Grana39: 90± 102, 2000 Pollenmorphological variation in ( ± )

FREDERIC LENS,STEVEN JANSEN,SUZY HUYSMANS, ELMAR ROBBRECHT andERIK SMETS

Lens, F., Jansen, S.,Huysmans,S .,Robbrecht,E . & Smets, E. 2000. Pollenmorphological variation in Vanguerieae ( Ixoroideae± Rubiaceae ) . ±Grana39: 90± 102 . ISSN0017-3134 . TheVanguerieae is alargetribe of the subfamily Ixoroideae ( Rubiaceae) andconsists ofabout 500 speciesin 27 genera . This studygives a detailedpollen morphological description of 30species from 16 genera,based on light microscopy and scanning electron microscopy . TheVanguerieae are considered tobe stenopalynous, although there is someevidence to question this . First, thereis adifferencein sexinetypes that clearly de® nes Keetia and ( bothreticulate ) , and and Vanguerieae ( bothperforate with verysmall perforations ) fromthe other Vanguerieae investigated . Second,pollen grainscorroborate the subgeneric delimitations of Canthium sensuBridson . Pollenmorphologically the tribeis characterisedby ( 1) theshape of theectoapertures which is intermediatebetween a colpusand a porus, and ( 2) thepresence of a costaoriented perpendicular to the ectoaperture and has a formwhich is correlatedwith theshape of the costa . FredericLens, Steven Jansen, Suzy Huysmans & Erik Smets,Laboratory of PlantSystematics, Institute ofBotany and Microbiology, K .U.Leuven,Kardinaal Mercierlaan 92, B-3001 Leuven; Elmar Robbrecht, NationalBotanic Garden of Belgium, Domein van Bouchout, B-1860 Meise; Belgium . E-mail: [email protected]. ( Manuscriptaccepted 29 September 2000 )

Withnearly 500 species arranged in 27 genera, the blanceswould most probably reduce the number of genera . Vanguerieaeis one of the larger tribes of the subfamily Afterthe revision of Canthium by Bullock ( 1932) , Bridson Ixoroideae ( Rubiaceae) . Itis widespread in tropical Africa, ( 1985,1986, 1987 a, b, 1992) refutedthe opinion of Asia,Australia and the isles of the Paci® c Ocean,but the Verdcourt. Sheaccepted and broadened, on the basis of largestnumber of species occurs in Africa and Madagascar morphologicalfeatures, several genera of Canthium s.l., ( Bridson1987 a) . Thehabit is generally shrubby, but also e.g. Psydrax, Pyrostria, Keetia and Multidentia . Further- fewlarge trees and lianas occur . Thetribe combines many more,she recognised four subgenera for the remaining distinctivefeatures, e .g. axillarypaired in¯ orescences, species ( ~ Canthium s.s.) , namely Canthium, Afrocanthium , valvateaestivation, secondary pollen presentation, one Lycioserissa and Bullockia. Thesetaxonomic changes are pendulousovule per locule, pyrenes with an apical alsosupported by otherauthors, such as Tilneyet al . ( 1988) germinationslit, and seeds with soft oily endosperm and whostudied anatomical features of young stems of South verylarge embryos ( Robbrecht1988 ) . Africanspecies of Canthium s.l. Accordingto the classi® cation of Schumann (1891) , the Themacromorphological homogeneity of the tribe becomes Vanguerieaewere placed in the supertribe ` Guettardinae’ alsoevident in the pollen morphology . Robyns ( 1928:6) togetherwith the Alberteae, Guettardeae, Retiniphylleae and mentionedthat pollen grains in the Vanguerieae are `tre Ás Knoxieae. Thisphylogenetic relationship was adopted by semblablesdans tout le groupe’ . Verdcourt ( 1958) came to the Robbrecht ( 1988, 1994) whounited these tribes in his sameconclusion and described the grains as 3-porate with an subfamilyAntirheoideae . Moleculardata have demonstrated unsculpturedsexine . However,Robbrecht ( 1980) found morerecently that the subfamily Antirheoideae is polyphyletic severalsexine types within the tribe . Moreover,Tilney & andthat the Vanguerieae and the closely related Alberteae van Wyk ( 1997) foundpollen morphological support for the belongto the Ixoroideae ( Bremer et al. 1995,Andreasen & division of Canthium s.s. intothree subgenera . Otherstudies Bremer1996, Bremer 1996, Bremer & Thulin1998, alsoreveal pollen morphological variation within the Andersson& Rova1999, Andreasen et al . 1999,Rova 1999 ) . Vanguerieae,although the tribe is still considered to be Althoughthe Vanguerieae are well characterisedmor- stenopalynous ( Verdcourt1987, Igersheim 1989 ) . phologically,the generic delimitation remains problematic . Theinside ornamentation of pollengrains in Rubiaceae is Robyns ( 1928) wasthe ® rsttaxonomist who dealt with this knownto have systematic value, as ® rstmentioned by problem. Herevised the tribe, without treating Canthium, Frenchresearchers ( e.g. Malplanche1971, Lobreau-Callen whichis the largest that most likely plays a keyrole in 1978,Van Campo 1978, Keddam-Malplanche 1980 ) . Since thetribe’ sphylogeny ( Bridson1992 ) . Thehigh degree of Huysmanset al . ( 1994) describeda newmethod for breaking homogeneitywithin the Vanguerieae is discussed by pollengrains with glass beads, observations of the inside of Verdcourt ( 1958,1987:123 ) whocommented on `the thegrains became more accessible ( e.g. Jansen et al. 1996, poornessof characters used for generic delimitation’ . DeBlock & Robbrecht1998, Bosser & Lobreau-Callen Accordingto Verdcourt, the strong morphological resem- 1998,Huysmans 1998 ) . Newfeatures, such as different

