Evidence of Interspecific Chromosomal Diversification in Rainbowfishes (Melanotaeniidae, Teleostei)

Rainbowfishes (Melanotaeniidae) are the largest monophyletic group of freshwater fishes occurring in Australia and New Guinea, with 112 species currently recognised. Despite their high taxonomic diversity, rainbowfishes remain poorly studied from a cytogenetic perspective. Using conventional (Giemsa staining, C banding, chromomycin A3 staining) and molecular (fluorescence in situ hybridisation with ribosomal DNA (rDNA) and telomeric probes) cytogenetic protocols, karyotypes and associated chromosomal characteristics of five species were examined. We covered all major lineages of this group, namely, Running River rainbowfish Melanotaenia sp., red rainbowfish Glossolepis incisus, threadfin rainbowfish Iriatherina werneri, ornate rainbowfish Rhadinocentrus ornatus, and Cairns rainbowfish Cairnsichthys rhombosomoides. All species had conserved diploid chromosome numbers 2n = 48, but karyotypes differed among species; while Melanotaenia sp., G. incisus, and I. werneri possessed karyotypes composed of exclusively subtelo/acrocentric chromosomes, the karyotype of R. ornatus displayed six pairs of submetacentric and 18 pairs of subtelo/acrocentric chromosomes, while C. rhombosomoides possessed a karyotype composed of four pairs of submetacentric and 20 pairs of subtelo/acrocentric chromosomes. No heteromorphic sex chromosomes were detected using conventional cytogenetic techniques. Our data indicate a conserved 2n in Melanotaeniidae, but morphologically variable karyotypes, rDNA sites, and heterochromatin distributions. Differences were observed especially in taxonomically divergent species, suggesting interspecies chromosome rearrangements.


Introduction
The genomes of teleost fishes display remarkable features (e.g., variability in size or basic chromosome organisation) that might be involved in the formation of their immense species diversity. Access to whole-genome sequences provides important insights into the gene occurrence and organisation within a species, and it revolutionises our understanding of how genetic information is We examined 25 individuals from five species as follows: three males and three females of Melanotaenia sp. (F1 captive bred fish from Running River, Queensland, Australia); five males and two females of I. werneri (obtained via the European aquarium trade); two males and two females from G. incisus (obtained via the Australian aquarium trade); two males and two females from R. ornatus (large captive population originally from Spring Creek, Carindale, Queensland, Australia); two males and two females from C. rhombosomoides (wild fish from Ninds Creek, Queensland, Australia). Wild samples were obtained under state fishery permits, and research was conducted with approval from the University of Canberra Ethics Committee (CEAE. . More information about the number of cells is listed in Table  1.

Chromosome Preparation and Staining
Metaphase chromosomes were prepared according to Bertollo et al. [19] with slight modifications. Briefly, fish were injected with 0.1% colchicine solution (1 mL/100 g of body weight) 45 min before being sacrificed using an overdose of anaesthetic (clove oil). The kidneys were dissected in 0.075 M KCl at room temperature. The cell suspension free of tissue fragments was hypotonised for 30 min in 0.075 M KCl, fixed in freshly prepared fixative (methanol: acetic acid 3:1, v/v), washed twice in fixative, and finally spread onto slides. Chromosomal preparations from all individuals were stained with conventional Giemsa solution (5%, 10 min) or DAPI (4′,6-diamidino-2-phenylindole) in order to confirm number and morphology of their chromosomes. C banding, visualising blocks of constitutive heterochromatin, was performed according to Sumner [20] with slight modifications described in Pokorná et al. [21]. After C-banding, the chromosomes were counterstained by DAPI to enhance the contrast, and the images were captured in the fluorescent regime and inverted. We applied reversed fluorescent staining with chromomycin A3 (CMA3) specific for GC-rich regions, counterstained with DAPI, with a higher affinity for AT-rich regions [22].

