﻿Complete mitochondrial genome sequences of Physogyralichtensteini (Milne Edwards & Haime, 1851) and Plerogyrasinuosa (Dana, 1846) (Scleractinia, Plerogyridae): characterisation and phylogenetic analysis

﻿Abstract In this study, the whole mitochondrial genomes of Physogyralichtensteini and Plerogyrasinuosa have been sequenced for the first time. The length of their assembled mitogenome sequences were 17,286 bp and 17,586 bp, respectively, both including 13 protein-coding genes, two tRNAs, and two rRNAs. Their mitogenomes offered no distinct structure and their gene order were the same as other typical scleractinians. Based on 13 protein-coding genes, a maximum likelihood phylogenetic analysis showed that Physogyralichtensteini and Plerogyrasinuosa are clustered in the family Plerogyridae, which belongs to the “Robust” clade. The 13 tandem mitogenome PCG sequences used in this research can provide important molecular information to clarify the evolutionary relationships amongst stony corals, especially at the family level. On the other hand, more advanced markers and more species need to be used in the future to confirm the evolutionary relationships of all the scleractinians.


Introduction
The order Scleractinia (Cnidaria, Anthozoa), including numerous reef-building coral species, is important as the constructors of coral reefs as an ecosystem. The mitogenome data of cnidarians contain important phylogenetic information for understanding their evolutionary history (Kayal et al. 2013). Single-or multiple-gene analysis of mitochondrial genes have already been used to infer phylogenetic relationships amongst scleractinians (Kitahara et al. 2016;Arrigoni et al. 2020).
In Scleractinia, three main clades have been defined based on molecular analyses, "Complex", "Robust", and "Basal" (Romano and Palumbi 1996;Kitahara et al. 2010;Stolarski et al. 2011). Plerogyridae Rowlett, 2020 is a small family of the "Robust" clade of corals containing four genera (Plerogyra Milne Edwards & Haime, 1848, Physogyra Quelch, 1884, Blastomussa Wells, 1968, and Nemenzophyllia Hodgson & Ross, 1982) (see Hoeksema and Cairns 2022), all from the Indo-West Pacific. Previously, the genera Plerogyra and Physogyra were placed in the Euphylliidae of the "Complex" group (Fukami et al. 2008), the family Plesiastreidae of the the "Robust" group (Dai and Horng 2009), and in Scleractinia incertae sedis (Budd et al. 2012;Benzoni et al. 2014;Waheed et al. 2015), but recently Rowlett (2020) placed them in the family Plerogyridae. Physogyra has one recently accepted species and four uncertain species, whereas Plerogyra has seven accepted species (Hoeksema and Cairns 2022). Through molecular analyses of two mitochondrial genes, Fukami et al. (2008) found that Plerogyra and Physogyra do not belong to the "Complex" clade of Scleractinia but to the "Robust" clade. Morphologically, plerogyrid species are characterised by mantle vesicles that are diurnally visible when the tentacles are partially retracted (Benzoni et al. 2014).
Physogyra lichtensteini (Milne Edwards & Haime, 1851) and Plerogyra sinuosa (Dana, 1846) are covered by round to irregularly bifurcating vesicles during the day and active, open polyps at night (Veron 2000;Benzoni et al. 2014). Physogyra lichtensteini is common in lagoons and reef slopes to deeper than 38 m (De Palmas et al. 2021). Colonies of Physogyra lichtensteini are generally massive. They are meandroid, with short, widely separated valleys interconnected with a light, blistery coenosteum. Septa are large, solid, smooth-edged, exsert, and widely spaced. Walls are solid. Columellae are absent. Tentacles are extended only at night. The septal vesicles of Physogyra are considerably smaller and more numerous when compared to the closely related Plerogyra. The colour of Physogyra lichtensteini is pale grey or sometimes dull green (Veron 2000), while in Plerogyra sinuosa, the colonies are flabello-meandroid with valleys somewhat connected by a light, blistery coenosteum. Living parts of colonies are sometimes separated by dead basal parts. Vesicles are the size of grapes and usually have the shape of grapes but may be tubular, bifurcated, or irregular, depending primarily upon the state of inflation. The colour of Plerogyra sinuosa is cream or bluish grey. Plerogyra sinuosa is a prominent species and reasonably common in protected reefs, and it is easily recognised by its bubbly appearance (Veron 2000;Rowlett 2020).
In this research, the complete mitochondrial genomes of Physogyra lichtensteini and Plerogyra sinuosa are sequenced and their genome structures are analysed for the first time. The phylogenetic analyses of these two species, based on 13 protein coding genes (PCGs) of the mitogenome, in combination with another 42 species of other genera of Scleractinia and two species of Corallimorphidae Hertwig 1882 (order Corallimorpharia) as outgroups, because they are closely related to Scleractinia in evolutionary terms. This helps determine their phylogenetic status and facilitate further study on stony coral evolutionary and phylogenetic relationships.

