Complete mitochondrial DNA sequence of the Psammocora profundacella (Scleractinia, Psammocoridae): mitogenome characterisation and phylogenetic implications

Abstract Complete mitochondrial DNA sequence data have played a significant role in phylogenetic and evolutionary studies of scleractinian corals. In this study, the complete mitogenome of Psammocora profundacella Gardiner, 1898, collected from Guangdong Province, China, was sequenced by next-generation sequencing for the first time. Psammocora profundacella is the first species for which a mitogenome has been sequenced in the family Psammocoridae. The length of its assembled mitogenome sequence was 16,274 bp, including 13 protein-coding genes, two tRNAs and two rRNAs. Its gene content and gene order were consistent with the other Scleractinia species. All genes were encoded on the H strand and the GC content of the mitochondrial genome was 30.49%. Gene content and order were consistent with the other Scleractinia species. Based on 13 protein-coding genes, Maximum Likelihood phylogenetic analysis showed that P. profundacella belongs to the “Robust” clade. Mitochondrial genome data provide important molecular information for understanding the phylogeny of stony corals. More variable markers and additional species should be sequenced to confirm the evolutionary relationships of Scleractinia in the future.


Introduction
Scleractinia (Cnidaria, Anthozoa), including numerous hermatypic corals, have always been highlighted by ecologists and taxonomists for their important role in maintaining the balance of ecosystems in shallow tropical and subtropical seas (Fukami et al. 2000). Traditionally, reef-building corals were described, using morphological characteristics of their skeletons, whereas the skeletal structures, especially of colonial corals, can be extremely variable due to environment-induced phenotypic plasticity. In addition, it was difficult to discriminate cryptic species and species that have arisen via introgression or hybridisation without genetic and ecological data (Niu et al. 2018,Combosch and Vollmer 2015, Richards and Hobbs 2015. Molecular technologies have changed the taxonomic landscape and the integration of morphological and molecular analyses have promoted a more rigorous classification and precise phylogeny of stony corals (Kitahara et al. 2016). Though mitochondrial genes have been known for producing erroneous phylogenetic inferences amongst anthozoans, mitochondrial phylogenies of scleractinians may still provide insight into mitochondrial and gene evolution and may provide preliminary insights into evolutionary relationships (Lin et al. 2014).
Cnidarian mitogenome data contain important phylogenetic information for understanding its evolutionary history (Kayal et al. 2013). Single or multiple gene analysis of mitochondrial genes have been used to infer phylogenetic relationships amongst scleractinians (Arrigoni et al. 2020, Kitahara et al. 2016. Data of complete mitochondrial genomes have also become important sources for assessing scleractinian phylogenies due to the declining cost of next-generation sequencing (NGS) technologies (Jex et al. 2010, Niu et al. 2018, Schuster 2008. Nevertheless, there are more than 1600 species, whereas only approximately 100 complete mitogenomes of Scleractinia species have been collected in NCBI (https://www.ncbi.nlm. nih.gov/) to date (Hoeksema and Cairns 2020).
Psammocora profundacella Gardiner, 1898 is a species of small-polyp stony coral with grey, brown, tan or cream colours, belonging to the family Psammocoridae (Chevalier and Beauvais 1987). Its colonies are sub-massive or encrusting, corallites are in short valleys and walls are rounded, although they may have a central ridge. Petaloid septa and enclosed petaloid septa are always present and rice-grain shaped , Benzoni et al. 2007. There is only one genus, Psammocora Dana 1846, in the Psammocoridae and is widely distributed in the Indo-Pacific. It had been placed in the family Siderastreidae until Benzoni et al. (2007) published strong evidence to distinguish Psammocora from other siderastreids and resurrected Psammocoridae to accommodate this genus.  revised all 24 species in Psammocora, reducing the number to seven species using a combination of morphological and molecular analyses; subsequently, Randall (2015) identified a new species, Psammocora eldredgei.
In the present research, the complete mitochondrial genome of P. profundacella was sequenced using NGS and its genome structure was analysed for the first time. Simultaneously, it was also the first species within the family Psammocoridae for which the mitogenome had been sequenced. The phylogenetic analyses of P. profundacella, based on protein coding genes (PCGs) of the mitogenome, combined with 81 other scleractinians, will help determine its taxonomic status and facilitate further study on stony coral evolutionary and phylogenetic relationships.

Sample collection and genomic DNA extraction
A specimen of P. profundacella ( Fig. 1) was collected from Yangmeikeng of Daya Bay in Guangdong Province and it was kept in our Coral Sample Repository with a special code, 20190718-D17. Identification was conducted according to the description of : calices are between 1.4 and 1.7 mm in diameter, the columella is made of one central process surrounded by 4-6 granules positioned at the inner end of the septa and up to 6 septa reaching the fossa are petaloid. Complete genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Shanghai, China). Electrophoresis with 1% agarose gel was used to measure the integrity of the genomic DNA and the spectrophotometer NanoDrop 2000 (Thermo Scientific, USA) was used to measure the genomic DNA concentration.

