Botanical Journal of the Linnean Society, 2008, 158, 556–566. With 1 figure Cytogeography of Gentianaceae–Exaceae in Africa, with a special focus on Sebaea: the possible role of dysploidy and polyploidy in the evolution of the tribe JONATHAN KISSLING1*, LOUIS ZELTNER1, PHILIPPE KÜPFER1 and GUILHEM MANSION2 1 Laboratoire de Botanique Évolutive, Institut de Botanique, Université de Neuchâtel, Rue Emile-Argand 11, CH-2007 Neuchâtel, Switzerland 2 Institute for Systematic Botany, University of Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland Received 25 February 2008; accepted for publication 23 April 2008 Unlike other tribes of Gentianaceae, Exaceae have so far received little attention regarding their karyological evolution. Indeed, only 35 chromosome number counts (19 species) have been referenced to date, representing only a negligible fraction of the tribal diversity. In this paper, we performed an intensive chromosome count on material collected in the field (South and central Africa, plus Madagascar), encompassing 155 populations and c. 60 species from four genera of Exaceae, including Exacum, Ornichia, Sebaea and Tachiadenus. Fifty nine species (14 Exacum, one Ornichia, 42 Sebaea and two Tachiadenus) were examined for the first time, revealing a broad set of chromosome numbers (2n = 18, 28, 32, 36, 42, 56) and the occurrence of polyploid systems within Exacum and Sebaea. These results allow us to postulate x = 7, 8 or 9 as possible base chromosome numbers for Exaceae and emphasize the importance of both dysploidy and polyploidy processes in the evolution of the tribe. Finally, chromosome numbers appear to be associated to some morphological or geographical traits, suggesting new systematic combinations and likely active speciation patterns in the group. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 556–566. ADDITIONAL KEYWORDS: chromosome numbers – Exacum – karyology – Ornichia – Tachiadenus. INTRODUCTION The particular knowledge of chromosome number in plants is critical in detecting processes enabling abrupt speciation such as polyploidy or aneuploidy/ dysploidy (Briggs & Walters, 1997). Polyploidy is an extremely important phenomenon in plants and occurs in, for example, 97% of ferns and c. 70% of angiosperms (Averett, 1980; Grant, 1981). The knowledge of chromosome numbers within a species or a polyploid system may help to differentiate between allopolyploidy (i.e. the merging of genomes that have diverged from one another before episodes of polyploidization) and autopolyploidy (i.e. the merging of similar genomes before polyploidization) (Stebbins, *Corresponding author. E-mail: Jonathan.Kissling@unine.ch 556 1947). Furthermore, establishing extensive karyological surveys on particular taxa may allow the detection of particular changes in chromosome number such as aneuploidy/dysploidy processes (loss or gain of chromosomes in a genome). The occurrence of both polyploidy and dysploidy has been recently demonstrated for certain groups of angiosperms, including, for example, Borago, Nonea (Boraginaceae; Selvi, Coppi & Bigazzi, 2006), Hypochaeris (Asteraceae; Cerbah et al., 1998), Centaurium, Gentiana or Zeltnera (Gentianaceae; Yuan, Küpfer & Zeltner, 1998; Mansion, Zeltner & Bretagnolle, 2005; Mansion & Zeltner, 2004). Despite the importance of karyological studies for understanding evolutionary processes in Gentianaceae or establishing systematic treatments (Favarger, 1949; Rork, 1949; Favarger, 1952), especially within Gentianeae (Shigenobu, 1983; Yuan, © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 556–566 CYTOGEOGRAPHY OF EXACEAE IN AFRICA 557 1 2 3 4 5 6 7 8 9 Figure 1. Chromosome of Sebaea. Pictures were taken at a magnification of ¥1000 and the scale bars on each image correspond to 10 mm. Fig. 1. S. thomasii, mitotic metaphase of root tip, 2n = 28. Fig. 2. S. bojeri, meiotic anaphase I, n = 14. Fig. 3. S. filiformis, mitotic metaphase, 2n = 28. Fig. 4. S. leiostyla, meiotic metaphase II, n = 14. Fig. 5. S. repens, meiotic metaphase II, n = 14. Fig. 6. S. micrantha, meiotic anaphase I, n = 28. Fig. 7. S. macrophylla, mitotic metaphase, 2n = 56. Fig. 8. S. sedoides, mitotic metaphase, 2n = 42. Fig. 9. S. rehmanii, meiotic anaphase I, n = 21. 