Pollen of African Spermacoce species (Rubiaceae) Morphology and evolutionary aspects

Steven Dessein; Suzy Huysmans; Elmar Robbrecht; Erik Smets

Abstract: The pollen morphology of 24 species of Compositae belonging to 18 genera was examined by light and scanning microscopy. The present data confirms the eurypalynous nature of the family. Delimitation of the genera on the basis of pollen characters is less marked. Pollen grains usually radially symmetrical, isopolar rarely apolar; tricolporate, often porate. Shape commonly oblate-spheroidal to prolate-spheroidal, with some suboblate to subprolate types. Tectum echinate to echinolophate, often non-echinate. Tectum in-between the spines or spinules is sub-psilate, or perforated to striate. On the basis of the exine pattern, 4 distinct pollen types are recognized viz.,

Pollen morphology of 24 species of 14 genera belonging to four families, viz., Amaranthaceae, Convolvulaceae, Malvaceae and Nyctaginaceae has been studied. The pollen grains of these taxonomically unrelated taxa, exhibit variation in colour, size, shape, exine ornamentation as well as in the number, position and character (NPC) of pores, but the pollen grains were usually polyporate and a few are polyzonocolporate. The pollen grains in the taxa of the family Amaranthaceae were small, non-spinous with polyporate exine, whereas, the pollen grains of taxa belonging to other three families were echinate. The maximum numbers of pores have been recorded in convolvulaceous pollens. Likewise, pollen grains of nyctagenaceous taxa can be distinguished from the pollens of other families on the basis of the size of pollen and spines. The pollens of the members of family Malvaceae exhibit different patterns of pore arrangements on the exine.

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To cite this article: St even Dessein , Suzy Huysmans , Elmar Robbrecht & Erik Smet s (2002) Pollen of Af rican Spermacoce species (Rubiaceae) Morphology and evolut ionary aspect s, Grana, 41: 2, 69-89, DOI: 10. 1080/ 001731302760156882 To link to this article: ht t p: / / dx. doi. org/ 10. 1080/ 001731302760156882 PLEASE SCROLL DOWN FOR ARTI CLE Taylor & Francis m akes every effort t o ensure t he accuracy of all t he inform at ion ( t he “ Cont ent ” ) cont ained in t he publicat ions on our plat form . However, Taylor & Francis, our agent s, and our licensors m ake no represent at ions or warrant ies what soever as t o t he accuracy, com plet eness, or suit abilit y for any purpose of t he Cont ent . Any opinions and views expressed in t his publicat ion are t he opinions and views of t he aut hors, and are not t he views of or endorsed by Taylor & Francis. 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Term s & Condit ions of access and use can be found at ht t p: / / www.t andfonline.com / page/ t erm s- and- condit ions Grana 41: 69±89, 2002 Pollen of African Spermacoce species (Rubiaceae) Morphology and evolutionary aspects Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 STEVEN DESSEIN, SUZY HUYSMANS, ELMAR ROBBRECHT & ERIK SMETS Dessein, S., Huysmans, S., Robbrecht, E. & Smets, E. 2002. Pollen of African Spermacoce species (Rubiaceae). Morphology and evolutionary aspects. ± Grana 41: 69±89. Pollen morphology of 43 African species of the genus Spermacoce has been investigated by scanning electron and light microscopy. The genus is eurypalynous, which is reected in the remarkable variation of almost all pollen characters. The average equatorial diameter ( E) ranges from 15.8 mm to 115.5 mm. Grains are colporate or pororate. The number of apertures varies from 3 up to more than 25. The majority of species has apertures situated only at the equator (being zonoaperturate), but a few species have pantoaperturate grains. The endoaperture is generally an endocingulum, often with a secondary lolongate or lalongate thinning at the ectocolpus; endocolpi and endopores are also observed. The sexine is usually perforate, but eutectate, foveolate, and (micro)reticulate tecta were also found. Supratectal elements are present as granules, microspines or spines. The inner nexine surface is granular, often with irregular grooves (endocracks). Among native African species, nine pollen types are recognized mainly on the basis of pollen size, aperture morphology and tectum peculiarities. In two of the pantoaperturate types, apertures are in a conguration not yet recorded for the angiosperms in general. Some evolutionary trends are proposed that await verication by further systematic study. Pollen morphological characters have a high taxonomic value in the genus Spermacoce. They provide almost unique identication marks for the species, which enables sharpening of species boundaries. Small groups of related species often share the same pollen type. The genus Borreria, previously separated from Spermacoce on the basis of its fruit morphology only, is not supported by pollen data. Steven Dessein, Suzy Huysmans & Erik Smets, Laborator y of Plant Systematics, Institute of Botany and Microbiology, K.U.Leuven, Kasteelpark Arenberg 31, B-3001 Leuven; Belgium: Elmar Robbrecht, National Botanic Garden of Belgium, Domein van Bouchout, B-1860 Meise; Belgium. E-mail: steven.dessein@bio.kuleuven.ac.be (Manuscript received 13 July 2001; accepted 11 April 2002) The Spermacoce-Borreria alliance (Rubiaceae: Spermacoceae) is a large complex with a worldwide distribution in the tropics and subtropics. The estimated number of species varies between 150 and 250 (Verdcourt 1989), depending on the authors. The plants typically grow in woodlands and grasslands, and are characterized by a herbaceous habit, mbriate stipules connected to the petioles, mostly small, isostylous owers arranged in compact lateral and terminal inorescences, valvate bud aestivation, uni-ovulate ovary locules, dry capsular fruits, and presence of raphides. The group is, along with 18 smaller genera, placed in the tribe Spermacoceae, subfamily Rubioideae (Robbrecht 1988). A molecular study of Andersson & Rova (1999) conrmed the monophyly of the tribe, but the group was nested within members of the tribe Hedyotideae, which makes the latter paraphyletic (see also Bremer et al. 1995, Natali et al. 1995, Bremer 1996, Bremer & Manen 2000). For this reason, Bremer (1996 ) proposed the Spermacoceae sensu lato, including the former tribes Hedyotideae, Knoxieae and Manettieae. In the present paper, the term Spermacoceae is used in its more traditional, narrow sense (cf. Robbrecht 1988). Generic delimitations within the complex have been debated for almost two centuries. Spermacoce L. broadly dened by some authors (e.g., Verdcourt 1975, 1976, 1983, 1989, Deb & Dutta 1984, Sivarajan et al. 1987), is divided by others (e.g., Bacigalupo 1972, Steyermark 1972, 1974, Bacigalupo & Cabral 1996) on the basis of peculiarities of the fruit dehiscence. The segregated genus Borreria G.Mey. is then characterized by having capsules with a septicidal opening and both fruit valves dehiscent, and Spermacoce sensu stricto by its fruits splitting into two valves, one dehiscent and the other indehiscent (Meyer 1818). These fruit characters are easy to observe, but no other features seem to be correlated with it (cf. Verdcourt 1975, 1976, Deb & Dutta 1984, Sivarajan et al. 1987). In the present article we accept Verdcourt’s view (1975) and relegate Borreria to the synonymy of Spermacoce. Only few, limited studies exist on the pollen of Spermacoce. The earlier ones were part of broader studies on the phylogeny of Rubiaceae (e.g., Bremekamp 1952, Verdcourt 1958). All reported that pollen of Spermacoce is multi-aperturate, but they did not fully survey the morphological variation within the genus. More recently, pollen studies of the American representatives of Spermacoceae demonstrated the systematic value of pollen characters in the tribe. Pollen characters were used to re-establish the genus Galianthe (Cabral 1991, Pire & Cabral 1992, Pire 1997a) and to support a new subgeneric classication for the American Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 70 S. Dessein et al. species of Psyllocarpus ( Kirkbride 1979 ) and Borreria (Bacigalupo & Cabral 1996, Pire 1996, Cabral & Bacigalupo 1999). Pollen morphology was also used to erect the genus Phylohydrax for the African and Madagascan Hydrophylax species (PuV 1986). Until now, pollen of the African Spermacoce species was never investigated. The present study aims (1) to survey the pollen morphology of the African representatives of Spermacoce, (2) to discuss its signicance in the taxonomy of the genus, and (3) to formulate a hypothesis for the evolution of pollen characters within the genus. The project is part of the ongoing research of the rst author to document the biodiversity of Spermacoce in Africa. MATERIAL AND METHODS This study is based on herbarium material from BR, K, WAG and ZT ( Table I ). Pollen of 92 specimens from 43 species were investigated (Table I). For the remaining African species (ca. 25 ) owering material in the above mentioned herbaria was not suYcient to carry out palynological research. Due to the problematic delimitation of several species of Spermacoce, we only selected specimens that fully correspond with the type specimen (if seen) or with the original descriptions; additional comments on some problematic