Abstract

INTRODUCTION

In gymnosperms, seed ferns, and anthers of flowering plants, tiny (<4 µm) granules may occur on the radial and innermost tangential walls of secretory tapetum cells. These granules are sometimes in close contact with the pollen grains, and are called orbicules (Erdtman et al., 1961) or Ubisch bodies (Kosmath, 1927). Orbicules were observed for the first time by Rosanoff (1865). Despite their early discovery, little attention was paid to these structures and in many palynological studies they have been overlooked.

Orbicules develop simultaneously with the growing pollen exine and are composed of sporopollenin, similar to the pollen exine. Orbicules may thus serve as a model for the study of sporopollenin biosynthesis and polymerization in comparison with the exine, since orbicules are acellular structures independent of cytoplasmic control (Clément and Audran, 1993a, b, c). Pro‐orbicules, which are produced by the endoplasmic reticulum of secretory tapetal cells, are the progenitors of orbicules (Echlin and Godwin, 1968). The pro‐orbicules are extruded through the radial and innermost tangential walls of the secretory tapetum cells, at the beginning of the tetrad stage (Christensen et al., 1972; El‐Ghazaly and Jensen, 1986; Clément and Audran, 1993a, b, c; Vijayaraghavan and Chaudry, 1993). Pro‐orbicules are spherical structures, which are composed of a complex organic mixture. They are surrounded by a glycocalyx. The deposition of sporopollenin precursors, produced by the protoplast of the secretory tapetum cells on the glycoprotein filaments of the glycocalyx and on the growing pollen exine, starts at the late tetrad stage. The mature orbicule wall is composed of structural and filling elements (Clément and Audran, 1993a, b, c). Proteins, pectins, polysaccharides and glycolipids, organized in a three‐dimensional polygonal frame (Rowley, 1990) are part of the structural elements. The filling elements consist of unsaturated lipids and polyphenols (Clément and Audran, 1993a, b, c). At the ultrastructural level, Clément and Audran (1993a, b, c) could distinguish four different zones of electron density in mature orbicules of Lilium L. ‘Enchantment’: (1) an electron‐translucent orbicule cavity; (2) a thin and very electron‐dense orbicule cavity–wall interface; (3) a homogeneous electron‐dense orbicule wall; and (4) a thin, very electron‐dense peripheral layer.

Orbicules are considered to be a general feature of species characterized by a secretory tapetum. Huysmans et al. (1998) reported, however, that orbicules are lacking in several taxa with such a tapetum. The predominance of the secretory tapetum among land plants was demonstrated by Furness and Rudall (2001). This observation supports the hypothesis of Pacini et al. (1985) that the secretory type is plesiomorphic in angiosperms. Species with an amoeboid tapetum, which is believed to be apomorphic, characteristically do not produce orbicules (Sporne, 1973; Pacini et al., 1985), although there are exceptions such as Gentiana acaulis L. (Lombardo and Carraro, 1976; Vinckier and Smets, 2002a, c). Tiwari and Gunning (1986) reported the presence of sporopollenin granules on the tapetal remnants in species with amoeboid tapeta, generally smaller in size than the orbicules produced by secretory tapeta. Orbicules have been recorded in some Cretaceous (Mesozoic) (Taylor and Alvin, 1984; Osborn et al., 1991; Serbet and Stockey, 1991; Archangelsky and Taylor, 1993) and Mioceneous (Cenozoic) (Zetter et al., 2002) seed plants which may indicate, along with the proposed plesiomorphic nature of the secretory tapetum (Pacini et al., 1985), that the presence of orbicules is a plesiomorphic feature of seed plants.

During the past 20 years different hypotheses have been suggested to attribute a function to orbicules. However, none of the proposed functions are satisfactorily proven. For a summary of some of the proposed functions see Huysmans et al. (1998).