Fluorescence in Situ Hybridisation (FISH) with Telomeric and rRNA Genes Probes
The organisation of the telomeric motif (TTAGGG)n, as well as rDNA genes, within the genomes was analysed by fluorescence in situ hybridisation (FISH). Telomeric FISH was performed using a Cy3-labelled PNA (peptide nucleic acid) probe according to the manufacturer's instructions (Telomere PNA FISH Kit/Cy3, Dako). Probes for FISH experiments were produced by PCR with primer pairs and thermal cycling conditions according to Komiya et al. [23] for 5S rDNA and Zhang et al. [24] for 28S rDNA. The PCR reactions were carried out in a final volume of 25 μL consisting of 100 ng of genomic DNA of C. rhombosomoides, 12.5 μL of PPP master mix, 0.01 mM of each primer, and PCR water to complete the volume (all reagents from TopBio, Prague, Czech Republic). Probes were indirectly labelled with biotin-16-dUTP (Roche, Mannheim, Germany) and digoxigenin-11-dUTP (Roche) through PCR reamplification of PCR products. Reamplification was carried out under ; 3 Genes 2020, 11, x FOR PEER REVIEW 3 of 12 We examined 25 individuals from five species as follows: three males and three females of Melanotaenia sp. (F1 captive bred fish from Running River, Queensland, Australia); five males and two females of I. werneri (obtained via the European aquarium trade); two males and two females from G. incisus (obtained via the Australian aquarium trade); two males and two females from R. ornatus (large captive population originally from Spring Creek, Carindale, Queensland, Australia); two males and two females from C. rhombosomoides (wild fish from Ninds Creek, Queensland, Australia). Wild samples were obtained under state fishery permits, and research was conducted with approval from the University of Canberra Ethics Committee (CEAE. . More information about the number of cells is listed in Table  1.

Chromosome Preparation and Staining
Metaphase chromosomes were prepared according to Bertollo et al. [19] with slight modifications. Briefly, fish were injected with 0.1% colchicine solution (1 mL/100 g of body weight) 45 min before being sacrificed using an overdose of anaesthetic (clove oil). The kidneys were dissected in 0.075 M KCl at room temperature. The cell suspension free of tissue fragments was hypotonised for 30 min in 0.075 M KCl, fixed in freshly prepared fixative (methanol: acetic acid 3:1, v/v), washed twice in fixative, and finally spread onto slides. Chromosomal preparations from all individuals were stained with conventional Giemsa solution (5%, 10 min) or DAPI (4′,6-diamidino-2-phenylindole) in order to confirm number and morphology of their chromosomes. C banding, visualising blocks of constitutive heterochromatin, was performed according to Sumner [20] with slight modifications described in Pokorná et al. [21]. After C-banding, the chromosomes were counterstained by DAPI to enhance the contrast, and the images were captured in the fluorescent regime and inverted. We applied reversed fluorescent staining with chromomycin A3 (CMA3) specific for GC-rich regions, counterstained with DAPI, with a higher affinity for AT-rich regions [22].

Fluorescence in Situ Hybridisation (FISH) with Telomeric and rRNA Genes Probes
The organisation of the telomeric motif (TTAGGG)n, as well as rDNA genes, within the genomes was analysed by fluorescence in situ hybridisation (FISH). Telomeric FISH was performed using a Cy3-labelled PNA (peptide nucleic acid) probe according to the manufacturer's instructions (Telomere PNA FISH Kit/Cy3, Dako). Probes for FISH experiments were produced by PCR with primer pairs and thermal cycling conditions according to Komiya et al. [23] for 5S rDNA and Zhang et al. [24] for 28S rDNA. The PCR reactions were carried out in a final volume of 25 μL consisting of 100 ng of genomic DNA of C. rhombosomoides, 12.5 μL of PPP master mix, 0.01 mM of each primer, and PCR water to complete the volume (all reagents from TopBio, Prague, Czech Republic). Probes were indirectly labelled with biotin-16-dUTP (Roche, Mannheim, Germany) and digoxigenin-11-dUTP (Roche) through PCR reamplification of PCR products. Reamplification was carried out under 50 13 31 10 15

