A molecular phylogenetic analysis based on internal transcribed spacer (ITS) sequences in 31 species, four varieties and four cultivars of Lachenalia, two species of Massonia and three species of Polyxena was made. These three genera have been considered as closely related with each other. The ITS sequence data confirmed that Massonia was clearly different from Lachenalia and Polyxena. Polyxena constructed one clade distinguished from Lachenalia. The species of Lachenalia used showed a correlationship between ITS sequence data and karyotypes, excepting the species with the basic chromosome numbers x=7 and x=8 did not show any correlationship with those with the other basic chromosome numbers. Similarities of their arrays of ITS sequence data among species with x=8 were greater than those among the species with x=7. Lachenalia pusilla and L. muirii which had x=7 formed a clade with the species with x=8. Differences in The species with the basic chromosome numbers of X=7 and 8 showed less differences in correlationship between ITS sequence data and karyotypes than those with other basic chromosome numbers It was suggested that the species of Lachenalia with the basic chromosome numbers of x=7 and 8 might be originated from a common ancestor, while the other species of the genus with basic chromosome numbers other than x=7 and 8 might be originated from other ancestor-else.

The structural comparison of somatic chromosomes of 13 species and one variety of Lachenalia (Liliaceae) that contained the chromosome numbers either of 2n=14 or 2n=16 (the basic chromosome numbers may be x=7 or x=8) was performed by DAPI staining and fluorescence in situ hybridization (FISH) using 5S rDNA and 18S rDNA probes. The five taxa which had the chromosome number of 2n=16 (x=8) displayed similar characters and close relationships to each other. Eight species and one variety which had the chromosome number of 2n=14 (x=7) showed a variety of patterns. The cases were suggested as traces of additions to the 18S rDNA region, translocations of chromosomes and hereditary exchanges among the taxa with x=7 and a taxa with x=8. It was suggested that the chromosome diversity in Lachenalia, especially in the taxa of basic chromosome number of x=7, was resulting a speciation reflected by hereditary influences between the taxa which had other patterns of chromosome structures.

ABSTRACT. The ploidy level and the phylogenic position of Lachenalia congesta W. F. Barker (Asparagaceae) were analyzed by karyomorphological, molecular cytogenetical and molecular phylogenetical studies. The chromosome number of L. congesta was 2n=28, and these chromosomes showed bi-modal karyotype with four large sized chromosomes and 24 small sized chromosomes. The ploidy level of this species was diploid, because each two chromosomes made 14 pairs on the karyotype analysis by acetoorcein staining, DAPI staining and FISH methods. In addition two signals of 18S rDNA sites were detected by the FISH method supported this assumption. All 28 chromosomes of this species did not show any clear DAPI-positive bands and four signals of 5S rDNA sites were detected near the centromeres of small sized chromosomes. These characters were quite different from the characters observed by karyomorphological and molecular cytogenetical studies on common Lachenalia species with basic chromosome number of x=7 or 8. The data from molecular phylogenetic analysis of internal transcribed spacer (ITS) region supported this hypothesis. KEYWORDS: DAPI, FISH, Karyotype, Lachenalia congesta, Phylogenetic analysis, ITSChromosome Botany (2012) Lachenalia is distributed in the western part of South Africa and the southern part of Namibia, and includes ca. 110 species. This genus was formerly classified in the Hyacinthaceae or Liliaceae, although now it revised in the Asparagaceae followed to the third Angiosperm Phylogeny Group (APG III) classification (The Angiosperm Phylogeny Group 2009).Lachenalia congesta W. F. Barker is a dwarf species and is found on mountain slopes of Eastern Cape interior at up to alt. 2400 m with large colonies (Duncan 2009). Duncan (2009) described that Lachenalia congesta was a very distinctive species which cannot be easily mistaken against any other member of Lachenalia during the flowering season. It was recognized by two prostrate, broadly lanceolate (or occasionally ovate), leathery leaves that are dark olive-green on the upper surface and deep magenta below, with prominent dark maroon margins (Duncan 2009). Johnson and Brandham (1997) reported that the chromosome number of L. congesta was 2n=26, 28, although any other karyomorphological information about this species were not described. The molecular phylogenetic analysis using chlorophyll (cp) DNA data of genus Lachenalia indicated that L. congesta was closely related to six Polyxena species (these were transferred to genus Lachenalia by Manning et al. 2004) and three Lachenalia species (Spies 2004), although phylogenetic information from nuclear DNA analysis were not reported.In this report, we attempt to identify the ploidy level of L. congesta by the karyomorphological analysis and fluorescence in situ hybridization (FISH) methods, and to clarify the molecular phylogenetic relationship of this species among genus Lachenalia on the basis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. MATERIALS AND METHODSPlant materials Some pla...

Molecular cytogenetic analysis in seven species of Lachenalia (Liliaceae) with 2n=18, 22, 23, 26 and 28 was studied using DAPI staining and fl uorescence in situ hybridization (FISH). Lachenalia latimerae (2n=18), L. juncifolia (2n=22) and L. zeyheri (2n=23) would be considered as diploids which had basic chromosome numbers of x=9, 11 and 11, respectively. They showed similarities in the characters of DAPI bands and FISH signals of 5S rDNA and 18S rDNA sites. Lachenalia aloides var. vanzyliae (2n=28) and L. orchioides (2n=28) showed tetraploidy (X=7). Lachenalia paucifolia (2n=26) and L. longituba (2n=28), which belonged to the genus Polyxena by different system, considered as diploids which might have in respect to their basic chromosome numbers of x=13 and 14. Lachenalia paucifolia and L. longituba showed different characters of DAPI bands and FISH signals of 5S rDNA and 18S rDNA and showed different characters from the other species of Lachenalia studied here.

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