The Mitogenome of the Subarctic Octocoral Alcyonium digitatum Reveals a Putative tRNAPro Gene Nested within MutS

We sequenced and analyzed the complete mitogenome of a Norwegian isolate of the octocoral Alcyonium digitatum using the Ion Torrent sequencing technology. The 18,790 bp circular mitochondrial genome was found to harbor the same set of 17 genes, which encode 14 protein subunits, two structural ribosomal RNAs and one tRNA, as reported in other octocorals. In addition, we detected a new tRNAPro-like gene sequence nested within the MutS protein coding region. This putative tRNA gene feature appears to be conserved among the octocorals but has not been reported previously. The A. digitatum mitogenome was also shown to harbor an optional gene (ORFA) that encodes a putative protein of 191 amino acids with unknown function. A mitogenome-based phylogenetic analysis, presented as a maximum likelihood tree, showed that A. digitatum clustered with high statistical confidence with two other Alcyonium species endemic to the Mediterranean Sea and the Southeast Pacific Ocean.


Introduction
Octocorallia (class Anthozoa) represents an important subclass of marine cnidarians that hosts the well-known orders Pennatulacea (sea pens) and Alcyonacea (soft corals and sea fans).The order Alcyonacea consists of about 2700 valid species organized into 30 families, mostly found in warm, shallow waters [1].Alcyonium digitatum (dead man's fingers), however, is a cold-water soft coral in the family Alcyoniidae.A. digitatum is confined to the North Atlantic Ocean and commonly present in Norwegian coastal waters [2].
Mitochondrial genomes (mitogenomes) in octocorals vary in size from approximately 18 to 20 kb and are highly economically organized [3][4][5].Rearrangements are relatively common and both strands are gene coding.The circular mitogenome codes for the same set of 13 oxidative phosphorylation (OxPhos) proteins and two ribosomal RNAs (rRNAs) as most metazoans.However, only one tRNA gene has been recognized, indicating an extensive mitochondrial import of cytoplasmic tRNAs to support mitochondrial translation.The highly reduced tRNA gene repertoire also challenges the mitochondrial mRNA processing pattern, which is complex and clearly different from the tRNA punctuation pathway known, for example, in vertebrates [6,7].
A hallmark of octocoral mitogenomes is the presence of a MutS homolog gene (mutS).Bacterial MutS proteins are involved in DNA mismatch repair and recombination [8], and similar roles have been suggested for the mitochondrial homolog [9].The mutS gene, along most mitochondrial protein-coding genes (PCGs) in octocorals, is under strong purifying selection [10,11] and encodes a multidomain protein of approximately 980 amino acids [12].Interestingly, several distinct mRNA variants are generated during RNA processing and maturation, which involve alternative 5 ′ ends and at least seven different polyadenylation sites [7].Why the mutS gene has become a compulsory feature in octocoral mitogenomes is currently not fully understood.Here, we report the complete mitogenome sequence from a subarctic specimen of A. digitatum (Nor-1).The analyses revealed the presence of a new tRNA gene feature nested within the MutS coding region, which may be conducive to the mutS gene retention among octocoral species.

DNA Extraction and Sequencing
We determined the complete mitogenome sequence from a subarctic specimen of A. digitatum (Nor-1) sampled at the coast of northern Norway (Bodø, Nordland County; 67 • 16 ′ N, 14 • 24 ′ E).Tissue sample from the body wall was preserved in absolute ethanol at −20 • C and subsequently stored in Nord University's tissue collection at −80 • C. Total DNA was extracted as described by the supplier using the Epicentre TM MasterPure TM Complete DNA and RNA Purification Kit (Biosearch TM Technologies, Hoddesdon, UK).DNA was subjected to whole-genome Ion Torrent PGM sequencing (Thermo Fisher Scientific, Waltham, MA, USA) using 316 v.2 chips as previously reported [13].In short, sequencing libraries were constructed from DNA fragment sizes selected to be approximately 400 bp, and a total of 3.0 million reads were obtained from A. digitatum.

Data Analysis
The assembly was based on the MITObim script [14] and revealed a contig of 18,790 bp, which corresponded to the circular mitogenome.The maximum likelihood (ML) treebuilding method in MEGA 11 was used to infer the molecular phylogeny, and the sequence alignment was model tested prior to tree construction [15].The best-fit model was the general time reversible (GTR) with gamma-distributed (+G) rates across sites.The tree topology was evaluated using 2000 bootstrap replicates.An unambiguous sequence alignment of approximately 11,700 nucleotide positions representing all 13 concatenated OxPhos protein-coding genes (PCGs) was used to reconstruct the mitogenome phylogeny.

