American Journal of Botany 91(7): 1007±1021. 2004.

INVITED SPECIAL PAPER

PHYLOGENETIC RECONSTRUCTION OF THE EVOLUTION OF STYLAR POLYMORPHISMS IN ()1

SEAN W. G RAHAM2,4 AND SPENCER C. H. BARRETT3

2UBC Botanical Garden and Centre for Research, 6804 SW Marine Drive, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4; and 3Department of Botany, University of Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2

We investigated the origin of stylar polymorphisms in Narcissus, which possesses a remarkable range of stylar conditions and diverse types of ¯oral morphology and pollination biology. Reconstruction of evolutionary change was complicated by incomplete resolution of trees inferred from two rapidly evolving chloroplast regions, but we bracketed reconstructions expected on the fully resolved plastid- based tree by considering all possible resolutions of polytomies on the shortest trees. Stigma-height dimorphism likely arose on several occasions in Narcissus and persisted across multiple speciation events. As proposed in published models, this rare type of stylar polymorphism is ancestral to distyly. While there is no evidence in Narcissus that dimorphism preceded tristyly, a rapid transition between them may explain the lack of a phylogenetic footprint for this evolutionary sequence. The single instances of distyly and tristyly in Narcissus albimarginatus and N. triandrus, respectively, are clearly not homologous, an evolutionary convergence unique to Amaryllidaceae. Floral morphology was likely an important trigger for the evolution of stylar polymorphisms: Concentrated-changes tests indicate that a long, narrow ¯oral tube may have been associated with the emergence of stigma-height dimorphism and that this type of tube, in combination with a deep corona, likely promoted, or at least was associated with, the parallel origins of heterostyly.

Key words: ancestral-state reconstructions; concentrated-changes test (CCT); ¯oral evolution; heterostyly; Narcissus; ndF; pol- lination biology; stigma-height dimorphism; stylar polymorphism; trnL-trnF.

Flowering possess extraordinary diversity in repro- arrangement of sex-organ heights. The reciprocal herkogamy ductive traits, even among closely related species. This vari- that characterizes heterostyly is a structural mechanism that ation is the result of the evolutionary lability of reproductive functions to increase the pro®ciency of cross-pollination in characters and implies that there are diverse functional solu- species with perfect ¯owers (Lloyd and Webb, 1992a, b). Al- tions for achieving mating and fertility. The ¯oral diversi®- though heterostyly (both distyly and tristyly) has been studied cation that has accompanied the coevolution of ¯owers and intensively since the mid-nineteenth century (reviewed in Bar- animal pollinators is particularly striking and has resulted in rett, 1992), remarkably little is known about its evolutionary contrasting suites of ¯oral characters associated with different history. Only two studies have explicitly used phylogenetic pollinator groups (e.g., Grant and Grant, 1965; Armbruster, approaches to investigate the origins of heterostyly (distylyÐ 1993; Johnston et al., 1998; Schemske and Bradshaw, 1999). Schoen et al., 1997; tristylyÐKohn et al., 1996); neither was Although diversifying selection has played a prominent role able to identify the ancestral states or the precise evolutionary in ¯oral evolution, pervasive convergence in pollination mech- pathways involved in the evolution of these complex polli- anisms also characterizes many unrelated animal-pollinated nation mechanisms. groups. The multiple origins of the heterostylous genetic poly- Narcissus a small genus of animal-pollinated geophytes in morphisms distyly and tristyly across at least 28 diverse ani- Amaryllidaceae (monocots: ) with exceptional ¯o- mal-pollinated angiosperm families are a classic example of ral diversity (e.g., Figs. 1, 2), provides an excellent opportunity the convergent evolution of plant sexual systems (Darwin, for examining the evolutionary history of heterostyly. Four 1877; Ganders, 1979; Lloyd and Webb, 1992a, b; Barrett et major classes of stylar condition, stylar monomorphism, stig- al., 2000b). Most heterostylous species are adapted for polli- ma-height dimorphism, distyly, and tristyly (Fig. 2), are rep- nation by long-tongued pollinators and commonly possess ac- tinomorphic, tubular ¯owers with a stereotypically reciprocal resented among the 10 sections recognized in the genus (Hen- riques, 1887; Fernandes, 1935; Dulberger, 1964; Bateman, 1968; Lloyd et al., 1990; Arroyo and Dafni, 1995; Barrett et 1 Manuscript received 2 September 2003; revision accepted 5 March 2004. al., 1996, 1997; Arroyo and Barrett, 2000; Baker et al., 2000b, The authors thank John Blanchard for sharing his broad expertise of the genus and for providing material from his live collection. Juan Arroyo and c; Arroyo et al., 2002; Thompson et al., 2003). No other het- Mark Chase also provided material. We also thank Juan Arroyo, Lawrence erostylous taxon displays this range of stylar variation. Nar- Harder, Angela Baker, John Thompson, and Luis Villar for helpful discussions cissus may therefore provide one of our best opportunities for and locality information. Hardeep Rai and Heath O'Brien provided substantial investigating the evolutionary pathways by which heterosty- assistance with laboratory work. Alan Meerow and Peter Endress provided lous polymorphisms originated. A diversity of ¯ower shapes helpful comments on the manuscript. This work was funded by Discovery Grants from the Natural Sciences and Engineering Research Council of Can- is found in Narcissus (Fig. 1), particularly associated with co- ada (NSERC) to S. W. G. and S. C. H. B. rona size and ¯oral tube length. It would therefore be valuable 4 E-mail: [email protected]. to assess the possible functional role of ¯oral morphology in 1007 1008 AMERICAN JOURNAL OF BOTANY [Vol. 91 stimulating evolutionary transitions among the different stylar tionary relationships and the selective mechanisms leading to conditions. evolutionary transitions among them. Species of Narcissus can be classi®ed as to whether they In Narcissus, the evolutionary sequence(s) in which the dif- are monomorphic, dimorphic, or trimorphic for style length ferent stylar polymorphisms originated from stylar monomor- (reviewed in Barrett et al., 1996). Here we use the term ``stylar phism is not known. A simple model based on increasing ¯oral polymorphism'' to refer to populations or species that are di- complexity posits an evolutionary transition series from an- morphic or trimorphic for style length, irrespective of the type cestral monomorphism, through stigma-height dimorphism, to of polymorphism (stigma-height dimorphism, distyly, tristyly) distyly, and then ®nally to tristyly. Theoretical support for that they exhibit. Monomorphic species are usually composed parts of this sequence of events comes from models for the of plants with long-styled ¯owers, with stigmas protruding be- evolution of distyly and tristyly. Lloyd and Webb's (1992a, b) yond the two anther levels that typically occur in members of ``pollen-transfer'' model for the evolution of distyly proposes this genus (e.g., Figs. 1d, h; 2a). This condition is referred to that it evolves from a monomorphic ancestor with approach as ``approach herkogamy'' (see Webb and Lloyd, 1986, for a herkogamous ¯owers, via an intermediate stage of stigma- review of herkogamy) and is the most common stylar condi- height dimorphism. The ``inbreeding avoidance'' model for the tion in Narcissus. In some monomorphic species (e.g., N. ser- evolution of distyly (Charlesworth and Charlesworth, 1979) otinus, Fig. 1m; N. viridi¯orus, Fig. 1n) the stigma is located also posits an intermediate stage of stigma-height dimorphism, just above or at the same level as the anthers. Populations with but the ancestral condition in this case is assumed to be a stylar monomorphism often display considerable quantitative homostylous population with long styles and long-level an- variation in style length, but do not exhibit the clear bimodality thers. Finally, Charlesworth's (1979) model of the evolution or trimodality that characterizes species with stylar polymor- of tristyly commences with an ancestral monomorphic long- phism (Barrett et al., 1996). styled population, with a transient dimorphic stage that then There are two types of stylar dimorphism in Narcissus. The becomes invaded by the M-morph, establishing stylar trimor- most common, stigma-height dimorphism (Fig. 2b), involves phism. The dimorphic stage involves a population with recip- populations with two ¯oral morphs that differ in style length rocal herkogamy, and therefore stigma-height dimorphism is (long- and short-styled plants, hereafter referred to as L- and not explicitly dealt with in this model. The particular evolu- S-morphs), but which have anthers placed at roughly the same tionary sequences in these three models are supported by de- position at the top of the ¯oral tube (Dulberger, 1964; Baker tailed theoretical arguments and are certainly plausible; how- et al., 2000c; Arroyo et al., 2002). This polymorphism occurs ever, empirical support from comparative analyses of heter- in approximately a dozen species distributed among three sec- ostylous groups of both the ancestral states and intermediate tions of the genus (Apodanthi, Fig. 1g; Jonquillae, Fig. 1e; conditions postulated in the models are currently lacking. Tazettae, Fig. 1k). The second dimorphic condition, distyly A common feature of the two models for the evolution of (Figs. 1b, 