Research Article |
Corresponding author: Irina N. Moreva ( irruz@yandex.ru ) Academic editor: Rafael Kretschmer
© 2021 Irina N. Moreva, Olga A. Radchenko, Anna V. Petrovskaya.
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.
Citation:
Moreva IN, Radchenko OA, Petrovskaya AV (2021) Differentiation of the frog sculpin Myoxocephalus stelleri Tilesius, 1811 (Actinopterygii, Cottidae) based on mtDNA and karyotype analyses. Comparative Cytogenetics 15(2): 179-197. https://doi.org/10.3897/CompCytogen.v15.i2.63207
|
A molecular genetic and karyological study of the frog sculpin Myoxocephalus stelleri Tilesius, 1811 was carried out on an extensive sample from a large area of the species’ range. A total of 42 specimens was sampled from the Sea of Japan, Sea of Okhotsk, and coastal waters off the southern Kuril Islands, which makes this sampling scheme the most comprehensive to date. The level of mtDNA polymorphism was found to be low. The haplotypes of the species formed three phylogenetic groups. The unique M. stelleri haplotype from the coast of Shikotan Island linked all the studied groups, indicating that it is likely ancestral. Robertsonian polymorphism was identified in the species. In all five cytotypes (I – 2n = 44, II – 2n = 43, III – 2n = 42, IV – 2n = 41, V – 2n = 40; NF = 44+2) were identified, all of which were present in the Sea of Japan. Only one (cytotype I) was found in the Sea of Okhotsk, which is probably the closest to the ancestral karyotype. The significant chromosomal polymorphism and the presence of common haplotypes in the studied samples indicate their recent origin from a common ancestor and/or relatively recent contacts within the range. The discrepancies between mtDNA and karyotypes in assigning the ancestral M. stelleri to the coastal waters off Shikotan Island (southern Kuril Islands) and the Sea of Okhotsk, respectively, can be explained by the different inheritance mechanisms and the rates of evolution of molecular genetic and karyological traits.
16S rRNA, COI, cytochrome b, cytotype, haplotype, Myoxocephalinae, Robertsonian polymorphism, Sea of Japan, Sea of Okhotsk
The genus Myoxocephalus Tilesius, 1811 is a large, taxonomically complex group of sculpins of the subfamily Myoxocephalinae (family Cottidae) (
Cytogenetic and genetic studies of M. stelleri have been described in two publications (
We conducted a study to determine the level of genetic and karyological differentiation within and between M. stelleri from the Sea of Japan, Sea of Okhotsk, and the southern Kuril Islands. Using this karyological (N = 42) and molecular genetic data (N = 34), we also aimed to find the centers of species diversification. Our extensive sample included individuals of M. stelleri captured from waters near the site of the original species description – the estuary of the Bolshaya Zapadnaya Kamchatka River (
Fig.
Specimens of M. stelleri and outgroup species examined (*specimens whose mtDNA was not studied).
Species (genetic voucher) | Locality | GenBank accession numbers | ||
---|---|---|---|---|
COI | Cyt b | 16S rRNA | ||
Sea of Japan | ||||
M. stelleri (1754) | Peter the Great Bay, Vostok Bay (15) | KY062754 | MH595735 | KY062665 |
M. stelleri (1755) | Peter the Great Bay, Vostok Bay (15) | MN115304 | MN115340 | MN097160 |
M. stelleri (1756) | Peter the Great Bay, Vostok Bay (15) | MN115305 | MN115341 | MN097161 |
M. stelleri (2097) | Peter the Great Bay, Russky Island (15) | MN115306 | MN115342 | MN097162 |
M. stelleri (1991) | Olga Bay (14) | MN115311 | MN115347 | MN097167 |
M. stelleri (2081) | Olga Bay (14) | MN115312 | MN115348 | MN097168 |
M. stelleri (2044) | Zolotaya Bay (12) | MN115313 | MN115349 | MN097169 |
M. stelleri (2083) | Zolotaya Bay (12) | MT258533 | MT253729 | MT251919 |
M. stelleri (2149) | Zolotaya Bay (12) | MN115314 | MN115350 | MN097170 |
M. stelleri (2150) | Zolotaya Bay (12) | MN115315 | MN115351 | MN097171 |
M. stelleri (2047) | Dzhigit Bay (13) | MN115316 | MN115352 | MN097172 |
