Grana ISSN: 0017-3134 (Print) 1651-2049 (Online) Journal homepage: https://www.tandfonline.com/loi/sgra20 Pollen of African Spermacoce species (Rubiaceae) Morphology and evolutionary aspects Steven Dessein, Suzy Huysmans, Elmar Robbrecht & Erik Smets To cite this article: Steven Dessein, Suzy Huysmans, Elmar Robbrecht & Erik Smets (2002) Pollen of African Spermacoce species (Rubiaceae) Morphology and evolutionary aspects, Grana, 41:2, 69-89, DOI: 10.1080/001731302760156882 To link to this article: https://doi.org/10.1080/001731302760156882 Published online: 05 Nov 2010. Submit your article to this journal Article views: 821 View related articles Citing articles: 6 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=sgra20
Grana 41: 69± 89, 2002 Pollen of African Spermacoce species (Rubiaceae) Morphology and evolutionary aspects 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 re ected 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 con guration not yet recorded for the angiosperms in general. Some evolutionary trends are proposed that await veri cation by further systematic study. Pollen morphological characters have a high taxonomic value in the genus Spermacoce. They provide almost unique identi cation 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, Laboratory 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: Sperm- 1989, Deb & Dutta 1984, Sivarajan et al. 1987), is divided acoceae) is a large complex with a worldwide distribution in by others (e.g., Bacigalupo 1972, Steyermark 1972, 1974, the tropics and subtropics. The estimated number of species Bacigalupo & Cabral 1996) on the basis of peculiarities of varies between 150 and 250 ( Verdcourt 1989), depending on the fruit dehiscence. The segregated genus Borreria G.Mey. the authors. The plants typically grow in woodlands and is then characterized by having capsules with a septicidal grasslands, and are characterized by a herbaceous habit, opening and both fruit valves dehiscent, and Spermacoce mbriate stipules connected to the petioles, mostly small, sensu stricto by its fruits splitting into two valves, one isostylous owers arranged in compact lateral and terminal dehiscent and the other indehiscent (Meyer 1818). These in orescences, valvate bud aestivation, uni-ovulate ovary fruit characters are easy to observe, but no other features locules, dry capsular fruits, and presence of raphides. The seem to be correlated with it (cf. Verdcourt 1975, 1976, Deb group is, along with 18 smaller genera, placed in the tribe & Dutta 1984, Sivarajan et al. 1987). In the present article Spermacoceae, subfamily Rubioideae ( Robbrecht 1988). A we accept Verdcourt s view (1975) and relegate Borreria to molecular study of Andersson & Rova (1999) con rmed the the synonymy of Spermacoce. monophyly of the tribe, but the group was nested within Only few, limited studies exist on the pollen of Spermacoce. members of the tribe Hedyotideae, which makes the latter The earlier ones were part of broader studies on the phylogeny paraphyletic (see also Bremer et al. 1995, Natali et al. 1995, of Rubiaceae (e.g., Bremekamp 1952, Verdcourt 1958). Bremer 1996, Bremer & Manen 2000). For this reason, All reported that pollen of Spermacoce is multi-aperturate, Bremer ( 1996) proposed the Spermacoceae sensu lato, including but they did not fully survey the morphological variation the former tribes Hedyotideae, Knoxieae and Manettieae. within the genus. More recently, pollen studies of the In the present paper, the term Spermacoceae is used in its American representatives of Spermacoceae demonstrated the more traditional, narrow sense (cf. Robbrecht 1988). systematic value of pollen characters in the tribe. Pollen Generic delimitations within the complex have been characters were used to re-establish the genus Galianthe debated for almost two centuries. Spermacoce L. broadly (Cabral 1991, Pire & Cabral 1992, Pire 1997a) and to de ned by some authors (e.g., Verdcourt 1975, 1976, 1983, support a new subgeneric classi cation for the American