species/ specimens are provided ( Table I ). Species introduced to Africa are marked with an asterisk; the table also indicates the specimens used for the illustrations. Mature ower buds were rehydrated in an Ageponâ (Agfa Gevaert) solution (1:200), and dissected using a stereomicroscope. Pollen was acetolysed for 9 minutes in a heating block at 90ßC. Pollen grains for SEM studies were suspended in ethanol (70%), pipetted onto specimen stubs, air-dried and coated with gold with a SPI-MODULETM sputter coater. Observations were made under a Jeol JSM-6400 SEM. Grains for LM studies were mounted in Kaiser’s glycerin jelly. The slides were observed using a Leitz Dialux 20 with a Ö100 oil immersion or Ö40 objective lens. Equatorial diameter ( E) was measured under LM in at least ten mature pollen grains (magnication 1000 ). We were not able to obtain accurate measurements for the polar axis (P) under LM, since most grains are suboblate. All other measurements were made on SEM-graphs. To express the relative length of the colpi, we used the length colpi/polar axis rate multiplied by 100 (= LC/P). Broken pollen grains were obtained by shaking a pollen and glass bead suspension as described by Huysmans et al. (1994 ). Terminology follows Punt et al. (1999) . RESULTS Pollen characters Pollen morphology of African Spermacoce species is remarkably heterogeneous. In all pollen characters, the group reveals a variation that is usually only observed at higher systematic levels. This diversity is described and discussed below for the diVerent diagnostic pollen characters and is illustrated by SEM-graphs. Detailed information at species level is presented in Table II. Size Pollen grain size of African Spermacoce species ranges from small to large, the average equatorial diameter (E ) varying from 15.8 mm in Spermacoce mauritiana to 115.5 mm in S. ivorensis. The size variability within a single specimen is considerable (often exceeding 10%); within a species the Grana 41 (2002) variation in E-averages frequently exceeds 20%. Sometimes, this variation can be explained in terms of intraspecic variation, as is the case for Spermacoce phyteumoide s: var. phyteumoides and var. caerulea have smaller pollen grains than ``var. longituba’’. Verdcourt informally recognized the latter variety on the herbarium sheets for its longer corolla tube. In general there is a positive correlation in the study group between size of the corolla tube and pollen size (Fig. 1 A; r2 = 0.43; p<0.01): larger owers tend to produce larger pollen grains. This correlation is also found in many other groups and may be correlated with diVerences in nutrition supply (Muller 1979). In the present study, however, the correlation between ower and pollen grain size is not strict. Flowers with a corolla tube smaller than 3 mm have pollen grains ranging from ca. 15 mm up to 60 mm, while medium sized owers (tube ca. 7 mm) show pollen grains between 55 mm and 105 mm. Moreover, very variable species, such as Spermacoce subvulgata, have corolla tubes ranging from 3 to 6 mm, but no correlated diVerences are observed in the size of their pollen. Probably, the true correlation lies in the pollen vector size, rather than in the size of the owers. Although only a few studies (cf. Muller 1979) have demonstrated that diVerent pollen vectors prefer diVerent ranges of pollen size, it seems likely that this partly explains the considerable interspecic pollen size variation. Another hypothesis to explain the pollen size variation is the level of polyploidy. Higher levels of polyploidy may express itself directly in a larger pollen size (Muller 1979). Herbaceous Rubiaceae are known to have high levels of polyploidy (cf. Lewis 1965, Kiehn 1986). Kiehn (1986, 1995 ) surveyed the chromosome numbers of the Rubiaceae; for the Spermacoceae, the basic chromosome number is predominantly 14, and tetraploidy and hexaploidy originated several times within the tribe ( Kiehn 1986). Moreover, diVerent ploidy levels are recorded for specimens of the same species; hence, giving support for the hypothesis that some of the intraspecic pollen size variation is caused by diVerences in ploidy levels. In many genera of Rubiaceae, diVerences in pollen size may be explained by heterostyly (Huysmans et al. 1998, Dessein et al. 2000). In general, the brevistylous form has larger pollen grains than the longistylous morph (cf. Ganders 1979). African Spermacoce species, however, are invariably homostylous, hence this hypothesis can not explain the intraspecic variation observed. Shape P/E. ± In equatorial view, pollen shape is described by the P/E rate. In the study group, subprolate (P/E is 1.14±1.33; Fig. 10), spheroidal (P/E is 0.88±1.14; Figs. 11±14) as well as suboblate (P/E is 0.75±0.88; Figs. 15±16 ) and oblate (P/E is 0.50 ±0.75; Fig. 17 ) pollen is found. The spheroidal condition can be further divided into oblate spheroidal (P/E is 0.88 ±1) and prolate spheroidal (P/E is 1±1.14). There is a negative correlation between the equatorial diameter and the P/E rate (Fig. 1 B; r2 = 0.29; p<0.01). The smaller the pollen grains the higher the P/E value. Spheroidal pollen grains, however, can be small as well as large. Pollen of African Spermacoce species (Rubiaceae) 71 Table I. Specimens examined including type- and gure references. Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 1 Spermacoce articularis L.f.: treated as a synonym of Spermacoce hispida L. by Verdcourt (1976 ) in FTEA and many others, but recognized again by Sivarajan et al. (1987). Native to Asia, but introduced in tropical Africa. 2 Cited in FTEA as Spermacoce hispida L. by Verdcourt (1976). 3 Spermacoce assurgens Ruiz & Pav.: There is much confusion about the identity of this species. It was treated under S. laevis Lam. by Verdcourt (1976 ) in FTEA and many others. Recently, Cabral & Bacigalupo (1999 ) placed S. assurgens in synonymy to Spermacoce remota Lam., which they transferred to Borreria subgenus Borreria section Borreria, series Laeves. Whether this is correct, needs conrmation; the pollen grains of the specimen studied here do not correspond with the pollen grains of S. remota (Pire 1996, treated under B. laevis (Lam.) Griseb.). 4 Spermacoce mauritiana Osia Gideon: the delimitation of this species has been debated for a long time, and it was often confused with Spermacoce ocymoides Burm.f. Verdcourt (1983 ) ± based on unpublished material of Gideon and Fosberg ± stated that ``the suggestedly cosmopolitan S. ocymoides is actually a complex of species distinguishable mainly on seed characters and published Gideon’s name Spermacoce mauritianta (a substitute name for Borreria repens DC.) for the form with two calyx lobes’’. Cabral & Bacigalupo (1996), however, accept ``Borreria ocymoides as one variable species until more African and Asiatic specimens can be studied’’. The specimens investigated here are provided with two sepals (as all African species seen by the rst author). 5 The specimen investigated was labeled by Verdcourt as Spermacoce phyteumoides var. longituba . This name, however, is still unpublished. * Species introduced to Africa. Taxon Collection Country Herb. Type Figure Spermacoce annua Verdc. *S. articularis L.f.1 , 2 S. arvensis (Hiern) Good Mutimushi 3356 Milne-Redhead & Taylor 7522 Bingham 8946 Pope, R-Smith & Goyder 2226 Bidgood, Monasunki & Vollesen 1062 Faulkner 943 Bidgood, Mbago & Vollesen 2528 Richards 12162 Vanden Berghen 6674 Zambia Tanzania Zambia Zambia Tanzania Tanzania Tanzania Tanzania Senegal K BR K K BR BR K BR BR 6 15 14 Fig. 25 Vanden Berghen 8522 Thomas 2731 Adam 12589 Berhaut 2933 Leeuwenberg & Beek 10375 Leippert 5071 Lisowski B-1172 Pawek 13957 Skarpe S-444 Bidgood, Mbago & Vollesen 2695 Senegal Sierra Leone Guinea Senegal Cameroon Kenya Tchad Malawi Botswana Tanzania BR K BR BR BR BR BR BR K BR 15 17 14 Fig. 41 Fig. 44 Figs. 8, 17, 20, 46, 49 Lejeune 197 Chapman 272 Troupin 14994 Pereira, Sarmento & Marques 1829 Lewalle 1694 Saintenoy 162 de Wilde & de Wilde-Duyfjes 3091 D.R. Congo Malawi Rwanda Mozambique Burundi Burundi Cameroon BR BR BR BR BR BR BR 12 Figs. 51-52 Geerling & Bokdam 984 Dalziel 212 Faulkner 2168 Drummond & Hemsley 3632 Lejoly 85/314 HombleÂ 796 Schaijes 1467 Schaijes 1960 Duvigneaud & Timperman B-2627 QuarreÂ 1957 Hess-Wyss 52/584 Bos, van der Laan & Nzabi 10676 Geerling & Bokdam 1701 Schlechter 12123 Lisowski 56863 Hepper & Maley 7760 Scott s.n. McClounie 84 Ivory Coast Nigeria Tanzania Tanzania Burkina Faso D.R. Congo D.R. Congo D.R. Congo D.R. Congo D.R. Congo Angola Gabon Ivory Coast Mozambique D.R. Congo Ivory Coast Malawi Zambia BR K BR BR BR BR BR BR BR BR ZT WAG BR BR BR BR K K 11 17 Fig. 12 *S. assurgens Ruiz & Pav. 3 S. azurea Verdc. S. bambusicola (Berhaut) J.-P.Lebrun & Stork S. chaetocephala DC. S. congensis (Bremek.) Verdc. S. deserti N.E.Br. S. dibrachiata Oliv. S. lifolia (Schumach. & Thonn.) J.