Complete Mitochondrial Genome and Its Phylogenetic Position in Red Algae Fushitsunagia catenata from South Korea

The mitogenome is an important tool in taxonomic and evolutionary studies. Only a few complete mitogenomes have been reported for red algae. Herein, we reported the complete mitochondrial genome sequence of Fushitsunagia catenata (Harvey) Filloramo, G.V. and Saunders, G.W. 2016, a monospecific genus. The genome was 25,889 bp in circumference and had a strongly biased AT of 70.4%. It consisted of 2 rRNAs, 23 tRNAs, and 24 protein-coding genes (PCGs). nad5 (1986 bp) was the largest and atp9 (231 bp) was the smallest PCG. All PCGs used ATG as an initiation codon and TAA as a termination codon, except TAG, which was the termination codon used in the sdh3, rps3, and rps11 genes. The general structure and gene content of the present findings were almost identical to those of other red algae genomes, particularly those of the Rhodymeniales order. The maximum likelihood analysis showed that F. catenata was closely related to Rhodymenia pseudopalmata. The mitochondrial genome data presented in this study will enhance our understanding of evolution in Rhodophyta species.

Recent studies that combined morphological and genomic data resulted in numerous taxonomic revisions.The genus Fushitsunagia was recently isolated from the genus Lomentaria [4].Fushitsunagia catenata is larger, measuring 10-15 cm in height, with straight apices, a turgid texture, and irregular branches [5].The taxonomy of the genus Fushitsunagia is still Life 2024, 14, 534 2 of 10 unclear because morphological traits have limited taxonomic relevance.To date, only a few mitochondrial genes (cob, cox1, and cox3) of F. catenata have been reported.Phylogenetic analysis using complete mitochondrial genomes is more informative for determining evolutionary relatedness than single-gene sequencing [6].Therefore, we analyzed the whole mitochondrial genome of F. catenata and discussed the evolutionary connections between rhodophytes.
Mitochondrial genes are valuable for phylogenetic research.However, a more precise understanding of phylogenetic relationships may be obtained by analyzing the full mitochondrial genome.There have been no reports of the full mitochondrial genome or phylogenetic analyses of F. catenata.This study included the construction of the first comprehensive mitochondrial genome of F. catenata using de novo assembly on an Illumina platform.The findings of this study will be important for future phylogenetic analysis, in-depth comprehension of gene content and structure, and comparative mitochondrial genome analyses.

Sample Collection and Genomic DNA Extraction
The red macroalga F. catenata sample (Figure S1) used in this study was collected from the coastal region of Gijang, Busan, South Korea (35.284634N, 129.259071 E) in August 2022.The samples were subsequently deposited in the Ecological Restoration Group, Marine Eco-Technology Institute, Busan, South Korea (specimen number PU-T01-S-MA-05).Genomic DNA was isolated using a DNeasy Blood and Tissue kit (Qiagen, Germany) according to the manufacturer's instructions.The concentration and purity of the extracted DNA were evaluated using a NanoDrop spectrophotometer (Thermo Fisher Scientific D1000, Waltham, MA, USA).The extracted genomic DNA was stored at a temperature of −4 • C and transported to Macrogen (Daejeon, South Korea; https://www.macrogen.com/ko/)for library creation and sequencing.

Mitochondrial Genome Sequencing
DNA libraries were created using the TrueSeq Nano DNA Kit and then subjected to sequencing on the Illumina platform (Illumina, HiSeq 2500, San Diego, CA, USA) using paired-end reads with a length of 150 bp.To reduce analytical bias, the acquired reads were trimmed using the Trimmomatic v0.36 (http://www.usadellab.org/cms/?page=trimmomatic, accessed on 15 October 2023) [12].This included the removal of adapter sequences and low-quality reads with quality scores below 20 (Q < 20).The trimmed reads were randomly sampled to assemble the mitochondrial genome.In this case, only the sampled reads were used for de novo assembly.The overall quality of sequencing reads was assessed using FastQC v0.11.5 (http://www.bioinformatics.babraham.ac.uk/ projects/fastqc, accessed on 15 October 2023) [13].High-quality reads were assembled using k-mers and SPAdes v3.15.0 (http://cab.spbu.ru/software/spades/,accessed on 15 October 2023) [14,15].After the complete genome was assembled, BLAST analysis was performed to identify the contigs containing the mitogenome sequences in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi,accessed on 15 October 2023).

Phylogenetic Analysis
The phylogenetic tree was constructed using the complete mitochondrial genome and the cox1, cox3, and cob gene sequences of 12 selected red algae from the subclass Rhodymeniophycidae, together with one outgroup member from the family Glaucocystaceae (Table 1).The mitochondrial genomes and gene sequences used in this study were retrieved from the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/,accessed on 10 November 2023).Multiple sequence alignments were performed using ClustalW [24], and a maximum likelihood (ML) phylogenetic tree was created using MEGA11 [25].ML analysis was conducted using the Tamura-Nei model with default settings and 1000 bootstrap replications [21].