1993; Yuan & Küpfer, 1993a, b; Küpfer & Yuan, 1996; Yuan, Küpfer & Zeltner, 1998; Liu, Ho & Chen, 2002; Chassot, 2003) and Chironieae (Favarger, 1960; Zeltner, 1970; Zeltner & Mansion, 2003; Mansion & Zeltner, 2004), only a few chromosome counts have been performed so far on tropical Exaceae (Table 1). Exaceae is a small tribe of Gentianaceae, with c. 180 species and five genera (Struwe et al., 2002; Klackenberg, 2006). Exacum L. (including the saprophytic Cotylanthera Blume) comprises 65 species, distributed in tropical Africa, Madagascar and Asia (Klackenberg, 1985, 1990). Gentianothamnus Humbert is a monotypic genus occurring in Madagascar (Klackenberg, 1990). Ornichia Klack. contains three species endemic to Madagascar (Klackenberg, 1986). Sebaea Sol. is the most species-rich genus with c. 90–150 species in South Africa, tropical Africa and Madagascar (Schinz, 1906; Boutique, 1972; Paiva & Nogueira, 1990). Finally, Tachiadenus Griseb. comprises 11 species endemic to Madagascar (Klackenberg, 1987). To date, most karyological studies have focused on Exacum (Riseman, Sumanasinghe & Craig, 2006), but not including African or Malagasian taxa. Early work on Exacum tenuis (under Cotylanthera tenuis; Oehler, 1927) failed to establish unambiguously a definite number (2n = 32–36). Several species from India (Borgmann, 1964; Subramanian, 1980; Mallikarjuna, Scheriff & Krishnappa, 1987; Riseman et al., 2006) and Socotra (Sugiura, 1936a, b; Post, 1967) have also been examined, showing an extensive range in © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 556–566 558 J. KISSLING ET AL. Table 1. Chromosome numbers documented for the tribe Exaceae Chromosome number Taxon n Exacum affine Balf.f. Exacum affine Balf.f. n = 18 Exacum Exacum Exacum Exacum Exacum Exacum Exacum Exacum Exacum Exacum Exacum Exacum 2n 2n = 36 affine Balf.f. affine Balf.f. atropurpureum Bedd. courtallens var courtallens Arn. courtallens var laxiflorum Gamble gracilipes Balf.f. grande Klack. Under E. perrotteti Griseb. lawii C.B.Clarke macranthum Arn. pallidum (Trimen) Klack. pedunculatum L. pedunculatum L. Exacum pedunculatum L. Exacum pedunculatum L. Exacum petiolare Griseb. Exacum pumilum Griseb. Exacum sessile L. Exacum tenue (Blume) Klack. under Cotylanthera tenuis Blume Exacum tetragonum Roxb. Exacum tetragonum Roxb. Under E. bicolor Roxb. Exacum tetragonum Roxb. Under E. perrottetii Griseb. Exacum travancoricum Bedd. Exacum trinervium (L.) Druce Exacum trinervium subsp. macranthum (Arn.) L.H.Cramer Exacum trinervium subsp. pallidum (Trimen) L.H.Cramer Exacum trinervium subsp. ritigalensis (Willis) L.H.Cramer Exacum trinervium subsp. ritigalensis (Willis) L.H.Cramer Exacum trinervium subsp. trinervium (L.) Druce Exacum trinervium subsp. trinervium (L.) Druce Exacum wightianum Arn. Exacum wightianum Arn. Under E. foliosum Griseb. Sebaea brachyphylla Griseb. Sebaea ovata (Labill.) R.Br. chromosome number range, e.g. 2n = 18, 30, 34, 36, 52, 54, 56, 62, 68. More recently, Riseman et al. (2006) reported karyological data in the Exacum trinervium complex, showing a large difference in chromosome number. Finally, only two chromosome counts are currently available for the large genus Sebaea, including S. brachyphylla from Africa (2n = 22) (Thulin, 1970) and S. ovata from New Zealand (n = c. 27) (Beuzenberg & Hair, 1983). In this context, the main goals of the present paper are: (1) to determine and confirm chromo- 2n = 36 2n = 36 2n = 34 n = 34 2n = 68 2n = 20 2n = 68 2n = 56 2n = 54 2n = 52 2n = 62 2n = (30), 54, (56) 2n = 56 n = 28 2n = 62 n = 31 n = 31 n = 16–18 2n = 18 2n = 62 2n = 68 2n = 68 2n = 60 2n = 54 2n = 52 2n = 60 2n = 60 2n = 60 2n = 60 2n = 68 2n = 68 2n = 22 n = c. 27 References Sugiura, 1936a, b; Post, 1967 Rork, 1949; Darlington & Wylie, 1955 Riseman et al., 2006 Sumanasinghe, 1986 Mallikarjuna et al., 1987 Mallikarjuna et al., 1987 Mallikarjuna et al., 1987 Villemoes, 2000 Mallikarjuna et al., 1987 Mallikarjuna et al., 1987 Sumanasinghe, 1986 Sumanasinghe, 1986 Mallikarjuna et al., 1987 Subramanian, 