2c), is restricted to a single species: N. albimargin- distyly is the existence of an intermediate stage of stigma- atus of section Apodanthi (Arroyo and Barrett, 2000; PereÂz et height dimorphism (and see Richards, 1997). This polymor- al., 2003). The only two known populations of this rare Mor- phism is a very rare condition in ¯owering plants, with oc- occan species contain two ¯oral morphs (L- and S-morphs) casional cases reported from both non-heterostylous (e.g., that differ reciprocally in stigma and anther height (i.e., recip- Epacris, KalmiopsisÐEricaceae; ChlorogalumÐAgavaceae) rocal herkogamy; heterostyly). The ®nal stylar condition, tris- and heterostylous families (e.g., Anchusa, LithodoraÐBora- tyly (Figs. 1a, 2d), is a more complex form of reciprocal her- ginaceae; reviewed in Barrett et al., 2000b). In the heterosty- kogamy in which populations contain three ¯oral morphs that lous families in which stigma-height dimorphism occurs, there differ in style length (L-, M- [mid-] and S-morphs). This poly- is no phylogenetic evidence linking this polymorphism to the morphism occurs in a single widespread species (N. triandrus, origin of distyly. Lloyd and Webb (1992a) proposed that stig- Fig. 1a) of section Ganymedes and its expression is unique ma-height dimorphism is rare or missing in heterostylous among the six ¯owering plant families that have tristylous taxa groups because the polymorphism is an unstable transitional (Amaryllidaceae, Connaraceae, Linaceae, Lythraceae, Oxali- state. According to this view, the polymorphism should be daceae, Pontederiaceae; Barrett, 1993; Thompson et al., 1996). easily modi®ed through selection for anther dimorphism, and Narcissus triandrus possesses imperfect sex-organ reciprocity it is therefore a short-lived stage that is quickly passed through (Fig. 2d) and a system of late-acting self-incompatibility that during the evolution of distyly (and see Charlesworth and permits both intermorph and intramorph mating (Barrett et al., Charlesworth, 1979). This argument is largely based on func- 1997, 2004; Sage et al., 1999). In contrast, typical tristylous tional grounds, because of the apparent maladaptiveness of species have a trimorphic incompatibility system that prevents uniform anther height for pollen transfer in populations with intramorph mating and three organ levels that are positioned two stigma heights (but see Stone and Thomson, 1994). If this reciprocally (reviewed in Barrett and Cruzan, 1994). The oc- scenario were true in Narcissus, we would not expect stigma- currence in Narcissus of these four contrasting patterns of sty- height dimorphism to be common and distyly rare, the sce- lar variation raises obvious questions concerning their evolu- nario that actually occurs. Stigma-height dimorphism in Nar-

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Fig. 1. Flowers of representative Narcissus species illustrating the wide diversity in ¯oral design. This variation is associated with different pollinator groups, as discussed in the text. Section names are indicated parenthetically: (a) N. triandrus var. cernuus (Ganymedes), long-styled morph; (b) N. albimarginatus (Apodanthi), short-styled morph; (c) N. alpestris (Pseudonarcissi); (d) N. hedraeanthus (Bulbocodii); (e) N. gaditanus (Jonquillae); (f) N. cavanillesii (Tapein- anthus); (g) N. rupicola (Apodanthi); (h) N. bulbocodium (Bulbocodii); (i) N. cyclamineus (Pseudonarcissi); (j) N. poeticus (Narcissus); (k) N. tazetta (Tazettae); (l) N. dubius (Tazettae); (m) N. serotinus (Serotini); (n) N. viridi¯orus (Jonquillae) Photographs are by S. C. H. Barrett, except (n), by Juan Arroyo (Universidad de Sevilla). July 2004] GRAHAM AND BARRETTÐNARCISSUS PHYLOGENY 1009 1010 AMERICAN JOURNAL OF BOTANY [Vol. 91

may be explained for the same reason that stigma-height di- morphism is rare in distylous groups; the intermediate dimor- phic stages were transient and are therefore not represented among extant taxa. The relationship between stylar dimor- phism and trimorphism may potentially be evaluated using phylogenetic approaches in Narcissus. Here we investigate the evolutionary history of stylar var- iation in Narcissus by constructing phylogenetic trees based on two relatively rapidly evolving regions of the plastid ge- nome. We use character optimizations of ¯oral traits to address the following speci®c questions arising from the theoretical and empirical work discussed in this section: (1) Is stylar monomorphism the ancestral condition in Narcissus? (2) How many times has stigma-height dimorphism evolved in the ge- nus? (3) Has distyly evolved from stigma-height dimorphism, as proposed by Lloyd and Webb (1992a, b)? (4) Has tristyly evolved from an intermediate dimorphic stage, as proposed by Charlesworth (1979)? (5) Is there evidence of reversion from stylar polymorphism to monomorphism? (6) What is the role of ¯oral morphology in promoting transitions among stylar conditions? We begin by providing a brief review of the sys- Fig. 2. Examples of the four classes of stylar variation in Narcissus: (a) tematics and natural history of Narcissus to provide a context stylar monomorphism in Narcissus longispathus; (b) stigma-height dimor- for our historical reconstructions. We then use new molecular phism in N. assoanus; (c) distyly in N. albimarginatus; (d) tristyly in N. triandrus. Narcissus longispathus exhibits approach herkogamy, with stigmas systematic data from the plastid genome to infer phylogenetic exserted beyond the two anther levels. The sex organs are located within a relationships in Narcissus and to reconstruct the evolution of funnel-like corona. Narcissus assoanus populations contain two ¯oral morphs stylar polymorphisms and associated ¯oral characters in the that differ in style length. Note that there are small differences in the height genus. of the lower anther levels. The ¯owers possess long ¯oral tubes with small coronas. Narcissus albimarginatus populations contain two ¯oral morphs that differ in style length and in anther position. Stigmas and anthers are recip- GENERAL BACKGROUND ON NARCISSUS rocally positioned, and ¯owers possess long, narrow ¯oral tubes and deep coronas. Narcissus triandrus populations usually contain three ¯oral morphs Narcissus includes all species known as daffodils and nar- that differ in style length. There are three anther levels that correspond in cissi and is comprised of approximately 65 species of peren- height to the three stigma heights, but unlike other tristylous species sex- nial geophytes, geographically concentrated in the Mediterra- organ reciprocity is imperfect, because of the anomalous position of the upper- nean region, particularly the Iberian Peninsula, southern level anthers of the long-styled morph. Flowers have long, narrow ¯oral tubes France, and Morocco (Blanchard, 1990). There are more than and deep coronas. 20 000 registered names that represent over a century's worth of breeding effort directed towards plant improvement, and a cissus appears to be a stable sexual strategy and not simply history of cultivation dating back at least to the seventeenth an ephemeral stage on the evolutionary pathway to distyly. century, and possibly substantially earlier (Wells, 1989; Blan- Whether the origin of distyly in Narcissus involved an ances- chard, 1990; Jefferson-Brown, 1991). Narcissus is one of the tor with stigma-height dimorphism, as theoretical models also most economically important ornamental plants (Jefferson- predict, needs to be determined. The occurrence of both stig- Brown, 1991; Hanks, 2002), and yet remarkably little is ma-height dimorphism and distyly in section Apodanthi may known about the ¯oral and pollination biology of members of permit the determination of whether this evolutionary se- the genus. Phylogenetic relationships in the genus also are quence has indeed occurred. largely unknown (e.g., PereÂz et al., 2003). In tristylous groups there is no comparative evidence that The of Narcissus is unsettled, and species cir- stylar dimorphism (a term that can include polymorphic con- cumscriptions vary widely. Early workers recognized up to ditions that range from stigma-height dimorphism to fully- 160 species or as few as 16 (reviewed in Blanchard, 1990). ¯edged distyly) is an intermediate stage in the evolution of Blanchard recognizes ϳ65 species. With some minor excep- tristyly. Distylous species (Lythraceae, Oxalidaceae) and di- tions, Blanchard (1990) used the classi®cation scheme of Fer- morphic populations (Pontederiaceae) occur in these well- nandes (1968a), and he also summarized recent taxonomic studied tristylous families. However, microevolutionary stud- work in the genus. We follow Blanchard's treatment (1990) ies clearly indicate that these are derived rather than ancestral here. Blanchard viewed Webb's (1980) classi®cation of Nar- conditions (reviewed in Weller, 1992). Evolutionary transitions cissus in Flora Europaea as overly conservative in the number from tristyly to distyly or other dimorphic conditions com- of taxa recognized at the species level. Webb's classi®cation monly result from the loss of style morphs from tristylous has the additional disadvantage that it does not deal with the populations, as a result of stochastic and/or deterministic forc- non-European taxa. The most recent classi®cation scheme of es (Ornduff, 1972; Weller, 1976; Barrett et al., 1989; Eckert Narcissus (Mathew, 2002) incorporates elements of Fernan- and Barrett, 1992, 1995). The loss of style morphs is often des' and Webb's schemes. Mathew (2002) also noted that sub- accompanied by evolutionary modi®cations to the remaining stantial revisionary