-P.Lebrun & Stork S. liformis Hiern S. lituba ( K.Schum.) Verdc. S. hepperana Verdc. S. hockii (De Wild.) Dessein S. huillensis (Hiern) Good S. intricans (Hepper) H.M.Burkill S. ivorensis Govaerts S. kirkii (Hiern) Verdc. *S. latifolia Aubl. S. latituba ( K.Schum.) Verdc. Fig. 7 11 14 Figs. 15, 27, 37 16 Fig. 42 11 Figs. 6, 13 11 14 Fig. 43 15 ?17 16 15 2 Fig. 21 Figs. 19, 32 15 Figs. 28, 39 Grana 41 (2002) 72 S. Dessein et al. Table I. (Continued). Taxon Collection Country Herb. Type S. mauritiana Osia Gideon4 Evrard 157 QuarreÂ 7867B Vanden Berghen 9164 Polhill & Paulo 1419 Thulin & Mhoro 3128 de Wilde 992 Lely 522 Simpson 7325 King 3 D.R. Congo D.R. Congo Senegal Tanzania Tanzania Ivory Coast Nigeria Sudan Zambia BR BR BR BR WAG BR K K BR 1 Richards 8431 Tanzania K Fig. 38 Carter, Abdullah & Newton 2582 Tanzania K Fig. 22 Ngoundai 250 Huxley 84 Malaisse 9691 Schmitz 7228 HombleÂ 930 Troupin 1407 Berhaut 435 Robbrecht & Leman 3377 Vanden Berghen 6197 Leeuwenberg 2323 van den Bossche 3473SerI Polhill & Paulo 2347 Tanner 659 de Wilde 8518 van Eijnatten 2077 Reekmans 10237 Bredo 1487 Michel 3058 Drake 92 Vanden Berghen 9184 Vanden Berghen 6119 Milne-Redhead 4338 Malaisse & Robbrecht 1996 Bamps, Martins & Maia 4175 Lewalle 5606 Tanzania Tanzania D.R. Congo D.R. Congo D.R. Congo D.R. Congo Senegal Niger Senegal Ivory Coast Togo Tanzania Tanzania Cameroon Nigeria Burundi D.R. Congo D.R. Congo Sudan Senegal Senegal Zambia R.D. Congo Angola Burundi BR BR BR BR BR BR BR BR BR BR BR BR BR BR BR BR BR BR BR BR BR K BR BR BR 15 Fig. 47 Lisowski 10476 Bredo 2262 Lisowski, Malaisse & Symoens 4139 Schlieben 2321 Milne-Redhead & Taylor 8821 D.R. Congo D.R. Congo D.R. Congo Tanzania Tanzania BR BR BR BR BR 15 Fig. 5 Humblot 599 Fay 3445 Gossweiler 2347 Pearson 2772 Hess-Wyss 52/1798 Chevalier 1026 Dessein 24 Madagascar R.C. Africa Angola Angola Angola Sudan Cultivated at BR K BR BR K ZT BR BR S. natalensis Hochst. Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 S. octodon (Hepper) Hakki S. phyteuma Schweinf. ex Hiern S. phyteumoides Verdc. var. phyteumoides S. phyteumoides Verdc. var. caerulea Verdc. S. phyteumoides Verdc. ``var. longituba ’’5 S. princeae ( K.Schum.) Verdc. S. pusilla Wall. S. quadrisulcata (Bremek.) Verdc. S. radiata (DC.) Hiern S. ruelliae DC. S. senensis ( Klotzsch) Hiern S. spermacocina ( K.Schum.) Bridson & PuV S. sphaerostigma (A.Rich.) Oliv. S. stachydea DC. S. stipularis Dessein S. subvulgata ( K.Schum.) J.G.Garcia S. taylorii Verdc. *S. tenuior L. S. tenuissima Hiern S. terminaliora Good. S. thymoidea (Hiern) Verdc. *S. verticillata L. Amb. ± In polar view, the pollen outline is usually circular and often lobed due to the longer columellae towards the centers of the mesocolpia or due to the thicker nexine around the apertures (cf. below). Only Spermacoce natalensis and S. mauritiana have a triangular or quadrangular polar outline (Figs. 2±3). Grana 41 (2002) 1 Figure Figs. 3, 10, 18, 35 Fig. 2 12 6 14 17 Figs. 23, 36 Figs. 9, 26, 33±34 Figs. 16, 45, 50 Fig. 40 11 11 11 17 15 Fig. 14 ?17 17 17 Figs. 29±30 14 1 12 13 13 3 Figs. 48, 53±54 Figs. 57±58 Figs. 24, 55±56 Figs. 4, 11, 31 Apertures Number. ± The number of colpi ranges from 3±4 in Spermacoce mauritiana (Figs. 2±3) to 16±21 in S. dibrachiata (Fig. 8). The pantopororate species have up to 30 pores. The higher the number of colpi, the higher the intraspecic variation observed. There is a weak positive correlation Pollen of African Spermacoce species (Rubiaceae) 73 Table II. Some variable pollen morphologica l characters for each species studied. Species are arranged by pollen type as discussed in the text. (x): number of specimens studied. Shape as seen in equatorial view: O = oblate; SO = suboblate; OS= oblate spheroidal; S = spheroidal; PS = prolate-spheroidal; SP = subprolate. Number of apertures: values between brackets were not found in all specimens investigated . Aperture type: PC = pantocolporate; PP = pantopororate; ZC= zonocolporate. Peculiarities : CM= colpus membrane; EP = equatorial plane. Types in Endo-column are discussed in the text. Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 Apertures Wall ornamentation Type Species Size Shape Number Type Endo Peculiaritie s Sexine Nexine 1 S. mauritiana (3) 14-(15.8)-1 8 PS 3-4 ZC II 1 S. natalensis (2) 14-(16.5)-1 9 SP 3-4 ZC II nely granular, endocracks indistinct nely granular, endocracks indistinct or absent 1 S. tenuior (1)* 15-(16.1)-1 7 PS-SP 6-8 ZC II perforate, margo of granules/ microspines eutectat e at apocolpium, perforate at mesocolpium, margo of granules perforate, margo of granules/ microspines 2 S. latifolia (2)* 44-(48.3)-5 0 SO-OS (7)-9-(11 ) ZC V perforate, granulate granular, broadÔdistinct endocracks 3 S. verticillata (2)* 23-(28.0)-3 0 OS (6-9 ) ZC IV perforate, granulate granular, narrowÔdistinct endocracks 6 S. annua (1) 62-(66.5)-7 4 S ca. 30 PP VI 6 S. phyteuma (1) 80-(87.0)-9 0 S ca. 24 PP VI microreticulate to reticulate, microechinate perforate-microreticulate , microechinate nely granular, no endocracks nely granular, no endocracks 11 S. assurgens (1)* 35-(36.8)-3 8 SO-OS 8-10 ZC II 11 38-(44.7)-5 2 SO-OS (9)-11 ZC II 11 S. chaetocephala (4) S. liformis (1) 30-(32.0)-3 4 S 9-10 ZC II 11 S. hepperana (1) 42-(46.5)-5 2 SO-OS 9-11 ZC II 11 11 35-(37.0)-3 9 37-(42.3)-4 5 SO-OS SO-OS 8-10 (10-13 ) ZC ZC II II perforate, granulate perforate, granulate 11 S. pusilla (1) S. quadrisulcata (3) S. radiata (3) 29-(36.2)-4 4 OS (9)-10(13) ZC II perforate, granulate 12 S. lifolia (2) 50-(56.6)-6 4 S ca. 15 PC III 12 S. octodon (2) 37-(40.9)-4 5 S ca. 14 PC III 12 S. tenuissima (1) 47-(54.1)-5 7 S ca. 15 PC III 13 S. terminaliora (1) 52-(54.4)-5 7 S ca. 24 PC III 13 S. thymoidea (2) 50-(56.9)-6 3 S 24-25 PC III 14 S. arvensis (3) 75-(81)-8 6 O (16)-17(18) ZC 14 S. azurea (2) 62-(69.6)-7 6 O-SO (13)-15(16) 14 S. dibrachiata (7) 71-(84.8)-9 6 O-SO 14 S. hockii (4) 67-(72.4)-8 2 14 S. phyteumoides 65-(66.7)-6 9 var. phyteumoides (1) operculum present operculum present endocingulum perforate, granulateindistinct microechinate perforate, granulate columellae slightly longer in EP columellae slightly longer in EP colpi arranged in a loop colpi arranged in a loop colpi arranged in a loop perforate, granulate perforate, granulate nely granular, endocracks indistinct or absent granular, narrowÔdistinct endocracks granular, indistinct endocracks granular, narrow irregular endocracks nely granular, narrow distinct or obscure endocracks ? granular, narrow indistinct endocracks granular, narrow indistinct endocracks perforate, granulate to microechinate granular, narrow indistinct endocracks perforate, granulate to microechinate granular, narrow distinct or obscure endocracks perforate, granulate to microechinate granular, narrowÔdistinct endocracks colpi perforate, microechinate arranged in a spiral pattern CM granular, perforate, microechinate colpi arranged in a spiral pattern granular, numerous irregular endocracks I columellae distinctly longer in EP ? ZC I columellae distinctly longer in EP (16-21 ) ZC I columellae distinctly longer in EP SO 13-(15 ) ZC I columellae longer in EP O-SO 14-16 ZC I columellae distinctly longer in EP perforate to perforatereticulate in mesocolpia, microechinate, surface undulating perforate to perforatereticulate in mesocolpia, microechinate, surface undulating perforate to perforatereticulate in mesocolpia, microechinate, surface undulating perforate to perforatereticulate in mesocolpia, microechinate, surface undulating perforate to perforatereticulate in mesocolpia, microechinate, surface undulating granular, numerous irregular endocracks nely granular, broad distinct endocracks nely granular, broadÔdeep endocracks nely granular, narrow distinct endocracks nely granular, broadÔdeep endocracks Grana 41 (2002) 74 S. Dessein et al. Table II. (Continued). Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 Apertures Wall ornamentation Type Species Size Shape Number Type Endo Peculiaritie s Sexine Nexine 14 S. phyteumoides var. caerulea (1) 68-(70.7)-7 3 O-SO 14-15 ZC I columellae distinctly longer in EP nely granular, broad undeep endocracks 14 S. phyteumoides ``var. longituba ’’ (1) 75-(83.2)-9 0 SO 13-14 ZC I columellae somewhat longer in EP 14 S. stipularis (3) 75-(83.0)-9 2 O-SO (14)-15(17) ZC I columellae distinctly longer in EP perforate to perforatereticulate in mesocolpia, microechinate, surface undulating perforate to perforatereticulate in mesocolpia, microechinate, surface undulating perforate-microreticulate , microechinate ?15 S. articularis (1)* 72-(76.8)-8 2 SO 12-14 ZC I/II 15 S. congensis (1) 95-(99.0)-10 5 SO 12-14 ZC II 15 S. huillensis (2) 78-(80.0)-8 2 SO-OS 9-11 ZC ? 15 S. kirkii (1) 88-(97.0)-10 9 SO-OS 10-11 ZC ? 15 S. latituba (2) 88-(99.2)-11 0 SO (10)-12(13) ZC I 15 S. senensis (2) 75-(80.0)-8 4 SO-OS 11 ZC II 15 S. subvulgata (5) 73-(86.6)-9 4 SO-OS 10-(12 ) ZC I/II 15 S. taylorii (1) 100-(105.3)-11 0 SO 10-12 ZC II CM granular; perforate, microechinate colpiÔloxocolporate, nexine slightly thicker around ectoaperture microreticulate to reticulate, microechinate nexine perforate, microechinate slightly thicker around ectoaperture CM granular; perforate-microreticulate , nexine microechinate to echinate thicker around ectoaperture CM granular; perforate-microreticulate , nexine microechinate to echinate thicker around ectoaperture CM granular; perforate-microreticulate , nexine microechinate thicker around ectoaperture CM granular; perforate-microreticulate , nexine microechinate thicker around ectoaperture nexine Microreticulat e to reticulate, thicker microechinate around ectoaperture 16 S. bambusicola (4) 74-(90.0)-10 5 O-SO (13)-16 ZC II columellae longer in EP 16 S. ivorensis (1) 105-(115.5)-12 5 OS 16-18 ZC ? columellae longer in EP 17 S. deserti (1) 58-(62.0)-6 8 SO 11-13 ZC II 17 S. lituba (2) 77-(81.2)-8 7 SO-OS 12-(14 ) ZC ? ?17 S. intricans (1) 70-(73.2)-7 6 SO 9-10 ZC ? 