Portioned pollen release and the syndromes of secondary pollen presentation in the Campanulales-Asterales-complex1)

Flora (1995) 190 323-338 ©by Gustav Fischer Verlag Jena

Portioned pollen release and the syndromes of secondary pollen presentation in the Campanulales-Asterales-complex 1 ) CLAUDIA ERBAR and PETER LEINS Institut fiir Systematische Botanik und Pflanzengeographie, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany Accepted: January 4, 1995

Summary The release of pollen in portions, i. e. limiting the pollen removed by individual pollinators during a single visit, is a widespread phenomenon. The biological significance of this phenomenon is that it helps to optimize the pollination. Parameters like number of ovules, number of application sites to the pollinator or spectrum of pollinators, which may be correlated with the size of the pollen portion, are illustrated as a net of possible correlations (Fig. 1). Apart from pollenkitt, viscin or cellulosic threads and the non-simultaneous opening of the anthers, the secondary pollen presentation is a special mechanism of pollen portioning. Secondary pollen presentation occurs in several dicotyledonous and in a few monocotyledonous families. Its occurrence is a distinctive feature in the Campanulales-Asterales-complex. The different mechanisms in the Campanulales-Asterales ( Asteraceae, Lobeliaceae, Campanulaceae, Goodeniaceae, Brunoniaceae, Calyceraceae) are briefly described. The differentiations of the "basic syndrome" (radial symmetry of the flower bud, complete androecial whorl, introrse anthers, the latter connivent at the time of dehiscence, proterandry, late and sequential growth of filaments and style) are presented in two diagrams (Figs. 30 + 31). In addition, a few remarks are given on pollen presentation in Rubiaceae, Cyphiaceae, Sphenocleaceae, Pentaphragmataceae, Stylidiaceae, of which the latter four are usually assigned to Campanulales-Asterales. Key words: Campanulales-Asterales, Rubiaceae, secondary pollen presentation, portioned pollen release, pollination syndromes, phylogeny

Introduction Animal-pollination has the advantage over windpollination that the directed pollen transport from flower to flower requires lower quantities of pollen. Nevertheless, loss of pollen cannot be discounted in animal-pollinated flowers. Loss occurs through pollen-eating, pollen collecting, pollen falling beneath the anthers during the pollinator's visit, grooming of the pollinator or its unreliability. The uncertainty of successful pollen transport is reduced by limiting the pollen removed by individual pollinators during a single visit. It is therefore not surprising that the issue of pollen in portions is a widerspread phenomenon. The division of a plant's total pollen production 1 ) Dedicated in friendship to Prof. Dr. Andreas Sievers on the occasion of his 65th birthday (June 21, 1996)

into separate doses which sequentially become available to pollinators can be achieved by pollenkitt only. The sticky secretion of the tapetum is responsible for the aggregation of pollen in smaller or larger packets. Pollenkitt may be replaced by viscin threads (consisting of sporopollenin) as, e. g., in Onagraceae. They provide a loose pollen aggregation, which looks like pollen presented on a clothes-line. A further possibility of pollen portioning is given by cellulosic threads formed by the disintegration of the inner layers of the pollen sacs (e. g. in Impatiens, VoGEL&Cocucci 1988). A common, often additional phenomenon concerning pollen release in portions is the successive dehiscence of the anthers in a flower. An example out of a long list is Parnassia palustris, in which the male phase of anthesis lasts several days, with one anther opening each day (see also HEss 1983). The wide distribution of portioned pollen release perhaps indicates that it is connected with the FLORA (1995) 190

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optimization of the pollination. Some parameters involved in the optimization of pollination through portioned pollen release are illustrated as a net of possible correlations (Fig. 1). The size of the pollen portion may be correlated, e. g., with the number of ovules, the number of application sites to the pollinator, and with the spectrum of pollinators. We want to touch shortly on the correlation of the size of pollen portion with the spectrum of pollinators. A large spectrum of different pollinators like beetles, bees, wasps and flies can be found on the inflorescences of the Apiaceae. The loss of pollen through the unreliability of flower-visiting animals, which behave like vagabonds, can be reduced by limiting the pollen removed by a single pollinator: In the Apiaceae; the small anthers contain only a small amount of pollen and open successively. In the other extreme, the pollen portion may be so large, that, in a strict sense, it is not possible to speak of portioning. The fact that the flower's total pollen production is removed as two pollinia in many orchids, is correlated with the high reliability and specifity of the pollinators. The flowers of Ophrys speculum, e. g., are highly adapted for the pollination by the males of Campsoscolia ciliata only (PAULUS & GACK 1980). Apart form pollenkitt, viscin or cellulosic threads and the non-simultaneous opening of the anthers, the secondary pollen presentation is a further and special mechanism of pollen portioning. Secondary pollen presentation covers mechanisms in which the pollen grains are not presented directly out of the anthers but are transferred to certain other flower structures just before or at the outset of anthesis. In the male phase of anthesis, different auxiliary floral structures may be involved in the exposure of the pollen deposit. From this auxiliary structures the pollen load is not