Grana 39 ( 2000) # 2000Taylor & Francis . ISSN0017-3134 Pollenmorphology of Vanguerieae 91 ornamentationtypes of the inner nexine and the large MATERIAL ANDMETHODS diversityof endoapertures, have shown their taxonomic importance. Thisis demonstrated for example in the Pollenof 30speciesout of 16 genera were removed from herbarium Coptosapelteae ( Huysmanset al . 1993, 1994) and in the specimensat the National Botanic Garden of Belgium ( BR) and Isertieae s.l. ofwhich characters from the inside of the wereacetolysed following the method of Reitsma ( 1969) . The pollenwere included for the ® rsttime in a cladisticanalysis specimensinvestigated are listed separately at the end of this paper . ( Huysmanset al . 1998) . InVanguerieae, however, inside LMpreparationswere made in glycerine jelly . For SEMobserva- observationsof pollen grains were only made for the small tions, pollengrains were mounted on an aluminium stub, coated with goldand observed with aJEOLJSM-5800 LV or JSM-6400 . genus Tapiphyllum ( Havard& Verdcourt1987 ) and few Pollengrains were broken using glass beads ( Huysmanset al . 1994) . . otherrepresentatives of the tribe ( Igersheim1989 ) Thepolar axis and equatorial diameter were measured on ten Thepresent study gives a detailedpollen morphological grainsin equatorial view with LMusinga cameralucida . Other descriptionof selected species of the Vanguerieae, including measurementswere made on SEM-micrographs . observationsof the inside ornamentation of the pollen Terminologyfollows theGlossary of Pollenand Spore Terminol- grains. ogy ( Punt et al. 1994) . Theterminology of pollen shape in polar

Figs. 1 ± 6. Pollenin equatorial view . SEM. ( 1) Cuvieralongi¯ ora ,perforatespheroidal grain with congruentapertures; ( 2) Canthium parasiebenlistii ,suboblateshape, microverrucae ( detailin Fig . 28) inbands around psilate apoporium; ( 3) Pyrostriabibracteata , subo- blateperforate grain; ( 4) Psydraxhorizontalis ,suboblatereticulate grain; ( 5) Keetiahispida , idem; ( 6) Psydraxsubcordata var. subcor- data,spheroidalreticulate pollen .

Grana 39 ( 2000) 92 F. Lens et al.

Figs. 7 ± 14. Pollenin polar view . SEM. ( 7) Canthiumseti¯ orum ,ambview circular, perforated sexine; ( 8) Pyrostriabibracteata , amb viewcircular, perforate tectum; ( 9) Canthiumparasiebenlistii ;polarview circular, granules ( detailin Fig . 28) inbands around psilate apoporium; ( 10) Pygmaeothamnuszeyheri ,circularamb view, perforate sexine with psilateapoporium; ( 11) Lagyniaspallidi¯ ora , amb viewnearly circular, perforate sexine; ( 12) Fadogiaschmitzii ,polarview triangular with convexsides, perforatetectum; ( 13) Tapiphyllum cinerascens var. laetum, idem; ( 14) Psydraxhorizontalis ,ambview triangular with convexsides, reticulatesexine .

