Research Article |
Corresponding author: Weerayuth Supiwong ( supiwong@hotmail.com ) Academic editor: Nina Bogutskaya
© 2021 Pun Yeesin, Phichaya Buasriyot, Sukhonthip Ditcharoen, Patcharaporn Chaiyasan, Chatmongkon Suwannapoom, Sippakorn Juntaree, Sitthisak Jantarat, Sucheela Talumphai, Marcelo de Bello Cioffi, Thomas Liehr, Alongklod Tanomtong, Weerayuth Supiwong.
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:
Yeesin P, Buasriyot P, Ditcharoen S, Chaiyasan P, Suwannapoom C, Juntaree S, Jantarat S, Talumphai S, Cioffi MB, Liehr T, Tanomtong A, Supiwong W (2021) Comparative study of four Mystus species (Bagridae, Siluriformes) from Thailand: insights into their karyotypic diversity. Comparative Cytogenetics 15(2): 119-136. https://doi.org/10.3897/CompCytogen.v15.i2.60649
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Karyotypes of four catfishes of the genus Mystus Scopoli, 1777 (family Bagridae), M. atrifasciatus Fowler, 1937, M. mysticetus Roberts, 1992, M. singaringan (Bleeker, 1846) and M. wolffii (Bleeker, 1851), were analysed by conventional and Ag-NOR banding as well as fluorescence in situ hybridization (FISH) techniques. Microsatellite d(GC)15, d(CAA)10, d(CAT)10 and d(GAA)10 repeat probes were applied in FISH. The obtained data revealed that the four studied species have different chromosome complements. The diploid chromosome numbers (2n) and the fundamental numbers (NF) range between 52 and 102, 54 and 104, 56 and 98, or 58 and 108 in M. mysticetus, M. atrifasciatus, M. singaringan or M. wolffii, respectively. Karyotype formulae of M. mysticetus, M. atrifasciatus, M. singaringan and M. wolffii are 24m+26sm+4a, 26m+24sm+2a, 24m+18sm+14a and 30m+22sm+6a, respectively. A single pair of NORs was identified adjacent to the telomeres of the short arm of chromosome pairs 3 (metacentric) in M. atrifasciatus, 20 (submetacentric) in M. mysticetus, 15 (submetacentric) in M. singaringan, and 5 (metacentric) in M. wolffii. The d(GC)15, d(CAA)10, d(CAT)10 and d(GAA)10 repeats were abundantly distributed in species-specific patterns. Overall, we present a comparison of cytogenetic and molecular cytogenetic patterns of four species from genus Mystus providing insights into their karyotype diversity in the genus.
Chromosomes, fluorescence in situ hybridization (FISH), karyotype, Mystus
Bagridae are the largest family of Thai catfishes, with six genera (Bagrichthys Bleeker, 1857, Batasio Blyth, 1860, Hemibagrus Bleeker, 1862, Mystus Scopoli, 1777, Pseudomystus Jayaram, 1968, and Sperata Holly, 1939) and 28 species in Thailand. They play an important role in the national economic value of the country, as they are kept in aquaria and contribute heavily to the aquaculture industry. Most species of the genus Mystus are booming in aquaculture, with some of them being kept in aquaria (
Cytogenetic studies on Thai bagrids are quite scarce; as yet only conventional cytogenetics have been applied to determine chromosome numbers and karyotype complements. Therefore, their chromosomal evolution is not clear, even though from family Bagridae up to 45 species have been karyotyped so far. The diploid chromosome number (2n) varies between 2n = 44 [Coreobagrus brevicorpus Mori, 1936)] and 2n = 80 [Batasio fluviatilis (Day, 1888)]. The fundamental number (number of chromosome arms, NF) varies between 64 [for M. tengara (Hamilton, 1822) and M. vittatus (Bloch, 1794)] and 116 [for Horabagrus brachysoma (Günther, 1864) and H. nigricollaris Pethiyagoda et Kottelat, 1994] (
Focusing on the genus Mystus, chromosomal diversity and chromosomal variations among populations can be found. The so far reported 2n for diploid chromosome numbers varies between 50 and 58 chromosomes and for NF from 64 to 110 (Table
Comparative cytogenetics of Mystus genus (2n = diploid chromosome number, m = metacentric, sm = submetacentric, st = subtelocentric, a = acrocentric, t = telocentric, NOR = nucleolar organizer regions, NF = fundamental number, and – = not available).