1980 Riseman et al., 2006 Sumanasinghe, 1986 Mallikarjuna et al., 1987 Mallikarjuna et al., 1987 Mallikarjuna et al., 1987 Oehler, 1927 Borgmann, 1964 Mallikarjuna et al., 1987 Mallikarjuna et al., 1987 Mallikarjuna et al., 1987 Sumanasinghe, 1986 Riseman et al., 2006 Riseman et al., 2006 Riseman et al., 2006 Sumanasinghe, 1986 Riseman et al., 2006 Sumanasinghe, 1986 Mallikarjuna et al., 1987 Mallikarjuna et al., 1987 Thulin, 1970 Beuzenberg & Hair, 1983 some numbers for taxa within Exaceae, with a special focus on the genus Sebaea; (2) to check the systematic relevance of the chromosome groups obtained; and (3) to infer patterns of chromosome evolution and speciation mechanisms within the tribe. MATERIAL AND METHODS Most samples come from wild populations collected in the field in 1995 and 2003–2006 (Table 2). All voucher © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 556–566 CYTOGEOGRAPHY OF EXACEAE IN AFRICA 559 Table 2. Accession information for taxa including name, collector, voucher code, origin and chromosome number. Chromosome numbers not reported for the first time are marked with an asterisk (*) while chromosome counts that are different from previous reports are marked with an exclamation mark (!). Collectors’ names are abbreviated as follow: Robert Archer (RA), Martin Callmander (MC), Petra DeBlock (PD), Steven Dessein (SD), Berit Gehrke (BG), Jonathan Kissling (JK), Brian Luwingu (BL), Michael Pirie (MP), J. C. Piso (JP), Elias Tembo (ET), Sébastien Wohlhauser (SW) and Louis Zeltner (LZ) Taxon Collector Voucher code Exacum affine Balf.f. Exacum appendiculatum Klack. Exacum appendiculatum Klack. Exacum dolichantherum Klack. Exacum exiguum Klack. Exacum exiguum Klack. Exacum exiguum Klack. Exacum exiguum Klack. Exacum exiguum Klack. Exacum exiguum Klack. aff. Exacum hoffmannii Schinz Exacum hoffmannii Schinz Exacum humbertii Klack. Exacum marojejyense Humbert Exacum marojejyense Humbert Exacum microcarpum Klack. Exacum microcarpum Klack. Exacum millotii Humbert Exacum millotii Humbert Exacum millotii Humbert Exacum millotii Humbert Exacum nummularifolium Humbert Exacum quinquenervium Griseb. Exacum quinquenervium Griseb. Exacum quinquenervium Griseb. Exacum quinquenervium Griseb. Exacum quinquenervium Griseb. Exacum quinquenervium Griseb. Exacum quinquenervium Griseb. Exacum quinquenervium Griseb. Exacum quinquenervium Griseb. Exacum quinquenervium Griseb. Exacum quinquenervium Griseb. Exacum spathulatum Baker Exacum stenophyllum Klack. Exacum stenophyllum Klack. Exacum stenopterum Klack. Exacum stenopterum Klack. Exacum stenopterum Klack. Exacum stenopterum Klack. Exacum stenopterum Klack. Exacum subteres Klack. Ornichia madagascariensis Klack. Sebaea ‘pentendra aff. X 35?’ Sebaea ‘repens X thodeana?’ Sebaea africana J.Paiva & I.Nogueria Sebaea albens (L.f.) Roem. & Schult. aff. Sebaea ambigua Cham. JK Cultivar 1 JP, SW & LZ M028 JP, SW & LZ M030 SW & MC M064 JP, SW & LZ M008 JP, SW & LZ M015 JP, SW & LZ M046 JP, SW & LZ M048 JP, SW & LZ M051 JP, SW & LZ M050 JP, SW & LZ 2 ème arrêt JP, SW & LZ M026 SW & J.-I. Pfund M052 JP, SW & LZ M033 SW & J.-I. Pfund M056 SW & J.-I. Pfund M054 SW & J.-I. Pfund M055 JP, SW & LZ M032 JP, SW & LZ M035 JP, SW & LZ M036 SW & J.-I. Pfund M057 SW & J.-I. Pfund M058 JP, SW & LZ – JP, SW & LZ – JP, SW & LZ – JP, SW & LZ 2 ème arrêt JP, SW & LZ 4 ème arrêt JP, SW & LZ M007 JP, SW & LZ M012 JP, SW & LZ M021 JP, SW & LZ M025 JP, SW & LZ M038 JP, SW & LZ M065 JP, SW & LZ M011 JP, SW & LZ M045 JP, SW & LZ M049 JP, SW & LZ Station 3 JP, SW & LZ M019 JP, SW & LZ M020 JP, SW & LZ M027 JP, SW & LZ M029 SW & J.-I. Pfund M053 SW M002 JK & LZ 36 JK & LZ 23 SD, RA, PD, JK, BL & ET 603 JK 93 JK 94 Origin Chromosome numbers Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar South Africa Leshoto Zambia South Africa South Africa n = 18* 2n = 36* n = 16 2n = 32 n = 16 2n = 32 2n = 32 n = 16 n = 16 2n = 32 2n = 32 2n = 32 n = 16 n = 16 2n = 32 2n = 32 2n = 32 2n = 32 n = 16 2n = 32 2n = 32 2n = 32 2n = 32 2n = 32 2n = 32 2n = 32 2n = 32 2n = 32 2n = 36 n = 16 2n = 36 2n = 36 2n = 36 2n = 36 2n = 36 2n = 36 n = 16 2n = 36 2n = 32 2n =32 n = 16 2n = 36? 