Grana 39 ( 2000) Pollenmorphology of Vanguerieae 93

Grana 39 ( 2000) 94 F. Lens et al. viewfollows Reitsma ( 1970) . Termsfor shapeclasses in equatorial instance Canthiumlactescens ( Fig. 25) and Psydraxsub- vieware adopted from Erdtman ( 1971) . cordata var. subcordata ( Fig. 26) . Ectocolpiwithin the Themeasurements are always mean values . Asummaryof the Vanguerieaeare scarcely present and always short with pollenmorphological features per species is listedin Table I . obtuse ends ( e.g. Vangueriainfausta ) oracute ends, such as in Fadogiastigmatoloba ( Fig. 15) . Themargin of the RESULTS ectoapertureis often obviously demarcated, except in Canthiumseti¯ orum ( Fig. 21) and Keetiagueinzii ( Fig. 22) ; Size sometimesthe ectoaperture has irregular margins because of Pollengrains are generally medium sized ( P: 44.9 mm and E: sexinebulges, especially in Canthiumlactescens ( Fig. 25) and 48.9 mm) . Thelargest pollen grains are observed in Cuviera Canthiummundianum . Thelargest ectoapertures are nigrescens ( P: 65.1 mm, E: 67.1 mm) ,thesmallest grains in observed in Cuvieranigrescens , Pachystigmapygmaeum , Canthiummundianum and C. parasiebenlistii ( P: ca 29 mm, E: Lagyniaspallidi¯ ora and Psydraxhorizontalis ( length ca ca 32.5 mm) . 16.5 mm;width ca 10 .5 mm) ;thesmallest ones in Canthium parasiebenlistii and Canthiummundianum ( length ca 5 mm; width ca 4 mm) . Ectoaperturemembranes are usually Shape coveredwith few microverrucae ( e.g. Fig. 15) . However, in species of Rytigynia and Vangueriopsis thesestructures are Pollengrains are oblate spheroidal in 57 % ofthe specimens absent. Theaperture membrane is sometimes perforated investigated,such as in Cuvieralongi¯ ora ( Fig. 1) . Suboblate ( Fig. 19) orcovered with sexine elements in Canthiuminerme pollengrains are found in Canthium subg. Afrocanthium ( Fig. 20) . ( Fig. 2) and subg. Bullockia, Pyrostria ( Fig. 3) , Keetia ( Fig. 4) , and Psydraxhorizontalis ( Fig. 5) ,whileother specieshave spheroidal pollen, such as Cuvieralongi¯ ora Endoaperture ( Fig. 1) , Fadogiaschmitzii and Psydraxsubcordata var. subcordata ( Fig. 6) . Thespecimen of Vangueriopsis sp. Theendoaperture, which is often incongruent with the investigatedis the only one with prolate spheroidal pollen . ectoaperture,is circular in Keetia ( Fig. 22) andsometimes in Thepolar view of the grains is circular, e .g. in Canthium Canthiumlactescens ( Fig. 25) , Psydrax ( Fig. 26) and in seti¯ orum ( Fig. 7) , Pyrostriabibracteata ( Fig. 8) , Canthium Pyrostria;usuallythe endoaperture is elongated, e .g. Cuviera parasiebenlistii ( Fig. 9) , Pygmaeothamnuszeyheri ( Fig. 10) , ( Fig. 1) ,orrectangular in Fadogiellastigmatoloba ( Fig. 15) ortriangular with convex sides, e .g. in Lagyniaspallidi¯ ora and Tapiphyllumcinerascens var. laetum ( Fig. 17) . ( Fig. 11) , Fadogiaschmitzii ( Fig. 12) , Fadogiellastigmato- loba ( Fig. 13) , and in Psydraxhorizontalis ( Fig. 14) . Sexine

Apertures Pollenin the Vanguerieae are mostly tectate, although semitectatein Keetia ( Fig. 33) and Psydrax ( Figs. 6, 26 & Pollen are ( 2) -3-( 4) -zonoaperturate . Theapertures are 34) . Thetectum is always even and smooth ( except in generallycompound consisting of two units . Congruent Canthiumparasiebenlistii , Figs. 2, 9 & 28) , but a bumpy aperturesare sometimes observed, e .g. in Cuvieralongi¯ ora tectumis observed in Keetiacornelia and K. hispida ( Fig. 1) ,andsometimes in Psydrax ( Fig. 24) and in ( Fig. 33) . Perforatetaxa are common within the Vanguer- Pyrostria. ieae,such as in Canthium ( Fig. 27) , Vangueria, Fadogia, Meyna, Cuviera, and Pygmaethamnus . Someof these genera havebaculae in the perforations ( Figs. 29 ± 31) . Perforations Ectoaperture areoften unequally distributed, variable in size, and have a Elongatedectopori are frequently present within the moreor less circular shape . Verysmall perforations are Vanguerieae . Theends of the ectopori are usually obtuse, foundin the genera Canthium and Vangueria. Some of the e.g. in Ancylanthosrubiginosus ( Fig. 16) and Tapiphyllum perforatedtaxa have a psilatetectum on theirpoles, such as cinerascens var. laetum ( Fig. 17) ,oracute such as in in Canthiumparasiebenlistii ( Fig. 28) , Pygmaeothamnus specimensinvestigated of Lagyniaspallidi¯ ora ( Fig. 18) zeyheri ( Fig. 10) , and Canthiummundianum . The former and Canthiuminerme ( Figs. 19 & 20) . Thetribe sometimes speciesis characterised by microverrucae which are densely alsoshows oval ectopori, e .g. Rytigyniaverruculosa ( Fig. 23) groupedin bands around the apo- and mesoporia ( Figs. 2, 9 and Psydraxhorizontalis ( Fig. 24) ,orcircular ectopori in for & 28) . Areticulatesexine type is illustrated by Keetia

