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Floral development in the legume tree Colophospermum mopane, Caesalpinioideae: Detarieae
Botanical Journal of the Linnean Society (1999), 131: 223–233. With 17 figures Article ID: bojl.1999.0278, available online at http://www.idealibrary.com on
Floral development in the legume tree Colophospermum mopane, Caesalpinioideae: Detarieae ¨ GER1∗, L. R. TIEDT2 AND D. C. J. WESSELS3 H. KRU 1
School for Environmental Sciences and Development and 2Laboratory for Electron Microscopy, Potchefstroom University for Christian Higher Education, 2520 Potchefstroom, South Africa 3 Department of Botany, University of the North, Private Bag X1106, 0727 Sovenga, South Africa Received January 1999; accepted for publication April 1999
Floral ontogeny of Colophospermum mopane (Kirk ex Benth.) Kirk ex J. Leonard, an apetalous member of the Crudia group with four sepals and a large number (20–25) of stamens, was studied as part of a larger project on reproductive biology of this much-utilized tree. The flowers have been described as being inserted in the axil of a bract, but lacking lateral bracteoles. Four outer, light cream or white ‘sepals’ are present. The first two sepals are initiated in a lateral position, where the bracteoles, if present, develop in other members of the Caesalpinioideae. The inner sepals arise simultaneously adaxially and abaxially. These four structures, conventionally regarded as sepals, enclose the bud. The outer two ‘sepals’ should be regarded as lateral bracteoles inserted at the apex of the pedicel. The inner structures represent the only two sepals. The large number of stamens arise on a large meristematic surface and different whorls were not observed. The filaments elongate within the bud and after anthesis become exposed outside the flowers. The filaments are of equal length and the large anthers form a suspended cluster. One carpel develops terminally and gives rise to an indehiscent one-seeded fruit. 1999 The Linnean Society of London
ADDITIONAL KEYWORDS:—bracteoles – flower – Hardwickia – mopane – ontogeny. CONTENTS
Introduction . . . . Material and methods Results . . . . . Mature flower . . Floral apex . . . Sepal initiation . . Stamen initiation . Carpel development Discussion . . . . Conclusion . . . . Acknowledgements . References . . . .
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∗ Corresponding author. Email: [email protected]. 0024–4074/99/110223+11 $30.00/0
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1999 The Linnean Society of London
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¨ GER ET AL. H. KRU INTRODUCTION
Colophospermum mopane (Kirk ex Benth.) Kirk ex J. Leonard was reduced to Hardwickia mopane (Kirk ex Benth.) Breteler, comb. nov (Breteler et al., 1997) within the Crudia group (tribe Detarieae DC.), despite differences in seed and fruit morphology between Hardwickia binata Roxb. and C. mopane, and their disjunctive distributions. Hardwickia Roxb. and Colophospermum J. Leonard are monotypic genera. C. mopane occurs in Africa and H. binata is only known from India. Floral differences between the two species mentioned by Breteler et al. (1997) include sepal number (4–5 in H. binata and 4 in C. mopane) and stamen number (10–11 in H. binata and 20–25 in C. mopane). Smith, Timberlake & Van Wyk (1998) proposed the conservation of the name Colophospermum mopane on several grounds and as it is such a well-known African monotypic genus, the name is used in this study, as the matter has apparently not yet been settled. If only conventional characters are used, the tribes Detarieae and Amherstieae are almost impossible to separate. Watson (1981) treated the Detarieae–Amherstieae (or main group 2) as separate from the other tribes in Caesalpinioideae with the Detarieae more heterogeneous than the Amherstieae. The presence of relatively well-developed bracteoles is a feature cited by Cowan & Polhill (1981) as characteristic for the Detarieae–Amherstieae, but they mention that bracteoles are absent in some members of the Crudia group of the Detarieae. This also applies to C. mopane, where bracteoles are regarded as absent. In their list of similarities between C. mopane and H. binata, Breteler et al. (1997) also mention the absence of bracteoles. Representatives of the Crudia group include unarmed trees with flowers varying from small to showy and regular to markedly zygomorphic. Bracteoles can be anything from small to large, imbricate or partly fused. The number of petals range from absent to five (Cowan & Polhill, 1981). Generic and tribal limits are difficult to fix, due to modifications of the flowers. The significance of ontogenetic evidence for the evaluation of phylogenetic relationships between the taxa of the Leguminosae is stressed by Tucker (1987) and Tucker & Douglas (1994). Using floral development as a source of characters and character states combined with conventional characters, the preliminary phylogenies produced through analysis by Tucker & Douglas (1994) indicate a monophyletic origin of the tribe Detarieae. Floral development of C. mopane has as yet received no attention and the information presented here may contribute to a better understanding of its taxonomic position within the Crudia group and its inclusion within the genus Hardwickia. The only other report on floral ontogeny within the Crudia group is that on Crudia choussyana where five petals are initiated but all fail to enlarge (Tucker, 1997). Endress (1990) recognized the lack or reduction of the perianth as one of the two most important conditions leading to instability in organ position in flowers. This also applies to Colophospermum mopane where no petals are present in mature flowers, in combination with the large number of stamens. The aims of this work were to determine the order of floral organ initiation in C. mopane, to determine whether the apetalous situation is due to loss or suppression of organs, to compare the ontogeny with those of other members of the Caesalpinioideae, to contribute to the investigation on the reproductive biology of C. mopane which is presently being conducted, and to contribute towards the floral ontogenetic information available on the Caesalpinioideae.
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Figure 1. Ground plan of main axis of panicle of Colophospermum mopane to show position of flowers and different arrangements of bract and sepals. Flowers numbered from oldest to youngest.
MATERIAL AND METHODS
Young inflorescences of C. mopane were collected at the Messina Experimental Farm (22°14″5.1′ S;29°55″8.1′ E) in the Northern Province, South Africa, fixed in formalin-acetic acid-ethanol ( Johansen, 1940) or 4% paraformaldehyde, pH7.4, dehydrated in acetone and critical point dried. Floral buds of different stages were opened, attached to scanning electron microscope stubs, coated with gold-palladium and studied with a Philips XL30 scanning electron microscope. Young fixed inflorescences were dehydrated in acetone, infiltrated with resin (Spurr, 1969), sectioned with glass knives and stained with toluidine blue O and neufuchsin to determine the structure of the inflorescence in serial sections. Herbarium specimens of material used are housed in UNIN (Potgieter & Wessels 140, Wessels & Jordaan 51 and Potgieter & Wessels 1).