2n = 32 2n =32 n = 16 2n = 32 2n =32 2n =32 2n =32 2n = 28 n = 14 2n = 28 2n = 28 n = 21 n = 14 2n = 28 n = 14 2n = 28 © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 556–566 560 J. KISSLING ET AL. Table 2. Continued Taxon Sebaea Sebaea Sebaea Sebaea Sebaea Sebaea Sebaea Sebaea Sebaea Sebaea Sebaea Sebaea Sebaea Sebaea Sebaea ambigua Cham. aurea (L.f.) Roem. & Schult aurea (L.f.) Roem. & Schult aurea (L.f.) Roem. & Schult aurea (L.f.) Roem. & Schult. baumiana (Gilg) Boutique baumiana (Gilg) Boutique baumiana (Gilg) Boutique baumiana (Gilg) Boutique baumiana (Gilg) Boutique baumiana (Gilg) Boutique baumiana (Gilg) Boutique baumiana (Gilg) Boutique baumiana (Gilg) Boutique bojeri Griseb. Collector Voucher code Origin JK & LZ JK JK JK JK & LZ SD, RA, PD, SD, RA, PD, SD, RA, PD, SD, RA, PD, SD, RA, PD, SD, RA, PD, SD, RA, PD, SD, RA, PD, SD, RA, PD, LZ 45 91 98 89 49 809 824 845 906 933 969 970 971 974 05.03.01 2a; I11_Jtr.X 05.03.02 1b; I11_Jtr_4 05.03.02 2b; H14_Jfr_2 M044 05.03.04 1b; I11_Lpr.3 656 South Africa South Africa South Africa South Africa South Africa Zambia Zambia Zambia Zambia Zambia Zambia Zambia Zambia Zambia South Africa JK, JK, JK, JK, JK, JK, JK, JK, JK, BL BL BL BL BL BL BL BL BL & & & & & & & & & ET ET ET ET ET ET ET ET ET Sebaea bojeri Griseb. LZ Sebaea bojeri Griseb. LZ Sebaea brachyphylla Griseb. Sebaea brachyphylla Griseb. JP, SW & LZ LZ Sebaea gracilis (Welw.) Paiva & Nogueira aff. Sebaea gracilis (Welw.) Paiva & Nogueira aff. Sebaea rehmannii Schinz aff. SD, RA, PD, JK, BL & ET Sebaea rehmannii Schinz aff. Sebaea scabra Schinz aff. Sebaea clavata J.Paiva & I.Nogueria Sebaea exacoides Schinz Sebaea exacoides Schinz Sebaea exacoides Schinz Sebaea exacoides Schinz Sebaea exacoides Schinz Sebaea exacoides Schinz aff. Sebaea fernandesiana J.Paiva & I.Nogueria Sebaea filiformis Schinz n = 14 n = 14 n = 21 n = 21 n = 21 n = 21 n = 21 n = 21 n = 21 2n = 28 2n = 42 2n = 28 2n = 28 2n = 42 2n = 42 2n = 42 2n = 42 2n = 42 2n = 42 2n = 42 2n = 28 South Africa 2n = 28 South Africa 2n = 28 South Africa South Africa 2n = c. 56! 2n = 42! Zambia n = 21 2n = 42 SD, RA, PD, JK, BL & ET 692 Zambia n = 21 2n = 42 LZ South Africa n = 21 05.03.02 1; K09_Tal.3 LZ 05.03.02 1a; K03_Jtr.4 JK, BG & MP 109 SD, RA, PD, JK, BL & ET 543 JK & LZ 81 JK & LZ 86 JK & LZ 87 JK & LZ 88a JK & LZ 88b JK, BG & MP 104 SD, RA, PD, JK, BL & ET 1011 South Africa Zambia South Africa South Africa South Africa South Africa South Africa South Africa Zambia LZ South Africa 2n = 28 South Africa 2n = 28 Sebaea filiformis Schinz LZ Sebaea Sebaea Sebaea Sebaea Sebaea Sebaea Sebaea Sebaea SD, RA, PD, JK, SD, RA, PD, JK, SD, RA, PD, JK, SD, RA, PD, JK, JK, BG & MP JK, BG & MP JK & LZ JK & LZ grandis Steud. grandis Steud. grandis Steud. grandis Steud. griesbachiana Schinz aff. griesbachiana Schinz aff. hymenosepala Gilg aff. hymenosepala Gilg aff. Chromosome numbers BL BL BL BL & & & & ET ET ET ET 05.03.03 1a; I11_Gre1 05.03.04 1a; H14_Jtr.1 657 752 764 815 112 115 3 4 South Africa n = 21 Zambia Zambia Zambia Zambia South Africa South Africa South Africa South Africa n = 21 n = 14 2n = 28 2n = 42 2n = 28 2n = 28 2n = 28 2n = 28 n = 14 n = 21 2n = 28 2n = 42 n = 21 2n = 42 n = 21 2n = 42 n = 14 n = 14 2n = 28 2n = 28 2n = 28 © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 556–566 CYTOGEOGRAPHY OF EXACEAE IN AFRICA 561 Table 2. Continued Chromosome numbers Taxon Collector Voucher code Origin Sebaea hymenosepala Gilg aff. Sebaea hymenosepala Gilg aff. Sebaea leiostyla Gilg JK & LZ JK & LZ LZ South Africa South Africa South Africa 2n = 28 2n = 28 2n = 28 Sebaea leiostyla Gilg LZ South Africa 2n = 28 Sebaea leiostyla Gilg LZ South Africa 2n = 28 Sebaea leiostyla Gilg Sebaea macrophylla Gilg Sebaea macrophylla Gilg Sebaea madagascariensis Klack. Sebaea marlothii Gilg Sebaea marlothii Gilg Sebaea marlothii Gilg Sebaea marlothii Gilg Sebaea marlothii Gilg Sebaea marlothii Gilg Sebaea marlothii Gilg Sebaea membranaceae Hill. aff. Sebaea micrantha aff. (Cham & Schlechdtl.) Schinz Sebaea minuta J.Paiva & I.Nogueria Sebaea minutiflora Schinz Sebaea minutiflora Schinz Sebaea oligantha Schinz Sebaea