Phylogenetic relationships of three rockfish: Sebastesmelanops, Sebastesciliatus and Sebastesvariabilis (Scorpaeniformes, Scorpaenidae) based on complete mitochondrial genome sequences

Abstract We characterise the complete mitochondrial genomes (mitogenomes) of Black rockfish (Sebastesmelanops Girard, 1856; n = 1), Dark rockfish (Sebastesciliatus Tilesius, 1813; n = 2) and Dusky rockfish (Sebastesvariabilis Pallas, 1814; n = 2). The lengths of the mitogenomes are 16,405 bp for S.melanops, 16,400 bp for both S.ciliatus and 16,400 and 16,401 bp for S.variabilis. We examine these species’ phylogenetic relationships using 35 previously published rockfish mitogenomes, representing 27 species. We find that S.melanops is sister to a clade consisting of S.rubrivinctus, S.nigrocinctus, S.umbrosus and S.oculatus, whereas S.ciliatus and S.variabilis are sister to a clade consisting of S.norvegicus, S.viviparus, S.mentella and S.fasciatus. We were unable to separate S.ciliatus and S.variabilis using their complete mitogenomes.


Introduction
Black rockfish (Sebastes melanops Girard, 1856), Dark rockfish (Sebastes ciliatus Tilesius, 1813) and Dusky rockfish (Sebastes variabilis Pallas, 1814) are members of Sebastes (Cuvier, 1829), a diverse genus of marine fishes comprising more than 110 species (Fig. 1 ).These commercially important rockfishes are found in the North Pacific Ocean, with sympatric geographic ranges.Sebastes melanops schools over high relief rocky outcrops from 0-366 m, S. ciliatus schools over high relief on rocky reefs and in kelp forests from 5-160 m and S. variabilis schools over high-relief sea floors from 6-675 m (Butler et al. 2012).Although S. ciliatus and S. variabilis were described separately in the early 1800s, they have long been considered a single variable species under the name S. ciliatus (Jordan andGilbert 1881, Eigenmann andBeeson 1894).However, the presence of two colour morphs within S. ciliatus, with associated ecological differences, led to speculation that S. ciliatus consisted of a dark, shallow-water morph (S. ciliatus) and a light, deep-water morph (S. variabilis; Eschmeyer andHerald (1983), Kessler (1985)).Orr and Blackburn (2004) officially resurrected S. variabilis from S. ciliatus using morphological and meristic data, but molecular analyses have produced conflicting results.Genetic differences were identified in S. ciliatus using allozymes (Tsuyuki et al. 1965, Seeb 1986) and microsatellites (Orr and Blackburn 2004); however, it is unclear if these differences resulted from species-level separation or population-level differences resulting from geographic separation of the samples.Tsuyuki et al. (1965) did not provide specific location data for their nine samples of S. ciliatus and the samples analysed by Seeb (1986) did not come from sympatric populations.Conversely, mitochondrial DNA, specifically NADH dehydrogenase subunits, was not significantly different (Orr and Blackburn 2004).We report the complete mitogenomes of S. melanops, S. ciliatus, and S. variabilis to provide new insight into the taxonomic relationships amongst these species.We aimed to determine if the lack of resolution in mitochondrial DNA was limited by the small portion of mitochondrial DNA examined in previous studies.

Results
The complete mitochondrial genome of Sebastes melanops (OK048741) was 16,405 bp in length, S. ciliatus (MZ420215, OK048740) were both 16,400 bp in length and S. variabilis (OK048743, OK048742) were 16,400 and 16,401 bp, respectively, in length.Consistent with previous studies (Zhang et al. 2012, Sandel et al. 2018, Campbell et al. 2022), the control region's length was highly variable because of repetitive DNA sequences (Fig. 2).The complete mitogenomes of S. ciliatus and S. variabilis were ~ 0.5% divergent.In comparison, the complete mitogenome of S. melanops was between 6.2% and 10.6% divergent with other members in its clade.In our phylogeny, S. melanops is sister to a clade including S. rubrivinctus, S. nigrocinctus, S. umbrosus and S. oculatus, whereas S. ciliatus and S. variabilis are sister to a clade including S. norvegicus, S. viviparus, S. mentella and S. fasciatus.We were unable to resolve the phylogenetic relationship between S. ciliatus and S. variabilis (Fig. 3).

Discussion
Previous molecular analyses of allozymes, microsatellites and mitochondrial DNA have produced inconsistent results about the relationship of Sebastes ciliatus and S. variabilis ( Tsuyuki et al. 1965, Seeb 1986, Orr and Blackburn 2004).However, in these studies, it is unclear if these differences result from species-level or population-level differences.In addition, for the studies that used mitochondrial DNA, only a small portion of mitochondrial DNA was examined (Orr and Blackburn 2004).By using samples of S. ciliatus and S.
Voucher, BioProject, BioSample, GenBank and SRA accession numbers for each sample of Sebastes used in the study.
variabilis from sympatric populations, as well as generating whole mitochondrial genomes, we provide new insight into the status of these species.Consistent with previous studies using partial mitochondrial sequences, we found minimal sequence divergence between S. ciliatus and S. variabilis.Assuming S. ciliatus and S. variabilis are distinct sister species, we would expect greater sequence divergence, as well as a monophyletic relationship in our phylogeny, with higher bootstrap values.Further research is needed.This research should include specimens from a wider range of locations across their geographical ranges, with both allopatric and sympatric populations, a suite of genetic markers (nuclear and mitochondrial), as well as ecological and morphological characteristics.Such information will be essential in resolving the complicated relationships between these two putative species.Mitogenome map of Sebastes melanops.Outer circle illustrates order of genes, tRNAs, rRNAs and control region.Inner circle represents GC content with darker shades indicating higher GC content.Sebastes melanop's mitogenome consists of 13 protein-coding genes, 22 tRNAs, two rRNAs and one control region.Order is identical in S. ciliatus and S variabilis (mitogenome maps not displayed).