Biodiversity Data Journal 11: e98167 OO)
doi: 10.3897/BDJ.11.e98167 open access
Short Communication

Phylogenetic relationships of three rockfish:
Sebastes melanops, Sebastes ciliatus and
Sebastes variabilis (Scorpaeniformes,
Scorpaenidae) based on complete mitochondrial

genome sequences

Peter C. Searle?, Andrea L. Kokkonen§, Jillian R. Campbell#, Dennis K. Shiozawal, Mark C. Belk?,
R. P. Evans$§

+ Department of Biology, Brigham Young University, Provo, UT, United States of America
§ Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT, United States of America
| Monte L. Bean Life Science Museum, Brigham Young University, Provo, UT, United States of America

Corresponding author: Peter C. Searle (petersearle94@qmail.com)
Academic editor: Rupert Collins
Received: 29 Nov 2022 | Accepted: 20 Feb 2023 | Published: 28 Feb 2023

Citation: Searle PC, Kokkonen AL, Campbell JR, Shiozawa DK, Belk MC, Evans RP (2023) Phylogenetic
relationships of three rockfish: Sebastes melanops, Sebastes ciliatus and Sebastes variabilis (Scorpaeniformes,
Scorpaenidae) based on complete mitochondrial genome sequences. Biodiversity Data Journal 11: e98167.

https://doi.org/10.3897/BDJ.11.e98167

Abstract

We characterise the complete mitochondrial genomes (mitogenomes) of Black rockfish
(Sebastes melanops Girard, 1856; n = 1), Dark rockfish (Sebastes ciliatus Tilesius, 1813; n
= 2) and Dusky rockfish (Sebastes variabilis 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.

© Searle P et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY
4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are
credited.

2 Searle P et al

Keywords

Sebastes, speciation, phylogenetics, rockfish, mitogenome

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).

Dusky rockfish (Sebastes variabilis)

Figure 1. EES]

Photographs of Black rockfish (Sebastes melanops), Dark rockfish (Sebastes ciliatus) and
Dusky rockfish (Sebastes variabilis). Photos taken by Mark C. Belk.

Phylogenetic relationships of three rockfish: Sebastes melanops, Sebastes ... 3

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
and Gilbert 1881, Eigenmann and Beeson 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. varniabilis; Eschmeyer and Herald (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.

Materials and Methods

Using hook-and-line sampling, we collected three rockfish specimens (Sebastes melanops,
S. ciliatus and S. variabilis) from Frederick Sound, near Admiralty Island (57.307504,
-134.133069) in 2018 and two rockfish specimens (S. ciliatus and S. variabilis) near
Excursion Inlet, Alaska (58.3159, -135.4592) in 2019 (IACUC-approved protocol
#15-0602). Upon capture, we euthanised specimens with tricaine methanesulfonate
(MS-222, MilliporeSigma, St. Louis, MO, USA), excised liver samples and placed the
samples in RNAlater (MilliporeSigma, St. Louis, MO, USA). Samples were flash-frozen at
-20°C, transported to Brigham Young University and stored at -80°C. Samples were
catalogued in the Monte L. Bean Life Science Museum under accession numbers: S.
melanops (BYU:1003048), S. ciliatus (BYU:1003050, BYU:267108) and S. variabilis (BYU:
1003082, BYU:267107), (Table 1). Morphological vouchers were retained for S. ciliatus and
S. variabilis collected in 2018 (BYU:1003050 and BYU:1003082, respectively). Total DNA
was extracted from 40 mg liver samples using a DNeasy Blood & Tissue kit (Qiagen,
Hilden, Germany). DNA concentration was measured by a Qubit Fluorometer (Thermo
Fisher Scientific, Waltham, MA, USA). Libraries of each sample were created according to
the Illumina Library prep protocol (Illumina 1003806 Rev. A) and sequenced on the Illumina
HiSeq 2500 (Illumina, San Diego, CA, USA; Paired End 150 bp) at Brigham Young
University’s DNA Sequencing Center (Provo, Utah, USA). We used FastQC (Andrews 2010
) to assess quality of raw reads. Mitogenomes were assembled with Geneious v. 2021.2
(Biomatters Ltd., Auckland, New Zealand) using S. fasciatus as a reference genome
(KX897946) and annotated with MitoAnnotator (Iwasaki et al. 2013). Raw reads and
assembled genomes were deposited in NCBI's Sequence Read Archive (raw data) and

4 Searle P et al

GenBank nucleotide (assembled mitogenomes) databases (Table 1). Mitogenome maps
were generated using Circos (Krzywinski et al. 2009). We included 35 rockfish
mitogenomes, representing 27 species, in our phylogenetic analysis. We used MAFFT v.
7.475 (Katoh et al. 2002) to generate multiple sequence alignments for each of the 13
protein-coding genes and concatenated the alignments. We generated a Maximum
Likelihood phylogeny with W-IQ-Tree (Trifinopoulos et al. 2016).

