Review of Palaeobotany and Palynology 112 (2000) 189–205 www.elsevier.nl/locate/revpalbo Pollen morphological survey of Pentas (Rubiaceae– Rubioideae) and its closest allies Steven Dessein a, *, An Scheltens a, Suzy Huysmans a, Elmar Robbrecht b, Erik Smets a a Laboratory of Plant Systematics, Institute of Botany and Microbiology, K.U.Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven, Belgium b National Botanic Garden, Domein van Bouchout, B-1860 Meise, Belgium Received 27 January 2000; received in revised form 13 June 2000; accepted for publication 22 June 2000 Abstract Pollen descriptions, based on LM and SEM observations, are provided for the Pentas complex, an African alliance within the Hedyotideae–Spermacoceae alliance, which includes the genera Pentas, Otomeria, Batopedina, Parapentas, and Chamaepentas. The close relationship among these genera is confirmed by pollen morphological data. The observed variation in pollen character states agrees well with the existing genera and subgenera. The combination of small spheroidal pollen grains with narrow, acute endocolpi, and a small apocolpium index distinguishes Batopedina from Otomeria and Pentas. Parapentas differs from Batopedina in having broad endocolpi with vague ends. Two of the three subgenera of Otomeria, Neotomeria and Volubilis, are supported by pollen data. Pollen of the subgenus Otomeria, however, is less differentiated and is similar to pollen of Pentas subgenus Pentas. The exact relationship between the genera Otomeria and Pentas remains unclear, and molecular data are needed to interpret the observed morphological variation. Chamaepentas has pollen that is nearly identical to that of the Pentas subgenus Chamaepentadoides and of the subgenus Megapentas, i.e. large spheroidal pollen grains with three, medium-length colpi and endocolpi with acute ends. Pollen morphology provides additional support for a close affinity between Pentas subgenus Phyllopentas and Pentas subgenus Vignaldiopsis. © 2000 Elsevier Science B.V. All rights reserved. Keywords: pollen morphology; Rubiaceae; sexine ornamentation; systematics 1. Introduction Pentas Bentham comprises 39 species, distributed in Africa, Arabia, Madagascar and the Comores. Four other African genera are generally considered closely related to Pentas: Otomeria Bentham (eight species, widely distributed in tropical Africa), Batopedina Verdcourt (three species, restricted to central and West Africa), Parapentas Bremekamp * Corresponding author. Tel.: +32-16-321536; fax: +32-16-321968. E-mail address: steven.dessein@bio.kuleuven.ac.be (S. Dessein) (three species, distributed in tropical Africa), and Chamaepentas Bremekamp (one species, an endemic from Tanzania). The separation of these genera from Pentas has been a matter of dispute (Bremekamp, 1952; Verdcourt, 1953a,b,c, 1976, 1989; Hepper, 1960; Robbrecht, 1981). All these genera were revised by Verdcourt (1953a,b,c). From the founding of Otomeria by Bentham (1849; fide Verdcourt 1953a), the close relationship between this genus and Pentas has been noticed by several authors, and some of them even proposed merging it with Pentas (e.g. Elliot, 1896; and Bär, 1923; fide Verdcourt, 1953a). Verdcourt 0034-6667/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0 0 3 4 -6 6 6 7 ( 0 0 ) 0 0 04 1 - 5 190 S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 (1953a) recognized seven species in his revision of Otomeria (divided in three subgenera) and confirmed its close affinity with Pentas; he especially stressed the similarity between the subgenus Neotomeria and Pentas subgenus Pentas section Coccineae and between Otomeria micrantha and Pentas herbacea. However, he maintained Otomeria because he considered merging it with Pentas as even less satisfying. The oblong fruit shape and the spicate infructescence were used as diagnostic features for Otomeria. Parapentas and Chamaepentas—both erected by Bremekamp (1952)—are also closely allied to Pentas. They share articulate hairs of the external indumentum, capsular fruits and colleter tipped fimbriate stipules. They differ mainly from Pentas in being straggling herbs, while Pentas is a more or less erect herb or subshrub, often woody at the base. A third genus, Tapinopentas, erected by Bremekamp (1952), has been merged with Otomeria by Hepper (1960). Verdcourt (1953a) erected Batopedina. This small genus only differs from Otomeria in its fewflowered inflorescence, depressed fruit apex and its ericoid growth form. Verdcourt (1989) later doubted the generic value of Batopedina. Since the ‘‘Prodromus’’ of de Candolle (1830), all classifications (Hooker, 1873; Schumann, 1897; Verdcourt, 1958; Bremekamp, 1966; Robbrecht, 1988) included Pentas and its allies in the tribe Hedyotideae (called Oldenlandieae by some of these authors). Bremekamp (1952) stressed that the knowledge of the Hedyotideae was still too incomplete to divide it into subtribes, but he was the first who explicitly recognized the close affinities between the above-mentioned genera of the Pentas complex. Verdcourt (1953c) proposed evolutionary schemes for the members of the Pentas complex, which are, however, merely based on an intuitive interpretation of observations. The homogeneity of the complex was also supported by the cytopalynological study of Lewis (1965). He made a limited study of the chromosomes and pollen of African Hedyotideae and recognized three groups, the Oldenlandia group, the genus Manostachya and the Pentas group. The latter (including Parapentas, Otomeria and Pentas; Batopedina