Genome sequence assembly and analyses
After DNA extraction and quality detection, the sequencing library was produced using the Illumina Truseq™ DNA Sample Preparation Kit (Illumina, San Diego, USA) according to the manufacturer's recommendations. Five µg of double-stranded DNA was sheared to ~ 300 bp using a M200 Focused-ultrasonicator (Covaris, Woburn, MA, USA). The prepared library was loaded on the Illumina HiSeq X Ten platform for PE 2 × 150 bp sequencing. The quality and quantity of data were assessed by FastQC (Andrews 2010). Filtered clean reads were obtained by removing reads containing poly-N regions, adapters and by eliminating low-quality reads using fastp (Chen et al. 2018). The median of insert sizes and average read length were used to reconstruct the mitochondrial genome via NOVOPlasty 3.8.3 (Nicolas et al. 2016). A total of 60,910 of 128,526,740 raw reads (approximately 0.05%) were de novo assembled to produce the mitogenome with the guidance of seed sequence and the average coverage was 565×.

Mitogenome analyses
The circularised contig was submitted to MITOS (Bernt et al. 2013) WebServer (http://mitos.bioinf.uni-leipzig.de/index.py) for preliminary mitochondrial genome annotation. We then identified and annotated all 13 PCGs and two rRNA genes by alignments of homologous mitogenomes with other scleractinians that had been reported through BLAST searches in NCBI. All the PCG codon usage and nucleotide frequencies were obtained through Molecular Evolutionary Genetics Analysis software MEGA7 (Kumar et al. 2016). tRNA genes were identified by comparing the results predicted by tRNAscan-SE 2.0, based on its unique cloverleaf secondary structure information and we also validated the result using ARWEN (Laslett andCanbäck 2008, Lowe andChan 2016).

Characteristics and composition of mitogenome
The mitochondrial genome size of P. profundacella (GenBank accession number: MT576637) was 16,274 bp, including 13 PCGs, 2 tRNA (tRNA , tRNA ) and 2 rRNA genes (see Table 1, Fig. 2). The mitogenome of P. profundacella offered no distinct structure and its gene order, gene identity and gene number were the same as those of published stony coral mitogenomes (Wang et al. 2013). The base composition of complete mitogenome was 26.34% A, 11.32% C, 19.17 G and 43.17% T, which showed a higher AT content (69.51%) than GC content (30.49%) (see Fig. 3, Table 2). In addition, all genes remained encoded on the H-strand. Organisation of the mitochondrial genome of P. profundacella.
Nucleotide composition in different regions of mitochondrial genome of P. profundacella.

Figure 2.
The mitochondrial genome of P. profundacella. Gene order and positions are shown. COI, COII and COIII refer to the cytochrome oxidase subunits, Cyt b refers to cytochrome b and ND1-ND6 refers to NADH dehydrogenase components. All the genes are encoded on the Hstrand.

Protein-coding genes and its codon usages
The length of all 13 protein-coding genes sequence was 11,511 bp, with base composition of 23.6%, 11.2%, 18.4% and 46.8% for A, C, G and T, respectively. ND5 gene had an intron insertion of 9,549 bp. The start codon of all PCGs used ATG, except for Cyt b using TTA and ND2 using ATT. Three PCGs (COII, ND4 and ND5) terminated with TAG, the other ten PCGs (ATP6, Cyt b, ND1, ND2, ND3, ND4L, ND6, ATP8, COII and COIII) stopping with TAA. The shortest gene was ATP8 (195 bp) and the longest gene was ND5 (1,812 bp). The intergenic region between cytb and ND2 was 208 bp (see Table 1). According to the results of AT-skew and GC-skew analysis (Fig. 4), all PCGs showed a stronger nucleotide asymmetry, with AT-skew higher than GC-skew. Amongst L, F, V, G and S, codon use frequency was higher, accounting for 52.5% of a total of 3837 codons. Amongst the 20 amino acids, the majority were were non-polar amino acids which accounted for 68.0%; the minority were polarity-charged amino acids accounting for 11.1% and the remainder were polar amino acids which accounted for 20.5% (Fig. 5).  The PCGs' AT-skew and GC-skew of mitochondrial genome of P. profundacella.

rRNA and tRNA genes
The encoding genes 12S and 16S rRNA in P. profundacella were 906 bp and 1,704 bp in size, respectively. Both the two rRNAs' base composition was 37.32% A, 10.15% C, 18.54% G and 33.98% T. There were also two tRNA encoding genes-tRNA (72 bp) and tRNA (70 bp). They were folded into the classic cloverleaf structure which included an amino acid accept arm, DHU loop, anticodon loop and TψC loop (Fig. 6).

Phylogenetic analyses
The ML tree topology of the 82 stony corals species showed that P. profundacella belongs to the "Robust" clade and is closely related to Polycyathus chaishanensis (Caryophylliidae) with high bootstrap support (Fig. 7). These findings are consistent with the results of Lin et al. (2012), which clearly showed that Polycyathus chaishanensis is closely related to a clade comprising Psammocora, Coscinaraea, Leptastrea and Fungiidae. The mitochondrial genome data have provided important molecular information for understanding evolutionary relationships amongst stony corals, but more variable markers and additional Met Trp Figure 5.
The PCGs' codons use frequency of mitochondrial genome of P. profundacella.

Figure 6.
Putative secondary structures of two tRNA of P. profundacella.
species should be sequenced to confirm the evolutionary relationships of Scleractinia in the future.

Conclusions
The complete mitochondrial genome of P. profundacella was sequenced for the first time and it was also the first species in the family Psammocoridae whose mitogenome had been sequenced. The mitogenome of P. profundacella is 16,274 bp in size and shows similar gene order and gene composition with other scleractinian mitogenomes. The phylogenetic analysis of P. profundacella, on the basis of its mitochondrial protein-coding genes along with 81 other scleractinians, while preliminary, will help facilitate further studies on stony coral evolutionary and phylogenetic relationships.