work is needed for some taxa in the genus. ¯oral morphs, including changes to sex-organ position, incom- Fernandes (1968a) divided Narcissus into two subgenera patibility relations, and pollen size (reviewed in Weller, 1992). (Hermione with base chromosome number x ϭ 5, and Nar- The absence of ancestral dimorphism in tristylous families cissus with x ϭ 7) and 10 sections (Apodanthi, Aurelia, Bul- July 2004] GRAHAM AND BARRETTÐNARCISSUS PHYLOGENY 1011 bocodii, Ganymedes, Jonquillae, Narcissus, Pseudonarcissi, are also visited by long-tongued bees, butter¯ies, and ¯ies. Serotini, Tapeinanthus, Tazettae) based on his extensive chro- Nectar serves as the main ¯oral reward in these species. The mosomal studies of the genus, conducted over more than four ®nal group combines structural elements from the ®rst two decades (summarized in Fernandes, 1967, 1968a, b, 1975). He groups, as ¯owers possess both well-developed and narrow also suggested pre-cladistic phylogenetic schemes for the ge- ¯oral tubes and extended coronas. This combination of ¯oral nus (summarized in Fernandes, 1951). Until very recently (see traits only occurs in two species: distylous N. albimarginatus Discussion), there have been no efforts to evaluate systematic (Fig. 1b) and tristylous N. triandrus (Fig. 1a). Narcissus trian- relationships in Narcissus using modern phylogenetic ap- drus is pollinated primarily by long-tongued solitary bees (pri- proaches. marily Anthophora and Bombus spp.), which forage for nectar All Narcissus species are winter-growing and summer-dor- and pollen. No pollinator information is currently available for mant that are commonly found in open sites from low the rare Moroccan endemic N. albimarginatus. elevation marshes, through rocky hillsides, to high elevation montane pastures. Most species ¯ower in late winter and MATERIALS AND METHODS spring, although ®ve species ¯ower in the autumn (N. brous- sonetii; N. cavanillesii, Fig. 1f; N. elegans; N. serotinus, Fig. Taxon sampling and DNA sequencingÐWe examined 32 species from the 10 sections of Narcissus recognized by Fernandes (1968a, 1975) and four 1m; N. viridi¯orus, Fig. 1n). Little detailed information is outgroup species for DNA sequence variation in two rapidly evolving plastid available on the pollination biology of species of Narcissus regions (see Appendix in Supplementary Data accompanying the online ver- (for an exception see Herrera, 1995). All species are insect- sion of this article). The outgroup species sampled include representatives pollinated, with the majority possessing showy ¯owers (Fig. from both genera in tribe Galantheae (ϭ Galanthus and Leucojum) and Nar- 1), some of which are highly scented (Dodson et al., 1997). cisseae (ϭ Narcissus and possibly Sternbergia) as de®ned by Dahlgren et al. The major pollinators visiting ¯owers of Narcissus species are (1985), who also speculated that these tribes deserve combination. A close bees, butter¯ies, ¯ies, and hawkmoths (Arroyo and Dafni, relationship between Narcissus and Sternbergia is supported by chromosomal 1995; Herrera, 1995; Barrett et al., 1996; Baker et al., 2000a; evidence (Flagg and Flory, 1962). Lapiedra martinezii was included because Worley et al., 2000; Arroyo et al., 2002; PereÂz et al., 2003; of its possible sister-group relationship to Narcissus as noted by Meerow et Thompson et al., 2003). Most species have white, pale yellow, al. (1999) based on plastid evidence. In Narcissus multiple populations were or deep yellow ¯owers (Fig. 2), although the only night-¯ow- sampled for a number of species, including three of its four monotypic sec- ering species in the genus, N. viridi¯orus (Fig. 1n), has highly tions (Aurelia, Ganymedes, Tapeinanthus, but not Serotini). Although noted scented green ¯owers that are pollinated by crepuscular moths in the Appendix, we excluded replicate populations from the phylogenetic (Vogel and MuÈller-Doblies, 1975). Flowers in Narcissus vary analysis, except for an unusual population of N. bulbocodium (see Results: in diameter from ϳ12 to 125 mm and are borne either soli- Phlyogenetics of Narcissus) and N. triandrus, for which we included repre- tarily (e.g., section Bulbocodii and most members of section sentatives of three of four varieties recognized by Blanchard (1990). Pseudonarcissi) or in in¯orescences that can contain as many One of the plastid noncontiguous regions examined is a rapidly evolving Ј as 15±20 ¯owers (N. papyraceus and N. tazetta, Fig. 1k). The portion at the 3 -end of ndhF, the gene for subunit F of plastid NADH de- most prominent features of Narcissus ¯owers are the corona, hydrogenase. The other region, referred to here as trnL-F, spans two plastid a cylindrical cone extending beyond the tepals, and the ¯oral tRNA transferase (trn) genes, trnL(UAA) and trnF(GAA), and consists pri- marily of two noncoding regions; the trnL(UAA) intron and an intergenic tube. Coronal morphology is variable, ranging from a tiny spacer between the two trn genes. We generated the majority of the ndhF ochre-to-yellow pigmented disk in N. serotinus (Fig. 1m) and sequences using a Sequenase version 2.0 kit (United States Biochemical, a rudimentary structure in N. cavanillesii (Fig. 1f) to long Cleveland, Ohio, USA) with 35-S labeled dATP (following manufacturer in- trumpet-like structures (e.g., N. alpestris, Fig. 1c; N. cyclam- structions) and using DNA ampli®cation and sequencing protocols outlined ineus, Fig. 1i) in section Pseudonarcissi (hence, the name in Graham et al. (1998). Automated sequencing was used to generate se- ``trumpet daffodils''). Floral tubes range from long and narrow quences for the trnL-F region, and a subset of those from ndhF, following in species of sections Apodanthi and Jonquillae (Fig. 1e, n) to protocols in Graham and Olmstead (2000). Primers used for ampli®cation and virtually absent in N. cavanillesii (Fig. 1f). Finally, ¯ower ori- sequencing were designed by Olmstead and Sweere (1994) and Graham et al. entation varies from pendant (N. triandrus, Fig. 1a; N. alpes- (1998) for ndhF, and by Taberlet et al. (1991) for trnL-F. tris, Fig. 1c) to horizontal (N. gaditanus, Fig. 1e; N. poeticus, Fig. 1j) or erect (N. cavanillesii, Fig. 1f; N. serotinus, Fig. AnalysisÐWe performed alignments using criteria outlined in Graham et 1m). al. (2000). Alignment gaps were coded as ``missing data'' for phylogenetic Field observations of pollinators visiting ¯owers of Narcis- analysis, but we included two binary characters representing parsimony-in- sus species over the past decade establish three primary func- formative insertion/deletions (indels) in the intergenic spacer region between tional groups (L. D. Harder, University of Calgary, and S. C. trnL and trnF. Across the 37 taxa of Narcissus considered in the ®rst phy- H. Barrett, personal observations). The ®rst group, exempli®ed logenetic analysis, representing 34 species, there are 72 parsimony-informa- by members of sections Pseudonarcissi (Figs. 1c, i) and Bul- tive characters. Of these, 37 are from ndhF. Using N. papyraceus as a ref- bocodii (Fig. 1d, h), possesses ¯owers with large funnel-like erence taxon, the ndhF region examined here is 490 base pairs [bp] long, and coronas and short, wide, or highly funnelform ¯oral tubes. the unaligned portion of the trnL-F region that we examined is 971 bp in length. Because we had to sample nearly twice as many characters in the These ¯owers are pollinated by a wide range of small- and trnL-F region compared to ndhF to obtain a comparable amount of parsi- large-bodied bees that generally forage for pollen from anthers mony-informative variation, the latter protein-coding region thus evolves ap- enclosed within the corona (Herrera, 1995). The second in- proximately twice as fast, per nucleotide, as the predominantly noncoding volves species with long, narrow ¯oral tubes, relatively shal- trnL-F region. low coronas, and horizontally orientated, highly fragrant ¯ow- We conducted maximum-parsimony analysis of the DNA sequence data ers (e.g., sections Jonquillae, Fig. 1e; Apodanthi, Fig. 1g; and using PAUP* version 4.0 beta 10 (Swofford, 2002). All characters and char- Narcissus, Fig. 1j). Members of these sections are primarily acter-state changes were equally weighted. Heuristic searches were conducted adapted for pollination by long-tongued Lepidoptera, mostly using 10 000 random addition replicates and tree bisection-reconnection sphingid moths (e.g., Macroglossum spp.), although ¯owers (TBR) branch-swapping, with the ``MulTrees'' option activated. We included 1012 AMERICAN JOURNAL OF BOTANY [Vol. 91 or excluded several taxa of known or suspected hybrid origin, including N. Reconstructions of character evolutionÐWe performed character map- dubius and N. tortifolius (see Results: Phylogenetics of Narcissus). Branch pings using MacClade 4.03 (Maddison and Maddison, 2001). All character support was evaluated using bootstrap analysis (Felsenstein, 1985), consid- reconstructions were performed with character-state changes equally weighted ering only a single random addition sequence for each of 100 replicates. (Fitch, 1971; Hartigan, 1973) and with taxa of known or suspected hybrid Analyses were performed on combined data from both regions, because in- origin excluded (i.e., with N. dubius and N. tortifolius and a suspected case spection of bootstrap pro®les from separate analysis of each region indicated of introgression involving N. bulbocodium removed; see Results). no serious con¯ict among them (i.e., there were no con¯icting regions with None of the most parsimonious (MP) trees we obtained were fully resolved; more than 50% bootstrap support in both data