THE SUBGENERIC DELIMITATION OF LACHENALIA (HYACINTHACEAE)

The genus Lachenalia consists of approximately 116 species. Many of these species are used in a breeding programme for pot plants at ARC-Roodeplaat, South Africa. Various basic chromosome numbers are present in the genus Lachenalia, i.e. x=5, 6, 7, 8, 9, 10, 11 and 13. Ploidy levels range from diploid to octoploid and polyploidy is present in many species. For breeding purposes it is necessary to know the chromosome number and the genetic diversity within and between species. Species delimitations in the genus are often poor and many specimens are misidentified. Three different subgeneric classification systems have been proposed for the genus. This study evaluates whether the three classification systems are natural. This is done by comparing the system to the basic chromosome number per group and by using sequence data to determine the relationships among the different species. This evaluation clearly indicates that the system proposed by Crosby (1986) is the most natural one and should be expanded to include all species in the genus. INTRODUCTION The genus Lachenalia Jacq.f. ex Murray consists of approximately 116 species. The genus contains beautiful ornamentals and is used in a breeding programme at ARCRoodeplaat, South Africa. Numerous bulbs are annually exported to especially the Netherlands, where they are sold as pot plants. During his revision of the genus, Baker (1897), described five subgenera, i.e. Eulachenalia, Coelanthus, Orchiops, Chloriza and Brachyscypha. These subgenera were based on morphological differences. Crosby (1986) reclassified the genus, using five groups, each based on a typical species, i.e. the L. aloides, L. orhioides, L. pusilla, L. unicolor and L. unifolia groups. In his documentation of Lachenalia, Duncan (1988) used the ratio between the lengths of the perianth and stigma, as well as the inflorescence form as criteria and divided the genus into 10 subgroups. Since the first subgeneric classification of Lachenalia, various additional taxonomical aids were described. This ranges from anatomy, to embryology, cytogenetics, chemical taxonomy and molecular systematics. The combination of these methods may culminate in the best natural classification possible for Lachenalia. In this preliminary investigation of the genus, cytogenetic and molecular methods will be emphasized. The aim of this study is to determine which of the three subgeneric classifications of Lachenalia correspond best with the phylogenetic relationships within this group. MATERIALS AND METHODS Bulbs, obtained from the Agricultural Research Institute, Roodeplaat, were grown in a nursery at the University of the Orange Free State, Bloemfontein. Material for the cytogenetic and molecular studies was collected from these plants. Squashes of mitotic Proc. 8th Int. Symp. on Flowerbulbs Eds. G. Littlejohn et al. Acta Hort. 570, ISHS 2002 226 (Spies et al. 2000) and meiotic material were made. Leaf material for the molecular study was collected and DNA extracted according to Rogstad (1992). Amplification of the trnL-F region was according to Taberlett et al. (1991) with the primers for the intergenic spacer, i.e. e-f (e – 5”-GGT TCA AGT CCC TCT ATC CC-3’; f – 5’-ATT TGA ACT GGT GAC ACG AG-3’) being used. An ABI 310 sequencer was used. The region was sequenced from both ends, edited and aligned with the eye after ClustalW (Thompson et al. 1994) was used. Phylogenetic analysis was done, using PAUP4* (Swofford 1998). The heuristic search option, with TBR branch swapping and MULPARS selected, was used. RESULTS AND DISCUSSION Cytogenetic studies revealed that various chromosome numbers are present in Lachenalia and chromosome numbers of 54 species are already known (Moffett 1936; De Wet 1957; Riley 1962; Mogford 1978; Ornduff and Watters 1978; Nordenstam 1982; Crosby 1986; Hancke and Liebenberg 1990; Hancke 1991; Johnson and Brandham 1996; Hancke and Liebenberg 1998; Kleynhans and Spies 1999; Spies et al. 2000). During this study 35 specimens, representing 19 species were studied cytogenetically. The chromosome number of 2n = 28 for L. variegata represents a first report on this species. The chromosome numbers observed in all other species corresponded with published numbers for those species (Table 1). Polyploidy is fairly common in Lachenalia. It is present in 24 (44%) species (Table 1). All known chromosome numbers of Lachenalia reveal that a high percentage (29.6%) of “deviating” chromosome numbers were described. Although the different basic chromosome numbers indicate that aneuploidy played a major role in the evolution of the genus, it is doubtful that so many species (16) may contain aneuploid representatives. These “deviating” numbers may rather be attributed to erroneous counts, the presence of B-chromosomes without recognizing the B-chromosomes as such, or most likely, the wrong identification of the specimen studied. The latter applies especially to seeds received as a particular species. Seeds of one species are often contaminated with seeds of another. Misidentification of specimens occur because species boundaries are often not well identified. According to the classification of Baker (1897) the subgenus Eulachenalia has a basic chromosome number of 7, Coelanthus has x = 7, Orchiops has x = 7, 8 and 10, Chloriza has x = 7, 8, 10, 11 and 13 and Brachyscypha has x = 7. The L. aloides group (Crosby 1986) shares x = 7 with the L. orchiodes and L. pusilla groups. The L. unicolor has x = 8 with one species (L. violacea) at x = 7 and one species (L. zeyherii) at x = 11. The L. unifolia group shows the most variation with x = 10, 11 and 13. In Duncan’s (1988) system group 1a has x = 5, 6, 7, 8, 9, 10 and 13, group 1b and 1c have x = 7, 1d has x = 7 and 8, 1e has x = 7, 8, 10, 11 and 13, 1f has x = 6, 7, 8 and 11, 2a has x = 8 and 9, 2b has x = 10 and 2c has x = 7, 8, 10 and 11. The basic chromosome numbers suggest that the subgeneric classification of Crosby (1986) is the most natural one. However, this system has an unfair advantage, since basic chromosome numbers were one of the criteria in the delimitation of the groups. In an attempt to determine the validity of the three classification systems, a cladogram of some of the species was used, obtained from sequences of the intergenic spacer between trnL and trnF genes. In this process the species L. aloides (L.f.) Engl. (data from Genbank), L. bulbifera (Cyrillo) Engl. , L. comptonii W.F. Barker, L. hirta (Thunb.) Thunb. , L. juncifolia Bak. , L. mediana Jacq. , L. namibiensis W.F. Barker (data from Genbank), L. orchioides (L.) Ait. , L. pallida Ait. (two specimens – one from Genbank and one from our own study), L. pusilla Jacq. , L. reflexa Thunb. , L. unifolia Jacq. , L. violacea Jacq. , were used. The intergenic spacer differs between 305 and 308 basepairs and produced 37 informative sites for phylogenetic purposes. The cladogram does not fully support any of the three classification systems. The most parsimonious cladogram was 59 steps (Figure 1). In order to support the Baker (1897) or the Crosby (1986) classification systems, the