17 S. princeae (2) 63-(71.9)-8 7 SO-OS 12-14 ZC II 17 S. ruelliae (2) 74-(79.1)-9 0 SO-OS ZC II ?17 S. spermacocina (2) 64-(70.1)-7 7 OS (13)-15(16) 8-9 ZC II 17 S. sphaerostigma (4) S. stachydea (2) 82-(91.0)-10 3 SO 11-(14 ) ZC II 72-(81.7)-9 1 SO-OS (12)-13(15) ZC II columellae longer in EP columellae longer in EP columellae slightly longer in EP columellae longer in EP columellae longer in EP columellae slightly longer in EP columellae longer in EP columellae longer in EP 17 Grana 41 (2002) nely granular, broad undeep endocracks granular, broad distinct endocracks nely granular, indistinct endocracks granular, indistinct endocracks ? ? granular, narrow deep endocracks granular, indistinct endocracks granular, narrow indistinct endocracks granular, narrow indistinct endocracks perforate-foveolate , margins of perforations bordered, echinate perforate, margins of perforations bordered, echinate nely granular, very broad distinct or obscure endocracks ? perforate, granulate to microechinate perforate, microechinate to echinate perforate, granulate to microechinate granular, narrowÔdistinct endocracks ? ? perforate-microreticulate , granulate to microechinate perforate-microreticulate , (micro)echinate perforate, granulate to microechinate nely granular, narrow deep endocracks granular, broad distinct endocracks nely granular, indistinct endocracks perforate, microechinate to echinate perforate, (micro)echinate granular, no endocracks granular, narrow distinct endocracks Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 Pollen of African Spermacoce species (Rubiaceae) 75 Fig. 1. Correlation analyzes between diVerent pollen and ower characters. (A) Correlation between corolla tube size and pollen size (r2 = 0.43; p<0.01); S. ivorensis was excluded because of its exceptional long corolla tube. (B) Correlation between P/E index and pollen grain size (r2 = 0.29; p<0.01) . (C ) Correlation between number of apertures and pollen size (r2 = 0.14; p<0.01) . (D) Correlation between number of apertures and pollen size with exclusion of the pantoaperturate species (r2 = 0.43; p<0.01). between pollen size and number of apertures (Fig. 1 C; r2 = 0.14; p<0.01). However, the pantoaperturate species have many more apertures than can be expected from their size. This is not surprising if we realize that the surface where the apertures can originate is much larger than for equally sized zonoaperturate species. Disregarding the pantoaperturate species, the positive correlation between number of apertures and pollen size is strong (Fig. 1 D; r2 = 0.43; p<0.01 ). This multi-aperturate condition is apomorphic within the family Rubiaceae, the 3-colporate pollen type being plesiomorphic (cf. Robbrecht 1988). Other genera of the tribe Spermacoceae also have multi-aperturate grains (Bremekamp 1952, Verdcourt 1958, Kirkbride 1979, Pire & Cabral 1992, Pire 1997 a, b), but it is rarely found in other Rubiaceae except in the tribe Rubieae (Bremekamp 1952). In this tribe, however, the number of colpi is less variable and mostly xed at 6 to 8 (Huysmans, pers. com.). Type. ± Apertures of Spermacoce are almost always compound, i.e. built up by two or more components that are situated in more than one layer of the pollen wall. In the species studied the apertures usually consist of an ectocolpus and an endocingulum (e.g., S. dibrachiata). In Spermacoce verticillata and S. latifolia the endoaperture is a colpus. Both species, however, are most probably introduced from America. Spermacoce annua and S. phyteuma are characterized by ecto- and endopores. Position. ± In most species, the apertures are ordered along the equator (zonoaperturate; Figs. 10±17), Spermacoce phyteuma and S. annua are pantoaperturate, the ectopores being evenly spread over the pollen surface (Fig. 9). Pire (1996: 422) noted in some Borreria species ``a tendency of the apertures to drift away from the equatorial plane; the apertural plane appears as suVering a torsion due to the presence of one or two pairs of convergent colpi’’. We clearly observed such loxocolporate colpi in Spermacoce articularis; in other species, e.g., S. chaetocephala, it was only sporadically observed. Two remarkable new colpi arrangements were also observed. The rst type, with the colpi arranged in a looplike pattern, similar to the line on a tennis ball, is found in Spermacoce lifolia, S. octodon, and S. tenuissima (Figs. 51±52 ). The second type, where the short colpi are arranged in a spiral pattern (Figs. 55±56 ), characterizes Spermacoce terminaliora and S. thymoidea. To our knowledge these two pollen types have not been previously observed in angiosperms. The second type can be considered as an intermediate form between zonoaperturate and pantoporate grains. An in-depth morphological and ontogenetic study is needed to further clarify these remarkable pollen types. Ectoaperture. ± As mentioned above, the ectoaperture is a colpus in most species (Figs. 18±24). The length of the colpi Grana 41 (2002) S. Dessein et al. Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 76 Figs. 2±9. Polar views of Spermacoce pollen grains. (2) 3-colporate grain, slightly triangular in outline, tectum eutectate and psilate (S. natalensis). (3) 4-colporate grain, sub-quadrangular in outline (S. natalensis). (4) 7-colporate grain, tectum perforate (S. verticillata). (5) 10-colporate grain, notice the elevation of the sexine around the ectoapertures (arrows), tectum microreticulate (S. taylorii ). (6) 11-colporate grain, columellae longer in the equatorial plane, tectum perforate (S. lituba). (7) 13-colporate grain, columellae longer in equatorial plane, tectum perforate-microreticulate (S. arvensis). (8) 19-colporate grain, columellae longer in equatorial plane, tectum perforate (S. dibrachiata). (9) pantopororate grain with ca. 24 apertures, tectum perforate-microreticulate and microechinate (S. phyteuma). Grana 41 (2002) 77 Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 Pollen of African Spermacoce species (Rubiaceae) Figs. 10±17. Equatorial views of Spermacoce pollen grains. (10) grain subprolate, margo of granules around ectoaperture (S. natalensis). (11) grain prolate-spheroidal (S. verticillata). (12) grain spheroidal, columellae in equatorial plane slightly longer (S. liformis). (13) grain suboblate, columellae longer in equatorial plane (S. lituba). (14) grain oblate-spheroidal, nexine thickened around ectoapertures forming a distinct margo, colpus membrane beset with large sexine particles (S. senensis). (15) grain suboblate, columellae longer in equatorial plane (S. azurea). (16) grain suboblate, columellae longer in equatorial plane, margins of colpi beset with microspines (S. phyteumoides). (17) oblate grain, columellae longer in equatorial plane, margins of colpi beset with microspines (S. dibrachiata). Grana 41 (2002) S. Dessein et al. Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 78 Figs. 18±26. Morphology of ectoapertures of Spermacoce pollen grains. (18) slit-like colpus with margo of granules (S. natalensis). (19) slit-like colpus, sexine undiVerentiated (S. latifolia). (20) slit-like colpus, margins beset with microspines, columellae longer in equatorial plane (S. dibrachiata). (21) colpus with margo of thickened nexine, colpus membrane beset with sexine particles (S. kirkii ). (22) slit like apertures with columellae slightly elongated in mesocolpium (S. phyteumoides). (23) detail of short colpus with margins beset with microspines (S. octodon). (24) short, spool-shaped aperture, colpus membrane beset with sexine particles (S. thymoidea). (25) three pores with operculum (S. annua). (26) pore with operculum of sexine particles (S. phyteuma). Grana 41 (2002) Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 Pollen of African Spermacoce species (Rubiaceae) compared with the polar axis (LC/P) ranges from 6 in Spermacoce terminaliora to 62 in S. tenuior. The colpi are often slit-like (Figs. 18±21), but sometimes more widened (Fig. 24 ). In the latter case, there is often a gap in the colpus membrane where the ecto- and endoapertures overlap. If present, the colpus membrane is beset with large cubicshaped elements. The margins of the ectocolpus are often beset with microspines (Figs. 20, 23 ). Sometimes, the exine around the colpi is diVerentiated (Figs. 18, 20±22). Most commonly, the columellae are longer in the equatorial zone, but a thicker nexine around the apertures and the presence of granules around the colpi (Fig. 18 ) were also observed. These features are further discussed below. The pantopororate species have pores up to 4 mm in diameter (Figs. 25±26). An operculum that covers the whole pore is present. In Spermacoce phyteuma it appears as an aggregate of angular, sexinous elements intermingled with smaller particles (Fig. 26); in S. annua it is made up of a granular aggregate that bears a central spine (Fig. 25). Endoaperture. ± The endoaperture is mostly an endocingulum, a ring-shaped endoaperture lying at the equatorial plane (Figs. 27, 29 ). The width of the endocingulum ranges from 1/3 to 2/3 of the length of the ectocolpi and often there are two triangular extensions (horns) in each mesocolpium. The inner surface is