Mitochondrial Genome Characterization
The F. catenata library was subjected to next-generation sequencing using an Illumina HiSeq 2500 sequencer, resulting in 22,066,120 raw reads.The GC content of the reads was 43.39%, with a Q20 score of 93.81% and a Q30 score of 87.08%.After removing lowquality sequences, 14,944,788 filtered reads were obtained with a GC content of 42.65%, Q20 accuracy of 98.86%, and Q30 accuracy of 95.78%.The reads were subjected to de novo assembly (100% coverage with a depth of 479.84), resulting in a contig consisting of 25,889 bases, with a GC content of 29.59%.The mitochondrial genome of F. catenata (GenBank: OR045827) was circular with a length of 25,889 bp (Figure 1).It contained 49 genes, consisting of 24 PCGs, 23 tRNAs, and 2 rRNAs.The H-strands contained 10 PCGs, 11 tRNAs, and 2 rRNAs.In contrast, the L strand consisted of 14 PCGs and 12 tRNAs.The nucleotide content of the whole genome was determined to be 37.1% A, 33.3% T, 15.1% G, and 14.5% C, as shown in Table 1.The analysis of nucleotide composition revealed a biased composition of A + T, which accounted for 70.4% of the total genome.The whole genome exhibited positive AT and GC skewness, suggesting a preference for using As over Ts and Gs over Cs.

Mitochondrial Genome Characterization
The F. catenata library was subjected to next-generation sequencing using an Illumina HiSeq 2500 sequencer, resulting in 22,066,120 raw reads.The GC content of the reads was 43.39%, with a Q20 score of 93.81% and a Q30 score of 87.08%.After removing low-quality sequences, 14,944,788 filtered reads were obtained with a GC content of 42.65%, Q20 accuracy of 98.86%, and Q30 accuracy of 95.78%.The reads were subjected to de novo assembly (100% coverage with a depth of 479.84), resulting in a contig consisting of 25,889 bases, with a GC content of 29.59%.The mitochondrial genome of F. catenata (GenBank: OR045827) was circular with a length of 25,889 bp (Figure 1).It contained 49 genes, consisting of 24 PCGs, 23 tRNAs, and 2 rRNAs.The H-strands contained 10 PCGs, 11 tRNAs, and 2 rRNAs.In contrast, the L strand consisted of 14 PCGs and 12 tRNAs.The nucleotide content of the whole genome was determined to be 37.1% A, 33.3% T, 15.1% G, and 14.5% C, as shown in Table 1.The analysis of nucleotide composition revealed a biased composition of A+T, which accounted for 70.4% of the total genome.The whole genome exhibited positive AT and GC skewness, suggesting a preference for using As over Ts and Gs over Cs.

Protein-Coding Genes
Twenty-four PCGs comprised 69.35% of the mitochondrial genome of F. catenata.These genes comprised a total length of 17,955 bp.There were clusters of genes such as NADH dehydrogenase subunits, succinate dehydrogenase, apocytochrome b, cytochrome c oxidase, ATP synthase, small-and large-subunit ribosomal proteins, independent protein translocase, and a gene encoding a hypothetical protein (Table 2).The nad5 and atp9 genes were the largest and smallest, respectively, in terms of length within the whole mitochondrial genome.Nad5 accounted for 7.67% (1986 bp) of the genome, whereas atp9 accounted for 0.89% (231 bp).ATG and TAA served as the initiation and termination codons for all PCGs, except TAG, which served as the termination codon specifically for the sdh3, rps3, and rps11 genes.The mitochondrial genome of F. catenata contained only rpl16 among the ribosomal protein genes (Table 3).The genes rpl5 and rpl20 do not exist, and no intronic coding sequences were identified.Note: H and L indicate that the genes were transcribed on the heavy and light strands, respectively; * denotes the number of nucleotides between a given gene and the next, with a negative value indicating an overlap.

RNAs
In the mitochondrial genome of F. catenata, the rRNA genes were located on the H-strand and identified as rnl (large subunit, 2604 bp) and rns (small subunit, 1361 bp) (Table 2).These genes had a combined length of 3965 bp, accounting for 15.32% of the whole mitochondrial genome.The rRNA genes were separated using the nad4L gene.A total of 23 tRNAs, ranging from 71 to 93 bp in length, were found in the mitochondrial genome; trnI was not identified in the F. catenata mitochondrial genome.Among these, arginine (trnR-TCT and trnR-ACG), glycine (trnG-TCC and trnG-GCC), leucine (trnL-TAA and trnL-TAG), methionine (trnM-CAT), and serine (trnS-TGA and trnS-GCT) had two copies with distinct anticodons, with the exception of methionine, which had the same anticodon.The tRNA cysteine (trnC-GCA, 71 bp) was the shortest and serine (trnS-GCT, 93 bp) was the longest.The total tRNA length was 1741 bp, accounting for 6.73% of the whole genome length, and no intronic RNA sequences were detected.

