HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplemental Data Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Olson, M. E. Search for Related Content PubMed Articles by Olson, M. E. Agricola Articles by Olson, M. E.

Ontogenetic origins of floral bilateral symmetry in Moringaceae (Brassicales)¹

Instituto de Biología, U.N.A.M., Departamento de Botánica, Circuito exterior s/n, Ciudad Universitaria, Copilco, Coyoacán, A.P. 70-367 México, D.F., C.P. 04510, Mexico

Received for publication February 26, 2002. Accepted for publication June 11, 2002.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Floral morphology of the 13 species of Moringa ranges from actinomorphic flowers with little hypanthium to highly zygomorphic flowers with well-developed hypanthia. Scanning electron and light microscopy were used to identify ontogenetic differences among two actinomorphic and eight zygomorphic species. All species show traces of zygomorphy between petal organogenesis and anther differentiation. At late organogenesis, zygomorphy is manifest by one petal being larger than the others, slight unidirectional maturation of the anthers, and in many species, some staminodes may be missing. At organ differentiation and beyond, the actinomorphic species show a trend toward increasing actinomorphy, whereas the zygomorphic features of early ontogeny are progressively accentuated throughout the ontogeny of the zygomorphic species. Because of the early traces of zygomorphy throughout the family, ontogeny in Moringa does not resemble that known from the sister taxon Caricaceae, which has flowers that are actinomorphic throughout ontogeny. Great intraspecific variation was found in floral plan in the actinomorphic-flowered species in contrast to the zygomorphic species. Each of the main clades in the family is distinguished by at least one feature of floral ontogeny. In general, ontogenetic differences that are congruent with deeper phylogenetic splits tend to occur earlier in ontogeny than those congruent with more recent divergences.

Key Words: Brassicales • development • evolution • floral symmetry • homology • Moringa • ontogeny • phylogeny

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Bilateral floral symmetry (zygomorphy) has repeatedly arisen from ancestors with radially symmetrical (actinomorphic) flowers (Endress, 1999 ) and is a character of major systematic significance. Likewise, floral symmetry is one of the primary factors determining the spectrum of pollinators that a flower attracts (Dafni and Kevan, 1996 ). As a result of the importance of this character, the evolutionary processes leading to symmetry differences have received attention from a variety of perspectives, from molecular genetics (Theissen, 2000 ; Shepard and Purugganan, 2002 ) to comparative morphological studies of ontogeny (e.g., Tucker, 1984 , 1997 , 2000 ). The latter approach is particularly valuable when a phylogenetic hypothesis for the group in question is available, because the phylogeny can be used to identify the polarity of evolutionary transformations within a clade. For example, Hufford (1995) evaluated his studies of comparative ontogeny in the snapdragon relative Besseya in the context of a phylogeny estimated from nonfloral data and arrived at the counterintuitive conclusion that evolutionary inversions and deletions of early and intermediate ontogenetic stages are as common as modifications to the terminal phases of ontogeny.

The 13 species of Moringa span the range from small, actinomorphic, cream-colored flowers with little hypanthium, to highly zygomorphic, intensely colored flowers with pronounced hypanthia (Figs. 1–13). Recent field work in dry tropical Asia, Africa, and Madagascar has made it possible to include ten of the 13 species in a comparative study of floral ontogeny with the following aims: (1) document floral organogeny, anatomy, and ontogeny in Moringa, the only genus in Moringaceae; (2) test the expectation that actinomorphic flowers in Moringa are entirely so in all stages, as in the the sister group Caricacae (Ronse Decraene and Smets, 1999 ), and that zygomorphy becomes manifest in the later stages of ontogeny; (3) examine the congruence of ontogenetic characters with a previous phylogenetic reconstruction (Fig. 14); and (4) examine the correlation of the timing of ontogenetic divergences between species groups with degree of phylogenetic relationship. Because perturbations to early ontogeny may drastically affect subsequent events, numerous workers (e.g., Takhtajan, 1972 ; Tucker, 1984 , 1997 ) have suggested that early stages should be more evolutionarily conserved while later stages are more evolutionarily labile. Therefore, divergences in early ontogeny should characterize deep phylogenetic splits, and divergences at later stages should characterize more recent evolutionary branching.

View larger version (61K): [in this window] [in a new window]   Figs. 1–7. Flowers of all species in the actinomorphic grade and slender tree clades of Moringa. Side views unless specified. 1–4. Members of the actinomorphic grade showing actinomorphic or nearly actinomorphic arrangement of parts. 1. Moringa drouhardii Jum., apical and side views, showing reflexed petals, and bud showing irregular shape at base. 2. Moringa hildebrandtii Engl., showing slightly unequal filaments 3. Moringa stenopetala (Baker f.) Cufod., with details showing shallow and deep lobing of style apex. 4. Moringa ovalifolia Dinter & A. Berger. 5–7. Members of the slender tree subclade of the zygomorphic clade. 5. Moringa concanensis Nimmo, flower with nonadhering filaments and reduced anthers, with detail of reduced anther. 6. Moringa oleifera Lam. with detail of three-tiered anther presentation. 7. Moringa peregrina (Forssk.) Fiori

View larger version (45K): [in this window] [in a new window]   Fig. 14. Phylogenetic distribution of floral actinomorphy and zygomorphy in Moringa based on phylogeny of Olson (2002) . The basal four species that make up the actinomorphic grade have radially symmetrical or nearly radially symmetrical flowers. The radially symmetrical symbol labelling this grade is repeated in the figures that follow to denote a member of this group. The rest of the family comprises the zygomorphic clade. The bilaterally symmetrical symbol shown labeling this clade is also repeated in the figures that follow. This clade is divided into two subclades: the slender trees (S) and the tuberous shrubs and trees of northeast Africa (T). The tuberous clade is further subdivided into the rivae clade (ri) and the red-flowered clade (rf). Species included in the microscopy component of the present study are indicated with an asterisk