Table 1. Occurrence of secondary pollen presentation in dicotyledonous and monocotyledonous families (after YEo 1993, modified) Families with secondary pollen presentation Proteaceae Me/iaceae Rafflesiaceae Vochysiaceae Polyga/aceae Apocynaceae Rubiaceae

Myristicaceae Fumariaceae Sterculiaceae Loasaceae Epacridaceae Fabaceae Rhizophoraceae Myrtaceae Campanulaceae Lobeliaceae Goodeniaceae Brunoniaceae Asteraceae Calyceraceae Hydrocharitaceae Xyridaceae Cannaceae

Marantaceae Liliaceae

handed over as a whole. Secondary pollen presentation occurs in several dicotyledoneous and in a few monocotyledonous families (Table 1). Its occurrence is a distinctive feature in the Campanulales-Asteralescomplex, which is the object of this study. Based upon a "basic syndrome" (see discussion), different modes of secondary pollen presentation have evolved in this complex. Part of the mechanisms of secondary pollen presentation have been known for a long time. In the light of the newest insights into systematics, especially through the use of molecular data, it seems advisable to do more, and more detailed, comparative reinvestigations. Cladistic analyses as well as "classical" phylogenetic considerations only make sense if precise morphological and ontogenetical data are available.

Material and methods We refer to earlier studies in which SEM -analyses had been prominent (ERBAR & LEINS 1988, 1989, LEINS&ERBAR 1989, 1990, ERBAR 1992, 1993). In addition, fresh flowers were observed under a stereomicroscope (Leitz SV8) for this study. The results originate from examination of fresh, of liquid-preserved and of herbarium material. Illustrations are given for the following taxa: Fig. 1. Net of possible correlations illustrating parameters involved in the optimization of pollination through portioned pollen release. 324

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Achillea clypeolata SIBTH. & SM. (Asteraceae; Botanical Garden of the University of Heidelberg= BGHD), Acicarpha tribuloides Juss. (Calyceraceae; BGHD), Barnadesia

polyacantha WEDD. (Asteraceae; Botanical Garden of the University of Munich, FAA fix.), Brunonia australis SM. (Brunoniaceae; BGHD), Catananche caerulea L. (Asteraceae; BGHD), Chuquiraga varians (GARDNER) RusBY (Asteraceae; WU: A. Miguel Bang 2335), Cichorium intybus L. (Asteraceae; BGHD), Codonopsis ussuriensis (RUPR.&MAXIM.) HEMSL. (Campanu/aceae; BGHD), Dampiera stricta (SM.) R. BR. (Goodeniaceae; Royal Botanical Gardens Kew 420.84.04495, FAA fix.), Dasyphyllum brasiliense (SPRENG.) CABRERA (Asteraceae; Herbar Walter Till, Wien: G. Hatschbach 48113), Grindelia squarrosa (PuRsH) DUNAL (Asteraceae; BGHD), Heliopsis helianthoides (L.) SWEET (Asteraceae; BGHD), Hoffmannia ghiesebreghtia (LEM.) HEMSL. (Rubiaceae; Palmengarten Frankfurt, FAA fix.), Isotoma axil/aris LINDL. (Lobeliaceae; BGHD), Ixora coccinea L. (Rubiaceae; Botanical Garden of the University of Mainz, FAA fix.), Lobelia erinus L. (Lobeliaceae; BGHD), Pentaphragma philippinense MERR. (Pentaphragmataceae; M: H. Koerper s. n.), Phuopsis stylosa (ThiN.) JACKS. (Rubiaceae; BGHD), Phyteuma spicatum L. (Campanulaceae; Handschuhsheim/Heidelberg: Fig. 15, photo T. HARTMANN; Helenental near Baden/Austria, FAA fix.: Fig. 16), Platycodon grandiflorus (JACQ.) A. DC. ( Campanulaceae; BGHD), Scaevo/a aemula R. BR. (Goodeniaceae; BGHD), Sphenoclea zeylanica GAERTNER (Sphenocleaceae; M: D. Podlech 32590), Stylidium adnatum R. BR. (Stylidiaceae; Botanical Garden of the University of Karlsruhe, FAA fix.: Fig. 44; BGHD: Fig. 45) Vangueria apicu/ata K. SCHUM. (Rubiaceae; leg. Puff 860920-1/1, FPA fix.; cf. IGERSHEIM 1993b).

