genus Mimulus, hybrid- phylogeny. In this analysis, 102 nodes, which represent spe- ization has been hypothesized to be an important contributor ciation events, were resolved. The role of ploidy in speciation to diversity (Thompson, 1993). Of the 115 Mimulus taxa sam- can be calculated in different ways. Adherents of the biological pled in this analysis, however, only one species demonstrated species concept would argue that polyploid and aneuploid strong evidence for discordance among gene regions from dif- forms represent distinct species due to reproductive isolation ferent genomes. These results indicate that hybridization in from diploid forms. Vickery (1978) has documented many ex- this group may not play as strong a role as previously believed amples of low compatibility between Mimulus with different in the establishment of persistent lineages. However, the rate ploidy levels. In this case, the numbers of polyploid and an- of hybridization in Mimulus may be underestimated because euploid events simply need to be summed and divided by the of the dif®culty in detecting ancient hybridization (Sang and total number of speciation events. In Mimulus, nodes would Zhong, 2001) or a lack of resolution at the species level in need to be added to the total number obtained for cases in some clades (e.g., Simiolus). For example, some of the 13 which only one individual was sampled for a named species reconstructed polyploid events may have been the result of that contains multiple ploidy levels. This would result in at allopolyploidy, though Ramsey and Schemske (1998) suggest least 11 additional nodes. Using this approach, polyploidy that rates of autopolyploid formation are often higher than the could have played a role in 13/113 (11.5%) speciation events rate of allopolyploid formation. Reconstructing individual and aneuploidy in 15/113 (13.3%) speciation events. It is also polyploid origins in Mimulus deserves more attention. possible that a switch to a polyploid or aneuploid lineage oc- curred after the initial divergence of two lineages and did not Patterns of genetic variationÐThe term cryptic genetic play a role in the speciation event. Our estimates may therefore variation has been applied to species that contain distinct ge- in¯ate the relative frequency of these two processes. netic breaks without phenotypic differentiation. These distinct It is also possible to argue that evolutionary biologists are genetic lineages are often geographically based. Patterns of most interested in the frequency with which polyploidy or an- cryptic genetic variation have been documented in a number euploidy gives rise to persistent lineages. An examination of of animal species (Avise, 2000; Omland et al., 2000) but such Figs. 4 and 5 reveals that a large majority of the inferred chro- phylogeographic studies have been infrequent in plants (re- mosomal events occur near the tips of the tree. Of the 13 viewed by Baldwin, 2000). Though sampling in this study was polyploid events reconstructed, only two give rise to lineages designed to investigate interspeci®c relationships within Mi- with greater than four descendant taxa. For aneuploidy, only mulus, multiple accessions from widespread, dif®cult taxa three aneuploid events out of 15 give rise to lineages with highlight the insights to be gained from collecting genetic data greater than four descendant taxa. Given these estimates, poly- in plant groups that have been dif®cult taxonomically (Bald- ploidy and aneuploidy have played a relatively small role in win, 2000). the evolution of persistent lineages in Mimulus, though a num- Diplacus provides an example in which patterns of genetic ber of extant taxa (e.g., Simiolus) are currently ``experiment- diversity do not match patterns of morphological variation or ing'' with polyploid and aneuploid populations. patterns suggested by named taxa, and, depending on how spe- cies are de®ned in the group, may be an example of a para- DiscordanceÐHybridization can be detected by systema- phyletic species. There has been considerable disagreement tists through careful analyses of discordance between different over taxon delimitation within Diplacus, with the number of gene trees (Rieseberg and Soltis, 1991; Sang and Zhong, species de®ned in the section varying from two to 13. Popu- 2001). Discordance for the relationship of M. evanescens ex- lations within Diplacus differ morphologically and ecologi- ists between the ETS tree and both the ITS and trnL/F tree cally (McMinn, 1951; Beeks, 1962). Diplacus is found in at (which are congruent with each other). The ITS and trnL/F least 12 of the approximately 29 Californian plant communi- data strongly support (100%) M. evanescens