Plant Syst. Evol. 234: 27–51 (2002) DOI 10.1007/s00606-002-0193-1 Phylogenetic analysis of Stylosanthes (Fabaceae) based on the internal transcribed spacer region (ITS) of nuclear ribosomal DNA J. Vander Stappen1, J. De Laet2, S. Gama-Lo´ pez3, S. Van Campenhout1, and G. Volckaert1 1Katholieke Universiteit Leuven, Laboratory of Gene Technology, Leuven, Belgium 2American Museum of Natural History, Division of Invertebrate Zoology, New York, USA 3Universidad Nacional Auto´ noma de Me´ xico, Laboratorio de Recursos Naturales, Municipio Tlalnepantla, Estado de Me´ xico, Me´ xico Received August 14, 2001; accepted March 4, 2002 Published online: November 14, 2002 Ó Springer-Verlag 2002 Abstract. Phylogenetic relationships in Stylosan- The genus Stylosanthes, established by Swartz thes are inferred by DNA sequence analysis of the in 1788, is a member of the family Fabaceae, ITS region (ITS1–5.8S–ITS2) of the nuclear ribo- subfamily Papilionoideae, tribe Aeschynome- somal DNA in 119 specimens, representing 36 neae, subtribe Stylosanthineae (Rudd 1981). species of Stylosanthes and 7 species of the Depending on the treatment, the genus includes Arachis Chapmannia outgroup genera and . In all 30 to 45 herbaceous or suffruticose species and a examined specimens of any particular diploid and variable number of subspecies and varieties of (allo)polyploid species, only a single ITS sequence type was observed. This allowed us to identify a neotropical origin (‘t Mannetje 1984). Most of parental genome donor for some of the poly- these are native to the tropical and subtropical ploids. In several diploid and polyploid species, regions of Central and South America, while a different specimens contained a different ITS few are distributed in the southeastern part sequence. Some of these sequence types were of the United States, the Antilles, the present in more than one species. Parsimony Galapagos Islands, central and southern Africa, analysis yielded several well-supported clades that Madagascar, and southern India and Ceylon agree largely with analyses of the chloroplast trnL (Mohlenbrock 1958, Williams et al. 1984). The intron and partially with the current sectional major centre of diversity for the genus is in the classification. Discordances between the nuclear southern neotropics, particularly Brazil, with a and cpDNA analyses are explained by a process secondary centre in the Mexico – Caribbean of allopolyploidization with inheritance of the basin (Stace and Cameron 1984). Several species cpDNA of one parent and fixation of the ITS sequences of the other. S. viscosa has been an of the genus are agronomically important in important genome donor in this process of speci- tropical and subtropical agriculture as pasture ation by allopolyploidy. and forage legumes (Burt and Miller 1975), as green manure (Gardener 1984), as cover crop Key words: Fabaceae, Stylosanthes. Internal trans- (Thomas 1984), and more recently in the cribed spacer region (ITS), molecular phylogeny, biological control of cattle tick (Khudrathulla allopolyploid, DNA sequence analysis. and Jagannath 1998). 28 J. Vander Stappen et al.: Phylogenetic analysis of Stylosanthes (Fabaceae) Stylosanthes, which is morphologically The more recent taxonomic studies of the genus close to the genera Arachis, Arthrocarpum, are by Ferreira and Costa (1979) and ‘t Man- Chapmannia and Pachecoa, is characterized by netje (1984). Ferreira and Costa (1979) concen- sessile flowers, monadelphous stamens which trated on the species of Brazilian origin and are often united in a closed tube, dimorphic emphasized the number of vascular bundles, the anthers, elongate receptacles, 3-foliate leaves, venation of the leaflets and the growth habit as and stipules that are fused with the petiole; the main diagnostic features. In contrast, ‘t Man- fruits are loments that develop aboveground netje (1984) relied mostly on fruit morphology, (Burkart 1939, Rudd 1981). Two subgeneric stressing in particular the shape and length of sections, sect. Stylosanthes Vog. and sect. the beak, the indumentation of the pod, and the Styposanthes Vog., have been recognized by width and venation of the outer bract. This the absence or presence of an axis rudiment, differential emphasis on different characteristics which is thought to be an aborted secondary may explain why several members of the floral axis (Taubert 1891), and the presence of S. guianensis complex have been classified as one or two inner bracteoles, respectively distinct species by Ferreira and Costa (1979) but (Kirkbride and Kirkbride 1987). as varieties by ‘t Mannetje (1977, 1984; Table 2). The basic chromosome number of Stylosan- The occurrence of major fertility barriers be- thes is x ¼ 10, with diploid, tetraploid and one tween members of this complex (Hacker et al. hexaploid species identified. While diploid spe- 1988), however, supports the former taxonomic cies are found in both sections, polyploid species