pentendra E.Mey. aff. Sebaea perparva Sileshi Sebaea procumbens Hill. Sebaea pusilla Eckl. Ex Cham. Sebaea repens Schinz aff. Sebaea repens Schinz aff. Sebaea scabra Schinz Sebaea scabra Schinz Sebaea schlechterii Schinz Sebaea schlechterii Schinz Sebaea sedoides var. confertiflora (Schinz) Marais Sebaea sedoides var. confertiflora (Schinz) Marais Sebaea sedoides var. sedoides Gilg LZ JK JK JP, JK JK JK JK JK JK JK JK JK 5 73 05.03.01 2b; I11_Aob_4 05.03.02 2a; I11_Jfr_3 05.03.03 2a; H14_Gac_2 05.03.01 3 72 74 M017 11 15 17 18 19 21 38 66 95 South Africa South Africa South Africa Madagascar South Africa Leshoto Leshoto Leshoto Leshoto Leshoto South Africa South Africa South Africa 2n = 28 2n = 56 SD, RA, PD, JK, BL & ET 623 JK 83 JK & LZ 46 SD, RA, PD, JK, BL & ET 499 JK & LZ 34 SD, RA, PD, JK, BL & ET 728 JK & LZ 10 JK & LZ 64 JK & LZ 14a JK & LZ 14b JK 85 JK, BG & MP 103 JK & LZ 50 JK & LZ 55 LZ 05.03.03 1b; H14_Lpr_2 LZ 05.03.03 2b; I11_Cun_2 LZ 05.03.03 2b; I11_Cun_2 JK, BG & MP 106a JK, BG & MP 107 JK, BG & MP 108 JK & LZ 35 JK & LZ 37 JK 99 JK, BG & MP 117 JK & LZ 30 Zambia South Africa South Africa Zambia South Africa Zambia South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa South Africa JK & LZ South Africa Sebaea sp. Sebaea sp. Sebaea sp. Sebaea sp. A Sebaea sp. A Sebaea sp. B Sebaea sp. C Sebaea sp. (Undeterminable, only very young buds present) Sebaea sp. (Undeterminable, only very young buds present) & LZ & LZ SW & LZ & LZ & LZ & LZ & LZ & LZ & LZ & LZ & LZ 69 n = 28 n=9 2n = 18 2n = 28 n = 14 n = 28 n = 21 n = 14 n = 14 n = 21 2n = 28 2n = 28 2n = 28 2n = 28 2n = 28 2n = 28 2n = 56 2n = 42 2n = 42 2n = 28 2n = 42 2n = 28 2n = 28 2n = 28 2n = 28 2n = 28 2n = 28 n = 14 2n = 28 2n = 42 South Africa 2n = 42 South Africa 2n = 42 South Africa South Africa South Africa South Africa South Africa South Africa South Africa Leshoto © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 556–566 n = 14 n = 14 n = 14 n = 14 2n = 28 2n = 28 2n = 28 2n = 28 2n = 28 2n = 28 562 J. KISSLING ET AL. Table 2. Continued Chromosome numbers Taxon Collector Voucher code Origin Sebaea sp. (Undeterminable, only very young buds present) Sebaea spathulata (E. Mey.) Steud. Sebaea spathulata (E. Mey.) Steud. Sebaea spathulata (E. Mey.) Steud. Sebaea spathulata (E. Mey.) Steud. Sebaea sulphurea Cham & Schlechtdl. Sebaea teucszii (Schinz) Taylor Sebaea teucszii (Schinz) Taylor Sebaea teucszii (Schinz) Taylor Sebaea teucszii (Schinz) Taylor Sebaea thodeana Gilg Sebaea thodeana Gilg. Sebaea thodeana Gilg. Sebaea thodeana Gilg. aff. Sebaea thodeana Gilg. aff. Sebaea thomasii (S.Moore) Schinz Sebaea thomasii (S.Moore) Schinz Tachiadenus carinatus (Desr.) Griseb. Tachiadenus longiflorus Bojer ex. Griseb. Tachiadenus longiflorus Bojer ex. Griseb. Tachiadenus longiflorus Bojer ex. Griseb. JK & LZ 71 South Africa 2n = 28 JK & LZ JK & LZ JK & LZ JK & LZ JK, BG & MP SD, RA, PD, JK, SD, RA, PD, JK, SD, RA, PD, JK, SD, RA, PD, JK, JK & LZ JK & LZ JK & LZ JK & LZ JK & LZ JK & LZ JK & LZ JP, SW & LZ JP, SW & LZ JP, SW & LZ JP, SW & LZ 12 25 26 40 100 557 599 701 771 20 16 22 31a 31b 29 70 M039 M006 M016 – South Africa Leshoto Leshoto South Africa South Africa Zambia Zambia n = 21 Zambia n = 21 Zambia Leshoto Leshoto Leshoto Leshoto Leshoto n = 14 Leshoto South Africa Madagascar Madagascar Madagascar Madagascar 2n = 28 2n = 28 2n = 28 2n = 28 2n = 28 2n = 42 specimens are deposited in the herbarium of the University of Neuchâtel, Switzerland (NEU). For karyological studies, flower buds were collected and directly fixed in the field in Carnoys solution (1/3 glacial acetic acid/absolute ethanol). When buds were not available, root tips of species cultivated in the botanical garden of Neuchâtel were used. In that case, suitable root tips were first pretreated with a saturated aqueous solution of a-bromonaphthalene for 1 h 20 min, fixed in Carnoys for 2 weeks and finally stained with aceto-carmine and squashed on temporary slides. Chromosome observations were performed either on meiotic plates from pollen mother cells (fixed buds) or on mitotic plates from young cells of the ovary wall (fixed buds) or root