sets; data not shown). three of the four MP trees (see Results: Phylogenetics of Narcissus) were We also estimated branch support using MrBayes 3.0 beta 3 (Ronquist and partial resolutions of the fourth (the least-resolved one). We interpret poly- Huelsenbeck, 2003). Modeltest version 3.06 (Posada and Crandall, 1998) was tomies on our trees as ``soft'' (corresponding to a lack of evidence for re- used to determine the DNA substitution model that best ®ts the data, among solving speciation events; see Maddison, 1989), rather than being ``hard'' (ϭ those available. The model chosen for our Narcissus data using hierarchical multiple and simultaneous speciation events). We removed some polytomies likelihood ratio tests was ``K81uf ϩ⌫.'' This model has three distinct sub- that are not relevant to the evolution of the morphological characters consid- stitution rate categories (one transition rate, two transversion rates) and ac- ered here prior to reconstructions of their evolution, by including only one counts for among-site rate variation using the gamma (``⌫'') distribution. The species in instances where two or more species had identical plastid sequences least complex substitution model implemented in MrBayes that permits at (or in the case of N. scaberulus parsimony-uninformative variation); such least three substitution rates is the general time reversible (GTR) model. We species were removed only if they possessed stylar and ¯oral character states ran four Markov chains on the combined plastid data for one million gener- identical to the representative species left behind. Two MP trees inferred from ations with the number of substitution types set to six and with among-site this reduced taxon set were identical to the four trees inferred from the more rate variation accounted for using the gamma distribution (i.e., the ``GTR ϩ complete set, when deleted taxa in the reduced data set are pruned from the ⌫'' model). Chains were heated using the default temperature (0.2), and were latter trees. However, the least resolved of these two trees still contains three polytomies (two trichotomies and a tetrachotomy). Different resolutions of sampled at intervals of 1000 generations. The log likelihood sum appeared to these polytomies can potentially result in different character reconstructions. stabilize before the ®rst 100 000 generations. We discarded trees sampled up We therefore bracketed the actual reconstructions that would be inferred on to this point to calculate posterior probabilities of individual clades. the fully resolved plastid-based tree by considering all possible resolutions of the different polytomies on the least resolved tree (requiring 135 different Character codings for ancestral-state reconstructionsÐBased on our ®eld resolutions [ϭ 3 ϫ 3 ϫ 15] from two trichotomies and one tetrachotomy). observations of the majority of taxa and herbarium surveys, we coded the Enumeration of character-state changes across this set of trees was performed stylar condition of taxa according to whether they are monomorphic, dimor- using the ``State Changes and Stasis'' tool in MacClade. Trees that represent phic for stigma height (but without reciprocal herkogamy), distylous, or tris- all possible polytomy resolutions were obtained by completely but randomly tylous. A few Narcissus species exhibit populations with two stylar conditions resolving polytomies on the least resolved MP tree, using the ``Create Trees'' (e.g., N. dubius, N. papyraceus, and N. tazetta have stigma-height dimorphic tool in MacClade. The ``Condense Trees'' option in PAUP* was then used to and monomorphic populations [Baker et al., 2000b; Arroyo et al., 2002; Ar- eliminate nonidentical topologies from a large pool of these resolutions, yield- royo and Dafni, 1995, respectively]; N. triandrus has trimorphic and dimor- ing the 135 possible resolved trees. phic populations [Barrett et al., 1997, 2004]). However, as discussed earlier for other tristylous groups, microevolutionary evidence suggests that in such Concentrated-changes testsÐThe concentrated-changes test (CCT; Mad- cases monomorphism (Arroyo et al., 2002) and dimorphism (Barrett et al., dison, 1990) can detect whether particular types of transition to a derived 2004) are likely to be recently derived intraspeci®c phenomena associated state in a dependent character are more frequent than would be expected by with the loss of style morphs from populations, and so we did not score these chance, with regards to the proportion of branches that possess a given state taxa as having among-population variation in the class of stylar polymor- for the independent character and the amount and type of change in the de- phism. Based on Fernandes' (1940) review of 19th century descriptions of N. pendent character. The CCT as implemented in MacClade requires binary broussonetii it is possible that this species also possesses a stigma-height characters, fully bifurcated trees, and unequivocal parsimony-based character dimorphism. However, our own examination of herbarium specimens of this reconstructions. The latter two conditions are not met in our case, but we species and the tendency of earlier workers to misclassify Narcissus species bracketed the probability estimates that would be obtained on the fully bifur- cated plastid tree by repeating all CCTs on the 135 possible resolutions of as heterostylous (see Barrett et al., 1996) lead us to believe that this species the polytomies on the least resolved MP tree and by assessing two contrasting does not have stigma-height dimorphism, and so we score it as monomorphic schemes of character optimization where reconstructions were partly equiv- for style length. All outgroup genera considered here are uniformly mono- ocal (see later). morphic for style length. An a priori decision must also be made on the state in the independent The ¯oral tube in Narcissus is the narrow, nearly cylindrical to funnelform character that may be associated with a particular type of change in the de- perianth structure formed by fusion of the six tepal bases. Floral tubes are pendent character. We used the CCT to examine whether certain types of coded here either as ``long and narrow'' (at least 10 mm and less than 5 mm stylar condition (dependent character) evolve more commonly in the context wide) or ``all other ¯oral tube types.'' We group in the latter functional cat- of particular types of ¯oral tube and corona (independent character). In one egory those taxa with very short tubes or that lack them completely (such as set of CCTs, we addressed whether polymorphic stylar conditions (stigma- N. cavanillesii), with taxa possessing long and wide tubes (most members of height dimorphism, distyly, and tristyly, considered collectively) are more section Pseudonarcissi) to highly funnelform tubes (section Bulbocodii). In likely to evolve when the ¯ower has a long, narrow ¯oral tube. If so, this our judgement none of these forms permit very precise depth-probing by would be consistent with the hypothesis (see Discussion) that a ¯oral mor- pollinators, in contrast to taxa with long, narrow tubes. The corona (paraper- phology that allows high-precision depth probe pollination is a necessary re- igone) in Narcissus is a tubular outgrowth above the ¯oral tube that develops quirement for the evolution of any stylar polymorphism. A second set of tests from the perianth (Arber, 1937; Singh, 1972). Coronas are coded here as either was used to investigate whether heterostyly is more likely to evolve when ``deep'' (at least 10 mm) or ``absent or shallow'' (``shallow'' coronas are ϳ5 ¯owers have both long, narrow ¯oral tubes and deep coronas. This ¯oral mm or less in depth). Our size cut-offs relate to what we understand to be morphology should permit greater segregation of pollen deposition on polli- functionally important with regard to plant±pollinator interactions and refer nator's bodies and therefore permit the evolution of reciprocal stigma-anther to natural measurement breaks across the taxa. We used the ¯oral measure- positioning (heterostyly) in Narcissus (Barrett et al., 1996; Arroyo and Barrett, ments provided by Blanchard (1990), supplemented with personal observa- 2000). tions. The probability of concentrated change was estimated in each case using July 2004] GRAHAM AND BARRETTÐNARCISSUS PHYLOGENY 1013

