A Comprehensive Description and Evolutionary Analysis of 22 Grouper (Perciformes, Epinephelidae) Mitochondrial Genomes with Emphasis on Two Novel Genome Organizations

<div><p>Groupers of the family Epinephelidae are a diverse and economically valuable group of reef fishes. To investigate the evolution of their mitochondrial genomes we characterized and compared these genomes among 22 species, 17 newly sequenced. Among these fishes we identified three distinct genome organizations, two of them never previously reported in vertebrates. In 19 of these species, mitochondrial genomes followed the typical vertebrate canonical organization with 13 protein-coding genes, 22 <i>tRNAs</i>, two <i>rRNAs</i>, and a non-coding control region. Differing from this, members of genus <i>Variola</i> have an extra <i>tRNA-Ile</i> between <i>tRNA-Val</i> and <i>16S rRNA</i>. Evidence suggests that this evolved from <i>tRNA-Val</i> via a duplication event due to slipped strand mispairing during replication. Additionally, <i>Cephalopholis</i><i>argus</i> has an extra <i>tRNA-Asp</i> in the midst of the control region, likely resulting from long-range duplication of the canonical <i>tRNA-Asp</i> through illicit priming of mitochondrial replication by tRNAs. Along with their gene contents, we characterized the regulatory elements of these mitochondrial genomes’ control regions, including putative termination-associated sequences and conserved sequence blocks. Looking at the mitochondrial genomic constituents, <i>rRNA</i> and <i>tRNA</i> are the most conserved, followed by protein-coding genes, and non-coding regions are the most divergent. Divergence rates vary among the protein-coding genes, and the three cytochrome oxidase subunits (<i>COI, II, III</i>) are the most conserved, while NADH dehydrogenase subunit 6 (<i>ND6</i>) and the ATP synthase subunit 8 (ATP8) are the most divergent. We then tested the phylogenetic utility of this new mt genome data using 12 protein-coding genes of 48 species from the suborder Percoidei. From this, we provide further support for the elevation of the subfamily Epinephelinae to family Epinephelidae, the resurrection of the genus <i>Hyporthodus</i>, and the combination of the monotypic genera <i>Anyperodon</i> and <i>Cromileptes</i> to genus <i>Epinephelus</i>, and <i>Aethaloperca</i> to genus <i>Cephalopholis</i><i>.</i></p> </div

Phylogenetic tree of 22 groupers in family Epinephelidae and 29 representatives from other families in suborder Percoidei.

<p>A species from the suborder Labrodei family Labridae, <i>Pseudolabrus sieboldi</i>, was selected as outgroup. Congruent tree topology was inferred from partitioned Maximum likelihood and Bayesian analyses using the concatenated nucleotide sequences of 12 mitochondrial protein-coding genes (excluding <i>ND6</i>). The Bayesian posterior probability values (top) and bootstrap values (bottom) are labeled at branch nodes. Branch length information from the Bayesian tree is shown. NCBI RefSeq or GenBank accession number of each species was listed on the right of the species name. Clade A indicates the derived epinephelid clade whose <i>ATP6</i> start codon is not ATG but CTG or TTG, different from most other teleosts and basal groupers.</p

Sequence variations among mitochondrial genes.

<p>Genes were ranked by their sequence identity percentages from low to high (left to right). (A) Sequence identities of 22 tRNA genes. (B) NT% (dark grey) and deduced AA% (light grey) of 13 protein-coding genes. Genes were ranked by the AA%.</p

Gene maps for mitochondrial genomes.

<p>Genes encoded on the heavy and light strand are shown outside and inside the circle, respectively. The inner grey ring indicates the GC content. This genome map was constructed via OrganellarGenomeDRAW [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073561#B81" target="_blank">81</a>] with manual modifications. (A) The mt gemone organization of <i>Aethaloperca rogaa</i>, <i>Anyperodon leucogrammicus</i>, <i>Cephalopholis sonnerati</i>, <i>Cromileptes altivelis</i>, <i>Epinephelus akaara</i>, <i>E</i>. <i>areolatus</i>, <i>E</i>. <i>awoara</i>, <i>E</i>. <i>bruneus</i>, <i>E</i>. <i>coioides</i>, <i>E</i>. <i>epistictus</i>, <i>E</i>. <i>fuscoguttatus</i>, <i>E</i>. <i>lanceolatus</i>, <i>E</i>. <i>moara</i>, <i>E</i>. <i>trimaculatus</i>, <i>Hyporthodus octofasciatus</i>, <i>H</i>. <i>septemfasciatus</i>, <i>Plectropomus areolatus</i>, <i>P</i>. <i>leopardus</i>, and <i>Triso dermopterus</i>. (B) The mt gemone organization of genera <i>Variola, V</i>. <i>albimarginata</i> and <i>V</i>. <i>louti</i>. (C) The mt gemone organization of <i>Cephalopholis argus</i>.</p

Schematic structures of mitochondrial control regions.

