Ruvimbo Jessy Mapaya
Supervisor: Prof. Glynis Goodman-Cron
School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, South Africa
Emilia (Cass.) Cass is an economically important plant genus in the Asteraceae.
With about 117 species (The Plant List, 2013), Emilia is indigenous to Africa,
south of the Sahara, and Asia (Fosberg, 1972; Nordenstam, 2007). The plants
are herbaceous, annual, biennial, and perennial with much variation in floral
features. Although the taxonomic history of Emilia dates back as early as 1817
(Cassini, 1817) no phylogenetic/phylogeographic study of Emilia has been done
to date. The aim of this study is therefore to investigate the phylogenetic
relationships of a good representative sample of species of Emilia, together
with the genera Emiliella, Bafutia and other closely related genera in the
Senecioneae, using DNA sequence data to provide a sound basis for future
taxonomic revisions of the genus.
The IAPT Research Grant awarded in 2013 contributed towards the molecular work (DNA extraction, PCR, and DNA sequencing needed to provide the molecular data for the phylogenetic analyses) essential for this project.
Fieldwork to collect fresh material of Emilia was done in Zimbabwe for different months from 2012 to 2014 when the species were known to be flowering. Voucher specimens are deposited at C. E. Moss herbarium (J). Most laboratory work was undertaken in 2014 and is being completed. Total genomic DNA was extracted from approximately 20 – 30 mg leaf material (dried in silica gel) or dried material taken with permission from herbarium specimens (EA, LISC, MA, MAL, MO, P, SRGH) using either the GenElute™ Plant Extraction Minikit (Sigma-Aldrich) or the DNeasy Plant Mini kit (QIAGEN®, Valencia, CA) following manufacturers’ protocol. So far, DNA has been extracted from 66 species of Emilia, one species of Emiliella, and one species of Bafutia. Additional DNA sample extractions of two Emilia and two Emiliella species were ordered and received from KEW.
In preliminary laboratory work, nuclear ITS and ETS regions, and plastid DNA
regions (trnL–trnF, matK and trnK Intron) were trialled. The nuclear ITS
and plastid trnL–trnF proved to be useful regions and were thus used in DNA
sequencing of a representative sample of Emilia species (from both sections
Spathulatae and Emilia), and representatives of Emiliella, and Bafutia.
Sequence data is now available for the following specimens: 52 accessions representing 43 Emilia, two Emiliella and one Bafutia species using ITS, 63 accessions representing 50 Emilia, three Emiliella and one Bafutia species using trnL–trnF, 17 using ETS, and seven using 5′ trnK intron. In addition, ITS and trnL–trnF sequences of five Emilia species and 14 species from 12 different genera in the tribe Senecioneae (to be used as outgroups) have been obtained from GenBank. Final laboratory work is continuing for other problematic species with different primer combinations being trialled as well as other PCR troubleshooting techniques being implemented.
Preliminary phylogenetic analyses are being done with 88 and 73 sequences in the plastid and nuclear datasets respectively including outgroups. These datasets have been aligned using BioEdit Sequence Alignment Editor (Hall, 1999) and coded manually for indels. A morphological character matrix with about 13 characters for the investigation/evaluation of evolutionary trends for each Emilia species and other related genera in the Senecioneae included in the molecular phylogeny is also being finalised. Preliminary results using maximum parsimony (MP) analyses (datasets of individual DNA regions) conducted in PAUP* 4.0b10 package for Macintosh (Swofford, 2002) or the program TNT 1.1 (Goloboff et al. 2008) and Tephroseris integrifolia to root the trees have so far indicated that Bafutia and three Emiliella species are nested within Emilia with relatively strong support in both plastid and nuclear datasets. The strict consensus of 44 MP trees using the plastid data revealed Emilia to be polyphyletic: in the first clade eighteen Emilia species from various countries are sister to a number of the outgroup taxa mostly forming a polytomy, Bethencourtia palmensis and S. lineatus are sister to the second clade consisting of 51 Emilia, one Bafutia, and three Emiliella species. Seven of the 18 species in clade 1 of consensus tree resulting from analysis of the plastid dataset were included in the nuclear dataset. MP results indicated that only one of these species (E. baumii) was placed outside of the main Emilia clade together with E. graminea. The three Emiliella species group together and their positions are congruent in the nuclear and plastid phylogenies. The eight species in the Emilia coccinea sensu lato complex do not form a monophyletic group, indicating that the taxonomic status of some species in this complex might be questionable. These preliminary results indicate that the monophyly of Emilia might be questionable and that hybridization and/or introgression has played a role in the history of Emilia, as with many other senecionoid genera. Phylogenetic analyses (including Bayesian analyses) are continuing in order to adequately address the objectives of the current study.
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