Aliso: A Journal of Systematic and Floristic Botany Aliso: A Journal of Systematic and Floristic Botany

Floral evolution requires reassessment in basal monocots, including species formerly assigned to Melanthiaceae, in the light of recent developments in the molecular phylogenetics of monocots. We have investigated flowers of Tofieldia (Tofieldiaceae), Japonolirion (Petrosaviaceae), and Narthecium (Nartheciaceae). We confirm Engler's (1888) hypothesis that orientation of lateral flowers in monocots is dependent on presence and position of additional phyllomes on the pedicel. The type of floral orientation that occurs in Tofieldia is unusual for monocots, since the additional phyllomes are rep resented by calyculus scales rather than a bracteole, and the outer whorl tepals are initiated alternating with the calyculus scales. In Japonolirion and Narthecium, a bracteole is inserted in an adaxial transverse or transverse position; either the outer median tepa! is adaxial or no single tepa! is inserted in the median position. In Tofieldia, the pedicel has a calyculus of an abaxial and two adaxial transverse phyllomes; the outer median tepa! is adaxial. Additional phyllomes on the pedicel are not adaxial, in contrast to adaxial prophylls in the vegetative regions. The presence or absence of a bracteole or calyculus is taxonomically important. Tofieldia pusilla differs from the other species of Tofieldia examined in the absence of a flower-subtending bract, but the calyculus demonstrates some bract-like features in position, structure and development, which can be interpreted as a hybridization of developmental pathways. The abaxial calyculus scale of T. coccinea is delayed in development.


INTRODUCTION
Prior to recent taxonomic rearrangements based partly on molecular data (e.g., Angiosperm Phylogeny Group [APG] 1998), genera that were formerly ascribed to Melanthiaceae s.l.(or Liliaceae-Melanthioideae) were considered to represent some of the most basal monocots because of their relatively "primitive" flowers with mostly free floral parts and at least partially apocarpous ovaries (Melchior 1964;Dahlgren et al. 1985;Takhtajan 1987Takhtajan , 1997;;Thorne 1992).Therefore, they were grouped partly on the basis of putatively plesiomorphic characters and partly because of their geographical distribution and habitat preferences, as they are mostly marsh-loving northern temperate herbs.
Plant material was fixed in FAA or 70% ethanol and stored in 70% ethanol.For scanning electron microscopy (SEM), parts of inflorescences and flowers were dehydrated in 100% ethanol and 100% acetone.Dehydrated material was critical point dried and sputter-coated with Pt/Pd.

Narthecium ossifragum (Nartheciaceae)
Inflorescences are racemose.Flowers are trimerous, actinomorphic, pedicellate, and subtended by a well-developed bract and have a single bracteole; the bract is slightly longer and broader than the bracteole (Fig. 2, 3).There are six tepals in two whorls of three, free at early stages of development and united in a floral tube at later stages (Fig. 4).
At early stages of flower development, the bract, bracteole, and tepals have secretory hairs on their tips (Fig. 2), but these are less prominent at later developmental stages (Fig. 3).The secretion products of these hairs hold the tepals together, probably to protect developing stamens and carpels.
Floral orientation in Narthecium ossifragum is variable due to the unstable position of the bracteole.The diagrams in Fig. 1 illustrate two limits of variation of bracteole orientation; both types and intermediate forms may be present in the same inflorescence.Bracteoles may be inserted to the left or the right of the flower.In some cases flowers possess the outer median tepa] in adaxial position; the bracteole occurs opposite any inner lateral tepal (Fig. lB).In other cases, the bracteole occupies an almost transverse position; one of the outer tepals is situated between the bract and the bracteole, another is opposite the bracteole, and the third occurs between the inflorescence axis and the bracteole (Fig. lC).

Japonolirion osense ( Petrosaviaceae)
Inflorescences are racemose.Flowers are trimerous, actinomorphic, pedicellate, and subtended by a well-developed bract and have a single bracteole; the bract is longer and broader than the bracteole (Fig. 5-7).The bracteole occurs at either the left or the right side of the flower, and occupies an intermediate transverse-adaxial o r a lmost transverse positio n.It is situated opposite an inner lateral tepal.There are six free tepals in two whorls of three, th e outer tepals shorter than the inner.In the o uter whorl of tepals, o ne tepa! is median adaxial and two are transverse-abaxial if the bracteole is inserted in tran sverse-adax ia l position (Fig. 1B).As in Narth.ecium,bracteole position is unstable, so flower orientation is also un stable (Fig. I B, C).

