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Anatomy of the unusual stigma in Orchidantha (Lowiaceae)¹

Botanical Institute, University of Copenhagen, Gothersgade 140, DK-1123 Copenhagen K, Denmark

Received for publication June 5, 2003. Accepted for publication October 7, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The stigma of Orchidantha is unlike any other stigma in the Zingiberales. It is zygomorphic and dorsiventral, and its complicated structure has confused botanists resulting in many different descriptions and interpretations. Basally and ventrally on the three-lobed stigma, a specialized "secretion tissue"—here called the viscidium—is found. When a pollinator enters the flower, mucilage from the viscidium becomes smeared over the dorsal side of the body, making it sticky so that pollen may adhere to it. The viscidium probably originates from secretory pollen-receptive epidermal cells, and in O. maxillarioides a gradual change in morphology between these cells and the viscidium is found. However, in O. fimbriata such a transition is lacking. In the "one-way" flower of O. fimbriata, the peripheral parts of the style consist of sclerenchymatous tissue making the style rigid. In O. maxillarioides, however, the pollinator enters and leaves the flower the same way, and to avoid self-pollination, the stigma is pushed upwards when the pollinator enters the flower. In this position, the pollinator cannot touch the receptive parts of the stigma when it leaves the flower. The flexibility of the style that maintains its dislocated position is accomplished by collenchymatous rather than sclerenchymatous tissue in the peripheral parts of the style.

Key Words: Orchidantha • stigma • viscidium • Zingiberales

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The genus Orchidantha was established in 1886 by N. E. Brown (Brown, 1886 ) based on a specimen collected in Borneo. Orchidantha (comprising 16 species) is the only genus in the family Lowiaceae (Zingiberales) and it is characterized by five functional stamens and a median petal, the labellum, that differs from the other petals. Although Lowiaceae is probably the least known of the families in Zingiberales, a remarkable number of morphological and anatomical studies have been made: general vegetative anatomy and morphology (Tomlinson, 1959 , 1969 ), inflorescence and floral morphology (Keng, 1969 ; Kunze, 1985 ; Kirchoff and Kunze, 1995 ; Liao et al., 1998 ; Wen and Liao, 1999 ), seed anatomy (Wen et al., 1997 ), and pollen morphology (Long and Wen, 1997 ). Several of the investigations have focused on floral morphology and anatomy, but the structure of the mature stigma has not been analyzed so far. This lack is strange because the stigma is highly unusual, specialized, and so variable that each species can be identified based on the stigma alone. The best insight into the structure of the stigma comes from the original descriptions of the species, but the authors have described the different parts of the stigma very differently and often incorrectly and insufficiently. Originally, the stigma in Orchidantha borneensis was described by Brown (1886) as being V-shaped (he probably referred to the "secretion tissue"; see Results). Scortechini (1886) did not mention the structure of the stigma in his description of Lowia longiflora (O. longiflora), and the drawing of the stigma is very inaccurate. Ridley (1893) in his description of Protamomum maxillarioides (O. maxillarioides) mentions that the stigma has teeth at the apex but did not mention that the stigma is trilobed. Larsen (1961) in his descriptions of O. siamensis and O. laotica characterizes the stigmas as trilobed with laciniate margins. Holttum (1970, p. 244) describes the receptive part of the stigma in O. fimbriataas "white at base beneath (receptive surface) divided almost to the base into three narrow, dark purple, shining lobes." Like Brown (1886) , the receptive white part that Holttum refers to is almost certainly the "secretion tissue" (see Results). Wu (1964) describes the stigmas of O. chinensis and O. insularis as trilobed with crenulated margins. Larsen (1973) , when describing O. vietnamica, questions whether the basal part of the tripartite laciniate stigma is glandular. In his description of O. holttumii, Larsen (1993) does not mention any glands on the tri-fid, minutely fimbriate stigma although the "secretion tissue" is clearly visible on the drawing. The first complete description of an Orchidantha stigma is found in the description of O. longisepala by Fang and Qin (1996) . They describe the stigma as being tripartite denticulate with a V-shaped protrusion on the basal ventral side. In the description of O. inouei, Nagamasu and Sakai (1999) give a perfect description of the stigma, and they mention that the base of the stigma is swollen with an obcordate "secretion tissue." Recently, Pedersen (2001) described four new species and Jenjitiikul and Larsen (2002) one new species of Orchidantha—all species descriptions include a full description of the stigma.