Glossolepis incisus 2
Genes 2020, 11, x FOR PEER REVIEW 3 of 12 We examined 25 individuals from five species as follows: three males and three females of Melanotaenia sp. (F1 captive bred fish from Running River, Queensland, Australia); five males and two females of I. werneri (obtained via the European aquarium trade); two males and two females from G. incisus (obtained via the Australian aquarium trade); two males and two females from R. ornatus (large captive population originally from Spring Creek, Carindale, Queensland, Australia); two males and two females from C. rhombosomoides (wild fish from Ninds Creek, Queensland, Australia). Wild samples were obtained under state fishery permits, and research was conducted with approval from the University of Canberra Ethics Committee (CEAE. . More information about the number of cells is listed in Table  1.

Chromosome Preparation and Staining
Metaphase chromosomes were prepared according to Bertollo et al. [19] with slight modifications. Briefly, fish were injected with 0.1% colchicine solution (1 mL/100 g of body weight) 45 min before being sacrificed using an overdose of anaesthetic (clove oil). The kidneys were dissected in 0.075 M KCl at room temperature. The cell suspension free of tissue fragments was hypotonised for 30 min in 0.075 M KCl, fixed in freshly prepared fixative (methanol: acetic acid 3:1, v/v), washed twice in fixative, and finally spread onto slides. Chromosomal preparations from all individuals were stained with conventional Giemsa solution (5%, 10 min) or DAPI (4′,6-diamidino-2-phenylindole) in order to confirm number and morphology of their chromosomes. C banding, visualising blocks of constitutive heterochromatin, was performed according to Sumner [20] with slight modifications described in Pokorná et al. [21]. After C-banding, the chromosomes were counterstained by DAPI to enhance the contrast, and the images were captured in the fluorescent regime and inverted. We applied reversed fluorescent staining with chromomycin A3 (CMA3) specific for GC-rich regions, counterstained with DAPI, with a higher affinity for AT-rich regions [22].

Fluorescence in Situ Hybridisation (FISH) with Telomeric and rRNA Genes Probes
The organisation of the telomeric motif (TTAGGG)n, as well as rDNA genes, within the genomes was analysed by fluorescence in situ hybridisation (FISH). Telomeric FISH was performed using a Cy3-labelled PNA (peptide nucleic acid) probe according to the manufacturer's instructions (Telomere PNA FISH Kit/Cy3, Dako). Probes for FISH experiments were produced by PCR with primer pairs and thermal cycling conditions according to Komiya et al. [23] for 5S rDNA and Zhang et al. [24] for 28S rDNA. The PCR reactions were carried out in a final volume of 25 μL consisting of 100 ng of genomic DNA of C. rhombosomoides, 12.5 μL of PPP master mix, 0.01 mM of each primer, and PCR water to complete the volume (all reagents from TopBio, Prague, Czech Republic). Probes were indirectly labelled with biotin-16-dUTP (Roche, Mannheim, Germany) and digoxigenin-11-dUTP (Roche) through PCR reamplification of PCR products. Reamplification was carried out under ; 2 Genes 2020, 11, x FOR PEER REVIEW 3 of 12 We examined 25 individuals from five species as follows: three males and three females of Melanotaenia sp. (F1 captive bred fish from Running River, Queensland, Australia); five males and two females of I. werneri (obtained via the European aquarium trade); two males and two females from G. incisus (obtained via the Australian aquarium trade); two males and two females from R. ornatus (large captive population originally from Spring Creek, Carindale, Queensland, Australia); two males and two females from C. rhombosomoides (wild fish from Ninds Creek, Queensland, Australia). Wild samples were obtained under state fishery permits, and research was conducted with approval from the University of Canberra Ethics Committee (CEAE. . More information about the number of cells is listed in Table  1.