MacClade, via simulation (Maddison and Maddison, 2001); actual changes were counted across 5000 simulations in each case. We considered both ac- celerated and delayed transformation (ACCTRAN and DELTRAN) resolu- tions (Swofford and Maddison, 1987) when equivocal character reconstruc- tions were encountered for a particular polytomy resolution, but used the same character optimization scheme across the two characters when both were part- ly equivocal. Although outgroups were included to permit character recon- struction, we conducted the CCT on Narcissus only, excluding all outgroups from the test group to reduce the proportion of white branches and hence minimize type I error (see Lorch and Eadie, 1999). Prior to performing each CCT, the dependent character (``stylar condition'' for the ®rst tests, ``heterostyly'' for the second) was scored visually to count the number of changes to the state of interest (``stylar polymorphism'' from ``stylar monomorphism'' for the ®rst tests, ``heterostyly'' [distyly or tristyly] from any other stylar condition for the second) on branches that also possessed the distinguished state in the independent character. The independent character for the ®rst set of CCTs is ``¯oral-tube,'' with a ``long and narrow'' tube its distinguished state (the alternative state is: ``all other ¯oral tube types''). In the second CCTs, the independent character re¯ects the combination of ¯oral tube and corona status, with the distinguished state being a ``deep corona and long, narrow tube'' (the alternative state is a placeholder for any other com- bination of ¯oral tube and corona type). One problem with performing the test concerns how to score cases where there is a ``simultaneous'' change to the derived state in both the dependent and the independent characters. Such event classes may or may not support the hypothesis of dependence, because there is no way with parsimony to determine the relative order of change on the branch (Donoghue, 1989; with parsimony these ``simultaneous'' changes can be considered as occurring in either order anywhere along the branch). It can be argued, therefore, that the count should be restricted to non-simultaneous cases. However, Donoghue Fig. 3. Phylogeny of Narcissus based on parsimony analysis of combined (1989) and others (Armbruster, 1993; Frumhoff and Reeve, 1994) have also ndhF and trnL-trnF DNA sequence data, including two indels. The tree (one pointed out that we might expect traits with the strongest evolutionary asso- of four most-parsimonious) is presented as a phylogram, with branch lengths ciations to result in rapid, successive changes: the stronger the evolutionary computed using ACCTRAN optimization (length ϭ 197 steps; consistency association, the more likely we would be to see ``simultaneous'' transitions index ϭ 0.878; retention index ϭ 0.945). Trees were rooted at Galanthus and in independent and dependent characters on the tree. We therefore performed Leucojum equivalent to tribe Galantheae (as circumscribed by Dahlgren et the ®rst set of CCTs (concerning whether polymorphic stylar conditions are al., 1985). The branch marked with an arrowhead collapses in two most- parsimonious trees, and two of three varieties of N. triandrus (cernuus and more likely to evolve when there is a long, narrow ¯oral tube) with ``simul- concolor) are resolved as a clade on two most-parsimonious trees (subtended taneous'' changes either excluded or included from the character change by a branch of length one step under DELTRAN optimization). Bootstrap score. For the second set of tests, which address whether heterostyly is more support values and Bayesian posterior probabilities presented as percentages likely to evolve when ¯owers have long, narrow ¯oral tubes and deep coronas, are indicated beside branches (the former either above branches or to the left only ``simultaneous'' changes were inferred and therefore used in CCTs. of paired values; the latter below branches or to the right of paired values). Several numbered clades (I±VI) correspond approximately or exactly to named infrageneric taxa; highlighted lineages disrupt the monophyly of in- RESULTS dividual sections (see text). The asterisk indicates putative allopolyploid spe- cies noted by other authors; an un®lled arrow indicates a probable introgres- Phylogenetics of NarcissusÐFour MP trees were inferred sant population of N. bulbocodium (individual 2 here). Gray bars indicate two from the full data set (Fig. 3), and two from the reduced taxon indels in the intergenic spacer region between trnL(UAA) and trnF(GAA) set used in reconstructions of character evolution (see Figs. 4, that act as unreversed synapomorphies for their respective clades. 5). The four MP trees from the full data set differ from each other only in two polytomous regions, one involving the three populations of N. triandrus (Fig. 3). The two MP trees inferred We brie¯y summarize our phylogenetic ®ndings within the using the reduced taxon set (not shown) are completely con- genus with respect to the infrageneric taxa recognized by Fer- sistent with the four MP trees from the larger data set, subject nandes (1968a, 1975). Two clades de®ned by the well-sup- to tree pruning in the latter case. Bootstrap support values and ported basal split in Narcissus correspond to subgenera Her- Bayesian posterior probabilities are presented in Fig. 3; the mione (clade I, 97% BV; Fig. 3) and Narcissus (clade VI, 70% two support measures generally appear to be strongly corre- BV; Fig. 3), respectively. Section Tazettae in subgenus Her- lated with each other, although the percentage values reported mione is not monophyletic, because it includes N. broussonetii for posterior probabilities are often somewhat higher that the and N. serotinus (corresponding respectively to two monotypic corresponding bootstrap values. To simplify the presentation sections, Aurelia and Serotini highlighted in clade I; Fig. 3). here, we generally note only bootstrap support values in the Narcissus elegans is identical to N. serotinus (ϭ section Ser- text. With the rooting among outgroups employed here (at otini; Fig. 1m) and N. tazetta (Fig. 1k) for the plastid regions tribe Galantheae), Narcissus and Sternbergia are depicted as examined here (Fig. 3). sister taxa (with good support; bootstrap support value, BV, Only one section in subgenus Narcissus (clade VI) is clearly 95%; Fig. 3) among the genera considered. The monophyly monophyletic (Apodanthi corresponding to clade III in Fig. 3; of Narcissus is moderately well supported (BV ϭ 86%; and 96% BV). The plastid data de®ne two major lineages within a Bayesian posterior probability estimate of 100%). Apodanthi. Narcissus calcicola is the sister group of the re- 1014 AMERICAN JOURNAL OF BOTANY [Vol. 91

Fig. 4. Relationship between ¯oral tube status and stylar condition in Narcissus. Some taxa with identical ¯oral conditions are reduced here to single termini; taxa of suspected hybrid origin are excluded (see text). The tree is one of 135 resolutions of the least resolved most parsimonious tree; resolved branches are indicated with arrowheads. Changes in ¯oral tube status are reconstructed on the left-hand reconstruction (with three or four origins of long, narrow tubes indicated under ACCTRAN and DELTRAN optimization, respectively). The right-hand reconstruction illustrates shifts in stylar condition. Six origins of ``stylar polymorphism'' (stigma-height dimorphism, distyly, and heterostyly; see text) are inferred. Numbers in brackets indicate ranges across all 135 possible polytomy resolutions for the derived character states. mainder of the section. This basal split is well supported by regions considered here (Fig. 3). Clade II also includes a rep- bootstrap analysis (100% BV) and by a unique, unreversed 4- resentative of a population of N. bulbocodium that presumably bp indel (Fig. 3) located in the intergenic spacer region be- was derived by introgression with an unknown member of tween trnL(UAA) and trnF(GAA). The indel is inferred to section Jonquillae and that gained its plastid genome in the represent an insertion. Three of ®ve remaining species of Apo- process. We hypothesize this introgression because of the dis- danthi considered here are indistinguishable from each other junct position of this N. bulbocodium sample in clade II rel- for the plastid regions examined (Fig. 3); the two species with ative to the other sampled representatives of section Bulbo- parsimony-uninformative variation in this polytomy are N. al- codii (clade IV, discussed later). The introgressant appears to bimarginatus (Fig. 1b) and N. scaberulus. be indistinguishable from other N. bulbocodium, although its A deep trichotomy observed in half of the MP trees for morphology and genetics warrant closer examination. Five subgenus Narcissus is resolved in the others (Fig. 3). This other individuals sampled in the same population possess the resolution is very poorly supported (32% BV). Clade II (99% same introgressed plastid genome (S. W. Graham, unpublished BV) corresponds partly to section Jonquillae, except that it data). The other members of section Jonquillae in clade II (N. excludes subsection Juncifoliae A. Fernandes (represented fernandesii, N. jonquilla, and N. viridi¯orus, Fig. 1n) corre- here by N. assoanus and N. gaditanus, part of clade V; Fig. spond to subsection Jonquillae. Subsection Jonquillae is 1e) and includes two intersectional allopolyploids (involving monophyletic if the putative hybrids (the allopolyploids and sections Jonquillae and Tazettae; Fernandes, 1937, 1967; presumed introgressant) are excluded from consideration (e.g., Romero et al., 1983); N. dubius (Fig. 1l) and N. tortifolius. Figs. 4, 5). The same basic tree topologies are inferred if these These two taxa are stable, fertile allopolyploids (Blanchard, taxa are excluded from tree searches (not shown). 1990) that are nearly identical to each other for the plastid The third major lineage in subgenus Narcissus is comprised July 2004] GRAHAM AND BARRETTÐNARCISSUS PHYLOGENY 1015