Because the form and function of the stigma is crucial for proper pollination (Sakai and Inoue, 1999 ; L. B. Pedersen and B. Johansen, personal observations), we here examine the stigma of Orchidantha maxillarioides and O. fimbriata anatomically for the first time in order to understand one of the most peculiar and complicated types of stigmas.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Plants of Orchidantha maxillarioides (Ridley) K. Schum. (four specimens) and O. fimbriata Holttum (two specimens) were grown in greenhouses in the Botanical Garden, University of Copenhagen. Vouchers are kept at C. Flowers were collected in the morning just after they opened, and stigmas and styles were fixed for 12 h at 4°C in freshly made 2.5% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.0. Following fixation, the tissue was rinsed in buffer before dehydration in acidified dimethoxypropane for 15 min. For light microscopy, the tissue was embedded in glycolmethacrylate according to standard methods (O'Brien and McCully 1981 ). Sections 2 µm thick were stained in periodic acid-Schiff's/aniline blue black (PAS/ABB) or toluidine blue before viewing in a Reichert Jung Polyvar microscope adapted with an Orca 4742-95 CCD-camera (Hamamatsu, Japan). Sections of four styles and stigmas from two specimens of O. maxillarioides and three styles and stigmas from two specimens of O. fimbriata were examined. Material for scanning electron microscopy was transferred to 100% acetone and critical point dried, sputtered with gold and viewed in a Philips SEM 515 scanning electron microscope (Eindhoven, The Netherlands). Two styles and stigmas from two specimens of O. maxillarioides were examined in the SEM, and additionally 10 styles and stigmas of O. maxillarioides from all specimens and eight styles and stigmas of O. fimbriata from both specimens were eaxmined in a Wild stereomacroscope.

	RESULTS

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The style in Orchidantha maxillarioides is 6–8 mm long and 0.3–0.4 mm thick (Fig. 1). A longitudinal furrow runs the length of the ventral side (Fig. 1). The stigma is zygomorphic, dorsiventral, and more or less globose, 2 mm long and 2 mm wide, three-lobed, with each lobe ending in two teeth. Each lobe has a slit on the adaxial side (Fig. 1). Where the lobes are joined to the base of the stigma, there is a depression with trichomes covered with a secreted material (Fig. 2). The rest of the stigma has a rather smooth epidermis.

View larger version (83K): [in this window] [in a new window]   Figs. 1–3. Stigmas of Orchidantha maxillarioides and O. fimbriata. 1. Scanning electron micrograph of O. maxillarioides stigma and style seen from the ventral side. A longitudinal furrow is seen in the style (solid arrows). Small arrows with figure numbers indicate the approximate position of anatomical sections illustrated in corresponding figures. The "secretion tissue" (viscidium) in the depression on the ventral side is marked with an asterisk. 2. Detail of stigma showing the "secretion tissue" (viscidium) in the depression on the ventral side (asterisk). 3. Micrograph of O. fimbriata stigma seen from the ventral side. The large bright V-shaped structure at the base of the stigma is the "secretion tissue" (viscidium). Only the upper part of the style is seen. Small arrows with figure numbers indicate the approximate position of sections

In cross section, the three stigmatic lobes of O. maxillarioides possess a secretory receptive epidermis in the slit on the adaxial side (Fig. 4), and the epidermal cells secrete a PAS-positive material into the slit (Figs. 4, 5). The receptive epidermis is also found outside the slit especially ventrally on the two lateral stigmatic lobes (Fig. 5). At the base of the lateral stigmatic lobes, the epidermal cells gradually change from receptive cells to trichome-bearing cells on the abaxial side of the stigmatic lobes, forming the depression at the base of the stigma (Fig. 6). A large amount of PAS-positive material produced by these trichomes fills the depression (Figs. 6, 7). The secretory trichomes are unicellular and up to 100 µm long with a swollen base (Fig. 16). They are apparently alive and active, with intact cytoplasm and vacuole. Further down the stigma, the stylar canal is sealed off from the ventral depression and its secretory trichomes by a thin layer of parenchyma cells that connect the lateral stigmatic lobes (Figs. 7–9). In the style, the cells of the epidermis and the peripheral parts are living, with a large central vacuole, and they possess uniformly thick PAS-positive cell walls (Fig. 17). However, the cell walls gradually become thinner towards the center of the style, so there is a smooth transition from collenchyma to parenchyma cells (Fig. 17).