Table 1.
Voucher, BioProject, BioSample, GenBank and SRA accession numbers for each sample of
Sebastes used in the study.

Species Voucher BioProject BioSample GenBank SRA

S. ciliatus BYU:267108 PRJNA741690 SAMN20892472 OK048740 SRX11870776
S. ciliatus BYU:1003050 PRJNA741690 SAMN20892468 MZ420215 SRX11870778
S. melanops BYU:1003048 PRJNA741690 SAMN20892467 OK048741 SRX11870777
S. variabilis BYU:267107 PRJNA741690 SAMN20892471 OK048742 SRX11870775
S. variabilis BYU:1003082 PRJNA741690 SAMN20892469 OK048743 SRX11870779
Results

The complete mitochondrial genome of Sebastes melanops (QK048741) 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.

Phylogenetic relationships of three rockfish: Sebastes melanops, Sebastes ... 5

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.

® —-tRNA-Phe

—tRNA-lle
~tRNA-GIn
IBNAMet

Figure 2. EES

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).

6 Searle P et al

. thompsoni KJ834064.1

. Inermis KF725093.1

. vulpes KJ525743.1

. trivittatus KJ834062.1

. schlegeli AY491978.1
taczanowskii KJ525744.1

. owstoni KJ834063.1

. koreanus KJ775792.1

. koreanus KF561237.1

. oblongus KF836441.1
pachycephalus KF836442.1
. longispinis KJ834061.1

. hubbsi KJ525745.1

. Maliger MW846222.1

. melanops OK048741.1

. rubrivinctus MH378777.1

. rubrivinctus MH378776.1

. Nigrocinctus MH378778.1

. umbrosus CM026822.1
oculatus MN218776.1
norvegicus MW846223.1

. viviparus LC493909.1

. Viviparus MT410865.1

. mentella LC493910.1

. fasciatus KX897946.1

S. ciliatus MZ420215.1

S. variabilis OK048743.1

S. variabilis OK048742.1

. ciliatus OK048740.1

. melanostictus MH378781.1
. melanostictus MH378780.1
. Steindachneri MH378779.1
. Steindachneri KJ834060.1
. Minor KJ834059.1

. Minor MH378782.1

73

ADNNNDNDDHDNDDHDDHDDHHDHDDHHDHDHDHHHDHHYD

53

by 8

NNNHHAHNnN

81

Tree scale: 0.01 #———+

Figure 3. EES

Phylogenetic tree inferred by Maximum Likelihood using W-lQ-Tree. Thirty-five Sebastes
mitogenomes, representing 27 species, were used in the phylogeny. Ultrafast bootstrap values
> 95 are not displayed and Sebastiscus tertius (1117231) was used as an outgroup, but is
not shown.

Acknowledgements

We thank Scott and Jody Jorgenson at Pybus Point Lodge, Mark and Kristina Warner and
staff at Doc Warner’s lodge, the Roger and Victoria Sant Foundation, the College of Life
Sciences, the Department of Biology at Brigham Young University and the Monte L. Bean
Life Science Museum for supporting this study.

Hosting institution

Department of Biology, Brigham Young University, Provo, UT, United States of America

Phylogenetic relationships of three rockfish: Sebastes melanops, Sebastes ... 7

Ethics and security

All samples were collected under Brigham Young Universities’ IACUC-approved protocol
#15-0602 for Dennis K. Shiozawa.

Author contributions

Conceptualisation: P.C.S., A.L.K., D.K.S. & R.PE.; Data Curation: P.C.S., A.L.K., &
J.R.C.; Formal Analysis: P.C.S. & J.R.C; Funding Acquisition: D.K.S., M.C.B., & R.P.E.;
Investigation: P.C.S., A.L.K., J.R.C., D.K.S., & R.P.E.; Methodology: P.C.S., A.L.K.,
J.R.C., D.K.S. & R.P.E.; Project Administration: R.P.E.; Resources: D.K.S., M.C.B., &
R.P.E.; Supervision: D.K.S., M.C.B., & R.P.E.; Validation: D.K.S., M.C.B., & R.P.E.;
Visualisation: P.C.S. & J.R.C.; Writing — Original Draft: A.L.K.; Writing — Reviewing &
Editing: P.C.S., J.R.C., D.K.S., M.C.B., & R.P.E.

Conflicts of interest

The authors declare no potential conflict of interest and the authors alone are accountable
for the content and composition of the paper.

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