Tofie ldia (To.fieldiaceae)
A common feature of all Tofie ldi aceae is the presence of a structure that is usuall y termed a calyculus (e.g ., Engler 1888;Zomlefer 1997;Rerni zova and Sokoloff 2003).The term "calyculus" can be defined as a group of phyllo mes on a ped ice l th at simulates an outer whorl of the perianth.In te rms of homology, the calyculus does not represent part of the flower.The term "involucre" is sometimes used as a synonym of "calyculus" (e.g., Engler 1888;Zomlefer 1997).
( 1) Tofieldia coccinea, T. okuboi (sect.Tofieldia) Inflorescences are racemose.Flowers are trimerous, actinomorphic, pedicellate, and subtended by a well-developed bract.Six basally united tepals occur in two distinct whorls.A three-lobed calyculus is inserted on the pedicel just below the flower.The calyculus normally has a radiate structure and consists of three connate scales, one median abaxial, the others transverse adaxial (Fig. I D), but rarely the fusion of scales is not complete (e.g., the median scale is free).The three scales are of equal size (or sometimes the median scale is shorter than the lateral ones) and alternate with the outer tepals; therefore, the median outer tepa! is adaxial.
Early stages of inflorescence and flower development were examined in T. coccinea, and later stages in the closely related species T. okuboi; these two species are similar in mature floral morphology.Flowers are initiated in an acropetal sequence on the inflorescence axis.A terminal flower is absent, and a residual meristem is visible at the inflorescence apex (Fig. 8).The subtending bract and flower are usually initiated as a common primordium that rapidly divides into two separate primordia by means of a latitudinal slit (Fig. 9); the resulting bract primordium and flower primordium of a given pair may be roughly equal in size, or in some cases the bract primordium is (much) smaller (bract primordia are larger in the lower part of the inflorescence).Occasionally, the subtending bract and the flower primordium are initiated separately and almost simultaneously.
Calyculus initiation is always unidirectional.Lateral scales arise before abaxial one.If floral primordia are not too densely aggregated (especially on the top of the raceme), lateral calyculus scales arise on the adaxial side of the floral meristem as a single crescent-shaped primordium (Fig. 10, II); each lateral scale forms as an outgrowth of this primordium (Fig. 12).If the floral primordia are densely arranged, each lateral calyculus scale is initiated as a separate primordium (Fig. 13).
The median scale is always initiated later as a separate primordium, either before (in the case of small bract primordia) or after (in the case of large bract primordia) formation of tepals and stamens (Fig. 14).The median scale fuses with two other scales at a later stage of flower development (Fig. 15-19) to form a calyculus tube, by means of "zonal growth" (as, for example, in calyx and stamen tubes of papilionoid legumes: Tucker 1987).
Tepals and stamens are often initiated as common primordia (PA: perianth plus androecium; Fig. 20, 21 ), although in some cases the tepa! and stamen of the same pair arise as separate primordia (Fig. 22,23).Mode of tepal/stamen initiation is sometimes difficult to determine at early stages of flower development, probably due to the presence of intermediate types.
The FA-primordia of the outer whorl alternate with the primordia of the lateral calyculus scales and the primordium of the subtending bract or the primordium of the median calyculus scale (Fig. 20).Initiation of the inner PA-primordia follows soon after.PA-primordia of the same whorl are initiated simultaneously or reveal unidirectional develop-ment, in which case they appear first on the adaxial side of the young flower (Fig. 24,25).Unidirectional development appears to be correlated with large bract primordium size: the larger the bract primordium, the more prominent the unidirectionality.Since bract primordium size is somewhat variable, the sequence of organ initiation is also relatively unstable.Here we illustrate two of the more common types: (I) The outer median FA-primordium and the inner transverse PA-primordia are initiated first (Fig. 24), followed by the outer transverse FA-primordia and the inner median FAprimordium.(2) All outer FA-primordia are initiated almost simultaneously, followed by the inner transverse FA-primordia and the inner median FA-primordium (Fig. 25).Soon after initiation, each FA-primordium divides into tepa!and stamen primordia (Fig. 21).The calyculus scales and tepals grow more rapidly, and soon completely cover the central part of the developing flower.