smooth or nely granular. Within the endocingulum, there is an additional thinning of the nexine at the ectocolpi. These thinnings are lalongate (Figs. 29±30) or lolongate (Figs. 27±28 ). In the former case, the thinning is oval- or diamond-shaped, and in the cut-aways, the surface is granular; in the latter case, the nexine abruptly declines near the ectocolpus and the surface becomes rough. Sometimes, this thinning is bordered by a granular thickening of the nexine within the endocingulum. In Spermacoce princeae, the endoaperture is not always continuous, the structure being disrupted by endocolpi that are not laterally fused. This transition between endocolpi and endocingulum has already been observed in other Rubiaceae (Dessein et al. 2000). In the species with a loop-like colpus arrangement, a continuous endoaperture connecting the ectocolpi is observed (Figs. 53±54 ). It cannot be named an endocingulum, since it is not situated at the equatorial plane. In each mesocolpium, two broad extensions are continuous with the pattern of endocracks. An additional, diamond-shaped thinning within this endopattern is present at the ectocolpus. In the cutaways a granular layer becomes visible. An equally complex endopattern is found in Spermacoce thymoidea and S. terminaliora (Figs. 57±58 ), where the ca. 24 colpi are interconnected by an irregular pattern of nexine thinnings: between two adjacent colpi a V-shaped or straight thinning of the nexine is present, with a triangular extension on one or both sides. A second, asymmetrical endocolpus perpendicular to the rst one can be present, often with branching tips. Around the ectocolpus, there is an oval thinning, revealing a perforated, granular layer. The endopores of Spermacoce phyteuma and S. annua are not distinct. In Spermacoce annua, it is only visible as a radiating pattern of ne endocracks within the inner surface of the nexine surrounding the ectopore; in S. phyteuma 79 (Figs. 33±34 ), the surface of the nexine enclosing the ectopore is pitted and coarser than the surrounding nexine. The lalongate endocolpus of Spermacoce verticillata is bordered by two islands of thickened nexine but the ends are vaguely delimited and more or less continuous with the endocracks (Fig. 31). In Spermacoce latifolia, an indistinct lolongate endocolpus is present (Fig. 32). In conclusion six types of endoapertures can be distinguished: Type I endocingulum with additional lolongate thinnings (Figs. 27, 28 ) Type II endocingulum with additional lalongate thinnings (Figs. 29, 30 ) Type III endopattern comprising an irregular pattern of nexine thinnings between the ectocolpi (Figs. 53, 54, 57, 58) Type IV lalongate endocolpus (Fig. 31) Type V lolongate endocolpus (Fig. 32 ) Type VI endopore (Figs. 33, 34 ) Sexine ornamentation The variation in sexine patterns within Spermacoce is considerable. The majority of species, however, have a perforated tectum. Perforation size ranges from 0.1 mm to 1 mm, and perforation density is species dependent (Figs. 36±40 ). In some species, small, circular perforations are intermingled with larger ones. In other species (e.g., S. latituba), there is a continuous transition into microreticulate tecta (Fig. 40). This character state is indicated as ``perforatemicroreticulate’’ in Table II. A true microreticulate to reticulate tectum (muri narrower than lumina; Fig. 41 ) only occurs in Spermacoce annua, S. congensis, and S. taylorii. The tectum of Spermacoce bambusicola tends to be foveolate (Fig. 42). In this species and in Spermacoce ivorensis the margins of the perforations are distinctly thickened. There is often a considerable diVerence between the sexine pattern observed at the mesocolpium and at the apocolpium. In Spermacoce natalensis, for example, the tectum is eutectate at the apocolpium and perforate at the mesocolpium. In Spermacoce dibrachiata the perforations are more elongated and much larger at the mesocolpium than at the apocolpium, the larger perforations being intermingled with smaller ones. The sexine pattern in which we have large lumina (often exceeding 1 mm) intermingled with small perforations with muri broader and smaller than the enclosed lumina is termed perforate-reticulate in Table II (cf. mesocolpia of Figs. 15±17). We preferred not to use the term heterobrochate because it refers to a true reticulate pattern (Punt et al. 1999), which is not the case here. In a number of species (e.g., S. azurea, S. dibrachiata) the tectum is undulating in the apocolpium (Fig. 38). The tectum is always beset with supratectal elements, except for Spermacoce natalensis, S. mauritiana, and S. tenuior where the apocolpium is smooth or slightly scabrate (Fig. 35). The supratectal elements can be granules, microspines or spines. They are scattered over the pollen surface, or more rarely restricted to the zone around the apertures to form a margo of granules or microspines (Fig. 18). Grana 41 (2002) S. Dessein et al. Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 80 Figs. 27±34. Endoaperture morphology of Spermacoce pollen grains. (27) inner view of a broken pollen grain showing endocingulum with additional lolongate thinnings at the ectoapertures (TE ) and in each mesocolpium an extension of the endocingulum (EE); notice the thickening of the nexine and the elongation of the columellae towards (arrow) the equatorial plane (S. azurea). (28) detail of additional lolongate thinning within endocingulum (S. latituba). (29) inside of pollen fragment with an endocingulum; nexine surface granular with numerous endocracks (S. stachydea). (30) detail of lalongate thinning within endocingulum (detail of 29). (31) detail of lalongate endocolpus with two islands of thickened nexine (TN) around endoaperture (S. verticillata). (32) detail of indistinct lolongate endocolpus (S. latifolia). (33) inside view at pollen half showing endopori (S. phyteuma). (34) detail of two endopori (detail of 33). Grana 41 (2002) 81 Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 Pollen of African Spermacoce species (Rubiaceae) Figs. 35±42. Ornamentation of the tectum of Spermacoce pollen grains. (35) detail of apocolpium, eutectate (S. natalensis). (36) detail of apocolpium, tectum with small perforations, granulate (S. octodon). (37) detail of apocolpium, tectum with large, elongated perforations, micro-echinate (S. azurea). (38) detail of apocolpium, tectum surface undulating, perforations large, micro-echinate (S. phyteumoides). (39) detail of apocolpium, tectum perforate-microreticulate, echinate (S. latituba). (40) detail of mesocolpium, tectum perforate-microreticulate, small perforations intermingled with larger ones, granulate to micro-echinate (S. princeae). (41) detail of mesocolpium, tectum microreticulate to reticulate, micro-echinate (S. congensis). (42) detail of apocolpium, tectum perforate to foveolate, margins of perforations thickened, (micro-)echinate (S. bambusicola). Grana 41 (2002) 82 S. Dessein et al. Columellae Columellae are always present and well developed (Figs. 43±46 ). Their length ranges from 0.2 to 2.6 mm at the apocolpium. In many species, the columellae are longer towards the centers of the mesocolpia, resulting in a protruding equatorial zone (Fig. 44 ). Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 Nexine The nexine is fairly thick ranging from 0.3 to 1.7 mm at the apocolpium. The inner surface of the nexine is nely or coarsely granular. Endocracks are mostly present; they can be narrow and supercial (Fig. 47 ), narrow and distinct (Fig. 48 ) or broad and deep (Figs. 49±50 ). Endocracks are mostly connected to the extensions of the endocingulum, but are absent in the zones between these extensions. In some species, the nexine is gradually thickened towards the equatorial plane (Fig. 27), in other species the nexine is distinctly thickened around the ectoapertures resulting in a protruding zone around the ectocolpi (Figs. 14, 21 ). The sexine/nexine rate varies between 0.9 and 2.1 in the apocolpium, but typically shows a value around 1.5. The value is strongly inuenced by the position of the measurement (even within the apocolpium) and is thus of minor importance in the characterization of the pollen grains. Pollen types Pollen of Spermacoce is so variable and the variation for each character so continuous, that it is diYcult to propose a matching typology without recognizing a large number of types. However, to make this study more compatible with the work of Pire (1996), we propose a typology based on the same characters she used: pollen size, aperture number, aperture position, relative length of the ectoaperture, and endoaperture type. In the key below, we also include the pollen types found among the American representatives of the Spermacoce/Borreria complex; the type numbers used correspond with the types of Pire (1996). Description of pollen types Only the pollen types found among species present in Africa are described here. The descriptions are based on the African material only. For a detailed description of the other pollen types keyed out below, see Pire (1996). Type 1 (Figs. 2±3, 10, 18, 35) Pollen zonocolporate (3±4) or (6±8), mean E 15.8±16.5 mm; mean P/E rate 1.1±1.2 (prolate-spheroidal, subprolate); polar outline triangular, quadrangular to almost circular. Ectocolpus long (LC/P 53±62 ), narrow, slightly sunken. Endoaperture narrow endocingulum. Tectum perforate to eutectate at apocolpium; margo of granules or microspines. Inner nexine surface nely granular. Species included: 3±4 colporate: S. mauritiana, S. natalensis. 