The first complete mitochondrial genome of the Mariana Trench Freyastera benthophila (Asteroidea: Brisingida: Brisingidae) allows insights into the deep‐sea adaptive evolution of Brisingida

Abstract Starfish (phylum Echinodermata) are ecologically important and diverse members of marine ecosystems in all of the world's oceans, from the shallow water to the hadal zone. The deep sea is recognized as an extremely harsh environment on earth. In this study, we present the mitochondrial genome sequence of Mariana Trench starfish Freyastera benthophila, and this study is the first to explore in detail the mitochondrial genome of a deep‐sea member of the order Brisingida. Similar to other starfish, it contained 13 protein‐coding genes, two ribosomal RNA genes, and 22 transfer RNA genes (duplication of two tRNAs: trnL and trnS). Twenty‐two of these genes are encoded on the positive strand, while the other 15 are encoded on the negative strand. The gene arrangement was identical to those of sequenced starfish. Phylogenetic analysis showed the deep‐sea Brisingida as a sister taxon to the traditional members of the Asteriidae. Positive selection analysis indicated that five residues (8 N and 16 I in atp8, 47 D and 196 V in nad2, 599 N in nad5) were positively selected sites with high posterior probabilities. Compared these features with shallow sea starfish, we predict that variation specifically in atp8, nad2, and nad5 may play an important role in F. benthophila's adaptation to deep‐sea environment.

All 13 protein-coding genes play key roles in oxygen usage and energy metabolism (Boore, 1999;Xu et al., 2007). Because variation in mitochondrial protein-coding genes that involved in oxidative phosphorylation can directly influence metabolic performance, an increasing number of researches related to adaptive evolution of these genes have been reported (Maliarchuk, 2011;Xu et al., 2007;Yu, Wang, Ting, & Zhang, 2011;Zhou, Shen, Irwin, Shen, & Zhang, 2014).
Here, we report the complete mitogenome of the starfish Freyastera benthophila, which collected from Mariana Trench at 5,463 m depth. Freyastera benthophila exist in abyssal and is mainly distributed in the southern Pacific, eastern Pacific off California, mid-Atlantic (between Azores and Spain), the Bay of Bengal, and Biscay, ranging from 4,250 to 5,000 m depth (Downey, 1986).
The mitogenome features, organization, codon usage, and gene arrangement information were presented. The phylogenetic relationships between F. benthophila and 19 other species from Echinodermata were analyzed. To infer the deep-sea adaptive evolution, positive selection analysis of mitochondrial genes was also performed.

| Sample collection and DNA extraction
The specimen was collected at Mariana Trench, in June 2016 (10°51.0971′N, 141°57.2705′E, at 5,463 m depth). The collection was accomplished by deep-sea human occupied vehicle (HOV) "Jiao Long" during an expedition. The specimen was preserved in 95% ethanol. Total genomic DNA was extracted from ethanol-fixed tissue with tissue DNA kit (Omega Bio-Tek) and stored at −20°C.

| PCR amplification and DNA sequencing
The universal metazoan primers for mtDNA were used in PCR.
PCRs were performed with a gradient machine (Applied Biosystems Inc.

| Phylogenetic analysis
Twenty echinoderm mt genomes including the one obtained in this study were used for phylogenetic analysis. All complete mtDNA sequences vailable in GenBank are listed in Table 2. Crinoidea is generally considered as the earliest diverged group of echinoderms (Scouras & Smith, 2006). In F I G U R E 1 Mitochondrial gene map of Freyastera benthophila. All of 37 genes are encoded on the both strands. Genes for proteins and rRNAs are shown with standard abbreviation. Genes for tRNAs are designated by a single letter for the corresponding amino acid with two leucine tRNAs and two serine tRNAs differentiated by numerals Tang et al.
Yasuda et al.
The amino acid sequence from each of 13 protein-coding genes was aligned separately using Clustal ×2.0 (Larkin et al., 2007), and then, the relatively poor homologous sequence was eliminated. The aligned amino acid sequences were concatenated into a single dataset. The phylogenetic reconstruction approach was performed using neighbor joining (NJ) and maximum likelihood (ML) with MEGA 5.0 (Tamura et al., 2011). The assessment of node reliability was performed using 1,000 bootstrap replicates.