The different mechanisms of secondary pollen presentation Brushing mechanism in Asteraceae (Fig. 2a) The introrse anthers are united into a tube by the more or less firm coherence of their cuticulae. The back of the ± long stylar branches and the upper part of the style bear short hairs. In old flower buds, the tip of the style is above the top of the anther tube. Shortly before anthesis, growth of the filaments and the stamen-corolla tube brings the anthers up to the same level as the hairy part of the style so that the style nearly fills the anther tube. The anthers open and the pollen is partly loaded onto the hairs of the style. By elongation of the style, the pollen grains are little by little completely swept out of the anthers by the brushing hairs, which now are somewhat spreading. Thus in the male phase of anthesis, the hairy part of the style emerges out of the anther tube and gradually presents the pollen on its outside (Fig. 3). Later on the style arms separate to expose the stigmatic surface (female phase). This mechanism is characteristic for the tribe Lactuceae throughout. It is also found in the tribes

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Fig. 2. Different mechanisms of secondary pollen presentatation: brushing mechanism in Asteraceae (a), pump mechanism in Asteraceae (b), pump mechanism in Lobeliaceae (c), deposition mechanism in Campanulaceae (d), brushing mechanism in Phyteuma (e), cup mechanism in Goodeniaceae, combination of cup with brushing mechanism in Brunoniaceae (g), deposition mechanism in Ca/yceraceae (h).

Arctotideae, Eupatorieae, and Vernonieae and sometimes in Cardueae, Heliantheae, Inuleae, and Mutisieae (cf. JUEL 1908, THIELE 1988).

Pump mechanism in Asteraceae (Fig. 2 b) In older flower buds, the tip of the style is more or less on the same level with the top of the anther tube. Shortly before anthesis, growth of the filaments - supported or even substituted by growth of a stamen-corolla tube- raises the anther tube higher than the stylar tip. The style branches mostly are truncate with hairs only at their very tips. They block the lower end of the anther tube. The anthers open and the pollen is released into the cavity of the anther tube as a temporary receptacle. In the male phase of an thesis, the growing style acts as a piston and pushes the pollen gradually out of the anther tube. When part of the pollen becomes visible at the top of the anther FLORA (1995) 190

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Fig. 3. Cichorium intybus. Brushing mechanism: pollen is gradually presented on the outside of the styles (arrows). Fig. 4. Heliopsis helianthoides. Pump mechanism: pollen grains protrude from five slits between the connivent appendages ofthe connectives. - Fig. 5. Lobelia erinus. Pump mechanism; anther tube opened: the stylar tip (arrow) is on the same level with the base of the anther tube at the time of anther dehiscence. - Figs. 6-8. /sotoma axillar is. -6, Anther tube with apical opening closed (by the bases of the two coherent trigger hairs; arrow). 7, Pollen grains have flowed out (arrow) after pushing the trigger hairs towards the anther tube.- 8, Anther tube opened: the elongating style accumulates the pollen grains in the upper part of the anther tube thus building up a pressure against the closed top of the anther tube. - Figs. 9-11. Codonopsis ussuriensis.- 9, Anthers deposit their pollen onto the style in the still closed flower.- 10, Early male phase of anthesis with pollen shed onto the stylar hairs. - 11, Style in female phase of anthesis (all stylar hairs withdrawn, stigma lobes spreading).- A= anther, AT= anther tube, F =filament, Sti =stigma, Sty= style, tH =trigger hairs.

tube, it may be carried away by any visiting pollina- in portions by the subsequent growth of the style tor. Pollen is pushed up further and the next portion with its collar of hairs below the appressed short may be dislodged by the next visitor. A refined pollen stigmas. release is accomplished by the apical appendages of We observed this mechanism in several species of the connectives: in many cases pollen grains protrude Lobelia (L. erinus: ERBAR&LEINS 1989, L. cardinalis, from five slits between the connivent appendages L. sessilifolius, L. splendens), Burmeistera brachyan(Fig. 4). Thus each portion of pollen is divided once dra, Downingia pulchella, and Hippobroma longiflora.1) more. Since the flowering period of a capitulum extends The lobeliaceous genus Isotoma exhibits an advanover more than one day with the single flowers ced elaboration with regard to the timing of the opening successively in centripetal direction, there is pollen release (BRANTJES 1983). The small opening at a "three-staggered pollen-dosing": a rough portion- the top of the anther tube is closed by the basal parts ing as regards the capitulum, a fine portioning by of two long hairs (Fig. 6), so that pollen cannot be gradually pushing the pollen out of the anther tube pushed out by the growing style. As a result, pressure and the refined portioning involving the connective is exerted on the pollen inside the anther tube (Fig. 7). appendages. The release of pollen is triggered by the pollinator In many members of the family (in those with when operating a valve consisting of the two long pump mechanism as well as in those with brushing hairs. When the visitor opens the valve, the pressure mechanism), the stames are irritable: on being built up in the anther tube causes the pollen to flow touched, the filaments shorten through loss of turgor, out (Fig. 8). thus drawing the anther tube downwards. This assures that part of the pollen is presented just at the Deposition mechanism in Campanulaceae (Fig. 2d) time when a pollinator is present. In most genera of the Campanulaceae, a long part of Within the Asteraceae, the pump mechanism can the style possesses long hairs, that are curved upbe found mainly in the Asteroideae s. str. (especially wards.2) Just before anthesis, the filaments elongate in Anthemideae, Calenduleae, and Senecioneae) alt- slightly in order to adjust the position of the free hough in this subfamily the mechanisms of secondary anthers to the hairy part of the style. The anthers pollen presentation are less uniform within the tribes dehisce in the bud stage and deposit their pollen than in those of the Cichorioideae s. I. (cf. THIELE among the stylar hairs (Fig. 9). The anthers and the 1988, YEO 1993). upper thin parts of the filaments wither quickly and curl away. The pollen deposit on the style often is moulded, reflecting the shape of the opened anthers Pump mechanism in Lobeliaceae (Fig. 2c) (Fig. 10). Like in Asteraceae, the anthers are firmly coherent The behaviour of the collecting hairs of the style is through their cuticles. The anther tube, however, is a feature peculiar to Campanulaceae: for the release of slightly curved towards the three-lobed lower lip (in pollen in the male phase of anthesis, they invaginate into a resupinate flower). The pump mechanism is very their own large bulbous base (cf., e. g., BRONGNIART similar to that of the Compositae: In late develop- 1893, JOST 1918, ERBAR&LEINS 1989, NYMAN 1993). mental stages, the tip of the style lies above the base of the anther tube. Shortly before anthesis, the 1) A more detailed analysis is in preparation. filaments elongate to bring the base of the anther 2) A few species of Wlhlenbergia, J asione, and Roella tube on the same level with the stylar tip (Fig. 5). have a sticky instead of a hairy pollen-collecting stylar area Pollen is shed into the anther tube and pushed out (SCHONLAND 1894, ECKARDT 1929, YEO 1993). 326