Figs. 15 ± 26. Detailof apertures . SEM. ( 15) Fadogiellastigmatoloba ,smallectocolpus, rectangular endoporus; ( 16) Ancylanthosrubigino- sus,elongatedporus with obtuseends, endoporus rectangular with curvedmargins; ( 17) Tapiphyllumcinerascens var. laetum, elongated ectoporuswith obtuseends, rectangular endoporus; ( 18) Lagyniaspallidi¯ ora ,elongatedectoporus with acuteends, endoaperture rec- tangular; ( 19) & ( 20) Canthiuminerme ,elongatedectoporus with acuteends, membrane of ectoporus perforated ( arrows; Fig. 19) or coveredwith sexineelements ( Fig. 20) ; ( 21) Canthiumseti¯ orum ,elongatedectoporus with diffuseends; ( 22) Keetiagueinzii , idem; ( 23) Rytigyniaverruculosa ,elongatedto oval ectoporus, small granules on the inside of the lumina ( arrows) ; ( 24) Psydraxhorizontalis , oval ectoporuscongruent with endoporus,margin present; ( 25) Canthiumlactescens ,sexinebulges form irregular margin of circular ecto- porus, circularendoporus; ( 26) Psydraxsubcordata var. subcordata,circularecto- and endoporus .

Grana 39 ( 2000) Pollenmorphology of Vanguerieae 95

Figs. 27 ± 34. SEM. Detailof different sexine types . ( 27) Canthiuminerme ,perforatedmesoporium with circularperforations; ( 28) Canthiumparasiebenlistii ,psilateapoporium with detailof microverrucae; ( 29) Fadogiaschmitzii ,presenceof one or morebaculae in theperforation of apoporium; ( 30) Tapiphyllumcinerascens var. laetum, idem; ( 31) Vangueriopsis sp., idem; ( 32) Keetiagueinzii , perfo- rateto reticulate mesoporium with irrgularlumina; ( 33) Keetiahispida ,bumpyreticulate apoporium; ( 34) Psydraxsubcordata var. sub- cordata,reticulatemesoporium with largepolygonal lumina .

Grana 39 ( 2000) 96 F. Lens et al.

Figs. 35 ± 42. Innernexine ornamentation and costae ( arrows pointto poles ) . ( 35) Cuvieranigrescens ,two crescent-shapedcostae perpen- dicularto ectoaperture; LM . ( 36) Vangueriainfausta ; LM. 37 ± 42. SEM. (37) Canthiumciliatum ,innernexin egenerallysmo oth,scab rae concentratednear endoaperture,two crescen t-shapedcostae; (38) Canthiumseti¯ orum , idem; (39) Fadogiaschmitzii , idem; (40) Pyg- maeothamnuszeyheri ,scabrae concentratedn ear endoaperture, thetwoend exinethick eningsof costa become fusedon equator ;(41) Keetiahispida ,ellipticalto circular costa; (42) Psydraxsubcordata var. subcordata,circular costa withgran ularsurface.