6±8 colporate: S. tenuior. Remark: Other species with same pollen type listed by Pire (1996). Type 2 (Figs. 19, 32) Pollen zonocolporate (7±11), mean E 48.3 mm; mean P/E rate 0.88 (suboblate, oblate-spheroidal ); polar outline circular. Ectocolpus long (LC/P 38 ), narrow, slightly sunken. Endoaperture lolongate. Tectum perforate, uniformly granulate. Inner nexine surface nely granular with very broad, irregular endocracks. Species included: S. latifolia. Remark: Other species with same pollen type listed by Pire (1996). Type 3 (Figs. 4, 11, 31) Pollen zonocolporate (6±9), mean E ca. 28.0 mm; mean P/E rate 0.95 (oblate-spheroidal ); polar outline circular. Ectocolpus short (LC/P 20), narrow, slightly sunken. Endoaperture lalongate. Tectum perforate, uniformly granulate. Inner nexine surface granular with numerous endocracks. Species included: S. verticillata. Remark: Other species with same pollen type listed by Pire (1996). Type 6 (Figs. 9, 25±26, 33±34 ) Pollen pantopororate (24±30 ), mean E 66.5±87.0 mm; mean P/E rate ca. 1 (spheroidal ); polar outline circular. Ectopore 2±5 mm in diameter (LC/P 4.5±5), operculum present. Endoaperture an indistinct endopore. Tectum perforatemicroreticulate or (micro)reticulate, microspines sparsely present on whole surface. Inner nexine surface nely granular, endocracks absent. Species included: S. annua, S. phyteuma. Remarks: Other species with same pollen type listed by Pire (1996). Although included in pollen type 6 of Pire (1996), Figs. 43±50. Pollen wall stratication and morphology of inner nexine surface of Spermacoce pollen grains. (43) wall stratication at apocolpium showing nexine, row of short columellae and tectum with microspines; note regular endocracks in nexine surface (S. hockii ). (44) broken pollen wall at mesocolpium showing the elongation of the columellae (S. deserti). (45) cross section of pollen wall in mesocolpium showing from top to bottom: tectum with microspines (T), columellae (C), nexine (N ), endocingulum (E), lolongate thinning within endocingulum (TE), extensions of endocingulum (EE ) and nexine surface with endocracks (NE) (S. phyteumoides). (46) detail of stratication: from top to bottom: perforated tectum with microspines (T ), columellae (C ), and nexine (N ); note granules on nexine (S. dibrachiata). (47) inner surface of granular nexine with indistinct endocracks (S. subvulgata ). (48) inner surface of granular nexine with distinct, narrow endocracks (S. tenuissima). (49) inner surface of nely granular nexine with broad deep endocracks (S. dibrachiata). (50) inner surface of nely granular nexine with broad deep endocracks (S. phyteumoides). Grana 41 (2002) 83 Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 Pollen of African Spermacoce species (Rubiaceae) the pollen described here are larger and the apertures more numerous. Type 11 (Fig. 12) Pollen zonocolporate (8±13 ), mean E 32.0±46.5 mm; mean P/E rate 0.80±1.00 (suboblate, oblate-spheroidal ); polar outline circular. Ectocolpus short (LC/P 15±25 ), narrow, slightly sunken. Endoaperture endocingulum with two horns in each mesocolpium. Tectum perforate, granules (or microspines) uniformly present. Inner nexine surface nely granular with numerous narrow endocracks. Species included: S. assurgens, S. chaetocephala, S. liformis, S. hepperana, S. pusilla, S. quadrisulcata, S. radiata. Remark: Other species with same pollen type listed by Pire (1996). Type 12 (Figs. 23, 36, 48, 51±54) Pollen pantocolporate (ca. 15 ), E 40.9±56.6 mm; mean P/E Grana 41 (2002) 84 S. Dessein et al. rate ca. 1 (spheroidal ); polar outline circular. Ectocolpus short (LC/P 6±14 ) and narrow. Endopattern complex, including an ``endocingulum’’ with horns, an additional lalongate thinning of the nexine at the ectocolpus, and numerous, often deep endocracks. Tectum perforate, granules or microspines uniformly present. Inner nexine surface granular, but smooth within endocracks. Species included: S. lifolia, S. octodon, S. tenuissima. Remark: The exact number of apertures is diYcult to determine. Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 Type 13 (Figs. 24, 55±58) Pollen pantocolporate (ca. 24), mean E 54.4±56.9 mm; mean P/E rate ca. 1 (spheroidal ); polar outline circular. Ectocolpus short (LC/P 6±11) and narrow or somewhat widened, colpus membrane (if present) beset with granules. Endopattern complex, including an ``endocingulum’’ with horns, an additional lalongate thinning of the nexine at the ectocolpus, and numerous endocracks. Tectum perforate, small perforations intermingled with larger ones, microspines uniformly present. Inner nexine surface granular, numerous endocracks. Species included: S. terminaliora, S. thymoidea. Remark: The exact number of apertures is diYcult to determine. Type 14 (Figs. 7±8, 15±17, 20, 22, 27, 37±38, 43, 45±46, 49±50) Pollen zonocolporate (13±21), mean E 66.7±84.8 mm; mean P/E rate 0.7±0.8 (oblate, suboblate); polar outline circular, clearly lobed due to longer columellae in the equatorial zone. Ectocolpus relatively long (LC/P 24±45 ), slit like, margins beset with microspines. Endoaperture a broad endocingulum with horns in the mesocolpia and with an additional lolongate thinning of the nexine at the ectocolpus. Tectum perforate, often perforate-reticulate at the mesocolpia, large perforations intermingled with smaller ones, surface often undulating; microspines uniformly present. Inner nexine surface nely granular with broad, undeep or deep, smooth endocracks. Species included: S. arvensis, S. azurea, S. dibrachiata, S. hockii, S. phyteumoides, S. stipularis. Remark: Pollen of Spermacoce dibrachiata diVers from the other species included in its high number of colpi. Type 15 (Figs. 5, 14, 21, 28, 39, 41, 47 ) Pollen zonocolporate (9±14 ), mean E 76.8±105.3 mm; mean P/E rate 0.82±0.88 (suboblate, rarely oblate-spheroidal ); polar outline circular, slightly or strongly lobed due to thickened nexine around ectocolpi. Ectocolpus short (LC/P 10±23) and relatively broad, colpus membrane (if present) beset with large cubic-shaped particles; nexine thickened around the ectocolpus, forming a distinct margo. Endoaperture a broad endocingulum with an additional lalongate or lolongate thinning of the nexine at the ectoaperture. Tectum perforate or perforate-microreticulate, rarely reticulate; margins of the perforations sometimes slightly thickened; microspines or spines uniformly present. Inner nexine surface granular with numerous, often indistinct endocracks. Species included: S. congensis, S. huillensis, S. kirkii, S. latituba, S. senensis, S. subvulgata, S. taylorii. Tentatively included: S. articularis. We found only one African collection with suYcient owers to carry out palynological research; the nexine around the ectocolpi is thickened, but only slightly so. Remark: In S. congensis, the margo is not distinct, but it ts very well within this pollen type for all other characters. Type 16 (Fig. 42 ) Pollen zonocolporate (13±18), mean E 90±115.5 mm; mean P/E rate 0.7±0.9 (oblate, oblate-spheroidal ); polar outline circular, lobed due to longer columellae in the equatorial plane. Ectocolpus short (LC/P 14.6±24.6), slit like. Endoaperture a broad endocingulum with horns in the mesocolpia and with a rather indistinct lalongate thinning at the ectocolpus. Tectum perforate or foveolate, margins of perforations thickened, spines uniformly present. Inner nexine surface granular with broad, undeep, smooth endocracks. Species included: S. bambusicola, S. ivorensis. Type 17 (Figs. 6, 13, 29±30, 40, 44) Pollen zonocolporate [(8±10 )±11±16 ], mean E 62.0±91.0 mm; mean P/E rate 0.8±0.92 (suboblate, rarely oblate-spheroidal ); polar outline circular, often (slightly) lobed due to longer columellae at the equatorial plane. Ectocolpus relatively short (LC/P 18±24), slit like. Endoaperture a broad endocingulum with or without horns at the mesocolpia but always with an additional, lalongate thinning at the ectocolpus. Tectum perforate or perforate-microreticulate, large perforations often intermingled with smaller ones; microspines or spines uniformly present. Inner nexine surface granular with or without distinct endocracks. Species included: S. deserti, S. lituba, S. princeae, S. ruelliae, S. sphaerostigma, S. stachydea. Tentatively included: S. intricans and S. spermacocina. Both diVer from the other species included in the lower number Figs. 51±58. Detailed morphology of pollen types 12 & 13 of Spermacoce. (51) pollen grain of type 12 showing two rows of ectocolpi (S. lifolia). (52) diVerent view of same pollen grain as in (51 ) showing colpi arranged in a loop. (53) view of inner side of an half of pollen showing complex endopattern with an ``endocingulum’’ (E ), lalongate thinnings within endocingulum (TE), extensions of endocingulum (EE), and nexine (NE ) surface with numerous endocracks (S. tenuissima). (54) Detail of (53). (55) pollen grain of type 13 showing colpi arranged in a spiral (S. thymoidea). (56) diVerent view of same pollen grain as in (55 ). (57) view of inner side of an half of pollen showing complex endopattern with cross-linked endocolpi, lalongate thinnings (TE ) within endocingulum (E ), extensions of endocingulum (EE), nexine surface (NE) with numerous endocracks (S. thymoidea). (58) detail of (57) showing the same elements. Grana 41 (2002) Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 Pollen of African Spermacoce species (Rubiaceae) 85 Grana 41 (2002) 86 S. Dessein et al. of apertures, the only weak elongation of the columellae in the EP, and in the granulate to microechinate ornamentation. They are intermediate between types 11 and type 17. Key to pollen types 1. 