Plant Syst. Evol. 223:1-13 (2000) Plant Systematics and Evolution © Springer-Vertag 2000 Printed in Austria Controversial taxonomy of Strumariinae (Amaryllidaceae) investigated by nuclear rDNA (ITS) sequences 1. Hessea, Namaquanula, Kamiesbergia, and Dewinterella Katja Weiehhardt-Kulessa 1, Thomas B6rner 2, Jiirgen Sehmitz 3, Ute Miiller-Doblies 4, and Dietrich Miiller-Doblies 1 ilnstitut ffir Okologie und Biologie, Technische Universitiit Berlin, Berlin, Germany 2Institut fiir Biologie (Genetik), Humboldt-Universit/it zu Berlin, Berlin, Germany SDeutsches Primatenzentrum, G6ttingen, Germany 4Berlin, Germany Received February 20, 1998 Accepted August 16, 1999 Abstract. Two opposing opinions concerning the phon (subgenus Kamiesbergia) is the sister taxon genetic differentiation of Amaryllidaceae subtribe of H. breviflora. Strumariinae two taxonomic opinions were pub- lished in the last decade. According to Mfiller- Key words: Amaryllidaceae, Strumariinae. Phy- Doblies and Mfiller-Doblies (1985, 1994) the logeny, ITS 1, ITS 2, 18S rDNA, 25S rDNA, Strumariinae includes eight genera, among them maximum parsimony analysis, distance analysis, Hessea, Namaquanula, and Dewinterella. Snijman maximum likelihood analysis. (1991) described the new genus Kamiesbergia and accepted Namaquanula (1992). Of the entire The cosmopolitan family Amaryllidaceae con- complex she recognized only Hessea (Snijman sists of nine tribes, some of them divided 1994). The section Myophila was simultaneously into subtribes (Dahlgren et al. 1985). The pre- described as the genus Dewinterella by Miiller- dominantly South African tribe Amaryllideae Doblies and Mfiller-Doblies. is the largest tribe of the family according to The possible phylogenetic relationships of eight the number of genera (11-16 genera) and the species belonging to these taxa are estimated from second in size according to the number of the sequences of the ITS regions of the 18S-25S species. It comprises two subtribes, Amarylli- rDNA. Two species of Nerine are used as the dinae (confined to Africa except for the outgroup. The sequences of these taxa are analyzed with maximum parsimony, distance, and maximum subcosmopolitan genus Crinum) and Strumar- likelihood methods. In all phylogenetic reconstruc- iinae (endemic to southern Africa). tions Namaquanula is confirmed to be an inde- Most genera of the Amaryllidinae have pendant clade aside from all other six species of the been revised, either provisionally or compre- Strumariinae. In this group Hessea and Dewinter- hensively, throughout the second and third ella turn out to be sister groups. Hessea stenosi- quarters of this century: Ammocharis and 2 K. Weichhardt-Kulessa: Controversial taxonomy of Strumariinae (Amaryllidaceae) Cybistetes (Milne-Redhead and Schweickerdt H. bruce-bayeri (D. & U. M-D.) Snijman and 1939), Brunsvigia (Dyer 1950, 1951), Nerine section Myophila with two species, H. pulcher- (Traub 1967), and Crinum in South and East rima (D. & U. M-D.) Snijman and H. mathewsii Africa (Verdoorn 1973, Nordal 1977) and W. E Barker (Fig. 1). When the latter sectional Cameroun (Nordal and WahlstrCm 1980). taxon was published in June 1994, the genus Numerous classifications from the 19th Dewinterella D. & U. M-D. with the same two century dealing with the Hessea-Strumaria species, D. pulcherrima (D. & U. M-D.) D. & U. group and its related genera can be found M-D. and D. mathewsii (W. F. Barker), was (Herbert 1837, Kunth 1850, Salisbury 1866, already in print. As both taxa, the section Baker 1888) which reveal a heterogeneous Myophila and the genus Dewinterella, do not picture of this cluster. Not until the middle of compete for priority, it is possible, therefore, to this century, however, did several botanists consider them as more or less simultaneously start working on it again: Barker (1948), published. Leighton (1948), Obermeyer (1963), Traub The second subgenus, H. subgen. Kamies- (1963), and Goldblatt (1976). In 1985 Miiller- bergia, is monotypic with Hessea stenosiphon Doblies and Mfiller-Doblies validated Traub's (Snijman) D. & U. M-D. (1957) Strumariinae as a subtribe including The subgenus H. subgen. Hessea includes seven genera: Carpolyza Salisb., Namaqua- nine species. Three of them, H. breviflora, nula D. & U. M-D., Strumaria Jacq., Bokke- H. stellaris, and H. cinnabarina, are analyzed veldia D. & U. M-D. (segregated from in the present paper of which the latter species Strumaria), Gemmaria Salisb. (segregated from deserves a taxonomic note. It was not Hessea), Hessea Herb., and Tedingea D. & U. recognized by Snijman and was treated as M-D. Nine years later another genus, Dewinter- synonymous with H. stellaris. The same ella D. & U. M-D., was added (Mfiller-Doblies problem occurred with H. longituba which and