RESULTS
Flowers are borne axillary in panicles (Fig. 2). The inflorescences have helical phyllotaxy (in a 38 helix in the inflorescences studied) of acropetally produced bracts and flowers (Fig. 1). Inflorescence size differs from season to season and also between localities. All the flowers studied were perfect. The angle between the ±5 mm long pedicel and the axis may be 90° or smaller, but seems to be relatively constant for a plant. The number of flowers per inflorescence reaching anthesis differs during different seasons and large numbers of flowers collected and fixed during dry seasons were infected by insect larvae (herbarium specimen Potgieter & Wessels 1). The bracts are initiated acropetally and each subtends a single flower (Fig. 1). In inflorescences of some trees, the youngest three to five floral buds were all enclosed
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in the large bract of the next older flower, even in inflorescences where the older flowers had already undergone anthesis or where almost fully developed fruits were present. In these inflorescences the younger flowers did not reach anthesis and remained as small buds after maturation of older flowers, or became senescent within the bud. Fewer fruits than flowers were produced per inflorescence. In some inflorescences only one fruit reached maturity. More than one flush of flowering occurs in some trees, apparently depending on precipitation. Different trees in the same locality may also flower at different times.
Mature flower Floral buds are enclosed by the bract and four cream or white glabrous structures, conventially regarded as sepals (Figs 1, 3). The inner (adaxial and abaxial) sepals often have acute apices, while the apices of the outer (lateral) sepals are rounded. At anthesis all four sepals reflex back completely (Fig. 3). The rounded apices of the outer lateral reflexed sepals are incurved, making the mature flower slightly zygomorphic. In flowers where all the sepals have rounded apices, the lateral sepals are always incurved. Two patterns of imbricate sepal arrangement were encountered (Fig. 1). The most common pattern seems to be the one where the outer sepal is to the left of the flower as viewed from the abaxial side (flower 1 in Fig. 1). A second pattern was observed in fewer flowers in the inflorescences investigated with the outer sepal to the right of the flower if viewed similarly (flower 3 in Fig. 1). In some inflorescences both patterns occurred (Fig. 1), while flowers of other inflorescences studied exhibited only the most common pattern. The 20–25 (with the most common number of 20 encountered in the material studied) free stamens have long filaments of equal length (Fig. 3). The anthers are tetrasporangiate, dorsifixed and dehisce by longitudinal slits. At anthesis the flowers hang downwards and the heavy anthers are exposed outside the flowers, suspended on the long, thin filaments (Fig. 3). The central uni-ovulate indehiscent ovary is sessile and bears a capitate stigma with a distinctly folded appearance, on a c. 2 mm long style (Fig. 17). Style length varied between different trees. The style is inserted on the adaxial side of the ovary (Fig. 17) and at anthesis is often flexed towards the abaxial side. The fruit is almost
Figures 2–9. Inflorescences and floral primordia of Colophosperimum mopane. Fig. 2. Three inflorescences. Scale bar=10 mm. Fig. 3. Inflorescence with one open pendulous flower with long anthers of equal length and floral buds enclosed by the bract and ‘sepals’. Scale bar=10 mm. Fig. 4. Tangentially elongated floral primordium after removal of bract (b); abaxial side of flower at base of figure. Scale bar=50 lm. Fig. 5. Floral primordium with one lateral ‘sepal’ (1) initiated after removal of bract (b); abaxial side of flower at base of figure. Position of initiation of second lateral ‘sepal’ is indicated (2). Scale bar=50 lm. Fig. 6. Floral primordium with two lateral ‘sepals’ (1, 2) and adaxial and abaxial sepal initials (3) after removal of bract (b), with abaxial side of flower at base of figure. Scale bar= 50 lm. Fig. 7. Floral primordium with two enlarging lateral ‘sepals’ (1, 2) and two inner sepal initials (3) after removal of bract (b), with abaxial side of flower at base of figure. Scale bar=50 lm. Fig. 8. Floral primordium with four ‘sepals’ (1, 2, 3, 3) after removal of bract (b), with abaxial side of flower at base of figure. Scale bar=50 lm. Fig. 9. Floral primordium with large lateral ‘sepal’, adaxial and abaxial sepals (3) and carpel initial (c); bract (b) and one lateral ‘sepal’ removed, with abaxial side of flower to the right of figure. Scale bar=50 lm.
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reniform, but the shape varies in different plants, sometimes markedly so. The style is persistent. Rarely, an extra smaller carpel is present, producing an ovule. Exceptional carpels with more than one ovule have also been observed. The bracts and sepals contain large secretory canals lined with epithelium. Floral apex The floral apex is convex and broader tangentially than anticlinally after bract initiation (Fig. 4). The apex becomes flattened, but retains its elongated shape after initiation of the sepal primordia, becoming rectangular before stamen initiation (Figs 9–11). Sepal initiation Four ‘sepals’ are initiated. The first sepal primordium is initiated in either of the two lateral positions (Fig. 5). The second lateral sepal primordium appears soon after the first, and both enlarge simultaneously to enclose the two inner sepals in the bud. The outer sepal (either of the lateral sepals) may enclose the other lateral sepal both adaxially and abaxially or only abaxially. Overlapping of the one lateral sepal by the other seems to be a matter of chance as no specific pattern was discernible in any of the inflorescences studied (Fig. 1). In some inflorescences all the flowers showed the same pattern, while both patterns occurred in other inflorescences. The adaxial and abaxial sepal primordia arise simultaneously (Figs 6, 7) and are completely enclosed by the lateral sepals in the bud (Fig. 3). The abaxial sepal was interior to the adaxial sepal primordium in all the flowers studied (Fig. 1). As in all other legume flowers, the bases of the sepals do not overlap and imbricate aestivation is manifested only when the sepals enlarge. Stamen initiation The large number of stamen primordia (usually 20) are formed on an enlarged meristematic surface without any specific pattern of initiation (Fig. 10). At initiation
Figures 10–17. Flowers of Colophospermum mopane at different stages of development. Fig. 10. Floral primordium with enlarging adaxial and abaxial sepals (3), stamen initials (s) and carpel initial (c); bract (b) and both lateral sepals removed, with abaxial side of flower at base of figure. Position of one lateral ‘sepal’ (1) visible. Scale bar=50 lm. Fig. 11. Developing stamens (s) and carpel (c); bract and all sepals (1, 2, 3, 3) removed, with abaxial side of flower to the right of figure. Scale bar=50 lm. Fig. 12. Lateral view of flower with developing stamens with short filaments and basifixed anthers (s); bract and most sepals removed. Stigma is visible as a flattened structure (c). Scale bar=100 lm. Fig. 13. Lateral view of flower with elongating filaments; bract and sepals removed. Scale bar=500 lm. Fig. 14. Lateral view of flower with enlarging stigma on short style and with enlarging ovary (c); bract, sepals and most stamens (s) removed. Scale bar=100 lm. Fig. 15. Lateral view of flower with developing gynoecium (c); bract, sepals and most stamens (s) removed; abaxial side of gynoecium to the left of figure. Scale bar=100 lm. Fig. 16. Abaxial view of flower with developing gynoecium showing enlarging folded stigma and elongating style; bract, sepals and most stamens removed. Scale bar=500 lm. Fig. 17. Developing gynoecium with folded capitate stigma and elongating style; abaxial side of gynoecium to the left of figure. Scale bar=1 mm.