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The retraction depends on a cohesion/adhesion mechanism. The invagination begins at the thinnest part of the cell wall at the base, and the tip sinks into the foot of the unicellular hair, which is embedded in the stylar tissue (see scheme Fig. 35inLEINs 1991). The hairs withdraw so completely that the style becomes nearly glabrous but dotted with pits (Fig. 11 ). The hairs do not retract simultaneously on the whole stylar surface, but successively in different regions, starting more or less at the upper part of the style. Due to the gradual progress of invagination of the hairs, plates of pollen grains can easily fall onto a visiting insect. This way, portion by portion of pollen can be carried away by different insect individuals. We observed a deposition mechanism in several species of Campanula (e. g., in C. garganica, C. medium, C. punctata, C. rotundifolia), in Codonopsis ussuriensis, Jasione montana, Legousia speculum-veneris, Platycodon grandiflorus, Wah/enbergia hederacea and W. simi/is (see ERBAR& LEINS 1989 for Campanula rotundifo/ia and Jasione montana). In Platycodon, the anthers also deposit their pollen onto the style (Fig. 12). However, the style starts to elongate just before the anthers curl away (Fig. 13). Thus there may be a partly sweeping action of the stylar hairs. In this context it needs to be mentioned that the hairy stylar part is longer than the anthers, and reaches beneath the anthers at the time they open (Fig. 12). At the end of the male phase of an thesis all hairs are invaginated in their own bulbous bases (the style appears hairless and is dotted with pits) and the stigmatic surfaces become exposed (Fig. 14). IGERSHEIM (1993 a) has investigated the deposition mechanism in Petromarula pinnata. It differs in that the stylar head is ± globose and is partially overtopped by the anther tube. Nevertheless, the pollen is shed onto the globose stylar top with its long retractile hairs. Brushing mechanism in Phyteuma ( Campanulaceae) (Fig. 2e) Within the Campanulaceae, an additional mechanism is found, which differs from the typical campanulaceous deposition mechanism. This brushing mecha-

nism in Phyteuma (e. g., P. spicatum), however, also differs from that in Compositae in that there is no stable tube of fused anthers. Pollen brushing is possible only with the aid of the corolla. At the time of anthesis, the long, laterally cohering corolla lobes separate in acropetal direction but remain- for some time - connected at their tips. The dehiscing anthers are pressed towards the hairy part of the style not only by the corolla hood but also because of a growth in the dorsal side of the filaments. While the open anthers are slipping down along the hairy zone of the style (by growth in the ventral side of the lower part of the filaments) the corolla hood is pulled downwards and the style starts to elongate. As a result, the style ruptures the corolla hood and gradually presents the pollen on its outside (Fig. 15). It is notable that in Phyteuma the invagination of the hairs is incomplete (Fig. 16). The hairs become lodged in the style like little swords because the bulbous base is to small. 1) Cup mechanism in Goodeniaceae (Fig. 2 f) This highly elaborated mechanism was studied by us in Selliera radicans (LEINs&ERBAR 1989), Dampiera stricta, and Scaevo/a aemula. The distinctive character is the cup-like outgrowth beneath the tip of the style, which envelops the young stigma. Shortly before anthesis, the border of the cup is above the five free anthers. Subsequent intensive filament growth raises the anthers above the stylar cup. The introrse anthers open and the whole pollen mass - the pollen grains of an anther sticking together by pollenkitt -slides into the stylar cup while the style elongates by rapid growth. The cup loaded with own pollen closes. The style bends downwards, so that the cup comes to face the abaxial petal. In open flowers, the style with its apical cup functions as an upper lip, whereas the entire zygomorphic, deeply slit corolla forms a lower These results were obtained by THoMAs HARTMANN in a study of Phyteuma spicatum for his Diplomarbeit (diploma thesis). A detailed description will be given in a further study. 1)

