Background: Characterization of the key factors determining gene expression level has been of significant interest. Previous studies on the relationship among evolutionary rates, codon usage bias, and expression level mostly focused on either nuclear genes or unicellular/multicellular organisms but few in chloroplast (cp) genes. Ophioglossum vulgatum is a unique fern and has important scientific and medicinal values. In this study, we sequenced its cp genome and transcriptome to estimate the evolutionary rates (dN and dS), selective pressure (dN/dS), gene expression level, codon usage bias, and their correlations. Results: The correlation coefficients between dN, dS, and dN/dS, and Transcripts Per Million (TPM) average values were -0.278 (P = 0.027 < 0.05), -0.331 (P = 0.008 < 0.05), and -0.311 (P = 0.013 < 0.05), respectively. The codon adaptation index (CAI) and tRNA adaptation index (tAI) were significantly positively correlated with TPM average values (P < 0.05). Conclusions: Our results indicated that when the gene expression level was higher, the evolutionary rates and selective pressure were lower, but the codon usage bias was stronger. We provided evidence from cp gene data which supported the E-R (E stands for gene expression level and R stands for evolutionary rate) anti-correlation.

Mitochondria are crucial for energy production in plant tissues, but their quantity and activity vary in different tissues and developmental processes. Determining the factors underlying differential molecular evolutionary rates has long been a central question in evolutionary biology, with expression level emerging as the prime predictor. Although we have previously observed an anti-correlation between expression level (E) and evolutionary rate (R) in chloroplast genes, it remains unclear whether such an anti-correlation exists in plant mitochondrial genes. Ophioglossum vulgatum is a typical plant belonging to the Ophioglossaceae, characterized by its unique morphology with only a single leaf above ground. It holds significant scientific and medicinal value. Using the mitochondrial genome and transcriptome data of O. vulgatum, we first analyzed the correlation between mitochondrial gene expression, codon usage bias, and evolutionary rates in different tissues. Our findings indicated that mitochondrial gene expression level was the strongest between stem and leaf, while the weakest was between sporangium and root. Kruskal-Wallis tests revealed significant differences across various tissue types. Codon usage bias was influenced by both mutation and selection, with selection exerting a greater impact. The Spearman’s rank correlation coefficients between codon adaptation index and expression levels of sporangium, stem, leaf, and root were 0.1178, 0.3926, 0.4463, and 0.2945, respectively, with significance in stem and leaf (P < 0.05). The correlation coefficients between the nonsynonymous substitution rate (dN) and expression levels in sporangium, stem, leaf, and root were -0.0840, -0.1786, -0.1714, and -0.0857, respectively, yet none are statistically significant. The correlation coefficient between the synonymous substitution rate (dS) and expression levels in sporangium was negative, whereas those between dS and the stem, leaf, and root were positive, although they were not significant. The dN/dS ratio exhibited a significant negative correlation with expression levels in both leaf and root (P < 0.05). For the first time, our study revealed differences in the correlation between mitochondrial gene expression and codon usage bias, as well as evolutionary rates, across various tissues of O. vulgatum. Moreover, we also provide novel insights into understanding the effects of plant mitochondrial gene expression on evolutionary patterns.

Many plant mitochondrial (mt) genomes have been sequenced but few in ferns. Ophioglossum vulgatum represents a typical species of fern genus Ophioglossum with medicinal and scientific value. However, its mt genome structure remains to be characterized. This study assembled and annotated the complete O. vulgatum mt genome and presented its structural characters and repeat sequences firstly. Its mt and chloroplast (cp) transfer sequences were explored, and the phylogenetic significance of both mt and cp genomes was also evaluated at the family level. Our results showed that the complete mt genome of O. vulgatum is a single circular genome of 369,673 bp in length, containing 5000 dispersed repetitive sequences. Phylogenetic trees reconstructed from cp and mt genomes displayed similar topologies, but also showed subtle differences at certain nodes. There exist 4818 bp common gene fragments between cp and mt genomes, of which more than 70% are located in tRNA intergenic regions (in mt). In conclusion, we assembled the complete mt genome of O. vulgatum, identified its remarkable structural characters, and provided new insights on ferns. The complementary results derived from mt and cp phylogeny highlighted that some higher taxonomic-level phylogenetic relationships among ferns remain to be resolved.

The mechanisms of species formation and diversity within the Orchidaceae have always been a topic of great research interest, and molecular biology-based explanations for these phenomena are lacking. Here, we present a comparative genomic analysis comprising 24 chromosome-level genome assemblies and genome skimming data for 204 species from Dendrobium. We identify that the genetic conservation in Dendrobium is associated with its environmental adaptability. And the formation of species diversity in the genus Dendrobium is driven by the compression of the Indian plate and changes in sea level and precipitation. The CYP734A50 might play important role in Dendrobium self-incompatibility (SI) species and the type-1 SI system lost through deletion of the entire single polymorphic locus (S-locus). Our results reveal the genetics background of genomic variation, biosynthesis, and adaptive evolution in Dendrobium, providing fundamental insights into the relationships between the genome and trait evolution of orchids and into angiosperm diversification.

Ophioglossum vulgatum is a rare and ancient fern. In this study, the chloroplast (cp) genome of O. vulgatum was completely sequenced. The genome size is 138,562 bp, which contains a large single-copy (LSC) region with 99,351 bp, a small single-copy (SSC) region with 19,661 bp, and two inverted repeats (IR) regions of 9,775 bp each. Additionally, the overall GC content is 42.14%. It encodes a total 129 genes, including 84 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The Bayesian phylogenetic tree shows that O. vulgatum and O. californicum formed a monophyletic branch. This study can provide a molecular basis for studying the phylogenetic genomics and population variation of Ophioglossaceae.