and Chamaepentas were not studied ) was characterized by relatively large chromosomes with chromosome complements based on x=10 and small, 3–4-colporate pollen grains. Bremer (1987) carried out a cladistic analysis of morphological data and could confirm Louis’ Oldenlandia (as the ‘Hedyotis group’) and Pentas group. A later cladistic analysis by Bremer (1996), based on rbcL sequences, included Pentas in the Spermacoceae sensu lato. Her wide concept of the Spermacoceae merges three former tribes, Hedyotideae, Knoxieae and Spermacoceae. Within this larger tribe, Bremer (1996) defined two groups: a Hedyotis/Spermacoce group (including the former tribe Spermacoceae sensu stricto) and a Pentas group (the former tribe Knoxieae and the genus Otiophora inclusive). The morphological evidence for these two large complexes is rather poor. Andersson (1999) considered the recognition of the Spermacoceae sensu lato as premature and stated that ‘‘it may prove possible, and more convenient, to split the heterogeneous Hedyotideae into monophyletic entities’’ (p. 180). The alliance of Pentas, Otomeria, Parapentas, Batopedina and Chamaepentas, further called the ‘Pentas complex’, can be considered as one of these monophyletic units. The purpose of the present paper is to provide detailed pollen morphological data for the Pentas complex to (1) test the generic value of Otomeria, Batopedina, Parapentas and Chamaepentas and (2) re-evaluate the subgenera and sections within Pentas and Otomeria. 2. Material and methods 2.1. Methods Pollen was acetolysed for 10 min in a heating block at 90°C. Pollen for SEM was mounted on a stub, air-dried and coated with gold. Observations were made with a Jeol 6400 SEM. The glycerine jelly slides have been observed with a Leitz Dialux 20. Polar axis (P) and equatorial diameter (E) were measured in at least 10 mature pollen grains with S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 LM (magnification 1000). Other measurements were made on SEM micrographs. Pollen grains were broken using glass beads, as described by Huysmans et al. (1993). The terminology follows Punt et al. (1994). 2.2. Material This study is based on herbarium material (BR) of 54 specimens of 42 taxa; ‘Congo’ stands for the Democratic Republic of Congo (ex Zaire). The list below also indicates which specimens were used for the illustrations. Batopedina linearifolia (Bremekamp) Verdcourt var. glabra Petit: de Witte 06615, Congo (BR 810054); de Witte 03379, Congo (BR 810140), figs. 5, 12. Batopedina linearifolia (Bremekamp) Verdcourt: Fanshawe 4143, Zambia (BR 810012). Batopedina pulvinellata Robbrecht: Duvigneaud 3018R2, Congo (BR 810097). Batopedina tenuis (A. Chevalier ex Hutchinson & Dalziel ) Verdcourt: Geerling & Bokdam 1087, Ivory Coast (BR 901718). Chamaepentas greenwayi Bremekamp var. glabra Bremekamp: Greenway 6570, Tanzania (BR 810053). Otomeria cameronica (Bremekamp) Hepper: Carvalho 2732, Equatorial Guinea (BR 807353), fig. 19; Rodenburg 84, Ghana (BR 810177). Otomeria elatior (A. Richard) Verdcourt Dewèvre 494, Congo (BR 810161); Quarré 2780, Congo (BR 809998), figs. 24, 29–30. Otomeria guineensis Bentham: Bouharmont 6799, Congo (BR 807311); Pauwels 5899, Congo (BR 807370). Otomeria lanceolata Hiern: Dacremont 154, Congo (BR 810123). Otomeria madiensis Oliver: Dewulf 177, Congo (BR 901528), figs. 3, 25. Otomeria micrantha K. Schumann: Ankei 79/108I, Congo (BR 807347); de Wilde 7761, Cameroon (BR 810126), fig. 8. Otomeria oculata S. Moore: Gillet & Faden 18257, Kenya (BR 810154), figs. 17, 26. Otomeria volubilis ( K. Schumann) Verdcourt: Louis 296, Congo (BR 810045), figs. 9, 13, 20; Troupin 2445, Congo (BR 810082). Parapentas battiscombii Verdcourt: Polhill & Verdcourt 283, Kenya (BR 810020). Parapentas setigera (Hiern) Verdourt: Louis 8519, Congo (BR 810056); Louis 13330, Congo (BR 810173). Parapentas silvatica ( K. Schumann) Bremekamp: Mabberley 1435, Tanzania (BR 809992), fig. 7. Pentas angustifolia (A. Richard) Verdcourt: Bamps, Raimundo & Matos 4027, Angola (BR 809990). Pentas arvensis Hiern: Tisserant 3616, Cameroon (BR 810117), fig. 21. 191 Pentas bussei K. Krause: Oxtoby s.n., without locality (BR 810122), figs. 10, 22, 27–28. Pentas caffensis Chiovenda: de Wilde & de Wilde-Duyfjes 6980, Ethiopia (BR 809991), fig. 11. Pentas cleistostoma K. Schumann: Luxen 404, Congo (BR 810058). Pentas decora S. Moore: Chambon 12, Congo (BR 810010), figs. 23, 31; Van der Ben 1805, Burundi (BR 810078). Pentas elata K. Schumann: Richards 24623, Tanzania (BR 810090). Pentas graniticola E.A. Bruce: Tanner 594, Tanzania (BR 810087). Pentas herbacea (Hiern) K. Schumann: De Graer 712, Congo (BR 810057). Pentas hindsioides K. Schumann: pickled material Dessein 23 from specimen 19981824-18 cultivated at National Botanic Garden of Belgium (origin Kenya), fig. 2. Pentas hindsioides K. Schumann var. williamsii Verdcourt: Shabani 308, Tanzania (BR 810083). Pentas ionolaena K. Schumann subsp. madagascariensis Verdcourt: Malcomber, Hutcheon, Razafimanantsoa & Zjhra 1410, Madagascar (BR 810024). Pentas lanceolata (Forsskal ) Deflers subsp. cymosa ( Klotzsch) Verdcourt: Schlieben 11253, Comoro Islands (BR 810022), figs. 1, 18. Pentas longiflora Oliver: Greenway & Eggeling 8569, Tanzania (BR 901618); Schlieben 4311, Tanzania (BR 810124). Pentas longituba K. Schumann ex Engler: Eggeling 6811, Tanzania (BR 810153). Pentas micrantha Baker subsp. wyliei (N.E.Br.) Verdcourt: Tweedi 1643, Tanzania (BR 810133), figs. 4, 16. Pentas mussaendoides Baker: Phillipson 1613, Madagascar (BR 810100). Pentas nobilis S. Moore: Bidgood, Mbago & Vollesen 2275, Tanzania (BR 810187). Pentas parvifolia Hiern: Gillett 12797, Kenya (BR 810067). Pentas pseudomagnifica M. Taylor: Schlieben 3379, Tanzania (BR 901619), fig. 14. Pentas pubiflora S. Moore: Graham 1105, Kenya (BR); Reekmans 6450, Burundi (BR 901683). Pentas purpurea Oliver: Tanner 3889, Tanzania (BR 810034). Pentas schimperiana (A.Richard ) Vatke: Louis 4877, Congo (BR 810023); Schlieben 945, Tanzania (BR 901617), fig. 32. Pentas schimperiana (A. Richard ) Vatke subsp. occidentalis (Hooker f.) Verdcourt: Alluaud 345, Afrique orientale (BR 810031). Pentas schumanniana K. Krause: Milne-Redhead & Taylor 9030, Tanzania (BR 810009), fig. 6. Pentas zanzibarica ( Klotzsch) Vatke: Haerdi 370/0, Tanzania (BR 810148); Smith 103, Tanzania (BR 810144). 