tips (Fig. 1). In a few cases, chromosomes were counted from the second pollen mitosis. All counts were made with a Leica light microscope either with or without phase contrast and a 100¥ oil immersion objective, given a total magnification of ¥1000. Microphotographs were taken and drawings were made using a camera lucida. Each chromosome number was determined from at least four different preparations by two of the authors (JK and LZ). BL BL BL BL & & & & ET ET ET ET 2n = 42 2n = 28 2n = 28 2n = 28 2n = 28 2n = 28 2n = 28 2n = 32 2n = 32 2n = 32 2n = 32 RESULTS AND DISCUSSION A total of 155 accessions representing 61 species and four genera (Exacum, Ornichia, Sebaea and Tachiadenus) collected in Lesotho, Madagascar, South Africa and Zambia were analysed (Table 2). Chromosome number ranged from n = 9 (Sebaea madagascariensis) to n = 28 (e.g. Sebaea brachyphylla, S. macrophylla), with most variability occurring in Sebaea (n = 9, 14, 21, 28) and Exacum (n = 8, n = 9). Chromosomes numbers for 59 taxa are reported for the first time, including species of Exacum (14 out of 69), Ornichia (one out of three), Sebaea (c. 40 out of c. 95; Schinz, 1906) and Tachiadenus (two out of 11 species). In the following, we provide an enumeration of the chromosome numbers hitherto found in Exaceae and interpret them in the light of the present results. KARYOLOGICAL REVIEW OF EXACEAE Based on new chromosome numbers evidenced in this study, several base numbers can be proposed for Exaceae: x = 7 (n = 14, n = 21, n = 28), x = 8 (n = 16) and x = 9 (n = 9, n = 18). The haploid numbers n = 17, 26, 27, 28, 30, 31 and 34, reported in the literature © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 556–566 CYTOGEOGRAPHY OF EXACEAE IN AFRICA (Table 1), may be interpreted as the result of dysploidy and/or allopolyploidy, two processes frequently occurring in some genera of Gentianaceae, including Centaurium (Zeltner, 1970; Mansion et al., 2005), Exacum (Darlington & Wylie, 1955; Riseman et al., 2006) or Gentiana section Chondrophyllae (Yuan et al., 1998). Base chromosome number x = 7 The chromosome number n = 14 occurs frequently within Sebaea (26 species investigated) but also in Ornichia (1 species), for which it is the first published report. Fourteen species of Sebaea from Zambia and South Africa share the haploid number n = 21, whereas one species, S. brachyphylla, was found with either n = 21 (Madagascar) or n = 28 (Drakensberg). In both cases, our results differ from a previous report from Kenya (n = 22, Thulin, 1970). Two other species, S. minutiflora (n = 21 or 28) and S. aurea (n = 14 or 21) were found to have different chromosome numbers. Finally, n = 28 is specific to two species of Sebaea collected in South Africa (S. macrophylla and S. aff. micrantha). In the absence of populations with n = 7, we can propose n = 14 to be either a tetraploid number or a secondary diploid one, in a series based on x = 7. In the latter case, the genus is presently represented by tetraploid (n = 14), hexaploid (n = 21) and octoploid (n = 28) species. Base chromosome number x = 8 This number, new for Exaceae, is reported here with the haploid chromosome number n = 16 and seems to characterize Malagasian species of Exacum (14 species). However, one species of Exacum from Indonesia was reported to have n = 16–18 (Table 1; under Cotylanthera tenuis). Two species of the Malagasian endemic Tachiadenus are reported here with a haploid number of n = 16. Base chromosome number x = 9 The haploid number n = 9 was previously reported for Exacum tetragonum from India (Borgmann, 1964), but was not confirmed by Mallikarjuna et al. (1987) who found 2n = 62, a chromosome number more frequent in Exacum (Table 1). Our results support the occurrence of n = 9 in only one species of Sebaea (S. madagascariensis), endemic to Madagascar. The haploid number n = 18 occurs in two species of Exacum from Madagascar. This study also confirms the previous reports of n = 18 for Exacum affine from Socotra (Sugiura, 1936a, b; Post, 1967; Riseman et al., 2006). Other chromosome numbers The haploid number n = 17, reported for Exacum atropurpureum from