Grana 39 ( 2000) Pollenmorphology of Vanguerieae 97 cornelia and K. hispida ( Fig. 33) , Psydrax ( Figs. 6, 14 & 34) , whichdescribed porate pollen grains as the most and Rytigyniaverruculosa . Thelatter species has granules commontype within the tribe ( e.g. Verdcourt1987, atthe inside of the lumina ( Fig. 23) . Theshape of the Igersheim1989 ) . Pororatepollen grains are otherwise luminais elongated in Keetiagueinzii ( Fig. 32) ,irregularin rarein the Rubiaceae and occur amongst others K. hispida ( Fig. 33) orpolygonal in Psydraxsubcordata inGardenieae-Gardeniinae ( Persson1993 ) , Isertieae var. subcordata ( Fig. 34) . An intermediatestate between ( Huysmanset al . 1998) andCoptosapelteae ( Huysmans perforateand reticulate pollen grains is shown by Keetia 1998) . gueinzii ( Fig. 32) . Amargois present in pollen of Psydrax Thetwo other pollen classes that occur in the Vanguer- horizontalis ( Fig. 24) , Pyrostria sp., and Vangueriopsis sp. ieae,namely porate and colporate pollen, are less common . Inthe latter case, the colpi of the ectoapertures are characteristicallyshort, which is in contrast to most Innernexine ornamentation and strati® cation of pollen wall Rubiaceaethat have long ectocolpi, such as the related Thenexine is generally smooth with scabrae usually Coffeeaeand Pavetteae ( Robbrecht1988, Stoffelen et al . concentratednear the endoapertures in e .g. Canthium 1997,De Block & Robbrecht1998 ) . Withinthe Vanguer- ciliatum ( Fig. 37) , Pygmaeothamnuszeyheri ( Fig. 40) , and ieae,it is sometimes dif® cult to distinguish ectocolpi from in Canthiumseti¯ orum ( Fig. 45) . However,the whole surface ectoporibecause the length/ widthratio of the ectoapertures ofthe inner nexine in Keetiahispida iscovered with equally ofthe same specimen sometimes varies around the critical distributedscabrae ( Fig. 43) . Thecostae, which lie perpen- value of two ( in e.g. Vangueria) . Furthermore,some species dicularto the ectoaperture ( Figs. 35 & 36) , are always of Canthium,showa tendencyto evenshorter ectoapertures . presentin the tribe . Costaehave the shape of two crescents C. lactescens ( Fig. 25) and C. mundianum for example orientedtoward another in e .g. Canthiumciliatum ( Fig. 37) , bothshow inward sexine bulges around the ectoaperture . Canthiumseti¯ orum ( Fig. 38) , Fadogiaschmitzii ( Fig. 39) , Asecondindication of the reduction trend is observed in and in Pygmaeothamnuszeyheri ( Fig. 40) ; costae are C. seti¯ orum,whichhas a porusmembrane that is covered ellipticalto circular in Keetiahispida ( Fig. 41) ,orcircular witha sexinelayer ( Fig. 21) . Thisvague transition between inspecies of Psydrax ( Fig. 42) and Pyrostria. thesexine and the porus membrane is also observed in Sexineis mostly thicker than nexine ( mean ratio 1.78) . Keetiagueinzii ( Fig. 22) . Canthiuminerme showsa third Thehighest ratio is nearly 2 .75 in Keetiacornelia ; the lowest evidencefor the shrinking ectoporus length because it values ( ca 1) areobserved in Cuvieralongi¯ ora , Fadogiella sometimesshows sexine elements on the porus membrane stigmatoloba ( Fig. 46) , and Tapiphyllumcinerascens var. ( Fig. 20) . Moreover,the porus membrane is sometimes laetum. Meanlength of the columellae is 0 .74 mm. The perforated ( conferperforations in tectum: Fig . 19) . shortestcolumellae are present in Vangueriopsis sp. Canthium ( Fig. 25) , Psydrax ( Fig. 26) , Pyrostria and ( ca 0.05 mm) , while Keetiagueinzii haslong columellae Keetia havecircular ectoapertures . Hence,it is clear that ( ca 2.7 mm; Fig. 44) . Wallsections show a highcolumella pollenof the tribe is characterised by a continuoustransition density in e.g. Fadgiellastigmatoloba ( Fig. 46) and Lagynias betweenshort ectocolpi, elongated ectopori and circular pallidi¯ora anda lowdensity in Psydrax ( Fig. 42) and Keetia ectopori. Thisevolutionary tendency is also discussed by ( Fig. 44) . Thethickness of the tectum is rather homo- Lobreau-Callen ( 1978) forRubiaceae and was also brie¯ y geneous ( on average 0.74 mm) . Thecolumella/ tectumratio is mentionedby Robbrecht ( 1980) . high in Psydraxsubcordata var. subcordata ( Fig. 42) and MostRubiaceae, such as the Pavetteae ( De Block & Keetiagueinzii ( ca 2.4; Fig. 44) ,whilemuch lower values Robbrecht1998 ) andCoffeeae ( Stoffelenet al . 1997) , have occur in Canthiumciliatum and Vangueriopsis sp. ( ca 0.1) . compoundapertures consisting of ecto-, meso-, and endoaperture ( Lobreau-Callen1978 ) . Inthe Vanguerieae, however,the structure of the compound aperture is very dif®cult to observe on the basis of LM andSEM . Two DISCUSSION interpretationsare possible: 1. Compoundapertures consist of twostrata, an ectocolpus/ Aperturecon® guration porusand an endoporus ( openingin the pollen wall ) . The Pollengrains in Vanguerieae are nearly always 3-zonoaper- areadelimited by the costa is not considered as an turate,which is the most common pollen class within the aperture. Rubiaceae ( Erdtman1952, Robbrecht 1988 ) . The pre- 2. Thearea within the costa is considered as the senceof 2- or 4-aperturate pollen in the tribe is endoaperture;the opening in the pollen wall then exceptionaland occurs in some grains of Pyrostria sp., correspondsto the mesoporus . Canthiummundianum , Pachystigmapygmaeum , Tapiphyl- Accordingto the latter interpretation, the endo- and lumcinerascens var. laetum, and Vangueriainfausta . mesoapertureare congruent when a circularcosta/ ecto- Pollenwith 2 or4 apertureswere also observed in apertureis present . We believethat the ® rsthypothesis is Canthium and Psydrax byTilney & vanWyk ( 1997) . mostplausible for the Vanguerieae because the region Ourresults clearly demonstrate that pororate pollen de®ned by thecosta is not likely to be aspecialisedregion of grainsare most common within the Vanguerieae, because thesporoderm ( Punt et al. 1994) . Furthermore,in case of a theectoaperture ( usuallya porus ) isoften not congruent circularcosta/ ectoaperture,the congruence between the withthe aperture lying beneath it . Thisis not in mesoporusand the endoaperture ( andpossibly the ecto- agreementwith earlier pollen morphological studies porus) ishard to prove on the basis of morphological