70 S. Dessein et al. â Ö Ö species of Psyllocarpus ( Kirkbride 1979) and Borreria variation in E-averages frequently exceeds 20%. Sometimes, ( Bacigalupo & Cabral 1996, Pire 1996, Cabral & Bacigalupo this variation can be explained in terms of intraspeci c 1999). Pollen morphology was also used to erect the genus variation, as is the case for Spermacoce phyteumoide s: var. Phylohydrax for the African and Madagascan Hydrophylax phyteumoides and var. caerulea have smaller pollen grains species (PuV 1986). Until now, pollen of the African than ``var. longituba. Verdcourt informally recognized the Spermacoce species was never investigated. latter variety on the herbarium sheets for its longer corolla The present study aims (1) to survey the pollen morpho- tube. In general there is a positive correlation in the study logy of the African representatives of Spermacoce, ( 2) to group between size of the corolla tube and pollen size discuss its signi cance in the taxonomy of the genus, and (3) (Fig. 1 A; r 2 =0.43; p<0.01): larger owers tend to produce to formulate a hypothesis for the evolution of pollen characters larger pollen grains. This correlation is also found in many within the genus. The project is part of the ongoing other groups and may be correlated with diverences in research of the rst author to document the biodiversity of nutrition supply (Muller 1979). Spermacoce in Africa. In the present study, however, the correlation between ower and pollen grain size is not strict. Flowers with a MATERIAL AND METHODS corolla tube smaller than 3 mm have pollen grains ranging from ca. 15 mm up to 60 mm, while medium sized owers This study is based on herbarium material from BR, K, WAG and (tube ca. 7 mm) show pollen grains between 55 mm and ZT (Table I ). Pollen of 92 specimens from 43 species were investi- 105 mm. Moreover, very variable species, such as Spermacoce gated (Table I). For the remaining African species (ca. 25 ) owering subvulgata, have corolla tubes ranging from 3 to 6 mm, but material in the above mentioned herbaria was not suycient to carry out palynological research. Due to the problematic delimitation of no correlated diverences are observed in the size of their several species of Spermacoce, we only selected specimens that fully pollen. Probably, the true correlation lies in the pollen vector correspond with the type specimen (if seen) or with the original size, rather than in the size of the owers. Although only a descriptions; additional comments on some problematic species/ few studies (cf. Muller 1979) have demonstrated that diverent specimens are provided ( Table I ). Species introduced to Africa are pollen vectors prefer diverent ranges of pollen size, it seems marked with an asterisk; the table also indicates the specimens used likely that this partly explains the considerable interspeci c for the illustrations. Mature ower buds were rehydrated in an Agepon (Agfa pollen size variation. Gevaert) solution (1:200), and dissected using a stereomicroscope. Another hypothesis to explain the pollen size variation is Pollen was acetolysed for 9 minutes in a heating block at 90ß C. the level of polyploidy. Higher levels of polyploidy may Pollen grains for SEM studies were suspended in ethanol (70%), express itself directly in a larger pollen size (Muller 1979). pipetted onto specimen stubs, air-dried and coated with gold with a Herbaceous Rubiaceae are known to have high levels of SPI-MODULE TM sputter coater. Observations were made under polyploidy (cf. Lewis 1965, Kiehn 1986). Kiehn ( 1986, 1995) a Jeol JSM-6400 SEM. Grains for LM studies were mounted in Kaiser s glycerin jelly. The slides were observed using a Leitz Dialux surveyed the chromosome numbers of the Rubiaceae; for the 20 with a 100 oil immersion or 40 objective lens. Equatorial Spermacoceae, the basic chromosome number is predomidiameter (E) was measured under LM in at least ten mature pollen nantly 14, and tetraploidy and hexaploidy originated several grains (magni cation 1000). We were not able to obtain accurate times within the tribe ( Kiehn 1986). Moreover, diverent measurements for the polar axis (P) under LM, since most grains ploidy levels are recorded for specimens of the same species; are suboblate. All other measurements were made on SEM-graphs. hence, giving support for the hypothesis that some of the To express the relative length of the colpi, we used the length intraspeci c pollen size variation is caused by diverences in colpi/polar axis rate multiplied by 100 (=LC/P). Broken pollen grains were obtained by shaking a pollen and glass bead suspension ploidy levels. as described by Huysmans et al. (1994). Terminology follows Punt In many genera of Rubiaceae, diverences in pollen size et al. (1999). may be explained by heterostyly (Huysmans et al. 1998, Dessein et al. 2000). In general, the brevistylous form has RESULTS larger pollen grains than the longistylous morph (cf. Ganders 1979). African Spermacoce species, however, are invariably 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 homostylous, hence this hypothesis can not explain the intraspeci c 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; r 2 =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.