3. Results: characters and variation Several pollen characters with systematic value within the Pentas complex are listed at the specimen level in Table 1. Taxon NC E (mm) P/E A.I. Ectocolpus Endoaperture ends Sexine 18–(22.2)–25 28–(29.2)–30 16–(20.2)–24 26–(27.0)–28 1.00–(1.10)–1.16 1.04–(1.08)–1.11 0.3 0.3 Rounded Rounded Microreticulate Granules in mesocolpium Microreticulate Granules in mesocolpium Fanshawe 4143 de Witte 03379 L de Witte 06615 3 26–(28.3)–32 20–(24.1)–27 1.08–(1.17)–1.30 0.3 Rounded Colpus, acute ends Microreticulate Granules in mesocolpium L Duvigneaud 3018R2 3 14–(14.3)–15 13–(14.2)–15 1.00–(1.00)–1,08 0.4 Acute Colpus, acute ends Microreticulate Granules around apertures L Geerling & Bokdam 1087 3 24–(26.6)–30 24–(26.6)–30 0.96–(1.00)–1.04 0.3 Rounded Colpus, acute ends Microreticulate Granules around apertures Greenway 6570 3 41–(45.0)–50 38–(42.4)–48 1.00–(1.06)–1.11 0.5–0.7 Acute Colpus with diffuse (acute) ends Microreticulate Granules on whole tectum Otomeria subg. Neotomeria Otomeria elatior I Dewèvre 494 3 35–(37.3)–40 27–(30.4)–33 1.15–(1.23)–1.30 0.2 Otomeria elatior I Quarré 2780 3 28–(32.0)–36 26–(30.0)–32 1.00–(1.07)–1.20 Otomeria madiensis B Dewulf 177 3 30–(31.4)–34 25–(26.2)–28 1.11–(1.19)–1.30 Otomeria oculata L Gillet & Faden 18257 3 26–(28.8)–30 20–(21.1)–22 1.27–(1.37)–1.45 0.2 Roundedacute 0.2–0.3 Roundedacute 0.4 Rounded Endocingulum branched in mesocolpium Endocingulum branched in mesocolpium Endocingulum Microreticulate Granules around apertures, muri with irregular margins Microreticulate Granules around apertures, muri with irregular margins Microreticulate Granules around apertures, to perforate muri with irregular margins Microreticulate Granules around apertures, to perforate muri with irregular margins Otomeria subg. Otomeria Otomeria cameronica Otomeria cameronica Otomeria guineensis Otomeria guineensis Otomeria micrantha Otomeria micrantha Otomeria lanceolata L L B L I I L Carvalho 2732 Rodenburg 84 Pauwels 5899 Bouharmont 6799 Ankei 79/108I de Wilde 7761 Dacremont 154 4 4 3 3 3–4 4 3 30–(31.9)–35 29–(26.8)–25 40–(41.0)–44 31–(32.5)–35 32–(36.1)–38 34–(37.8)–40 30–(31.4)–32 24–(27.7)–29 27–(25.3)–24 36–(38.0)–40 32–(29.4)–26 32–(34.4)–38 32–(33.1)–34 26–(26.7)–28 1.07–(1.15)–1.25 1.00–(1.06)–1.13 1.00–(1.05)–1.19 1.07–(1.11)–1.23 0.94–(1.06)–1.19 1.00–(1.15)–1.24 1.23–(1.17)–1.14 0.7–0.8 0.7 0.7 0.7–0.8 0.5–0.8 0.7–0.8 0.5–0.6 Acute Rounded Acute Acute Obtuse Acute Acute Endocingulum Endocingulum Colpus Endocingulum (branched) Colpus, broad ends Colpus Endocingulum Microreticulate Microreticulate Microreticulate Microreticulate Microreticulate Microreticulate Microreticulate Granules around apertures Granules around apertures Granules around apertures Granules on whole tectum Granules covering whole tectum Granules covering whole tectum Granules around aperture Otomeria subg. Volubilis Otomeria volubilis Otomeria volubilis I I Troupin 2445 Louis 296 3 3 24–(24.3)–26 20–(21.8)–25 24–(24.3)–26 20–(21.3)–24 1.00 0.95–(1.02)–1.05 0.3 0.3 Acute Acute Endocingulum Endocingulum Reticulate Reticulate None Granules around aperture Parapentas Parapentas battiscombii B 28–(29.7)–32 26–(27.5)–30 0.97–(1.08)– 1.12 0.5 Acute Broad colpus, vague ends Microreticulate Granules covering whole tectum L L I Polhill & Verdcourt 283 Louis 13330 Louis 8519 Mabberley 1435 3 Parapentas setigera Parapentas setigera Parapentas silvatica 3 3 3 24–(25.3)–29 24–(24.8)–26 no accurate measurements 21–(23.0)–25 22–(23.0)–24 no accurate measurements 1.00–(1.1)–1.16 1.04–(1.08)–1.13 1? 0.5 Acute 0.5–0.6 Acute 0.5 Acute Broad colpus, vague ends Microreticulate Granules around aperture Broad colpus, vague ends Microreticulate Granules around aperture Colpus, vague ends Microreticulate Granules covering whole tectum 3 40–(42.0)–48 38–(41.0)–47 0.98–(1.02)–1.05 0.4–0.5 Rounded Colpus 3 39–(42.8)–46 35–(40.0)–45 1.00–(1.09)–1.18 0.5 Colpus Pentas subg. chamaepentadoides Pentas hindsioides I Shabani 308 var. williamsii Pentas hindsioides I Dessein 23 Obtuse Rounded Colpus, acute ends Colpus, acute ends SE L B Chamaepentas Chamaepentas greenwayi I 1 var. glabra 3 3 P (mm) Endocingulum Microreticulate Granules sparsely covering whole tectum Microreticulate Granules sparsely covering whole tectum S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 Batopedina Batopedina linearifolia Batopedina lineari linearifolia var. glabra Batopedina lineari linearifolia var. glabra Batopedina lineari pulvinellata B. tenuis FM Collection 192 Table 1 List of species examined, with collecting data, flower morph (FM; B: brevistylous; L: longistylous; I: isostylous), number of colpi (NC ), length of polar axis (P), equatorial diameter (E ), shape (P/E), apocolpium index (A.I.), colpus ends, endapertures, sexine, and supratectal elements (SE) Pentas subg. Longiflora Pentas decora I Chambon 12 4 56–(50.7)–58 42–(46.7)–50 1.00–(1.09)–1.17 0.5–0.6 Acute Pentas decora I Van der Ben 1805 4 40–(46.0)–55 35–(42.3)–50 1.02–(1.10)–1.14 0.5 Pentas longiflora I 3–4 38–(42.6)–46 38–(40.8)–44 1.00–(1.03)–1.10 Pentas longiflora I Greenway & Eggeling 8569 Schlieben 4311 4 40–(42.1)–44 37–(38.3)–40 Tanner 594 3 58–(60.8)–64 Pentas subg. Megapentas Pentas graniticola I 0.7–0.8 Acute Colpus-endocingulum costa Colpus-endocingulum costa Broad endocingulum Perforatemicroreticulate Perforatemicroreticulate Microreticulate 1.05–(1.1)–1.16 0.7 Broad endocingulum Microreticulate Granules are sparsely present 52–(56.3)–60 1.07–(1.09)–1.13 Colpus, vague ends Microreticulate Granules covering whole tectum Colpus Colpus Microreticulate Granules around aperturess Microreticulate Granules sparsely present to perforate Microreticulate Granules covering whole tectum Acute Acute No granules, interwoven muri Granules sparsely present, inerwoven muri Granules around apertures I I Eggeling 6811 Bidgood et al. 2275 3 3 70–(75.0)–80 44–(48.0)–52 60–(65.0)–70 42–(45.0)–50 1.07–(1.15)–1.20 1.00–(1.07)–1.14 Pentas pseudomagnifica I Taylor & Schlieben 3379 3 69–(72.7)–74 60–(64.3)–70 1.07–(1.13)–1.16 Pentas subg. Pentas Section Coccinea Pentas bussei Pentas parvifolia L L Oxtoby s.n. Gillett 12797 3 3 22–(23.8)–26 24–(25.3)–26 20–(20.6)–22 20–(20.6)–21 1.10–(1.15)–1.18 1.18–(1.23)–1.29 0.3 Rounded 0.4–0.5 Rounded Colpus+fishtail ends Colpus-endocingulum Section Monomorphi Pentas angustifolia Pentas herbacea Pentas micrantha I I I Bamps et al. 4027 De Graer 712 Tweedi 1643 3 3 3 20–(22.7)–24 22–(23.9)–26 26–(26.9)–28 20–(22.2)–24 20–(20.8)–24 24–(24.9)–26 0.91–(1.10)–1.04 1.08–(1.15)–1.25 1.04–(1.08)–1.13 0.3–0.4 Acute 0.4 Rounded 0.5 Rounded Endocingulum (branched) Microreticulate Granules around aperture Endocingulum Microreticulate Around aperture Colpus Microreticulate Granules around apertures Section Hirtistylus Pentas caffensis L de Wilde & de Wilde Duyfjes 6980 3–4 34–(36.1)–40 32–(35.5)–40 1.00–(1.02)–1.12 0.7 Acute Endocingulum (branched) Microreticulate Granules present Section Pentas Pentas arvensis Pentas cleistostoma Pentas lanceolata Pentas pubiflora B L L B Tisserant 3616 Luxen 404 Schlieben 11253 Reekmans 6450 3 3 3 3 28–(29.1)–30 25–(27.0)–29 25–(25.9)–26 29–(31.6)–34 25–(28.3)–30 22–(23.5)–26 25–(25.3)–26 25–(27.5)–29 1.00–(1.05)–1.12 1.08–(1.15)–1.20 1.00–(1.02)–1.08 1.07–(1.15)–1.28 0.5 0.5–0.6 0.5–0.6 n.a. Obtuse Acute Rounded Acute Endocingulum (LM ) Colpus Endocingulum (branched) Endocingulum Pentas pubiflora L Granam 1105 3 25–(26.4)–27 22–(22.7)–24 1.13–(1.18)–1.28 Acute Endocingulum (LM ) Pentas purpurea Pentas zanzibarica L B Tanner 3889 Haerdi 370/0 3 3 23.5–(26.0)–28.5 20–(21.9)–24.8 1.10–(1.20)–1.29 24–(26.2)–28 20–(21.4)–23 1.18–(1.21)–1.25 0.5–0.6 Obtuse 0.5 Rounded Pentas zanzibarica L Smith 103 3 20–(21.6)–24 16–(19)–20 1.00–(1.14)–1.25 0.4 Malcomber et al. 1410 Phillipson 1613 4 19–(21.1)–22 17–(18.6)–20 1.10–(1.13)–1.20 0.5–0.6 Acute Endocingulum (branched) Microreticulate Granules sparsely present 4–(5) 28–(29.5)–32 24–(27.0)–30 1.20–(1.11)–1.07 0.5 Endocingulum (branched) Microreticulate Granules sparsely present, abundantly around apertures Endocingulum (branched) Microreticulate Granules around apertures Endocingulum (LM ) Endocingulum (branched) Endocingulum Endocingulum (branched) Pentas subg. Phyllopentas Pentas ionolaena subsp. L madagascariensis Pentas mussaendoides L? Rounded Pentas schumanniana L Milne-Redhead & Taylor 9030 4 26–(27.3)–30 22–(23.6)–25 1.12–(1.15)–1.20 Somewhat rounded 0.5–0.6 Acute Pentas subg. Vignaldiopsis Pentas elata Pentas schimperiana Pentas schimperiana Pentas schimperiana subsp. occidentalis L B L L Richards 24623 Louis 4877 Schlieben 945 Alluaud 345 4 3–4 3–4 4 32–(34.1)–36 24–(24.7)–27 26–(27.5)–30 27–(29.6)–32 30–(31.8)–34 22–(24.4)–27 26–(27.2)–30 25–(27)–28 1.13–(1.08)–1.13 0.96–(1.01)–1.09 1.00–(1.02)–1.08 1.00–(1.1)–1.20 0.7 0.5–0.6 0.5–0.6 0.5–0.7 Acute Obtuse Rounded Obtuse Colpus Perforate Granules around apertures Microreticulate Granules around apertures to perforate Microreticulate Microreticulate Perforate Microreticulate perforate Microreticulate to perforate Perforate Microreticulate Granules Granules Granules Granules throughout tectum around aperture around apertures around apertures Granules around apertures Endocingulum Granules around apertures EndocingulumGranules covering whole tectum colpus+fish tail Endocolpus-endocingulum Microreticulate Granules around apertures Microreticulate Microreticulate Microreticulate Microreticulate S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 Pentas longituba Pentas nobilis 0.3–0.4 Acuterounded n.a.m. Acute 0.4–0.5 Rounded or blunt 0.5–0.6 Rounded Granules covering whole tectum Granules covering whole tectum Granules around apertures Granules around apertures 193 194 PLATE I S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 3.1. Polarity and symmetry All species investigated have isopolar and radially symmetrical pollen. 195 or sub-quadrangular (Plate I, 3, 6), due to the sunken colpi. All species investigated are angulaperturate. 