Chromosome Preparation and Staining
Metaphase chromosomes were prepared according to Bertollo et al. [19] with slight modifications. Briefly, fish were injected with 0.1% colchicine solution (1 mL/100 g of body weight) 45 min before being sacrificed using an overdose of anaesthetic (clove oil). The kidneys were dissected in 0.075 M KCl at room temperature. The cell suspension free of tissue fragments was hypotonised for 30 min in 0.075 M KCl, fixed in freshly prepared fixative (methanol: acetic acid 3:1, v/v), washed twice in fixative, and finally spread onto slides. Chromosomal preparations from all individuals were stained with conventional Giemsa solution (5%, 10 min) or DAPI (4′,6-diamidino-2-phenylindole) in order to confirm number and morphology of their chromosomes. C banding, visualising blocks of constitutive heterochromatin, was performed according to Sumner [20] with slight modifications described in Pokorná et al. [21]. After C-banding, the chromosomes were counterstained by DAPI to enhance the contrast, and the images were captured in the fluorescent regime and inverted. We applied reversed fluorescent staining with chromomycin A3 (CMA3) specific for GC-rich regions, counterstained with DAPI, with a higher affinity for AT-rich regions [22].

Fluorescence in Situ Hybridisation (FISH) with Telomeric and rRNA Genes Probes
The organisation of the telomeric motif (TTAGGG)n, as well as rDNA genes, within the genomes was analysed by fluorescence in situ hybridisation (FISH). Telomeric FISH was performed using a Cy3-labelled PNA (peptide nucleic acid) probe according to the manufacturer's instructions (Telomere PNA FISH Kit/Cy3, Dako). Probes for FISH experiments were produced by PCR with primer pairs and thermal cycling conditions according to Komiya et al. [23] for 5S rDNA and Zhang et al. [24] for 28S rDNA. The PCR reactions were carried out in a final volume of 25 μL consisting of 100 ng of genomic DNA of C. rhombosomoides, 12.5 μL of PPP master mix, 0.01 mM of each primer, and PCR water to complete the volume (all reagents from TopBio, Prague, Czech Republic). Probes were indirectly labelled with biotin-16-dUTP (Roche, Mannheim, Germany) and digoxigenin-11-dUTP (Roche) through PCR reamplification of PCR products. Reamplification was carried out under 22 21 13 12 16

Iriatherina werneri 2
Genes 2020, 11, x FOR PEER REVIEW 3 of 12 We examined 25 individuals from five species as follows: three males and three females of Melanotaenia sp. (F1 captive bred fish from Running River, Queensland, Australia); five males and two females of I. werneri (obtained via the European aquarium trade); two males and two females from G. incisus (obtained via the Australian aquarium trade); two males and two females from R. ornatus (large captive population originally from Spring Creek, Carindale, Queensland, Australia); two males and two females from C. rhombosomoides (wild fish from Ninds Creek, Queensland, Australia). Wild samples were obtained under state fishery permits, and research was conducted with approval from the University of Canberra Ethics Committee (CEAE. . More information about the number of cells is listed in Table  1.

Chromosome Preparation and Staining
Metaphase chromosomes were prepared according to Bertollo et al. [19] with slight modifications. Briefly, fish were injected with 0.1% colchicine solution (1 mL/100 g of body weight) 45 min before being sacrificed using an overdose of anaesthetic (clove oil). The kidneys were dissected in 0.075 M KCl at room temperature. The cell suspension free of tissue fragments was hypotonised for 30 min in 0.075 M KCl, fixed in freshly prepared fixative (methanol: acetic acid 3:1, v/v), washed twice in fixative, and finally spread onto slides. Chromosomal preparations from all individuals were stained with conventional Giemsa solution (5%, 10 min) or DAPI (4′,6-diamidino-2-phenylindole) in order to confirm number and morphology of their chromosomes. C banding, visualising blocks of constitutive heterochromatin, was performed according to Sumner [20] with slight modifications described in Pokorná et al. [21]. After C-banding, the chromosomes were counterstained by DAPI to enhance the contrast, and the images were captured in the fluorescent regime and inverted. We applied reversed fluorescent staining with chromomycin A3 (CMA3) specific for GC-rich regions, counterstained with DAPI, with a higher affinity for AT-rich regions [22].

