Studies on South African and New Zealand species of bulbinella using nuclear and chloroplast sequence data
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The genus Bulbinella Kunth consists of geophytes occurring in South Africa and New Zealand and includes a number of beautiful, conspicuous, mostly threatened flowering species. The genus is composed of about 23 species and is taxonomically related to Bulbine Wolf and Kniphofia Moench. There are six species in New Zealand and 17 species in South Africa. The genus represents one of the most understudied genera in South Africa. The species relationships and complexes are poorly understood due to morphological homogeneity and it has been flagged as a priority to study due to its ethnomedicinal value. The aim of this thesis was to establish the first set of DNA sequence data for phylogenetic studies complimenting previous morphological and taxonomic studies because molecular techniques offers increased precision by permitting assessment of additional characters. This was done using a number of conventional phylogenetic genes for plants, as well as following a phylogenomic approach of the chloroplast. In the thesis the taxonomy, morphology and importance of species in Bulbinella were reviewed. The 94 specimens were sampled, of which 86 specimens were in-group and eight outgroup sequences, using either sequences obtained from GenBank or those generated in this study. DNA sequencing of four gene regions (ITS, rbcL, matK and psbA-trnH) was conducted to resolve some of the major questions in the phylogeny of Bulbinella in South Africa and New Zealand. Due to the fact that South African species relationships needed more definition, a subsequent phylogenetic analysis based on 34 protein-coding genes from 16 taxa was done in a phylogenomic approach to improve resolution and give a better understanding of the evolutionary process of Bulbinella. Phylogenies were constructed using Maximum Likelihood (ML) conducted in Garli v2 and Bayesian Inference (BI) using Mr Bayes v3.2, with consensus topologies generated using PHYLIP v3.695. For chloroplast draft genome assembly, the filter reads were processed in a bioinformatics pipeline, annotated and used in phylogenetic analyses. In each of the gene analyses (separate and combined) New Zealand species always grouped on their own but in the overall group of Bulbinella. New Zealand and South African species included distinct, polyphyletic or possible synonymous species. The standard DNA barcode region matK (but not rbcL), were able to distinguish most South African and New Zealand species, but not others. The psbA-trnH spacer and ITS could be used as a supplementary barcode. Based on the genome data, phylogenetic trees confirmed the gene tree results and conclusions but provided greater statistical support and could distinguish between previously indistinguishable species. The results suggested that the following genes can be used or recognized as barcode genes to distinguish Bulbinella species and these are atpA, atpF, atpI, rbcL, ndhI, ndhH, ndhF, rpl2, rpoC, rpoC2, rps15, orf188, rps2, matK, ndhE, ndhG, ccsA, psaC, ycf2, psbA, rpoB and ndhD. The study has established multigene phylogenies for the genus for the first time which will strengthen the taxonomy of the genus, aid identifications for users of the plants for medical applications, the ornamental industry, as well as facilitate biodiversity and conservation efforts to protect the diversity of this genus. However, our results showed that there is a great need for increased sampling and morphological supported studies for these species, while the genes identified in the whole genome sequencing approach will be helpful to support the phylogeny of this genus.