Evolutionary Dynamics and Phylogeny of Family Malvaceae

Abstract

The plant family Malvaceae (eudicot, angiosperms) belongs to order Malvales. This family comprises of 244 genera and 4225 species, which are distributed in tropical to temperate regions of the world. Malvaceae possesses plastic morphology which leads to taxonomic discrepancies in the classification. The subdivision of family Malvaceae into nine subfamilies is the most accepted classification. The complete chloroplast genome sequences are used to evaluate evolutionary dynamics and phylogeny of plant lineages that help to resolve taxonomic discrepancies. The current study aimed to evaluate the evolutionary dynamics and phylogenetic relationships within the family Malvaceae. We sequenced and assembled chloroplast genome sequences of four species of family Malvaceae including Hibiscus rosa-sinensis, Hibiscus mutabilis, Malvastrum coromandelianum and Malva parviflora. The whole genomic DNA shotgun was generated through Illumina Hiseq2500 from pair end run with 150 bp short read and 350 bp insert size. Moreover, we also assembled chloroplast genome sequences of four species from the Sequence Read Archive (SRA) of National Center for Biotechnology Information (NCBI) including Firmiana colorata, Sterculia monosperma, Pterospermum truncatolobatum, and Urena procumbens. The quality of raw reads was accessed by fastQC, and chloroplast genomes were de novo assembled by Velvet 1.2.10 with various kmer values. The coverage analysis was performed with Burrow wheel aligner (BWA)/Bowtie and Tablet was used for visualisation. Chloroplast genomes were annotated with Dual Organellar genome annotator (DOGMA) and GeSeq. The genomic features, genes content, codon usage, and amino acids frequency were analysed using Geneious R8.1. RNA editing sites were analysed with Predictive RNA editor for plant chloroplast genes (PREP-cp), simple sequence repeats (SSRs) were analysed with MIcroSAtellite identification tools (MISA), and oligonucleotide repeats were analysed with REPuter program. The rate of synonymous and non-synonymous substitutions of protein coding genes was analysed in DnaSP 5.10 after pairwise alignment through Geneious R8.1 using Theobroma cacao as reference. Correlation among substitutions, InDels, and oligonucleotide repeats were analysed at family and genus level. At family level comparisons, one species, each from 13 genera was pairwise aligned to Theobroma cacao, a species basal to Malvaceae, to find substitutions and InDels across species in Malvaceae. Using coordinate positions of forward and reverse oligonucleotide repeats in T. cacao chloroplast genome, we evaluated correlations among direct and reverse repeats, substitutions and InDels in these Malvaceae chloroplast genomes. At the genus level, we investigated these correlations in five genera by taking one species of the respective genus as a reference and comparing it with another species of the same genus. The phylogenetic tree was reconstructed based on chloroplast genome sequences using the IQ-tree program. Our results showed that the sequencing of whole genomic DNA with low coverage depth provides quality genomic resources for the assembly of chloroplast genome with high coverage depth due to 100-1000 times higher chloroplast genomic DNA in the plant cell as compared to the nuclear genome. The comparative analyses of the chloroplast genomes of family Malvaceae from basal lineages to crown groups revealed high similarity in gene content, gene organisation, intron content, and GC content. We observed differences in the length of these chloroplast genomes due to variations in the length of intergenic spacer regions and contraction and expansion of IRs regions. The rate of synonymous and non-synonymous substitutions revealed about 95% similarities among Malvaceae species. However, the rate of synonymous substitutions was higher than non-synonymous substitutions. The IRs contraction and expansion not only lead to generation of pseudogenes at junctions of chloroplast genomes, but also lead to duplication or deletions of a single copy of some genes as observed in Durio zibethinus and Abelmoschus esculentus. The correlation analyses of substitutions with repeats, substitutions with InDels, and repeats with InDels revealed weak to strong correlations. High regression value was observed for substitutions on InDels, substitutions on repeats, and InDels on repeats. We hypothesize that such correlations are a common characteristic of chloroplast genomes in all plant lineages as these were also previously observed in the family Araceae (monocots, angiosperms) and Cephalotaxaceae (gymnosperms). The comparative analyses of three genera including Theobroma, Firmiana, and Hibiscus revealed high similarities in inter-genus and intra-genus level comparison for codon usage, amino acid frequency, RNA editing sites, and simple sequence repeats. We found two times higher oligonucleotide repeats in the crown group of family Malvaceae (Hibiscus) than basal groups (Theobroma and Firmiana). Thirty mutational hotspots were identified in each genus. The phylogenetic inference of family Malvaceae based on complete chloroplast genome sequences attests the previous classification of family Malvaceae into nine subfamilies. Our results revealed high suitability of coding sequences and complete chloroplast genome in inferring of phylogeny and resolve species at the subfamilies level with 100 bootstrapping. In conclusion, this study provides a broad insight into evolutionary dynamics and phylogeny of family Malvaceae. The identified mutational hotspots could be used for development of robust and cost-effective markers to infer phylogeny of these genera, specifically, in the genus Hibiscus