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all stamen primordia are of the same size and separate whorls were not discernible. At this stage the meristematic surface becomes distinctly rectangular (Fig. 9). It often seems as if stamen primordia do not develop to completely fill the available lateral space (Fig. 11). The anthers are basifixed at first (Figs 12, 14), as are all legume anthers (Tucker, 1996). The filaments elongate after initiation to form long slender structures, folded in the bud (Fig. 13). The anthers become dorsifixed and at anthesis they protrude from the flower, hanging downwards in a close group (Fig. 3). Carpel development The terminal carpel primordium appears before the stamen primordia as a single rounded protrusion (Fig. 9). Before the appearance of the filaments, an adaxial cleft is formed (Fig. 11). The stigma appears after the filaments have been formed (Fig. 12) and enlarges simultaneously with the abaxial part of the ovary to become flattened, with the adaxial cleft still evident (Fig. 14). The position of the stigma and style, that form at a later stage, thus becomes subapical (Fig. 15). The style forms after the stigma has enlarged significantly (Fig. 15) and elongation of the style occurs as the bud enlarges. The stigma enlarges to become capitate and folded (Figs 16, 17).
DISCUSSION
Sepal initiation in members of the Caesalpinioideae is helical or rarely unidirectional with the median sepal in the abaxial position (Tucker, Stein & Derstine, 1985; Tucker, 1989). According to descriptions of C. mopane flowers, the outer floral whorls are characterized by loss of at least one sepal and complete absence of petals (see Breteler et al., 1997). Few floral ontogenetic studies have as yet been done on other members of the Detarieae where only four sepals are present at anthesis. Sepal suppression occurs in Schotia brachypetala (tribe Detarieae) where five sepals are initiated in helical order. The last-initiated sepal (on the adaxial side) remains small. In Saraca declinata five sepals are initiated of which two become fused before anthesis, appearing as only one (Tucker, 1989). In Crudia choussyana, the only member of the Crudia group reported on as yet, five petals are initiated but none enlarge and the mature flower is apetalous (Tucker, 1997). Complete absence of several sepals was reported for the first time in Aphanocalyx djumensis, Monopetalanthus durandii and Brachystegia species of the Brachystegia group, tribe Detarieae (Tucker, 1997). As no rudimentary organs were observed in any of the Colophospermum mopane flowers studied, the four ‘sepals’ must be regarded as the result of loss of one sepal. The position and structure of the mature ‘calyx’ make the flower weakly zygomorphic. This only becomes evident after enlargement of the four sepals. The lateral position of the primordia of the first formed floral structures (‘sepals’) in C. mopane differs from any of those in caesalpinioid taxa yet reported (see Tucker et al., 1985; Tucker, 1998). Sepal initiation in C. mopane must be regarded as bidirectional, starting from one lateral to the other lateral and then followed by the last two (adaxial and abaxial) sepals. The outer, lateral primordia develop before initiation of the inner (adaxial and abaxial) sepals. Bidirectional sepal initiation has
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been described for the mimosoid legumes Wallaceodendron celebicum Koord., Acacia smallii Iseley and A. pinetorum Benth. (Ramı´rez-Domenech & Tucker, 1990) and the caesalpinioid legumes (tribe Cassieae) Petalostylis labicheoides R. Br. and Dialium guianense Steud. (Tucker, 1998). In C. mopane however, only four sepals are initiated, while the calyx in W. celebicum, A. pinetorum, P. labicheoides and D. guianense is pentamerous (Ramı´rez-Domenech & Tucker, 1990; Tucker, 1998). As is the case in Colophospermum mopane, the two lateral sepals in W. celebicum arise first, but then the abaxial sepals enlarge to partly cover the other sepals. In C. mopane the four sepal primordia arise equidistant on the flanks of the floral meristem, and no indication of a retarded sepal primordium was observed in any of the flowers investigated. The lateral sepals always enclose the adaxial and abaxial sepals. The imbricate arrangement and position of the sepals in the bud are according to their order of initiation; the firstformed sepals enlarging first to enclose the others. In Dialium guianense the lateral sepals are initiated last, after the other three sepal primordia have been formed, and these lateral sepals overlap the other three imbricately (Tucker, 1998). In mopane, the lateral sepals are the first-formed sepal primordia, but remain in the outer position due to their enlargement before the two inner sepal primordia. Laterally placed primordia which arise after initiation of the bract usually give rise to bracteoles in caesalpinioid legumes (see Tucker, 1996, 1998). It would thus seem that the first two structures initiated in C. mopane flowers, directly after initiation of the bract, should be regarded as bracteoles and not as sepals. The position of the bracteoles at the apex of the pedicel would then be determined by absence of elongation of the pedicel above the point of insertion of the bracteoles. This brings C. mopane in line with the other members of the tribe Detarieae where bracteoles are usually present as mentioned by Cowan & Polhill (1981). If this is the case, only two sepals, initiated adaxially and abaxially, are present in C. mopane. The imbricate position of the bracteoles would be similar to that regarded as typical for other members of the Detarieae, in contrast with valvate bracteole position in the tribe Amherstieae (Cowan & Polhill, 1981). The flowers of C. mopane must then be regarded as zygomorphic as only two sepals are present. The two lateral bracteoles supplement the protective function of the two sepals as is the case in some caesalpinioids (Tucker, 1987). Floral ontogeny of related taxa, including Hardwickia binata, should be studied to determine the number and position of the sepals in flowers of other representatives of the Crudia group. Initially the floral meristem is broader tangentially and somewhat convex, but becomes rectangular at stamen initiation. The large number of stamens, positioned in a seemingly single whorl, may be due to an increased apical meristematic surface as suggested by Sattler & Singh (1977) for Limnocharis flava (L.) Buch. However, Tucker (1989, 1991, 1998) found that disruption of other subsequent floral whorls often occurs in flowers with missing organs. No petal primordia were present in any of the flowers of C. mopane investigated, and this disrupts the meristematic floral apex, resulting in the unusual pattern of stamen initiation. A meristematic ring was not observed in any of the flowers studied.
