Comparative Analysis of the Chloroplast Genomes of Eight Species of the Genus Spach with Its Generic Delimitation Implications.

Based on Sima and Lu's system of the family Magnoliaceae, the genus Spach s. l. includes approximately 25 species, each with exceptional landscaping and horticultural or medical worth. Many of these plants are considered rare and are protected due to their endangered status. The limited knowledge of species within this genus and the absence of research on its chloroplast genome have greatly impeded studies on the relationship between its evolution and systematics. In this study, the chloroplast genomes of eight species from the genus were sequenced and analyzed, and their phylogenetic relationships with other genera of the family Magnoliaceae were also elucidated. The results showed that the chloroplast genome sizes of the eight species ranged from 159,548 to 159,833 bp. The genomes consisted of a large single-copy region, a small single-copy region, and a pair of inverted repeat sequences. The GC content was very similar across species. Gene annotation revealed that the chloroplast genomes contained 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes, totaling 130 genes. Codon usage analysis indicated that codon usage was highly conserved among the eight species. Repeat sequence analysis identified 42-49 microsatellite sequences, 16-18 tandem repeats, and 50 dispersed repeats, with microsatellite sequences being predominantly single-nucleotide repeats. DNA polymorphism analysis revealed 10 highly variable regions located in the large single-copy and small single-copy regions, among which , , and were the recommended candidate DNA barcodes for the genus species. The inverted repeat boundary regions show little variation between species and are generally conserved. The result of phylogenetic analysis confirmed that the genus s. l. is a monophyletic taxon and the most affinal to the genera, and , in Sima and Lu's system and revealed that the genus s. s. is paraphyletic and the genus s. l. polyphyletic in Xia's system, while subsection is paraphyletic and subsection polyphyletic in Figlar and Nooteboom's system. Morphological studies found noticeable differences between species in aspects including leaf indumentum, stipule scars, floral orientation, tepal number, tepal texture, and fruit dehiscence. In summary, this study elucidated the chloroplast genome evolution within and laid a foundation for further systematic and taxonomic research on this genus.