| Summary: | Clivia (family Amaryllidaceae; tribe Haemantheae) with its six species is closest related to the genus Cryptostephanus. These genera share the highest chromosome numbers (2n = 22 & 24 respectively) in the tribe and has similar 2C values. Evolution in Clivia correlates with geographical distribution from west to east and genome size from small to large.
The tree, character and distance-based analyses are effective in the identification process of three Clivia species. Clivia mirabilis, C. nobilis, C. miniata and C. caulescens / C. Ã nimbicola can be identified with DNA-barcoding. The best barcoding regions regarding discriminatory power are the trnL-F chloroplast region, the matK chloroplast gene and the ITS2 nuclear spacer region. A single DNA-barcoding locus is insufficient to be used in Clivia barcoding.
Hybridization events in Clivia may lead to false positive or false negative identifications. Analysis of an unknown sample resulting in a C. miniata, C. gardenii or C. robusta identification should need further analysis on additional data (e.g. morphology, distribution) to confirm the results. All the C. Ã nimbicola natural hybrids included in this study shared the chloroplast DNA of C. caulescens. Alternative methods should be developed to effectively distinguish and identify hybrid species.
The genus Lachenalia has 133 species and shows extensive morphological variation and exceptional diverse chromosome numbers (x = 5, 6, 7, 8, 9, 10, 11, 12, 13 and 15). Lachenalia might have evolved from a common ancestor and the two largest basic chromosome number groups, x = 7 and 8 have evolved from a common predecessor. It seems as if the higher basic numbers (x = 9, 10, 11 and 13) evolved independently from the lower numbers.
Several speciation events were involved in the evolution of Lachenalia, resulting in the morphological and chromosomal diversity. Many of the species with the same basic chromosome numbers share a common ancestor, and it is expected that there may be incomplete lineage sorting in some species resulting in non-specific DNA-barcodes.
The tree-based and character-based analyses are effective methods to identify all the focus species (L. unifolia, L. bifolia, L. punctata and L. mediana) in this study. The matK, trnL-F and ITS2 regions results in the positive identification of an unknown specimen (tree-based analyses). SNP analyses can be used in the analyses of matK and atpH-I. The combination of atpH-I + trnL-F and trnL-F + ITS2 will effectively distinguish an unknown sample of the focus species. Due to the large size of the genus, a two to three DNA-barcode locus is preferred over a single DNA-barcoding locus. The nuclear ITS2 gene region has to be included as a DNA-barcode to detect hybrid species and plastid capture.
The chloroplast trnL-F region together with the nuclear ITS2 region can be universally used for barcoding the small genus Clivia and the large genus Lachenalia. Both these regions require an third region (or more in the case of Lachenalia) to be effective for identification of species.
Ancient hybridization, introgression and incomplete lineage sorting occurs in both genera. Therefore, certain species will not be effectively identified based on barcodes alone.
There are some small differences between the DNA-barcoding of the small (Clivia) and the large genus (Lachenalia). However, the similarities between the genera are that identifications in both genera are influenced by the degree of hybridization (ancient or recent) and the time of divergence (thus incomplete lineage sorting).
The classification of Clivia and Lachenalia must be properly resolved before barcodes can be implemented for the species-level identification of these genera.
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