Canonical Mitochondrial Gene Features
The 18,790 bp mitogenome was sequenced with high coverage (159 times average coverage), has a low GC content (37%), a high gene density (97%), and contains the same set of 13 OxPhos PCGs as most metazoan mitochondrial genomes, as well as the mutS (Figure 1A; Table S1).The mitogenome also encodes small subunit (SSU) and large subunit (LSU) rRNAs and tRNA Met .No introns are present, which is a distinguishing feature of the subclass Octocorallia compared to the related Anthozoan subclass Hexacorallia [16].In A. digitatum, most mitochondrial genes are encoded by the same strand, but with five exceptions (Figure 1A).Thus, the gene order and organization appear similar to that of most Alcyonacea mitochondrial genomes [3][4][5].

Non-Canonical Mitochondrial Gene Features
We noted two additional and previously unannotated putative genes in the A. digitatum mitogenome; a putative tRNA Pro gene and an ORFA gene encoding a 191 aa of unknown function.

Non-Canonical Mitochondrial Gene Features
We noted two additional and previously unannotated putative genes in the A. digitatum mitogenome; a putative tRNA Pro gene and an ORFA gene encoding a 191 aa protein of unknown function.

Putative tRNA Pro Gene
A tRNA-like gene was found nested within the MutS coding region (Figure 1A).The derived tRNA structure resembles that of tRNA Pro and appears to be highly conserved within the order Alcyonacea (Figure 1B; Figure S1; Table S2), corresponding to an evolutionary time of approximately 500 million years [17].Furthermore, its folding pattern and level of conservation is similar to that of tRNA Met (Figure 1C).Two closely related Scleractinian hexacoral species, Porites rus and Porites lutea, are also reported to encode a putative tRNA Pro gene in their mitogenomes [18,19].Interestingly, the putative tRNA gene is embedded in a mitochondrial non-canonical homing endonuclease gene within the COI group I intron.The dual coding functions of DNA segments are also known from other hexacoral mitogenomes.Specifically, the COI group I introns in several sea anemones and mushroom corals species encode both a catalytic RNA and an endonuclease protein from the same sequence [20][21][22].Another well-characterized example is the vertebrate mitochondrial-derived peptides encoded within the SSU rRNA and LSU rRNA genes [23][24][25].The octocoral mutS evolution and function appear complex and obscure [12], including different mRNAs with alternative 5 ′ and 3 ′ ends [7].Thus, it appears plausible that mutS may have a dual coding capacity of protein and structural RNA, which would also be conducive to its retention among octocoral species, but this feature needs to be further investigated in a functional context.

Phylogenetic Considerations
An alignment of 11,700 nucleotide positions representing all 13 concatenated OxPhos PCGs was used to reconstruct the mitogenome phylogeny of nine species representing six families of octocorals.The sea pen Pennatula grandis (order Pennatulacea) was used as the outgroup in the analysis.SSU and LSU rRNA genes and MutS gene sequences were excluded from the alignment due to frequent indels among the octocoral mitochondrial genomes assessed.The resulting phylogenetic tree (Figure 2) showed that the A. digitatum isolates cluster with high confidence (bootstrap value 100%) to the congeneric A. acaule (endemic to the Mediterranean Sea) and A. haddoni (endemic to the Southeast Pacific Ocean).

Conclusions
We report new structural features in the mitogenome sequence of A. digitatum with general relevance to octocorals that include a putative tRNA gene and a protein-coding gene with an unknown function.A biological role of the putative tRNA Pro is currently not known, but we speculate that it could act as a regular tRNA in mitochondrial translation.The MutS coding region in A. digitatum contains as much as 44 proline codons, and an internal tRNA Pro gene may thus compensate for an increased demand of tRNA Pro during translation.Alternatively, the putative tRNA Pro structure may be involved in RNA processing and the maturation of the mutS mRNA variants by recruiting mitochondrial RNaseP.
S.D.J.; visualization, T.E.J. and S.D.J.; supervision, Å.E., T.M. and S.D.J.; project administration, T.M. and S.D.J.; funding acquisition, T.M. and S.D.J.All authors have read and agreed to the published version of the manuscript.

Figure 2 .
Figure 2. Molecular phylogeny of Alcyonium digitatum and some related octocoral species based on concatemeric mitochondrial protein-coding sequences (11,700 positions) and maximum likelihood analysis (2000 bootstrap replicates).Bootstrap support values are indicated at the branch nodes.The sea pen Pennatula grandis was used as the outgroup.Specimens from family Alcyoniidae are boxed.The scale bar indicates the fraction of substitutions per site.