as sister to M. ties (Beeks, 1962). When growing in undisturbed, native hab- brevi¯orus Piper whereas the ETS data strongly support itats, morphologically divergent populations are hypothesized (100%) a sister-group relationship between M. evanescens and to be ecologically isolated (Tulig and Clark, 2000). However, M. latidens. Discordance in this case may be attributed to hy- where populations are sympatric in disturbed areas or when bridization followed by a recombination event in the rDNA morphologically divergent populations co-occur in areas near which was subsequently ®xed through the process of concerted ecological transition zones, hybridization is suggested to be evolution (Arnheim, 1983). When Meinke (1995a) ®rst de- common (Waayers, 1996). Beeks (1962) demonstrated that scribed M. evanescens, he proposed a close relationship to three Diplacus taxa in southern California were speci®c to both M. latidens and M. brevi¯orus. In a common garden certain environments but hybridization was common in dis- study, Meinke measured 18 morphological characters in a total turbed habitats. Similarly, Waayers (1996) found that M. pun- of 38 populations of the three species. In a principal compo- iceus (Nutt.) Steudel and M. aurantiacus ssp. australis formed nents analysis, M. evanescens was consistently and clearly a hybrid zone only in transitional habitats and that strong se- morphologically intermediate between the other two species. lection for species-speci®c ¯oral characters existed outside the These results led Meinke (1995a) to suggest that M. evanes- transition zone. Some Diplacus populations are suggested to cens may be a hybrid between M. brevi¯orus and M. latidens. have morphological traits that are typically associated with dif- The discordant gene trees that we recovered support this hy- ferent pollinators leading to the proposition that pollinator pothesis. An alternative explanation is that lineage sorting of preference may maintain these taxa (Grant, 1993). However, ancestral polymorphisms has occurred, though the survival of the effectiveness of pollinator preference as a potential repro- this polymorphism through a minimum of ®ve speciation ductive isolating barrier in Diplacus is unknown. events is unlikely. Though a much more extensive survey of populations of The frequency and importance of hybridization in plants has Diplacus is necessary to accurately represent patterns of evo- 486 AMERICAN JOURNAL OF BOTANY [Vol. 91 lution in Diplacus, our results suggest some preliminary con- ical change. All of these plants are small (mostly less than 10 clusions. Patterns of genetic diversi®cation are not consistent cm, though M. cusickii tends to be larger than the other taxa with named taxa, a hypothesis supported by preliminary data [Thompson, 1993]), annuals, and possess similarly shaped co- from AFLPs (P. Beardsley, unpublished data). Four of Munz's rollas that are different shades of magenta. Differences (1974) species (M. puniceus, M. bi®dus Pennell [including ssp. amongst taxa are primarily in leaf margins, leaf shape, pedicel fasciculatus Pennell], M. aurantiacus [including ssp. austral- length, general plant size, and the degree of anther exertion. is], and M. longi¯orus [including ssp. calycinus (Eastw.) A greater amount of morphological change has occurred in the Munz]) were represented by more than one individual, yet the mostly Californian clade. Two species, M. brevipes Benth. and two representatives of the same species clustered together only M. bolanderi Gray, have individuals that can be up to 90 cm in M. longi¯orus. Mimulus aurantiacus sensu Thompson does in size. In addition, corolla color varies from white to yellow not correspond to a monophyletic group because M. clevelan- to many shades of magenta. Corolla shape also varies from dii, which is the only other species in Diplacus recognized by the nearly rotate corollas of M. mohavensis to the large, Thompson, is strongly supported as being derived from within strongly zygomorphic corolla of M. brevipes. it, making M. aurantiacus sensu Thompson a paraphyletic spe- Observations in bird lineages (e.g., ravens [Omland et al., cies. A comparable lack of concordance between patterns of 2000]; and chickadees [Gill et al., 1999]) suggest that cryptic genetic diversity and named taxa have been documented in genetic variation and paraphyletic species may be more com- other perennial, shrubby groups, such as oaks (Whittmore and mon in groups with some widespread species that possess few Schall, 1992; Petit et al., 1997) and rhododendrons (Kron et morphological changes. The widespread species M. nanus was al., 1993; Milne et al., 1999). found to be paraphyletic