Halophila beccarii is known as a "living dinosaur". It is one of the oldest two generations of seagrass plants and one of the 10 species of seagrass currently at risk of extinction worldwide. It is usually distributed in areas with an average sea surface of 0.9-1.5 meters. Unfortunately, due to habitat loss, the population of this species has decreased. Therefore, how to effectively protect the H. beccarii resources from extinction is a huge challenge. Molecular biology research can provide scientific basis for species conservation. However, research on the genetics of H. beccarii is still in its early stages. So far, there has been no detailed analysis of the mitochondrial genome of the genus Halophila. Based on readings from the Illumina Novaseq6000 platform, the mitochondrial genome of H. beccarii was assembled. This genome is a multi-ring genome, consisting of 28 free loops. The total length is 1,964,072bp, with a GC content of 46.71%. It contains 39 genes, including 26 protein coding genes, 10 tRNA genes, and 3 rRNA genes. Repeat sequence analysis and prediction of RNA editing sites revealed a total of 850 dispersed repeats, 1,205 simple repeats, 61 tandem repeats, and 120 RNA editing sites. Analysis of codon usage indicates that codons ending in A/U are preferred. Gene migration between mitochondrial genome and chloroplast genome was observed through homologous fragment detection. In addition, Ka/Ks analysis showed that most protein coding genes in the mitochondrial genome experienced negative selection, while only the nad3 gene experienced potential positive selection in most Alismatales. Nucleotide polymorphism analysis revealed variations in each gene, with rpl10 being the most significant. In addition, comparative analysis shows that the GC content is conserved, but there are significant differences in the size and structure of mitochondrial genomes among different species of Alismatales. The phylogenetic analysis based on mitochondrial genome reflects the exact evolutionary and taxonomic status of H. beccarii.
