A Synopsis of Melanthiaceae (Liliales) with Focus on Character Evolution in Tribe Melanthieae

Melanthiaceae s.l. comprises five tribes: Chionographideae, Heloniadeae, Melanthieae, Parideae, and Xerophylleae--each defined by distinctive autapomorphies. The most morphologically diverse tribe Melanthieae, now with seven genera, had not been subject to rigorous phylogenetic character study prior to the current series of investigations that also include an overview of the family. Data from our publications and studies underway are here assessed and integrated, providing a useful overview of Melanthiaceae, and especially of Melanthieae. The results of parsimony analyses of ITS (nuclear ribosomal) and trnL-F (plastid) DNA sequence data correlate with potentially synapomorphic phenotypic characters for genera of Melanthieae, including habit form, rootstock type, bulb shape, inflorescence structure, indumentum type, tepa! shape, nectary morphology, and ovary position. Sequence data also correlate well with the pattern of variation in chromosome number. The molecular and morphological data support generic recircumscription in Melanthieae and also validate several generalizations concerning character evolution within the tribe, as well as among the tribes of the family.


INTRODUCTION
Recent molecular studies over the past decade (e.g., Chase et al. 1995aChase et al. , b, 2000;;Rudall et al. 2000;Soltis et al. 2000;Hilu et al. 2003;Davis et al. 2004) have resulted in reevaluation of monocot phylogeny, particularly of the polyphyletic Liliaceae s.l., the "petaloid" or "lilioid monocots"so named because the flowers have conspicuous petaloid tepals and, therefore, superficially resemble true lilies (species of Lilium L. ).These reassessments include revision of Melanthiaceae, a family with a long history of problematic circumscription (comprehensive summaries in Zomlefer 1997a and Zomlefer et al. 2001 ).Before availability of DNA sequence data, the most contemporary treatment of the family by Dahlgren and associates (Dahlgren and Clifford 1982;Dahlgren et al. 1985) divided the family into six tribes, some now shown to be misplaced and/or polyphyletic (Table I).

Parideae/Xerophylleae Clade
The infrageneric phylogeny of Parideae has been recently extensively investigated via cladistic analyses of morphological and molecular data (e.g., Kato et al. 1995a, b;Kawano and Kato 1995;Osaloo and Kawano 1999;Osaloo et al. 1999;Fukuda 2001;Farmer and Schilling 2002).Evident morphological autapomorphies for this distinctive tribe include the unusual habit (a simple stem terminating in a relatively large, solitary flower subtended by a single whorl of net-veined leaves or leaf-like bracts; Fig. 1), the differentiated perianth of calyx and corolla, the unusual fruit (see Discussion), and arillate seeds.The monophyly of Parideae is also supported by a unique karyology with a basic complement of five, morphologically similar chromosomes (n = 5) comprising the large genome size of the 2n, 3n, 4n, 6n, and 8n plants of the tribe (summary in Zomlefer 1996).
Autapomorphies for the monogeneric Xerophylleae (Xerophyllum) include several anatomical features, including a thick pericycle of two or three cell layers, in addition to several unique characters of the highly modified, sclerified leaf: annular thickenings on the mesophyll cells, two distinct phloem poles in the vascular bundles, and sunken stomata that lack aperture lips (Ambrose 1975).A morphological synapomorphy is the rootstock comprising a bulb terminating a stout rhizome (Table 2; Fig. 1) Parideae, often traditionally maintained as segregate family Trilliaceae (e.g., Hutchinson 1959Hutchinson , 1973;;Dahlgren et al. 1985;Takhtajan 1997;Tamura 1998), are embedded within Melanthiaceae as sister to Xerophylleae (summary in Zomlefer 1996).Presently, no morphological synapomorphies for the clade comprising these two tribes are known.However, Xerophylleae are linked morphologically with Melanthieae by a raphide-styloid combination, a bulb plus rhizome rootstock, and a unique susceptibility to certain rust fungi (Table 2 ;Goldblatt 1995;Zomlefer 1997a).Additional study is needed to reconcile these morphological and molecular results, particularly to carefully examine characters supporting the presumed phylogenetic relationship of Parideae and Xerophylleae.