View larger version (137K): [in this window] [in a new window]   Figs. 4–9. Transverse sections through the Orchidantha maxillarioides stigma. Periodic acid-Schiff's/aniline blue black (PAS-ABB) stained. Same magnification for all figures. 4. Slightly oblique section through the apical parts of the three stigmatic lobes. The dark epidermis on the ventral side is secreting and receptive, and a small amount of PAS-positive material is seen in the slit of the median and left stigmatic lobe (asterisk). 5. Section through the three stigmatic lobes. The dark epidermis on the adaxial side is secreting and receptive, and plenty PAS-positive material is seen in the slit of all three stigmatic lobes (asterisks). Notice that the receptive epidermis is also covering a small area outside the slits. 6. Section through the area where the three stigmatic lobes join. The secretory trichomes (viscidium) of the two lateral stigmatic lobes can be seen in the ventral depression of the stigma. The entire depression is filled with a PAS-positive material (asterisk). The trichomes gradually become shorter towards the edge of the furrow of the lateral stigmatic lobes, and a connection between the trichomes and the receptive epidermis in this furrow is present. 7. Section through the upper part of the stylar canal and the "secretion tissue" (viscidium). The entire depression on the ventral side of the stigma is covered with secretory trichomes, and the secretion fills the depression (asterisk). 8–9. At the base of the stigma, a thin style is gradually formed

View larger version (142K): [in this window] [in a new window]   Figs. 16–19. Details of "secretion tissue" (viscidium) and supporting tissue in the style. Figs. 16–18 periodic acid-Schiff's/aniline blue black (PAS-ABB) stained. 16. Secretory trichomes from O. maxillarioides. 17. Transverse section through the style of O. maxillarioides showing collenchymatous tissue with thick cellulose walls in the peripheral parts of the style. All cells are apparently alive. 18. Secretory trichomes from O. fimbriata. 19. Toluidine-blue-stained transverse section through the style of O. fimbriata showing sclerenchymatous tissue with thick lignified walls in the peripheral parts of the style. All cells are apparently dead

View larger version (164K): [in this window] [in a new window]   Figs. 10–15. Transverse sections through the Orchidantha fimbriata stigma and style. Figs. 10–14 periodic acid-Schiff's/aniline blue black (PAS-ABB) stained. 10. Section through the frimbriae of the median stigmatic lobe. The darkly stained epidermis on the adaxial side of the lateral fimbriae is secreting and receptive (arrows). 11. Section through the area where the two lateral stigmatic lobes unite with the median one. The receptive/transmitting tissue of the three stigmatic lobes is stained very darkly. The "secretion tissue" (viscidium) is visible at the edges of the two lateral stigmatic lobes (V). 12. The two lateral stigmatic lobes are fused completely with the median one, but they are not united with each other. The "secretion tissue" (viscidium) is visible at the edges of the two lateral stigmatic lobes (V). 13. The two lateral lobes are united. There is no connection between the "secretion tissue" (viscidium, V) and the secretory epidermis of the stylar canal. 14. "Secretion tissue" (viscidium, V) fills the entire depression on the ventral side of the style. 15. Sections through the style. Left: Toluidine-blue-stained section showing the large amount of sclerenchymatous tissue in the peripheral parts of the style. Right: PAS-ABB stained section showing the PAS-positive cells lining the stylar canal