3.4. Apertures 3.2. Size Pollen size in the Pentas complex varies from small to large. P ranges from 14 to 80 mm respectively in Batopedina pulvinellata and Pentas longituba. E values are generally slightly smaller and range from 13 to 70 mm. Most species have P and E values in the range between 25 and 35 mm. In some species, we noticed a considerable (more than 10%) size variation within a given species. In some cases, this variation could be correlated with the infraspecific classification (e.g. Pentas schimperiana subsp. schimperiana: P=27.5; P. schimperiana subsp. occidentalis: P=29.6) or with flower dimorphism (e.g. Otomeria guineensis, longi: 32.5; O. guineensis, brevi: 41.0). In other cases (e.g. Pentas decora Chambon 12: P=50.7; P. decora Van der Ben 1805: P=46.0), the observed size variation could only be explained in terms of a natural size variation, which is probably correlated with growth conditions. Such variation detracts from the systematic value of these characters. 3.3. Shape The majority of species has spheroidal pollen (0.88<P/E>1.14); however, subprolate grains (1.14<P/E>1.33) are also common. A remarkable infraspecific variation is observed in Otomeria elatior, having both spheroidal and subprolate pollen grains. In a polar view, pollen of the Pentas complex is circular (Plate I, 1–2, 4–5) or slightly triangular The apertures in the Pentas complex are compound, i.e. consisting of openings on top of each other in different layers of the wall. Ectoaperture: All specimens investigated have ectocolpi; the length and width vary considerably. Most species have narrow ectocolpi, often not exceeding 1 mm in width (Plate III, 13, 16–18). Ectocolpus ends are mostly acute (Plate I, 3; Plate III, 16) or rounded (Plate I, 1, 5; Plate III, 17, 18); in some species, both acute and rounded ends are present. Rounded ectocolpus ends tend to appear as acute when pollen grains are not fully hydrated; this makes it difficult to distinguish both character states. Endoaperture: Endoapertures have an important systematic value within Rubiaceae. In the Pentas complex, two major types were observed: an endocolpus and an endocingulum. The endocolpus ends are acute (Plate V, 27), blunt, only vaguely delimited or with two diverging tips ( like a fish tail, Plate V, 28, 32). Small pointed extensions on the lateral sides of the endocolpi were also observed (e.g. Pentas bussei, Plate V, 27). In some species, endocolpi as well as endocinguli were observed, suggesting a trend from distinct endocolpi towards an endocingulum by fusion of the colpi (e.g. P. bussei, Plate V, 28). Endocinguli sometimes have extensions in the mesocolpium (e.g. Otomeria elatior, Plate V, 29, 30). Fig. 1 illustrates the different endoapertures of the Pentas complex and indicates the hypothesized relations between the character states. Only PLATE I Polar views (SEM ). 1. Pentas lanceolata, 3-colporate grain showing perforate tectum and ectocolpi with rounded ends. 2. Pentas hindsioides, 3-colporate grain showing microreticulate sexine and ectocolpi with acute ends. 3. Otomeria madiensis, 3-colporate grain showing perforate to microreticulate tectum and long ectocolpi with acute ends. 4. Pentas micrantha, 3-colporate grain showing irregularly shaped lumina and fastigium, ectocolpi with rounded ends. 5. Batopedina linearifolia, 3-colporate grain showing microreticulate sexine and ectocolpi with rounded ends. 6. Pentas schumanniana, 4-colporate grain showing microreticulate sexine, ectocolpi with acute ends. 196 PLATE II S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 Fig. 1. Hypothetical scheme (theoretical 2-aperturate grains, broken line=ectoaperture) illustrating the different endoapertures within the Pentas complex. Arrows indicate the hypothesized relations between the observed types. A—endocolpi with acute ends; B—endocolpi, the left one with a small extension; C—endocolpi, the left one with a fish-tail end; D—endocingulum with fish-tail extensions in mesocolpium; E—endocolpus with vague ends; F—endocingulum; G—endocingulum with extensions in the mesocolpium. Pentas decora (Plate V, 31) is characterized by the presence of a costa (as defined by Huysmans et al., 1998). Where the ectocolpus and the endocolpus or endocingulum overlap, a gap is present in the pollen wall (in acetolysed grains). We did not consider this gap as a mesoaperture because it results from the wall thinnings of both the ectoand endoaperture. The gap is sometimes rounded, being porus (Plate III, 15), or elongated (with the length/breadth ratio exceeding 2) and lolongate (Plate III, 14). In general, pollen grains of the Pentas complex are 3-colporate (Plate I, 1–5), though 4-colporate grains are also present (Plate I, 6). Some species (e.g. Otomeria micrantha, Pentas longiflora) have both 3- and 4-colporate grains, mostly in equal 197 quantities. In Pentas mussaendoides, a small number of 5-colporate grains were observed. The colpus membrane always has granular elements, mostly small (Plate III, 13–14, 16), but occasionally large and cube-shaped (Plate III, 15). The apocolpium index (an indirect measure of the length of the colpi) ranges from 0.2 (e.g. Otomeria madiensis, Plate I, 3) to 0.6–0.8 (e.g. Chamaepentas, Plate I, 2). There is limited infraspecific variation, which makes the character very interesting from a systematic point of view. A fastigium is present in Pentas micrantha (Plate I, 4; Plate III, 16). In the apertural region, the inner part of the exine is separated from the domed sexine. In some species, there is a distinct thickening of the colpus membrane around the gap; this character, however, is only sporadically met, even within one species; except for Otomeria volubilis where an indistinct thickening is invariably present (Plate II, 9; Plate III, 13). 