Melanthieae
Systematists have long recognized Melanthieae (properly designated "Veratreae" when within Liliaceae s.l.), as a cohesive and natural group.As listed in Table 2 (details in Zomlefer 1997a), the tribe is validated by several potential synapomorphies, including unique alkaloids ("veratrum alkaloids"), distinctive floral anatomy, bulbs, conspicuous tepal nectaries, andromonoecism, unusual anther dehiscence, operculate pollen (Colasante and Rudall 2000), and incompletely fused carpels maturing into a "ventricidal" capsular fruit type (splitting along the ventral sutures).A base chromosome number of x = 8 has also been postulated as potentially synapomorphic for the tribe (detailed below under Discussion).
parsimony analyses of ITS (nuclear ribosomal) and trnL-F (plastid) DNA sequence data (Zomlefer et al. 2001).The strongly supported cladograms are summarized in the phylogeny shown in Fig. 2. Based on our study, Stenanthium, as traditionally defined, is biphyletic (and embedded within Zigadenus s.l.), Zigadenus s.l. is polyphyletic, and Amianthium is a distinct entity only distantly related to the other Zigadenus species.
Fig. 2 .-Phylogeneticrelationshjps of the seven genera in Melanthieae, based on ITS and trnL-F molecu lar data for 170 samples representing 90 taxa (Zomlefer et a t. 2001(Zomlefer et a t. , 2003, in prep.), in prep.).Veratrum is further divided into two monophyletic ections, sec t.Veratrum and sect.Fuscoveratrum.data resolved Amianthium/Veratrum s.I. and Stenanthium/ Anticlea as si ter taxa, and Zigadenus glaberrimus, as sister to the rest of the tribe.
Veratrum!Melanthium complex.-Wenext investigated the infrageneric phylogeny and monophyly of Veratrum s.I., the most diverse genus in the tribe.Veratrum, a problematic group of 20-45 species with a complicated taxonomic history, has been variously circumscribed w ith Melan.thiumsubmerged totally or in part (detailed summaries in Zimmerman 1958 ; Kupchan et al. 1961 ;Bodkin 1978;ZomJefer 1997a).Contemporary authors generally recognize both genera, with Melanthium typically composed of two to four eastern North American species as in the Flora of North America (Bodkin and Utech 2002;McNeal and Shaw 2002).We sought to evaluate whether segregation of Melanthium (however defined) created a paraphyletic Veratrum.In addition , Veratrum s.l. had also formally (Loesener 1926(Loesener , 1927(Loesener , 1928) ) and informally (Zimmerman 1958) been subdivided into several subgenera and sections (all of doubtful monophyly) .
According to our analyses of ITS data (Zomlefer et a!. 2003) , the recognition of the traditional Melanthium with any combination of the four North American species makes the rest of Veratrum paraphyletic, and we concluded that the Veratrum/Melanthium complex is best treated conservatively as one monophyletic genus, divided into two sections (and two subsections) that are supported by several morphological synapomorphies.The two morphologically distinct subsections of Veratrum, sect.Veratrum and sect.Fuscoveratrum 0 .Loes., are indicated in Fig. 2 and below in our discussion of character evolution in the family (Fig. 3, 4).
Schoenocaulon.-Currently in progress (Zomlefer et al. in prep.) are morphological and ITS analyses of the species and intraspecific taxa in the last genus in the tribe, Sch.oenocaulon.Schoenocaulon is a distinctive group, well defined by several synapomorphies (Table 3).Our preliminary results thu s far support recircumscription and placement of several problematic species and also allow insight into the evolution some unusual characters within the genus, such as nectary and tepa! margin type (see Discussion).

MATERJALS AND METHODS
Our cladograms of Melanthieae in this review paper (Fig. 2-4) is based on cladistic analyses of trnL-F and ITS data (Zomlefer et al. 2001(Zomlefer et al. , 2003) ) in addition to current, unpublished studies on Schoenocaulon (Zomlefer et al. in prep.).Laboratory protocols and search strategies are detailed in Zomlefer et al . (2001Zomlefer et al . ( , 2003)).For our preliminary study (Zomlefer et al. 2001), plant material was collected fresh or silica dried (Chase and Hills 1991 ).Herbarium specimens, ranging in age from 2-127 years, provided supplemental material for subsequent, more thorough study involving littlecollected taxa.Successful extractions in volved leaf bl ades and/or tepals.Specimens have been carefully chosen to represent variation within species complexes, as well as multiple collections of variable taxa.Sequences and vo ucher information for all taxa in these publications have been deposited in GenBank (tmL-F: AF303663-AF30370 I; ITS: AF303702-AF303731, AF494297-AF494336).