Table I. Specimens examined including type- and gure references. Pollen of African Spermacoce species (Rubiaceae) 71 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 con rmation; 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. Mutimushi 3356 Zambia K 6 Fig. 25 *S. articularis L.f. 1, 2 Milne-Redhead & Taylor 7522 Tanzania BR 15 S. arvensis (Hiern) Good Bingham 8946 Zambia K 14 Pope, R-Smith & Goyder 2226 Zambia K Bidgood, Monasunki & Vollesen 1062 Tanzania BR Fig. 7 *S. assurgens Ruiz & Pav. 3 Faulkner 943 Tanzania BR 11 S. azurea Verdc. Bidgood, Mbago & Vollesen 2528 Tanzania K 14 Richards 12162 Tanzania BR Figs. 15, 27, 37 S. bambusicola (Berhaut) Vanden Berghen 6674 Senegal BR 16 J.-P.Lebrun & Stork Vanden Berghen 8522 Senegal BR Thomas 2731 Sierra Leone K Fig. 42 Adam 12589 Guinea BR S. chaetocephala DC. Berhaut 2933 Senegal BR 11 Leeuwenberg & Beek 10375 Cameroon BR Leippert 5071 Kenya BR Lisowski B-1172 Tchad BR S. congensis (Bremek.) Verdc. Pawek 13957 Malawi BR 15 Fig. 41 S. deserti N.E.Br. Skarpe S-444 Botswana K 17 Fig. 44 S. dibrachiata Oliv. Bidgood, Mbago & Vollesen 2695 Tanzania BR 14 Figs. 8, 17, 20, 46, 49 Lejeune 197 D.R. Congo BR Chapman 272 Malawi BR Troupin 14994 Rwanda BR Pereira, Sarmento & Marques 1829 Mozambique BR Lewalle 1694 Burundi BR Saintenoy 162 Burundi BR S. lifolia (Schumach. & Thonn.) de Wilde & de Wilde-Duyfjes 3091 Cameroon BR 12 Figs. 51-52 J.-P.Lebrun & Stork Geerling & Bokdam 984 Ivory Coast BR S. liformis Hiern Dalziel 212 Nigeria K 11 Fig. 12 S. lituba ( K.Schum.) Verdc. Faulkner 2168 Tanzania BR 17 Drummond & Hemsley 3632 Tanzania BR Figs. 6, 13 S. hepperana Verdc. Lejoly 85/314 Burkina Faso BR 11 S. hockii (De Wild.) Dessein Homble 796 D.R. Congo BR 14 Schaijes 1467 D.R. Congo BR Fig. 43 Schaijes 1960 D.R. Congo BR Duvigneaud & Timperman B-2627 D.R. Congo BR S. huillensis (Hiern) Good Quarre 1957 D.R. Congo BR 15 Hess-Wyss 52/584 Angola ZT S. intricans (Hepper) H.M.Burkill Bos, van der Laan & Nzabi 10676 Gabon WAG?17 S. ivorensis Govaerts Geerling & Bokdam 1701 Ivory Coast BR 16 S. kirkii (Hiern) Verdc. Schlechter 12123 Mozambique BR 15 Fig. 21 *S. latifolia Aubl. Lisowski 56863 D.R. Congo BR 2 Figs. 19, 32 Hepper & Maley 7760 Ivory Coast BR S. latituba ( K.Schum.) Verdc. Scott s.n. Malawi K 15 Figs. 28, 39 McClounie 84 Zambia K
72 S. Dessein et al. Table I. (Continued). Taxon Collection Country Herb. Type Figure S. mauritiana Osia Gideon 4 Evrard 157 D.R. Congo BR 1 Quarre 7867B D.R. Congo BR Vanden Berghen 9164 Senegal BR S. natalensis Hochst. Polhill & Paulo 1419 Tanzania BR 1 Figs. 3, 10, 18, 35 Thulin & Mhoro 3128 Tanzania WAG Fig. 2 S. octodon (Hepper) Hakki de Wilde 992 Ivory Coast BR 12 Lely 522 Nigeria K Figs. 23, 36 S. phyteuma Schweinf. ex Hiern Simpson 7325 Sudan K 6 Figs. 9, 26, 33± 34 S. phyteumoides Verdc. var. King 3 Zambia BR 14 Figs. 16, 45, 50 phyteumoides S. phyteumoides Verdc. var. Richards 8431 Tanzania K Fig. 38 caerulea Verdc. S. phyteumoides Verdc. ``var. Carter, Abdullah & Newton 2582 Tanzania K Fig. 22 longituba 5 S. princeae ( K.Schum.) Verdc. Ngoundai 250 Tanzania BR 17 Fig. 40 Huxley 84 Tanzania BR S. pusilla Wall. Malaisse 9691 D.R. Congo BR 11 S. quadrisulcata (Bremek.) Verdc. Schmitz 7228 D.R. Congo BR 11 Homble 930 D.R. Congo BR Troupin 1407 D.R. Congo BR S. radiata (DC.) Hiern Berhaut 435 Senegal BR 11 Robbrecht & Leman 3377 Niger BR Vanden Berghen 6197 Senegal BR S. ruelliae DC. Leeuwenberg 2323 Ivory Coast BR 17 van den Bossche 3473SerI Togo BR S. senensis ( Klotzsch) Hiern Polhill & Paulo 2347 Tanzania BR 15 Fig. 14 Tanner 659 Tanzania BR S. spermacocina ( K.Schum.) de Wilde 8518 Cameroon BR?17 Bridson & PuV van Eijnatten 2077 Nigeria BR S. sphaerostigma (A.Rich.) Oliv. Reekmans 10237 Burundi BR 17 Bredo 1487 D.R. Congo BR Michel 3058 D.R. Congo BR Drake 92 Sudan BR S. stachydea DC. Vanden Berghen 9184 Senegal BR 17 Figs. 29± 30 Vanden Berghen 6119 Senegal