View larger version (55K): [in this window] [in a new window]   Figs. 8–13. Flowers of all species of the tuberous subclade of the zygomorphic clade in Moringa. Side views unless specified. 8–11. Members of the rivae subclade. 8. Moringa arborea Verdc.; sepals may be entirely reflexed. 9. Moringa borziana Mattei, showing nearly actinomorphic calyx. 10. Moringa rivae Chiov. subsp. rivae; lateral views of flower from above and one side as it is oriented at anthesis. 11. Moringa pygmaea Verdc. from type specimen. 12–13. Members of the red-flowered subclade. 12. Moringa ruspoliana Engl. 13. Moringa longituba Engl

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Inflorescences of ten species were collected from the field or from cultivated specimens (see http://ajbsupp.botany.org/v90/ for voucher information; species studied are noted with an asterisk in Fig. 14) and preserved in FAA (formalin-acetic acid-alcohol) or locally available alcohol. All specimens were transferred to 70% aqueous ethanol for storage. For scanning electron microscope (SEM) observation, samples were moved to 95% aqueous ethanol at least 24 h before dissection to make the tissue more brittle, facilitating the removal of sepals and petals. After dissection, the material was passed through three changes of absolute ethanol, critical-point dried, mounted on stubs, sputter coated in a Polaron E-5000 (Polaron, Warrington, Pennsylvania, USA) and observed in an Hitachi S-450 SEM (Hitachi Scientific Instruments, Pleasanton, California, USA) at 20 kV. For light microscope (LM) observation, samples were dehydrated in an ethanol-tertiary butyl alcohol series, embedded in paraffin, sectioned on a rotary microtome at 13 µm, and stained with a safranin-fast green combination. Hand sections stained with cresyl violet acetate were also employed to confirm observations made with SEM and LM, particularly for the later stages of ontogeny. The ontogenetic stages studied with each method are summarized for each species at http://ajbsupp.botany.org/v90/. Herbarium material of all 13 Moringa species was consulted from the following herbaria: BM, EA, FT, K, MO, ON, P, PAL, RHT, TAN, TEF, UPS, WIND. The phylogenetic hypothesis used here is the preferred topology presented in Olson (2002) and is based on variation in three DNA loci and morphological data. The morphological data set included seven floral characters whose main role was the placement of the unsequenced M. pygmaea.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In many cases it is possible to generalize regarding ontogenetic patterns in more than one species and avoid cumbersome descriptions of each species individually. Thus in many cases I refer to development in the "tuberous clade" to summarize observations in those species when I have found homogeneity among samples. The samples and stages on which these generalizations are based can be found at http://ajbsupp.botany.org/v89/.

Inflorescence structure
All of the species of Moringa bear axillary panicles. The simplest inflorescence is found in M. ruspoliana, which has one flower borne at the apex of the inflorescence (the "terminal" flower), with up to five additional flowers ("primary" flowers) borne on pedicels branching from the main inflorescence axis (diagram in Fig. 17; a very young inflorescence is shown in SEM images in Figs. 15–16). The pedicel of a primary flower is subtended by a bract on the main inflorescence axis (particularly conspicuous are those labeled "6c" and "7c" in Fig. 15). Each pedicel also bears two bracteoles (e.g., those labeled "2b" in Fig. 16). The terminal flower of the inflorescence may rarely be subtended by a bract (e.g., the flower labeled "1a" and its bract "1c" in Fig. 16; this bract is shown in gray in Fig. 17). Although they are indicated as such in the schematic Figs. 17–22 to distinguish them from the bracteoles, in all species the bracts are not always larger than the bracteoles at anthesis.

View larger version (126K): [in this window] [in a new window]   Figs. 15–22. Inflorescence structures in Moringa. 15–16. Moringa ruspoliana. Entire panicle with some floral buds partially dissected, SEM micrographs. Structures marked "a" are flowers, "b" are bracteoles, and "c" are bracts. The numbers indicate the members subtended by the same bract: 1a is the top flower in the panicle, and 1c is its bract, while 2a is the next flower down in the inflorescence, 2b is its bracteoles, and 2c is its bract, etc. In some cases, bracts or bracteoles have been removed. Bars = 500 µm. 15. Oblique view of panicle. 16. Apical view of the same panicle. 17–22. Schematic representations of inflorescences in Moringa. The main inflorescence axis is shown in gray; in black are groups of flowers consisting of one bract, a pedicel with two to three bracteoles, one flower borne at the end of the pedicel (the primary flower, indicated by a 1), flowers borne in the axils of the bracteoles (2 = secondary flowers), flowers borne in the axils of the bracteoles of the secondary flowers (3 = tertiary flowers), flowers borne in the axils of the bractoles of the tertiary flowers (4 = quaternary flowers); t = the terminal flower of the inflorescence; t1 = the terminal flower of of an inflorescence branch; bracts are shown as being larger than bracteoles, though this is often not the case. 17. Basic inflorescence structure consists of an apical flower that rarely is subtended by a bract (shown in gray). Bracts on the main axis subtend pedicels that bear usually two bracteoles and a flower. 18. Subsidiary flowers are often borne in the axils of the bracts (not shown) or bracteoles (as shown here). The pedicels of these flowers bear two bracteoles. 19. Dichasium-like inflorescence common in some members of the tuberous clade. 20. Similar inflorescence in M. arborea and M. rivae with multiple tertiary flowers in the axils of bracteoles of secondary flowers. 21. Compound inflorescence composed of repetitions of a module like that in Fig. 18 . along a main axis shown as a gray dashed line. Bracts at the base of inflorescence branches are shown in gray. Tertiary and quaternary flowers are shown on the right side of the inflorescence. For clarity, only primary and secondary flowers are shown on the left side. 22. Inflorescence structure in M. ovalifolia

Except for M. ruspoliana and the small tuberous shrub species M. longituba, M. borziana, and M. pygmaea, inflorescences in Moringa are often characterized by three or more repetitions of inflorescence units similar to those in Fig. 18 along a main axis (represented by the dashed line in Fig. 21). The main axis of such an inflorescence has a terminal flower, as do the branches (labeled as "t1" in Figs. 21 and 22). Often glandular bracts subtend the branches along the main inflorescece axis (shown in gray in Fig. 21). The inflorescences of the three species of slender tree and the actinomorphic-flowered species correspond to this type. The inflorescences of the slender trees are characterized by abundant initiation of flowers in the axils of the bracteoles of secondary and tertiary flowers (Fig. 21), and pedicels that elongate more markedly than those in the rest of the family. The inflorescence subunits close to the terminal flowers may be reduced with respect to those in the rest of the inflorescence. In the actinomorphic-flowered species, some of the internodes in the inflorescence may be very short, a characteristic most conspicuous in M. ovalifolia. The very short internodes in each congested group of ten flowers gives them a nearly fascicular appearance (Fig. 22 shows such clusters with slightly elongated internodes for clarity).