In angiosperms, the ornamentation of the pollen exine and that of the orbicule wall often show striking analogies (Nilsson and Robyns, 1974; El‐Ghazaly and Jensen, 1986; Hesse, 1986). These parallels are explained by Hesse (1985) as rooted in the homology of tapetum and sporogeneous tissue. Since ornamentation of the pollen exine offers useful characters for systematics, orbicules might also have taxonomic value. In the subfamily Cinchonoideae of Rubiaceae (Huysmans et al., 1997) an orbicule typology has been worked out based on the morphological and ultrastructural orbicule diversity. It was concluded that orbicule characteristics can be useful for systematics at the generic and tribal level. Apart from the study on Cinchonoideae taxa (Huysmans et al., 1997), the taxonomic usefulness of orbicule characters has so far only been studied in four angiosperm taxa: the genera Dioscorea L. (Schols et al., 2001) and Euphorbia L. (El‐Ghazaly, 1989; El‐Ghazaly and Chaudhary, 1993), the Chloanthaceae, now included in Lamiaceae (Raj and El‐Ghazaly, 1987), and the order Gentianales (Vinckier et al., 2000; Vinckier and Smets, 2002a, b, c; Vinckier, 2003).

Recently a phylogenetic, genus‐level classification of the Gentianaceae, based on cladistic analyses of the trnL intron, matK and internal transcribed spacer sequence (ITS) data, was presented by Struwe et al. (2002). In this new classification, the family Gentianaceae consists of approx. 87 genera with approx. 1615–1688 species distributed among six tribes: Saccifolieae, Exaceae, Chironieae, Helieae, Potalieae and Gentianae (Struwe et al., 2002). It is the third largest family in Gentianales, with Rubiaceae and Apocynaceae s.l. being distinctly larger. They have a cosmopolitan distribution from temperate to tropical regions. The representatives of this family are trees, shrubs, vines, or perennial and annual herbs. Flowers are usually bisexual (unisexual in Veratrilla Baill. ex Franch.), hypogynous, 4‐ or 5‐merous, or rarely 3‐ or 6–16‐merous. The calyx is actinomorphic, rarely zygomorphic (Exacum L.). Inflorescences are terminal or axillary, and are usually cymose, but less often racemose, capitate, clustered or spicate; the flowers are seldom solitary. Members of the family do not produce latex and stipules are absent (Struwe et al., 2002). Interesting to the scope of the present paper is the highly variable tapetum; both secretory and amoeboid tapetum types occur, as well as very variable pollen exine characteristics (Nilsson, 1967; Sankara Rao and Chinnappa, 1983). Pollen grains may be released as monads, tetrads or polyads (Nilsson, 2002).

The present study has five major aims: (1) to investigate the presence of orbicules in Gentianaceae; (2) to provide detailed descriptions of the morphology and ultrastructure of orbicules present; (3) to construct an orbicule‐typology; (4) to investigate pollen exine characters by examining the possible parallels between pollen exine and orbicule wall ornamentation; and (5) to discuss the systematic usefulness of the orbicule typology in comparison with the latest systematic insights within the family Gentianaceae.

MATERIALS AND METHODS

This study is based on herbarium material: 53 species of 34 Gentianaceae genera were selected for examination (Table 1; Appendix). This selection covered all different tribes and subtribes recognized in Gentianaceae (Struwe et al., 2002).

For scanning electron microscopy (SEM) of orbicules and pollen, dried flowers or buds were rehydrated in the wetting agent Agepon® (Agfa Gevaert, Leverkusen, Germany) for 1–2 h. The anthers were picked out from the flowers and the tips of the anthers were removed with a razor blade to facilitate rehydration of the locules. After dissection, the anthers remained for another hour in the wetting agent. Following dehydration in a graded acetone series, the material was critical point dried (CPD 030, Balzers) and mounted on stubs with double‐sided adhesive tape before further preparation. The pollen grains were slightly removed from the opened locules with a fine cactus spine to facilitate observation of the locule surface. The removed pollen grains were collected on the same stubs for further observations. The stubs were sputter coated with gold (SPI‐MODULE™ Sputter Coater; SPI Supplies, West Chester, PA, USA). We used a Jeol JSM‐6400 microscope, at 10–20 kV, for morphological observations. Comparative size measurements of orbicules were ascertained from SEM micrographs using Carnoy 2·0 (Schols et al., 2002). From each anther investigated, 100 orbicules were measured, and mean values and standard deviations were calculated (Table ​(Table11).

For observations using transmission electron microscopy (TEM) anthers of selected flowers were fixed with 2 % glutaraldehyde at pH 7·4, buffered with 0·05 m sodium cacodylate. Selected anthers are embedded in LR‐White Resin (Polysciences Inc., Warrington, PA, USA). Prior to embedding in LR‐White Resin the material was dehydrated in a graded ethanol series. Semi‐thin (±1 µm) sections were cut with a Reichert Jung Ultracut E microtome and stained with 0·1 % thionin/0·1 % methylene blue. The semi‐thin sections were observed with a Leica DM LB light microscope. The ultra‐thin (±70 nm) sections, on copper grids, were stained with uranyl acetate and lead citrate in a LKB 2168 Ultrostainer and were observed in a Zeiss EM 900 transmission electron microscope at 50–80 kV.