India (Mallikarjuna et al., 1987), has 563 not been found in the present study in which mostly Malagasian species of Exacum were investigated. The series n = 26, n = 27 and n = 30, published recently for several subspecies of Exacum trinervium from Sri Lanka (Riseman et al., 2006), underlies the great chromosome instability of this taxon. This report might also support x = 10 and x = 13 as a possible base number for Exaceae. Finally, n = 31 and n = 34 have been reported for eight species of Exacum from India (Mallikarjuna et al., 1987) supporting x = 17 as another possible base number for the tribe. CHROMOSOME NUMBER AND SYSTEMATICS OF EXACEAE Exacum In the last monograph of the genus (Klackenberg, 1985), Exacum has been divided in two sections based on phenotypic and biogeographic evidence. Section Exacum (21 species, including four saprophytes previously included in Cotylanthera) is restricted to Sri Lanka, India and the Himalayas. This section comprises biennial robust plants with large flowers (c. 2–7 cm in diameter; Klackenberg, 1985) and is characterized by a wide range of published chromosome numbers inferred as n = 9, 16, 17, 18, 26, 27, 28, 30, 31 and 34. If we exclude the approximate n = 16, reported with caution for Exacum tenuis (under Cotylanthera), of which confirmation is needed, and the intraspecific variation (n = 26, 27, 28, 30) observed in the E. trinervium complex, which might be the result of allopolyploidy/dysploidy events (Riseman et al., 2006), the more frequent haploid numbers observed in Exacum are n = 17, 18, 28, 30, 31 and 34. Possible primary or secondary base numbers for this section might then be x = 7, 9, 10 and 17 (31). Section Africana (44 species) is distributed in Madagascar, Socotra and the African mainland. All the investigated members of this section, generally small annual plants with a tiny corolla (c. 0.8– 1.5 cm in diameter; Klackenberg, 1985), show either n = 16 (14 species, Madagascar) or n = 18 (1 species, Socotra), except the variable E. quinquinervium (n = 16 or 18), a fact which supports x = 8 and x = 9 as possible base numbers for this group. Some systematic support can be drawn if the respective Madacascan and Socotran groups are determined to be monophyletic (morphology or molecular data). Overall, the range in base chromosome number detected in the genus, x = 7, 8, 9, 10 and 17 (31), does not allow us to propose an unambiguous scenario for karyotype evolution within Exacum. Nonetheless, the high karyotypic diversity encountered in section Exacum might indicate rapid evolutionary episodes within this group, a fact supported by recent phylogenetic studies (Yuan et al., 2005), showing multiple © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 556–566 564 J. KISSLING ET AL. out-of-Madagascar dispersals of Exacum species, with further extensive radiation into Asia. Sebaea Although the morphologically variable Sebaea represents the most important genus in term of species number (c. 95 species; Schinz, 1906), no convincing taxonomic treatment has been proposed so far, mainly because of a lack of global studies integrating phylogenetic hypotheses, biogeographical data and cytological evidence. The present karyological investigation and the range of chromosome variation detected (n = 9, 14, 21, 28) help elucidate the possible evolutionary history of the genus. The lowest chromosome number detected so far in Sebaea (n = 9), indeed in Exaceae, occurs in S. madagascariensis. This species, endemic to the northwestern part of Madagascar, differs from all other Sebaea by having a ‘raceme-like’ inflorescence and particular floral features (Klackenberg, 1990). Furthermore, recent phylogenetic studies (Yuan et al., 2003) support the exclusion of S. madagascariensis from Sebaea and indicate affinities with Ornichia and Exacum. As also evidenced by our karyological data, S. madagascariensis probably deserves generic ranking. The most common chromosome number in Sebaea (n = 14, 2n = 28; Table 2) is found in a group of species distributed from the Cape region of South Africa to the Drakensberg mountains. These species are morphologically