|Characterization of Lachenalia, the Liliaceae based on karyomorphological, molecular
phylogenetical and molecular cytogenetical studies*
Shuichi Hamatani 1)
1. Lachenalia Jacq. ex Murray, the Liliaceae is a perennial herb and endemic to the western part of southern Africa. The
plants belonging to this genus are used as half-hardy and fall planting bulbs for horticultural use in Japan, and some of
which bloom in late fall and others in early spring. About 110 species of this genus are known, and they are expected to
become popular for ornamental interior use because of their varied colors and compact plant forms. The production of
artifi cial hybrids has begun, however, it has not yet become popular because there have been some diffi culties in cross
breeding. Analysis of the relationships of plants within the genus is desired for the purpose of choosing effi cient parent
plants for cross breeding to obtain excellent strains. Then, 40 species and four varieties of Lachenalia were characterized
using karyomorphological studies, molecular phylogenetical studies, and molecular cytogenetical studies.
2. In the karyomorphological studies, karyotype was analyzed using the aceto-orcein staining and squashed method.
Chromosome numbers were counted as 2n=14, 15, 16, 17, 18, 22, 23, 24, 26, 28, and 42. L. algoensis (2n=14), L.
aloides var. vanzyliae (2n=28), L. aloides ePearsoniif(2n=15), L. latimerae (2n=18), L. longibracteata (2n=14) , L.
longituba (2n=28) and L. thomasiae (2n=14) have been reported here for the fi rst time, whereas L. arbuthnotiae (2n=15),
L. capensis (2n=16), L. purpureocoerulea (2n=15), L. variegata (2n=14), and L. zeyheri (2n=23) have been reported
previously with another chromosome numbers.
On the bases of an analysis of resting nuclei during somatic cell division and somatic chromosomes in the mitotic
prophase and metaphase, the taxa studied were categorized into six groups based on the basic chromosome numbers of
x=7, x=8, x=9, x=11, x=12, and x=13 or 14.
3. In the molecular phylogenetical studies, sequencing of the bases of internal transcribed spacer (ITS) regions on
nuclear ribosomal (nr)DNA was performed, and phylogenetical analysis was conducted using data from the ITS regions
of selected 34 species and four varieties. In the phylogenetic tree based on the ITS sequences, the taxa studied were
roughly divided into the three species (L. hirta, L. latifolia, and L. latimerae) showing no relationships among them, two
clades (Clade I and Clade II) had high confi dence values and each was constructed from three species, and one clade
(Clade III) had a low confi dence value and was constructed from 25 species and four varieties. Clade III was further
divided into one clade with a high confi dence value and was constructed from 12 species (Clade IV), and the remaining
13 species and four varieties showed no relationships with each other.
The interrelation was confi rmed by analyzing variations in basic chromosome numbers and results of comparisons
between ITS regions. L. hirta showed a basic chromosome number of x=11, L. latifolia showed x=12, L. latimerae
showed x=9, the three species of Clade I showed x=13 or 14, the three species of Clade II showed x=11, almost all of
the taxa of Clade IV showed x=8, and L. muirii and L. pusilla of Clade IV and the other 13 species, four varieties, and
two cultivars showed x=7. Whereas some questions were remained; some variations were observed in chromosome
numbers between the species of Clade I, which were transferred from genus Polyxena to genus Lachenalia by Manning
et al. (2004) and showed close correlations in the comparison of ITS sequences; Clade IV which was constructed from
almost all of the taxa of x=8, but also included L. muirii and L. pusilla of x=7; the relationships between the taxa of x=7
were not determined in both the karyomorphological or molecular phylogenetical studies. Therefore, the molecular
cytogenetical methods were applied to somatic cells to solve these assignments.
4. In the molecular cytogenetical studies, 4f6-diamidino-2-phenylindole (DAPI) staining and fluorescence in situ
hybridization (FISH) with 5S rDNA and 18S rDNA probes were used for analyzing somatic chromosomes in the mitotic
metaphase of selected 20 species and two varieties of Lachenalia.
Some taxa showed clear DAPI bands on all chromosomes, some on partial chromosomes, and others not on all
chromosomes. Two FISH signals of 5S rDNA sites were observed in almost all taxa, but in some taxa, four signals were
observed. Similarly, two signals of 18S rDNA sites were observed in almost all taxa, but in some taxa, four or six signals
were observed. In L. aloides var. aloides, L. aloides var. aurea, L. aloides var. vanzyliae, L. longibracteata, L. muirii, L.
orchioides var. orchioides, and L. viridifl ora with a basic chromosome number of x=7, similarities in the features of the
DAPI bands and the signals of 5S rDNA sites were observed in all of these taxa, and in which similarities were also
observed in the signals of 18S rDNA sites in addition to the features of the DAPI bands except in case of L. muirii. In all
fi ve taxa of x=8, there were similarities in the features of DAPI bands and the signals of 5S rDNA and 18S rDNA sites.
In L. pusilla of x=7, L. latimerae of x=9, and L. hirta, L. juncifolia and L. zeyheri of x=11, which did not show clear
DAPI band in all chromosomes, there were similarities in the features of the signals of 5S rDNA and 18S rDNA sites.
The analysis using DAPI staining and FISH showed similar features in L. pusilla of x=7 and the taxa of x=9 and 11,
which did not show any similarities on karyomorphological analyses and on the comparing the sequences of ITS
regions. The taxa of x=8 had very similar characteristics, whereas the taxa of x=7 had similarities but also some
differences. Therefore, it was suggested that the taxa of x=7 be speciated from plural ancestral species, and done with
crossing each other, transformation of chromosomal structures, and so on. Moreover, it was suggested that there were
mutual elements between L. muirii of x=7 and the taxa of x=8. DAPI staining and FISH showed diferent results between
L. longituba and L. paucifolia, which were transferred from Polyxena to Lachenalia, and there were different results
between these two taxa and the other taxa.
5. It was concluded that Lachenalia were divided into roughly three groups. A: The group consisted of the taxa showing
clear DAPI bands on their chromosomes. This group included most of the taxa of x=7 and x=8, which had descended
from one or more ancestral species of x=8 and from plural ancestral species of x=7, and take the hereditary affects each
other. Furthermore, this group showed small amount of variations in the ITS regions. B: The group consisted of taxa
with no clear DAPI bands on their chromosomes. This group had clear variations in the sequences of the ITS regions
and included the taxa of x=11, a portion of the taxa of x=7 (L. pusilla), and the taxa of x=9. C: The group consisted of
taxa transferred from the genus Polyxena by Manning et al. (2004). A complex correlations in this group was suggested.
The chromosomal observations using DAPI staining were very useful for researching the relationships among
species belonging to Lachenalia. It was expected that the results of this study would be utilized as the important basic
information, for the future increasing ornamental uses