in the Paci®c Northwest because M. Diplacus exhibits a relatively high amount of genetic di- cusickii, M. jepsonii, and M. clivicola have been derived from vergence among accessions. The ITS sequences exhibit 1±2% within it. Rieseberg and Brouillet (1994) suggest that para- divergence and ETS sequences show 5±7% divergence. Data phyletic species may be more common in plants due to a high- from the chloroplast trnL/F show less diversity with 0±1% er frequency of different modes of speciation. The discovery (®ve parsimony informative sites) and one prominent inser- of more highly polymorphic gene regions in plants will enable tion. This level of divergence is comparable to that seen in more comparisons at the intraspeci®c level, which will allow Simiolus (ITS 0±2%, ETS 0±6%, and trnL/F 0±2%) and great- greater opportunities to understand the nature of plant species. er than that in Erythranthe (ITS 0±2%, ETS 0±2%, and trnL/ The taxonomic status of M. nanus and its related species is F 0±1%). These data indicate that populations of Diplacus dif®cult and controversial (Ezell, 1970; Thompson, 1993, have experienced signi®cant periods of reproductive isolation. 1997). The results of this study indicate that species bound- The lack of correlation between the genetic data and mor- aries need to be reconsidered. A species concept that requires phology suggested by named taxa combined with the relatively a strict adherence to monophyly for species recognition (Mish- high levels of genetic divergence support the hypothesis that ler and Donoghue, 1982; de Queiroz and Donoghue, 1988) previously separated populations of Diplacus, representing dif- would result in a very different classi®cation and may have ferent named taxa, are now expanding their range and expe- implications for taxa that are now listed for protection on the riencing secondary contact. basis of the Endangered Species Act (e.g., Mimulus clivicola The patterns of genetic variation in Eunanus recovered in [Mancuso and Cooke, 2002]). this analysis demonstrate signi®cant cryptic genetic diversity Thompson (1993) reduced M. dudleyi and M. arenarius that does not correlate with current species delimitations based Grant to synonymy under M. ¯oribundus. An accession of M. on morphology. With only one exception, all of the Eunanus dudleyi was sampled in this study and was strongly supported taxa in the Cascade mountains of the Paci®c Northwest and (98%) to be in a clade with M. ¯oribundus and M. norrisii, in the Rocky Mountains, including M. nanus, form a clade which were strongly supported (99%) as being sister to each that is distinct from all the Eunanus taxa that occur mostly in other, making M. ¯oribundus sensu Thompson paraphyletic. In the Sierra Nevada and southern California, including a cluster light of these results, we support the recognition of M. dudleyi of three M. nanus accessions from the Sierra Nevada. The only as distinct from M. ¯oribundus. The status of M. arenarius, exception is M. jepsonii, sampled from Placer County, Cali- unsampled in this study but likely allied to M. ¯oribundus due fornia, in the northern-central Sierra Nevada, which groups to its glandular pubescence, cup-shaped calyx, and southern with the Paci®c Northwest/Rocky Mountain clade. In Eunan- Sierra Nevada distribution, should also be reexamined. us, patterns of genetic diversity more closely match geograph- ical patterns than would be suggested by the current taxonomy. RarityÐThe clade of Mimulus in western North America Strong phylogeographic clustering of accessions that contra- contains many species that are rare or threatened (Table 1). dict morphological and taxonomic patterns are reported in Las- Soltis and Gitzendanner (1999) suggest that systematists need thenia (Chan et al., 2002) and Downingia (Schultheis, 2001). to play a larger role in the conservation of rare species by A similar phylogeographic pattern involving a Sierran/Paci®c identifying lineages worthy of protection. Large-scale analyses Northwest and Rocky Mountain division has also been docu- with complete or nearly complete sampling at the species level mented for populations of M. lewisii (Beardsley et al., 2003). of groups that are dif®cult taxonomically are important for the Using AFLPs, nearly all of the M. lewisii from the Sierra Ne- identi®cation of species or clades that are evolutionarily iso- vada form a well-supported clade and nearly all the M. lewisii lated and patterns of cryptic biodiversity, as discussed in the from the Cascades and the Rockies form a separate well-sup- preceding section. Species in Mimulus in the western United ported clade. The species of the M. moschatus alliance also States that are classi®ed by the Natural Heritage Program as follow strong phylogeographic patterns (discussed later). globally imperiled with ®ve or fewer occurrences (G1), 6±20 Morphological changes differ between the geographically occurrences (G2), or very rare and local throughout its range separate clades in Eunanus. Eunanus taxa from the Paci®c with 21±100 occurrences (G3) are mapped onto the inferred Northwest and the Rockies have undergone little morpholog- phylogeny (Figs. 1 and 2). Rare species are found in many March 2004] BEARDSLEY ET AL.