Figs.12-14. Platycodon grandiflorus. -12, Deposition of pollen onto the stylar hairs; note that the hairy stylar part is longer than the anthers (arrows indicate stylar hairs below the anthers). -13, Elongation of the style just before the anthers curl away.- 14, Female phase of anthesis (stylar hairs withdrawn, stigma lobes spreading). - Figs. 15-16. Phyteuma spicatum.- 15, Brushing mechanism; the elongating style ruptures the corolla hood presenting the pollen on its outside (arrow) while the corolla hood is pulled downwards; the empty anthers are already visible between the outwards bending lower parts of the petals. -16, Incompletely retracted stylar hairs; SEMgraph. -Figs. 17-18. Scaevola aemula. -17, Open flower; the style with the apical cup (arrow) functions as upper lip and faces the zygomorphic corolla forming the lower lip; the natural position of the flower is turned in an angle of90 degrees. -18, Pollen (arrows) is presented at the cup aperture in portions- Fig. 19. Brunonia australis. Combination of cup and brushing mechanism: pollen grains are visible in the stylar cup (white arrow) as well as on hairs on the outside of the cup and the upper half ofthe style (black arrows).- A= anther, F =filament, P = petal, Sti = stigma, Sty = style. 328

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lip (Fig. 17). It is the continuous growth of the stigma at the bottom of the cup by which pollen is gradually pressed out of the stylar cup and presented at the cup aperture in portions (Fig. 18). At last the stigma itself emerges from the cup (female stage of anthesis). Combination of cup with brushing mechanism in Brunoniaceae (Fig. 2 g) In the mono typic Australian family Brunoniaceae, as in Goodeniaceae, the stylar cup is involved in the capturing of own pollen (Fig. 19). In contrast to the Goodeniaceae, the pollen is first shed into the firmly coherent anther tube (the anthers stick together laterally by their cuticles) when it is above the border of the cup, due to intensive filament growth. Afterwards, when the style elongates, not all pollen grains are caught in the stylar cup. The remaining pollen is swept out of the anther tube by long hairs on the outside of the cup and the upper half of the style (ERBAR&LEINS 1988). Deposition mechanism in Calyceraceae (Fig. 2h) The family Calyceraceae is considered in this study because recent investigations strongly indicate a close relationship with the CampanulalesjAsterales (see ERBAR 1993). Acicarpha tribuloides exhibits a simple deposition mechanism. 1) In older flower buds the style tip is half way through the cavity formed by the free but connivent anthers. Shortly before anthesis the anthers are raised (due to growth mainly in the stamencorolla tube) until only the club-like part of the style exceeds the bases of the anthers. The upper part of the undivided style exhibits a papillate epidermis. The anthers open in the still closed flower. The pollen grains sticking together by pollenkitt are discharged onto the top of the style. Now the corolla lobes begin to unfold, the anthers spread somewhat (Fig. 20), the style elongates and the pollen hood on top of the style is elevated high above the corolla lobes and presented to the pollinators (Fig. 21). The pollen mass exhibits 1) The description of the mechanism in Acicarpha as a "pump-mechanism" (BREMER 1994), comparable to that in Lobeliaceae (LADD 1994), is not correct (for detailed description see ERBAR 1993). HOWELL et al. (1993) summarize the families Lobeliaceae, Asteraceae, and Calyceraceae in the same secondary pollen presentation system ("terminal stylar presenters with active pollen placement"). This classification, however, disregards the different behaviour of the anthers after the delivery of the pollen and the different place of pollen presentation in Calyceraceae compared to the other two families as well as the fact, that the mechanisms of secondary pollen presentation are not uniform in Asteraceae. HoWELL et al. (1993) do not consider the brushing mechanism in Asteraceae.

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five grooves, each corresponding to an imprint of the walls of the inner pollen sacs. The spreading anthers wither and then surround the style like a cuff. Pollen portioning in Acicarpha is only possible by a gradual decrease in the stickiness of the pollenkitt. The fact that no other floral structures are involved in pollen delivery in measured doses distinguishes the secondary pollen presentation in Acicarpha from the mechanisms observed in the remaining taxa of the Campanulales-Asterales-complex analysed so far (LEINS & ERBAR 1990). Deposition mechanism in Rubiaceae The Rubiaceae are included in this comparison, because secondary pollen presentation similar to that in Acicarpha is widerspread in the family, namely in the subfamilies Ixoroideae and Antirheoideae (see IGERSHEIM 1993 b for further literature), and occasionally in the Cinchonoideae (C. PUFF, pers. com.). In the subfamily Rubioideae, secondary pollen presentation is known so far only in the monotypic genus Phuopsis (ERBAR 1993, YEO 1993). Like in Acicarpha, secondary pollen presentation is a simple deposition mechanism in the Rubiaceae: Pollen grains are deposited and presented on the outside of the uppermost part of the style. The style may show no obvious modifications, or it can be of a more elaborate shape in order to increase the surface for holding the pollen. The stylar apex may be club-shaped, obovate or capitate; the surface of the upper stylar part may be papillate by enlarged epidermal cells or structured by longitudinally arranged ridges, furrows or short hairs, or occasionally provided with sticky substances (see IGERSHEIM 1993 b, YEo 1993; see Figs. 22-24 for differently shaped styles in taxa with secondary pollen presentation). In Phuopsis stylosa, the upper part of the style is clavate with an epidermis of broad, roundish papillae; the uppermost stylar part exhibits smaller stigmatic papillae (Fig. 28). The stamens are attached near the throat of the slender corolla tube, i. e., the long tube of the adult flower is formed mainly by the stamen-corolla tube (ERBAR 1991). Just before anthesis - at this stage flowers can easily be opened by touching the connivent corolla lobes - the just opening anthers enclose the upper part of the style like a dome (Figs. 25, 29). When the pollen has been deposited, the anthers spread (Fig. 26), and the pollen-loaded top of the style is lifted up high above the corolla lobes and presented to the pollinators (Fig. 27). There are no special modifications for pollen issue in measured doses.

