Complete Organellar Genomes And Molecular Phylogeny Of Hypnea Cervicornis (Gigartinales,Florideophyceae)

Hypnea cervicornis belongs to Rhodophyta,Florideophyceae,Gigartinales,Cystocloniaceae,and Hypnea.It is mainly distributed in the intertidal and subtidal zones of tropical and warm temperate waters.Due to its high carrageenan and agglutinin content,H.cervicornis was widely used in food,industry,and medical applications.It is an economically important algae.The simple morphological structure of H.cervicornis,without complex tissue-organ differentiation,makes the classification system based on traditional morphological characters extremely challenging in terms of scientificity and objectivity.Organellar genomes as independent genetic elements with matrilineal inheritance have become a powerful tool to reveal the taxonomic status of algae and to reshape evolutionary relationships.Up to December 2022,no organellar genomes data were available for species of the genus Hypnea in the public database of NCBI.In this study,we sequenced,assembled and annotated the mitochondrial genome and the plastid genome of H.cervicornis using high-throughput sequencing technology,and studied the structural features,comparative genomics,and phylogenetic analysis of red algae with their divergence times,which provided more data and theoretical support for the molecular phylogeny of red algae.The main findings are as follows:(I)Structural characteristics of the organellar genomes of H.cervicornisIn this study,we obtained the complete sequences of the mitochondrial genome and plastid genome of H.cervicornis through high-throughput sequencing,then annotated and analyzed the characteristics through bioinformatics methods.The mitochondrial genome encodes 50 genes including 24 CDS,2 r RNA genes and 24 t RNA genes with total length of 25,060 bp and GC content of 27.04 %.There is a type II intron(Intron)located in the gene trn I.While the plastid genome is 176,446 bp in length and encodes a total of 230 genes including 194 protein-coding,30 t RNA,3 r RNA genes,2 misc＿RNAs and 1 tm RNA gene.There is a type II intronic insertion in gene trn M.And the average GC contents is 28.09 %.Secondary structures were present in the mitochondrial genome of H.cervicornis: one stem-loop structure existed between genes trn S2 and trn A,and one hairpin structure was found between genes cob and trn L2.It is speculated that these regions might be involved in replication,transcription or translation.The results of codon preference and ENC-plot plots showed that the codon preference of both mitochondrial and plastid genomes about H.cervicornis was weak and affected by both mutational and other factors such as environmental selection pressures.(II)Comparative genomics of the order GigartinalesThe comparative analysis and collinearity analysis of 11 published Gigartinales mitochondrial genomes species and 8 plastid genomes showed that the mitochondrial genome and plastid genome of all Gigartinales algae were highly conservative in gene number,gene type and arrangement,but there were also differences,mainly reflected in(1)the inversion of trn Y and trn R genes in the mitochondrial genome;(2)There is a gene rearrangement in the 12.5 kb region between ycf21 and psa M genes in the plastid genome.RSCU,ENC,and Ka/Ks analyses were conducted on the codon usage of 11 mitochondrial genomes and 8 plastid genomes.The results showed that the RSCU differences in these algae were small and most of them were larger than 1,indicating a preference for codon ending with A/T bases.The Ka/Ks analysis showed that most protein-coding genes in the organellar genomes of H.cervicornis had Ka/Ks values below 1,indicating that the purifying selectivity remained strongly stable and the gene-related functions were more conserved,reflecting the importance of these genes in maintaining the conserved organellar genomes.(III)Phylogenetic analysis and divergence time estimation of red algaeBased on the mitochondrial genome and plastid genome data of red algae in the Gen Bank database,as well as the data obtained from this study,phylogenetic tree was reconstructed using BI and ML methods at the levels of Rhodophyta and the order Gigartinales,respectively.The results showed that:(1)the topological structure of the phylogenetic tree obtained using two methods based on the mitochondrial genome and plastid genome datasets was basically consistent,and all species were clearly divided into four branches:Floridophyceae,Bangiophyceae,Compsopogonophyceae,and Cyanidiophyceae,which were consistent with the traditional classification of the "class";(2)Combined with the divergence time analysis,the Cyanidiophyceae differentiated earliest and are the most primitive group of red algae with a divergence time of 1,275.19 million years ago(Mya)(mitochondrial genome)or 1,779.68 Mya(plastid genome),and the Floridophyceae are the most advanced group of red algae with a divergence time of 916.01 Mya(mitochondrial genome)or 1.568.97 Mya(plastid genome);(3)Combined with the results from the phylogenetic tree of Gigartinales,the species in Gigartinales were clearly divided into two branches,Cystocloniaceae,Caulacanthaceae and Solieriaceae were clustered into one branch,and Gigartinaceae,Phyllophoraceae and Endocladiaceae clustered into another branch.This indicates that the species of Caulacanthaceae originated from multiple lineages and that different species within the family evolved independently,forming independent genera such as Hypnea and Caulacanthus,and that the algae of Hypnea diverged relatively early.The structural characterization of the organellar genomes,the comparative analysis of the genomes of the order Gigartinales and the reconstruction of the phylogenetic tree of red algae with divergence time in this study provided molecular biological data for the study of the taxonomic status of the order Gigartinales,and also laid the theoretical foundation for the phylogenetic and genetic study of red algae.