Fluorescence in Situ Hybridisation (FISH) with Telomeric and rRNA Genes Probes
The organisation of the telomeric motif (TTAGGG)n, as well as rDNA genes, within the genomes was analysed by fluorescence in situ hybridisation (FISH). Telomeric FISH was performed using a Cy3-labelled PNA (peptide nucleic acid) probe according to the manufacturer's instructions (Telomere PNA FISH Kit/Cy3, Dako). Probes for FISH experiments were produced by PCR with primer pairs and thermal cycling conditions according to Komiya et al. [23] for 5S rDNA and Zhang et al. [24] for 28S rDNA. The PCR reactions were carried out in a final volume of 25 μL consisting of 100 ng of genomic DNA of C. rhombosomoides, 12.5 μL of PPP master mix, 0.01 mM of each primer, and PCR water to complete the volume (all reagents from TopBio, Prague, Czech Republic). Probes were indirectly labelled with biotin-16-dUTP (Roche, Mannheim, Germany) and digoxigenin-11-dUTP (Roche) through PCR reamplification of PCR products. Reamplification was carried out under ; 5 Genes 2020, 11, x FOR PEER REVIEW 3 of 12 We examined 25 individuals from five species as follows: three males and three females of Melanotaenia sp. (F1 captive bred fish from Running River, Queensland, Australia); five males and two females of I. werneri (obtained via the European aquarium trade); two males and two females from G. incisus (obtained via the Australian aquarium trade); two males and two females from R. ornatus (large captive population originally from Spring Creek, Carindale, Queensland, Australia); two males and two females from C. rhombosomoides (wild fish from Ninds Creek, Queensland, Australia). Wild samples were obtained under state fishery permits, and research was conducted with approval from the University of Canberra Ethics Committee (CEAE. . More information about the number of cells is listed in Table  1.

Chromosome Preparation and Staining
Metaphase chromosomes were prepared according to Bertollo et al. [19] with slight modifications. Briefly, fish were injected with 0.1% colchicine solution (1 mL/100 g of body weight) 45 min before being sacrificed using an overdose of anaesthetic (clove oil). The kidneys were dissected in 0.075 M KCl at room temperature. The cell suspension free of tissue fragments was hypotonised for 30 min in 0.075 M KCl, fixed in freshly prepared fixative (methanol: acetic acid 3:1, v/v), washed twice in fixative, and finally spread onto slides. Chromosomal preparations from all individuals were stained with conventional Giemsa solution (5%, 10 min) or DAPI (4′,6-diamidino-2-phenylindole) in order to confirm number and morphology of their chromosomes. C banding, visualising blocks of constitutive heterochromatin, was performed according to Sumner [20] with slight modifications described in Pokorná et al. [21]. After C-banding, the chromosomes were counterstained by DAPI to enhance the contrast, and the images were captured in the fluorescent regime and inverted. We applied reversed fluorescent staining with chromomycin A3 (CMA3) specific for GC-rich regions, counterstained with DAPI, with a higher affinity for AT-rich regions [22].

Fluorescence in Situ Hybridisation (FISH) with Telomeric and rRNA Genes Probes
The organisation of the telomeric motif (TTAGGG)n, as well as rDNA genes, within the genomes was analysed by fluorescence in situ hybridisation (FISH). Telomeric FISH was performed using a Cy3-labelled PNA (peptide nucleic acid) probe according to the manufacturer's instructions (Telomere PNA FISH Kit/Cy3, Dako). Probes for FISH experiments were produced by PCR with primer pairs and thermal cycling conditions according to Komiya et al. [23] for 5S rDNA and Zhang et al. [24] for 28S rDNA. The PCR reactions were carried out in a final volume of 25 μL consisting of 100 ng of genomic DNA of C. rhombosomoides, 12.5 μL of PPP master mix, 0.01 mM of each primer, and PCR water to complete the volume (all reagents from TopBio, Prague, Czech Republic). Probes were indirectly labelled with biotin-16-dUTP (Roche, Mannheim, Germany) and digoxigenin-11-dUTP (Roche) through PCR reamplification of PCR products. Reamplification was carried out under 25 35 30 35 28