RESULTS

Shape

There are three distinct shapes: (1) more or less rounded oblate (Figs 1C and G, ​G,2C,2C, E and F and ​and3B,3B, F and H); (2) irregular‐shaped granular orbicules (Fig. 5B–F); and (3) irregular angular and ‘folded’ orbicules (Fig. 4B, C, E and H; Table 1). Apart from these clearly definable shapes, the orbicular elements were generally amorphous.

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Fig. 1. SEM of species with Type I orbicules (A and B) and SEM and TEM of Type IIIa (C–H) orbicules. Abbreviations used in figures: E, endothecium; ED, electron dense; ET, electron translucent; PE, pollen exine; SEM, scanning electron microscopy; TEM, transmission electron microscopy. A and B, Hockinia montana. A, SEM observation of the spiny orbicules (arrow), covering the locule wall surface. B, Detail of the exine ornamentation. Microspines (arrow) are present upon the tectum. C–E, Gentianopsis procera. C, SEM observation of the small spherical orbicules (arrow). D, TEM observation of the orbicules. An electron‐transparent orbicule cavity is absent. The orbicule wall is delimited by a very electron‐dense and granular peripheral layer (arrow). Bar = 0·6 µm. E, Detail of the reticulate exine. The lumina are beset with granules (white arrow). The muri are sharply crested (black arrow). F, Gentianella bellidifolia. SEM observation of the striato‐reticulate exine. The muri are oriented parallel and criss‐crossed with smaller muri. The muri are rounded (arrow). G and H, Ixanthus viscosus. G, SEM observation of single or compound orbicules. Sporopollinous threads occur between the orbicules (arrow). H, General view of the locule wall surface, imprints of pollen grains are striking. Orbicules (black arrow) are present on the edges between the imprints.

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Fig. 2. SEM and TEM of species with Type IIIa orbicules. A–D, Chironia purpurascens. A, General observation of six pollen grains lying on the locule wall surface, which consists of wavy ridges (arrow). B, Detail of the striate exine ornamentation in the polar region. The muri possess granules (arrow) on their side walls. C, SEM observation of the small spherical orbicules (arrow) covering the locule wall surface. D, TEM observation of the small orbicules (arrow). An electron‐transparent orbicule cavity is lacking. Bar = 0·6 µm. E, Eustoma grandiflorum. SEM observation of the small spherical orbicules (arrow). F–H, Blackstonia perfoliata. F, SEM observation of small orbicules (arrow) present on the locule wall surface. G, Equatorial view of a striate pollen grain lying on the locule wall surface. The muri are oriented in various directions. H, TEM observation of the orbicules, lacking an electron‐transparent orbicule cavity. Bar = 0·6 µm.

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Fig. 3. SEM and TEM of species with Type IIIa orbicules. A and B, Tachia guianensis. A, Equatorial view of a granulate pollen grain lying on the locule wall surface. The granulate exine consist of numerous granules (arrow) on the smooth sexine. B, Detail of the spherical orbicules (arrow), resembling the granules present on the exine. C and D, Prepusa montana. C, SEM observation of a granulate tetrad lying on the locule wall surface. The granulate exine consist of numerous macroprocesses (black arrow) intermingled with microprocesses (white arrow). D, Detailed observation of the spherical orbicules (arrow), resembling the macroprocesses present on the exine. E–G, Frasera parryi. E, Equatorial view of a striate pollen grain lying on the locule wall surface. F, Observation of the small orbicules (arrow) present on the locule wall surface at a very low distribution density. G, TEM observation of a part of a pollen grain, close to the tapetal remnants on which orbicules (arrow) are present. Bar = 0·6 µm. H, Swertia bimaculata. SEM observation of the small orbicules (arrow), distributed upon the locule wall surface at a very low distribution density.