Sample collection and genomic DNA extraction
Samples of Physogyra lichtensteini (Fig. 1A, C) and Plerogyra sinuosa (Fig. 1B, D) were obtained in 2019 from a coral mariculture company in China, which originally obtained mother stock from Negeri Sabah of Malaysia. Their specimens were maintained in our Coral Sample Repository with the codes 20191207-J2 (Physogyra lichtensteini) and 20191207-Y1 (Plerogyra sinuosa). Total 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 their genomic DNA, and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA) was used to measure their genomic DNA concentration.

Mitogenome sequencing, annotation, and analyses
In this study, two methods were used to obtain the mitogenomes of Physogyra lichtensteini and Plerogyra sinuosa, respectively. The complete mitogenome of Plerogyra sinuosa was obtained through a PCR approach using the primer pairs designed by Lin et al. (2011). The complete mitogenome of Physogyra lichtensteini was obtained from high-throughput sequencing with a HiSeqX Ten platform (Illumina, San Diego, CA, USA) with a paired-end 150 bp approach according to Tian et al. (2021), and a total of 102,074 of 116,026,504 raw reads (approximately 0.09%) were de novo assembled to produce a single, circular form of the complete mitogenome with an average coverage of 892 X. The circularised contig of these two species were then submitted to the MITOS WebServer (Bernt et al. 2013; http://mitos.bioinf.uni-leipzig.de/index.py) for preliminary mitochondrial genome annotation. We identified and annotated their 13 PCGs and two rRNA genes by alignments of homologous mitogenomes from other scleractinians that had been recovered through BLAST searches in NCBI, and we also validated the tRNA genes using ARWEN (Laslett and Canbäck 2008). The genome structures were mapped using the online CGView Server (Stothard and Wishart 2005; https://proksee.ca/). Base composition, nucleotide frequencies, and codon usage were obtained through MEGA7 (Kumar et al. 2016). The skewing of the nucleotide composition was measured in terms of AT skews and GC skews according to the following formulae: AT skew = (A -T) / (A + T) and GC skew = (G -C) / (G + C) (Perna and Kocher 1995). The mitogenome sequences of Physogyra lichtensteini and Plerogyra sinuosa are available in GenBank under accession numbers MW970409 and MW936598.

Phylogenetic analyses
The phylogenetic positions of Physogyra lichtensteini and Plerogyra sinuosa were inferred using 13 tandem mitogenome PCG sequences (ND5 + ND1 + Cytb + ND2 + ND6 + ATP6 + ND4 + COIII + COII + ND4L + ND3 + ATP8 + COI) (Tian et al. 2021) together with another 42 species of other genera of Scleractinia and two species belonging to two genera of Corallimorpharia that we obtained from GenBank (Table 1). We used MEGA 7 to select the best-fitting model based on the Akaike Information Criterion (AIC) and then constructed a maximum likelihood (ML) tree with 500 bootstrap replicates.