Variation in floral plan
Little variation in floral plan was observed in the flowers of the zygomorphic clade. The most common sepal arrangement in Moringa is found in two equally common enantiomorphs (Figs. 23, 24). Both consist of two outer sepals that are initiated first and second in ontogeny and usually remain larger than the others, two internal sepals, and another that is overlain only by the first sepal. Which enantiomorph a given flower displays does not appear to be correlated with terminal or lateral position in an inflorescence.

View larger version (31K): [in this window] [in a new window]   Figs. 23–28. Floral diagrams in Moringa. Black arc "B" = subtending bract; black lateral arcs are bracetoles subtending secondary flowers (white ovals with black outlines). Small black circle = inflorescence axis. Black numbered arcs = sepals with order of initiation; gray arcs = petals. 23, 24. The most common enantiomorphs in Moringa. In the zygomorphic clade, the petal between sepals 3 and 5 is usually larger than the others and is borne at the top of the flower at anthesis (the banner = b). The other four petals are lateral with respect to the banner and are referred to as the upper petal pair (between sepals 2/5 and 1/3) and the lower petal pair (between sepals 1/4 and 2/4). 25. Rare variations in the zygomorphic clade, with differences from Fig. 23 indicated by arrows. 26. An arrangement with imbricate petals and identically oriented anthers that is common in the actinomorphic grade. 27, 28. Common petal arrangement variants found in the actinomorphic grade. External petals are marked "b."

In all members of the zygomorphic clade examined, the anthers point toward the banner in bud (Fig. 23). The variation that was observed mostly consisted of between-species differences in anther inclination with most species having the long axes of the anthers more or less parallel with the long axis of the banner in bud. In other species, such as M. borziana, the distalmost anthers (farthest at left in Fig. 9) are held strongly obliquely in bud.

Sepal arrangements in the actinomorphic species resemble those in the zygomorphic clade. However, the actinomorphic species show much more intraspecific variation in petal and anther arrangement. In the actinomorphic species, as in the zygomorphic species, one petal is usually external and larger throughout the early stages of ontogeny. But rather than the consistent location of this petal between sepals 3 and 5 as in the zygomorphic-flowered species, it was observed equally frequently between the sepal pairs 3/5, 1/3, or 2/4 (Figs. 27, 28). Because this petal resembles the banner of the zygomorphic species in its larger size and external position but is of inconsistent location, such a petal is referred to as a "banner-like" petal. Just as commonly, petal aestivation was observed to be entirely imbricate (Fig. 26). The petal arrangement shown in Fig. 28 was the one most commonly observed in M. ovalifolia, in which one external petal overlaps two petals, one of which is entirely internal and the other that is overlain only by the external petal. No consistent pattern was observed as to the handedness of these two petals (the internal petal could be on either side of the external petal). Likewise, no consistent orientation of this petal arrangement with respect to the sepals was observed, with the external petal having been observed between sepal pairs 1/3 (slightly more common than the other positions), 1/4, 2/5, and 3/5.

Anther orientation shows even more variation in these species, from the arrangement typical of the zygomorphic species (Fig. 23) to all having the same oblique orientation perpendicular to the floral axis (Fig. 26). The orientation most commonly observed consisted of four anthers facing the same side of their respective petals and one anther pointing to the other side (Fig. 27).

Floral ontogeny: stages
Floral ontogeny is summarized below for the ten species examined. The account of the continuous process of floral ontogeny is divided into the following phases for convenience: (1) organogenesis, consisting of the appearance of primordia; (2) differentiation, in which the primordia become distinguished from one another in shape and continue to enlarge; (3) enlargement, in which differentiated floral parts enlarge and change positions; (4) anthesis, which mostly involves flexion of the sepals, petals, and stamens, and elongation of the gynoecium. These four categories correspond, respectively, to Tucker's (1997) early-, mid-, late-stage, and anthesis categories. In Figs. 29–86, radially symmetrical symbols surround the figure number in images of actinomorphic-flowered species, and bilaterally symmetrical symbols surround those of zygomorphic species.

View larger version (208K): [in this window] [in a new window]   Figs. 29–37. Organogenesis in Moringa flowers, SEM micrographs. Labels on sepals and petals correspond to Fig. 23 and as follows: p = non-banner petal primordia; st = stamen primordia; so = staminode primordia. Bars = 50 µm. 29. Moringa arborea bud at sepal initiation. 30. Moringa longituba floral bud prior to petal initiation showing sepals beginning to cover the floral apex shortly after sepal initiation. 31. Moringa longituba bud with sepals removed. 32. Moringa ruspoliana bud with sepals 1 and 2 removed to show that petals have not been initiated. 33. Moringa ruspoliana bud with sepals removed to show that petals and stamens form at angles of the pentagonal field only after sepals completely cover bud. 34. Moringa peregrina bud with sepal 1 removed and petal, stamen, and staminode primordia emerging. 35. Moringa longituba bud showing simultaneous emergence of petal, stamen (in the antepetalous positions), and ovary primordia. 36. Moringa peregrina bud showing bilateral symmetry in the form of elongation in the direction of the banner and suppression of the staminode opposite sepal 4. 37. Moringa drouhardii bud with sepals removed showing bilateral symmetry in the form of a banner that is larger than the other petals and a lost staminode opposite sepal 4

As the sepals converge over the floral apex, petal primordia appear simultaneously on the apices of the pentagonal field, in alternate positions with respect to the sepals (Figs. 33–37). The stamen primordia, which are antepetalous, form immediately after the appearance of petal primordia. Similar patterns of petal and stamen initiation were observed in all samples studied, including members of the tuberous clade (e.g., M. longituba in Fig. 35), the slender tree clade (e.g., M. peregrina in Fig. 36), and the actinomorphic grade (e.g., M. drouhardii Fig. 37).