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Fig. 4. SEM and TEM of species with irregular and folded Type IV orbicules. A and B, Sebaea ovata. A, Equatorial view of a striato‐micro‐reticulate pollen grain. B, Detail of the irregular and folded orbicules (arrow), resembling parts of the muri building up the exine. C and D, Sebaea albidiflora. C, SEM observation of the irregular and folded orbicules (arrow). D, TEM observation of the irregular orbicules positioned on the remnants of the tapetum cells. The orbicules posses an elongated electron‐transparent cavity and the homogeneous ED orbicule wall is delimited by a electron‐dense peripheral layer (arrow). Bar = 0·6 µm. E and F, Triptospermum fasciculatum. E, SEM observation of the irregular and folded orbicules (arrow) covering the locule wall surface. F, TEM observation of the irregular orbicules, lacking an electron‐transparent cavity. The homogeneous ED orbicule wall is delimited by an ED peripheral layer (arrow). Bar = 1 µm. G and H, Calolisianthus pedunculatus. G, SEM observation of a tetrad lying on the locule wall surface. The muri of the reticulate pattern are organized in heterobrochate reticulate islands. The lumina are beset with granules (arrow). H, SEM observation of the orbicules on the locule wall surface. The orbicules consist of columellae‐like particles (white arrow) which support the muri‐like part (black arrow) of the orbicules.

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Fig. 5. SEM of species with irregular Type V (A–F) and strongly embedded Type VI (G–H) orbicules. A, Anthocleista scandens. Polar view of a tri‐porate perforate pollen grain. Orbicules (arrow) are attached to the exine. B, Anthocleista amplexicaulis. Detail of the irregular orbicules (arrow) covering the locule wall surface. C, Fagraea racemosa. SEM observation of the irregular and compound orbicules (arrow). D, Exacum oldenlandioides. SEM observation of the irregular orbicules covering the locule wall surface. On the orbicule wall small sporopollinous granulae are present (arrow). E and F, Exacum macranthum. E, SEM observation of a rugulate pollen grain lying on the locule wall surface which is covered by many orbicules (arrow). F, Detailed SEM observation of the orbicules (black arrow) covering the locule wall surface. Intermingled with the orbicules small granules of sporopollenin (white arrow) may cover the locule wall surface. G, Centaurium spicatum. SEM observation of the distinctly embedded orbicules (arrow). H, Hoppea fastigiata. SEM observation of the distinctly embedded orbicules (arrow).

Orbicule types

The typology (Table ​(Table1)1) follows the treatment of orbicule types described for the rubiaceous subfamilies, Cinchonoideae (Huysmans et al., 1997) and Ixoroideae (Vinckier et al., 2000). In the Cinchonoideae, Huysmans et al. (1997) defined four orbicule types: spiny orbicules (Type I), microrugulate orbicules (Type II), smooth and rounded oblate orbicules (Type III), and irregular and folded orbicules (Type IV). Two additional orbicule types were defined by Vinckier et al. (2000) in the Ixoroideae: large flattened and irregular orbicules (Type V), and distinctly embedded orbicules (Type VI). In Ixoroideae (Vinckier et al., 2000) Type III orbicules were split up into two subtypes: rounded oblate orbicules (Subtype IIIa), and doughnut‐shaped orbicules (Subtype IIIb) which are characterized by a central indentation in the orbicule wall. For a more extensive description of the different orbicule types, refer to Huysmans et al. (1997), Vinckier et al. (2000), and Vinckier and Smets (2002a).

Orbicules in Gentianaceae fit well into the types described previously in Rubiaceae, and hence these are used in the present paper (Table ​(Table1).1). Orbicule aggregation does not affect their placement into types, due to the fact that single orbicules or aggregations may occur within one species (Fig. ​(Fig.1G).1G). Of the six orbicule types, Type II orbicules are lacking in the study group. In the majority of species (17 species) Type III orbicules are recorded (Figs ​(Figs1C–H,1C–H, 2 and ​and3;3; Table ​Table1).1). Hockinia Gardner is the only representative with Type I orbicules (Figs ​(Figs1A1A and B). The number of representatives with orbicules belonging to the other orbicule types are equally distributed among the species studied: seven species possess Type IV orbicules (Fig. ​(Fig.4A–H),4A–H), six species Type V (Fig. ​(Fig.5A–F)5A–F) and seven species Type VI (Figs5G and H and ​and6A6A and B; Table1).