Mi.iller-Doblies 1994), accommodating two was placed into the synonymy of H. breviflora. isolated species of Gemmaria. The new genera Thus within the subclade A 1 none of the were accepted by Gunn et al. (1992), Brummitt three additional genera published between (1992), and Greuter et al. (1993). Two of them, 1985 and 1994 are recognized, not even the Tedingea and Namaquanula, were also genus Kamiesbergia of 1991. A single genus, approved by Snijman and Perry (1987) and Hessea, is left (Fig. 1). Snij man (1992). At the beginning of the nineties There are some molecular studies of the a new genus of the Strumariinae, Kamiesbergia Amaryllidaceae and closely related families, Snijman, was published (Snijman 1991) which such as Liliaceae (Shinwari et al. 1994, Fay Miiller-Doblies and Mfiller-Doblies had pub- and Chase 1996). Snijman (1994) presented a lished as a new species of Hessea at the scheme of the phylogenetic relationships of same time. Shortly afterwards Kamiesbergia the Strumariinae based on morphological and stenosiphon was changed to Hessea stenosiphon anatomical data. Because of the controversial (Miiller-Doblies and Mfiller-Doblies 1992). discussion concerning the phylogeny of the Snijman, (1994) who did not recognize Strumariinae, it is useful to look for a new the subtribe Strumariinae of Amaryllideae, source of information for the phylogenetic divided it into two subclades, A 1 (= Hessea reconstruction, based on the results in several sensu Snijman) and subclade A 2 ( --- Carpolyza publications dealing with ITS sequence data as and Strumaria sensu Snijman). The subclade a successful tool to reconstruct the phylogeny A 1 contains three subgenera, Namaquanula, of certain plant groups (e.g. Poaceae by Hsiao Kamiesbergia, and Hessea. Hessea subgen. et al. 1994, 1995a, b; Fabaceae by Wojcie- Namaquanula consists of two sections: section chowski et al. 1993; Asteraceae by Baldwin Namaquanula (formally) with the single species 1992, 1993), this region of the nuclear rDNA, K. Weichhardt-Kulessa: Controversial taxonomy of Strumariinae (Amaryllidaceae) 3 Section Subgenus U Namaq,amda ~ H. bruce.bayeri Nanlaquantda -]__ Section H. pulcherrima Myophila H. mathewsii Subgenus H. stenosiphon Hessea Kamiesbergia H. breviflora Subgenus ._~ Hessea Fig. 1. Snijman's dendrogram con- H. stellaris taining the modified subclade A 1 the internal transcribed spacer (ITS) with the of different species and individuals were isolated on intervening 5.8S gene, was chosen. Therefore, different days. it is hoped that this study provides a new and About 1 mg fresh or frozen leaves were homo- conclusive phylogenetic scenario of the Stru- genized in 400gl Wilson-buffer (100raM Tris, mariinae. 10ram EDTA, 100raM NaC1, 0.1% SDS, pH 8.0) with a pestle. The homogenate was treated with RNAse, proteinase K (50 lag/ml), and 1% SDS for Material and methods 2 h at 55 °C. Proteins and cell debris were extracted once with a phenol/chloroform/isoamylalcohol Taxon sampling. Ten species, all arranged in the mix (25 : 24 : 1), once with a chloroform/isoamyl- tribus Amaryllideae of Amaryllidaceae, were used alcohol mix (24: 1) and precipitated in ethanol for the sequence determination of the 5.8S gene (Sambrook et al. 1989). and flanking transcribed spacers (ITS 1 and ITS 2 The investigated DNA region was amplified with regions): Dewinterella pulcherrima, Hessea brevi- primers described by Hsiao et al. (1995a, b). The flora, H. cinnabarina, H. Iongituba, 1t. stellaris, primer ITSL (5 t-TCGTAACAAGGTTTCCG- H. stenosiphon, Namaquanula bruce-bayeri, N. ete- TAGGTG-3') anneals to the 3 ~ end of the 18S sionamibensis, Nerine humilis, and N. sarniensis. rDNA near the ITS 1 border, and the primer ITS4 Both species of Nerine were chosen as the out- (5~-TCCTCCGCTTATTGATATGC-3 ~) is comple- group, because they belong to the Amaryllidinae mentary to the 5 ~ end of the 25S rDNA near the and are therefore closely related to the genera of ITS 2 border. ITSL and ITS4 cover the entire ITS the Strumariinae. Moreover, Nerine was used as region which is divided by the 5.8S gene into the the outgroup in Snijman's work (1994), so that the ITS 1 and ITS 2 regions. results are well comparable. To confirm each sequence, the DNA of two This study was based on living materials of different individuals were sequenced and compared. cultivated plants from wild provenance. The plants The sequences of each individual were also done in were collected in southern Africa and grown as both directions by using complementary strands. flowers in a greenhouse of the Institute of Ecology The PCR was carried out in a thermal cycler of the Technical University of Berlin. The species, (Biometra) and set for initial 93°C for 3min their abbreviations, taxonomic positions, cultiva- (denaturation),