The polyphyletic genus Sebaea (Gentianaceae): A step forward in understanding the morphological and karyological evolution of the Exaceae
Introduction
The Exaceae (Gentianaceae) in their current circumscription (Struwe et al., 2002, Klackenberg, 2006) are a small tribe that includes ca. 181 species distributed in the respective genera Exacum L. (69 spp. including Cotylanthera Blume), Gentianothamnus Humbert (1 sp), Klackenbergia Kissling (2 spp.), Ornichia Klack. (3 spp.), Sebaea Sol. (c. 95 spp.), and Tachiadenus Griseb. (11 spp.) (Schinz, 1906, Hill and Prain, 1909, Klackenberg, 1985, Klackenberg, 1986, Klackenberg, 1987a, Klackenberg, 1990, Klackenberg, 2002, Klackenberg, 2006, Kissling et al., 2009). The Exaceae mainly differ from the five other gentian tribes by the star-shaped testa cells of the seeds and the rounded-convex shape of the epidermis cells of petals (Klackenberg, 1985, Klackenberg, 2002, Bouman et al., 2002). A wide range of morphological variation has been found within the tribe (Klackenberg, 2002), including general habit (herb versus subshrub), gynoecium structure (ovary truly or pseudo-bilocular, style straight or bent downwards), androecium (insertion of filaments, opening mechanism of the stamens), corolla color (white, yellow, orange, blue, and purple), corolla-merosity (4 or 5 lobes), corolla size (a few mm up to 7 cm in diameter), or length of the corolla tube (a few mm up to 20 cm). To date, important morphological investigations have been carried out on Exacum, Ornichia, and Tachiadenus (Klackenberg, 1983, Klackenberg, 1985, Klackenberg, 1986, Klackenberg, 1987a), while only a few molecular studies have been performed on the Exaceae, most of them focusing on Exacum (Yuan et al., 2003, Yuan et al., 2005). Surprisingly, systematic studies on Sebaea, the most species-rich genus of the tribe, remained anecdotal (Klackenberg, 2002, Yuan et al., 2003).
Sebaea comprises annual to perennial, erect to procumbent herbs, or more rarely achlorophyllous saprophytes (S. oligantha). The genus size is still a matter of speculation. Indeed, early estimates ranged from 60 to 159 species (Wielgorskaya, 1995, Schinz, 1906, Boutique, 1972, Paiva and Nogueira, 1990a, Mabberley, 1997, Dyer, 1975, Adams, 1996), while recent field observations and examination of ca. 3000 herbaria specimens (Jonathan Kissling, pers. obs.), better support ca. 95 species, making Sebaea the most species-rich genus within the Exaceae.
Sebaea is widely distributed in the Old World, with two centers of diversity in Tropical Africa and Southern Africa (Schinz, 1906, Hill, 1908, Marais and Verdoorn, 1963, Boutique, 1972, Paiva and Nogueira, 1990a, Paiva and Nogueira, 1990b, Nemomissa, 2002). Tropical species (ca. 25) are mainly distributed on the Katanga plateau at the intersection between the Democratic Republic of Congo, Angola, and Zambia, while the Southern African species (ca. 70 spp.) mostly occur in the Cape Region and the Drakensberg escarpment (Lesotho and South Africa). Four species extend their range eastward to Madagascar, two of them being endemic to the island (Klackenberg, 1987b, Klackenberg, 1990). Finally, two species are endemic to the Australia/New Zealand region (Adams, 1996), whereas one species extends its range from Africa to the Indo-Malaysian area (Ho and Pringle, 1995, Suksathan and Sasirat, 2000).
Sebaea species grow from sea level (e.g. S. minutiflora, S. ambigua) to high elevations (3000–3500 m) in the Drakensberg (S. marlothii and S. thodeana) or in the East African mountains (S. brachyphylla and S. leiostyla). They can also be encountered in most parts of Sub-Saharan Africa in a wide variety of habitat including dense tropical forests, grasslands, and savannas, marshes or waterlogged areas, alpine meadows, or rocks and sandy riverbanks.