M. stelleri (2082) | Dzhigit Bay (13) | MN115317 | MN115353 | MN097173 |
M. stelleri (2148) | Dzhigit Bay (13) | MN115318 | MN115354 | MN097174 |
M. stelleri (1985) | Aleksandrovsky Bay (11) | MN115319 | MN115355 | MN097175 |
M. stelleri (2084) | Aleksandrovsky Bay (11) | MN115320 | MN115356 | MN097176 |
M. stelleri (2151) | Chikhachev Bay (10) | MN115321 | MN115357 | MN097177 |
M. stelleri (2152) | Chikhachev Bay (10) | MN115322 | MN115358 | MN097178 |
M. stelleri (2049) | Chikhachev Bay (10) | MN115323 | MN115359 | MN097179 |
M. stelleri (1973) | Chikhachev Bay (10) | MN115324 | MN115360 | MN097180 |
M. stelleri (2153) | Chikhachev Bay (10) | MN115325 | MN115361 | MN097181 |
Pacific Ocean, Shikotan Island | ||||
M. stelleri (1736) | Gorobets Bay (9) | MN115307 | MN115343 | MN097163 |
M. stelleri (1737) | Krabovaya Bay (9) | MN115308 | MN115344 | MN097164 |
M. stelleri (1739) | Otradnaya Bay (9) | MN115309 | MN115345 | MN097165 |
M. stelleri (1740) | Otradnaya Bay (9) | MN115310 | MN115346 | MN097166 |
Sea of Okhotsk | ||||
M. stelleri* | Kunashir Island, Pervukhin Bay (8) | – | – | – |
M. stelleri (1748) | Taui Bay, Uta River estuary (6) | MN115326 | MN115362 | MN097182 |
M. stelleri (1749) | Taui Bay, Shestakov Bay (6) | MN115327 | MN115363 | MN097183 |
M. stelleri (1778) | Taui Bay, Shestakov Bay (6) | MN115328 | MN115364 | MN097184 |
M. stelleri (1780) | Taui Bay, Shestakov Bay (6) | MN115329 | MN115365 | MN097185 |
M. stelleri (2077) | Taui Bay, Odyan Bay (5) | MN115330 | MN115366 | MN097186 |
M. stelleri (2078) | Taui Bay, Odyan Bay (5) | MN115331 | MN115367 | MN097187 |
M. stelleri (2133) | Taui Bay, Odyan Bay (5) | MN115332 | MN115368 | MN097188 |
M. stelleri (2134) | Taui Bay, Odyan Bay (5) | MN115333 | MN115369 | MN097189 |
M. stelleri (2131) | Taui Bay, Nedorazumeniya Island (5) | MN115334 | MN115370 | MN097190 |
M. stelleri (2132) | Taui Bay, Nedorazumeniya Island (5) | MN115335 | MN115371 | MN097191 |
M. stelleri* | Feklistov Island (7) | – | – | – |
Feklistov Island (7) | – | – | – | |
Shelikhov Bay, Gizhigin Bay (4) | – | – | – | |
Shelikhov Bay, Gizhigin Bay (4) | – | – | – | |
Shelikhov Bay, Penzhina Bay (3) | – | – | – | |
Western Kamchatka, Kvachin Bay (2) | – | – | – | |
Western Kamchatka, Kvachin Bay (2) | – | – | – | |
Western Kamchatka, Pichgygyn Bay (1) | – | – | – | |
Outgroups | ||||
M. jaok | Sea of Japan | MN115336 | MN115372 | MN097192 |
M. brandtii | Shikotan Island | MN115337 | MN115373 | MN097193 |
M. polyacanthocephalus | Shikotan Island | MN115338 | MN115374 | MN097194 |
M. ochotensis | Sea of Okhotsk | MN115339 | MN115375 | MN097195 |
This study utilizes samples collected with all applicable international, national and/or institutional guidelines for sampling, care and experimental use of organisms. The fishes studied here are not included in the IUCN Red List of Threatened Species, nor are they are listed as endangered, vulnerable, rare, or protected species in the Russian Federation. The sampling points are located beyond any protected areas.
We obtained sequences for three mtDNA markers: COI, cytochrome b, and 16S rRNA. Total DNA was extracted from muscle tissues by standard phenol extraction (
DNA sequences were aligned in Clustal W (MEGA version X:
Chromosomes were prepared by the air-drying technique (
Chromosomes were classified according to the nomenclature of
For M. stelleri, the length of the partial COI was 1,009 base pairs (bp), including 16 variable sites, 8 parsimony informative sites, and 5 non-synonymous substitutions. The length of the partial cytochrome b was 747 bp, including 16 variable sites, 10 parsimony informative sites, and 1 non-synonymous substitution. The length of the partial 16S rRNA was 600 bp, including 1 variable site and 1 parsimony informative site. All sequence data are deposited in GenBank/NCBI (www.ncbi.nlm.nih.gov) (for accession numbers, see Table
Haplotypes of M. stelleri (nucleotide substitutions indicating phylogenetic groups are highlighted in bold).
