Composition of seagrass organelles
In this study, the mt genome of R. sinensis was sequenced, while its cp genome was re-sequenced (Fig. 1, Fig. S1). The sequenced cp genome was compared with the previously published genome using a dot plot. The results showed that the two cp genomes were highly collinear and only differed in two SNP sites(Figs. 2 and 3). Therefore, the cp genome is highly conserved in R. sinensis.
A single circular molecule was resolved in R. sinensis, Z. japonica, and P. iwatensis. However, the mt genome of Z. marina was linear. The sizes of the four seagrass mt genomes ranged from 178,929 bp in P. iwatensis to 256,174 bp in R. sinensis (Table 1). In total, 44 genes were annotated in the R. sinensis genome, including 28 PCGs, 15 tRNA, and 6 rRNA genes; 50 genes were annotated in Z. japonica, including 29 PCGs, 18 tRNA and 3 rRNA genes; 44 genes were annotated in P. iwatensis, including 26 PCGs, 15 tRNA and 3 rRNA genes; and 38 genes were annotated in Z. marina, including 25 PCGs, 10 tRNA and 3 rRNA genes. The overall GC content in the mt genome of these four seagrass species is 49.63%, 46.26%, 48.69%, 45.14%, and, respectively. It was notable that, although most of the rRNA genes occurred in single copies, the R. sinensis mt genome possessed two copies of the rRNA genes (Table 2).
Table 1
General information of complete mt and cp genomes of species analyzed in this study.
Species | Total genome size(bp) | GC content (%) | Total number of genes | Protein coding genes | tRNA genes | rRNA genes | GenBank |
Mitochondria | | | | | | | |
Ruppia sinensis | 256,174 | 49.63 | 44 | 28 | 10 | 6 | This study |
Zostera marina | 191,481 | 45.14 | 38 | 25 | 10 | 3 | NC_035345 |
Zostera japonica | 221,614 | 46.26 | 50 | 29 | 18 | 3 | NC_068803 |
Phyllospadix iwatensis | 178,929 | 48.69 | 44 | 26 | 15 | 3 | NC_068804 |
Chloroplast | | | | | | | |
Ruppia sinensis | 158,897 | 35.84 | 136 | 88 | 37 | 8 | This study |
Phyllospadix iwatensis | 152,726 | 36.18 | 132 | 86 | 38 | 8 | NC_058622 |
Zostera marina | 143,877 | 35.46 | 116 | 78 | 30 | 8 | NC_036014 |
Zostera japonica | 146,090 | 35.92 | 127 | 85 | 34 | 8 | NC_058623 |
Table 2
Annotated genes in the R. sinensis mt genome.
Category | Gene group | Gene name |
Core genes | ATP synthase | atp1, atp4, atp6, atp8, atp9 |
| Subunits of NADH dehydrogenase | ccmB, ccmC, ccmFc*, ccmFn |
| Ubichinol cytochrome c reductase | cob |
| Cytochrome c oxidase | cox1, cox2, cox3 |
| Maturases | matR |
| NADH dehydrogenase | nad1****, nad2****, nad3, nad4*** nad4L, nad5****, nad6, nad7**** nad9 |
Variable genes | Ribosomal proteins (LSU) | |
| Ribosomal proteins (SSU) | rps1, rps12, rps3*, rps7 |
rRNA genes tRNA genes | Succinate dehydrogenase Ribosomal RNAs Transfer RNAs | rrn18 (2), rrn26 (2), rrn5 (2) trnC-GCA, trnD-GUC, trnE-UUC trnH-GUG, trnM-CAU (3), trnQ-UUG, trnW-CCA, trnY-GUA |
Note: The numbers after gene names represent the number of copies, and the superscripts * indicate the number of introns. |
Gene loss in seagrass organelles
To investigate gene losses and gains in the organelle genomes of seagrasses, a gene content analysis was performed on seagrasses along with other downloaded monocotyledons. This study revealed that most cp-ndh genes were absent in the cp genomes of the Hydrocharitaceae family, and the cp-rps19 gene was missing in the cp genomes of the Zosteraceae family. Specifically, only the cp genome of Z. marina lacked cp-pet and cp-psb genes (Fig. 2A). Compared to cp genomes of four seagrass species, mt genomes experienced major gene loss. The mt-rpl genes were absent in all examined seagrasses except for the mt-rpl16 gene in P. iwatensis. Additionally, four ribosomal genes and mt-rps genes were lost in all seagrasses during the evolutionary process (Fig. 2B).