(2) Tofieldia pusilla (sect.Unibracteatae) Inflorescences are racemose.Flowers are trimerous and actinomorphic, with six free tepals in two distinct whorls.The flower-subtending bract is absent, and the calyculus occupies the position of the bract at the base of the pedicel (Fig. IE,26,27).The calyculus of T. pusilla has a bilateral structure and consists of three scales, as in members of sect.Tofieldia.Normally the lateral scales are fused with the median abaxial one but free from each other (Fig. 26, 27); the median scale is usually longer than the lateral ones.This calyculus structure is atypical for both To.fieldia and Tofieldiaceae, in general.The outer tepals alternate with the calyculus scales.The median outer tepa! is adaxial.The calyculus of T. pusilla is highly variable both in structure and size of scales (Remizova and Sokoloff 2003).
Flowers are initiated in an acropetal sequence along the inflorescence axis as hemispherical bulges (Fig. 28).A terminal flower or flower-like structure is absent.A subtending bract is absent at all stages of development.The first part to be initiated is the calyculus, which arises as a single hemispherical or slightly transversely extended bulge on the abaxial side of the floral primordium (Fig. 29).Soon after initiation, the calyculus becomes horseshoe-shaped.The lateral scales arise as appendages on the common horseshoe-shaped primordium (Fig. 30, 31; Remizova and Sokoloff 2003).In some flowers the lateral scales arise as separate primordia (Fig. 32, 33).
Tepals and stamens are initiated as common FA-primordia or as distinct primordia, but always from the same sector of the floral meristem (Fig. 34-37).Organs of the same whorl arise simultaneously; unidirectional flower development is lacking in T. pusilla.Outer FA-primordia alternate with the calyculus scales.Inner FA-primordia are initiated soon after the outer ones.In some cases of distinct tepa! and stamen primordial development, the outer stamens and inner tepals are initiated almost simultaneously.

DISCUSSION
In general, floral orientation in Iilioid monocots is highly dependent on the presence of a bracteole (Eichler 1875;Engler 1888).In trimerous lateral flowers subtended by a bract, if the bracteole is absent the median tepa! of the outer whorl Basal part of mature flower.Calyculus is recurved to show floral tube. (BR = subtending bract; FP = floral primordium; L = lateral calycu lu s scale or its primordium; M = median calyculus scale or its primordium ; OT = ou ter tepa!; IT = inner tepal .Scale bars: Fig. 14, 15 = 100 f.Lm, Fig. 16, 18, 19 = 300 f.Lm, Fig. 17  occupies an abaxial position (see also Rudall and Bateman 2004).This type of floral orientation (Type 1) is most common in lilioid monocots (Fig. lA).If a bracteole is present, it occurs in either an intermediate (between median adaxial and transverse) or transverse position.The bracteole is always situated in the same radius as one of the inner whorl of tepals.Due to the unstable bracteole position, floral orientation is also unstable (Type 2; Fig. 1B, C).This variability of floral orientation due to various bracteole positions has been described for Dianella longifolia R. Br. (Hemerocallidaceae) (Eichler 1875 ;Engler 1888).Type 3 floral orientation is characteristic of Tofieldia and its relatives (Tofieldiaceae), in which the median outer tepa! is adaxial due to insertion of a trimerous calyculus below the perianth (Fig. lD, E).