BR S. stipularis Dessein Milne-Redhead 4338 Zambia K 14 Malaisse & Robbrecht 1996 R.D. Congo BR Bamps, Martins & Maia 4175 Angola BR S. subvulgata ( K.Schum.) Lewalle 5606 Burundi BR 15 Fig. 47 J.G.Garcia Lisowski 10476 D.R. Congo BR Bredo 2262 D.R. Congo BR Lisowski, Malaisse & Symoens 4139 D.R. Congo BR Schlieben 2321 Tanzania BR S. taylorii Verdc. Milne-Redhead & Taylor 8821 Tanzania BR 15 Fig. 5 *S. tenuior L. Humblot 599 Madagascar K 1 S. tenuissima Hiern Fay 3445 R.C. Africa BR 12 Figs. 48, 53± 54 S. terminali ora Good. Gossweiler 2347 Angola BR 13 S. thymoidea (Hiern) Verdc. Pearson 2772 Angola K 13 Figs. 57± 58 Hess-Wyss 52/1798 Angola ZT Figs. 24, 55± 56 *S. verticillata L. Chevalier 1026 Sudan BR 3 Figs. 4, 11, 31 Dessein 24 Cultivated at BR BR ± ± Amb. In polar view, the pollen outline is usually circular Apertures and often lobed due to the longer columellae towards the Number. The number of colpi ranges from 3± 4 in centers of the mesocolpia or due to the thicker nexine around Spermacoce mauritiana ( Figs. 2± 3) to 16± 21 in S. dibrachiata the apertures (cf. below). Only Spermacoce natalensis and S. ( Fig. 8). The pantopororate species have up to 30 pores. The mauritiana have a triangular or quadrangular polar outline higher the number of colpi, the higher the intraspeci c (Figs. 2± 3). variation observed. There is a weak positive correlation
Table II. Some variable pollen morphological characters for each species studied. Pollen of African Spermacoce species (Rubiaceae) 73 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. Apertures Wall ornamentation Type Species Size Shape Number Type Endo Peculiarities Sexine Nexine 1 S. mauritiana (3) 14-(15.8)-18 PS 3-4 ZC II perforate, margo of granules/ nely granular, endocracks microspines indistinct 1 S. natalensis (2) 14-(16.5)-19 SP 3-4 ZC II eutectate at apocolpium, nely granular, endocracks perforate at mesocolpium, indistinct or absent margo of granules 1 S. tenuior (1)* 15-(16.1)-17 PS-SP 6-8 ZC II perforate, margo of granules/ nely granular, endocracks microspines indistinct or absent 2 S. latifolia (2)* 44-(48.3)-50 SO-OS (7)-9-(11 ) ZC V perforate, granulate granular, broadô distinct endocracks 3 S. verticillata (2)* 23-(28.0)-30 OS (6-9) ZC IV perforate, granulate granular, narrowô distinct endocracks 6 S. annua (1) 62-(66.5)-74 S ca. 30 PP VI operculum microreticulate to reticulate, nely granular, no present microechinate endocracks 6 S. phyteuma (1) 80-(87.0)-90 S ca. 24 PP VI operculum perforate-microreticulate, nely granular, no present microechinate endocracks 11 S. assurgens (1)* 35-(36.8)-38 SO-OS 8-10 ZC II endocingulum perforate, granulate- granular, narrowô distinct indistinct microechinate endocracks 11 S. chaetocephala 38-(44.7)-52 SO-OS (9)-11 ZC II perforate, granulate granular, indistinct (4) endocracks 11 S. liformis (1) 30-(32.0)-34 S 9-10 ZC II columellae perforate, granulate granular, narrow irregular slightly endocracks longer in EP 11 S. hepperana (1) 42-(46.5)-52 SO-OS 9-11 ZC II columellae perforate, granulate nely granular, narrow slightly distinct or obscure longer in EP endocracks 11 S. pusilla (1) 35-(37.0)-3 9 SO-OS 8-10 ZC II perforate, granulate? 11 S. quadrisulcata 37-(42.3)-45 SO-OS (10-13 ) ZC II perforate, granulate granular, narrow indistinct (3) endocracks 11 S. radiata (3) 29-(36.2)-44 OS (9)-10- ZC II perforate, granulate granular, narrow indistinct (13) endocracks 12 S. lifolia (2) 50-(56.6)-64 S ca. 15 PC III colpi perforate, granulate to granular, narrow indistinct arranged in a microechinate endocracks loop 12 S. octodon (2) 37-(40.9)-45 S ca. 14 PC III colpi perforate, granulate to granular, narrow distinct or arranged in a microechinate obscure endocracks loop 12 S. tenuissima (1) 47-(54.1)-57 S ca. 15 PC III colpi perforate, granulate to granular, narrowô distinct arranged in a microechinate endocracks loop 13 S. terminali ora 