CONCLUSION
Floral ontogeny in C. mopane follows the usual pattern found in most caesalpinioid taxa only as far as the bract and gynoecium are concerned. The sequence of sepal
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initiation differs from the usual pattern and may be interpreted in either of two ways. Traditionally four outer sepals and absence of bracteoles are included in all descriptions of Colophospermum mopane and Hardwickia mopane (see Breteler et al., 1997 and literature cited therein). The position of the primordia of the outer two structures however, conforms to that of bracteoles in other members of the Leguminosae (see Tucker, 1987, 1988a) and although bracteole initiation has been studied only in C. mopane and in no other member of the Crudia group, it must be assumed that the ontogenetic position of the bracteoles will not differ from that of the other caesalpinioids. If this is confirmed by other studies on members of the Crudia group, it must be assumed that only two sepals are present, due to loss of three sepals. As no indication of petal primordia was observed in any of the flowers studied and as the shape of the floral apex gave no indication of initiation of any such primordia, the apetalous situation in C. mopane is regarded as due to complete loss of the petal whorl and not to petal suppression. The large number of stamens are initiated as separate primordia, but not in separate whorls. This is a special feature observed in C. mopane flowers and conforms to the observation of Tucker (1988b) that loss of petals affects subsequent events such as stamen initiation. Floral ontogeny of C. mopane may shed more light on the relationship between this species and H. binata. If a similar bracteole and sepal initiation pattern to that observed in C. mopane is present in H. binata, it may serve as further evidence for their inclusion in one genus.
ACKNOWLEDGEMENTS
The interest shown in this study by Prof. Shirley Tucker, her review of the manuscript and critical comments are gratefully acknowledged. The support and facilities of the Potchefstroom University made this study possible. The project was partially funded by FRD.
REFERENCES
Breteler FJ, Ferguson IK, Gasson PE, Ter Welle BJH. 1997. Colophospermum reduced to Hardwickia (Leguminosae-Casalpinioideae). Adansonia se´r. 3. 19: 279–291. Cowan RS, Polhill RM. 1981. Detarieae. In: Polhill RM, Raven PH, eds. Advances in legume systematics, Part 1. Kew: Royal Botanic Gardens, 117–134. Endress PK. 1990. Patterns of floral construction in ontogeny and phylogeny. Biological Journal of the Linnean Society 39: 153–175. Johansen DA. 1940. Plant microtechnique. New York: McGraw-Hill. Ramı´rez-Domenech JI, Tucker SC. 1990. Comparative ontogeny of the perianth in mimosoid legumes. American Journal of Botany 77: 624–635. Sattler R, Singh V. 1977. Floral organogenesis of Limnocharis flava. Canadian Journal of Botany 55: 1076–1086. Smith PP, Timberlake JR, Van Wyk AE. 1998. Proposal to conserve the name Colophospermum against Hardwickia (Leguminosae, Caesalpinioideae). Taxon 47: 751–752. Spurr AR. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructure Research 26: 31–43. Tucker SC. 1987. Floral initiation and development in legumes. In: Stirton CH, ed. Advances in legume systematics, Part 3. Kew: Royal Botanic Gardens, 183–239.
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Tucker SC. 1988a. Dioecy in Bauhinia resulting from organ suppression. American Journal of Botany 75: 1584–1597. Tucker SC. 1988b. Loss versus suppression of floral organs. In: Leins P, Tucker SC, Endress P, eds. Aspects of floral development. Stuttgart: Gebru¨der Borntraeger, 69–82. Tucker SC. 1989. Evolutionary implications of floral ontogeny in legumes. In: Stirton CH, Zarucchi JL, eds. Advances in legume biology. Monographs in Systematic Botany from the Missouri Botanical Garden, Vol. 29. St. Louis: Missouri Botanical Garden, 59–75. Tucker SC. 1991. The role of floral development in studies of legume evolution. Canadian Journal of Botany 70: 692–700. Tucker SC. 1996. Stamen structure and development in legumes, with emphasis on poricidal stamens of ceasalpinioid tribe Cassieae. In: D’Arcy WG, Keating RC, eds. The anther form, function and phylogeny. Cambridge: Cambridge University Press, 236–254. Tucker SC. 1997. Evolutionary loss of sepals or petals in flowers of ceasalpinioid tribe Detarieae (Leguminosae). American Journal of Botany 84: 238. Tucker SC. 1998. Floral ontogeny in legume genera Petalostylis, Labichea, and Dialium (Caesalpinioideae: Cassieae), a series in floral reduction. American Journal of Botany 85: 184–208. Tucker SC, Douglas AW. 1994. Ontogenetic evidence and phylogenetic relationships among basal taxa of legumes. In: Ferguson IK, Tucker SC, eds. Advances in legume systematics, Part 6. Kew: Royal Botanic Gardens, 11–32. Tucker SC, Stein OL, Derstine KS. 1985. Floral development in Caesalpinia (Leguminosae). American Journal of Botany 72: 1424–1434. Watson L. 1981. An automated system of generic descriptions for Caesalpinioideae, and its application to classification and key-making. In: Polhill RM, Raven PH, eds. Advances in legume systematics, Part 1. Kew: Royal Botanic Gardens, 65–80.
