The entire internal transcribed spacer (ITS) region sequencing was used to determine the phylogenetic relationship of 44 Eastern Himalayan Dendrobium species using the Sanger method and MEGA 11. The combined length of ITS-I and ITS-II varies between 623 and 644 base pairs (bp). Among them, there are 403 polymorphic, 91 unique, and 223 conserved nucleotides. In addition, 337 nucleotides are parsimony informative, with a mean of 0.51 that supports the presence of genetic variation in the genome of the selected species. Overall composition distance is 0.32, transition and transversion bias (R) 1.444, and mean evolutionary divergence (d) 0.16. The length of ITS-2 sequences is bigger than that of ITS-I. ITS-II exhibits a higher G+C% content, and the K2+G model is the most suitable, with the lowest penalty for nucleotide substitutions. The average consistency, retention, composite value, and parsimony-informative sites (in parentheses) are 0.5028 (0.4571), 0.6545 (0.6545), and 0.3290 (0.2992), respectively. The mean value of the disparity indices is 0.192, which measures the variation in evolutionary trends between two sequences. In addition, the ratio of non-synonymous to synonymous substitutions (dN/dS) is −0.33 promotes purifying species selection. The prevalence of the TGT codon is abundant across the sequences, with the UCG codon exhibiting the highest relative synonymous codon usage (RSCU). Conversely, the GAC codon has the lowest RSCU value. The greatest genetic divergence occurred between Dendrobium aphyllum and Dendrobium denudans. The Breviflorus species group has the most genetic diversity, whereas Formosae has the least. Between Grastidium and Stachyobium group sequences had the highest evolutionary distance, whereas Dendrobium and Grastidium had the least. The negative value of Tajima’s D, Fu and Li’s D*, F*, and Fu’s Fs tests indicated current population is growing after facing a recent bottleneck. This study suggests that ITS sequencing could be a major focus of comparative sequencing at the generic level in Dendrobium plants and could provide valuable phylogenetic reconstruction for clarifying the evolutionary relevance of the taxa studied.
Key words: Dendrobium spp, Genetic diversity, Internal transcribed spacer (ITS), Neutral evolution theory, Tajima's D.
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