Character states were manually mapped on our dadograms (Fig. 2-4) so that character state transitions were minimized (i.e., application of parsimony).For our investigations, we recognize taxa above the rank of species based on criteria o utlined by Backlund and Bremer (1998) for general principles of classification: first and foremost, they are monophyletic but secondarily, they should have strong statistical support and also be more or less recognizable based on morphological characters.Secondary criteria, including the size of the clade, nomenclatural stability, and issues relating to minimizing redundancy in classification, are addressed in Kellogg and Judd (2002) and APG II (2003).

RESULTS AND DISCUSSION
Besides supporting generic recircumscription in Melanthieae, our investigations also validate several generali zations concerning character evoluti on within the tribe, as well as among the tribes of the fami ly (Fig. 1).Variation in habit, tepals, and perigonal glands have traditionally been used to distingui sh taxa within Melanthieae, but several features, such as the extension of the tepal base into a claw, are not discrete, and some character states, such as those associated with a particular habit, may be strongly correlated.Formal comprehensive cladistic analyses of morphological (and other) characters are in progress (Zomlefer and Judd, in prep.) to confirm putative synapomorphies and to identify addi- "' (1)   Ul 572 Zomlefer, Judd, Whitten, Willi ams ALISO  Zomlefer (1997aZomlefer ( , 2003)).? =questionable or unverified report; V = Veratrum sect.Veratrum; F = Veratrum sect.Fuscoveratrum; Ami = Amianthium; Ver = Veratrum s.l.; Ste = Stenanthium; Ant = Anticlea; Tox = Toxicoscordion; Sch = Schoenocaulon; Zig = Zigadenus s.s.tiona!ones.Our summary here outlines the pattern of a few distinctive exemplar character state changes within Melanthieae inferred by hypothesis of phylogeny (Fig. 2-4).Lack of resolution in cladogram topology sometimes prohibited determination of the precise level of universality for particular states, and these uncertainties, along with unequivocal character transitions, are discussed below.

Characters
Rootstock.-Theplants of Chionographideae, Heloniadeae, and Parideae have rhizomes (Table 2; Fig. 1)-in contrast to the tunicate bulbs (characterized by an outer coat of membranous leaf bases) that have evolved within Melanthieae (Fig. 3A).Zigadenus s.s.(sister to the rest of Melanthieae) has a bulb less rhizome covered in persistent leaf bases but not developing the tunicate apex, whereas a bulb occurs in all other genera (Ambrose 1975(Ambrose , 1980)).The consistently narrow (cylindrical) bulb of Stenanthium is apomorphic for the genus (Zomlefer and Judd 2002), as is the distinctive dark fibrous bulb (covered with dark brown to black scales or fibers) for Schoenocaulon (Table 3;Zomlefer 1997a;Zomlefer et al. 2001).In Veratrum sect.Veratrum, the bulb terminates a well-developed rhizome, an unusual arrangement synapomorphic for the genus.A somewhat similar rhizome-bulb combination also characterizes Xerophylleae (Table 2; Fig. 1), but the swollen leaf bases do not encircle the stem as in the bulbs of Veratrum (Ambrose 1975).According to our phylogeny (Fig. 1), the rootstock type of Xerophylleae is autapomorphic for this tribe and has been independently derived, a conclusion supported by the morphological differences in the rhizome-bulb configurations of the two clades.The well-developed bulbs of some members of Veratrum sect.Fuscoveratrum (and several spe-cies of Schoenocaulon) terminate a very reduced rhizome.Based on parsimony, the rhizome likely has been secondarily reduced/lost in these taxa (Fig. 3A).
Habit features.-Ourphylogeny allows development of hypotheses concerning evolution of some distinctive plant forms in Melanthiaceae (especially within Melanthieae), although the appearance of a plant is generally difficult to quantify.For example, an obvious autapomorphy for the Parideae is the unusual habit: a simple stem terminating in a relatively large, solitary flower subtended by a single whorl of net-veined leaves or leaf-like bracts (Fig. 1F).Reduced leaves cover the stem in Xerophylleae (a possible synapomorphy), so the plants appear scapose (Fig. lG;Zomlefer 1997a).The majority of the remainder of the family are characterized by leafless stems (Fig. 1A, D, E) and the basal leaves, typically with a conspicuous midvein, are generally narrow, sessile, and taper gradually to a sheathing base.These features likely are synapomorphic for Melanthiaceae (or at least in part synapomorphic, as some may have evolved in more inclusive clades; Fig. 1).