Grana 39 ( 2000) 98 F. Lens et al.

observationsonly . Onlyadditional TEM studiesmay clarify havea circularor elongated shape, and usually vary in size this. ( Figs. 27 & 29 ± 31) . Thepresence of baculae in the Sincethe endoapertures in our circumscription are usually perforationsis a ratherunusual feature in rubiaceous notentirely visible on broken grains in SEM, theshape is pollen,but clearly present in most of the perforate describedbased on external observations . Thissometimes taxawith larger perforations ( seealso Igersheim 1989, givesan unusual picture of the endoaperture, such as a Figs. 29 ± 31) . Perforationswith baculae were also observed rectangularshape with straight ( Figs. 15 & 17) or curved by Huysmans ( 1998) insome Simireae and Cinchoneae . The ( Figs. 16 & 18) borders. Onlywhen the endoapertures are perforationswithin the Coffeeae differ from those in entirelyvisible from the outside of a pollengrain, it is clear theVanguerieae by their elongated and irregular shape thatthey are circular ( Figs. 25 & 26) ,elliptical ( Fig. 1) or ( Stoffelenet al . 1997) . Pollen of Keetia and Psydrax are oval ( Figs. 23 & 24) . distinguishedfrom other Vanguerieae by their reticulate sexine ( seealso Tilney & vanWyk 1997 ) . Incontradiction to theobservations of these authors, who found oblate pollen Sexinepattern witha ®nelyreticulate tectum in Psydrax,we observedthat Pollenof the Vanguerieae have two main sexine types . thegrains of Psydraxsubcordata var. subcordata are Perforatepollen grains are most common . On thebasis of spheroidaland have very large polygonal lumina ( on thesize of the perforations, one can easily distinguish average 2.75 mmonapoporium and 3 mmonmesoporium; Canthium and Vangueria fromthe remaining perforated taxa Fig. 34) . However,according to the same authors, this ( Figs. 29 ± 31) ;bothgenera have very small perforations sexinepattern is characteristic of Keetia. Furthermore,they ( Tilney& vanWyk 1997, Fig . 27) . In Canthiumparasie- didnot observe a bumpytectum in Keetia. benlistii ( Fig. 2) , C. mundianum, and Pygmaeothamnus zeyheri ( Fig. 10) ,perforationsare present in the mesoporia, Innernexine ornamentation butabsent on the poles ( psilateapoporium ) . Canthium parasiebenlistii ischaracterised by circular bands of micro- Pollengrains in the tribe generally have a smoothinner verrucaearound the apo- and mesoporia ( Figs. 2 & 9) . The surfacewith scabrae near the endoapertures, in contrast to perforationsare nearly always unequally distributed, they mostof the Rubiaceae which have a granularinner nexine

Grana 39 ( 2000) Pollenmorphology of Vanguerieae 99

( Robbrecht1988 ) . Sometimesthe whole inner surface is Strati®cation of the pollen wall coveredwith scabrae ( Fig. 43) . Thepresence of a costain all Rubiaceaeare characterised by a sexine/nexineratio 1 thespecimens investigated is typical of pollen of the ( e.g. Huysmans1998 ) . Thisis also observed in the study Vanguerieae . Correlatedwith the shape of the ectoaperture, group,which has a meanratio of 1 .75. Variationin the threecosta types are observed . Ina ®rsttype, present in columellaeis found in the length ( on average 0.75 mm) and pollenwith short ectocolpi or elongated ectopori ( e.g. in the density. Vangueriopsis sp. illustratesshort columellae, Fadogiaschmitzii ) ,twocrescent-shaped thickenings occur while Keetiagueinzii haslong columellae ( Fig. 44) . In parallelto the equator plane ( Figs. 35 & 36) . These Ancylanthosrubiginosus , Canthiumseti¯ orum and Lagynias thickeningsmay be enlarged on the mesoporium/ colpium pallidi¯ora ,thecolumellae are grouped closely together, . . sideby the presence of verrucae in e g Pygmaeothamnus while Keetia and Psydrax havewidely scattered columellae . zeyheri ( Fig. 40) . Whenthe two thickenings become fused, Sincethe thickness of the tectum layer is rather constant, the anelliptical costa ( second type) isformed, which is found columella/tectumratio varies together with the length of the forinstance in Canthiumlactescens . Theelliptical costa columellae. usuallyis correlated with elongated ( ormore or less elliptical) ectopori. Thethird costa type, possibly evolved byshrinking of the elliptical costa, has a circularshape . A Dispersalof the pollen grains transitionis observed in Keetiahispida ( Fig. 41) . The circularcostae occur in Keetia, Psydrax and Pyrostria, the Nearlyall rubiaceous pollen is dispersed as monads . Pollen sametaxa that also have circular ectopori . This costa grainsare united in permanent tetrads in some genera typeis also characterised by its granular surface, in ofGardenieae and in Gleasonia ofthe Henriquezieae contrastto the ® rsttwo costa types which are more ( Robbrecht1988, Persson 1993, Robbrecht & Puff1986, massiveand have a coarsesurface ( Fig. 42) . Robbrechtet al . 1996) . In the genus Massularia ( Garden- Costaeare also seen in many other tribes of the family, ieae) thetetrads are further united into massulae ( Persson suchas the related Pavetteae ( DeBlock & Robbrecht1998 ) 1993) ,whichis a veryrare feature within the angiosperms andGardenieae ( Persson1993 ) ,orthe more distantly ( Walker& Doyle1975 ) . Massulaewere also observed by relatedCoptosapelteae ( Huysmans1998 ) . Igersheim ( 1989) in Psydrax subgenus Psydrax and in