Figs. 20-21. Acicarpha tribuloides. Deposition mechanism: the pollen grains sticking together by pollenkitt are discharged onto the top of the style, the anthers spread somewhat, the style elongates and the pollen hood (arrow in Fig. 21) is elevated high above the corolla lobes.- A= anther, P =petal.

Comparative discussion When comparing the different modes of secondary pollen presentation in the Asterales-Campanulalescomplex s. 1., a "basic syndrome" may be formulated. As already emphasized in earlier papers (ERBAR & LEINS 1989, LEINS & ERBAR 1990), all these mechanisms may have developed on the basis of three sequential processes before and/or during an thesis: 1. elongation of the filaments (or stamen-corolla tube or both), 2. opening of the anthers, 3. elongation of the style. Characters like radial symmetry of the flower bud, complete androecial whorl, introrse anthers, the latter connivent at the time of dehiscence as well as the pronounced proterandry are further components of the "basic syndrome". We try to show the differentiations of this "basic syndrome" in two diagrams (Figs. 30-31 ). A first dichotomy of the mechanisms may be achieved by the character "absence or presence of a stable anther tube" in which the anthers are firmly coherent through their cuticles. In diagram A (Fig. 30) the character "stylar cup" is regarded as having developed only once, uniting the Goodeniaceae and Brunoniaceae. This requires to postulate a "loss of anther fusion" in the Goodeniaceae with free anthers. Presence of an anther tube, however, is reported for the genera Anthotium, Dampiera, Diaspasis, and Leschenaultia (KRAUSE 1912, MoRRISON

1992, RAJPUT&CAROLIN 1992). The illustrations (KRAUSE 1912, MORRISON 1992, RAJPUT&CAROLIN 1992), however, seem to show that an anther tube is not a constant feature in these genera or that the anthers may separate during anthesis. We observed that the coherence of the anthers is not firm throughout in Dampiera stricta. Anthers may be joined or free even within a flower (Figs. 32-34). There are two possible interpretations: Within the family Goodeniaceae, the coherence of the anthers either is a primitive character, in the sense of an occasional remnant of strictly coherent anthers in a possible common ancestor with Brunonia. Or it is an advanced character, possibly a preadaption leading to a firm coherence of the anthers in Brunonia. At any rate, the Lechenaultia-Anthotium-Dampiera group seems to be distinct from the rest of the Goodeniaceae, and rather is to be associated with Brunonia, not only because of their anther tubes but also because of a common base chromosome number, as suggested by CAROLIN et al. (1992). If we want to transfer the Goodeniaceae to the other side of the main branching (diagram B, Fig. 31) we must regard the stylar cup as having arisen twice. Then the cup mechanism would have evolved from a simple deposition mechanism, which we can find in Calyceraceae. The position of Goodeniaceae next to Calyceraceae is supported by molecular data (JANSEN et al. 1991, OLMSTEAD et al. 1992) and morphological characters (ERBAR 1993, BREMER 1994). The undivided stigma and FLORA (1995) 190

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pollen aggregated by plenty of pollenkitt are, e. g., common characters of the two families. Staying with the right main branch of the diagrams (A+ B; Figs. 30-31), it has to be mentioned that transitions between pump and brushing mechanism occur within the Asteraceae: Part of pollen is shed into the anther tube, another part onto the style. Correspondingly, part of pollen is pushed out, the other part brushed out. There is a correlation between the presentation mechanism (or the position of the style tip at the time of anther dehiscence) and the arrangement of the hairs: Species with hairs only at the very tip of the style (or upper part of the style

branches) exhibit the pump mechanism, while those with hairs reaching below the branching of the style exhibit the brushing mechanism. Species with an intermediate mechanism (and intermediate style position at anther dehiscence) have a corresponding distribution of the hairs (see JUEL 1908, THIELE 1988; compare Figs. 35-37). In a recent study we presumed that a pump mechanism has evolved several times within the Asteraceae, and that reversals may have occurred as well (LEINS & ERBAR 1990). The tribe Barnadesieae, recently given the rank of subfamily Barnadesioideae (BREMER&JANSEN 1992), is of particular interest since it is regarded as a sister