Graceful, scapose plants with basal leaves characterize most of Melanthieae (Bodkin 1978; Fig. 3A).The robust leafy stems characterizing Veratrum sect.Veratrum are a striking contrast to typical plant form for the rest of the tribe (Fig. !B).Several obvious correlated leaf characteristics contribute to this so-called veratrum habit-large, elliptic, sessile blades strongly plicated along several prominent primary veins (conspicuous midvein lacking) and closed, tubular, overlapping basal sheaths forming a hollow pseudostem around the true stem (Zomlefer 1997a).Some members nested within sect.Fuscoveratrum have evolved some similar features.For example, V. nigrum L. has wide sessile leaves that form a weak pseudostem at the plant base, and V. parviflorum Michx.and V. woodii J. W. Robbins have somewhat broad and weakly plaited (but petiolate) leaves.According to our results (Zomlefer et a!. 2003), these developments are likely autapomorphic at the species rank.Thus, broad plicated leaves evolved more than once within Veratrum but are best developed in sect.Veratrum, characterized by the synapomorphic "veratrum habit." Inflorescence structure also contributes to the overall appearance of a plant.In Parideae, the inflorescence comprises a single terminal flower ( Tepals.-A perianth differentiated into three foliose sepals and three colorful petals characterizes the Parideae (Fig. lF), but the other tribes have a perianth of six, generally petaloid tepals [all sepaloid in some Melanthieae, e.g., Veratrum viride (L.) Aiton].As with habit type, certain aspects of tepal morphology are most variable in Veratrum.The tepal base is generally cuneate to gradually tapered for most of Melanthieae (Table 3), with the notable exceptions of distinctively clawed tepals characterizing Toxicoscordion and some species of Veratrum sect.Fuscoveratrum.According to our cladogram (Fig. 3B), the clawed tepals synapomorphic for Toxicoscordion (Zomlefer et al. 2001) likely evolved independently from those in Veratrum.In addition, on the basis of parsimony this specialized character either evolved at least twice within Veratrum sect.Fuscoveratrum or may be synapomorphic for the section and lost in certain species (see cladograms in Zomlefer et al. 2003).In sect.Fuscoveratrum, the degree of filament adnation to the tepal is correlated with tepal shape: arising at the base of unclawed tepals to conspicuously epitepalous on clawed tepals.Additionally, the epitepalous filaments strongly incurve in the species with the most well-differentiated claws [e.g., V. latifolium (Desr.)Zomlefer].These correlations (i.e., of tepal shape and stamina!adnation/curvature) are not found in Toxicoscordion, reinforcing our hypothesis that clawed tepals evolved in parallel for Toxicoscordion and Veratrum sect.Fuscoveratrum.
The tepal margin of most members of the family are entire.In Veratrum, however, the entire (sometimes undulate) plesiomorphic condition occurs only in species of sect.Fuscoveratrum and a synapomorphic nonentire form (erose, denticulate, to deeply fimbriate) diagnoses sect.Veratrum (Fig. 3B; Table 3).The erose to dentate tepal margins in most species of Schoenocaulon have an independent origin, and according to our preliminary cladograms from ITS data (Zomlefer et al., in prep.), may be synapomorphic for the genus and lost in some species.In addition, several species of Schoenocaulon (such asS.calcicola Greenm.)have well-developed hyaline auricles at the tepa) base (Fig. 3B; Brinker 1942; Frame 1990).
Perigonal glands.-Nectaries of Melanthiaceae (Table 2), when present, vary from a generalized nectariferous area or depression at the filament-tepa!base junction (some Heloniadeae and most Parideae), septal nectaries (some Parideae), or the well-defined, often fleshy, perigonal glands synapomorphic for Melanthieae.The distinctive melanthioid glands occur on the adaxial surface of the tepals, usually near the base (Leinfellner 1961;Daumann 1970), and vary in configuration depending on the genus (Table 3; Fig. 3B).The plesiomorphic condition for the tribe, i.e., in Zigadenus s.s., comprises paired ovate glands (see Fig. 1: outgroup condition = no perigonal glands).Since the hypothesis of a single event is most parsimonious, fused tepal glands are likely a synapomorphic for the rest of the tribe (i.e., for the clade containing all genera except for Zigadenus; Fig. 3B).Therefore, the bilobed (partially fused) gland characterizing Anticlea is possibly plesiomorphic; the obovate gland shape (but not one gland per tepal) is autapomorphic for Toxicoscordion.