Floral development in the legume tree Colophospermum mopane, Caesalpinioideae: Detarieae ¨ GER1∗, L. R. TIEDT2 AND D. C. J. WESSELS3 H. KRU 1
School for Environmental Sciences and Development and 2Laboratory for Electron Microscopy, Potchefstroom University for Christian Higher Education, 2520 Potchefstroom, South Africa 3 Department of Botany, University of the North, Private Bag X1106, 0727 Sovenga, South Africa Received January 1999; accepted for publication April 1999
Floral ontogeny of Colophospermum mopane (Kirk ex Benth.) Kirk ex J. Leonard, an apetalous member of the Crudia group with four sepals and a large number (20–25) of stamens, was studied as part of a larger project on reproductive biology of this much-utilized tree. The flowers have been described as being inserted in the axil of a bract, but lacking lateral bracteoles. Four outer, light cream or white ‘sepals’ are present. The first two sepals are initiated in a lateral position, where the bracteoles, if present, develop in other members of the Caesalpinioideae. The inner sepals arise simultaneously adaxially and abaxially. These four structures, conventionally regarded as sepals, enclose the bud. The outer two ‘sepals’ should be regarded as lateral bracteoles inserted at the apex of the pedicel. The inner structures represent the only two sepals. The large number of stamens arise on a large meristematic surface and different whorls were not observed. The filaments elongate within the bud and after anthesis become exposed outside the flowers. The filaments are of equal length and the large anthers form a suspended cluster. One carpel develops terminally and gives rise to an indehiscent one-seeded fruit. 1999 The Linnean Society of London
ADDITIONAL KEYWORDS:—bracteoles – flower – Hardwickia – mopane – ontogeny. CONTENTS
Introduction . . . . Material and methods Results . . . . . Mature flower . . Floral apex . . . Sepal initiation . . Stamen initiation . Carpel development Discussion . . . . Conclusion . . . . Acknowledgements . References . . . .
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∗ Corresponding author. Email: [email protected]. 0024–4074/99/110223+11 $30.00/0
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1999 The Linnean Society of London
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¨ GER ET AL. H. KRU INTRODUCTION
Colophospermum mopane (Kirk ex Benth.) Kirk ex J. Leonard was reduced to Hardwickia mopane (Kirk ex Benth.) Breteler, comb. nov (Breteler et al., 1997) within the Crudia group (tribe Detarieae DC.), despite differences in seed and fruit morphology between Hardwickia binata Roxb. and C. mopane, and their disjunctive distributions. Hardwickia Roxb. and Colophospermum J. Leonard are monotypic genera. C. mopane occurs in Africa and H. binata is only known from India. Floral differences between the two species mentioned by Breteler et al. (1997) include sepal number (4–5 in H. binata and 4 in C. mopane) and stamen number (10–11 in H. binata and 20–25 in C. mopane). Smith, Timberlake & Van Wyk (1998) proposed the conservation of the name Colophospermum mopane on several grounds and as it is such a well-known African monotypic genus, the name is used in this study, as the matter has apparently not yet been settled. If only conventional characters are used, the tribes Detarieae and Amherstieae are almost impossible to separate. Watson (1981) treated the Detarieae–Amherstieae (or main group 2) as separate from the other tribes in Caesalpinioideae with the Detarieae more heterogeneous than the Amherstieae. The presence of relatively well-developed bracteoles is a feature cited by Cowan & Polhill (1981) as characteristic for the Detarieae–Amherstieae, but they mention that bracteoles are absent in some members of the Crudia group of the Detarieae. This also applies to C. mopane, where bracteoles are regarded as absent. In their list of similarities between C. mopane and H. binata, Breteler et al. (1997) also mention the absence of bracteoles. Representatives of the Crudia group include unarmed trees with flowers varying from small to showy and regular to markedly zygomorphic. Bracteoles can be anything from small to large, imbricate or partly fused. The number of petals range from absent to five (Cowan & Polhill, 1981). Generic and tribal limits are difficult to fix, due to modifications of the flowers. The significance of ontogenetic evidence for the evaluation of phylogenetic relationships between the taxa of the Leguminosae is stressed by Tucker (1987) and Tucker & Douglas (1994). Using floral development as a source of characters and character states combined with conventional characters, the preliminary phylogenies produced through analysis by Tucker & Douglas (1994) indicate a monophyletic origin of the tribe Detarieae. Floral development of C. mopane has as yet received no attention and the information presented here may contribute to a better understanding of its taxonomic position within the Crudia group and its inclusion within the genus Hardwickia. The only other report on floral ontogeny within the Crudia group is that on Crudia choussyana where five petals are initiated but all fail to enlarge (Tucker, 1997). Endress (1990) recognized the lack or reduction of the perianth as one of the two most important conditions leading to instability in organ position in flowers. This also applies to Colophospermum mopane where no petals are present in mature flowers, in combination with the large number of stamens. The aims of this work were to determine the order of floral organ initiation in C. mopane, to determine whether the apetalous situation is due to loss or suppression of organs, to compare the ontogeny with those of other members of the Caesalpinioideae, to contribute to the investigation on the reproductive biology of C. mopane which is presently being conducted, and to contribute towards the floral ontogenetic information available on the Caesalpinioideae.
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Figure 1. Ground plan of main axis of panicle of Colophospermum mopane to show position of flowers and different arrangements of bract and sepals. Flowers numbered from oldest to youngest.
MATERIAL AND METHODS
Young inflorescences of C. mopane were collected at the Messina Experimental Farm (22°14″5.1′ S;29°55″8.1′ E) in the Northern Province, South Africa, fixed in formalin-acetic acid-ethanol ( Johansen, 1940) or 4% paraformaldehyde, pH7.4, dehydrated in acetone and critical point dried. Floral buds of different stages were opened, attached to scanning electron microscope stubs, coated with gold-palladium and studied with a Philips XL30 scanning electron microscope. Young fixed inflorescences were dehydrated in acetone, infiltrated with resin (Spurr, 1969), sectioned with glass knives and stained with toluidine blue O and neufuchsin to determine the structure of the inflorescence in serial sections. Herbarium specimens of material used are housed in UNIN (Potgieter & Wessels 140, Wessels & Jordaan 51 and Potgieter & Wessels 1).
