The Complete Mitochondrial Genome of Homophyllia bowerbanki (Scleractinia, Lobophylliidae): The First Sequence for the Genus Homophyllia

Reef-building coral species of the order Scleractinia play an important role in shallow tropical seas by providing an environmental base for the ecosystem. The molecular data of complete mitochondrial genome have become an important source for evaluating phylogenetic and evolutionary studies of Scleractinia. Here, the complete mitogenome of Homophyllia bowerbanki (Milne Edwards and Haime, 1857), collected from Nansha Islands of the South China Sea, was sequenced for the first time through a next-generation sequencing method. H. bowerbanki is the first species of its genus for which the mitogenome was sequenced. This mitogenome was 18,154 bp in size and included two transfer RNA genes (tRNAs), 13 protein-coding genes (PCGs), and two ribosomal RNA genes (rRNAs). It showed a similar gene structure and gene order to the other typical scleractinians. All 17 genes were encoded on the H strand and the total GC content was 33.86% in mitogenome. Phylogenetic analysis (maximum likelihood tree method) showed that H. bowerbanki belonged to the “Robust” clade and clustered together with other two species in the family Lobophylliidae based on 13 PCGs. The mitogenome can provide significant molecular information to clarify the evolutionary and phylogenetic relationships between stony corals and to facilitate their taxonomic classification; it can also support coral species monitoring and conservation efforts.


Introduction
Reef-building coral species of the order Scleractinia play a critical role in shallow tropical waters by supporting diverse and abundant marine life and providing an important environmental base for the ecosystem. The molecular data of complete mitochondrial genomes have become an important source for evaluating phylogenetic and evolutionary studies of Scleractinia as the cost of next-generation sequencing (NGS) technology decreases [1][2][3][4]. Multiple-gene analysis of nuclear and mitochondrial genes has already been used to infer phylogenetic relationships amongst scleractinians [5,6]. Nevertheless, only less than 130 complete mitogenomes of Scleractinia species can be obtained through the NCBI database (https://www.ncbi.nlm.nih.gov/ (accessed on 17 February 2023)) to date, even though there are more than 1600 stony coral species [7].
H. bowerbanki (Milne Edwards and Haime, 1857) is a species of scleractinian coral with encrusting colonies. Its corallites are cerioid and usually with irregularly angular. Its septa are compact and columellae are small. It usually inhabits lower reef slopes protected from wave action. It shows different colours, such as pale grey, brown, or rust-coloured, and is often mottled [8]. H. bowerbanki was placed in the genus Acanthastrea by Veron (2000) and Budd  belongs to the same family as Acanthastrea, Lobophylliidae based on a combination of morphological and three molecular data sets [8][9][10][11].
In this study, the complete mitochondrial genome of H. bowerbanki was sequenced and assembled for the first time through a next-generation sequencing method (NGS), in which its genome structure and nucleotide composition were characterized and analysed. Phylogenetic analyses of H. bowerbanki, based on 13 protein-coding genes (PCGs) of the mitogenome, will help determine its taxonomic classification and facilitate ongoing taxonomic revisions of scleractinian taxa [12]; analyses can also clarify the evolutionary and phylogenetic relationships amongst stony corals in favor of coral species monitoring and conservation efforts [13].

Sample Collection and DNA Extraction
A specimen (Figure 1) of H. bowerbanki was collected from Nansha Islands of the South China Sea (9.9 • N, 115.5 • E) in 2020. Its fresh polyp was stored at −20°C in 95% ethanol and then used to extract total genomic DNA (gDNA); its skeleton was kept in the Coral Sample Repository of Third Institute of Oceanography, Ministry of Natural Resources, with a unique code, 20200501-L1. The gDNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Shanghai, China). The integrity and concentration of the gDNA were measured using 1% agarose gel electrophoresis and NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).
Homophyllia bowerbanki (Milne Edwards and Haime, 1857) is a species of scleractinian coral with encrusting colonies. Its corallites are cerioid and usually with irregularly angular. Its septa are compact and columellae are small. It usually inhabits lower reef slopes protected from wave action. It shows different colours, such as pale grey, brown, or rustcoloured, and is often mottled [8]. Homophyllia bowerbanki was placed in the genus Acanthastrea by Veron (2000) and Budd et al. (2012) until Arrigoni et al. (2016) transferred it to Homophyllia. This genus belongs to the same family as Acanthastrea, Lobophylliidae based on a combination of morphological and three molecular data sets [8][9][10][11].
In this study, the complete mitochondrial genome of H. bowerbanki was sequenced and assembled for the first time through a next-generation sequencing method (NGS), in which its genome structure and nucleotide composition were characterized and analysed. Phylogenetic analyses of H. bowerbanki, based on 13 protein-coding genes (PCGs) of the mitogenome, will help determine its taxonomic classification and facilitate ongoing taxonomic revisions of scleractinian taxa [12]; analyses can also clarify the evolutionary and phylogenetic relationships amongst stony corals in favor of coral species monitoring and conservation efforts [13].