Despite extensive sampling of the early stages of floral ontogeny, no samples were encountered in which only one or two or even only three staminodes appear. When one staminode can be seen, at least three others can be detected. Therefore, it appears that the staminodes, like the petals and stamens, emerge for all practical purposes simultaneously. In the slender trees, the staminode opposite sepal 4, which is the lowermost staminode at anthesis, is often the smallest or fails to form entirely (e.g., Figs. 36, 39, but present in Fig. 44; "lost" in the sense of Tucker, 2000 ). The lack of a staminode in the actinomorphic clade is also common, but is not consistent in its location with respect to the sepals and petals (Figs. 37, 38, 56, 58). No flowers from members of the tuberous clade were observed to lack staminodes.

View larger version (155K): [in this window] [in a new window]   Figs. 38–43. Organogenesis in Moringa flowers, SEM micrographs. Labels on sepals and petals correspond to Fig. 23 unless noted. Bars = 50 µm. 38. Moringa drouhardii bud. Bilateral symmetry is manifest in the suppression of the staminode opposite sepal 4. 39. Moringa peregrina bud showing banner overtopping its corresponding stamen and the lack of a staminode opposite sepal 4. 40. Moringa longituba bud showing bilateral symmetry in the much larger banner and adjacent stamen primordia. 41–43. Moringa arborea flowers showing massive primordia of the rivae clade

View larger version (154K): [in this window] [in a new window]   Figs. 44–49. Late organogenesis and beginning of differentiation in Moringa flowers, SEM micrographs. Labels on sepals and petals correspond to Fig. 23 unless noted. Bars = 50 µm. 44. Moringa peregrina flower polar view with petals beginning to overtop the stamens. This flower has five staminodes. Sepal arrangement corresponds to Fig. 24 . 45–47. Moringa ruspoliana buds showing packing of petals and anthers (especially conspicuous in the banner of Figs. 45 and 47 and the petal between sepals 1 and 4 in Fig. 46 ) and, in Fig 47, the ovary cavity deepening in Fig. 47 . 48. Moringa drouhardii bud showing scant zygomorphy and irregular packing. 49. Moringa oleifera bud showing bilateral symmetry in the symmetrical movements of the lower (l) and middle (m) petal pairs. In addition, the staminode opposite sepal 4 is lost

View larger version (199K): [in this window] [in a new window]   Figs. 56–64. Organ development in Moringa flowers, SEM micrographs. Labels on sepals and petals correspond to Fig. 23 unless noted; b = banner petal or its opposite stamen. Petals and sepals are removed in all figures. Bars = 50 µm in Figs. 56–61, 63 ; = 500 µm in Figs. 62, 64. Figs. 56–58. Moringa drouhardii, showing incipient anther differentiation. 56. Anther and gynoecium primordia enlarging. Bilateral symmetry is manifest as a lost staminode opposite sepal 4. 57. Bud with the most common anther arrangement in the family, in which the middle petal pair (m) and the lower petal pair (l) are symmetrically arranged and the anther opposite the banner does not form a symmetrical pair with any other anther. 58. Appearance of the anther dehiscence seam and anther packing pattern that differs from that depicted in Fig. 57 . 59. Moringa longituba showing rotation of the anthers very early in ontogeny. 60. Moringa peregrina anthers rotating to face the banner simultaneously with differentiation into anthers and filaments. 61. Moringa peregrina showing characteristic apiculi on young anthers that are differentiating from the filaments. 62. Moringa arborea oblique view of young flower showing anthers that have already assumed mature orientation. 63. Moringa arborea close view of differentiating anther and filament. 64. Moringa peregrina flower with anthers differentiated from filaments and dorsifixed condition established. Gynoecium has differentiated into an ovary with a short style. Asterisks mark the very small anthers opposite the upper petal pair

The floral buds of all species are zygomorphic at the end of organogenesis. The banner or banner-like petal and its associated stamen and flanking staminodes are larger than other similar groups in the flower, with the banner or banner-like petal extending farther radially from the axis of the flower than do the other petals (e.g., Figs. 35, 37, 40). This group of primordia is also distinctive in being bilaterally symmetrical, whereas the lateral petals are mirror images of one another (e.g., Figs. 36, 38, 40).

Floral ontogeny: early differentiation
During early differentiation, the sepals are already covering the more internal parts and are showing their pubescence patterns, the petal primordia continue to change shape, and the petals and anthers are involved in torsion apparently associated with the packing of the massive anthers in the confined space of the bud and the ultimate orientation of the anthers at anthesis.

The petals flatten ventrally, with the broad face appressed to the opposite stamen primordium. If present, the banner is usually conspicuously larger than the rest of the petals (Figs. 38–49) and often maintains its abaxial face perpendicular to the axis of symmetry of the flower (e.g., Figs. 39, 40). Moringa arborea in SEM images (Figs. 41–43) and M. borziana and M. rivae in LM sections are characterized by massive primordia and young organs and very compact packing in the buds. Nevertheless, bilateral symmetry is apparent in the greater radial extent of the banner and associated anther (Fig. 41).

In the zygomorphic-flowered species, the features of bilateral symmetry established in the early stages of ontogeny, such as a larger banner and lost staminode opposite sepal 4, are maintained through the early stages of differentiation. Furthering the aspect of zygomorphy in these species, the petals and stamens undergo characteristic patterns of torsion or packing ("twisting" of Ronse Decraene, De Laet, and Smets, 1998 ) symmetrically on either side of the axis of symmetry. This first becomes manifest with the asymmetrical growth of the lateral petals. The lateral petals closest to the banner incline away from it concurrently with the orientation of their associated anthers to face the banner (Figs. 45–47, 49). In the process, the bulk of an anther is shifted away from the bulk of its opposite petal, thereby maximally occupying the space available in the bud. A similar packing of forms takes place among the members of the lower petal pair. In this case, the petals move toward the banner while the anthers shift the majority of their bulk toward sepal 4, the bottom of the flower as viewed at anthesis (particularly conspicuous in Fig. 47, but also visible in Figs. 40, 45, 46). Similarly, the banner and the anther opposite to it may shift in opposite directions (Figs. 43, 47) or may show only minimal shifting (e.g., the anther opposite the banner in Fig. 45 points to the right of the figure, but the petal shows little torsion).