RESULTS
Flowers are borne axillary in panicles (Fig. 2). The inflorescences have helical phyllotaxy (in a 38 helix in the inflorescences studied) of acropetally produced bracts and flowers (Fig. 1). Inflorescence size differs from season to season and also between localities. All the flowers studied were perfect. The angle between the ±5 mm long pedicel and the axis may be 90° or smaller, but seems to be relatively constant for a plant. The number of flowers per inflorescence reaching anthesis differs during different seasons and large numbers of flowers collected and fixed during dry seasons were infected by insect larvae (herbarium specimen Potgieter & Wessels 1). The bracts are initiated acropetally and each subtends a single flower (Fig. 1). In inflorescences of some trees, the youngest three to five floral buds were all enclosed
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in the large bract of the next older flower, even in inflorescences where the older flowers had already undergone anthesis or where almost fully developed fruits were present. In these inflorescences the younger flowers did not reach anthesis and remained as small buds after maturation of older flowers, or became senescent within the bud. Fewer fruits than flowers were produced per inflorescence. In some inflorescences only one fruit reached maturity. More than one flush of flowering occurs in some trees, apparently depending on precipitation. Different trees in the same locality may also flower at different times.
Mature flower Floral buds are enclosed by the bract and four cream or white glabrous structures, conventially regarded as sepals (Figs 1, 3). The inner (adaxial and abaxial) sepals often have acute apices, while the apices of the outer (lateral) sepals are rounded. At anthesis all four sepals reflex back completely (Fig. 3). The rounded apices of the outer lateral reflexed sepals are incurved, making the mature flower slightly zygomorphic. In flowers where all the sepals have rounded apices, the lateral sepals are always incurved. Two patterns of imbricate sepal arrangement were encountered (Fig. 1). The most common pattern seems to be the one where the outer sepal is to the left of the flower as viewed from the abaxial side (flower 1 in Fig. 1). A second pattern was observed in fewer flowers in the inflorescences investigated with the outer sepal to the right of the flower if viewed similarly (flower 3 in Fig. 1). In some inflorescences both patterns occurred (Fig. 1), while flowers of other inflorescences studied exhibited only the most common pattern. The 20–25 (with the most common number of 20 encountered in the material studied) free stamens have long filaments of equal length (Fig. 3). The anthers are tetrasporangiate, dorsifixed and dehisce by longitudinal slits. At anthesis the flowers hang downwards and the heavy anthers are exposed outside the flowers, suspended on the long, thin filaments (Fig. 3). The central uni-ovulate indehiscent ovary is sessile and bears a capitate stigma with a distinctly folded appearance, on a c. 2 mm long style (Fig. 17). Style length varied between different trees. The style is inserted on the adaxial side of the ovary (Fig. 17) and at anthesis is often flexed towards the abaxial side. The fruit is almost
Figures 2–9. Inflorescences and floral primordia of Colophosperimum mopane. Fig. 2. Three inflorescences. Scale bar=10 mm. Fig. 3. Inflorescence with one open pendulous flower with long anthers of equal length and floral buds enclosed by the bract and ‘sepals’. Scale bar=10 mm. Fig. 4. Tangentially elongated floral primordium after removal of bract (b); abaxial side of flower at base of figure. Scale bar=50 lm. Fig. 5. Floral primordium with one lateral ‘sepal’ (1) initiated after removal of bract (b); abaxial side of flower at base of figure. Position of initiation of second lateral ‘sepal’ is indicated (2). Scale bar=50 lm. Fig. 6. Floral primordium with two lateral ‘sepals’ (1, 2) and adaxial and abaxial sepal initials (3) after removal of bract (b), with abaxial side of flower at base of figure. Scale bar= 50 lm. Fig. 7. Floral primordium with two enlarging lateral ‘sepals’ (1, 2) and two inner sepal initials (3) after removal of bract (b), with abaxial side of flower at base of figure. Scale bar=50 lm. Fig. 8. Floral primordium with four ‘sepals’ (1, 2, 3, 3) after removal of bract (b), with abaxial side of flower at base of figure. Scale bar=50 lm. Fig. 9. Floral primordium with large lateral ‘sepal’, adaxial and abaxial sepals (3) and carpel initial (c); bract (b) and one lateral ‘sepal’ removed, with abaxial side of flower to the right of figure. Scale bar=50 lm.
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reniform, but the shape varies in different plants, sometimes markedly so. The style is persistent. Rarely, an extra smaller carpel is present, producing an ovule. Exceptional carpels with more than one ovule have also been observed. The bracts and sepals contain large secretory canals lined with epithelium. Floral apex The floral apex is convex and broader tangentially than anticlinally after bract initiation (Fig. 4). The apex becomes flattened, but retains its elongated shape after initiation of the sepal primordia, becoming rectangular before stamen initiation (Figs 9–11). Sepal initiation Four ‘sepals’ are initiated. The first sepal primordium is initiated in either of the two lateral positions (Fig. 5). The second lateral sepal primordium appears soon after the first, and both enlarge simultaneously to enclose the two inner sepals in the bud. The outer sepal (either of the lateral sepals) may enclose the other lateral sepal both adaxially and abaxially or only abaxially. Overlapping of the one lateral sepal by the other seems to be a matter of chance as no specific pattern was discernible in any of the inflorescences studied (Fig. 1). In some inflorescences all the flowers showed the same pattern, while both patterns occurred in other inflorescences. The adaxial and abaxial sepal primordia arise simultaneously (Figs 6, 7) and are completely enclosed by the lateral sepals in the bud (Fig. 3). The abaxial sepal was interior to the adaxial sepal primordium in all the flowers studied (Fig. 1). As in all other legume flowers, the bases of the sepals do not overlap and imbricate aestivation is manifested only when the sepals enlarge. Stamen initiation The large number of stamen primordia (usually 20) are formed on an enlarged meristematic surface without any specific pattern of initiation (Fig. 10). At initiation
Figures 10–17. Flowers of Colophospermum mopane at different stages of development. Fig. 10. Floral primordium with enlarging adaxial and abaxial sepals (3), stamen initials (s) and carpel initial (c); bract (b) and both lateral sepals removed, with abaxial side of flower at base of figure. Position of one lateral ‘sepal’ (1) visible. Scale bar=50 lm. Fig. 11. Developing stamens (s) and carpel (c); bract and all sepals (1, 2, 3, 3) removed, with abaxial side of flower to the right of figure. Scale bar=50 lm. Fig. 12. Lateral view of flower with developing stamens with short filaments and basifixed anthers (s); bract and most sepals removed. Stigma is visible as a flattened structure (c). Scale bar=100 lm. Fig. 13. Lateral view of flower with elongating filaments; bract and sepals removed. Scale bar=500 lm. Fig. 14. Lateral view of flower with enlarging stigma on short style and with enlarging ovary (c); bract, sepals and most stamens (s) removed. Scale bar=100 lm. Fig. 15. Lateral view of flower with developing gynoecium (c); bract, sepals and most stamens (s) removed; abaxial side of gynoecium to the left of figure. Scale bar=100 lm. Fig. 16. Abaxial view of flower with developing gynoecium showing enlarging folded stigma and elongating style; bract, sepals and most stamens removed. Scale bar=500 lm. Fig. 17. Developing gynoecium with folded capitate stigma and elongating style; abaxial side of gynoecium to the left of figure. Scale bar=1 mm.