Sample Collection and DNA Extraction
A specimen (Figure 1) of H. bowerbanki was collected from Nansha Islands of the South China Sea (9.9° N, 115.5° E) in 2020. Its fresh polyp was stored at −20 ℃ in 95% ethanol and then used to extract total genomic DNA (gDNA); its skeleton was kept in the Coral Sample Repository of Third Institute of Oceanography, Ministry of Natural Resources, with a unique code, 20200501-L1. The gDNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Shanghai, China). The integrity and concentration of the gDNA were measured using 1% agarose gel electrophoresis and NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Mitogenome Sequencing, Annotation, and Analyses
The gDNA sample was sent to Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) for next-generation sequencing. The sequencing library was generated using NEBNext ® UltraTM DNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA, Catalog #: E7370L) according to the standard protocols. Genomic DNA was sheared into fragments about 400 bp using a Focused-ultrasonicator M200 (Covaris, Woburn, MA, USA). Then, DNA fragments were end-polished, A-tailed, and ligated with the full-length adapter for Illumina sequencing, followed by further PCR amplification. Next, PCR products were purified by AMPure XP system (Beckman Coulter, Beverly, MA, USA). Library

Mitogenome Sequencing, Annotation, and Analyses
The gDNA sample was sent to Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) for next-generation sequencing. The sequencing library was generated using NEBNext ® UltraTM DNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA, Catalog #: E7370L) according to the standard protocols. Genomic DNA was sheared into fragments about 400 bp using a Focused-ultrasonicator M200 (Covaris, Woburn, MA, USA). Then, DNA fragments were end-polished, A-tailed, and ligated with the full-length adapter for Illumina sequencing, followed by further PCR amplification. Next, PCR products were purified by AMPure XP system (Beckman Coulter, Beverly, MA, USA). Library quality control was performed using Agilent 5400 system (Agilent, Santa Clara, CA, USA) and qPCR (1.5 nM). Then, the library was pooled based on the effective concentration and targeted data amount (12 Gb of raw data). Next, 5 -end of the library was phosphorylated and cyclized. Subsequently, loop amplification was performed to generate DNA nanoballs. These DNA nanoballs were finally loaded into flow cell with DNBSEQ-T7 for paired-end 150 bp sequencing. FastQC was used to assess the quality and quantity of raw data [14]. Fastp was used to remove reads containing poly-N regions, adapters, and low-quality reads; then, a series of filtered clean reads was obtained [15]. These clean reads were applied to reconstruct the mitochondrial genome via NOVOPlasty 4.3.1 [16] with a parameter of K-mer 33. A total of 66,914 of 79,646,476 raw reads (approximately 0.08%) were de novo assembled to produce the mitogenome with the guidance of seed sequence (COI gene from GenBank: MG792550); the average sequencing coverage was 676×.

Phylogenetic Analyses
The phylogenetic positions of H. bowerbanki were inferred using thirteen tandem mitogenome PCGs together with another forty-one species of Scleractinia and two species of Corallimorpharia (outgroup), which we downloaded from https://www.ncbi.nlm.nih. gov/genbank/ (accessed on 10 February 2023) ( Table 1) [22]. We used MEGA11 to select the best-fitting model based on Akaike Information Criterion (AIC); subsequently, a maximum likelihood (ML) tree was constructed with 500 bootstrap replicates under GTR + G + I model.