ÐWESTERN NORTH AMERICAN MIMULUS 487 places throughout the phylogeny. The rare species M. gem- BALDWIN, B. G. 1992. Phylogenetic utility of the internal transcribed spacers miparus (G2) and M. pictus (G2) appear to be relatively iso- of nuclear ribosomal DNA in plants: an example from the Compositae. lated genetically. Both taxa possess morphological and eco- Molecular Phylogenetics and Evolution 1: 3±16. BALDWIN, B. G. 2000. Roles for modern plant systematics in discovery and logical attributes not found in any other Mimulus species. Mi- conservation of ®ne-scale biodiversity. MadronÄo 47: 219±229. mulus gemmiparus, known from only eight populations in the BALDWIN,B.G.,AND S. MARKOS. 1998. Phylogenetic utility of the external Colorado Rocky Mountains (Beardsley, 1997), reproduces ex- transcribed spacer (ETS) of 18S±26SrDNA: congruence of ETS and ITS clusively by asexual bulbils formed from supernumerary buds trees of Calycadenia (Compositae). Molecular Phylogenetics and Evo- in the axils of leaves (Moody et al., 1999). This particular lution 10: 449±463. method of reproduction and development is unique within Mi- BALDWIN, B. G., M. J. SANDERSON,J.M.PORTER,M.F.WOJCIECHOWSKI, mulus and probably all angiosperms. C. S. CAMPBELL, AND M. J. DONOGHUE. 1995. The ITS region of nu- clear ribosomal DNA: a valuable source of evidence on angiosperm phy- The M. moschatus alliance contains a relatively large num- logeny. Annals of the Missouri Botanical Garden 82: 247±277. ber of rare or threatened taxa that have strongly supported BALDWIN, B. G., AND B. L. WESSA. 2000. Origin and relationships of the phylogeographic patterns. One well-supported clade (100%) tarweed±silversword lineage (Compositae±Madiinae). American Journal contains M. ampliatus Grant (G1), M. hymenophyllus Meinke of Botany 87: 1890±1908. (G1), and M. patulus Pennell (G2/G3), which are all located BARRACLOUGH,T.G.,AND S. NEE. 2001. Phylogenetics and speciation. in the Snake River drainage of the Paci®c Northwest. A second Trends in Ecology and Evolution 16: 391±399. clade (100%) combines M. jungermannioides Suksd. (G2) and BEARDSLEY, M. R. 1997. Colorado's rare endemic plant, Mimulus gemmi- parus, and its unique mode of reproduction. Master's thesis, Colorado M. washingtonensis Gand., previously identi®ed as a sensitive State University, Fort Collins, Colorado, USA. species but no longer listed by the Oregon Natural Heritage BEARDSLEY, P. M., AND R. G. OLMSTEAD. 2002. Rede®ning Phrymaceae: the Program (Kagan et al., 2001), which both occur in the Colum- placement of Mimulus, tribe Mimuleae, and Phryma. American Journal bia river drainage. Mimulus evanescens (G2), a putative hybrid of Botany 89: 1093±1102. between M. latidens and M. brevi¯orus, is also closely related BEARDSLEY, P. M., A. YEN, AND R. G. OLMSTEAD. 2003. AFLP phylogeny to the M. moschatus alliance and is only known from 10 his- of Mimulus section Erythranthe and the evolution of hummingbird pol- toric localities that are threatened by private land use practices lination. Evolution 57: 1397±1410. BEAUVAIS, G., P. ORTEGON,G.JONES,D.KEINATH,B.HEIDEL,A.REDDER,R. (Meinke, 1995a). SMITH, AND J. BENNETT. 2002. Wyoming natural diversity database: species A mostly southern California clade of Paradanthus taxa of special concern. Wyoming Natural Diversity Database, website: http:// also contains a relatively large number of rare taxa. In this uwadmnweb.uwyo.edu/wyndd/PDF࿞®les/Other/Plants࿞Animals࿞ group, one well-supported clade (100%) contains the wide- 12June02.pdf. spread M. androsaceus and the rare M. shevockii (G2) and M. BEEKS, R. M. 1962. Variation and hybridization in Southern California pop- purpureus (G2). Mimulus shevockii is known from fewer than ulations of Diplacus (Scrophulariaceae). Aliso 5: 83±122. 10 populations (Heckard and Bacigalupi, 1986) and occurs in BENTHAM, G. 1876. Scrophulariaceae. In G. Bentham and J. D. Hooker [eds.], Genera Plantarum, vol. 2, 946±958.Reeve and Company, London, Joshua tree-xeric conifer forests within the vicinity of Lake UK. Isabella in the southern Sierra Nevada-desert transition zone. BRADSHAW, H. D., JR., K. G. 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