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all stamen primordia are of the same size and separate whorls were not discernible. At this stage the meristematic surface becomes distinctly rectangular (Fig. 9). It often seems as if stamen primordia do not develop to completely fill the available lateral space (Fig. 11). The anthers are basifixed at first (Figs 12, 14), as are all legume anthers (Tucker, 1996). The filaments elongate after initiation to form long slender structures, folded in the bud (Fig. 13). The anthers become dorsifixed and at anthesis they protrude from the flower, hanging downwards in a close group (Fig. 3). Carpel development The terminal carpel primordium appears before the stamen primordia as a single rounded protrusion (Fig. 9). Before the appearance of the filaments, an adaxial cleft is formed (Fig. 11). The stigma appears after the filaments have been formed (Fig. 12) and enlarges simultaneously with the abaxial part of the ovary to become flattened, with the adaxial cleft still evident (Fig. 14). The position of the stigma and style, that form at a later stage, thus becomes subapical (Fig. 15). The style forms after the stigma has enlarged significantly (Fig. 15) and elongation of the style occurs as the bud enlarges. The stigma enlarges to become capitate and folded (Figs 16, 17).
DISCUSSION
Sepal initiation in members of the Caesalpinioideae is helical or rarely unidirectional with the median sepal in the abaxial position (Tucker, Stein & Derstine, 1985; Tucker, 1989). According to descriptions of C. mopane flowers, the outer floral whorls are characterized by loss of at least one sepal and complete absence of petals (see Breteler et al., 1997). Few floral ontogenetic studies have as yet been done on other members of the Detarieae where only four sepals are present at anthesis. Sepal suppression occurs in Schotia brachypetala (tribe Detarieae) where five sepals are initiated in helical order. The last-initiated sepal (on the adaxial side) remains small. In Saraca declinata five sepals are initiated of which two become fused before anthesis, appearing as only one (Tucker, 1989). In Crudia choussyana, the only member of the Crudia group reported on as yet, five petals are initiated but none enlarge and the mature flower is apetalous (Tucker, 1997). Complete absence of several sepals was reported for the first time in Aphanocalyx djumensis, Monopetalanthus durandii and Brachystegia species of the Brachystegia group, tribe Detarieae (Tucker, 1997). As no rudimentary organs were observed in any of the Colophospermum mopane flowers studied, the four ‘sepals’ must be regarded as the result of loss of one sepal. The position and structure of the mature ‘calyx’ make the flower weakly zygomorphic. This only becomes evident after enlargement of the four sepals. The lateral position of the primordia of the first formed floral structures (‘sepals’) in C. mopane differs from any of those in caesalpinioid taxa yet reported (see Tucker et al., 1985; Tucker, 1998). Sepal initiation in C. mopane must be regarded as bidirectional, starting from one lateral to the other lateral and then followed by the last two (adaxial and abaxial) sepals. The outer, lateral primordia develop before initiation of the inner (adaxial and abaxial) sepals. Bidirectional sepal initiation has
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been described for the mimosoid legumes Wallaceodendron celebicum Koord., Acacia smallii Iseley and A. pinetorum Benth. (Ramı´rez-Domenech & Tucker, 1990) and the caesalpinioid legumes (tribe Cassieae) Petalostylis labicheoides R. Br. and Dialium guianense Steud. (Tucker, 1998). In C. mopane however, only four sepals are initiated, while the calyx in W. celebicum, A. pinetorum, P. labicheoides and D. guianense is pentamerous (Ramı´rez-Domenech & Tucker, 1990; Tucker, 1998). As is the case in Colophospermum mopane, the two lateral sepals in W. celebicum arise first, but then the abaxial sepals enlarge to partly cover the other sepals. In C. mopane the four sepal primordia arise equidistant on the flanks of the floral meristem, and no indication of a retarded sepal primordium was observed in any of the flowers investigated. The lateral sepals always enclose the adaxial and abaxial sepals. The imbricate arrangement and position of the sepals in the bud are according to their order of initiation; the firstformed sepals enlarging first to enclose the others. In Dialium guianense the lateral sepals are initiated last, after the other three sepal primordia have been formed, and these lateral sepals overlap the other three imbricately (Tucker, 1998). In mopane, the lateral sepals are the first-formed sepal primordia, but remain in the outer position due to their enlargement before the two inner sepal primordia. Laterally placed primordia which arise after initiation of the bract usually give rise to bracteoles in caesalpinioid legumes (see Tucker, 1996, 1998). It would thus seem that the first two structures initiated in C. mopane flowers, directly after initiation of the bract, should be regarded as bracteoles and not as sepals. The position of the bracteoles at the apex of the pedicel would then be determined by absence of elongation of the pedicel above the point of insertion of the bracteoles. This brings C. mopane in line with the other members of the tribe Detarieae where bracteoles are usually present as mentioned by Cowan & Polhill (1981). If this is the case, only two sepals, initiated adaxially and abaxially, are present in C. mopane. The imbricate position of the bracteoles would be similar to that regarded as typical for other members of the Detarieae, in contrast with valvate bracteole position in the tribe Amherstieae (Cowan & Polhill, 1981). The flowers of C. mopane must then be regarded as zygomorphic as only two sepals are present. The two lateral bracteoles supplement the protective function of the two sepals as is the case in some caesalpinioids (Tucker, 1987). Floral ontogeny of related taxa, including Hardwickia binata, should be studied to determine the number and position of the sepals in flowers of other representatives of the Crudia group. Initially the floral meristem is broader tangentially and somewhat convex, but becomes rectangular at stamen initiation. The large number of stamens, positioned in a seemingly single whorl, may be due to an increased apical meristematic surface as suggested by Sattler & Singh (1977) for Limnocharis flava (L.) Buch. However, Tucker (1989, 1991, 1998) found that disruption of other subsequent floral whorls often occurs in flowers with missing organs. No petal primordia were present in any of the flowers of C. mopane investigated, and this disrupts the meristematic floral apex, resulting in the unusual pattern of stamen initiation. A meristematic ring was not observed in any of the flowers studied.