2(1). Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 3(1). 4(3). 5(3). 6(5). 7(5). 8(7). 9(8). 10 (9). 11 (8). 12 (11). 13 (7). 14 (13). 15 (14). 16 (15). Ectoapertures pores ............................................. 2 Ectoapertures colpi .............................................. 3 Pollen zonopo(ro)rate ............. Type 5 (American) Pollen pantopo(ro)rate ........................................... .................................. Type 6 (American+ African) Pollen pantocolporate ......................................... 4 Pollen zonocolporate ........................................... 5 Colpi arranged in a loop ........... Type 12 (African) Colpi arranged in a spiral ......... Type 13 (African) E usually smaller than 20 mm; margo of granules .. .............................................................................. 6 E usually larger than 20 mm; granules evenly scattered over pollen surface ..................................... 7 Endoaperture an endocingulum .............................. .................................. Type 1 (American+ African) Endoaperture an endocolpus .. Type 7 (American) Endoaperture an endocolpus .............................. 8 Endoaperture an endocingulum ......................... 13 Colpi relatively long (L/P>35 ) .......................... 9 Colpi relatively short (L/P<35 ) ........................ 11 E smaller than 30 mm; 6-7-colporate ....................... .................................................. Type 8 (American) E larger than 30 mm; 8±10-colporate .................. 10 Tectum perforate ..................... Type 2 (American) Tectum reticulate and heterobrochate ..................... .................................................. Type 9 (American) Colpi relatively broad ............. Type 4 (American) Colpi slit-like ...................................................... 12 E smaller than 20 mm ............. Type 10 (American) E larger than 20 mm ................. Type 3 (American) E usually smaller than 50 mm .................................. ................................ Type 11 (American+ African) E larger than 50 mm ........................................... 14 Colpi relatively long (LC/P 24±45 ); number of apertures mostly more than 14 (rarely 13 ); columellae distinctly longer in equatorial zone; endocingulum with an additional lolongate thinning .............. .................................................... Type 14 (African) Colpi shorter; number of apertures usually less than 14; columellae not so distinctly longer in equatorial zone; endocingulum usually with an additional lalongate thinning ............................................... 15 Nexine distinctly thicker around the ectoapertures, visible as a distinct margo, if margo indistinct, colpi very short (LC/P < 15) and wide; colpus membrane (if present) beset with cubic-shaped particles ......... .................................................... Type 15 (African) Margo absent, colpi usually relatively long (LC/P>15) and slit-like; colpus membrane sometimes beset with cubic-shaped particles .............. 16 Margins of perforations thickened .......................... .................................................... Type 16 (African) Margins of perforations not thickened ................... .................................................... Type 17 (African) Grana 41 (2002) DISCUSSION African vs. American Spermacoce species In comparing our results with the palynological research of Pire (1996) on the American representatives of the Spermacoce-Borreria alliance, some diVerences can be observed. Pire recognized 11 pollen types, of which only three are observed among native African species, namely type 1 with small pollen grains and long colpi, type 6 with pantopo(ro)rate pollen grains, and type 11 with medium sized grains with an endocingulum. Six additional pollen types are described here, which are exclusively found in the African Spermacoce species. Two of these pollen types have aperture congurations not previously recorded for the angiosperms. In general, the following diVerences between the pollen grains of African and American species are observed: 1. Sexine patterns are more heterogeneous: perforate to (micro)reticulate or foveolate in African species, vs. perforate in most American species; 2. The apertural region is more complex in African than in American species (presence of margines and multiple endoapertures), and the pantoaperturate condition is more diVerentiated in African species; 3. Pollen size range is much larger among African species, and correlated with this also the variation in the number of apertures; 4. The number of pantopo(ro)rate pollen grains is lower among African species (5% vs. 25%); 5. In all but the pantopororate African species, the endocolpi are laterally joined to form an endocingulum. These diVerences support the recognition of additional ``patterns of pollen variation’’ for the evolutionary scheme proposed by Pire (1996). These are discussed in the following paragraph. Patterns of pollen variation The pollen variation among the Spermacoce-Borreria alliance perfectly illustrates Van Campo’s statement that ``amplitude of pollen variations within a homogenous assemblage pregures, in some cases, the possible pollen types within an assemblage higher in the classication’’ (Van Campo 1976: 126). Indeed, the diVerent pollen types found in Spermacoce/ Borreria cover to a large extent the pollen variation observed in Rubiaceae as a whole. Mathew & Philip (1983) surveyed the pollen morphology of the Indian representatives of the family and found ve major apertural types: colpate, inaperturate, colporate, porate and pororate. The latter three were all found in the genus Spermacoce sensu lato. At this point an evolutionary hypothesis for the Spermacoce sensu lato species based on molecular and morphological data or even a genus circumscription everyone agrees on is not available. Hence, a detailed analysis of the evolution of pollen characters is not possible. Nevertheless, in the light of the present study of the African taxa, we can consider the evolutionary sequence of pollen types proposed for Borreria by Pire (1996). Her evolutionary sequence postulated four ``tendencies’’: Pollen of African Spermacoce species (Rubiaceae) Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 1. extension of the apertural area; from zonoaperturate pantoaperturate; 2. reduction of the ectoapertures; from long colpishort colpipores; 3. narrowing of the endoapertures; from zonorate lalongatelolongate; 4. increase of the number of apertures; from few apertures many apertures. Many authors have previously proposed these trends in other groups of angiosperms ( Keddam-Malplanche 1985, Kuprianova 1969, Lewis 1965, Punt 1976, Thanikaimoni 1986, Van Campo-Duplan 1949, Van Campo 1966, 1976), and they are possibly present in Spermacoce species native to Africa. However, comparison between Pire’s (1996) proposed evolutionary sequences and the pollen features observed in the African Spermacoce species, raises some additional points. First of all pollen grains of the African species are signicantly larger on average than those of the neotropical species. Increase of pollen size is thought to represent an evolutionary tendency of simple grains ( Van Campo-Duplan 1949, Keddam-Malplanche 1985). Within the African Spermacoce species, this tendency is correlated with an increase of the number of apertures (see above). Furthermore, at least some tendencies ± or as called by Van Campo (1976 ) ``patterns of variation’’ ± should be added here: 1. lumina smalllumina larger; 2. ectocolpi without margoectocolpi with margo; 3. spiralization. The rst two patterns of variation are illustrated in Figs. 35±42 and Figs. 18±26 respectively, and were inter alia proposed by Punt (1976). The third one, spiralization, was introduced by Van Campo (1976) to describe a pattern in which the line joining the centers of the apertures shows spiralization. In this pattern, a process that involves shortening, spiralization of the position, and symmetrization of the ectoapertures leads to the pantoaperturate condition. The pattern was observed in Malvaceae ( Van Campo 1976), and can partly explain the two special pollen types (type 12 & 13 ) observed in Spermacoce. In conclusion, one can hypothesize that notwithstanding the supercial resemblance between pollen grains of neotropical and African species, they developed independently. The ancestral pollen type in Spermacoce is most probably a 3-colporate pollen grain that resembles type 1. Such 3-colporate pollen grains are also found in most members of the Hedyotideae, and it is thought to be a primitive Rubiaceae pollen grain type (Mathew & Philip 1983, Robbrecht 1988). In this context, it is of special interest that the most ubiquitous (and less specialized) Spermacoce species are characterized by these 3-colporate pollen grains. Other widespread species, such as Spermacoce assurgens also have relatively ``primitive’’ pollen grains. Pollen types which are considered more derived ( Types 6, 12, 13 ) often characterize species with a very narrow distribution. 