Haplotype | Parsimony informative nucleotide sites | Locality (see sampling site nos. in Table |
||
---|---|---|---|---|
COI | Cytb | 16S rRNA | ||
1a | CTAATTTC | TCTTGCTTCT | A | 9 |
2a | CTAACTTC | TCTTGCTTCT | A | 9, 10, 12, 13, 14 |
3a | CTAACTTC | ACTTGCTTCT | A | 11, 14 |
1b | CCAATCTC | TCTTACTTCT | G | 9 |
2b | TCAATCTC | TCTCATTTCT | G | 5, 6, 10 |
3b | CCAATCTC | TCTTATCTCT | G | 5, 9, 11, 12, 13 |
4b | CCAATCTC | TCTTATCCCT | G | 5, 6 |
5b | CTAATCTC | TCTTATCTCT | G | 12 |
6b | CCAATCTC | ACTTATCTCT | G | 5 |
1c | CCGATCTC | TTCTGCTTCC | G | 12 |
2c | CCGATCCC | TTCTGCTTCC | G | 13, 15 |
3c | CCGATCCT | TTCTGCTTCC | G | 10, 15 |
4c | CCGGTCCC | TTCTGCTTTC | G | 15 |
Haplotype polymorphism is determined by single-nucleotide mutations. The minimum difference (one substitution) was found between haplotypes 3b vs. 4b, 3b vs. 5b, 1c vs. 2c, and 2c vs. 3c; the maximum difference (14 substitutions) was found between haplotypes 2a vs. 4c and 3a vs. 4c (Fig.
A median network of the M. stelleri haplotypes based on mtDNA sequences. Each haplotype is represented by a circle; its size corresponds to the number of individuals with this haplotype. Black circle indicates a hypothetical (unsampled) haplotype. Colors designate the geographic distribution of haplotypes. Markings on the branches are nucleotide substitutions. Ellipses are the haplotype groups: NSJ (Northern Sea of Japan group); SO (Sea of Okhotsk group); WSJ (Western Sea of Japan group).
The haplotype network for M. stelleri is a star-shaped structure with the central haplotype (1b) from Shikotan Island (Fig.
We identified the nucleotide substitutions that distinguished the haplogroups. In Group NSJ, there is one substitution in the 16S rRNA gene (G → А at position 313) and one in COI (C → Т at position 495). In Group SO, there is only one substitution in the cytochrome b gene (А → G at position 195). In Group WSJ, there are three substitutions: one in the COI gene (G → А at position 318) and two in cytochrome b (Т → C at position 36, C → Т at positions 75 and 639; nucleotide positions are according to our matrix).
The Bayesian tree (Fig.
Five cytotypes were found in M. stelleri. Their major characteristics were described based on the analysis of 1,520 metaphase plates (Table
The cytotypes of M. stelleri and their geographic distribution (juv = juvenile individuals).
No. of cytotype | Number / sex of individuals | Number of metaphase plates | Locality (see sampling sites nos. in Table |
Region |
---|---|---|---|---|
I | 22 | 551 | 1–7, 10–12 | Sea of Okhotsk; northern Sea of Japan |
(10♀, 10♂, 2 juv) | ||||
II | 5 | 271 | 10, 12, 13 | Northern Sea of Japan |
(1 ♀, 2 ♂, 2 juv) | ||||
III | 3 | 130 | 10, 13, 14 | Northern Sea of Japan |
(1 ♀, 1 ♂, 1 juv) | ||||
IV | 3 | 148 | 11, 13, 14 | Northern Sea of Japan |
(2 ♂, 1 juv) | ||||
V | 9 | 420 | 8, 9, 15 | Western Sea of Japan; coastal waters off the southern Kuril Islands |
(3 ♀, 5 ♂, 1 juv) |
Karyograms of M. stelleri: (a) cytotype I, 2n = 44; (b) cytotype II, 2n = 43; (c) cytotype III, 2n = 42; (d) cytotype IV, 2n = 41; (e) cytotype V, 2n = 40 (according to
Cytotype I: 2n = 44 chromosomes (Fig. 4А), with the first two chromosomes identified as submeta-subtelocentrics; the row of uni-armed chromosomes contains 28 subtelocentrics (pairs 2 to 15) and 14 acrocentrics (pairs 16 to 22).