3.5. Sexine The most common sexine pattern in the Pentas complex is microreticulate (Plate IV, 19). In some taxa (e.g. Otomeria subgenus Neotomeria, Pentas subgenus Pentas section Pentas), there is a tendency towards a perforate tectum (Plate I, 1, 3; Plate IV, 22, 25). The distinction between these two states is often difficult to determine. Moreover, the sexine pattern can differ between the apocolpium and mesocolpium region, resulting in pollen grains that are both microreticulate and perforate. Perforations or lumina vary from 0.1 to 1.5 mm in diameter; the lumina are rounded or roundedangular. Reticulate pollen is only present in Otomeria volubilis (Plate II, 9; Plate IV, 20). In PLATE II Equatorial views (SEM ). 7. Parapentas silvatica, 3-colporate grain showing microreticulate sexine with granulate muri and lolongate porus. 8. Otomeria micrantha, 3-colporate grain showing microreticulate sexine with smooth muri. 9. Otomeria volubis, 3-colporate grain with reticulate sexine, narrow ectocolpus and indistinct annulus around porus. 10. Pentas bussei, 3-colporate grain with mesocolpium showing variably sized lumina. 11. Pentas caffensis, 4-colporate grain, mesocolpium showing elongated lumina, endocingulum visible through exine. 12. Batopedina linearifolia, 3-colporate grain showing microreticulate sexine. 198 PLATE III S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 Pentas caffensis the lumina are more elongated (Plate II, 11). Large lumina are often intermingled with smaller lumina or with perforations (Plate II, 10; Plate IV, 19–20). Two variations of the sexine pattern were observed: (1) in Pentas decora, the muri are slightly interwoven, resulting in a microreticulate to perforate tectum (Plate IV, 23); (2) in Otomeria subgenus Neotomeria, the muri (or perforations) have irregular margins (Plate IV, 24–26). In almost all species, the muri around the apertures have minute granules. In some species, the muri are granular throughout (Plate II, 7; Plate IV, 21). 3.6. Columellae and nexine All species are simplicolumellate, i.e. one row of columellae is supporting the muri. The inner nexine is always granular (Plate V, 32). 4. Discussion 4.1. Pollen morphology and heterostyly Heterostyly is very common in the Pentas complex. It occurs in all genera, but not in all species (e.g. heterostyly absent in members of Pentas subgenus Megapentas, and of subgenus longiflora, and Otomeria elatior). Most heterostylous species are distylous, but tristyly has been reported for Pentas lanceolata (Bahadur, 1970). The taxonomic value of heterostyly is rather poor, as stated by Verdcourt (1958, p. 227): ‘‘complete heterostyly occurs only in the Rubioideae but it is a character of limited value. It may have arisen on numerous occasions, independently which destroys the value of any character’’. 199 Both morphs of Batopedina linearifolia var. glabra, Otomeria guineensis, Pentas pubiflora, Pentas schimperiana and Pentas zanzibarica were investigated here. Pollen from the two morphs only differs in pollen size and sexine ornamentation ( Table 1). According to Ganders (1979), pollen of the brevistylous morph is generally larger than pollen of the longistylous flowers. This was confirmed for B. linearifolia, O. guineensis, P. pubiflora, and P. zanzibarica. In P. schimperiana, pollen of the brevistylous morph is slightly smaller than that of the longistylous morph. It is very difficult to draw any conclusion from these measurements, since pollen size may also be influenced by the growth conditions. In Otomeria volubilis, an isostylous species, the pollen size differs between the two investigated specimens by more than 10%. In assessing pollen differences associated with heterostyly, the two morphs should come from the same population. Because of the known polyploidy of the Hedyotideae (see Lewis, 1965), such a study should also include chromosome counts. Other pollen morphological differences, which are often associated with heterostyly of Rubiaceae, such as larger lumina in pollen from longistylous flowers (Robbrecht and Puff, 1989), and scabrate tecta in brevistylous flowers versus glabrous muri in longistylous flowers, were not observed except for Pentas schimperiana, where the brevistylous morph had scabrate muri throughout, while in the longistylous morph, scabrate muri are restricted to the aperture region. 4.2. Taxonomic discussion of pollen data Verdcourt (1958) and Lewis (1965) provided the only pollen data available for the Pentas complex. Both studies are based on LM observations PLATE III Variation in apertures (SEM ). 13. Otomeria volubilis, showing thickening of ectocolpus membrane around aperture, colpus membrane finely granular. 14. Pentas pseudomagnifica, lolongate aperture. 15. Chamaepentas greenwayi, porus, large cubic granules on colpus membrane. 