Characteristics and composition of mitogenome
The mitochondrial genome sizes of Physogyra lichtensteini and Plerogyra sinuosa are 17,286 bp and 17,586 bp, respectively, both including 13 PCGs, two tRNA (tRNA Met , tRNA Trp ), and two rRNA genes (Tables 2, 3; Fig. 2). Their mitogenomes offer no distinct structure and their gene orders are same as other typical scleractinians (Lin et al. 2012). All genes are encoded on the H-strand. The base composition of the complete mitogenome is 24.75% A, 13.32% C, 21.75% G, and 40.17% T for Physogyra lichtensteini, and 24.87% A, 13.16% C, 22.01% G, and 39.96% T for Plerogyra sinuosa. Both species show a higher AT content than GC content ( Fig. 3; Table 4).

Protein-coding genes
The lengths of all 13 protein-coding genes (PCGs) are 11,598 bp and 11,574 bp for Physogyra lichtensteini and Plerogyra sinuosa, respectively. In both species, the ND5 gene and COI gene have intron insertions, and the start and stop codon of all 13 PCGs are exactly the same except for the COI gene. Their shortest gene is in both ATP8, and their longest gene is ND5 (Tables 2, 3). According to the AT-skew and GC-skew analyses     ( Fig. 4), both PCGs of Physogyra lichtensteini and Plerogyra sinuosa show a stronger nucleotide asymmetry, with AT skew higher than GC skew. Amongst L, F, V, G, and S in Physogyra lichtensteini and Plerogyra sinuosa, codon use frequency was higher, accounting for 53.5% and 53.4%, respectively. In their 20 amino acids, the majority are non-polar amino acids, and a minority are polarity-charged amino acids (Fig. 5).
rRNA and tRNA genes The encoding genes 12S and 16S rRNA in Physogyra lichtensteini are 911 bp and 1,698 bp in size, and in Plerogyra sinuosa they are 907 bp and 1,698 bp in size. The base composition of rRNA in Physogyra lichtensteini was 35.19% A, 12.5% C, 20.62% G, and 31.7% T, and in Plerogyra sinuosa it was 35.12% A, 12.48% C, 20.73% G, and 31.67% T. The two tRNA encoding genes, tRNA Met (72 bp) and tRNA Trp (69 bp), are exactly the same in Physogyra lichtensteini and Plerogyra sinuosa (Tables 2, 3). They are folded into the classic cloverleaf structure which includes an amino acid accept arm, DHU loop, anticodon loop, and TψC loop (Fig. 6).

Phylogenetic analyses
There are three distinct clades of Scleractinia in our ML tree, including "Complex", "Robust", and "Basal" clade. The ML topology tree of all the 47 species shows that Physogyra lichtensteini and Plerogyra sinuosa are clustered in family Plerogyridae which belong to the "Robust" clade with high bootstrap support (Fig. 7). Our finding is consistent with the results of Fukami et al. (2008) who placed Plerogyra and Physogyra in the "Robust" clade. From the ML tree we also find that Physogyra lichtensteini and Plerogyra sinuosa are a sister group with Astrangia poculata, which belongs to the family Astrangiidae Milne Edwards & Haime, 1857. Our MT tree of the Plerogyridae shows the same classification as used by Rowlett (2020). Single-or multi-gene analyses of mitochondrial genes have already been used to infer phylogenetic relationships amongst scleractinians (Kitahara et al. 2016;Arrigoni et al. 2020). The 13 tandem mitogenome PCG sequences we used in this research can provide important molecular information to understand    the evolutionary relationships amongst stony corals, especially at the family level. As fewer than a tenth of stony coral species have been sequenced at this time, more mitogenomes of other scleractinians are necessary before accurate family-level evolutionary relationships can be reconstructed. In the future, more advanced markers and more species should be used to confirm the evolutionary relationships among all scleractinians.

Conclusions
The complete mitochondrial genomes of Physogyra lichtensteini and Plerogyra sinuosa were sequenced for the first time. Their mitogenomes show a similar gene order and composition with other typical Scleractinia. Our phylogenetic analysis of Physogyra lichtensteini and Plerogyra sinuosa, based on their 13 tandem mitochondrial protein-coding genes and including another 42 species of Scleractinia and two species of Corallimorpharia, help us to understand the evolutionary relationships amongst stony corals and facilitate further studies on stony coral evolutionary and phylogenetic relationships.