All three types of floral orientation recognized by Eichler (1875) and Engler (1888) are present in Melanthiaceae s.l., supporting their polyphyletic origin.Type I occurs in Veratrum L. (Melanthiaceae s.s.; e.g., Endress 1995), and Type 2 in Japonolirion and Narthecium.The sequence of organ initiation has been studied in detail in two lilioid genera with trimerous flowers and presence of a bracteole: Allium L. (Jones and Emsweller 1936) and Lilium L. (Greller and Matzke 1970).In both cases, tepa! initiation occurs in a spiral direction.The first outer tepa! arises opposite the bracteole, and the second between the first outer tepa! and bracteole, close to the bracteole.The direction of initiation of the inner tepal s is the reverse of the outer.In basal monocots, when the bracteole is absent, the sequence of perianth initiation is either simultaneous in each whorl or unidirectional, but seemingly never spiral (e.g., Endress 1995;Buzgo and Endress 2000;Buzgo 2001; our data).We have not yet observed sequences of perianth development in Japonolirion and Narthecium, but predict that they have a similar pattern of floral initiation to that of Allium and Lilium.Melchior ( 1964) illustrated a floral diagram of Petrosavia with reverse floral orientation to that of Japonolirion, with the outer median tepa! situated in an abaxial position and a bracteole in the radius of another outer tepa!.However, our investigations have demonstrated that the bracteole occurs in the same radius as one of inner tepals i.n both Petrosavia (unpubl.data) and Japonolirion.This supports recent analyses of molecular data that place Japonolirion as closely related to Petrosavia (Cameron et al. 2003).Another feature shared by both Petrosavia and Japonolirion is that the tepals of the outer whorl are smaller than those of the inner whorl.
Tofieldia has a structure that is unusual for monocots, termed a calyculus, which is common to all Tofieldiaceae.As a consequence, the flower of Tofieldia has almost the same orientation as some flowers of Type 2. There are significant differences between the different taxonomic sections of Tofieldia, both in calyculus morphology and developmental pattern, but their calyculi are probably homologous (Remizova and Sokoloff 2003).In Tofieldia sect.Tofieldia, the calyculus is situated just below the flower and has a radial structure (Fig. 1D,(15)(16)(17)(18)(19).By contrast, in T. pusilla (sect.Unibracteatae), a true snbtending bract is absent, and the calyculus is inserted at the base of pedicel , and has a bilateral structure (Fig. IE,26,27).The median organ of the calyculus not only occupies the position of the bract but also shows some bract-like features, both in morphology and de- (FP = tl ora l primordium ; M = medi an ca lyculus scale primo rdium ; L = late ra l calyculu s scale primo rd ium.Sca le ba rs: Fi g. 28, 3 1 = 100 f.Lm , Fi g. 29 = 50 f.Lm , Fi g. 30 = 300 f.Lm , Fi g. 32 = 80 f.Lm , Fi g. 33 = 30 fJ.m .)velopment (Remizova and Sokoloff 2003).In T. coccinea (sect.Tofieldia) the calyculus often develops as two separate primordia: the common primordium of the lateral scales, and the primordium of the median scale.This pattern of lateral calycu lus scale initiation in T. coccinea resembles prophyll initi ation in the vegetative bud.However, the lateral scale cannot be interpreted as a prophyll because in basal monocots with racemose inflorescences prophylls in the floral region (i.e., bracteoles), if present, are not adaxial.Moreover, th e modification of prophyll development in sect.Unibrac-teatae (T.pusilla) wou ld be un clear if we interpreted the lateral scales of sect.To.fieldia as a two-keeled prophyll.Another possibility is that the two lateral calyculus scales represent two lateral prophylls.We consider such an interpretation unlikely, because of the presence of a third (abaxial) calyculus sca le, which shou ld be interpreted as the next phyllome on the lateral shoot.In other monocots, we do not know any example of fusion between lateral prophylls and the next phyllome on the lateral shoot.
Formation of " hybrid " organs that comb ine characters of the flower-subtending bract and the first median abaxial phyllome on the pedicel is a feature common to T. pusilla and some other Alismatales, such as Aponogetonaceae, Juncaginaceae and Potamogeton densus L. (Potamogetonaceae), in addition to the putatively basal monocot, Acarus (Posluszny and Sattler 1973;Buzgo and Endress 2000;Buzgo 2001;Remizova and Sokoloff 2003).Formation of similar bract-like organs may be regarded as additional morphological evidence for a close relationship between these taxa.However, this similarity apparently represents a homoplastic tendency (or even a possible symplesiomorphy) rather than a synapomorphy.In Tofieldia pusilla, the first abaxial phyllome exhibiting bract-like features is the median calyculus scale, whereas in Acoraceae (Fig. 10), Aponogetonaceae, Juncaginaceae, and Potamogeton L. this is the outer median tepal.
