The complete mitochondrial genome of Indo-Pacific soft coral Sinularia acuta Manuputty and van Ofwegen, 2007 (Octocorallia: Alcyonacea)

Abstract The complete mitochondrial genome (mitogenome) of the soft coral Sinularia acuta Manuputty and van Ofwegen, 2007 was sequenced and annotated using Illumina next-generation sequencing (NGS). The mitogenome of S. acuta was 18,730 bp in length and consisted of 14 protein-coding genes (PCGs), two ribosomal RNA genes (rRNA), and only one transfer RNA gene (tRNA-Met). The base composition was 30.18% A, 16.46% C, 19.35% G, and 34.00% T, with a total A + T content of 64.19%. The phylogenetic analysis demonstrated a close evolutionary relationship among Sinularia acuta, Sinularia penghuensis, and Sinularia maxima.


Introduction
The soft coral genus Sinularia May 1898, also called short fingered soft coral, belongs to Alcyonacea: Alcyoniidae. The species of Sinularia are distributed in extensive environmental fields in Indo-Pacific open water, from shallow to deep reefs. They have various growth patterns and different sizes of colonies (Fabricius and Alderslade 2001). Sinularia acuta Manuputty and van Ofwegen, 2007 was described by Manuputty and van Ofwegen from Moluccas (Indonesia) in 2007. The South China Sea is one of the habitats of this species in Southeast Asia (Benayahu et al. 2012(Benayahu et al. , 2018. The soft coral Sinularia, with more than 170 described species, is a large genus in Octocorallia; therefore, its mitochondrial genome information can draw a clear evolutionary relationship among species, but few mitochondrial genomes have been sequenced Chen et al. 2019;Shen et al. 2021). In this study, we sequenced and annotated the complete mitochondrial genome of S. acuta (GenBank no. MW987591), and a phylogenetic analysis was performed to investigate its relationships with other Sinularia species.

Materials and methods
To collect the sample, permission was obtained from the Hainan Province government (Department of Science and Technology, reference number ZDYF2019154). Additionally, the specimen collection and experimental protocol of this study were approved by the Ethical Review Department of Science and Technology of Hainan Province (China) (reference number ZDKJ2019011-03-02). A live specimen of S. acuta was collected in West Island (Sanya, Hainan Province, China; 18 14 0 5.93 00 N, 109 22 0 46.46 00 E), and its taxonomic status was confirmed based on the morphology of sclerites following McFadden et al. (2009). The specimen and its DNA were deposited at the Hainan Tropical Ocean University Museum of Zoology (specimen voucher number: 0008-Sp; DNA ID number: 0008-D; Chaojie Yang: duanduan1986@outlook.com). Figure 1 shows the specimen reference image and morphology of sclerites.
Total genomic DNA was extracted from 30 mg of tissue using Genomic Animal DNA Isolation (Sangon Biotech Co., Kit NO. B518221, Shanghai, China) (Asem et al. 2021). A pairedend genomic library was arranged (10 GB; two pair reads: 150 bp) utilizing the Illumina HiSeq X-ten. FastQC software was used to control the quality of the reads (Andrews 2010). Information on classification of raw reads and quality score distribution along reads were shown in Figure S1. The complete mitochondrial genome of Sinularia ceramensis (NC_044122) was chosen to perform the de novo assemblies by Geneious v.9.1 (Kearse et al. 2012). The GenomeVx online platform (http://wolfe.ucd.ie/GenomeVx/) was used to draw the mitogenomic circular map of S. acuta (Figure 2). The ARWEN online platform was used to determine the position of tRNA-Met (http://130.235.46.10/ARWEN/). BioEdit software was employed to annotate the positions of protein-coding genes and ribosomal RNA genes using the reference mitogenome (Hall 1999). Additionally, the position and orientation of all PCGs were reconsidered by translation of amino acid sequences (ExPASy online tool: https://web.expasy.org/translate/) and the position of both the start codon and stop codon in each PCG. Maximum likelihood (ML) phylogenetic analysis was performed utilizing the software MEGA X (Kumar et al. 2018) with 1000 bootstrap replicates and a GTR model, which was determined by jmodeltest v.2.1.10 (Darriba et al. 2012). The concatenated sequences of 14 PCGs were used to draw a phylogenetic tree.

Results
Mean depth information for the assembly of complete mitochondrial genome of S. acuta was represented in Figure S2. The mitogenome of S. acuta was 18,730 bp in length. The overall base compositions were 30.18% A, 16.46% C, 19.35% G, and 34.00% T, with a total A þ T content of 64.19%. Similar to the other published Alcyoniidae mitogenomes, 17 genes were detected in the mitogenome of S. acuta, including 14 PCGs, two rRNAs, and one tRNA (tRNA-Met). Ten PCGs (MutS, cox1, cytb, nad1, nad2, nad3, nad4, nad4L, nad5, and nad6) and two rRNAs (12S and 16S) were encoded on the heavy chain. The other four PCGs (cox2, cox3, atp6, and atp8) and tRNA-Met were located on the light chain.
All PCGs used ATG as the initiation codon. Five PCGs, including nad4L, MutS, nad4, atp6, and atp8, had TAA as a stop codon, while cox1 consisted of a non-complete codon (TÀ). There were 16 intergenic spacers in the present mitogenome. The major non-coding region was between cox2 and cox1 (112 bp), and a 13 bp overlap was detected between nad2 and nad5.

Discussion
For the first time, in the present study, we reported the complete mitochondrial genome mitogenomes of Sinularia acuta which has been successfully sequenced, assembled, and annotated. The constructed phylogenetic tree revealed that Sinularia penghuensis, Sinularia maxima, and S. acuta were located in the same clade, while Sinularia ceramensis and Sinularia peculiaris were clustered together in another clade (Figure 3).

Conclusion
Generally, taxonomy and phylogenetic of genus Sinularia are problematic. We reported the first mitogenomes assembly and annotation of Sinularia acuta. The complete mitochondrial genome presented here could be utilized as a genomic  data for further studies on phylogenetic analysis, evolutionary biology, and population genetics.