Characteristics and Composition of Mitogenome
The complete mitogenome of H. bowerbanki was identified as a circular molecule with a similar gene order and gene structure to other typical scleractinians [23,24], which included thirteen PCGs (ND5, ND1, Cyt b, ND2, ND6, ATP6, ND4, CO III, CO II, ND4L, ND3, ATP8, and COI), two tRNA genes (tRNA Met and tRNA Trp ), and two rRNA genes (12S and 16S). The mitochondrial genome size of H. bowerbanki was 18,154 bp. The base composition was 24.75% for A, 13.32% for C, 21.75% for G, and 40.17% for T, which showed a higher AT (66.14%) bias. All of the seventeen genes were encoded on H strand and the total GC content was 33.86% in mitogenome. The GC content was one of the important compositional features of different genome regions. Identifying the driving force that shaped the GC content and deciphering the biological meaning of variations in the GC content will help us understand genome evolution [25]. The mitogenome GC content of most scleractinians ranged from 30% to 40% [22,[26][27][28]. The intergenic regions of different genes ranged from −1 to 1506 bp. Moreover, four overlaps were detected. ND6 overlapped ATP6 by 1 bp, ATP6 overlapped ND4 by 1 bp, COII overlapped ND4L by 19 bp, and ND5 overlapped tRNA Trp by 2 bp (Figures 2 and 3; Tables 2 and 3).

Protein-Coding Genes
The total length of all 13 PCGs was 11,595 bp, with a base composition of 21.61% (A), 13.2% (C), 20.56% (G), and 44.63% (T), which showed that the PCGs preferred base AT. All the PCGs started with ATG, except for COIII, COI, and ND2. COIII started with GTG, COI and ND2 started with ATT. Five PCGs (ND1, Cyt b, ND4, COII, and ND5) terminated with TAG; the other eight PCGs (ND2, ND6, COIII, ATP6, ND3, ND4L, ATP8, and COI) stopped with TAA. Like other stony corals, the ND5 gene of H. bowerbanki also had an intron insertion with a length of 10,461 bp. ATP8 (198 bp) was the shortest gene and the longest gene was ND5 (1,815 bp) (Tables 2 and 3). According to the analysis results of AT-skew and GC-skew, we can see both were negative (Table 3, Figure 4); meanwhile, all PCGs showed a stronger nucleotide asymmetry, with a higher AT-skew than GC-skew. Codon use frequency was higher amongst L, F, V, G, and S, accounting for 53.04% of a total of 3865 codons ( Figure 5). PCGs showed a stronger nucleotide asymmetry, with a higher AT-skew than GC-skew. Codon use frequency was higher amongst L, F, V, G, and S, accounting for 53.04% of a total of 3865 codons ( Figure 5).   PCGs showed a stronger nucleotide asymmetry, with a higher AT-skew than GC-skew. Codon use frequency was higher amongst L, F, V, G, and S, accounting for 53.04% of a total of 3865 codons ( Figure 5).

Transfer RNAs and Ribosomal RNAs
The 12S ribosomal RNA was 910 bp in size and located between ND4 and COIII; the 16S ribosomal RNA was 1,697 bp in size and located between tRNA Met and ND5. The base composition of two rRNAs was 35.52% A, 12.5% C, 20.02% G, and 31.95% T. The AT content of two rRNAs was 67.47%, and both AT-skew (0.053) and GC-skew (0.231) were positive. The two tRNA encoding genes (tRNA Met and tRNA Trp ) were 72 bp and 69 bp in size, respectively. The base composition of two tRNAs was 32.39% A, 19.01% C, 22.54% G, and 26.06% T. The AT content of two tRNAs was 58.45%, and both AT-skew (0.108) and GC-skew (0.085) were also positive. Both of the two tRNAs were folded into a typical cloverleaf structure, which included an amino acid accept arm, D loop, anticodon loop, and TψC loop (Table 3, Figure 6).

Transfer RNAs and Ribosomal RNAs
The 12S ribosomal RNA was 910 bp in size and located between ND4 and COIII; the 16S ribosomal RNA was 1,697 bp in size and located between tRNA Met and ND5. The base composition of two rRNAs was 35.52% A, 12.5% C, 20.02% G, and 31.95% T. The AT content of two rRNAs was 67.47%, and both AT-skew (0.053) and GC-skew (0.231) were positive. The two tRNA encoding genes (tRNA Met and tRNA Trp ) were 72 bp and 69 bp in size, respectively. The base composition of two tRNAs was 32.39% A, 19.01% C, 22.54% G, and 26.06% T. The AT content of two tRNAs was 58.45%, and both AT-skew (0.108) and GCskew (0.085) were also positive. Both of the two tRNAs were folded into a typical cloverleaf structure, which included an amino acid accept arm, D loop, anticodon loop, and TψC loop (Table 3, Figure 6).