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Fig. 6. SEM of species with strongly embedded Type VI (A and B) orbicules, and species lacking orbicules (C–H). A and B, Lisianthius nigrescens. A, General observation of the strongly embedded orbicules (arrow). B, Equatorial view of a reticulate pollen grain. C, Lomatogonium carinthiacum. General view of five pollen grains lying on the locule wall surface, which lacks orbicules. Deep grooves in the locule surface reveal the position of the underlying endothecial cells, the U‐shaped endothecial thickenings (arrow) can be distinguished. D, Schultesia guianensis. SEM observation of two reticulate tetrads lying on the smooth locule wall surface. E and F, Irlbachia purpurascens. E, General view of a micro‐reticulate polyad. F, Detailed observation of the muri of the reticulate pattern, at the distal regions, which are loop‐like. The loops of reticulum (arrow) are raised from the general surface of the pollen grain. G and H, Curtia tenuifolia. G, SEM observation of the locule surface consisting of wavy ridges (arrow). H, Equatorial view of a perforate pollen grain lying on the locule wall surface.

Pollen characters

The pollen grains are released as monads (Figs ​(Figs2A2A and G, ​G,3A3A and E, ​E,4A,4A, ​A,5A5A and E, ​E,6B,6B, C and H and ​and7D),7D), tetrads (Figs ​(Figs3C,3C, ​C,4G,4G, ​G,6D6D and ​and7A)7A) or polyads (Fig. ​(Fig.6E6E and F). Pollen grains are mostly 3‐colporate (Figs ​(Figs2A2A and G, ​G,3A3A and E, ​E,4A,4A, ​A,5E5E and ​and6B,6B, C and H) to 3‐porate (Fig ​(Fig5A),5A), but 2‐porate pollen (Fig. ​(Fig.7D)7D) also occurs in the Gentianaceae. The colpi or pores are bordered with a distinct margo or annulus. The characteristics of the pollen sexine are summarized in Table ​Table1,1, according to the terminology of Punt et al. (1994). In the palynologically diverse Gentianaceae a considerable variation in pollen ornamentation types is observed among the species studied. Most species possess a striate (Fig. ​(Fig.3E),3E), striato‐reticulate (Figs ​(Figs1F,1F, ​F,2B2B and G and ​and4A)4A) to reticulate exine pattern (Figs ​(Figs1E,1E, ​E,4G,4G, ​G,6B6B and D and ​and7A).7A). Apart from these ornamentation types microreticulate (Fig. ​(Fig.6F),6F), microspinulose (Fig. ​(Fig.1B),1B), perforate (Figs ​(Figs5A5A and ​and6H),6H), psilate (Fig. ​(Fig.7D),7D), rugulate (Fig. ​(Fig.5E)5E) and granulate (Figs ​(Figs3A3A and C) exine types are present. Of the Gentianaceae species studied by Sankara Rao and Chinnappa (1983), Hoppea fastigata (Griseb.) Clarke and Frasera albicaulis Douglas ex Griseb were also investigated in the present study. For both species the exine data substantiate the results of Sankara Rao and Chinnappa (1983). The granulate sexines may consist of numerous macroprocesses intermingled with microprocesses (Fig. ​(Fig.3C),3C), or only of granules present on a psilate exine (Fig. ​(Fig.3A).3A). The reticulate patterns can be heterobrochate (Figs ​(Figs4G,4G, ​G,6F6F and ​and7A)7A) or, as in most cases, homobrochate. The columellae of the reticulate patterns may be of variable length and the sexine organized in heterobrochate reticulate islands (Fig. ​(Fig.4G).4G). In Irlbachia purpurascens (Aubl.) Maas (Fig. ​(Fig.6E6E and F) polar loops of reticulum that is raised from the general surface of the pollen grain are present. Lumina can be beset with granules (Figs ​(Figs1E1E and ​and4G).4G). Muri are either sharply crested (Fig. ​(Fig.1E)1E) or rounded (Fig. ​(Fig.1F).1F). The muri are oriented parallel (Fig. ​(Fig.3E)3E) or in various directions (Fig. ​(Fig.2G),2G), or criss‐crossed and interlaced (Figs ​(Figs1F,1F, ​F,2B2B and ​and4A),4A), in a few cases with granules present on the sidewalls of the muri (Fig. ​(Fig.22B).