52-(54.4)-57 S ca. 24 PC III colpi perforate, microechinate granular, numerous irregular (1) arranged in a endocracks spiral pattern 13 S. thymoidea (2) 50-(56.9)-63 S 24-25 PC III CM granular, perforate, microechinate granular, numerous irregular colpi endocracks arranged in a spiral pattern 14 S. arvensis (3) 75-(81)-86 O (16)-17- ZC I columellae perforate to perforate-? (18) distinctly reticulate in mesocolpia, longer in EP microechinate, surface undulating 14 S. azurea (2) 62-(69.6)-76 O-SO (13)-15- ZC I columellae perforate to perforate- nely granular, broad (16) distinctly reticulate in mesocolpia, distinct endocracks longer in EP microechinate, surface undulating 14 S. dibrachiata (7) 71-(84.8)-96 O-SO (16-21 ) ZC I columellae perforate to perforate- nely granular, broadô deep distinctly reticulate in mesocolpia, endocracks longer in EP microechinate, surface undulating 14 S. hockii (4) 67-(72.4)-82 SO 13-(15 ) ZC I columellae perforate to perforate- nely granular, narrow longer in EP reticulate in mesocolpia, distinct endocracks microechinate, surface undulating 14 S. phyteumoides 65-(66.7)-69 O-SO 14-16 ZC I columellae perforate to perforate- nely granular, broadô deep var. phyteumoides distinctly reticulate in mesocolpia, endocracks (1) longer in EP microechinate, surface undulating
74 S. Dessein et al. Table II. (Continued). Apertures Wall ornamentation Type Species Size Shape Number Type Endo Peculiarities Sexine Nexine 14 S. phyteumoides 68-(70.7)-73 O-SO 14-15 ZC I columellae perforate to perforate- nely granular, broad var. caerulea (1) distinctly reticulate in mesocolpia, undeep endocracks longer in EP microechinate, surface undulating 14 S. phyteumoides 75-(83.2)-90 SO 13-14 ZC I columellae perforate to perforate- nely granular, broad ``var. longituba somewhat reticulate in mesocolpia, undeep endocracks (1) longer in EP microechinate, surface undulating 14 S. stipularis (3) 75-(83.0)-92 O-SO (14)-15- ZC I columellae perforate-microreticulate, granular, broad distinct (17) distinctly microechinate endocracks longer in EP?15 S. articularis (1)* 72-(76.8)-82 SO 12-14 ZC I/II CM granular; perforate, microechinate nely granular, indistinct colpiô loxocol- endocracks porate, nexine slightly thicker around ectoaperture 15 S. congensis (1) 95-(99.0)-10 5 SO 12-14 ZC II microreticulate to reticulate, granular, indistinct microechinate endocracks 15 S. huillensis (2) 78-(80.0)-82 SO-OS 9-11 ZC? nexine perforate, microechinate? slightly thicker around ectoaperture 15 S. kirkii (1) 88-(97.0)-10 9 SO-OS 10-11 ZC? CM granular; perforate-microreticulate,? nexine microechinate to echinate thicker around ectoaperture 15 S. latituba (2) 88-(99.2)-11 0 SO (10)-12- ZC I CM granular; perforate-microreticulate, granular, narrow deep (13) nexine microechinate to echinate endocracks thicker around ectoaperture 15 S. senensis (2) 75-(80.0)-84 SO-OS 11 ZC II CM granular; perforate-microreticulate, granular, indistinct nexine microechinate endocracks thicker around ectoaperture 15 S. subvulgata (5) 73-(86.6)-94 SO-OS 10-(12 ) ZC I/II CM granular; perforate-microreticulate, granular, narrow indistinct nexine microechinate endocracks thicker around ectoaperture 15 S. taylorii (1) 100-(105.3)-11 0 SO 10-12 ZC II nexine Microreticulate to reticulate, granular, narrow indistinct thicker microechinate endocracks around ectoaperture 16 S. bambusicola (4) 74-(90.0)-10 5 O-SO (13)-16 ZC II columellae perforate-foveolate, margins nely granular, very broad longer in EP of perforations bordered, distinct or obscure echinate endocracks 16 S. ivorensis (1) 105-(115.5)-12 5 OS 16-18 ZC? columellae perforate, margins of? longer in EP perforations bordered, echinate 17 S. deserti (1) 58-(62.0)-68 SO 11-13 ZC II columellae perforate, granulate to granular, narrowô distinct longer in EP microechinate endocracks 17 S. lituba (2) 77-(81.2)-87 SO-OS 12-(14 ) ZC? columellae