Fig. 5. Relationship between ¯oral morphology and the origin of heterostyly in Narcissus. The tree is one of 135 resolutions of the least resolved most parsimonious tree (see Fig. 4). Flowers are characterized on the left-hand reconstruction as possessing either a deep corona and long, narrow ¯oral tube or any other combination of tube and corona type (¯oral tube scores correspond to those used in Fig. 4; ®lled circles represent deep coronas and un®lled circles absent or shallow coronas). A reconstruction of the origin of heterostyly is depicted on the right; all taxa with stigma-height dimorphism or monomorphism are bracketed under a state encompassing these non-heterostylous conditions. All reconstructions across the 135 tree resolutions (see text) depict non-homologous origins of heterostyly in Narcissus (distyly in N. albimarginatus; tristyly in N. triandrus) and origins of heterostyly that occur ``simultaneously'' with the origin of the long-narrow ¯oral tubes and deep corona combination. Numbers in brackets indicate ranges across all 135 possible resolutions for the derived character states. of clades IV and V. This lineage is well supported in bootstrap ¯orescences are weakly supported (56% BV) as part of a clade analysis (100% BV) and by an additional unique and unrev- with N. assoanus and N. gaditanus (Fig. 1e). This clade is part ersed 19-bp indel located in the intergenic spacer region be- of a trichotomy in clade V, along with the morphologically tween trnL(UAA) and trnF(GAA), inferred to be a deletion. distinctive N. cavanillesii (Fig. 1f) and a moderately well sup- Clade IV corresponds approximately to section Bulbocodii ported lineage (84% BV) that comprises the remainder of the (98% BV; Fig. 3). This section can be regarded to be mono- clade. The latter lineage includes the sole tristylous species of phyletic if the putative introgressant N. bulbocodium in clade Narcissus, N. triandrus (Fig. 1a), along with N. poeticus (Fig. II is excluded from consideration. 1j, section Narcissus) and the remainder of section Pseudon- The ®nal major lineage considered here (clade V, supported arcissi. While interrelationships are generally poorly resolved by 95% BV; Fig. 3) incorporates members of section Pseu- and supported among these taxa, several well-supported clades donarcissi and several additional lineages (section Ganymedes are apparent. These include a small clade comprising the three [N. triandrus, Fig. 1a], section Narcissus [N. poeticus, Fig. 1j], varieties of N. triandrus considered here (100% BV) and sev- section Tapeinanthus [N. cavanillesii, Fig. 1f], and N. assoan- eral clades of Pseudonarcissi (with 66±88% BV). The species us and N. gaditanus, the two species that correspond to sub- that is perhaps the most distinctive trumpet daffodil ¯orally, section Juncifoliae). Some of the substructure in clade V is N. cyclamineus (Fig. 1i), is indistinguishable from two other well supported by bootstrap analysis. Two members of section members of Pseudonarcissi sampled here (N. asturiensis and Pseudonarcissi that are morphologically distinctive (N. lon- N. jacetanus) at the level of the two plastid regions that we gispathus and N. nevadensis) in possessing multi-¯owered in- considered. 1016 AMERICAN JOURNAL OF BOTANY [Vol. 91

Reconstructions of character evolutionÐOur reconstruc- tubes are potentially inferred to be homologous across all taxa tions across 135 resolutions of the least-resolved MP trees in- with this state, no simultaneous changes are inferred, and in dicate that stylar monomorphism is ancestral in Narcissus and these cases there is a signi®cant concentration of change (P Ͻ that stylar polymorphisms arose on multiple occasions within 0.05). In the remaining 121 polytomy resolutions, the origin the genus (5±6 to six origins across the 135 resolutions). An of stylar polymorphism is also inferred to be in¯uenced by the uncertainty in reconstructions of stylar condition concerns presence of a long and narrow ¯oral tube (P Ͻ 0.05) when whether or not stigma-height dimorphism is homologous with- simultaneous changes are included in the CCTs. In the ex- in several groups. Depending on how the deepest trichotomy ample shown in Fig. 4, four origins of stylar polymorphism in Narcissus is resolved (the branch marked with an arrowhead are inferred following the origin of a long, narrow ¯oral tube in Fig. 3), stigma-height dimorphism may or may not be ho- under DELTRAN optimization, with no concentrated change mologous between N. jonquilla-N. fernandesii and the instanc- of the former character state if two simultaneous changes are es of its occurrence in section Apodanthi (i.e., in those reso- excluded (P ϭ 0.2756), but signi®cantly concentrated change lutions with Apodanthi and N. jonquilla-N. fernandesii-N. vir- (P ϭ 0.0054) if the simultaneous origins are included. Under idi¯orus depicted as sister taxa and using DELTRAN vs. ACCTRAN optimization of ¯oral tube status, ®ve origins of ACCTRAN optimization, respectively). However, when it is stylar polymorphism are inferred to arise following the origin inferred to be homologous, stylar monomorphism in N. viri- of a long, narrow ¯oral tube (P ϭ 0.1066 if simultaneous di¯orus then represents the only possible instance of reversion changes are excluded; P ϭ 0.0080 if one simultaneous origin from stylar polymorphism to monomorphism in Narcissus. is included). Figure 4 (right-hand reconstruction) illustrates a tree resolution Only simultaneous changes to heterostyly (distyly or tris- in which stigma-height dimorphism is unequivocally not ho- tyly) and ¯owers with a combination of a long, narrow ¯oral mologous between these two clades. tube and a deep corona are observed. The two instances of The two instances of heterostyly in the genus, distyly in N. heterostyly (distyly in N. albimarginatus, tristyly in N. trian- albimarginatus and tristyly in N. triandrus, originated inde- drus) evolved independently of each other, and both evolved pendently of each other (Fig. 5). Because of the relatively on terminal lineages that also exhibited switches to ¯owers nested position of N. albimarginatus in section Apodanthi the with long, narrow tubes and deep coronas (Fig. 5). There is a distylous condition is inferred to be derived from stigma- highly signi®cant concentration of ``simultaneous'' switches to height dimorphism (e.g., Figs. 4, 5, right-hand reconstruc- distyly or tristyly, and a long, narrow ¯oral tube with deep tions). Tristyly in N. triandrus is inferred to have evolved di- coronas (P Ͻ 0.005 across all arbitrary resolutions examined). rectly from stylar monomorphism across all 135 resolutions This provides further support for a direct causal relationship (e.g., Figs. 4, 5, right-hand reconstructions). between perianth and corona morphology and the evolution of It is not clear whether instances of long, narrow ¯oral tubes heterostyly in Narcissus. However, this ®nding is nearly in- are homologous in Narcissus. Between one and ®ve origins dependent of our knowledge of Narcissus phylogeny: so long and 0±4 losses of a long tube can be inferred across the 135 as these two taxa are not each other's closest relatives, similar polytomy resolutions, depending on how equivocal optimiza- CCT probability values can be found on fully resolved random tions are addressed. Consequently, the primitive ¯oral tube trees with this number of taxa and two simultaneous origins condition in Narcissus is equivocal (one possibility is shown of these traits (data not shown). in Fig. 4). However, a long, narrow ¯oral tube is always found to be homologous within subgenus Hermione (clade I) and DISCUSSION between section Apodanthi (clade III) and the portion of clade II included in character reconstructions (e.g., Fig. 4). The evolution of stigma-height dimorphismÐOur histori- Deep coronas evolved at least twice in Narcissus and per- cal reconstructions of the evolution of stylar polymorphisms haps up to ®ve times, with 0±3 losses (reconstructions not in Narcissus provide evidence of multiple independent origins shown here, but see state codings in Fig. 5). Some resolutions of stigma-height dimorphism in the genus (Fig. 4). Four or of polytomies on the MP trees result in a deep corona being ®ve origins of stigma-height dimorphism are implied by the inferred to be homologous across all instances of its occur- plastid-based trees (5±6 origins of stylar polymorphism, minus rence among clades IV and V (not shown). However, the deep one non-nested origin of heterostyly), with either one or no corona in N. albimarginatus is not homologous with that seen reversions to stylar monomorphism. The primitive condition in the rest of the genus on any of the polytomy resolutions. in the genus is inferred to be stylar monomorphism, and so As a consequence, the combination of a long ¯oral tube and stylar polymorphism is inferred not to be homologous between deep corona clearly evolved twice; once in N. albimarginatus the two subgenera. Within subgenus Hermione stigma-height and once in N. triandrus (Fig. 5; left-hand reconstruction). dimorphism evolved twice (Fig. 4), once in N. papyraceus and once in N. tazetta; stylar polymorphisms also evolved twice Hypotheses of concentrated changeÐBetween three and or more in subgenus Narcissus (the precise number of origins six origins of stylar polymorphism in Narcissus arose in lin- depends on how polytomies are resolved). eages that possessed long and narrow ¯oral tubes. Whether or One of the most intriguing questions this raises is why stig- not there was a signi®cantly elevated number of origins of ma-height dimorphism arose so readily in Narcissus, and per- stylar polymorphism in these lineages depends mostly on sisted across multiple speciation events in some cases (Fig. 4), whether instances with ``simultaneous'' change in these two when it is so rare across the angiosperms as a whole. Stigma- character states are included in the CCTs. When these changes height dimorphism represents a relatively simple ¯oral poly- are excluded, in most cases no signi®cantly concentrated ori- morphism when compared to the heterostylous condition. In gin of stylar polymorphism is inferred in lineages with long, the majority of heterostylous species the ¯oral morphs are usu- narrow ¯oral tubes (P Ͼ 0.08). However, in a subset of po- ally distinguished by incompatibility reactions, stigma-anther lytomy resolutions (14 of 135; not shown) where long, narrow position, and pollen and stigma polymorphisms (reviewed in July 2004] GRAHAM AND BARRETTÐNARCISSUS PHYLOGENY 1017