Species | 2n | NF | Karyotype | NOR | Locality | Reference | |
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Mystus albolineatus Roberts, 1994 | 56 | 108 | 28m+6sm+12st+10a | - | Thailand (Ayutthaya) |
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M. atrifasciatus Fowler, 1937 | 54 | 92 | 30m+8sm+16a | - | Thailand (Nakhon Phanom) |
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54 | 96 | 24m+18sm+12st/a | Thailand (Bueng Kan) | Supiwong et al. (2014 a, b) | |||
54 | 104 | 24m+26sm +4a/t | 2 | Thailand (Maha Sarakham) | Present study | ||
M. bleekeri (Day, 1877) | 56 | 90 | 20m+14sm+10st+12a | - | India (Jammu) |
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56 | 102 | 32m+14sm+10a | - | India |
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M. bocourti (Bleeker, 1864) | 56 | 104 | 24m+18sm+6st+8a | - | Thailand (Nong Khai) |
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56 | 100 | 22m+22sm+12st/a | - | Thailand (Sing Buri) |
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M. cavasius (Hamilton, 1822) | 58 | 102 | 18m+16sm+10st+14a | 2 | India (Jammu) |
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58 | 108 | 18m+22sm+8t | - | India (Orissa) |
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58 | 102 | 14m+26sm+4st+14a | - | India (Bihar) |
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M. gulio (Hamilton, 1822) | 58 | 102 | 30m+12sm+2st+14a | 2 | India (West Bengal) |
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Female | 58 | 108 | 12m+34sm+4st+8t | - | India (Orissa) |
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Male | 58 | 110 | 13m+33sm+4st+8t | - | India (Orissa) |
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M. multiradiatus Roberts, 1992 | 54 | 98 | 30m+10sm+4st+10a | - | Thailand (Kanchana-buri) |
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54 | 96 | 18m+24sm+12st/a | - | Thailand (Maha Sarakham) | Supiwong et al. (2014 a, b) | ||
M. mysticetus Roberts, 1992 | 50 | 92 | 28m+14sm+8a | - | Thailand (Ayutthaya) |
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52 | 100 | 26m+22sm+4st/a | - | Thailand (Maha Sarakham) |
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52 | 102 | 26m+24sm+2a | 2 | Thailand (Bueng Kan) | Present study | ||
M. ngasep Darshan et al., 2011 | 56 | 90 | 12m+22sm+8st+14t | - | India (Manipur) |
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M. singaringan (Bleeker, 1846) | 56 | 94 | 24m+14sm+10st+8a | - | Thailand (Nakhonsawan) |
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56 | 98 | 24m+18sm+14a | 2 | Thailand (Sing Buri) | Present study | ||
M. tengara (Hamilton, 1822) | 54 | 64 | 10m+44a | - | India (Haryana) |
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Female | 54 | 101 | 9m+38sm+7a | - | India |
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Male | 54 | 102 | 10m+38sm+6a | - | (Haryana) |
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Female | 54 | 97 | 25m+18sm +11a | - | India |
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Male | 54 | 98 | 26m+18sm +10a | - | (Haryana) |
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M. vittatus (Bloch, 1794) | 54 | 108 | 22m+26sm+6st | - | India (Orissa) |
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54 | 108 | 22m+20sm+12st | - | India (Jammu) |
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58 | 110 | 10m+30sm+12st+6t | - | India (Orissa) |
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54 | 108 | 20m+24sm+10st | - | India |
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50 | 64 | 14m+36a | - | India |
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58 | 104 | 16m+10sm+20st+12a | - | India (West Bengal) |
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54 | 106 | 28m+22sm+2st+2a | 2 | India (Orissa) |
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54 | 78 | 6m+18sm+30a | - | India (Tamilnadu) |
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M. wolffii (Bleeker, 1851) | 58 | 100 | 26m+10sm+6st+16a | - | Thailand (Tak) |
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58 | 108 | 30m+22sm+6a | 2 | Thailand (Nakhon Sri Thammarat) | Present study |
Conventional cytogenetics may be sufficient to identify intra- and interspecific variations and is an inexpensive approach. However, it has restrictions, and accordingly the use of molecular cytogenetic analyses plays an increasing role for more precise characterization of the structure of genomes, including that of fishes. Especially, fluorescence in situ hybridization (FISH) for mapping of repetitive DNA sequences provided important contributions to the characterization of biodiversity and evolution in divergent fish groups (
In the present study, chromosomal structures and genetic markers for Thai populations of M. atrifasciatus Fowler, 1937, M. mysticetus, M. singaringan (Bleeker, 1846) and M. wolffii (Bleeker, 1851) (Fig.
Ten males and ten females of each species were collected from the Chi (Maha Sarakham Province), Songkhram (Bueng Kan Province), Chao Phraya (Sing Buri Province) and Pak Phanang Basins (Nakhon Sri Thammarat Province), Thailand from 2016–2018. The procedures followed ethical protocols as approved by the Institutional Animal Care and Use Committee of Khon Kaen University, based on the Ethics of Animal Experimentation of the National Research Council of Thailand ACUC-KKU-15/2559. Preparation of fish chromosomes from kidney cells was done as previously reported (
Microsatellites d(GC)15, d(CAA)10, d(CAT)10 and d(GAA)10 repeat probes (
The four studied Mystus species have different diploid chromosome numbers (2n) and fundamental numbers (NF) as follows: the 2n (NF) were 52 (102), 54 (104), 56 (98) and 58 (108) in M. mysticetus, M. atrifasciatus, M. singaringan and M. wolffii, respectively. The karyotypes of M. atrifasciatus (24m+26sm+4a), M. mysticetus (26m+24sm+2a), M. singaringan (24m+18sm+14a) and M. wolffii (30m+22sm+6a) were species-specific (Fig.