In Schoenocaulon, nectariferous tissue typically lines a shallow to conspicuous concavity at the tepal base except in S. officinale A. Gray, which has a single, oval, and pad-like gland (Zomlefer 1997a).Since our ITS phylogeny (Zomlefer et al. in prep.)strongly supports S. officinale as embedded within the genus, the unusual nectariferous pit is synapomorphic for Schoenocaulon, and the fleshy nectary of S. officinale is autapomorphic for that species.
Several perigonal nectary forms occur in Veratrum, including two unique nonsucculent types that have evolved within the genus (Table 3; Fig. 3B).According to our comprehensive sampling (Zomlefer et a!. 2003), the marginal and basally confluent (V-shaped) nectariferous zone characterizing most species in sect.Veratrum is likely synapomorphic for the section, and the nectariferous lateral band across the median of the tepal in several members of sect.Fuscoveratrum (V.maackii Regel complex and V. nigrum) is synapomorphic for or has evolved within this section.Presently, the level of universality is also uncertain for the paired succulent glands present in both sections of Veratrum (e.g., V. fimbriatum A. Gray in sect.Veratrum; V. woodii in sect.Fuscoveratrum) due to unresolved portions of our ITSbased cladogram.These may represent the plesiomorphic condition of the tribe or reversal to the plesiomorphic state.
According to our phylogeny (Fig. 3B), the reduction (or absence) of glands is apomorphic for and independently evolved in Amianthium and Stenanthium.Reduced/absent glands also occur in some species in Veratrum sect.Fuscoveratrum [e.g., V. anticleoides Trautv.& C. A. Mey.) H. Takeda & T. Miyake] and probably represent independent secondary reductions that may be autapomorphic for particular species in some instances (Zomlefer et al. 2003).
Ovary position is consistently superior in all tribes (Table 2) except within Melanthieae where partial epigyny has evolved in Anticlea and Stenanthium (half-inferior), and to a lesser extent, in Amianthium (partly inferior) and some Veratrum (superior to one-third inferior; Table 3).We had initially suggested the half-inferior ovary as synapomorphic for the Stenanthium/Anticlea clade (Zomlefer et al. 2001), but a partially inferior ovary may be a synapomorphy for the Amianthium/Veratrum-Stenanthium/Anticlea clade (Fig. 2).
Fruit type.-Theplesiomorphic loculicidal capsule is characteristic for Heloniadeae, Chionographideae, and Xerophylleae, while Parideae and Melanthieae each have unusual fruits (Fig. 1; Table 2) that deviate from the out group condition (loculicidal capsule for Liliaceae, berry for Smilacaceae).[The report of septicidal fruit in Chionographideae by Tamura (1998) is in error: the fruits are loculicidal but may also split deeply along the septa (Zomlefer and Judd, pers.

obs.).]
In Parideae, the often colorful fruit varies from baccate to non-fleshy and may be indehiscent (berry-like) or dehiscent (capsule-like) with irregular to regular dehiscence occurring at the base and/or along the septa and/or into the locules (Zomlefer 1996).According to Berg (1958), a few species (e.g., Trillium erectum L., T. undulatum Willd., T. camschatcense Ker-Gawl.)produce a "true berry" (i.e., indehiscent with fleshy mesocarp).The fruit of most members of the tribe, however, do not conform to classical definitions.The baccate fruit of Daiswa, for example, has loculicidal dehiscence.In Trillium, however, the dehiscent fruits evidently lack a definite type of valvate-dehiscence mechanism, and in some baccate fruits (e.g., T. maculatum Rafin.), the mesocarp is thin or mealy and the fleshy tissue comprises succulent placentae plus seed arils ("arilloid berry, " Zomlefer 1994).Baccate (sometimes dehiscent) fruit may be synapomorphic for Parideae, with loculicidal dehiscence evolving within the tribe and synapomorphic for Daiswa.The pattern of variation in fruit texture, coloration, and dehiscence in Parideae requires rigorous assessment in relation to various phylogenetic hypotheses postulated by Farmer and Schilling (2002).