The actinomorphic-flowered species show a less consistent pattern. The packing of anthers and petals in M. drouhardii and M. ovalifolia was observed at times to follow a pattern similar to that seen in the bilaterally symmetrical species (e.g., M. drouhardii in Fig. 38). Just as commonly, flowers were found that deviated from this pattern (e.g., M. drouhardii in Fig. 48, in which the following members are shifted in opposite directions from the arrangement in the zygomorphic-flowered species: the petal between sepals 1 and 3 and its opposite anther, the anther opposite the petal between sepals 2 and 4; the banner is also shifted to its left. Note the presence of all five staminodes).

Floral ontogeny: late differentiation
This stage is defined as the period in which the petals begin to converge and establish aestivation, the stamen primordia differentiate into anthers and filaments, and the gynoecium becomes differentiated into an ovary and a short style.

In all species, the petals remain thickest at the midrib and acquire broad margins as they overtop the stamens. The largest petals may bear apiculi or small trichomes (Figs. 50–55). The gynoecium continues to elongate apically, being shaped both by the triangular form of the three carpels that compose it and also by the pressure of the surrounding organs (Figs. 50, 55). As the petal margins converge on each other, the torsion associated with packing that was previously apparent is now present as slight inclinations of the petals along their long axes (Fig. 54). It is at this point, when the buds are nearly 1 mm in diameter, that the aestivation of the petals begins to be established.

View larger version (159K): [in this window] [in a new window]   Figs. 50–55. Organ development in Moringa flowers, SEM micrographs. Labels on sepals and petals correspond to Fig. 23 unless noted. Bars = 50 µm. 50. Moringa longituba. Bud showing deepening ovary cavity and banner markedly larger than other petals. 51. Moringa drouhardii bud with petals overtopping stamens. Sepal configuration corresponds to Fig. 24 . The petal marked "b" corresponds in sepal position to the banner in the zygomorphic clade. This image illustrates a variant that is common in actinomorphic species, in which the petal between sepals 1 and 3 is the largest and is external. 52. Moringa peregrina flower showing gynoecium emergence well after the organs of outer whorls have formed. 53. Moringa ruspoliana flower with the last sepal to develop left intact, with the inflorescence axis visible below the flower. 54–55. Moringa ruspoliana lateral and polar views of the same flower showing bilateral symmetry

View larger version (163K): [in this window] [in a new window]   Figs. 65–73. Organ development in transections of young Moringa buds, LM micrographs. Labels on sepals and petals correspond to Fig. 23 , unless noted otherwise. Bud diameters are widest dimension. 65–67. Moringa drouhardii. 65. Bud, 1.5 mm in diameter, sectioned at mid-anther shortly after anther differentiation, showing sepal arrangement corresponding to Fig. 24 and petal and anther arrangement as depicted in Fig. 26 . 66. Receptacle of young flower showing the extent of sepal 1. Diameter is 1 mm. 67. The same flower sectioned at the hypanthium apex to show the obliquity of the hypanthium. Diameter is 2 mm. 68. Moringa peregrina showing the most common configuration in Moringa at the hypanthium apex, with the banner conspicuous and separate from the lowermost edge of the hypanthium. Sepal configuration corresponds to Fig. 24 . Diameter is 3 mm. 69. Moringa rivae bud, 3 mm in diameter, sectioned at mid-ovary and mid-filament, showing robust filaments and staminodes and developing ovules. Sepal arrangement corresponds to Fig. 24 ; the right margin of the banner is internal. 70. Moringa borziana bud, 2.5 mm in diameter, sectioned toward the bases of the filaments, showing the abundant trichomes that cover the empty space formed by the hypanthium. The filaments and staminodes opposite sepal 4 are adherent to one another. 71. Moringa arborea sectioned at mid-filament. The filaments and staminodes opposite sepal 4 are adherent. Diameter is 3 mm. 72. Moringa ruspoliana bud, 2 mm in diameter, at anther differentiation. Anther orientation is established before completion of microsporogenesis. 73. Moringa longituba bud, 2.2 mm in diameter, showing that the anthers toward the banner are larger than the others at the early enlargement stage. Sepal arrangement corresponds to Fig. 24

View larger version (103K): [in this window] [in a new window]   Figs. 81–86. Organ positions in transections of Moringa buds, LM micrographs. Labels on sepals and petals correspond to Fig. 23 . The banner is shown at the top in the images of the bilaterally symmetrical-flowered species in Figs. 83–86 . 81. Transection of M. drouhardii at mid-filament of the same 2-mm flower in Fig. 67 to show irregular symmetry. The petal between sepals 1 and 4 is markedly larger than the others, and the staminode opposite sepal 2 is lost. 82. Moringa ovalifolia, showing the most common anther orientation in the actinomorphic species, with four anthers facing the same direction and one facing the opposite direction. Diameter is 3 mm. 83. Moringa peregrina bud, at 4 mm, showing the crown of adherent filaments and staminodes that forms opposite the banner and does not include the staminode opposite sepal 4. The petals of the lower lateral pair are markedly folded. The lateral opening of the gynoecium is atypical. 84. Moringa borziana, showing arrangement of androecium and petal folding. Diameter is 3 mm. 85. Moringa longituba. The crown of usually adherent filaments and staminodes in this species encircles the gynoecium and includes the staminode opposite sepal 4. The most highly reflexed petals in this species are those in the upper lateral pair. Diameter is 4 mm. 86. Moringa ruspoliana. As in M. longituba, the crown of filaments and staminodes encirles the gynoecium, but the lower lateral petal pair in this species is reflexed and it is these petals that are most folded. Diameter is 4 mm