Grana 39 ( 2000) 100 F. Lens et al.

Figs. 43 ± 46. Pollenwall strati® cation . ( 43) Keetiahispida ,innernexine wholly covered with scabrae,long and widely spread columellae; ( 44) Keetiagueinzii , idem; ( 45) Canthiumseti¯ orum ,short columellaestanding close to each other; ( 46) Fadogiellastigmatoloba , nexine hasnearly the same thickness as the sexine, columellae standing close to each other .

Keetia. Accordingto Igersheim, acetolysed pollen of these Bullockia inthe smaller mean size ( nospecimens of generaare held together by sexine bridges so that large C. subgenus Canthium wereavailable to us ) . Hence, this aggregatesof pollen can be visualisedby SEM-micrographs . studycorraborates some of Bridson’shypothesis,e .g. a close Inthe specimens of Psydraxsubcordata var. subcordata and relationshipbetween Keetia and Psydrax,andthe division of Keetia investigated,however, we foundno massulae but Canthium s.s. intosubgenera . Nevertheless,the delimitation monads. ofmost of the genera cannot be based on pollen morphologyonly .

Taxonomicvalue of pollen morphology within the Vanguerieae ACKNOWLEDGEMENT As alreadyconcluded in earlier studies ( Verdcourt1987, Wethank Marcel Verhaegen for technicalassistance during the Igersheim1989 ) ,pollenmorphology does not provide much SEM observations . This researchwas supportedby a grantfrom the usefulinformation for the generic delimitations within the ResearchCouncil of the K .U.Leuven ( OT/97/23) . S. Huysmansis a Vanguerieae . Nevertheless,based upon an investigation of PostdoctoralFellow of the Fund for Scienti®c Research-Flanders SouthAfrican representatives, Tilney & vanWyk ( 1997) ( Belgium) ( FWO) . de®ned three subgenera of Canthium sensuBridson ( 1992) onthe basis of the size and shape of the grains, the exine thicknessand the ratio of sexine/ nexine . Inour study, these SPECIMENSINVESTIGATED featuresalso proved to besystematically informative . Grains Allspecimens are from the herbarium of the National Botanic of Canthium subgenus Lycioserissa forinstance, represented Gardenof Belgium ( BR) . by C. ciliatum and C. inerme,areoblate spheroidal and theirsexine/ nexineratio is rather similar ( see Table I) . On Ancylanthosrubiginosus Desf.,ANGOLA:Huila, Quilemba, J .B. theother hand, subgenus Bullockia ( representedby Teixeira2817 ( Fig. 16) C. seti¯ orum) andsubgenus Afrocanthium ( representedby Canthiumciliatum ( D.Dietr.) Kuntze,SOUTH AFRICA: Zoutpans- C. lactescens, C. mundianum and C. parasiebenlistii ) have berg,Farm Rustfontein, H .J. Schlieben7351 ( Fig. 37) pollenwhich tend to be suboblate . Furthermore,the sexine C. inerme ( L.f.) Kuntze,ZIMBABWE: Umtali,A .C. Chase 5351 ofthese two subgenera is about two times the thickness of ( Figs. 19, 20 & 27) the nexine. Subgenus Afrocanthium differsfrom subgenus C. lactescens Hiern,RWANDA: Muhero, G . Michel 4821 ( Fig. 25)