The more or less apocarpous gynoecium of all Melanthieae matures into a capsule that is not strictly septicidal: the mature carpels open ventricidally (i.e., along the inner or ventral faces of the carpels) from the apex and base and then along the central column of the ovary-hence, the designation as "ventricidal capsule" by Dahlgren and Clifford (1982) for this unusual synapomorphic dehiscence.
Due to the small size of the chromosomes (ca.2.0-4.0JJ.m in length), the few comprehensive karyological studies (e.g., Lee 1985) lack the detail to infer mechanisms of chromosomal evolution, although these chromosome numbers indicate the prevalence of polyploidy and/or aneuploid variation of the prospective basic number.Polyploidy has been well documented in Veratrum sect.Veratrum (2n = 32).Based on documented secondary pairing of metaphase I bivalents in V. oxysepalum Turcz.and V. stamineum Maxim., Tokumoto (1940) hypothesized a tetraploid origin for the diploid number of this section.In addition, a polyploid sequence of 2n = 32, 64, 80, 96 occurs in V. oxysepalum (Sokolovskaya 1969;Zhukova 1969;Zhukova and Tikhonova 1971;Zhukova andPetrovsky 1976, 1980).Confirmation of the chromosome number for Zigadenus glaberrimus (a possible polyploid) and the reassessment of the base number for the Melanthieae merit further investigation (Zomlefer, in prep.), especially in relation to chromosomal evolution for the other tribes.

Phylogeny as Testable Hypotheses
As discussed above, our hypothesis of generic relationships and circumscription in Melanthieae, based on ITS and trnL-F molecular data (Fig. 2), is supported by several phenotypic and chromosomal character states.Our preliminary molecular analyses (Zomlefer et al. 2001) included 30 samples of 29 taxa in the tribe, and subsequently we have sequenced almost all taxa (total: 170 samples of 90+ taxa; Zomlefer et al. 2003 in prep.).The increased sampling for our current investigations represents the complete range of morphological variation, especially within Veratrum (Zomlefer et al. 2003) and Schoenocaulon (Zomlefer et al. in prep.).All species have resolved within the generic clades as we predicted based on morphology correlated with our preliminary cladogram.For example, the four segregate genera of the former Zigadenus complex consistently correlate with four distinct nectary types (Zomlefer et al. 2001;Zomlefer and Judd 2002), thereby supporting the circumscription of Toxicoscordion sensu Rydberg (1903) and novel delimitations of Anticlea [including Stenanthium frigidum (Schltdl.& Cham.)Kunth and S. occidentale A. Gray] and Stenanthium (including Zigadenus densus and Z. leimanthoides).
Using our phylogeny as a predictive tool, we have also extrapolated the chromosome number for certain taxa in Melanthieae.For example, new chromosome counts strengthen support for the monophyly of Stenanthium and Toxicoscordion, as circumscribed by Zomlefer and Judd (2002).The ALISO synapomorphic mitotic chromosome number of 2n = 20, reported for S. gramineum, has now been verified for the two Zigadenus species transferred to Stenanthium (Zomlefer and Smith 2002).The chromosome number 2n = 22 (or n = 11) had been reported for all species now placed in Toxicoscordion, except for one anomalous report by Zakharieva and Makushenko (1969) of 2n = 32 forT.nuttallii (A.Gray) Rydb.based on an undescribed and unvouchered plant then growing at the Munich Botanical Garden (original source not cited).Zomlefer (2003) confirmed the mitotic chromosome number of 2n = 22 for T. nuttallii with plants from populations in two counties in Texas, thereby validating the diploid number of 22 as a consistent synapomorphy for the genus.
Although earlier systematists had recognized some morphological characters that correlate with monophyletic groups within Melanthieae (e.g., spicate inflorescence for Schoenocaulon), many features, such as the non-entire tepals in Veratrum sect.Veratrum, had not been used to define supraspecific groups.In addition, the pattern of variation of these features had not previously been assessed from a phylogenetic perspective.In our investigations, clarification of polarity and level of universality of these phenotypic and chromosomal characters has supported clades resolved by our molecular analyses.In particular, the major supraspecific clades of Melanthieae (and Melanthiaceae) recognized in our studies have morphological, anatomical, chromosomal, and/ or chemical synapomorphies and are, thus, easily diagnosable.In our exploration of character evolution within Melanthiaceae, we will continue to examine potentially synapomorphic characters that may increase support of molecular based phylogenies and also facilitate recognition of these taxa.