The complete mitochondrial genome of Plesiastrea versipora (Scleractinia, Plesiastreidae) sheds light on its phylogeny and taxonomy of the family Plesiastreidae

The genus Plesiastrea used to be a member of the traditional family Faviidae, falling into the challenging ‘Bigmessidae’ clade, and was re-established until recent molecular phylogenies published. The entire mitogenome of the symbiotic coral Plesiastrea versipora (Lamarck, 1816), the type species of the family Plesiastreidae, was sequenced. The length of the mitochondrial genome is 15,320 bp and it includes thirteen protein-coding genes (PCGs), two rRNAs and two tRNAs. The nucleotide composition of GC is 32%. We perform phylogenetic reconstruction based on maximum likelihood (ML) and Bayesian analysis(BI) using all PCGs. Our result indicates that P. versipora clusters closely with species which belong to Mussidae, Merulinidae and Lobophylliidae. Our phylogenetic analyses provide solid evidence for phylogenetic placement of P. versipora and the evolutionary relationships among different families within the traditional robust clade of Scleractinia. In addition, the mitogenome data provide useful information for further molecular systematic investigations on Plesiastreidae as well as conservation biology research of P. versipora.


Introduction
By integrating genetic data and new morphological features, modern coral taxonomy has been greatly developed (Kitahara et al., 2016;Richards et al., 2019;Oku et al., 2019). Conventional morphology-based phylogenetic classification does not show all the phylogenetic relationships in Scleractinia groups, which accordingly has resulted in ambiguity about the taxonomic position of these groups. Molecular phylogenetic studies have therefore become increasingly important to provide solid evidence to organize coral taxa among scleractinian (Benzoni et al., 2011;Huang et al., 2014;Kang et al., 2020). The advantages of mtDNA over nuclear DNA (nDNA) in phylogenetics are the asserted orthology of all genes and the small genome structure being relatively conserved (Kayal et al., 2013). Due to their intrinsic properties, the mitochondrial genome played an important role in scleractinian corals phylogeny (In-Young et al., 2020;Niu et al. 2020).
Milne Edwards and Haime (1848) originally depicted the genus Plesiastrea on the basis of a specimen from the Indian Ocean which was named Astrea versipora. Traditionally, the genus Plesiastrea was considered belonging to the family Faviidae and morphologically closely related to the genus Montastraea (Benzoni et al., 2011). However, genetic data have shown that there is no close phylogenetic relationship between them and Montastraea is polyphyletic (Huang et al., 2014). Plesiastrea versipora (Lamarck, 1816), one of the two extant species of this genus, is evolutionarily contrasting to other robust group in Scleractinia (Fukami et al., 2008). Based on the molecular phylogenetic studies, a new family Plesiastreidae (Dai and Horng, 2009) including the genera of Plesiastrea, Physogyra, Plerogyra, and Blastomussa was originally established (Dai and Horng, 2009). Benzoni et al. (2014) moved the three genera(Physogyra, Plerogyra and Blastomussa) to Incertae sedis based on the morphology and molecular data. There is only one genus Plesiastrea in the Plesiastreidae now.
The phylogenic history of the genus Plesiastrea was well summarized in Benzoni et al. (2011), together with a series of analyses on the species P. versipora based on morphological and genetic https://doi.org/10.1016/j.sjbs.2020.04.041 1319-562X/Ó 2020 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). characteristics. Here, the whole mitogenome sequence of P. versipora has been sequenced and assembled for the first time using next-generation sequence (NGS). The phylogenetic status and taxonomy of the family Plesiastreidae was analyzed based on the protein-coding genes (PCGs) of the mitogenomes.

Sampling collection and genomic DNA extraction
The specimen of P. versipora (Fig. S1) was collected by SCUBA diving from Daya Bay in Guangdong Province, China. Specimen was determined according to identification guides (Chan et al., 2005;Dai and Horng, 2009). DNA extraction was carried out according to the manual of the Qiagen's DNeasy Tissue Kit.

Genome sequencing and analyses
The next generation sequencing was in accordance with the protocol (Niu et al. 2020). Purity and concentration of PCR products were tested with NanoDrop 2000 and Qubit 2.0 fluorometer. A total of 2 mg DNA after the quality control steps were fragmented to~550 bp by M220 system. DNA fragment's size proportion was quantified with the Agilent Bioanalyzer 2100. According to instruction manual of manufacturer's, library for MiSeq was prepared by TruSeq DNA PCR-free Sample Preparation Kit. Concentration of the library was confirmed by real-time qPCR. A de novo assembly for raw reads was performed by using Geneious V9, to yield a single stranded, circular configuration of entire mitochondrial genome.