View larger version (114K): [in this window] [in a new window]   Figs. 74–80. Organ development in longisections of young Moringa buds, LM micrographs. Labels on sepals correspond to Fig. 23 , unless noted otherwise. Bud diameters are widest dimension. 74, 75. Moringa drouhardii. 74. Young flower at ovule initiation. The androecium is differentiatied into anthers on very short filaments and the receptacle is massive relative to the rest of the flower. The pedicel and stalk between pedicel and bracteole are very short. Diameter is 1.2 mm. 75. Bud, 2.5 mm in diameter, showing elongation of the receptacle. Ovule development and microsporogenesis are advanced as the bud elongates markedly. 76–78. Moringa longituba, showing development of anthers and hypanthium. 76. Anther differentiation and ovule inititation within the gaping ovary cavity take place long before marked growth of the hypanthium. Diameter is 1.5 mm. 77. Medial longitudinal section of a bud 2.1 mm in diameter with slightly advanced ovule development and marked enlargement of the anthers and only a very slight deepening of the hypanthium. 78. Tangential section of the same flower to show the anthers nearly filling the entire space within the bud. 79. Moringa arborea showing the elongation of filaments occurring before zonal growth of the hypanthium. Diameter is 2.2 mm. 80. Moringa peregrina tangential section of a 2.1-mm bud showing differentiation of anthers before deepening of hypanthium

At organ differentiation, bilateral symmetry in the zygomorphic clade is enhanced by the rotation of the anthers and the continued asymmetric growth of the petals. The members of the tuberous clade establish the mature anther orientation depicted in Fig. 23 earlier in development than the other species. For example, in a flower of M. longituba that is scarcely 0.5 mm in diameter, all but one anther has already assumed the conformation seen at anthesis, and the remaining anther has nearly rotated to its final position (Fig. 59). Likewise, Fig. 62 shows the just-differentiating anthers of M. arborea in their mature orientation. In contrast, anthers in the slender trees are still rotating to their final orientation at mid-differentiation (Figs. 60, 61).

After elongating apically into a tubular structure (Figs. 60, 61), the gynoecium in all species differentiates into a squat ovary with a short, tubular style (Figs. 62–64, 74, 75). Ovule initiation coincides with or closely follows this differentiation (Figs. 68, 73, 74, 76). Concurrently, differentiation of the sporangia and pollen formation occurs (Figs. 72, 73). Aberrant androecial development was observed in the collection Olson 700 of M. concanensis, in which there were no flowers with functional anthers. The distal parts of the stamen primordia differentiate into apiculate bodies with a papillate texture distinct from the smooth surface of the filament (depicted in Fig. 5) but scarcely enlarge and remain without sporangia. The gynoecium of these flowers is of normal size.

Floral ontogeny: enlargement
This stage is characterized by enlargement of parts before the flower opens. The stamens are differentiated into anthers and filaments. Ovule and pollen formation may still be ongoing during this stage. It is at the early part of this stage that characteristics such as pubescence begin to become widespread on the central flower parts.

If they have not already done so, flowers in the members of the actinomorphic grade usually lose clear traces of zygomorphy during the enlargement phase. If a banner-like petal was present in the preceding stages, it is equalled in size by the other petals during enlargement. Moringa drouhardii and M. ovalifolia commonly show imbricate petal aestivation with petals of equal sizes and anthers that may all have the same oblique orientation (Fig. 65) or sets of four anthers facing the same side of their opposite petals and another that faces the opposite side (Fig. 82). The filaments of M. hildebrandtii may fail to elongate equally in this phase.

In all species, this stage is characterized by the continued appearance and growth of pubescence (e.g., Figs. 67–70). Members of the rivae clade still bear very thick, compact petals and androecial members (Figs. 69–71). This phase in all species is characterized by the enlargement and continued torsion of the stamens. As in the previous stage, enlargement in the zygomorphic clade appears to be slightly unidirectional, with the anther opposite the banner remaining larger than the others (Figs. 72, 73). In all species, just-differentiatied anthers rapidly become massive structures tightly filling the available space within the bud (Figs. 74–80). As anthesis approaches, the epidermal texture of the anthers become papillate and the anthers elongate markedly, as do the filaments to a lesser degree. (Fig. 75 shows a shortly preanthetic bud of M. drouhardii, and Fig. 79 shows an intermediate stage in M. ruspoliana.)

The enlargement of the gynoecium is accompanied by a marked constriction at the apex of the ovary and the elongation of the hollow style (Figs. 74, 75). After the appearance of the ovules as peg-like ingrowths of the inner wall of the ovary, the integuments of the ovules become differentiated. In the actinomorphic-flowered species, the ovary and style remain centered along the polar axis of the flower. In most of the zygomorphic-flowered species, the ovary is displaced toward the banner (Figs. 83, 84, 86, 87–90). The exception is M. longituba, in which the ovary is displaced toward sepal 4 (Fig. 85; cf. Figs. 1–13 and Figs. 91–94). The style may culminate in three stigmatic lobes (e.g., M. stenopetala in Fig. 3), but these are usually absent or very small.

View larger version (31K): [in this window] [in a new window]   Figs. 87–94. Drawings of longisections to show hypanthium development in Moringa arborea and M. longituba. The only androecial member shown is the left lower lateral stamen. All figures are 3x life size. 87–90. Moringa arborea. As the hypanthium deepens, the gynophore elongates, maintaining the ovary base at the hypanthium apex. The gynoecium and lower lateral stamens are reflexed toward the banner, at top. 91–94. Moringa longituba. A gynophore forms, but hypanthial growth is so extensive that the ovary becomes completely enclosed by the hypanthium. The gynoecium and androecium are deflected toward the bottom of the flower, a unique condition in the family

Two differing patterns of filament-staminode adhesion were observed in the two subclades of the tuberous clade. In the members of the rivae subclade, as in the flowers of the slender trees, the filaments and staminodes form a crown opposite the banner (incipient stages of adhesion can be seen in a young bud of M. arborea in Fig. 71 and in a nearly mature bud of M. borziana in Fig. 84) but with filaments often directly adhering to one another and staminodes adhering to their outer surfaces (Fig. 84). In the red-flowered subclade, the stamens and filaments adhere in a continuous ring that always includes the staminode opposite sepal 4 (Figs. 85, 86).