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deposition

........................ .. . brushing

~·································· ··········~ =-=--==~~cc==-=-=

complete retraction of stylar hairs

retractile stylar hairs long hairy stylar part

stamens shrivel or bend outwards ±before elongation of the style

long hairy part of style on same level with anthers at beginning of anthesis

.

hairs only at the very tip of the style

.

anthers coherent rigid stylar hairs stamens shrivel after elongation of the style radial symmetry of the flower bud complete androecial whorl introrse anthers anthers connivent at time of dehiscence proterandry 1. filament elongation 3. style elongation

2. dehiscence of anthers

Fig. 30. Diagram A. One possibility for phylogenetic interpretation of secondary pollen presentation mechanisms in the Campanulales-Asterales (Rubiaceae are included in the comparison). For discussion see text.

Figs. 22-24. Differently shaped style tips in members of Rubiaceae with se~ondary pollen pre~entation. - 22. I;:?ra coccinea. - 23. Hoffmannia ghiesebreghtia. - 24. Vangueria apiculata. - F1gs. 25-29. Phuopszs stylosa. Depos1t1on mechanism.- 25, Deposition of the pollen onto the stylar head; the still closed flower bud was artificially opened.- 26, The flower has opened, the anthers spread. - 27, The pollen-loaded style becomes lifted up high above the corolla. - 28, Epidermis of the upper stylar part with relatively broad, roundish papillae, the uppermost part (arrow) shows small stigmatic papillae; SEMgraph. - 29, The opening anthers enclose the upper stylar part like a dome. - A= anther, P =petal. FLORA (1995) 190

333

mechanisms

r..~::~.~~J!~~~---~ •

.......................... brushing

c1p

lCALYCERACEAEI IGOODE.NIACEAEI

CAMPANULACEAE

cup

IBRUNONIACEAEI

(P·P·)

stylar cup 'shrivelling" of the anthers delayed

complete retraction of stylar hairs

incomplete retraction of stylar hairs corolla lobes coherent at anthesis

long hairy part of style on same level with anthers at beginning of anthesis

hairs only at the very tip of the style

stylar cup

...

retractile stylar hairs long hairy stylar part

stylar tip at base of anther tube at beginning of anthesis

stamens shrivel or bend outwards ±before elongation of the style stamens shrivel after elongation of the style radial symmetry of the flower bud complete androecial whorl introrse anthers anthers connivent at time of dehiscence proterandry

1. filament elongation 3. style elongation

2. dehiscence of anthers

Fig. 31. Diagram B. Another possibility for phylogenetic interpretation of secondary pollen presentation mechanisms in the Campanulales-Asterales (Rubiaceae are included in the comparison); see text.

34.,

Figs. 32-34. Dampiera stricta.- 32, Five anthers surround the style with its apical cup; SEMgraph.- 33-34, Cross sections through the anthers showing the more or less firm cuticular coherence or neighbouring thecae. -A = anther, F =filament, Sty= style, dPS =dorsal pollen sac, vPS =ventral pollen sac; bar in Figs. 33-34 = 100 lJ.m.

Figs. 35-41. Differently shaped style tips in the Asteraceae; SEMgraphs. - 35, Achillea clypeolata. - 36, Grindelia squarrosa. - 37, Catananche caerulea. - 38, Chuquiraga varians. - 39, Dasyphyllum brasiliense. - 40-41, Barnadesia polyacantha; note the two transverse bulges (arrows) beneath the two short style branches; see text.

group of all other Asteraceae (JANSEN&PALMER 1987, BREMER et al. 1992, BREMER 1994). Styles of Barnadesioideae are shortly bilobed, sometimes more deeply bilobed but not with long style branches, and glabrous or only papillose (BREMER 1994; examples are shown in Figs. 38-39). In Barnadesia polyacantha we observed two transverse bulges beneath the two short style branches (Figs. 40-41). In cladograms based on rbcL gene sequences (OLMSTEAD et al. 1992, CHASE et al. 1993), the Goodeniaceae (together with the Calyceraceae) constitute the sister group of the Asteraceae. The formation of a bulge beneath short style branches in Barnadesia goes well with this view. The bulge can be

interpreted either as remnant of a stylar cup or as preadaption. In the former case, the Barnadesioideae would have had Goodeniaceae-like precursors. The process of secondary pollen presentation in Barnadesioideae has not been described so far, merely a pump mechanism is assumed by BREMER (1994, p. 37). As regards the pump mechanism in Lobeliaceae, which is very similar to that in Asteraceae, we may assume that it has developed independently (perhaps from a brushing mechanism; see LEINs&ERBAR 1990). In the left main branch of the diagrams (A+ B; Figs. 30-31) we have placed mechanisms, in which free anthers shrivel immediately after having opened and discharged their pollen. The deposition FLORA (1995) 190