Overlapping and Intergenic Spacer Regions
An examination of the intergenic nucleotides of the F. catenata mitochondrial genome sequence revealed that only two gene junctions exhibited an overlap of 21 bp: trnL-nad6 (1 bp overlap) and TatC-rps12 (20 bp overlap).In addition, we observed intergenic gaps ranging from 1 to 607 bp.The largest intergenic gap, measuring 607 bp, was observed between nad4 and nad5 genes (Table 2).

Phylogenetic Analysis
ML phylogenetic trees were constructed with complete mitochondrial genome sequences based on single-gene sequences of the species within the Rhodymeniales order.The ML phylogenetic analysis indicated that F. catenata was most closely related to Rhodymenia pseudopalmata with strong bootstrap support (Figure 2).Rhodymeniales species (F.catenata and R. pseudopalmata) formed a monophyletic clade with Halymeniales species (Grateloupia elliptica, G. turuturu) with high bootstrap support but not with other species.
don.The tRNA cysteine (trnC-GCA, 71 bp) was the shortest and serine (trnS-GCT, 93 was the longest.The total tRNA length was 1741 bp, accounting for 6.73% of the w genome length, and no intronic RNA sequences were detected.

Overlapping and Intergenic Spacer Regions
An examination of the intergenic nucleotides of the F. catenata mitochondrial gen sequence revealed that only two gene junctions exhibited an overlap of 21 bp: trnL-(1 bp overlap) and TatC-rps12 (20 bp overlap).In addition, we observed intergenic ranging from 1 to 607 bp.The largest intergenic gap, measuring 607 bp, was obse between nad4 and nad5 genes (Table 2).

Phylogenetic Analysis
ML phylogenetic trees were constructed with complete mitochondrial genom quences based on single-gene sequences of the species within the Rhodymeniales o The ML phylogenetic analysis indicated that F. catenata was most closely related to dymenia pseudopalmata with strong bootstrap support (Figure 2).Rhodymeniales sp (F.catenata and R. pseudopalmata) formed a monophyletic clade with Halymeniales sp (Grateloupia elliptica, G. turuturu) with high bootstrap support but not with other spec A phylogenetic analysis, using gene sequences of cox1 (Figure S2), cox3 (Figure and cob (Figure S4), revealed differences in the relationships among the species in group.However, the bootstrap values supporting each node were often modest, ex for a substantial bootstrap value that supported the relationship between sister tax Grateloupia and Gelidium species.Most algal orders formed a monophyletic group in phylogenies of cox1, cox3, and cob, except for Gigartinales and Rhodymeniales in the phylogeny.A phylogenetic analysis, using gene sequences of cox1 (Figure S2), cox3 (Figure S3), and cob (Figure S4), revealed differences in the relationships among the species in the group.However, the bootstrap values supporting each node were often modest, except for a substantial bootstrap value that supported the relationship between sister taxa of Grateloupia and Gelidium species.Most algal orders formed a monophyletic group in the phylogenies of cox1, cox3, and cob, except for Gigartinales and Rhodymeniales in the cox1 phylogeny.

Discussion
The mitochondrial genome of F. catenata conformed to characteristics often observed in red algae, and the quality of the sequenced genome was comparable to that of other species belonging to the Rhodymeniophycidae subfamily (Table 1).The size and base composition of monospecific F. catenata were consistent with those of a previously reported Rhodymeniales species, R. pseudopalmata (KC875852; 26,166 bp, 29.5% GC) [29].

Figure 1 .
Figure 1.The circular mitochondrial genome of Fushitsunagia catenata.The arrow direction shows gene orientation, and the different colors reflect the groupings of functional genes together with their acronyms.

Figure 2 .
Figure 2. Maximum likelihood phylogenetic tree based on complete mitochondrial genom quence of Rhodymeniophycidae.The sequence generated in this study is in bold.The support on each node represents the bootstrap value.

Figure 2 .
Figure 2. Maximum likelihood phylogenetic tree based on complete mitochondrial genome sequence of Rhodymeniophycidae.The sequence generated in this study is in bold.The support value on each node represents the bootstrap value.

Table 1 .
List of algal mitochondrial genomes with nucleotide compositions.