16. Pentas micrantha, fastigium around porus, ectocolpus with acute ends. 17. Pentas oculata, ectocolpus with vague to rounded ends. 18. Pentas lanceolata, ectocolpus with rounded ends. 200 PLATE IV S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 201 S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 Table 2 Summary of important character states of the Pentas complexa Genus/subgenus NC PSi P/E A.I. Sexine Endoaperture Batopedina Chamaepentas Otomeria subg. Neotomeria Otomeria subg. Otomeria Otomeria subg. Volubilis Parapentas Pentas subg. Chamaepentatoides Pentas subg. Longiflora Pentas subg. Megapentas Pentas subg. Pentas Pentas subg. Phyllopentas Pentas subg. Vignaldiopsis 3 3 3 3 3 3 3 3 3 3 4 3 S L S-M M S S L L L S-(M ) S S-(M ) S-(Sp) S (S)–Sp S-Sp S S S S S-(Sp) S-Sp S-(Sp) S S L S L S M M M-L S-M S-M M M-L Microreticulate Microreticulate Microreticulate-perforate Microreticulate Reticulate Microreticulate Microreticulate Microreticulate-interwoven muri Microreticulate Microreticulate-perforate Microreticulate Microreticulate Colpus, acute ends Colpus, acute ends Endocingulum Colpus or endocingulum Endocingulum Broad colpus, vague ends Colpus, acute ends Colpus to/or endocingulum Colpus, acute ends Colpus to/or endocingulum Endocingulum Endocingulum or 4 and 4 (4) and 4 a NC: number of colpi; Psi: pollen size (S<30 mm; 30<M>40 mm; L>40 mm); P/E: pollen shape (S: spheroidal; Sp: subprolate); A.I.: apocolpium index (S<0.4; 0.4<M>0.6; L>0.6). and treat only few species. Lewis (1965) confirmed the relationship between Otomeria, Parapentas and Pentas, as suggested by Verdcourt (1953b,c). The results of the present study further corroborate the close relationship between the genera of the Pentas complex. Pollen grains of Batopedina, Chamaepentas, Otomeria, Parapentas, and Pentas are isopolar, radially symmetrical, 3- or 4-colporate; spheroidal to subprolate; microreticulate to perforate (rarely reticulate) and granules are almost always present around the aperture. The variation observed in size, number of colpi, A.I., and endoaperture type coincides, to a large extent, with the different genera and subgenera ( Table 2) recognized by Verdcourt (1953a,b,c). Pollen grains of Batopedina and Parapentas can be distinguished from those of Pentas: Batopedina by the small pollen grains with a small A.I. and a narrow endocolpus with acute ends (Plate I, 5), and Parapentas (Plate II, 7) by its small pollen grains with a broad endocolpus with vague ends. In Otomeria, two subgenera have distinct pollen types, which agree with Verdcourt’s subgenera Neotomeria and Volubilis. Otomeria subgenus Neotomeria has medium-sized 3-colporate pollen grains. The ectocolpi are very long and relatively broad with rounded ends. The lumina or perforations have ragged margins; the endoaperture is a branched or unbranched endocingulum (Plate I, 3; Plate IV, 24–26). Otomeria volubilis, the only species of subgenus Volubilis, has small, spheroidal, 3-colporate pollen grains. The ectocolpi are long and small with acute ends. The tectum is reticulate (Plate II, 9); the endoaperture is an endocingulum. Pollen of subgenus Otomeria is less distinctive, medium-sized, and 3- or 4-colporate. The ectocolpus is relatively short, and the ends are acute. The PLATE IV Variation in sexine pattern (SEM ). 19. Otomeria cameronica, microreticulate sexine with rounded-angular lumina, muri smooth. 20. Otomeria volubilis, reticulate sexine. 21. Pentas arvensis, microreticulate sexine with granulate muri. 22. Pentas bussei, perforate tectum. 23. Pentas decora, interwoven muri, lumina slit-like narrowed. 24. Otomeria elatior, microreticulate to reticulate tectum with minute granules on the margins of the lumina. 25. Otomeria madiensis, perforate tectum with mintue granules on the margins of the perforations. 26. Otomeria oculata, microreticulate to perforate tectum, lumina irregularly shaped, margins of the muri ragged. 202 PLATE V S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 tectum is microreticulate, and the endoaperture is a short colpus or an endocingulum. Otomeria cameronica (recognized as Tapinopentas by Bremekamp in 1952) differs from the other species included in having four colpi. Otomeria micrantha has both 3- and 4-colporate pollen grains. Pollen of Otomeria micrantha, Otomeria guinensis and Otomeria lanceolata is difficult to distinguish from some pollen grains of Pentas subgenus Pentas section Monomorphi, but they tend to be larger. This palynological resemblance confirms the morphological evidence mentioned by Verdcourt (1953c). However, the close affinity between Otomeria subgenus Neotomeria and Pentas subgenus Pentas section Coccineae, suggested by habit and flower morphology, is not further supported by our data (cf. below). Verdcourt (1953c) recognized six subgenera in Pentas. The observed palynological variation confirms the value of at least some of these subgenera. Pentas subgenus Megapentas has large 3-colporate spheroidal grains, large or mediumsized colpi with acute or rounded ends, and an endocolpus with acute ends. The same pollen (but slightly smaller) is found in Pentas subgenus Chamaepentadoides, confirming the close relationship between these two subgenera, as proposed by Verdcourt (1953c). This type of pollen also occurs in Chamaepentas (Plate I, 2). Bremekamp (1952) erected this monospecific genus based on its straggling habit, conspicuous colleters and ebracteate inflorescences. Verdcourt (1953c, 1976) doubted the generic value of the genus and pointed to an affinity with Pentas hindsioides. Pollen features further support the close relationship between Chamaepentas and Pentas hindsioides. Although there is morphological evidence for merging 203 Chamaepentas with Pentas, we will reconsider the genus when our molecular data are available for this group. Pollen grains of Pentas subgenus Longiflora are large and spheroidal. The two species investigated differ remarkably: Pentas decora has an endocolpus with a thick costa around the mesoaperture (Plate V, 31); sometimes the endocolpi are fused and form an endocingulum. Pentas longiflora has an endocingulum with extensions into the mesocolpium. Pollen of this subgenus is most similar to pollen of Pentas subgenus Megapentas. It differs from the latter in the presence