Phylogenetic Analyses
In this study, the 13 PCGs in mitochondrial genomes of H. bowerbanki were tandem and established a phylogenetic relationship of Scleractinia. Numbers above the ML tree branches indicated bootstrap percentages (Figure 7). There were three distinct clades ("Complex," "Robust," and "Basal") of Scleractinia in the tree; the ML topology tree showed that H. bowerbanki belonged to the "Robust" clade and clustered together with two other species in the family Lobophylliidae based on 13 PCGs. Homophyllia bowerbanki and Sclerophyllia maxima were once placed in the genus Acanthastrea, while a molecular method based on several tandem nuclear and mitochondrial DNA has helped to clarify their taxonomic classification [9,10,29,30]. As only fewer than a tenth of mitogenomes of scleractinians can be obtained through NCBI to date, more mitogenomes of other Scleractinia species should be sequenced. Furthermore, the continuous reduction of costs of NGS technologies can facilitate further studies on stony coral evolutionary and phylogenetic relationships and taxonomic classification.

Phylogenetic Analyses
In this study, the 13 PCGs in mitochondrial genomes of H. bowerbanki were tandem and established a phylogenetic relationship of Scleractinia. Numbers above the ML tree branches indicated bootstrap percentages (Figure 7). There were three distinct clades ("Complex," "Robust," and "Basal") of Scleractinia in the tree; the ML topology tree showed that H. bowerbanki belonged to the "Robust" clade and clustered together with two other species in the family Lobophylliidae based on 13 PCGs. H. bowerbanki and S. maxima were once placed in the genus Acanthastrea, while a molecular method based on several tandem nuclear and mitochondrial DNA has helped to clarify their taxonomic classification [9,10,29,30]. As only fewer than a tenth of mitogenomes of scleractinians can be obtained through NCBI to date, more mitogenomes of other Scleractinia species should be sequenced. Furthermore, the continuous reduction of costs of NGS technologies can facilitate further studies on stony coral evolutionary and phylogenetic relationships and taxonomic classification. Genes 2023, 14, x FOR PEER REVIEW 9 of 11

Conclusions
Homophyllia bowerbanki is the first species of its genus for which the mitogenome was sequenced. This mitogenome was 18,154 bp in size and included two transfer RNA genes (tRNAs), 13 protein-coding genes (PCGs), and two ribosomal RNA genes (rRNAs). It showed a similar gene structure and gene order to other typical scleractinians. All 17 genes in the mitogenome were encoded on the H strand and the total GC content of the mitogenome was 33.86%, which showed a higher AT bias. Phylogenetic analysis showed that H. bowerbanki belonged to the "Robust" clade and clustered together with two other species in the family Lobophylliidae based on 13 PCGs. The mitogenome can provide significant molecular information to clarify the evolutionary and phylogenetic relationships

Conclusions
H. bowerbanki is the first species of its genus for which the mitogenome was sequenced. This mitogenome was 18,154 bp in size and included two transfer RNA genes (tRNAs), 13 protein-coding genes (PCGs), and two ribosomal RNA genes (rRNAs). It showed a similar gene structure and gene order to other typical scleractinians. All 17 genes in the mitogenome were encoded on the H strand and the total GC content of the mitogenome was 33.86%, which showed a higher AT bias. Phylogenetic analysis showed that H. bowerbanki belonged to the "Robust" clade and clustered together with two other species in the family Lobophylliidae based on 13 PCGs. The mitogenome can provide significant molecular information to clarify the evolutionary and phylogenetic relationships among stony corals and to facilitate their taxonomic classification; it can also support coral species monitoring and conservation efforts.
Author Contributions: J.X. and W.N. designed this experiment and B.C., Z.X., F.S. and Z.J. took part in investigation, sample processing, and data analysis. The original draft of the manuscript was written by P.T. and W.W. J.X. and W.N. contributed to the revision of the manuscript. All authors have read and agreed to the published version of the manuscript.