Cytotype II: 2n = 43 (Fig.
Cytotype III: 2n = 42 (Fig.
Cytotype IV: 2n = 41 (Fig.
Cytotype V: 2n = 40 (Fig.
All cytotypes have an equivalent number of chromosome arms: 44 + 2 (Fig.
The markers of cytotypes III–V are two submetacentrics (Fig.
Our DNA data show that M. stelleri is genetically diverse at various levels: within the same locality, between geographically close localities, and between geographically distant areas. Each sample had 2 to 4 haplotypes, some of which were unique and others widespread; however, there were no haplotypes common to all studied samples. Several samples had specific haplotypes, e.g. haplotypes 4b (frequency 14.7%) and 6b were found only in the Sea of Okhotsk, while haplotype 4c (frequency 5.9%) was found only in Peter the Great Bay. Similarly, the unique haplotypes 1a and 1b were found in M. stelleri from the coastal waters off the southern Kuril Islands, and haplotypes 1c and 5b were found only in the Zolotaya Bay sample.
The most common haplotype (2a; frequency of 20.6%) was found in M. stelleri from the northern Sea of Japan (Chikhachev Bay, Zolotaya Bay, Dzhigit Bay, and Olga Bay) and from the coastal waters off the southern Kuril Islands. Haplotype 3b (17.7%) was distributed wider across the species range, from the southern Kuril Islands and the northern Sea of Japan to the Sea of Okhotsk. The less common haplotypes (3a, 2b, 2c, and 3c) were also found in more than one sample. The common haplotypes from the Sea of Okhotsk, Sea of Japan, and the southern Kuril Islands can be explained either by their origin from a common ancestor followed by dispersal from the same center, or by recent contact in various parts of the species range. A pattern of haplotype distribution with a few haplotypes being very common and others being rare or unique is frequently found in marine fish (
In general, M. stelleri from the northern Sea of Japan exhibits higher genetic variation: eight haplotypes, many of which are shared with other geographic areas, and a low frequency of unique haplotypes. The variation between mtDNA sequences of M. stelleri from different localities is low, with the genetic distance between samples being approximately at the same level (Table
Localities (see sampling site nos. in Table |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
---|---|---|---|---|---|---|---|---|---|
1 | Odyan Bay + Nedorazumeniya Island (5) | ||||||||
2 | Shestakov Bay + Uta River estuary (6) | 0.10 | |||||||
3 | Peter the Great Bay (15) | 0.45 | 0.47 | ||||||
4 | Olga Bay (14) | 0.40 | 0.43 | 0.52 | |||||
5 | Dzhigit Bay (13) | 0.29 | 0.31 | 0.34 | 0.33 | ||||
6 | Zolotaya Bay (12) | 0.22 | 0.24 | 0.36 | 0.30 | 0.27 | |||
7 | Aleksandrovsky Bay (11) | 0.21 | 0.24 | 0.46 | 0.22 | 0.28 | 0.23 | ||
8 | Chikhachev Bay (10) | 0.36 | 0.35 | 0.40 | 0.24 | 0.30 | 0.28 | 0.26 | |
9 | Shikotan Island (9) | 0.24 | 0.26 | 0.44 | 0.24 | 0.28 | 0.24 | 0.21 | 0.26 |
The mtDNA haplotypes form three haplogroups, congruent with the three clades formed in the phylogenetic tree. These haplotypes belong to different phylogenetic groups: NSJ, SO, and WSJ. There were more differences within the Sea of Japan (groups NSJ and WSJ: d = 0.47%) than between the Sea of Okhotsk and the Sea of Japan (SO and NSJ: d = 0.36%; SO and WSJ: d = 0.42%). The geographic distribution of haplotypes is not uniform (Fig.
Two unique haplotypes, 1a and 1b, were found in the coastal waters off Shikotan Island. In the Bayesian tree (Fig.
The variation between haplotypes from different parts of the geographic range is most clear between the most distant localities: Shikotan Island vs. the western Sea of Japan, or the Sea of Okhotsk vs. the western Sea of Japan. Similar DNA differentiation has been reported for Cottidae species found in the Sea of Okhotsk and the Sea of Japan, documented both at the subspecies level, e.g. Megalocottus platycephalus platycephalus (Pallas, 1814) vs. M. platycephalus taeniopterus (Kner, 1868) (
The results of the karyological analysis are consistent with the conclusion drawn from the genetic data: M. stelleri is a heterogeneous species (Figs
All cytotypes of M. stelleri differed in the 2n (Fig.