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Although the stigmas of O. maxillarioides and O. fimbriata are very different in size, they are apparently rather similar. In O. maxillarioides, the fusion of the three gynoecial primordia is incomplete (Kirchoff and Kunze, 1995 ). However, in the mature style, there is no evidence that the style consisted of three separate primordia because they have become completely fused and only the furrow along the style is present as evidence of the incomplete fusion during early development. At the same young bud stage, the conduplicate stigmatic lobes are already present (Kirchoff and Kunze, 1995 ). Although the stylar canal is filled with a PAS-positive substance in both species of Orchidantha, the stylar canal is open as in Strelitzia (Kronestedt and Walles, 1986 ), and specialized transmitting tissue is not found. However, the style of both Orchidantha species differs from the style of Strelitzia (Kronestedt and Walles, 1986 ) in not having large intercellular spaces, the supporting tissue (collenchyma or sclerenchyma) appears more organized, and the stylar canal is open throughout the length of the style. The styles in O. maxillarioides and O. fimbriata differ in the type of supporting tissue found in the peripheral parts of the style. In O. maxillarioides, collenchyma with uniformly thickened cell walls is present in the outer parts of the style, while in O. fimbriata there are several layers of sclerenchyma. This difference may be related to the way the two plants are pollinated. In O. maxillarioides, the pollinating fruit beetle enters the flower and pushes the stigma upwards after it has delivered pollen to the receptive apical parts of the stigmatic lobes. The stigma is held in this position for up to 10 min, making it impossible for the pollinator to deliver self-pollen to the stigma when the beetle leaves the way it entered (L. B. Pedersen and B. Johansen, personal observations). Thus the style in O. maxillarioides is flexible and maintains the position it is pushed into for a while to avoid self-pollination. Orchidantha fimbriata on the other hand possesses a "one-way" flower. The pollinating fruit beetle leaves the flower between the exposed stamens, and the stigma does not need to be held in a dislocated position in order to avoid self-pollination (L. B. Pedersen and B. Johansen, personal observations). We believe that flexibility is due to the collenchymous tissue in the short style of O. maxillarioides and that the much longer style of O. fimbriata remains stiff due to the presence of sclerenchymous tissue.

It is rather unfortunate that the term "secretion tissue" has been used for the secretory trichomes at the base of the lateral stigmatic lobes (Nagamasu and Sakai, 1999 ; Sakai and Inoue, 1999 ; Pedersen, 2001 ; Jenjittikul and Larsen, 2002 ) as the receptive tissue of the stigmatic lobes, and the tissue lining the stylar canal is secretory tissue as well. Considering the special function of the mucilage-secreting trichomes—smearing mucilage on the dorsal side of the pollinator for pollen adherence (Sakai and Inoue, 1999 ; L. B. Pedersen and B. Johansen, personal observations) we think that a more specific term is appropriate. In Orchidaceae (the family after which Orchidantha was named because of the general appearance of the flower), the apical part of the median stigmatic lobe (rostellum) often forms a structure—the viscidium—that glues the pollinia to the pollinator (e.g., Dressler, 1981 ). Because the same function is performed by the base of the lateral stigmatic lobes in Orchidantha, we propose that the term viscidium should be used for the secretory trichomes in future descriptions of the Orchidantha stigma.

The viscidium develops only from the lateral stigmatic lobes. Kirchoff and Kunze (1995) found no indication of such a tissue in young buds, indicating that this tissue is formed during late bud development. Based on the observations in O. maxillarioides in which a gradual transition between the receptive tissue of the stigmatic lobes and the viscidium is found, we assume that the viscidium originally was receptive and gradually evolved into a highly specialized organ without contact with the receptive tissue as seen in O. fimbriata. The observation that the viscidium probably is able to sustain pollen tube growth in several Orchidantha species indicates that it is receptive in origin (L. B. Pedersen and B. Johansen, unpublished data). In fact, because of the continuum between the viscidium and receptive tissue of the stigma in O. maxillarioides, the viscidium may serve dual functions in this species—produce a sticky substance for pollen adherence to the body of the pollinator and be receptive. However, as there is no continuum between viscidium and the receptive tissue of the stigma in O. fimbriata, the viscidium cannot act as receptive tissue in this species (as in most other Orchidantha species); pollen tubes will not be able to cross the smooth epidermis that lies between the viscidium and the secretory receptive epidermal cells.

The deeply trilobed, zygomorphic, and dorsiventral stigma of Orchidantha is unlike any other stigma found in the Zingiberales, and a specialized structure—the viscidium—has probably evolved from secreting receptive epidermal cells on the lateral stigmatic lobes. Mucilage from the viscidium is smeared over the dorsal surface of the pollinating beetles and enables pollen to stick to the otherwise often smooth surface of these animals. While the "one-way" flower of O. fimbriata possesses plenty of sclerenchymatous tissue in the peripheral parts of the style, the style of O. maxillarioides contains collenchymatous tissue. The collenchymatous tissue enables the style of this species to be pushed upwards when the pollinator enters the flower and to be held in this position where the pollinator will not touch the receptive parts of the stigma when it leaves the flower.