Barrett, 1992). In contrast, for Narcissus species with stigma- with incomplete sex-organ reciprocity. Second, a recent ex- height dimorphism the main distinguishing feature that sepa- perimental study of N. assoanus comparing reproductive suc- rates the ¯oral morphs is a simple difference in style length, cess in monomorphic vs. dimorphic arrays indicated improved although minor variations in anther height have also been doc- cross-pollen transfer in1:1arrays (Thompson et al., 2003). umented in N. assoanus (Baker et al., 2000c). Studies of the Therefore, the close association between stigma-height dimor- genetic control of style length in N. tazetta indicate that a phism, elongated ¯oral tubes, and nectar-feeding, long- single diallelic locus with dominance controls the inheritance tongued pollinators seems likely to have a functional basis of style length (Dulberger, 1967). In common with most dis- related to effective cross-pollination. If this is true, it may also tylous species, the dominant S allele at the style-length locus explain the absence of stigma-height dimorphism from sec- governs the expression of short styles (Ss), with the long- tions of Narcissus without long, narrow ¯oral tubes (and with styled morph homozygous recessive (ss). If we assume a large coronas) (e.g., Pseudonarcissi [Herrera, 1995]; Bulbo- monomorphic ancestor with long styles, as seems likely for codii [L. D. Harder, and S. C. H. Barrett, personal observa- Narcissus, and a similar genetic control in other species, then tions]). Species in these sections are visited primarily by short- the origin of stigma-height dimorphism requires only that a tongued pollen-collecting bees, which enter the corona and single dominant mutation establish and spread. The genetic forage in a nonstereotypical manner. The behavior of these and developmental changes required for this event to occur are bees and their interaction with ¯oral morphology is thus likely likely to be much easier than for the evolution of distyly, in not conducive to the segregated pollen transfer required to which the functional grouping of associated morphological and maintain stylar polymorphism. physiological traits may often be dif®cult to achieve, because of constraints arising from the linkage of genes controlling The evolution of heterostylyÐFew studies have been able multiple traits associated with the polymorphism (Charles- to address whether heterostyly has originated on multiple oc- worth and Charlesworth, 1979). Nevertheless, despite the mor- casions among closely related taxa. In taxa with many heter- phological simplicity of stigma-height dimorphism, it is a very ostylous species, this seems quite plausible, but as yet no at- rare condition in angiosperms, suggesting that the appropriate tempt has been made to explicitly test this hypothesis using conditions for selection to maintain the polymorphism may be character reconstructions in such groups, despite the avail- limited in most taxa. This is presumably because in these in- ability of molecular phylogenies in Rubiaceae (e.g., Bremer stances stylar polymorphism provides only minimal ®tness and Manen, 2000) and Primulaceae (Mast et al., 2001), for bene®ts associated with more pro®cient cross-pollination, in example, two families in which heterostyly is especially well comparison with stylar monomorphism. Why then is stigma- represented. Although heterostyly is rare in Narcissus, our height dimorphism common in Narcissus, and what features phylogenetic analysis indicates that distyly and tristyly were of their ¯owers might favor the maintenance of this polymor- independently derived in the genus. Both polymorphisms are phism? restricted to a single species (distylous N. albimarginatus and Stigma-height dimorphism in Narcissus is strongly associ- tristylous N. triandrus). Sporadic origins of heterostyly are ated with extended, narrow ¯oral tubes (at least 10 mm and also known from other families (e.g., Lamiaceae, Barrett et al. less than 5 mm wide) and pollination by long-tongued Lepi- [2000a]; Polemoniaceae, Cochrane and Day [1994]), but no doptera, particularly hawkmoths (e.g., N. assoanus, Baker et other family contains both distyly and tristyly, with each re- al. [2000a]; N. dubius, Worley et al. [2000]; N. papyraceus, stricted to a single species. Arroyo et al. [2002]; N. tazetta, Arroyo and Dafni [1995]). Historical reconstructions of character evolution have pro- These associations characterize all species with stigma-height vided us with some insight into the evolutionary pathways by dimorphism, although several species with stylar monomor- which these polymorphisms arose in Narcissus. They also en- phism also possess long and narrow ¯oral tubes and are pol- able an assessment of theoretical models for the evolution of linated by hawkmoths (e.g., N. viridi¯orus, Vogel and MuÈller- heterostyly. The evolutionary pathway by which distyly orig- Doblies [1975]; N. poeticus, L. D. Harder and S. C. H. Barrett, inated in Narcissus is consistent with Lloyd and Webb's personal observations). Lloyd and Webb (1992a, b) proposed (1992a, b) theoretical model. In section Apodanthi, distyly that heterostyly is more likely to evolve from ¯oral mono- evolved from ancestors with stigma-height dimorphism and morphism in species with ¯oral tubes and depth-probed pol- these in turn evolved from monomorphic species that most lination. This is because of the greater opportunity for segre- likely possessed approach herkogamy. Because most Narcis- gated pollen deposition on the tongues and bodies of polli- sus species are self-sterile (Barrett et al., 1996; Sage et al., nators and the role of segregation in facilitating cross-polli- 1999), the evolution of heterostyly likely occurred in ancestors nation when stigmas and anthers are reciprocally positioned. that were largely outcrossing. If this is correct, the selective This argument also seems likely to apply to species of Nar- forces responsible for the evolution of distyly were unlikely cissus with stigma-height dimorphism, although precisely how to be related to rates of sel®ng and inbreeding depression. segregated pollen deposition and intermorph pollination is fa- Instead, as proposed by Lloyd and Webb (1992a, b), reciprocal vored without clear sex-organ reciprocity among short-level herkogamy in N. albimarginatus was probably selected to in- sex organs (see Fig. 2b) is unclear. crease the pro®ciency of cross-pollen transfer. Below we con- However, evidence that stigma-height dimorphism does in- sider why this may have occurred in N. albimarginatus and deed promote effective cross-pollen transfer between the ¯oral not other species in the genus with stigma-height dimorphism. morphs comes from two sources. First, populations of several The evolutionary pathway that gave rise to tristyly in sec- species visited by long-tongued pollinators are characterized tion Ganymedes is less clear. Unlike the situation discussed for by1:1¯oral morph ratios (Baker et al., 2000c; Arroyo et section Apodanthi, heterostylous N. triandrus is nested among al., 2002). Equality of morph ratios demonstrates clearly that monomorphic members of section Pseudonarcissi. Thus, there symmetrical disassortative mating arising from cross-pollina- is no phylogenetic evidence that tristyly evolved via an inter- tion between the ¯oral morphs can be achieved in populations mediate dimorphic stage (the pathway predicted by the model 1018 AMERICAN JOURNAL OF BOTANY [Vol. 91 of Charlesworth [1979]). Because it seems rather unlikely that tubes (Fig. 2c, d) may have been the structural innovations tristyly would evolve de novo from a monomorphic condition, that promoted the evolution of heterostyly in Narcissus. it is worth speculating on the reason for the absence of an intermediate dimorphic stage. The most probable explanation, Caveats with regard to reconstructions of character evo- mentioned earlier in relation to other tristylous families (see lutionÐOne assumption that is commonly used when recon- Introduction), is that any dimorphic stage (distyly or stigma- structing character evolution is that plastid phylogenetic his- height dimorphism) that preceded the origin of tristyly was tory is a reasonable estimate of organismal phylogeny. If taxa ephemeral and is consequently not represented among extant of hybrid origin are not recognized and removed from analy- taxa. This seems particularly likely in Narcissus because of sis, this can seriously mislead phylogenetic inference (e.g., several morphological and physiological features unique to Doyle, 1992). As we only have genetic evidence from one this instance of tristyly. The only difference between the L- linkage group (the plastid genome), our only sources of ex- and M-morphs of N. triandrus is style length, because these ternal evidence for making inferences about the existence of two morphs share similar anther heights (Fig. 2d) and have hybrids in Narcissus come from chromosomal data or from the same intramorph compatibility system. The transition from ``unusual'' phylogenetic placements. We removed two putative dimorphism to trimorphism may therefore have simply in- hybrids from analysis that were recognized previously on the volved a shortening of style length in the L-morph to create basis of chromosomal evidence (N. dubius and N. tortifolius: the third morph. In common with the establishment of stigma- Fernandes, 1937, 1967; Romero et al., 1983) and one that is height dimorphism (discussed in the preceding section), this phylogenetically disjunct from the main clade of section Bul- change may have been achieved in a relatively straightforward bocodii and that we therefore predict arose from an introgres- manner, leading to the replacement of dimorphism by trimor- sion event (N. bulbocodium, individual 2, Fig. 3). There are phism. This may well provide an example of a case where several records of natural hybrids involving members of sec- phylogenetic reconstruction is not adequate for detecting tion Bulbocodii (with sections Pseudonarcissi and Ganymedes; changes that occurred on microevolutionary timescales and Fernandes, 1951) and N. bulbocodium is one of the few taxa that involved relatively simple genetic and developmental with extensive variation in ploidy level (e.g., Fernandes, modi®cations. 