Mitochondrial genome annotation and analyses
The assembled mitogenome was preliminarily annotated with MITOS (Bernt et al., 2013) web servers and DOGMA (Wyman et al., 2004). Genes annotation of protein-coding and rRNA refer to homologous genes of other available scleractinian mitogenomes. Annotation of the tRNA genes were conducted with tRNA scan-SE. MEGA 7.0 (Kumar et al., 2016;Gao et al., 2017aGao et al., , 2017bLiang et al., 2018;Wang et al., 2017) was used for statistical analysis of nucleotide frequencies and codon usage. AT-skew and GC-skew analyses were used to indicate the nucleotide composition.

Phylogenetic analyses
To verify phylogenetic relationships, BI (Bayesian inference) and ML (maximum likelihood) were performed to reconstruct a tree based on the amino acid alignment of 13 PCGs using MEGA7.0. The entire mitogenome sequences of P. versipora and additional 15 representative robust clade species of scleractinian (Table S1) were used together for phylogenetic analysis. Three species of complex clade of Scleractinia including Acropora aspera, Agaricia humilis and Galaxea fascicularis were used as the outgroups (Table S1). 1000 bootstrap replicates were performed to estimate the node reliability for ML analysis. 4 chains (1 cold and 3 heated chains) were set to run at the same time for 10 million generations for the BI procedure. Each set was sampled every 100 generations after the initial 25% run were abandoned, then the consensus tree was obtained from the remaining samples.

General characteristics of the mitochondrial genome
The length of the mitochondrial genome sequence obtained from P. versipora is 15,320 bp (GenBank accession number: MH025639) including unique 13 PCGs, 2 rRNA and 2 tRNA genes (tRNA Met , tRNA Trp ) (Table 1, Fig. 1). Actually, P. versipora had the shortest mitogenome excepting Astrangia poculata (14,853 bp, NC008161) in all sequenced Scleractinia species. The variation in intergenic nucleotides led to the length differences. The base composition is A 25%, C 12.1%, G 19.9% and T 43%, The A + T content (68%) is distinctly higher than the GC content (32%) ( Table 2). The lowest level of base in the mitochondrial genome is C (Fig. S2). The mitogenome of P. versipora doesn't provide special construction; its gene feature, number and arrangement are same as most of other available hard coral mitochondrial genomes (Niu et al., 2020). According to the results of GC-/ AT-skews analysis (Fig. S3), the absolute value of GC-skew is smaller than AT-skew.

Protein-coding genes and codon usages
The overall length of all PCGs was 11,556 bp, with base composition of A 21.9%, C 12.2%, G 20% and T 45.9%. The intron insertion of ND5 was 9,396 bp. Most of the PCGs started with ATG. in addition to COI and ND2starting with ATT. and Cyt b starting with TTA. Four of the 13 PCGs have the conventional stop codons TAG, 9 PCGs have the stop codons TAA. Among thirteen PCGs, the longest and the shortest were ND5 gene (1,815 bp) and ATP8 gene (198 bp), respectively. Among 3852 codons for twenty amino acids, use frequency of codons for L, V, F, G and S accounting for 53.12% was higher. Nonpolar amino acid (A, G, I, L, M, F, V, Y, W) was the maximum, accounting for 67.86%, followed by polar amino acid (C, N, P, Q,S, T) and polarity charged amino acids (D, E, H,K, R),accounting for 20.56% and 11.24% (Fig. S4).

Ribosomal and transfer RNA genes
The genes which encode 16S rRNA and 12 rRNA were 1,702 bp and 910 bp in size, respectively. The length of total rRNA was 2,612 bp, and its base composition was A 36%, T 32.7%, G 19.6% and C 11.7% . The two transfer RNA length were 70 bp tRNA Trp and 72 bp for tRNA Met respectively. They included amino acid accept arm, anticodon arm, TwC arm and DHU arm (Fig. S5), which is the classic cloverleaf structure.

Phylogenetic analyses
Both of BI and ML tree, which included the newly sequenced mitogenomes of P. versipora and other 15 robust species and three outgroup taxa retrieved from GenBank, were done based on nucleotide data. The results of the two analyses gave similar tree topologies (Fig. 2). The resultant topology supported that P. versipora clusters closely with species which belong to Mussidae, Merulinidae and Lobophylliidae. Then Oculinidae, Pocilloporidae and Caryophylliidae clustered into the main clade successively. We can easily get the relationship information among different families through phylogenetic tree. Indeed, the combination of molecular markers and classical morphological characteristics can help enhance modern taxonomic system within scleractinian corals (Kitahara et al., 2016).

Conclusions
We have sequenced the whole mitogenome of P. versipora for the first time, one of the two extant species of the family Plesiastreidae (Hoeksema and Cairns, 2020). The mitogenome of P. versipora is 15,320 bp in size. The composition in gene order, low GC content and size is similar to other mitochondrial genomes which have been sequenced in Scleractinia. The phylogenetic analysis based on the entire mitogenomes of P. versipora and related species shed light on its phylogeny and taxonomy of the family Plesiastreidae. Lastly, the whole mitochondrial genome of the P. versipora obtained in the study provides important and essential molecular data for scleractinian corals phylogenetic and evolutionary analysis in the further.