Comparative Chloroplast Genome Analyses of Six Hemlock Trees in East Asia: Insights into Their Genomic Characterization and Phylogenetic Relationship

Hemlocks (Pinaceae: Tsuga) are widely distributed in North America and East Asia, forming a reticulate evolutionary structure in East Asia with significant ecological importance. To clarify the chloroplast genome characteristics and phylogenetic relationships among Tsuga species, we analyzed the chloroplast genomes of T. chinensis var. tchekiangensis, T. chinensis, T. diversifolia, T. dumosa, T. forrestii, and T. sieboldii, performing associated phylogenetic analyses. The results reveal that the chloroplast genome lengths among the six Tsuga species vary from 120,520 to 121,010 bp, encompassing about 108 to 112 genes, including 35/32 tRNA genes and 4 rRNA genes. A codon usage analysis highlighted a preference for A/U-ending codons, and all six nucleotide types have A/T bases and a prevalence of mononucleotides. Notably, all Tsuga species exhibit inverted repeat (IR) contractions and possess unique hexanucleotides absent in the other species of Pinaceae, potentially making them more susceptible to gene recombination or rearrangement during evolution. While most variations are observed in non-coding regions, particularly in intergenic fragments, substantial variation sites are also present within the genes. The phylogenetic tree, constructed using chloroplast genomes, substantiates the sister taxa relationship between Tsuga and Nothotsuga. Furthermore, it confirms that T. chinensis var. tchekiangensis exhibits a closer relationship with T. forrestii than with T. chinensis. These findings not only provide partial evidence that T. chinensis may not constitute a monophyletic species but also underscore the necessity of reevaluating the taxonomic status of T. chinensis var. tchekiangensis. In addition, while the RSCU cluster analysis is basically consistent with the phylogenetic analysis, it also highlights a distinct differentiation between Nothotsuga and Tsuga. This study not only provides molecular-level phylogenetic classification evidence of Pinaceous genera via chloroplast genome analyses but also offers compelling evidence for further exploring the relationships and species delimitation among the hemlocks of East Asia.