characterized by a bilobed stigma and a flowering period mainly between September and December. In addition, members of this group formed a well-supported clade in a phylogenetic analysis of the tribe based on plastid DNA and nrDNA markers (Yuan et al., 2003). Species of Sebaea with n = 21 generally occur in the Zambesian region of tropical Africa, are morphologically characterized by a clavate to linear stigma and come into flower in March–April (Paiva & Nogueira, 1990). Finally, S. brachyphylla shows intraspecific variation, with n = 21 found in African populations and n = 28 in the Malagasian ones (Table 2), adding to the confusing taxonomy reported within this taxon (Hedberg, 1955). Hence, at least four different taxa are referred to as S. brachyphylla (Boutique, 1972; Paiva & Nogueira, 1990), with the type species described from Madagascar. It would be interesting to survey additional taxa for chromosome counts from both tropical Africa and Madagascar, including the type locality, to confirm or discuss the observed karyological pattern. Finally, the report from Thulin (1970) for S. brachyphylla (2n = 22) is not confirmed by our study, either in Sebaea or in other Exaceae and might be taken with caution. Overall, if we exclude S. madagascariensis from Sebaea (Yuan et al., 2003), we can postulate a base chromosome number of x = 7 for Sebaea, with a group of tetraploid species (n = 14) centered in South Africa and a group of tetra/hexaploid species in Central Africa. The role of polyploidy in the evolution of Sebaea is highlighted for the first time in this study. KARYOTYPE EVOLUTION IN THE EXACEAE In our review, including present and past karyological reports for Exaceae, the following haploid chromosome numbers have been detected: n = 9 (Sebaea, 1 sp.; Exacum, 1 sp.), n = 14 (Sebaea, c. 29 spp.; Ornichia, 1 sp.), n = 16 (Exacum, 14 spp.; Tachiadenus, 2 spp.), n = 17 (Exacum, 1 sp.), n = 18 (Exacum, 2 spp.), n = 21 (Sebaea, 13 spp.), n = 26 (Exacum, 2 spp.), n = 27 (Exacum, 3 spp.), n = 28 (Sebaea, 4 spp.; Exacum, 2 spp.), n = 30 (Exacum, 1 sp.), n = 31 (Exacum, 5 spp.), n = 34 (Exacum, 5 spp.). These numbers confirm the polybasic and dysploid nature of both Exacum and Sebaea s.l. karyotypes. Hence, several intraspecific cytotypes have been detected for, e.g., Sebaea brachyphylla, S. minutiflora, Exacum trinervium and E. pedunculatum, which may indicate cryptic speciation in the absence of detectable morphological variation. Our current sampling does not allow further speculation on the topic. Considering the present data, we can propose a combination of both dysploidy and polyploidy events in the karyotypic evolution of Exaceae. If we accept x = 7 as a possible base number for Sebaea (n = 14, 21, 28), Exacum (n = 28) and Ornichia (n = 14), and x = 8 as another base number for Exacum (section Africana), we can infer a primarily dysploid series x = 7, 8 and 9 for Exaceae. In the absence of strong phylogenetic hypotheses and complete karyotype reconstruction for all investigated species, both the determination of a putative ancestral number and the polarity of dysploid series within Exaceae remain challenging. Nevertheless, external evidence can lead us to postulate x = 7 as a possible ancestral number for Exaceae and thus an ascending dysploid series for the tribe: (1) the haploid numbers n = 14, 21 and 28 (x = 7) occur in most of the species investigated in this study (44 of 75), followed by n = 16 (x = 8; 16 of 75) and n = 9, 18 (x = 9; 5 of 75); (2) Exaceae species based on x = 7 generally show a wide distribution; and (3) x = 7 occurs in morphologically distinct genera, including Sebaea, Exacum and Ornichia. This hypothesis is further supported by molecular data indicating that members of Sebaea with n = 14 form a primarily derived, well-supported clade in a global phylogenetic analysis of the tribe (Yuan et al., 2003). © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158, 556–566 CYTOGEOGRAPHY OF EXACEAE IN AFRICA ACKNOWLEDGEMENTS We would like to thank the anonymous reviewers for their useful suggestions and comments. 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