I

335

Fig. 42. Sphenoclea zeylanica. Flower bud with introrse anthers surrounding the short style; corolla and stamens partly removed; SEMgraph. - Fig. 43. Pentaphragma philippinense. Extrorse anthers surround the style; SEMgraph. -Figs. 44-45. Stylidium adnatum.- 44, Top view of the column with the immature stigma, which is partly concealed by the two anthers; SEMgraph.- 45, Column in set (a) or triggered (b) position.- A= anther, Col= column, P =petal, Sti = stigma, Sty = style, Th = theca.

mechanisms of Rubiaceae and Calyceraceae do not show considerable variation of the "basic syndrome". Therefore the possibilities of pollen portioning are limited. Pollen portioning by successive retraction of pollen-collecting stylar hairs, which faciliates the pollen removal by insects, is a feature peculiar to most Campanulaceae. In Phyteuma, the mechanism is modified to a special brushing mechanism. It can be distinguished from that in Asteraceae by the absence of a firm anther tube. Thus a brushing mechanism has arisen at least twice within the Campanulales-Asterales. The question, which of the mechanisms mentioned 336

FLORA (1995) 190

might be the most primitive, is difficult to answer. In an earlier study (LEINS & ERBAR 1990) we postulated that the combination of deposition and brushing mechanism could be a hypothetical starting point for the Campanulales-Asterales s. str. Considering new results in the Calyceraceae (ERBAR 1993), the starting point of all mechanisms in the Campanulales-Asterales s. 1. may have been a simple deposition mechanism. We have included the Rubiaceae in our diagrams only because of their secondary pollen presentation mechanism which agrees with the "basic syndrome". We do not believe that this family is closely related to the Campanulales-Asterales.

Pollen presentation in further families assigned to Campanulales-Asterales Cyphiaceae

The position of Cyphiaceae within the Campanulales is not questioned, but their precise relationships within the order are not clear. There are similarities with Campanulaceae, Lobeliaceae and Goodeniaceae (for detailed discussion see THULIN 1978, LAMMERS 1992). As regards secondary pollen presentation, the mechanism is not specialized. The free anthers shed their pollen onto the stigmatic cavity at the top of the style. The style with the pollen deposit does not elongate and remains within the throat of the corolla (see Fig. 18 in LADD 1994). Further information is needed to clarify the relationships of the Cyphiaceae. Sphenocleaceae

The monogeneric family is regarded to be closely related to the Campanulaceae (e. g., TAKHTAJAN 1980, 1987, CRONQUIST 1981, THORNE 1992). Sphenoclea, however, has no secondary pollen presentation: The anthers are introrse, but pollen is not deposited onto the short style with its capitate stigma (Sphenoclea zeylanica, Fig. 42). From the viewpoint of floral development, the formation of sympetaly, e. g., supports a position near the Campanulales. Sympetaly starts with an early corolla ring primordium. 1) Pentaphragmataceae

The monogeneric family is likewise usually associated with the Campanulaceae (e.g., TAKHTAJAN 1980, 1987, CRONQUIST 1981, THORNE 1992). Only AmY SHAW (1954) questioned this relationship. LAMMERS (1992) listened a number of characters in which Pentaphragma differs from Campanulaceae. As regards secondary pollen presentation, Pentaphragma philippinense has extrorse anthers and thus an important precondition for pollen deposition onto the style is missing (Fig. 43). Stylidiaceae It is the peculiar column in the centre of the flower that distinguishes the family Stylidiaceae from any other group. The column bears at its top both the bilobed terminal stigma and the two lateral anthers (Fig. 44). The column is irritable in Stylidium and Levenhookia. Upon stimulation, the column flips up from its set position against the modified anterior petal until it comes to rest against the posterior petals (Fig. 45). In the male phase of anthesis, pollen is 1) A study on the early floral development in Sphenoc/ea zey/anica is in preparation.

presented to visiting insects whilst still contained within the open anthers. If one emphasizes the secondary pollen presentation in the Campanulales, then the Stylidiaceae would not fit into the Campanulales s. str. Nevertheless, the Stylidiaceae are obviously closely related to the Campanulales, and it is a matter of judgement whether to treat them as a family of the Campanulales or as a distinct order Stylidiales near the Campanulales (ERBAR 1992).

Final remarks We realize that it is still a long way from our diagrams to the real history of secondary pollen presentation in Campanulales-Asterales. Further systematic investigations, at different levels and with all available methods, as well as further morphological and functional in-depth studies of the processes of secondary pollen presentation are necessary.

Acknowledgements We are grateful to the Directors of M and WU and to Dr. WALTER TILL (Vienna) for the loan of herbarium material. The Directors of the Botanical Gardens of Karlsruhe, Kew, Mainz, Munich and the Palmengarten Frankfurt kindly allowed us to collect flower buds. We wish to express our sincere thanks to Dr. lENS ROHWER for correcting the English text. Expert technical assistance was provided by BIRGIT VoLz (critical point drying, SEM and sectioning), INGEBORG ENDERS (diagrams), and UWE WAGENFELD (processing the photographs).

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