| Summary: | The genus Clivia Lindl., which belongs to the family Amaryllidaceae J. St-Hil.
(1805), is comprised out of seven different species. Clivia nobilis, C. caulescens,
C. miniata, C. gardenii, C. mirabilis, C. robusta and the natural hybrid Clivia
xnimbicola all forms part of this genus. Clivia mirabilis is found in the Northern
Cape Province and is geographically isolated from the six other species which
grows along the Eastern Coast and escarpment of South Africa. Conrad et al.
(2003) proved that C. mirabilis and C. nobilis were the two most primitive species
in the genus Clivia.
During this study sequencing results were used to detect barcodes/SNPs for C.
nobilis and C. mirabilis and to reveal genetic variation between the Clivia
species. Clivia nobilis and C. mirabilis were tested with cross-species
microsatellite makers to reveal intraspecific variation. Seven different gene
regions were sequenced. Six were chloroplast regions, namely the atpH-I, matK,
rpoB, rpoC1, rpl16, the trnL-F regions and one was a nuclear region, ITS1. The
regions used for sequencing were evaluated as potential barcoding/SNP regions
for future use. They were also used to infer the evolutionary development of C.
nobilis and C. mirabilis. All seven Clivia species were analysed but this study
focused mainly on morphologically different specimens of C. nobilis and C.
mirabilis. The sequences were aligned and edited with Geneious Pro. A total of
forty-seven polymorphic sites were observed between al seven species. Within
the rpl16 region eleven parsimony informative sites were observed. The matK
and trnL-F regions each had eight parsimony informative sites. ITS1 had three
sites and rpoB and rpoC1, one parsimony informative site each. Within the atpHI
region no parsimony informative sites were observed. The sequencing data
obtained could be used for species identification and, therefore, showed great
potential as barcoding regions. We propose that matK, rpl16 and trnL-F are used
as a barcode in C. nobilis and C. mirabilis because they had the most parsimony
informative sites. The cladogram obtained from the combined data set (atpH-I, rpoB, rpoC1, matK
and trnL-F) confirmed that C. nobilis and C. mirabilis are two separate species.
Clivia caulescens and C. xnimbicola forms a monophyletic group. Within the
rpl16 chloroplast region intraspecific variation in C. mirabilis and interspecific
variation between C. nobilis and C. mirabilis were observed. The phylogentic
tree representing the sequencing results of the rpl16 region revealed three
distinctive groups within the four different C. mirabilis populations. Two plants
within one of the Donkerhoek populations showed more variation than the rest of
the population. The rpl16 gene region proved to be ideal in order to test
intraspecific variation in C. nobilis and C. mirabilis.
To evaluate the use of cross-species markers, microsatellite makers designed for
Phaedranassa tunguraguae, Hymenocallis coronia and Clivia miniata were
tested on C. nobilis and C. mirabilis. Although amplification was obtained, in
most cases the results could not be optimized in order to provide reliable
analysis. In future species specific primers for C. nobilis and C. mirabilis will be
developed.
This study undoubtedly identified barcodes/SNPs for C. nobilis and C. mirabilis
which can be used to eliminated mistaken identity. Gene regions specific for
intra- and interspecific variation were identified and can be used in future for
population studies.
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