One of the most conspicuous features of late ontogeny is hypanthium formation. After the anthers differentiate and begin to enlarge and the gynoecium begins to differentiate, growth of the rim on which the petals, sepals, filaments, and staminodes are inserted begins. Likewise, growth ensues at the base of the ovary. In this way, a hyphanthial cup and a short gynophore are formed. The species with the least tendency to form hypanthia, if they can be considered as such, are the actinomorphic-flowered species. In M. drouhardii, the gynophore elongates as the margins of the receptacle grow slightly apically (Figs. 74, 75). In the zygomorphic-flowered species, a similar pattern occurs initially. Slight deepening of the hypanthial cup accompanies anther differentiation and the beginning of anther enlargement. For example, in M. longituba, the species with the longest hypanthium, little hypanthium is visible at ovule initiation (Fig. 76). The same is true in the slender tree species M. peregrina (Fig. 80). As the anthers enlarge and the ovules develop, a slight extension of the gynophore and deepening of the hyphanthial cup occurs (Figs. 77, 79, 87, 91). Moringa arborea illustrates an ontogenetic pattern common in the zygomorphic clade, in which the hypanthium deepens to 1–2 mm, with the gynophore elongating sufficiently to maintain the base of the ovary even with the hypanthium apex (Figs. 87–90). Unique in the family, M. longituba progresses well beyond this stage, with the hypanthium reaching up to 2 cm in length. In this species, hypanthial growth outpaces the elongation of the gynophore, and the ovary is drawn into the hypanthium and completely enclosed by it at maturity (Figs. 91–94).

In the zygomorphic species, the apex of the hypanthium reaches its farthest distal extent between the petals alternate with sepal 4, whereas the banner at this level remains free (Fig. 68 shows the hypanthium apex in M. peregrina in transection). Thus, the aperture of the hypanthial cup is tilted slightly upward at anthesis (e.g., Figs. 5, 12, 90, 94). The inclination of the hypanthium is less consistent in the actinomorphic-flowered species. Slight inclination in the insertion of the sepals can be seen in a bud of M. drouhardii in Fig. 1; Fig. 66 shows that sepal 1 is the first sepal to be separate from the receptacle as one sections up from the pedicel in M. drouhardii; Fig. 67 shows the inclination of the hypanthium from the area of sepal 5 at its distal extent to the area of the petal between sepals 1 and 4. The arrangement of the flower shown in Fig. 67 seems analogous to that in the zygomorphic flowered species because the greatest distal extent of the hypanthium is on the opposite side of the flower from a banner-like petal that is larger than the others. However, not only is this not the usual position of the banner with respect to the sepals, but flowers of M. drouhardii and M. ovalifolia were observed with the distalmost edge of the hypanthium anywhere from between the sepal pairs 3/5, 2/5, 2/4, or 1/4.

Floral ontogeny: anthesis
As the flowers open, each species is characterized by a unique degree of petal and sepal deflection, anther orientation, and style behavior. The illustrations of mature flowers in Figs. 1–13 may be used to represent the span of these differences, with a few generalizations noted here.

As anthesis approaches in the zygomorphic-flowered species, the petals that show the most flexion perpendicular to their longitudinal axes at anthesis begin to fold inward along their longitudinal axes. In all species but M. longituba, the members of the lower petal pair most exemplify this phenomenon as do, to a lesser extent, the members of the upper petal pair (Figs. 83, 84, 86). At anthesis in the zygomorphic clade, with the exception of M. longituba, the banner remains projecting forward, while the rest of the petals are downswept in a variety of arrangements, from slightly (e.g., M. rivae and M. pygmaea in Figs. 10, 11) to strongly reflexed (e.g., M. oleifera and M. arborea in Figs. 6, 8). Moringa longituba shows an opposite pattern, with the banner being borne upright or swept back and the lower petal pair projecting forward (Fig. 13). Likewise, it is the upper three petals in M. longituba that show the greatest extent of folding along their longitudinal axes (Fig. 85).

There is a greater tendency for sepals than petals to display actinomorphy at anthesis. The actinomorphic-flowered species have radially symmetrical calyces at anthesis (Figs. 1–4). Moringa arborea and M. borziana also have calyces that are radially symmetrical or nearly so (Figs. 8, 9), though sometimes both sepals of a sepal pair are not deflected equally (not shown). The calyces of M. rivae and M. pygmaea are zygomorphic mainly due to slight differences in flexion among the sepals (Figs. 10, 11). The members of the slender tree clade, in addition to M. longituba and M. ruspoliana, have backswept sepals that mirror the patterns of flexion seen in the petals (Figs. 5–7, 12–13).

In the actinomorphic species, there may be a very slight tendency for the members of one petal pair to have a greater angle between them than that found between the other petals (Fig. 1). However, this is impossible to detect in pressed material and the pickled material available was distorted by compression of the flowers against one another during fixation, so this subtle difference in petal angle may be artefactual. In contrast, petal flexion is very marked in the members of the zygomorphic clade.

At anthesis, any anthers not in their final positions rotate to their mature orientations. In the actinomorphic-flowered species, the anthers are more or less obliquely introrse at anthesis (Fig. 1). In the zygomorphic-flowered species, the anthers are borne in three tiers: the anther opposite the banner forms the distalmost element in all species but M. longituba. The anthers opposite the lower petal pair are in a rank together closest to the base of the flower, with the anthers opposite the upper petal pair being intermediate (these tiers can be seen in the illustrations of mature flowers in Figs. 5–12 and are highlighted in M. oleifera in Fig. 6). In M. longituba the pattern is reversed, with the anther opposite the banner borne closest to the base of the flower, with those of the lower petal pair being the distalmost elements (Fig. 13; cf. differences in flexion between lower lateral stamens in Figs. 90 and 94). Observations of M. borziana, M. concanensis, M. longituba, and M. oleifera in cultivation indicate that the anthers dehisce over the first 2 d after anthesis, after which the style elongates markedly and projects well above the anthers.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The ontogenetic origin of bilateral symmetry
It might be suspected that radial symmetry in the basal four species of Moringa is a symplesiomorphy with the sister family Caricaceae. Floral development in Carica papaya has been examined in detail by Ronse Decraene and Smets (1999) and was found to be actinomorphic at all stages. In contrast, all species of Moringa, including the actinomorphic ones, are zygomorphic in early ontogeny (cf. Bersama, Ronse Decraene et al., 2001 ). This difference, along with many other floral differences between the families (see Ronse Decraene and Smets, 1999 ; Olson, 2002 ), may tentatively be thought of as autapomorphic for Moringaceae. However, the finding that a floral symmetry gene is expressed asymmetrically in the early stages of Arabidopsis flowers (Cubas, Coen, and Martínez Z., 2001 ), which are actinomorphic throughout ontogeny, suggests that other examples of early zygomorphy may be undetected in Brassicales.