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Fig. 7. SEM of species lacking orbicules. A and B, Coutoubea spicata. A, General observation of a heterobrochate reticulate tetrad. B, General observation of the locule wall surface lacking orbicules. Smooth grooves (arrow) in the locule surface reveal the position of the underlying endothecial cells. C, Halenia corniculata. The locule surface possesses ridges (arrow) forming more or less a reticulum. D, Voyria caerulea. General view of a psilate 2‐porate (arrows) pollen grain.

DISCUSSION

The 53 species studied out of approx. 1650 in Gentianaceae represent only 3·2 % of the species in this family. Despite the limited sample size, a good representation of the family is presented due to the fact that the selected species cover all the different tribes and subtribes recognized recently in the family (Struwe et al., 2002).

CONCLUSIONS

Orbicule typology has proved to be useful for evaluating tribal delimitation within Rubiaceae (Huysmans et al., 1997; Vinckier et al., 2000) and Loganiaceae s.l. (Vinckier and Smets, 2002a); however, they seem not to be useful for tribal delimitation in Gentianaceae. In the tribes Potalieae and Gentianeae, orbicule typology may be useful at the subtribal level.

In the crown clade comprising Helieae, Potalieae and Gentianeae, no particular sister‐group relationships is well supported in the three‐gene analysis of Struwe et al. (2002). Further data are thus required to resolve these relationships. Despite the lack of congruence between orbicule and molecular data, individual orbicule characters along with pollen and other morphological characters can be used in a future combined analysis. Orbicule types should not be used in a cladistic analysis because they are aggregations of characters.

ACKNOWLEDGEMENTS

We thank the following people for excellent technical assistance: Dr Eric Schoeters (TEM, Leuven), Koen Collart (TEM, Leuven), and Ir. Marcel Verhaegen (SEM, National Botanic Garden of Belgium). Thanks are due to the curator of The National Botanic Garden of Belgium (BR) for the generous supply of anther samples. We are grateful for the valuable suggestions on the manuscript by Dr Carol Furness and Dr Favio Gonzàlez. This research was financially supported by projects No. 2·0038.91 (SEM) and G.0104.01 (general research project) from the Fund for Scientific Research‐Flanders (FWO) and a grant from the Research Council of the K.U.Leuven (OT/01/25)

APPENDIX

Alphabetical list of Gentianaceae specimens giving species, country, collector, specimen number and herbarium.

Anthocleista amplexicaulis Baker, Madagascar, A.J.M. Leeuwenberg & G.R. Rafamantanantsoa 14437 (BR)

Anthocleista scandens Hook. f., Madagascar, Bioko Fernando‐Po (s.n.) (BR)

Blackstonia perfoliata (L.) Huds., Algeria, D. Podlech 37064 (BR)

Calolisianthus pedunculatus (Cham. & Schltdl.) Gilg, Brazil, G. Hatschbach 47336 (BR)

Canscora decussata (Roxb.) Schult. & Schult. f., India, H.B. Naithouri 3954 (BR)

Canscora heteroclita (L.) Gilg, Sri Lanka, A.G. Robyns 7206 (BR)

Centaurium serpentinicola Carlström, Turkey, M. Nydegger 44139 (BR)

Centaurium spicatum (L.) Fernald, France, Berger 2310 (BR)

Centaurium tenuiflorum (Hoffmgg. & Link) Fritsch, France, C. Van den Berghen (s.n.) (BR); France, P. Bamps 7463 (BR); Portugal, J. Bouharmont 27871 (BR)

Chironia pegleriae Prain, South Africa, J. Lambinon & M. Reekmans 82/301 (BR)

Chironia purpurascens (E. Meyer) Benth. & Hook. f., South‐Africa, E.S. Kemp 818 (BR)

Coutoubea spicata Aubl., French Guyana, F. Billiet & B. Jadin 1145 (BR)

Curtia tenuifolia (Aubl.) Knobl., Brazil, G. Hatschbach 48278 (BR)

Enicostema verticillatum (L.) Engl. ex Gilg, Sri Lanka, N. Balakrishnan 53619 (BR)

Eustoma grandiflorum (Raf.) Shinners, USA, Geerinck‐Coutrez 4373 (BR)

Exacum gracilipes Balf. f., Oman, S.A. Ghazanfar 2337 (BR)

Exacum macranthum Arn., Sri Lanka, P.L. Comanor 419 (BR)

Exacum oldenlandioides (S. Moore) Klack, Guinea‐Bissau, Centro de botanico Lisboa 3761 (BR)