Horizontal gene transfer (HGT) of seagrass organelles
To investigate whether the lost cp genes were transferred to the mt genomes, analysis of HGT among their organelle genomes was conducted. The searches revealed that the deleted cp genes were not transferred to the mt genomes. In total, 17–48 MTPTs of homologous sequences were identified between cp and mt genomes from four seagrass species, with the total length ranging from 18,520 bp to 55,880 bp. The longest sequence (5,839 bp) was transferred from rps7, ndhB, trnL-CCA, and ycf2 in the cp genome to IGS in the mt genome. The fragments that migrated from the cp genome accounted for a variable proportion (29.18%, 25.13%, 13.80%, and 7.2%) of the mt genomes of the four seagrass species, with the highest proportion in the Z. marina mt genome. These migrated sequences were mainly located in the IGS or rRNA gene of the mt genomes (Table S1-S4). Notably, several intact plastome-derived PCGs were identified in mt genomes, including atpH, rbcL, atpB, atpE, atpF, ndhB, petD, rpoC1, and rpl23. Additionally, several fragments were annotated as part of the plastidial protein-coding sequences, namely ycf, rpl, rpo, atp, and psa genes (Fig. 3). These results demonstrate extensive intracellular DNA transfer from plastids to mitochondria in seagrasses.
Analyses of genomic syntenic regions and rearrangements
To explore the genome structure evolution of the four seagrass species, an analysis of syntenic regions among their organelle genomes was conducted. The cp genome structures of R. sinensis, Z. marina, Z. japonica, and P. iwatensis exhibited a high degree of collinearity. Almost the entire genome was collinear, except for an inversion that resulted from inverted repeat regions (Fig. 4A). The mt genomes of the four seagrass species displayed greater complexity than their cp genomes. A total of 27 collinear blocks (134,109 bp) were identified between the mt genomes of Z. marina and Z. japonica, 28 collinear blocks (147,068 bp) between the mt genomes of Z. japonica and P. iwatensis, and 28 collinear blocks (146,834 bp) between the mt genomes of P. iwatensis and R. sinensis. These collinear blocks were dispersed throughout the mt genome, suggesting four multiple structural rearrangement events (Fig. 4B). Subsequently, the genomic locations of all orthologous genes in the three mt genomes were compared to determine whether rearrangement events would disrupt the gene clusters. The results showed that gene order is poorly conserved in the seagrass species. Only five colinear gene clusters were found, including two or three genes. Two gene clusters (nad5- trnC-GCA and rrn18-rrn5) were shared by all four species and three gene clusters (atp6- trnE-UUC- trnQ-UUG, ccmFn- rps7, and ccmFc- trnD-GUG- cox1) were shared by two species (Fig. 4C).
Variations and evolutionary rates of core PCGs
To explore gene variations between the four seagrass species, calculations of the K2p, Ka/Ks, and pi among their mt genomes were performed. Across the 23 shared PCGs examined, atp4 exhibited the greatest genetic distance among the four seagrass species, followed by the matR. The nad1 gene exhibited the smallest genetic distance among the four seagrass species, indicating that this gene was highly conserved (Fig. 5A). The overall Ka/Ks values for most core PCGs were lower than 1, suggesting that these genes were subjected to purifying selection. However, the Ka/Ks values for atp4, atp8, ccmFc, and nad5 were more than 1 for seagrass species, indicating that these genes were subjected to positive selection pressure (Fig. 5B). As shown in Fig. 5C, a global comparison of the homologous gene sequences showed that matR exhibits the highest degree of variation, followed by atp4, atp8, rps7, and ccmFn. Both ccmC and nad1 exhibit a relatively low degree of variation and thus are more conserved.