That the flowers of all Moringa species are indistinguishable at early organogenesis is represented at the top of the gray branching diagram of ontogenetic similarity between species in Fig. 95 (ontogenetic differences between the actinomorphic and zygomorphic species are given in greater detail in Table 1). At this point all species show traces of zygomorphy. The first divergence in Fig. 95 represents the point in ontogeny when a species group can first be distinguished from the rest of the family, with the rivae clade being conspicuous because of its massive primordia (Fig. 41). The next branch in Fig. 95 indicates when the rest of the zygomorphic species become distinguished from the actinomorphic species, most notably by a banner that is always located between sepals 3 and 5 that remains the largest petal throughout ontogeny, consistent patterns of packing of stamens and petals, and a missing staminode, if any, that is always opposite sepal 4. In Fig. 95, the rivae clade becomes "reunited" with the rest of the species of the tuberous clade to indicate that these species become indistinguishable from one another at mid-differentiation. Similarities shared by these species include early assumption of mature anther position and the consistent full development of all staminodes. In all species, the position of a flower in an inflorescence appeared to have little effect on floral symmetry.

View larger version (69K): [in this window] [in a new window]   Fig. 95. Summary of ontogenetic programs in Moringa. The branching gray arrows indicate the potential to differentiate species groups based on a sample of a given ontogenetic stage (ontogenetic stages are labeled at right). For example, a sample of the rivae clade can be distinguished from the rest of the family at later organogenesis; a sample of the red-flowered clade can be distinguished from the rest of the family during the enlargement stage, etc. Abbreviations are as follows: A = anthers, F = filaments, G = gynoecium, H = hypanthium, P = petals, Se = sepals; Si = staminodes; St = stamens

View larger version (34K): [in this window] [in a new window]   Fig. 96. Venn-type diagram showing that the floral arrangements found in the early ontogeny of the zygomorphic species are a subset of those found in the ontogeny of the actinomorphic species. Stages found in the actinomorphic species are boxed in black; those found in the zygomorphic species are boxed in gray. Many symbols are shown in the black box in the intermediate stages of ontogeny to contrast the large variety of possible arrangements in this group with the low variation seen in the zygomorphic species.

Development in zygomorphic flowers can thus be thought of as a stabilization and accentuation of characteristics present in the highly variable ontogeny of the actinomorphic-flowered species (serving as a possible example of the "recruitment" of incipient zygomorphy suggested by Cubas, Coen, and Martínez Z., 2001 ). The high variability observed in the actinomorphic-flowered species is consistent with the hypothesis that differences in aestivation, floral orientation, and presence or absence of staminodes have little effect on pollination in these species and are therefore subject to little selective pressure. These features, however, appear to be under strong selection in zygomorphic-flowered species based on the low degree of variability observed in these same characteristics in the zygomorphic clade.

Phylogenetic distribution of ontogenetic patterns
Numerous congruences are noted when the distribution of ontogenetic differences between species groups is superimposed on the phylogenetic hypothesis favored here (Fig. 97). Selected congruences are reviewed here, as are the smaller number of incongruences. Differences in ontogeny between species groups are summarized in Table 1.

View larger version (68K): [in this window] [in a new window]   Fig. 97. Phylogenetic distribution of ontogenetic differences between species groups of Moringa. Major ontogenetic differences distinguishing the actinomorphic and zygomorphic species are shown in the shaded boxes at top. Differences diagnosing smaller groups are listed by arrows originating at the nodes of interest. Abbreviations are as in Fig. 95

Subclades within the zygomorphic clade each have distinguishing ontogenetic features (Fig. 97). In the slender trees, it is the position opposite sepal 4 that remains uninitiated, whereas in the actinomorphic-flowered species the staminode may be missing from a variety of locations. The pattern of stamen and filament adhesion observed in M. peregrina (Fig. 83) and M. oleifera (Fig. 6) is peculiar to the slender trees. The tuberous clade is distinguished, among other features, by always producing all five staminodes and anthers that differentiate nearly simultaneously, as opposed to the staggered or unidirectional differentiation shown by the rest of the family. Within the tuberous clade, the rivae clade is distinguished by buds with massive primordia (Figs. 42–43; see also later stages in Figs. 69–71). This clade also displays a distinctive pattern of filament and staminode adhesion (Fig. 84). The two red-flowered species share the third distinctive pattern of filament and staminode adhesion, with a continuous ring of alternating filaments and staminodes (Figs. 85, 86). Similarities that may be symplesiomorphies were noted between the slender trees and the actinomorphic-flowered species, most notably the tendency to lack a staminode and the early appearance of sutures on the anthers.

Moringa peregrina showed species-specific features earlier in ontogeny than any other species. In M. peregrina, initiation of the gynoecium lags distinctly behind that of the other floral organs (Figs. 39, 44, 52). This staggered emergence of the ovary well after the stamen primordial was not observed in any other species. Moringa peregrina also has anthers with apiculi that distinguish this species at anther differentiation (Figs. 61, 64). Both the emergence of the gynoecium and the anther apiculi in M. peregrina seem likely to be additional examples of the many autapomorphies of M. peregrina (e.g., bluish leaves with deciduous leaflets).

Timing of ontogenetic divergence as correlate of phylogenetic branching
If ever-earlier ontogenetic divergences characterize ever-deeper phylogenetic splits, then the branching diagram in Fig. 95 should approximate the major nodes of the phylogeny in Fig. 14. This would be the case in Moringa were it not for one main incongruence, which is the early divergence of the rivae clade at organogenesis. These species are distinguished at this early stage by having larger primordia than the rest of the species, a quantitative difference that does not represent a major departure in ontogenetic pattern such as differences in anther rotation patterns or staminode loss. Divergences deep in the tree, such as that between the actinomorphic and zygomorphic species, occur very early in ontogeny. Those more distal in the tree, such as the split between the rivae and red-flowered clades, are manifested late in ontogeny. The phylogenetic distribution of ontogenies in Moringa thus appears largely consistent with the expectation that ontogenetic differe