Despite series of systematic works performed on the Exaceae (Brown, 1810, Rafinesque, 1837, Meyer, 1838, Grisebach, 1845, Bentham, 1876, Schinz, 1891, Schinz, 1903, Schinz, 1906, Gilg, 1895a, Gilg, 1898, Hill, 1908), the generic circumscription of Sebaea remains controversial. Most of the confusion relies on the frequent use of homoplastic characters (Marais, 1961), including e.g. the form (straight or recurved) and disposition (free or connate) of the anthers, the presence of appendages (glands) on the anthers, and the height of the filament insertion. The circumscription of Sebaea is difficult because a large amount of morphological plasticity seems to occur depending on the varying environmental conditions, overall obscuring species boundaries (Hedberg, 1955) and resulting in a high systematic and taxonomic uncertainty (e.g. Chamisso and Schlechtendal, 1826, Rafinesque, 1837, Meyer, 1838, Grisebach, 1849, Bentham, 1876, Schinz, 1891, Knochblauch, 1894, Gilg, 1898, Schinz, 1903, Schinz, 1906, Hill, 1908, Marais, 1961, Marais and Verdoorn, 1963).
In addition to its poorly understood systematic history, the phylogenetic position of Sebaea is still a matter of speculation. Most molecular studies published so far support the inclusion of the genus in the Exaceae (Struwe et al., 2002, Yuan et al., 2003; Kissling et al., unpublished), and a possible monophyletic status if the newly described Klackenbergia is excluded from Sebaea s.l. (Kissling et al., 2009, Yuan et al., 2003), but the overall sampling prevents further phylogenetic or systematic conclusions.
In this paper, we used an extensive dataset of Sebaea species collected in the field to provide a phylogenetic hypothesis aimed at revealing the morphological and karyological evolution of the genus. More specifically, the goals of this study are to: (1) confirm the monophyly of the tribe Exaceae, taking into account the large geographical and morphological diversity of Sebaea; (2) test the monophyly vs. polyphyly of the genus; (3) identify synapomorphic characters supporting molecular clades; and (4) reconstruct the evolution of relevant non-molecular characters within the tribe.
Section snippets
Taxon sampling and outgroup choice
Plant samples were collected in 2004–06 in South Africa and Zambia (Sebaea) and Madagascar (Exacum and Tachiadenus), and were determined by the first author using local floras (Marais and Verdoorn, 1963, Taylor, 1963, Boutique, 1972, Klackenberg, 1990, Paiva and Nogueira, 1990a, Nemomissa, 2002). Voucher specimens are deposited in the herbarium of the University of Neuchâtel, Switzerland (NEU). But duplicates can also be found in the following herbaria BOL, BR, PRE, TAN and Mt Makulu Research
Sequence and alignment characteristics
The trnL intron sequences ranged from 334 to 539 bp in length. Within Gentianaceae, the Exaceae, and the Saccifolieae are characterized by longer sequences (from 449 to 539 bp), resulting in an insertion of 122 bp in the aligned matrix. The trnL-F spacer sequences ranged from 335 to 478 bp in length, with 36 indels in the aligned matrix, the longest one being a deletion of 93 bp occurring in some accessions of Sebaea. The atpB-rbcL spacer sequences ranged from 710 to 781 bp in length, and the partial
Discussion
Our phylogenetic analyses of a large dataset, including most species ascribed to Exaceae (71 out of c. 181) and particularly 45% of the diversity encountered in Sebaea (43 out of c. 95 species), support the monophyly of the tribe (BS 100) and the paraphyly of Sebaea, currently split into three separate “clades” (S1–S3; Fig. 1). The remaining five genera form a strongly supported (BS 82) clade, sister to S3 (BS 55), while S1 + S2 clade (BS 99), are sister to the rest of the tribe.
Acknowledgments
The authors thank Dirk Bellstedt, Benny Bytebier, Sven Bürki, and Louis Zeltner for providing leaf tissues and samples, Robert Archer, Petra DeBlock, Steven Dessein, Berit Gehrke, Brian Luwingu, Mike Pirie, Elias Tembo, Julie Würfel and Louis Zeltner for their help in the field, Xavier Bernet, Maxime Pujol and Fanny Schupfer for laboratory help, and the curators of the following herbaria: BM, BOL, BR, C, G, K, MO, NEU, PRE, TAN, S, UPS, WAG, and Z. Finally, we are grateful to the Western Cape
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