Memecylon section Buxifolia (Melastomataceae): alignments of nuclear rDNA spacer sequences (ITS, 5' ETS)
Aligned sequences (NEXUS file format) of the internal transcribed spacers (ITS1, ITS2) and 5’ external transcribed spacer (ETS) of nuclear ribosomal DNA, sampled from representatives of Memecylon section Buxifolia and outgroups (Melastomataceae).
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Twenty-seven samples of Memecylon were collected from forested localities in eastern South Africa, including sites in the Eastern Cape, KwaZulu-Natal, Mpumalanga and Limpopo. Leaf samples were rapidly dried in silica-gel (Chase & Hills 1991), except for two additional samples from Mozambique (Burrows 10766 & 10856) in which genomic DNAs were extracted from herbarium material. Voucher specimens are deposited in the Bews Herbarium, University of KwaZulu-Natal, Pietermaritzburg. In addition to the nuclear rDNA sequences newly obtained for this study (GenBank accession nos. KY040305 to KY040360), the phylogenetic analysis includes previously published sequences from 32 samples (Stone 2014). The outgroup consists of sequences from Memecylon sections Tenuipedunculata, Cauliflora and Montana, representing other lineages within a larger, monophyletic species-group centred in East Africa (Stone 2014). Laboratory materials and procedures were the same as those previously described by Stone & Andreasen (2010). PCR products were purified and directly sequenced at Stellenbosch University (Central Analytical Facilities, DNA Sequencing Unit). For each sample the forward and reverse sequences were assembled into contigs using the software Chromas Pro version 1.5 (Technelysium). Site-specific additive polymorphisms when present were captured using IUPAC ambiguity codes. Separate alignments of the ITS and 5’ETS regions were completed using the software ClustalW2 (Larkin et al. 2007) followed by minor manual adjustments. After alignment of the ITS region, the middle part (i.e., the highly conserved 5.8S rRNA gene) was excluded leaving separate ITS1 and ITS2 alignments for further analysis. The 5′ and 3′ ends of ITS1 were determined by comparison with published sequences of the 18S and 5.8S ribosomal RNA genes of Arabidopsis (Unfried et al. 1989; Unfried & Gruendler 1990). The boundaries of ITS2 were determined by modelling of stem hybridisation between the 3′ end of the 5.8S and 5′ end of the 26S (Keller et al. 2009), implemented in the online ITS2 Database (http://its2.bioapps.biozentrum.uni-wuerzburg.de/). A 40-nt motif representing the highly conserved 5′ end of the 18S rRNA gene was excluded from the ETS alignment.