<p>ETAS, extended termination associated sequences; CCD, central conserved domain; CBSs, conserved sequence blocks. This figure does not show the structure for the CR of <i>Cephalopholis argus</i>.</p

Li H, Mishra M, Ding S, Miyamoto MM (2018) Diversity and dynamics of “Candidatus Endobugula” and other symbiotic bacteria in Chinese populations of the bryozoan, Bugula neritina. Microb Ecol: in press

The metagenomics datasets for the geographic and life cycle samples of Li et al. (2018) are presented here in four separate folders: (OR_GEO) original reads for the geographic samples; (FFS_GEO) final filtered sequences for the geographic samples; (OR_LC) original reads for the life cycle samples; and (FFS_LC) final filtered sequences for the life cycle samples. Each separate dataset is labelled with the abbreviation of its sample (see Table 2 and Figure 1 of Li et al., 2018) followed by either “OR” or “FFS” (for original reads or final filtered sequences, respectively). For example, the “SY3_OR” dataset includes the original reads for the SY₃ geographic sample, whereas the “S6_FFS” dataset contains the final filtered sequences for the S6 life cycle collection

Geomean of ranking values of the 11 candidate reference genes calculated by refFinder for <i>V</i>. <i>alginolyticus</i> challenged <i>E</i>. <i>akaara</i>.

<p>The red diamond indicates the gene expression was unstable as determined by geNorm, NormFinder, or BestKeeper. Ranking of the gene expression stability was performed for the (A) liver, (B) intestine, (C) stomach, (D) spleen, (E) heart, (F) head kidney, (G) gills, and (H) white muscle.</p

Determination of the expression stability for salinity treatment samples according to different programs.

<p>(A) geNorm, the red line indicates the geNorm cut-off value of 1.5. (B) NormFinder. (C) BestKeeper, the red line indicates the BestKeeper cut-off value of 1.0. (D) refFinder, the red indicates the gene expression was unstable.</p

Selection and evaluation of new reference genes for RT-qPCR analysis in <i>Epinephelus akaara</i> based on transcriptome data

<div><p>Groupers are an economically important fish species in world fishery markets. Because many studies using RT-qPCR have addressed gene expression in groupers, appropriate reference genes are required to obtain reliable and accurate results. In this study, the most suitable reference genes were identified from eleven candidate genes of one of the most valuable species, <i>Epinephelus akaara</i>, in a range of different experimental conditions. Using the software packages geNorm, NormFinder, BestKeeper and refFinder, three traditionally used reference genes, <i>β</i>-actin (<i>β-ACT</i>), glyceraldehyde-3-phosphate dehydrogenase (<i>GAPDH</i>), and <i>beta-2-microglobulin</i> (<i>B2M</i>), were identified as not suitable for <i>E</i>. <i>akaara</i> gene expression studies, whereas two newly identified reference genes, <i>conserved oligomeric Golgi complex subunit 5</i> (<i>Cog5</i>) and <i>brefeldin a-inhibited guanine nucleotide-exchange protein 1</i> (<i>ARFGEF1</i>), could be universally applied under all the tested conditions. These data provide the foundation for more precise results in RT-qPCR studies of gene expression in <i>E</i>. <i>akaara</i> and other Epinephelus species.</p></div

Validation of the recommended reference genes.

<p>Expression profiles of <i>MSTN</i> and <i>IL-1β</i> were analysed using the most stable, the recommended universal and the least stable reference genes during different gonad development phases (A), during the early ontogenetic development stage (B), for the salinity treatment of <i>E</i>. <i>akaara</i> (C), and for <i>V</i>. <i>alginolyticus</i> challenged <i>E</i>. <i>akaara</i> (D). Bars represent the means and standard error of three biological replications. HS: high salinity, LS: low salinity, HK: head kidney, GS: gills.</p