perforate, microechinate to? longer in EP echinate?17 S. intricans (1) 70-(73.2)-76 SO 9-10 ZC? columellae perforate, granulate to? slightly microechinate longer in EP 17 S. princeae (2) 63-(71.9)-87 SO-OS 12-14 ZC II columellae perforate-microreticulate, nely granular, narrow deep longer in EP granulate to microechinate endocracks 17 S. ruelliae (2) 74-(79.1)-90 SO-OS (13)-15- ZC II columellae perforate-microreticulate, granular, broad distinct (16) longer in EP (micro)echinate endocracks?17 S. spermacocina 64-(70.1)-77 OS 8-9 ZC II columellae perforate, granulate to nely granular, indistinct (2) slightly microechinate endocracks longer in EP 17 S. sphaerostigma 82-(91.0)-10 3 SO 11-(14 ) ZC II columellae perforate, microechinate to granular, no endocracks (4) longer in EP echinate 17 S. stachydea (2) 72-(81.7)-91 SO-OS (12)-13- ZC II columellae perforate, (micro)echinate granular, narrow distinct (15) longer in EP endocracks
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 (r 2 = 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 (r 2 =0.29; p<0.01). (C ) Correlation between number of apertures and pollen size (r 2 =0.14; p<0.01). (D) Correlation between number of apertures and pollen size with exclusion of the pantoaperturate species (r 2 =0.43; p<0.01). ± ± ± between pollen size and number of apertures (Fig. 1 C; r 2 = Position. In most species, the apertures are ordered along 0.14; p<0.01). However, the pantoaperturate species have the equator (zonoaperturate; Figs. 10± 17), Spermacoce phymany more apertures than can be expected from their size. teuma and S. annua are pantoaperturate, the ectopores being This is not surprising if we realize that the surface where the evenly spread over the pollen surface ( Fig. 9). Pire (1996: apertures can originate is much larger than for equally sized 422) noted in some Borreria species ``a tendency of the zonoaperturate species. Disregarding the pantoaperturate apertures to drift away from the equatorial plane; the apertu- species, the positive correlation between number of apertures ral plane appears as suvering a torsion due to the presence and pollen size is strong (Fig. 1 D; r 2 =0.43; p<0.01 ). of one or two pairs of convergent colpi. We clearly observed This multi-aperturate condition is apomorphic within the such loxocolporate colpi in Spermacoce articularis; in other family Rubiaceae, the 3-colporate pollen type being plesiomorphic species, e.g., S. chaetocephala, it was only sporadically (cf. Robbrecht 1988). Other genera of the tribe observed. Spermacoceae also have multi-aperturate grains (Bremekamp Two remarkable new colpi arrangements were also 1952, Verdcourt 1958, Kirkbride 1979, Pire & Cabral 1992, observed. The rst type, with the colpi arranged in a looplike Pire 1997 a, b), but it is rarely found in other Rubiaceae pattern, similar to the line on a tennis ball, is found except in the tribe Rubieae (Bremekamp 1952). In this tribe, in Spermacoce lifolia, S. octodon, and S. tenuissima however, the number of colpi is less variable and mostly ( Figs. 51± 52). The second type, where the short colpi are xed at 6 to 8 (Huysmans, pers. com.). arranged in a spiral pattern (Figs. 55± 56), characterizes Type. Apertures of Spermacoce are almost always compound, Spermacoce terminali ora and S. thymoidea. To our knowledge these two pollen types have not been previously i.e. built up by two or more components that are observed in angiosperms. The second type can be considered situated in more than one layer of the pollen wall. In the as an intermediate form between zonoaperturate and panto- species studied the apertures usually consist of an ectocolpus porate grains. An in-depth morphological and ontogenetic and an endocingulum (e.g., S. dibrachiata). In Spermacoce study is needed to further clarify these remarkable pollen verticillata and S. latifolia the endoaperture is a colpus. Both types. species, however, are most probably introduced from America. Spermacoce annua and S. phyteuma are character- Ectoaperture. As mentioned above, the ectoaperture is a ized by ecto- and endopores. colpus in most species ( Figs. 18± 24). The length of the colpi
