Abstract
Species of the genus Nicotiana (Solanaceae), commonly referred to as tobacco plants, are often cultivated as non-food crops and garden ornamentals. In addition to the worldwide production of tobacco leaves, they are also used as evolutionary model systems due to their complex development history tangled by polyploidy and hybridization. Here, we assembled the plastid genomes of five tobacco species: N. knightiana, N. rustica, N. paniculata, N. obtusifolia and N. glauca. De novo assembled tobacco plastid genomes had the typical quadripartite structure, consisting of a pair of inverted repeat (IR) regions (25,323-25,369 bp each) separated by a large singlecopy (LSC) region (86,510-86,716 bp) and a small single-copy (SSC) region (18,441- 18,555 bp). Comparative analyses of Nicotiana plastid genomes with currently available Solanaceae genome sequences showed similar GC and gene content, codon usage, simple sequence and oligonucleotide repeats, RNA editing sites, and substitutions. We identified 20 highly polymorphic regions, mostly belonging to intergenic spacer regions (IGS), which could be suitable for the development of robust and cost-effective markers for inferring the phylogeny of the genus Nicotiana and family Solanaceae. Our comparative plastid genome analysis revealed that the maternal parent of the tetraploid N. rustica was the common ancestor of N. paniculata and N. knightiana, and the later species is more closely related to N. rustica. Relaxed molecular clock analyses estimated the speciation event between N. rustica and N. knightiana appeared 0.56 Ma (HPD 0.65-0.46). Biogeographical analysis supported a south-to-north range expansion and diversification for N. rustica and related species, where N. undulata and N. paniculata evolved in North/Central Peru, while N. rustica developed in Southern Peru and separated from N. knightiana, which adapted to the Southern coastal climatic regimes. We further inspected selective pressure on protein-coding genes among tobacco species to determine if this adaptation process affected the evolution of plastid genes. These analyses indicate that four genes involved in different plastid functions, including DNA replication (rpoA) and photosynthesis (atpB, ndhD and ndhF), came under positive selective pressure as a result of specific environmental conditions. Genetic mutations in these genes might have contributed to better survival and superior adaptations during the evolutionary history of tobacco species.