1967). It is possible that there are other unrecognized hybrid What aspects of the ¯oral biology of N. albimarginatus and taxa in our analysis. Con®rming the putative N. bulbocodium N. triandrus distinguish them from other Narcissus species and introgressant and sorting out whether other taxa are misplaced might have favored the independent evolution of heterostyly due to other undetected hybridization events will require evi- in sections Apodanthi and Ganymedes? Despite occurring in dence from additional multiple unlinked loci from the nuclear different clades, N. albimarginatus and N. triandrus have re- genome. Until these data are produced, the current results markably similar ¯owers that differ in shape from other Nar- should be viewed with this strong caveat. cissus species (Figs. 1, 2). Both have pendant yellow ¯owers In previous analyses of the evolution of heterostyly, we used of similar size and shape, with deep coronas and long, narrow biased weighting schemes to reconstruct the evolution of this ¯oral tubes, and ¯owers are produced in similar numbers each complex polymorphism (Graham and Barrett, 1995; Kohn et season (1±3 per plant). Flowering times are comparable al., 1996; Barrett and Graham, 1997). Here we limited con- (March±April) and, interestingly, the ¯owers of both species sideration of different schemes to the use of ACCTRAN and have a distinctive fragrance (Arroyo and Barrett, 2000) that is DELTRAN optimization in situations where we observed unique within Narcissus. These similarities exhibit all the hall- equivocal reconstructions. However, using complexity argu- marks of functional convergences associated with pollination ments to justify the use of biased weighting schemes for the biology. origin of stigma-height polymorphisms is less defensible in Arroyo and Barrett (2000) proposed that the striking con- Narcissus. Genetic evidence suggests that stigma-height di- vergence between the ¯owers of N. albimarginatus and N. morphism can arise relatively easily; for example, in N. tazetta triandrus may be associated with a shift from lepidopteran to it involves only a simple style length change governed by a long-tongued bee pollination. Pendant ¯owers with deep co- single allelic difference (Dulberger, 1967). It is more dif®cult ronas prevent long-tongued Lepidoptera from contacting sex to explain why stigma-height dimorphism persists in the ge- organs during nectar foraging, and only bees visit Narcissus nus, because on theoretical grounds (Lloyd and Webb, 1992a, species with large coronas and/or pendant ¯owers. This change b) we would expect it to transform into distyly relatively rap- in pollination biology may also have been causally associated idly. The explanation for the stubborn persistence of the di- with the evolution of heterostyly in Narcissus. According to morphism in the genus is likely to come from further analysis this hypothesis, the unique combination of deep coronas and of pollinator biology. long, narrow ¯oral tubes in N. albimarginatus and N. triandrus Heterostyly is one of the most complex sexual systems in facilitated the segregated pollen deposition required for ef®- ¯owering plants (Barrett, 1992), and we have argued previ- cient cross-pollen transfer between the ¯oral morphs in het- ously that this justi®es the use of a biased weighting scheme erostylous species. This is because extended coronas provide when reconstructing its evolutionary origin (e.g., Kohn et al., a longer vertical area for separation of the two stamen levels 1996). Homology of the two instances of heterostyly in the within a ¯ower. Deeper ¯owers should enable greater segre- genus (distyly in N. albimarginatus, tristyly in N. triandrus) gation of pollen deposition than is possible in species with can be forced during character reconstruction (if they are stigma-height dimorphism and shallow coronas. In the latter scored simply as ``heterostyly''), but the bias against the origin case (Fig. 2b), both anther levels are positioned close together of heterostyly would have to be very strong (around ®ve- to at the top of the narrow ¯oral tube, and packing constraints seven-fold heavier than its loss, across the 135 trees considered may limit opportunities for selective divergence in anther po- in the CCTs; data not shown), and multiple instances of stig- sition (Barrett et al., 1996). Thus, according to this view, the ma-height dimorphism in section Apodanthi and elsewhere evolution of deep coronas combined with long, narrow ¯oral would then have to be interpreted as stable evolutionary re- July 2004] GRAHAM AND BARRETTÐNARCISSUS PHYLOGENY 1019 versions to stigma-height dimorphism. The latter scenario is and pollinators in dimorphic Narcissus tazetta L. (Amaryllidaceae) in not supported in any current evolutionary scheme. The weight Israel. New Phytologist 129: 135±145. of evidence therefore supports convergent origins of hetero- BAKER, A. M., S. C. H. BARRETT, AND J. D. THOMPSON. 2000a. Variation in pollen limitation in the early ¯owering Mediterranean geophyte Nar- styly in N. albimarginatus and N. triandrus. cissus assoanus (Amaryllidaceae). Oecologia 124: 529±535. Since we submitted this paper, we became aware of another BAKER, A. M., J. D. THOMPSON, AND S. C. H. BARRETT. 2000b. Evolution study that attempted to reconstruct the evolution of stylar poly- and maintenance of stigma-height dimorphism in Narcissus: II. Fitness morphisms in Narcissus using phylogenetic approaches (PereÂz comparisons between style morphs. Heredity 84: 514±524. et al., 2003). However, the phylogenetic inferences in this BAKER, A. M., J. D. THOMPSON, AND S. C. H. BARRETT. 2000c. Evolution study were based on very limited taxonomic and genomic and maintenance of stigma-height dimorphism in Narcissus: I. Floral samples. Only a few sections of Narcissus were represented variation and style morph ratios. Heredity 84: 502±513. BARRETT,S.C.H.[ED.]. 1992. Evolution and function of heterostyly. Spring- by multiple species in their study, and their main phylogenetic er-Verlag, Berlin, Germany. analysis was based on a single, short plastid region (the inter- BARRETT, S. C. H. 1993. The evolutionary biology of tristyly. In D. Futuyma genic spacer region between trnL[UAA] and trnF[GAA]; they and J. Antonovics [eds.], Oxford surveys in evolutionary biology, 283± did not sample the trnL[UAA] intron), which provided only 326, vol. 9. Oxford University Press, Oxford, UK. limited information on relationships. The consensus tree in- BARRETT,S.C.H.,W.W.COLE,J.ARROYO,M.B.CRUZAN, AND D. G. ferred from this region (their ®g. 5) was consequently largely LLOYD. 1997. Sexual polymorphisms in Narcissus triandrus: is this spe- unresolved. PereÂz et al. (2003) used this tree to infer conver- cies tristylous? Heredity 78: 135±145. BARRETT, S. C. H., W. W. C OLE, AND D. H. WILKEN. 2000a. Heterostyly in gent origins of heterostyly in N. albimarginatus and N. trian- the Lamiaceae: the case of Salvia brandegeei. Plant Systematics and drus. Unfortunately, their tree lacks the robustness needed to Evolution 223: 211±219. validate such a conclusion. Indeed, polytomies and poorly sup- BARRETT,S.C.H.,AND M. B. CRUZAN. 1994. Incompatibility in heteros- ported branches (Ͻ50% bootstrap support) on their consensus tylous species. In E. G. Williams, A. E. Clarke, and R. B. Knox [eds.], tree can be resolved in such a way that it is ambiguous whether Genetic control of self-incompatibility and reproductive development in heterostyly is convergent across Narcissus (S. W. Graham, un- ¯owering plants, 189±219. Kluwer Academic, Amsterdam, Netherlands. published data). BARRETT,S.C.H.,AND S. W. GRAHAM. 1997. Adaptive radiation in the aquatic plant family Pontederiaceae: insights from phylogenetic analysis. In T. J. Givnish and K. J. Sytsma [eds.], Molecular evolution and adap- General conclusionsÐAlthough not completely resolved, tive radiation, 225±258. Cambridge University Press, Cambridge, UK. our plastid-based phylogeny of the genus Narcissus provides BARRETT, S. C. H., L. D. HARDER, AND W. W. C OLE. 2004. Correlated evo- substantial new insights into the evolutionary history of stylar lution of ¯oral morphology and mating-type frequencies in a sexually polymorphisms in this genus. Stylar monomorphism is the an- polymorphic plant. Evolution 58: 964±975. BARRETT, S. C. H., L. K. JESSON, AND A. M. BAKER. 2000b. The evolution cestral condition in Narcissus and stigma-height dimorphism and function of stylar polymorphisms in ¯owering plants. Annals of Bot- appears to have evolved multiple times, perhaps with one loss, any 85(supplement A): 253±265. and yet it clearly has also persisted across many speciation BARRETT, S. C. H., D. G. LLOYD, AND J. ARROYO. 1996. Stylar polymor- events in the genus. The single instance of distyly in the genus, phisms and the evolution of heterostyly in Narcissus (Amaryllidaceae). in the rare Moroccan endemic N. albimarginatus, likely In D. G. Lloyd and S. C. H. 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