The differences in the number of subtelocentrics between the chromosome sets (Fig.
The low level of genetic differentiation in mtDNA between the studied M. stelleri (Fig.
The significant chromosomal variability of individuals from the northern Sea of Japan may indicate their later divergence as compared to individuals from the western part of the sea. The assumption about different divergence times from the Okhotsk Sea is confirmed by the fact that the formation of submetacentrics of similar size in their cytotypes (II–IV and V) occurred because of different Robertsonian translocations. We assume that the polymorphism observed in individuals from the northern Sea of Japan could arise in the relatively recent past in the following way: the chromosomal rearrangement that took place in one or more individuals (Fig.
The frog sculpins from the coastal waters off the southern Kuril Islands deserve special attention. In the geological history of the basins of the Far Eastern seas, several long-lasting barriers existed during the regressions of the level of the World Ocean, separating the unified area of the species. One of them, extending along Sakhalin/Hokkaido/Honshu, kept the Japanese Sea and South Kuril populations separated for a long time. During this period of geographic isolation, the chromosome sets of individuals from the western Sea of Japan and the southern Kuril Islands could have formed independently of each other, despite their visual identity (cytotype V). One of the southern Kuril specimens has haplotype 1b, which connects all other haplotypes found in M. stelleri, and may thus be the closest to the ancestral haplotype. Contrary to the results of mtDNA analysis, the karyological data point to a significant divergence of M. stelleri (2n = 40) from the coastal waters off the southern Kuril Island. This discrepancy may be caused by the high rate of change in karyological traits compared to that of DNA markers (
Our results do not support the assumption that the individuals of M. stelleri from the Sea of Japan and the Sea of Okhotsk belong to different species (
Our data have revealed a similarity in chromosome sets, as well as low levels of differentiation in mtDNA between M. stelleri from the Sea of Okhotsk, the Sea of Japan, and the coast of Shikotan Island (southern Kuril Islands), thus, confirming that these represent a single, yet variable, species across its geographic range. The significant chromosomal polymorphism and the presence of common haplotypes in the studied samples indicate their recent origin from a common ancestor and/or relatively recent contacts within the range. The contribution of different Robertsonian translocations to the formation of cytotypes (II–IV and V) of individuals from the northern and western Sea of Japan allows us to conclude that they diverged from the Sea of Okhotsk M. stelleri independently of each other. The star-shaped structure of the haplotype network with a central ancestral haplotype indicates a connection between all constituent haplotypes and the ancestral position of the southern Kuril Islands haplotype (1b). The discrepancy in assessments of the divergence of individuals from the Sea of Okhotsk and waters off the southern Kuril Islands can be attributed to the different mechanisms of inheritance and rates of evolution of karyological traits and mtDNA markers.
The results of our study demonstrate the necessity of further detailed analysis of the widely sampled M. stelleri populations from the Pacific part of the species range and from the southern Sea of Okhotsk. Such studies should include differential chromosome staining and SNP markers, as well as comparative morphological and osteological analyses using up-to-date methods.
We are grateful to Dr. Igor A. Chereshnev, Andrey A. Balanov, and Vladimir V. Zemnukhov, as recognized experts in ichthyology, for their help with fish species identification, and also to Ruslan R. Yusupov for his assistance in preparing material for DNA analysis. We thank Aleksej V. Chernyshev, Evgeniy P. Shvetsov and Jacquelin De Faveri for proofreading the manuscript and useful comments.
The present study received a budgetary support within the framework of the Research Works “Fauna, systematics and ecology of marine and freshwater hydrobionts of the North-East of Russia” (State registry no. 117012710032-3, Ministry of Science and Higher Education of the Russian Federation, no. 0290-2019-0004), Institute of Biological Problems of the North, Far Eastern Branch, Russian Academy of Sciences, Russia, and “Biodiversity of the World Ocean: Composition and distribution of biota” (State registry no. 115081110047, Ministry of Science and Higher Education of the Russian Federation, no. 0268-2019-0007), A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russia.
I. N. M., collection and processing of material for karyological analysis, analysis of the obtained data and interpretation of the results. O. A. R., collection of material for DNA analysis, analysis of the obtained data and interpretation of the results. A. V. P., processing of material for DNA analysis (DNA isolate, amplify and sequence). I. N. M. and O. A. R. wrote and revised the manuscript. All authors discussed the results and approved the final manuscript.