	FOOTNOTES

 
¹ The authors thank Lis Mathorne, Lis Munk Frederiksen, and Kate Jensen for technical assistance. Dr. Lise Bolt Jørgensen is thanked for many valuable comments on the manuscript.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Brown N. E. 1886 Orchidantha borneensis, a new genus of Scitaminae. Gardens Chronicle N. S 26: 519.

Dressler L. R. 1981 The orchids. Natural history and classification. Harvard University Press, Cambridge, Massachusetts, USA

Fang D. D. H. Qin 1996 Five new species of monocotyledonae from Guangxi. Guihaia 16: 3-8

Holttum R. E. 1970 The genus Orchidantha (Lowiaceae). Gardens Bulletin Singapore 25: 239-246

Jenjitikul T. K. Larsen 2002 Orchidantha foetida (Lowiaceae) a new species from Thailand. Nordic Journal of Botany 22: 405-408[ISI]

Keng H. 1969 Notes on the flowers of Orchidantha longiflora. Gardens Bulletin Singapore 24: 347-349

Kirchoff B. K. H. Kunze 1995 Inflorescence and floral development in Orchidantha maxillarioides (Lowiaceae). International Journal of Plant Sciences 156: 159-171[CrossRef]

Kronestedt E. B. Walles 1986 Anatomy of the Strelitzia reginae flower (Strelitziaceae). Nordic Journal of Botany 6: 307-320[ISI]

Kunze H. 1985 Infloreszenz- und Blütenmorphologie von Orchidantha maxillarioides (Ridl.) K. Schum. (Lowiaceae). Beiträge zur Biologie der Pflanzen 61: 221-234

Larsen K. 1961 New species of Veratrum and Orchidantha from Thailand and Laos. Botanisk Tidsskrift 56: 345-350

Larsen K. 1973 New species of Orchidantha (Lowiaceae) from Vietnam. Adansonia 13: 481-482

Larsen K. 1993 A new species of Orchidantha (Lowiaceae) from Borneo. Nordic Journal of Botany 13: 285-288[ISI]

Liao J. Y. Wen Q. Wu 1998 Studies on vascular system anatomy of the flower of Orchidantha chinensis T. L. Wu. Journal of Tropical and Subtropical Botany 6: 275-282

Long H. Y. Wen 1997 Pollen morphology of Lowiaceae from China. Journal of Tropical and Subtropical Botany 5: 6-9

Nagamasu H. S. Sakai 1999 Orchidantha inouei (Lowiaceae), a new species from Borneo. Nordic Journal of Botany 19: 149-152[ISI]

O'Brien T. P. M. E. McCully 1981 The study of plant structure: principles and selected methods. Termarcarphi, Melbourne, Victoria, Australia

Pedersen L. 2001 Four new species of Orchidantha (Lowiaceae) from Sabah. Nordic Journal of Botany 21: 121-128

Ridley H. N. 1893 On the flora of the eastern coast of the Malay Peninsula. Transactions of the Linnean Society of London, series 2 3: 267-408.

Sakai S. T. Inoue 1999 A new pollination system: dung-beetle pollination discovered in Orchidantha inouei (Lowiaceae, Zingiberales) in Sarawak, Malaysia. American Journal of Botany 86: 56-61[Abstract/Free Full Text]

Scortechini B. 1886 Descrizione di nuove Scitaminee trovate nella Peninsola Malese. Nuovo Giornale Botanico Italiano 33: 308-311.

Tomlinson P. B. 1959 An anatomical approach to the classification of the Musaceae. Journal of the Linnean Society, Botany 55: 779-808

Tomlinson P. B. 1969 Commelinales-Zingiberales. In C. R. Metcalfe [ed.], Anatomy of the monocotyledons. III. Clarendon Press, Oxford, UK

Wen Y. J. Liao 1999 Floral morphology and anatomy of Orchidantha chinensis (Lowiaceae). Journal of Tropical and Subtropical Botany 7: 329-336

Wen Y. J. Liao Q. Wu 1997 Anatomy and histochemistry of the seed of Orchidantha chinensis T. L. Wu. Guihaia 17: 235-241

Wu T. L. 1964 Lowiaceae, a family new to the flora of China. Acta Phytotaxonomica Sinica 9: 335-345

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