| Positive selection analysis
To evaluate the variation in selective pressure between deep-sea F. benthophila and other eight shallow sea starfish, we used a codonbased likelihood approach implemented in the CODEML program of the pamlX package (Xu & Yang, 2013;Yang, 2007). All models correct the transition/transversion rate and codon usage biases (F3 × 4). The branch model tests were used to analyze the difference of selective pressure between the deep-sea and shallow sea starfish. The "oneratio" model (model 0), "free-ratio" model (model 1), and "two-ratio" model were used in the combined dataset of 13 protein-coding genes.
Considering that positive selection may occur in some amino acids during the evolution of a protein, we used two branch site models (A and A null). Bayes empirical Bayes (Yang, Wong, & Nielsen, 2005) analysis was used to calculate the posterior probabilities of a specific codon site.

| General features
The mitogenome of the F. benthophila is a 16,175-bp circular molecule ( Figure 1)  in Acanthaster planci (Table 3). Freyastera benthophila has the smallest complete mitogenome found in Asteroidea thus far. The size of Asteroidea mitogenomes ranged from 16,524 bp in Luidia quinaria to 16,175 bp in F. benthophila (Table 3). The synteny and identity level between F. benthophila and each of the other seven starfish mitogenomes is shown in Figure 2. The lack of similarity between F. benthophila and L. quinaria is the most obvious feature in the plot.
The genome encodes 37 genes including 13 protein-coding genes A total of 22 noncoding regions were found, with the largest continuous region (284 bp, A + T = 67.25%) located between trnT and 16S. Due to its AT richness, we predict that this part is mitochondrial control region.

| Protein-coding genes
With regard to PCGs,nad4L,cob,atp6,and atp8) are encoded by the positive strand, and the remaining three (nad1, nad2, and nad6) are encoded by the negative strand.
These features have been observed in all Asteroidea mitogenomes published so far. Thirteen PCGs initiate with the standard start codon ATG. Most of PCGs terminate with the stop codon TAA (9 of 13), and three genes terminate with the stop codon TAG. Incomplete termination codon T is used by cob. However, mitogenomes often use a variety of nonstandard initiation codons (Wolstenholme, 1992). Nonstandard initiation codon GTG and incomplete termination codon TA are also used in other starfish (Table 3). The lengths of PCGs are 11,506 bp, and the A + T content is 67.15% higher than that of other Asteroidea species (Table 3).
The codon usage of F. benthophila is shown in Figure 3. Among PCGs, leucine (15.85%) and cysteine (0.99%) are the most and the least frequently used amino acids, respectively. Codons, UUA (leucine 6.67%) and ACG (threonine 0.08%), are the most and the least frequently used, respectively. We predict that the richness of A and F I G U R E 3 Codon usage in Freyastera benthophila. All codons for amino acids have been classified. Each amino acid is designated by a single letter for the corresponding codon. x-axis and y-axis represent the used times of each codon T occurrence frequency of the mitogenome caused the corresponding amino acid bias to some extent. It is obvious that the A + T content of the third codon position (74.10%) is higher than that of the first (63.43%) and second positions (63.67%).

| Ribosomal RNA and transfer RNA genes
Boundaries of both the small and the large ribosomal genes were for Asteroidea, whereas the AT contents are higher than those of other starfish (Table 3).
We analyzed the entire mitogenome sequence of F. benthophila and successfully identified 22 tRNA genes based on their potential secondary structures using the tRNAscan-SE, ARWEN, and MITOS Web server (Table 4, Supporting Information Figure S1).