Fagraea racemosa Jack, Borneo, Veldkamp 8516 (BR)

Faroa affinis De Wild., Democratic Republic of Congo, Dikumbwa, Kisimba & Muzinga 3591 (BR)

Frasera albicaulis Douglas ex Griseb., USA, E.B. Babcock 1790 (BR)

Frasera parryi Torr., USA, V. Durand 3488 (BR)

Gentiana acaulis L., Switzerland, C. Vanden Berghen 30 (BR); France, G.F. De Witte 16457 (BR)

Gentiana algida Pall., Russia, Kharkevich (s.n.) (BR)

Gentiana alpina Adam, Spain, J. Auoyo, I. Fernandez & J. Pastor 11518 (BR)

Gentianella amarella (L.) Borner, Poland, S. Lisowski 57556 (BR)

Gentianella bellidifolia (Hook.f.) Holub, New Zealand, M.T. Mangaweka, P.D. Buckley & K.W. Julley 15/2/77 (BR)

Gentianopsis barbata (Froel.) Ma, Russia, Kharkevich & Buch (s.n.) (BR)

Gentianopsis procera (Th. Holm) Ma, Canada, F. Marie‐Victorin & F. Rolland‐Germain 49464 (BR)

Halenia corniculata (L.) Cornaz, Russia, Primorskiy, Kharkevich & Buch (s.n.) (BR)

Hockinia montana Gardner, Brazil, unknown 17150 (BR)

Hoppea fastigiata (Griseb.) Clarke, Sri Lanka, A.G. Robyns 7206bis (BR)

Irlbachia purpurascens (Aubl.) Maas, French Guyana, F. Billiet & B. Jadin 1550 (BR)

Ixanthus viscosus Griseb., Canary Islands, J. Bouharmont 25535 (BR)

Lisianthius areopolus B.L. Rob., Mexico, R.L. Wilbur 35600 (BR)

Lisianthius nigrescens Schltdl. & Cham., Mexico, Arnold arboretum, Universidad Nacional Autonoma de Mexico 446 (BR)

Lomatogonium carinthiacum (Wulfen) Rchb., Kazach stan, V. Zuev 6649 (BR)

Lomatogonium rotatum (L.) Fries ex Fern., Russia, S. Kharkevich & T. Buch (s.n.) (BR)

Macrocarpaea rubra Malme, Brazil, R. Kummrow 2385 (BR)

Obolaria virginica L., USA, H.E. Ahles & J. Haesloop 53200 (BR)

Potalia amara Aubl., French Guyana, collector of the Orstom centre 1188 (BR); French Guyana, F. Billiet & B. Jadin 1821 (BR); F. Billiet & B. Jadin 1900 (BR)

Prepusa montana Mart., Brazil, G. Hatschbach 47930 (BR)

Sabatia bartramii Wilbur, USA, S. Mc Daniel 6455 (BR)

Sabatia brachiata Elliott, USA, A.E. Radford 45410 (BR)

Sabatia quadrangula Wilbur, USA, J.F. Matthews, L. Kesler, P. Campbell & W. Weston (s.n.) (BR)

Schultesia guianensis (Aubl.) Malme, French Guyana, F. Billiet & B. Jadin 1157 (BR)

Schultesia pachyphylla Grisrb., Brazil, G. Hatschbach 47936 (BR)

Sebaea albidiflora F Muell., Australia, A. Mubold 7276 (BR)

Sebaea ovata (Labill.) R. Br., D.J.E. Whibley 8862 (BR)

Swertia bimaculata (Siebold & Zucc.) Hook. f. & Thomson ex C.B. Clarke, China, Sino‐American Guizhou Botanical Expedition 402 (BR)

Swertia perennis L., France, J. Bouharmont 7718 (BR)

Tachia guianensis Aubl., Brazil, P. Bamps 5416 (BR); Peru, F. Woytkowski 5348 (BR); Peru, S. Mc Daniel & M. Rimachi 16483 (BR)

Tripterospermum fasciculatum (Wall.) A.O. Chater, India, G. Panigrahi 19807 (BR)

Voyria caerulea Aubl., French Guyana, F. Billiet & B. Jadin 4381 (BR)

Supplementary Material

Notes

Received: 14 April 2003;; Returned for revision: 14 May 2003. Accepted: 28 July 2003; Published electronically: 19 September 2003

References