approach. Due to the lack of stable morpho- are restricted to sect. Styposanthes and they are logical characters, the identification and delim- thought to be products of intersectional hybrid- itation of several other species is still ization between diploids, followed by polyplo- problematic (‘t Mannetje 1984). For instance, idization (Stace and Cameron 1984). One S. scabra, S. tuberculata and S. nervosa are very exception to the intersectional origin of allo- difficult to distinguish because they have virtu- polyploids in Stylosanthes has been observed by ally identical pods and because the vegetative Liu et al. (1999) for the species S. capitata, which characters used to separate them are very is most probably formed by hybridization variable, raising the question of whether the between the diploid species S. pilosa and latter two species should be subsumed into S. macrocephala, both of sect. Styposanthes. S. scabra. With exception of S. hamata and S. mexicana, Given the difficulties in species delimitation polyploids are not known to occur as sub- and the agronomic importance of Stylosanthes specific races (Stace and Cameron 1984, in tropical agriculture, it comes as no surprise S. Gama-Lo´ pez, unpubl. data). The allopoly- that other techniques have been used to com- ploid origin of the polyploids has been con- plement the morphological approach. Most of firmed by biochemical and molecular data these studies, however, were not intended to be (Table 1). full taxonomic surveys and/or have been Apart from the division into two sections, on restricted to widespread, well-known or agro- which all authors agree, the taxonomic treat- nomically important Stylosanthes species, while ment of Stylosanthes is very ambiguous. A first minor species have been neglected (e.g. Edye et detailed study of the genus was done by Vogel al. 1974, Williams et al. 1984, Stace and Cam- (1838), who recognized fifteen species. Later eron 1987, Vieira et al. 1993). As a first step several new species, varieties and forms were toward a phylogenetic overview of the whole described and the classification became rather genus, Gillies and Abbott (1996) performed an confusing (Bentham 1859, Taubert 1891, Hass- analysis of chloroplast DNA (cpDNA) restric- ler 1919, Blake 1920, Burkart 1939). This is well tion fragment length polymorphisms (RFLP) in illustrated by the long list of synonyms in the 18 Stylosanthes species. A second phylogenetic revisions of Mohlenbrock (1958, 1960, 1963). study is based on sequence analysis of the J. Vander Stappen et al.: Phylogenetic analysis of Stylosanthes (Fabaceae) 29 Table 1. List of polyploid Stylosanthes species, with their putative parental genome donors Polyploid species Putative parental genome donorsa Referencesb S. capitata (4·) S. macrocephala ð$Þ Gillies and Abbott (1996), Vander Stappen et al. (1999a) S. macrocephala · S. sp. Vander Stappen et al. (1999b) S. macrocephala · S. pilosa Liu et al. (1999) S. macrocephala This study S. erecta (6·) S. sp. (clade 3) ($) J. Vander Stappen (unpubl.data) S. sp. (clade 3) · S. viscosa Vander Stappen et al. (1999b) S. seabrana/S. hamata · S. aff. Liu et al. (1999) viscosa · S. sp. S. viscosa This study S. fruticosa (4·) S. sp. (clade 3) ð$Þ Vander Stappen and Volckaert (1999) S. sp. (clade 3) · S. viscosa Vander Stappen et al. (1999b) S. viscosa This study S. hamata s. l. (4·) S. humilis · S. hamata s. str. Stace and Cameron (1984, 1987), Curtis et al. (1995) S. humilis ð$Þ Gillies and Abbott (1996), Vander Stappen et al. (1999a) S. humilis · S. hamata s. str. Vander Stappen et al. (1999b) S. humilis This study S. hamata s. str. This study S. viscosa This study S. ingrata (4·) S. sp. (clade 3) ð$Þ Vander Stappen et al. (1999a) S. sp. (clade 3) · S. viscosa Vander Stappen et al. (1999b) S. viscosa This study S. mexicana (4·) S. sp. (clade 3) ð$Þ · S. viscosa J. Vander Stappen (unpubl. data) S. viscosa This study S. scabra (4·) S. viscosa ð$Þ or S. hamata Gillies and Abbott (1996) s. str. ð$Þ S. seabrana · S. viscosa Liu and Musial (1997) S. sp. (clade 3) Vander Stappen et al. (1999a) S. sp. (clade 3) · S. viscosa Vander Stappen et al. (1999b) S. viscosa This study, Stace and Cameron (1984) S. sericeiceps (4·) S. sp. (clade 3) ð$Þ J. Vander Stappen (unpubl. data) S. seabrana/S. hamata · S. aff. Liu et al. (1999) viscosa S. viscosa This study S. subsericea (4·) S. sp. (clade 3) ð$Þ · S. viscosa Gama-Lo´ pez et al. (2001) S. viscosa This study S. sundaica (4·) S. humilis · S. hamata s. str. Stace and Cameron (1984), Liu et al. (1999) S. humilis ð$Þ · S. hamata s. str. J. Vander Stappen (unpubl. data) S. hamata This study S. sympodialis (4·) S. humilis ð$Þ Gillies and Abbott (1996) S. sp. · S. seabrana/S. hamata Liu et al. (1999) S. sp. (clade 1) ð$Þ Vander Stappen et al. (1999a) S. sp. (clade 1) · S. sp. (clade 3) Vander Stappen et al. (1998b) S. sp. (clade 1) This study S. tuberculata (4·) S. seabrana/S. hamata · S. aff. Liu et al.