| Gene arrangement
Mitochondrial gene arrangement has been demonstrated to be an effective means to solve the deep phylogenetic studies (Boore, 1999;Boore & Brown, 1998). In recent years, some research on mt gene arrangement of echinoderms has been reported (Arndt & Smith, 1998;Perseke et al., 2008Perseke et al., , 2010Scouras et al., 2004).
In this study, mitochondrial gene order of echinoderm was Echinoidea in Figure 4 (Sodergren et al., 2006). However, mitochondrial gene order has undergone significant changes in the classes of Holothuroidea, Ophiuroidea, and Crinoidea. Scouras et al. (2004) suggested that it is difficult to resolve the echinoderm phylogeny using the mitochondrial gene rearrangement.
It is interesting that mt gene order of the species in the classes of Asteroidea and Echinoidea is completely identical to each other.
If the tRNA is not considered, gene order of PCGs in species within the class Holothuroidea is also the same. This raises the questions: As these species are distributed throughout the world's oceans, why had the mt gene order not been changed and how do they evolve over time. More studies of mt genome species are needed to further investigate whether this pattern is common among starfish, sea urchins, and sea cucumbers.

| Phylogenetic analysis
The gene order and transcriptional orientation of the eight Asteroidea species are completely identical to each other, so the mt genome structures would not provide the phylogenetic information.
Thus, we performed the phylogenetic analysis using all amino acids of mt protein-coding genes ( Figure 5) (1987) recognized that Brisingida and Forcipulatida are the two orders within the Forcipulatacea and suggested that they were the most primitive asteroids (Blake, 1987(Blake, , 1988. Mah and Foltz (2011) described that the largest clade within the Forcipulatacea is formed  (Glover et al., 2016;Mah & Foltz, 2011). However, the number of Brisingida species with complete mitogenome is still limited, and more mitogenomes and analysis are necessary to determine the phylogenetic relationship among members of Brisingida.

| Positive selection analysis
We examined the potential positive selection in Brisingida lineage because of the colonization of deep-sea environments which may affect the function of mitochondrial genes. The results of selective pressure analyses are shown in Table 5. When the ω ratios for the 13 concatenated mitochondrial protein-coding genes were tested between the deep-sea F. benthophila and other eight shallow sea starfish, we failed to find a significant difference in their ω ratios, which may be due to the large bias of sample sizes (p > 0.05) ( Table 5). In addition, in the analyses of individual genes, we found five residues with high posterior probabilities in the atp8 (8 N, 16 I), nad2 (47 D, 196 V), and nad5 (599 N), respectively (Table 5). Similar results have been observed in deep-sea animals, and the authors concluded that it may be related to the adaptation to environment (Sun et al., 2018;Zhang et al., 2017). Under the deep-sea extreme environment, survival may require a modified and adapted energy metabolism (Sun et al., 2018).

| CON CLUS IONS
In this study, we determined the mitogenome of the deep-sea member F. benthophila, which is 16,175 bp in length and encodes 37 genes including 13 PCGs, two rRNA genes, and 22 tRNA genes on the both strands. We described the mitogenome features, codon usage, gene arrangement, phylogenetic analysis, and positive selection of the starfish F. benthophila. This study is the first determination of the mitogenome of a deep-sea member of the order Brisingida and may shed light on the adaptive evolution of Brisingida species to the deep-sea environment.

ACK N OWLED G EM ENTS
The authors thank the captains and crews of the R/V Xiangyanghong 09 and the pilots of HOV "Jiao Long" for their technical support.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Haibin Zhang and Wendan Mu designed the study. Haibin Zhang contributed to the project coordination and collected the samples.
Wendan Mu conducted the sequence analyses and drafted the manuscript. Haibin Zhang and Jun Liu helped to draft the manuscript. All authors read and approved the final manuscript.

DATA ACCE SS I B I LIT Y
The complete mitochondrial DNA sequence has been deposited in GenBank (Accession Number: MG563681).