of four apertures, shorter colpi (A.I. larger), and the fusion of the endocolpi into an endocingulum. Pentas subgenus Phyllopentas also has characteristic pollen grains. The grains are small and 4-colporate, and the endocolpi are fused to form a branched endocingulum (Plate I, 6). This subgenus is characterized by enlarged, membranous calyx lobes and is distributed in Madagascar and East Africa, indicating that this genus could have emerged relatively early in evolution, at a time when Madagascar was still a part of Africa. Pollen of Pentas subgenus Vignaldiopsis (widely distributed on the African continent) resembles the pollen of subgenus Phyllopentas, but the grains are both 3- and 4-colporate (50/50) or exclusively 4-colporate (Pentas schimperiana subsp. occidentalis). Verdcourt (1953c; p. 248) stated that ‘‘It is likely that Vignaldiopsis and Phyllopentas are closely related despite the strong calyx differences and the fact that only at the northern end of Lake Nyasa do their distributions overlap’’. The two subgenera also share the essentially montane distribution of the species. Pollen grains of subgenus Pentas have no shared PLATE V Variation in endoapertures shown on broken pollen grains (SEM ). Two small triangles indicate the equatorial orientation of the endoaperture. 27. Pentas bussei, half pollen grain showing endocolpus with acute ends, small lateral extensions present (arrow). 28. Pentas bussei, broken pollen grain showing fusion of an endocolpus with acute end with an endocolpus with fishtail ends. 29. Otomeria elatior, broken grain showing endocingulum with lateral extensions in mesocolpium. 30. Otomeria elatior, pollen grain showing endocingulum with regularly shaped, large extensions in mesocolpium. 31. Pentas decora, costa around endoaperture. 32. Pentas schimperiana, view of granular nexine and endocolpus with fishtail ends. 204 S. Dessein et al. / Review of Palaeobotany and Palynology 112 (2000) 189–205 features that distinguish it from the other subgenera. However, differences correspond very well with the sections proposed by Verdcourt (1953c). Section Coccineae has small, subprolate, microreticulate to perforate pollen with a variable endoaperture: ranging from a small endocolpus with acute ends to a large endocolpus having the shape of a fish tail and almost fused. No true endocingulum was observed in Pentas bussei (Plate II, 10; Plate V, 27–28). Pollen grains of section Coccineae are similar to pollen of subgenus Neotomeria, differing only in their small size and the absence of muri with irregular margins; thus, the close relationship between the two taxa, as proposed by Verdcourt (1953a), is not fully confirmed. Section Monomorphi is characterized by small, spheroidal 3-colporate pollen grains with an endocingulum (Pentas angustifolia, Pentas herbacea) or an endocolpus (Pentas micrantha). Pentas caffensis, the only species from section Hirtistylus, has medium sized, 3–4-colporate, microreticulate pollen (Plate II, 11). The endocingulum is broad and branched in the mesocolpium. Section Pentas has small, subprolate (or more seldom spheroidal ) pollen with a tendency towards a perforate tectum (Pentas lanceolata, Pentas pubiflora). The endoaperture is a branched endocingulum (Plate V, 29– 30) or a small endocolpus (Pentas cleistostoma). Pollen of this section agrees well with that of section Coccineae. Pollen of section Papillopilosa was not investigated. Phyllopentas, Megapentas, Longiflora, Hindsioides, and Vignaldiopsis, have more or less characteristic pollen. Pollen of subgenera Megapentas and Chamaepentadoides, on the one hand, and Phyllopentas and Vignaldiopsis, on the other hand, strongly resemble each other and support the relationships between these subgenera as proposed by Verdcourt (1953c). Pollen of the genus Chamaepentas is almost identical to that of Pentas subgenus Chamaepentadoides and supports Verdcourt’s (1976) view that the two genera probably should be merged. Pentas subgenus Pentas and Otomeria subgenus Otomeria have less differentiated pollen; pollen features appear to have a scattered distribution over these taxa. This indicates the close relationship between these two subgenera, but additional data, both molecular and morphological, are needed to support merging Otomeria subgenus Otomeria with Pentas. Acknowledgements Technical support by Anja Vandeperre and Marcel Verhaegen is gratefully acknowledged. We are also grateful to Dr. W. Punt and an anonymous reviewer for their detailed comments. This study was supported by the National Fund for Scientific Research—Flanders (F.W.O.)—project numbers 2.0038.91 and G.0143.95 and by a grant form the Research Council of the K.U.Leuven (OT/97/23). 5. Conclusion References Palynological characters clearly support the generic validity of Batopedina and Parapentas. There is thus neither any new evidence to merge Batopedina with Otomeria as proposed by Verdcourt (1989) nor any evidence to merge Parapentas with Pentas. Otomeria subgenus Volubilis and subgenus Neotomeria have pollen unique for the study group—reticulate pollen in the former and muri with irregular margins in the latter—which differentiate them from pollen of Pentas and Otomeria subgenus Otomeria. Five of the six subgenera of Pentas, namely Andersson, L., 1999. The rps16 intron and the phylogeny of the Rubioideae (Rubiaceae). Pl. Syst. Evol. 214, 161–186. Bahadur, B., 1970. Heterostyly and homostyly in Pentas lanceolata ( Forsk.). Defl. J. Genet. 60, 199–204. 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