CONCLUSION
Floral ontogeny in C. mopane follows the usual pattern found in most caesalpinioid taxa only as far as the bract and gynoecium are concerned. The sequence of sepal
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initiation differs from the usual pattern and may be interpreted in either of two ways. Traditionally four outer sepals and absence of bracteoles are included in all descriptions of Colophospermum mopane and Hardwickia mopane (see Breteler et al., 1997 and literature cited therein). The position of the primordia of the outer two structures however, conforms to that of bracteoles in other members of the Leguminosae (see Tucker, 1987, 1988a) and although bracteole initiation has been studied only in C. mopane and in no other member of the Crudia group, it must be assumed that the ontogenetic position of the bracteoles will not differ from that of the other caesalpinioids. If this is confirmed by other studies on members of the Crudia group, it must be assumed that only two sepals are present, due to loss of three sepals. As no indication of petal primordia was observed in any of the flowers studied and as the shape of the floral apex gave no indication of initiation of any such primordia, the apetalous situation in C. mopane is regarded as due to complete loss of the petal whorl and not to petal suppression. The large number of stamens are initiated as separate primordia, but not in separate whorls. This is a special feature observed in C. mopane flowers and conforms to the observation of Tucker (1988b) that loss of petals affects subsequent events such as stamen initiation. Floral ontogeny of C. mopane may shed more light on the relationship between this species and H. binata. If a similar bracteole and sepal initiation pattern to that observed in C. mopane is present in H. binata, it may serve as further evidence for their inclusion in one genus.
ACKNOWLEDGEMENTS
The interest shown in this study by Prof. Shirley Tucker, her review of the manuscript and critical comments are gratefully acknowledged. The support and facilities of the Potchefstroom University made this study possible. The project was partially funded by FRD.
REFERENCES
Breteler FJ, Ferguson IK, Gasson PE, Ter Welle BJH. 1997. Colophospermum reduced to Hardwickia (Leguminosae-Casalpinioideae). Adansonia se´r. 3. 19: 279–291. Cowan RS, Polhill RM. 1981. Detarieae. In: Polhill RM, Raven PH, eds. Advances in legume systematics, Part 1. Kew: Royal Botanic Gardens, 117–134. Endress PK. 1990. Patterns of floral construction in ontogeny and phylogeny. Biological Journal of the Linnean Society 39: 153–175. Johansen DA. 1940. Plant microtechnique. New York: McGraw-Hill. Ramı´rez-Domenech JI, Tucker SC. 1990. Comparative ontogeny of the perianth in mimosoid legumes. American Journal of Botany 77: 624–635. Sattler R, Singh V. 1977. Floral organogenesis of Limnocharis flava. Canadian Journal of Botany 55: 1076–1086. Smith PP, Timberlake JR, Van Wyk AE. 1998. Proposal to conserve the name Colophospermum against Hardwickia (Leguminosae, Caesalpinioideae). Taxon 47: 751–752. Spurr AR. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructure Research 26: 31–43. Tucker SC. 1987. Floral initiation and development in legumes. In: Stirton CH, ed. Advances in legume systematics, Part 3. Kew: Royal Botanic Gardens, 183–239.
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Tucker SC. 1988a. Dioecy in Bauhinia resulting from organ suppression. American Journal of Botany 75: 1584–1597. Tucker SC. 1988b. Loss versus suppression of floral organs. In: Leins P, Tucker SC, Endress P, eds. Aspects of floral development. Stuttgart: Gebru¨der Borntraeger, 69–82. Tucker SC. 1989. Evolutionary implications of floral ontogeny in legumes. In: Stirton CH, Zarucchi JL, eds. Advances in legume biology. Monographs in Systematic Botany from the Missouri Botanical Garden, Vol. 29. St. Louis: Missouri Botanical Garden, 59–75. Tucker SC. 1991. The role of floral development in studies of legume evolution. Canadian Journal of Botany 70: 692–700. Tucker SC. 1996. Stamen structure and development in legumes, with emphasis on poricidal stamens of ceasalpinioid tribe Cassieae. In: D’Arcy WG, Keating RC, eds. The anther form, function and phylogeny. Cambridge: Cambridge University Press, 236–254. Tucker SC. 1997. Evolutionary loss of sepals or petals in flowers of ceasalpinioid tribe Detarieae (Leguminosae). American Journal of Botany 84: 238. Tucker SC. 1998. Floral ontogeny in legume genera Petalostylis, Labichea, and Dialium (Caesalpinioideae: Cassieae), a series in floral reduction. American Journal of Botany 85: 184–208. Tucker SC, Douglas AW. 1994. Ontogenetic evidence and phylogenetic relationships among basal taxa of legumes. In: Ferguson IK, Tucker SC, eds. Advances in legume systematics, Part 6. Kew: Royal Botanic Gardens, 11–32. Tucker SC, Stein OL, Derstine KS. 1985. Floral development in Caesalpinia (Leguminosae). American Journal of Botany 72: 1424–1434. Watson L. 1981. An automated system of generic descriptions for Caesalpinioideae, and its application to classification and key-making. In: Polhill RM, Raven PH, eds. Advances in legume systematics, Part 1. Kew: Royal Botanic Gardens, 65–80.