87 Systematic value Species delimitation Pollen of Spermacoce s.l. is very heterogeneous, so that in many cases species can be determined by their pollen grains only. As a consequence, pollen characters provide stable and reliable characters for dening and delimiting species. This is a rather uncommon situation in Rubiaceae. Except for other europalynous tribes (e.g., Psychotrieae), pollen grains have only limited value in species delimitation. Up to now Spermacoce species were mainly dened on the basis of general habit, peculiarities of the owers, seed morphology and type of fruit dehiscence. These characters, however, can be very variable, hence making the limits of some species unclear. Pollen grains often provide evidence to sharpen species boundaries and to place ``diYcult’’ specimens. An example is given by the delimitation of Spermacoce senensis and S. sphaerostigma (see Verdcourt 1976). The former is a very variable annual (its height can range from a few cm to over half a meter), and in its owering state it is diYcult to separate from Spermacoce sphaerostigma. Pollen grains, however, clearly separate the two species: Spermacoce senensis has a margo of thicker nexine, while S. sphaerostigma has not. The present study also provides additional support for taxonomic decisions at the species level made by other investigators. A clear example is given by ``Borreria bambusicola’’, a species described by Berhaut (1973) based on material which had been identied as Borreria compressa (correct name: Spermacoce hepperana). He mainly distinguished Borreria bambusicola on the basis of its larger owers and its seeds bearing an elaiosome. Our pollen data give further support to this view: Spermacoce bambusicola has large pollen grains with a foveolate tectum and with bordered perforations, while Spermacoce hepperana has small pollen grains with a less diVerentiated perforated tectum. Pollen data sometimes refute taxonomic hypotheses; this is illustrated by the problematic delimitation of Spermacoce thymoidea. Verdcourt (1975) relegated ``Borreria hockii’’ (R.D. Congo) to the synonymy of Spermacoce thymoidea (Angola), and also used the name for some Zambian specimens. His decision was mainly based on habit and ower morphology, and was at rst sight fairly convincing. After observing pollen (and seeds), however, we were able to demonstrate that three distinct species are involved, the two cited ones and a new one (Dessein et al. in press). Generic and subgeneric delimitation Pire & Cabral (1992 ) recognized Galianthe at the generic level inter alia based on the peculiarities of its pollen grains, and Pire (1996 ) presented pollen morphological support for the subgeneric delimitation of Borreria proposed by Bacigalupo & Cabral (1996). So, at rst sight, pollen has value in delimiting genera and subgenera within the Spermacoceae. This consideration, however, should be handled with care. In our study, we were not able to nd any correlation between the described types of fruit dehiscence and the pollen morphology observed. This can be an indication that the fruit typology is not useful, or that the Grana 41 (2002) Downloaded by [Ingenta Content Distribution (Publishing Technology)] at 19:03 13 October 2014 88 S. Dessein et al. pollen morphology is too plastic to cluster groups at the generic level. The former hypothesis is supported by the observation that pollen type 1 is found in three species, each with a diVerent type of fruit dehiscence. Spermacoce mauritiana, a widespread species in Africa, Asia, as well as the neotropics, has fruits dehiscing from the apex downwards into four valves with the remnants of the septum remaining attached to the valves, which is in agreement with the denition of Borreria. Spermacoce natalensis has fruits splitting septicidally into tardily dehiscent mericarps, and for this reason it was once described under Diodia, a genus traditionally dened as having fruits splitting into indehiscent mericarps, but recently (Bacigalupo & Cabral 1996, 1999) redened as having indehiscent fruits. Spermacoce tenuior has fruits splitting into one dehiscent and one indehiscent fruit valve, and in this corresponds to the strict denition of Spermacoce (cf. above). In all other morphological characters, however, the three species are very similar. They all have small owers (corolla tube usually shorter than 2 mm) grouped in lateral inorescences, and the anthers are not or only slightly exserted. The same pollen type 1 is found in morphologically similar American ``Borreria’’ species (included by Bacigalupo & Cabral (1996) in subgenus Borreria subgenus Borreria section Pseudodiodia). The strong possibility that these species form a natural group, hence refuting the value of fruit characters in the delimitation of genera within the tribe, awaits support from molecular studies. What becomes clear, however, is that pollen, as well as other morphological characters do not correlate with the types of fruit dehiscence, an observation that strongly questions the taxonomic value of this carpological character. Pollen characters on the contrary are rather well supported by other morphological data. Pollen type 1 is also found in the neotropical Psyllocarpus sect. Psyllocarpus ( Kirkbride 1979). The latter genus is distinguished from the Spermacoce/Borreria group on the basis of the laterally compressed fruits and seed peculiarities. In other characters section Psyllocarpus is similar to the species mentioned above. Kirkbride (1979 ) already remarked that section Psyllocarpus is probably more closely related to Borreria by its vegetative structures and is less specialized than the other section, i.e., sect. Amazonica. Other groups of morphologically distinctive and similar species also share pollen types: 1. Type 12 (with the colpi arranged in a loop-like pattern) is limited to three species. Two of these, namely Spermacoce octodon and S. lifolia, once formed a genus on their own, namely Octodon. Its rounded, minute calyx lobes, the cup-shaped stipules that hold the owers, and the very slender habit, characterized the genus. The third species, Spermacoce tenuissima, has never been assigned to Octodon, but shares the same habit and the minute calyx lobes. As a consequence, pollen morphology clearly oVers arguments for resurrecting the (sub)genus Octodon. 2. Type 15 is found in Spermacoce congensis, S. huillensis, S. kirkii, S. latituba, S. senensis, S. subvulgata, and S. taylorii. Verdcourt (1976) already pointed to the close relationship between Spermacoce kirkii and S. subvulgata, Grana 41 (2002) but a close relationship between the other species was never hypothesized. Nevertheless Spermacoce congensis, S. kirkii, S. subvulgata, and S. taylorii all have a spikelike inorescence, two calyx lobes, and relatively large owers. Spermacoce huillensis, S. latituba, and S. senensis mainly diVer in the presence of four calyx lobes. Verdcourt (1975, 1976), however, described a form of Spermacoce subvulgata with a four-lobed calyx as variety quadrisepala, hypothesizing (and thereby suggesting implicitly possible relationship) that the specimen may well be a hybrid between S. senensis and S. subvulgata. 3. Another group of related species that share the same pollen type is formed by Spermacoce azurea, S. hockii, and S. stipularis. All three have a woody taproot, lanceolate leaves, two calyx lobes, large owers, seeds with an elaiosome (except the seeds are unknown for S. azurea), and type 14 pollen grains. All three are conned to high plateaus of the Zambezian regional center of endemism. 4. Spermacoce thymoidea and S. terminaliora are both narrow endemics of the Angolan high plateaus. They share the same habit, but in all other characters they diVer considerably. Whether the shared pollen type 13 is an adaptation to the specic environmental conditions or whether the species are sister taxa needs further study. In conclusion, pollen characters provide almost unique markers for many species in this group, enabling sharpening of species boundaries. Whether this variation is also useful to identify some groups of related species remains to be conrmed by incorporation of additional data, but there is good reason to believe it often is. The groups characterized by the same pollen type turn out to be in serious conict with the established generic concepts based on fruit characters only. ACKNOWLEDGEMENTS This study was supported by a grant form the Research Council of the K.U.Leuven (OT/01/25) and by a grant from the F.W.O.Vlaanderen (G.104.01) . Suzy Huysmans is a postdoctoral fellow, Steven Dessein a research assistant of the F.W.O. We are indebted to the curators of the herbaria of WAG, K and ZT for providing pollen material for this investigation. Special thanks are due to Anja Vandeperre for technical assistance and to Marcel Verhaegen for kindly taking the scanning electron micrographs. 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