Grana 39 ( 2000) Pollenmorphology of Vanguerieae 101

C. mundianum Cham. & Schltdl.,SOUTHAFRICA: Zoutpansberg, Andreasen,K ., Baldwin, B. G. & Bremer, B. 1999. Phylogenetic FarmRustfontein, H .J. Schlieben7596 utilityof the nuclear rDNA ITS regionin subfamily Ixoroideae C. parasiebenlistii Bridson, TANZANIA:Iringa region, Mufundi, ( Rubiaceae) :comparisonswith cpDNA rbcLsequencedata . ± PennyPennis Farm, R .K. Brummittet al . 18165 ( Figs. 2, 9 & 28) PlantSystematics and Evolution 217: 119 ± 135 . C. seti¯ orum Hiern,ETHIOPIA: Sidamo, J . Ash 2444 ( Figs. 7, 21, Andreasen,K . & Bremer, B. 1996. Phylogenyof the subfamily 38 & 45) Ixoroideae ( Rubiaceae) . ±OperaBotanica Belgica 7: Cuvieralongi¯ ora Hiern,CONGO-KINSHASA: Yangambi, J . 119 ± 138. Louis 823 ( Fig. 1) & 1255 Bosser, J. &Lobreau-Callen,D . 1998. Landiopsis Capurion et C. nigrescens Wernham,NIGERIA: Sapoba, J .D. Kennedy2319 Bosser, genrenouveau de Rubiace Âesde Madagascar . ± ( Fig. 35) Adansonia20: 131 ± 137 . . . Fadogiaschmitzii Verdc.,CONGO-KINSHASA:Kolwezi, Schmitz Bremer, B 1996 Phylogeneticstudies within Rubiaceaeand relationshipsto other families based on molecular data . ± 2958 ( Figs. 12, 29 & 39) OperaBotanica Belgica 7: 33 ±50 . Fadogiellastigmatoloba ( K.Schum.) Robyns,CONGO-KIN- Bremer, B.,Andreasen,K &Olsson,D 1995 . Subfamilialand tribal SHASA:Katanga, Kalukuluku, F . Malaisse9350 ( Figs. 15 & 46) relationshipsin the Rubiaceae based on rbcLsequencedata . ± Keetiacornelia ( Cham. & Schltdl.) Bridson, IVORYCOAST: Annalsof the Missouri BotanicalGarden 82: 383 ± 397 . ComoeÂriver,Bouin, C . Geerling & J. Bokdam 2153 Bremer, B. & Thulin, M. 1998. Collapseof the Isertieae, re- K. gueinzii ( Sond.) Bridson, CONGO-KINSHASA:Yalibwa, J . establishmentof Mussaendeae, and a newgenus of Sabiceeae . Louis 1198 ( Figs 22, 32 & 44) ( Rubiaceae) ;phylogeneticrelationships based on rbcL data. ± . . . K hispida ( Benth ) Bridson, CONGO-KINSHASA:Yangambi, J PlantSystematics and Evolution 211: 71 ± 92 . Louis 2944 ( Figs. 5, 33, 41 & 43) Bridson, D. M. 1985. Thereinstatement of Psydrax ( Rubiaceae, Lagyniaspallidi¯ ora Bullock,TANZANIA: Mahenge, H .J. Schlie- subfam. Cinchonoideaetribe Vanguerieae ) anda revisionof the ben 2135 ( Figs. 11 & 18) Africanspecies . ±KewBulletin 40: 687 ± 725 . Meynatetraphylla ( Schweinf.) Robyns,SUDAN: Imatong moun- Bridson, D. M. 1986. Thereinstatement of theAfrican genus Keetia tains, I. Friis & K. Vollesen1185 ( Rubiaceae,subfam . Cinchonoideaetribe Vanguerieae ) . ± Kew Pachystigmapygmaeum ( Schltr.) Robyns,CONGO-KINSHASA: Bulletin41: 965 ± 994 . Katanga,plateau of Marungu, S . Lisowiski 9669 Bridson, D. M. 1987a. Studiesin African Rubiaceae - Vanguerieae: Psydraxhorizontalis ( Schumach. & Thonn.) Bridson, IVORY anewcircumscription of Pyrostria anda newsubgenus, COAST:mount Niangbo, C . Geerling & J. Bokdam 1035 Canthium subgen. Bullockia. ±KewBulletin 42: 611 ± 639 . ( Figs. 4, 14 & 24) Bridson, D. M. 1987b. Therecognition and recircumscription of the P. subcordata ( DC.) Bridson var. subcordata, RWANDA:Rangiro, Africangenus Multidentia ( Rubiaceae- Vanguerieae ) . ± Kew D. Bridson 337 ( Figs. 6, 26, 34 & 42) Bulletin42: 641 ± 654 . Pygmaeothamnuszeyheri ( Sond.) Robyns,SOUTH AFRICA: near Bridson, D. M. 1992. The genus Canthium ( Rubiaceae- Vanguer- Josefsdal ( border S. Africa/Swaziland ) , C. Puff 2631 ( Figs. 10 & ieae) intropical Africa . ±KewBulletin 47: 353 ± 401 . 40) Bullock, A. A. 1932. Canthium inBritish EastAfrica . ±Bulletinof Pyrostria sp.,MADAGASCAR:Fianarantsoa Anosimasina, S . theMiscellanous Information of the Royal Botanic Gardens, Malcomberet al . 1622 Kew8: 353 ±389 . P. bibracteata Hiern,TANZANIA: Zanzibar, H .G. Faulkner2248 De Block, P. &Robbrecht,E . 1998. Pollenmorphology of the ( Figs. 3 & 8) Pavetteae ( Rubiaceae,Ixoroideae ) andits taxonomicsigni® - Rytigyniabagshawei ( S. 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