76 S. Dessein et al. 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).
Pollen of African Spermacoce species (Rubiaceae) 77 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).
78 S. Dessein et al. 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).
Pollen of African Spermacoce species (Rubiaceae) compared with the polar axis (LC/P) ranges from 6 in Spermacoce terminali ora 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. terminali ora (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).
80 S. Dessein et al. 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).
Pollen of African Spermacoce species (Rubiaceae) 81 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).
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 ). 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 super cial (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 in uenced 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 strati cation and morphology of inner nexine surface of Spermacoce pollen grains. (43) wall strati cation 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 strati cation: 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).
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) 83 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
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. 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. terminali ora, 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.
Pollen of African Spermacoce species (Rubiaceae) 85
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). 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) 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 con gurations 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 pre gures, in some cases, the possible pollen types within an assemblage higher in the classi cation (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) 1. extension of the apertural area; from zonoaperturate pantoaperturate; 2. reduction of the ectoapertures; from long colpi short colpi pores; 3. narrowing of the endoapertures; from zonorate lalongate lolongate; 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 signi cantly 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 small lumina larger; 2. ectocolpi without margo ectocolpi 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 super cial 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 de ning 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 de ned 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 identi ed 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
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 de nition 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 de ned as having fruits splitting into indehiscent mericarps, but recently (Bacigalupo & Cabral 1996, 1999) rede ned as having indehiscent fruits. Spermacoce tenuior has fruits splitting into one dehiscent and one indehiscent fruit valve, and in this corresponds to the strict de nition 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 in orescences, 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, 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 in orescence, 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 con ned to high plateaus of the Zambezian regional center of endemism. 4. Spermacoce thymoidea and S. terminali ora 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 speci c 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 con rmed 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 con ict 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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