One single pair with NOR-bearing chromosomes was present in all four species analyzed. NOR positions were observed at regions adjacent to the telomere of the short arm of the chromosome pairs 3 (metacentric), 20 (submetacentric), 15 (submetacentric), and 5 (metacentric) in M. atrifasciatus, M. mysticetus, M. singaringan and M. wolffii, respectively (Figs
Idiograms representing shapes and sizes of chromosomes (haploid set) of A Mystus atrifasciatus B M. mysticetus C M. singaringan and D M. wolffii; arrows indicate NOR carrying chromosomes.
Cytogenetic and FISH studies on four Mystus fishes in Thailand (2n = diploid chromosome number, NF = fundamental number or number of chromosome arm, m = metacentric, sm = submetacentric, a = acrocentric, NOR = nucleolar organizer region, I = interstitial site, T = telomere, W = whole chromosome).
Mystus Species | 2n | NF | Chromosome type | Ag-NOR pair (type) | Microsatellite patterns | |||||
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m | sm | a | (GC)15 | (CAA)10 | (CAT)10 | (GAA)10 | ||||
M. atrifasciatus Fowler, 1937 | 54 | 104 | 24 | 26 | 4 | 3 (m) | T&I | T&I | T&I | I |
M. mysticetus Roberts,1992 | 52 | 102 | 26 | 24 | 2 | 20 (sm) | T&I | T&I | T&I | T&I |
M. singaringan (Bleeker, 1846) | 56 | 98 | 24 | 18 | 14 | 15 (sm) | T&I | T&I | T | T&I |
M. wolffii (Bleeker, 1851) | 58 | 108 | 30 | 22 | 6 | 5 (m) | W&T | T | W&T | T&I |
The mapping of d(GC)15, d(CAA)10, d(CAT)10 and d(GAA)10 microsatellites showed different hybridization signals among the species. The repeats of d(GC)15 and d(CAA)10 are abundantly distributed in the telomeric regions of several pairs and in interstitial sites of some chromosomes in M. atrifasciatus, M. mysticetus, M. singaringan. In contrast, in M. wolffii, d(GC)15 repeats are dispersed throughout all chromosomes, while d(CAA)10 repeats are accumulated at telomeric positions of some chromosome pairs with more density in only one pair. The d(CAT)10 repeats in M. atrifasciatus and M. mysticetus display high accumulations at the telomeric regions of almost all chromosomes and interstitial sites in some pairs whereas they have high accumulations at only the telomeric regions of almost all chromosomes in M. singaringan, and highly distributed in some chromosome pairs in M. wolffii. The d(GAA)10 repeats are abundantly distributed at interstitial and telomeric regions of several chromosome pairs in M. mysticetus, M. singaringan and M. wolffii, while they are highly accumulated in some chromosome pairs of M. atrifasciatus (Fig.
The diploid chromosome numbers (2n) in all analyzed species confirmed previous cytogenetic studies (
Karyotypes of the genus Mystus in Thailand showed high diversification (Table
The localization of nucleolus organizer regions (NORs) is a simple method to determine chromosomal marker. NORs are specific positions on the chromosome that consist of tandemly repeated sequences of ribosomal genes (rRNA). In eukaryotes, each unit is composed of three genes coding for 18S, 5.8S and 28S ribosomal RNA (
If these loci are active during the interphase before mitosis, they can be detected by silver nitrate staining (
Repetitive DNAs like microsatellites can be used to spot genomic evolution as previously been reported for different fish groups (
Here, four bi- and tri-nucleotide microsatellite sequences were mapped on chromosomes of four Mystus species. The patterns of microsatellites d(GC)15 and d(CAA)10 repeats in three species in the present study (M. atrifasciatus, M. mysticetus, M. singaringan) are similar to those found in Channa micropeltes (Cuvier, 1831) (
From previous reports, it may be carefully deduced that most heterochromatin in fish genomes consist of microsatellites (
Indeed, the distribution of microsatellite motifs in fish genomes could be biased to some specific noncoding regions, as found in the Asian swamp eel, M. albus (
The present research is the first report on NOR and microsatellites d(GC)15, d(CAA)10, d(CAT)10 and d(GAA)10 mapping in M. atrifasciatus, M. mysticetus, M. singaringan and M. wolffii. There are differences in the diploid chromosome number, the fundamental numbers, karyotypes, pairs having NORs, and patterns of microsatellite distributions on chromosomes. These results indicated that (molecular) cytogenetic data can be used for classification in related fish species and to explain karyotype diversification.
This work was financially supported by the Research Fund for DPST Graduate with First Placement Year 2015, Development Promotion of Science and Technology Talents project (DPST), the Post-Doctoral Training Program from Research Affairs and Graduate School (Grant no 59255), Khon Kaen University, and Unit of Excellence 2020 on Biodiversity and Natural Resources Management, University of Phayao (UoE63005), Thailand.