According to Buzgo (2001), in Alismatales s.l., unidirectional development is often correlated with structures resembling subtending bracts (and absence of a true bract), and with formation of peloria (or more precisely, terminal flower-like structures: Buzgo et al. 2004).Our data show no such correlation in Tofieldia.Among species examined here, unidirectional floral development occurs in Tofieldia coccinea (sect.Tofieldia), but this species has a true subtending bract and no terminal flower-like structures.In T. pusilla the true bract is absent, being replaced by the median organ of the bract-like calyculus, but organ initiation is simultaneous in each whorl.
There is some similarity between calyculus development in Tofieldia sect.Tofieldia and tepal development in some lilioid monocots.In T. coccinea the median abaxial calyculus scale is considerably delayed in development.In Veratrum (Melanthiaceae s.s., Liliales) and Bulbine Wolf (Asphodelaceae, Asparagales) there is no calyculus or bracteole on the pedicel, but the development of the median outer tepal is delayed (Endress 1995).Thus, unidirectional development is probably a result of the presence of a closely situated large bract primordium in these cases (see also Endress 1999).
Our data show that the presence or absence of a calyculus and bracteole are stable characters within relatively large taxonomic groups of basal monocots.The presence of a bracteole and calyculus seems to be more stable than the presence of a bract, since a bract may be either present or absent in both Tofieldia and Potamogeton.Characters such as presence of bracteoles and calyculus can be used as taxonomic characters at the family level, but are homoplastic at higher taxonomic levels.For example, bracteoles are similar in members of Liliaceae, Nartheciaceae, and Petrosaviaceae, which are not closely related.The calyculus of Tofieldiaceae can be compared to a whorl of leaves below the flower in Paris L. and Trillium L., which belong to the relatively distantly related family Melanthiaceae s.s.(Liliales).The calyculus can also be compared to (pseudo)whorls of bracts in Alismataceae and the "spathe" of Hydrocharitaceae (Remizova and Sokoloff 2003).These two families are more closely related to Tofieldiaceae than Melanthiaceae (Chase et al. 2000;APG II 2003), but there is currently no direct evidence that these organs represent a synapomorphy for these families.CONCLUSIONS Among basal monocots, floral orientation is often a conservative feature in groups of family rank such as Acoraceae (Buzgo and Endress 2000), Araceae (Buzgo 2001), Tofieldiaceae (Remizova and Sokoloff 2003), and many others.Interestingly, Araceae and Tofieldiaceae, which belong in the same clade, share a similar type of floral orientation that is otherwise rare in monocots (Fig. 1).When a bracteole is present and its position on the pedicel is unstable, floral orientation is also unstable, often within a given inflorescence.In general, the orientation of lateral flowers in monocots is highly dependent on the presence and position of additional phyllomes on the pedicel.In Japonolirion, Lilium, Narthecium, and many others, the additional phyllome is represented by a bracteole.During flower development, an outer whorl tepal is never initiated in the radius of the bracteole; an inner whorl tepal is initiated there instead.In Tofieldia, the additional phyllomes are represented by calyculus scales and the outer whorl tepals are initiated alternating with the calyculus scales.Thus, the type of floral orientation that occurs in Tofieldia and other Tofieldiaceae is unusual for monocots.
Our investigation reveals striking differences in pattern of calyculus development within Tofieldia between members of sect.Tofieldia and sect.Unibracteatae: phyllome primordia surround the floral primordium in a very different manner, as flower subtending bract and lateral scale primordia in T. coccinea, and as a calyculus primordium in T. pusilla.The difference between the two sections is especially clear in position of lateral calyculus scales.In sect.Tofieldia the scales are initiated in transverse-adaxial position, but in almost transverse position in sect.Unibracteatae.Nevertheless, in both T. coccinea and T. pusilla the type of floral orientation is relatively stable (i.e., the median outer tepal is adaxial).Thus, it is possible that floral orientation in Tofieldia represents more than the result of the presence of a calyculus.
As might be expected in view of their polyphyletic origin, there are significant differences in inflorescence and flower morphology, floral orientation and flower development between members of the species examined here.Data on flower and inflorescence morphology and development are important for investigations of the systematics and phylogeny of basal monocots, even if many of the similarities represent homoplastic tendencies or symplesiomorphies rather than true synapomorphies.

Table I .
Remizova and Sokoloff 2003) examined (in addition to material listed inRemizova and Sokoloff 2003).