Rhadinocentrus ornatus 2
Genes 2020, 11, x FOR PEER REVIEW 3 of 12 We examined 25 individuals from five species as follows: three males and three females of Melanotaenia sp. (F1 captive bred fish from Running River, Queensland, Australia); five males and two females of I. werneri (obtained via the European aquarium trade); two males and two females from G. incisus (obtained via the Australian aquarium trade); two males and two females from R. ornatus (large captive population originally from Spring Creek, Carindale, Queensland, Australia); two males and two females from C. rhombosomoides (wild fish from Ninds Creek, Queensland, Australia). Wild samples were obtained under state fishery permits, and research was conducted with approval from the University of Canberra Ethics Committee (CEAE. . More information about the number of cells is listed in Table  1.

Chromosome Preparation and Staining
Metaphase chromosomes were prepared according to Bertollo et al. [19] with slight modifications. Briefly, fish were injected with 0.1% colchicine solution (1 mL/100 g of body weight) 45 min before being sacrificed using an overdose of anaesthetic (clove oil). The kidneys were dissected in 0.075 M KCl at room temperature. The cell suspension free of tissue fragments was hypotonised for 30 min in 0.075 M KCl, fixed in freshly prepared fixative (methanol: acetic acid 3:1, v/v), washed twice in fixative, and finally spread onto slides. Chromosomal preparations from all individuals were stained with conventional Giemsa solution (5%, 10 min) or DAPI (4′,6-diamidino-2-phenylindole) in order to confirm number and morphology of their chromosomes. C banding, visualising blocks of constitutive heterochromatin, was performed according to Sumner [20] with slight modifications described in Pokorná et al. [21]. After C-banding, the chromosomes were counterstained by DAPI to enhance the contrast, and the images were captured in the fluorescent regime and inverted. We applied reversed fluorescent staining with chromomycin A3 (CMA3) specific for GC-rich regions, counterstained with DAPI, with a higher affinity for AT-rich regions [22].

Fluorescence in Situ Hybridisation (FISH) with Telomeric and rRNA Genes Probes
The organisation of the telomeric motif (TTAGGG)n, as well as rDNA genes, within the genomes was analysed by fluorescence in situ hybridisation (FISH). Telomeric FISH was performed using a Cy3-labelled PNA (peptide nucleic acid) probe according to the manufacturer's instructions (Telomere PNA FISH Kit/Cy3, Dako). Probes for FISH experiments were produced by PCR with primer pairs and thermal cycling conditions according to Komiya et al. [23] for 5S rDNA and Zhang et al. [24] for 28S rDNA. The PCR reactions were carried out in a final volume of 25 μL consisting of 100 ng of genomic DNA of C. rhombosomoides, 12.5 μL of PPP master mix, 0.01 mM of each primer, and PCR water to complete the volume (all reagents from TopBio, Prague, Czech Republic). Probes were indirectly labelled with biotin-16-dUTP (Roche, Mannheim, Germany) and digoxigenin-11-dUTP (Roche) through PCR reamplification of PCR products. Reamplification was carried out under ; 2 Genes 2020, 11, x FOR PEER REVIEW 3 of 12 We examined 25 individuals from five species as follows: three males and three females of Melanotaenia sp. (F1 captive bred fish from Running River, Queensland, Australia); five males and two females of I. werneri (obtained via the European aquarium trade); two males and two females from G. incisus (obtained via the Australian aquarium trade); two males and two females from R. ornatus (large captive population originally from Spring Creek, Carindale, Queensland, Australia); two males and two females from C. rhombosomoides (wild fish from Ninds Creek, Queensland, Australia). Wild samples were obtained under state fishery permits, and research was conducted with approval from the University of Canberra Ethics Committee (CEAE. . More information about the number of cells is listed in Table  1.

Chromosome Preparation and Staining
Metaphase chromosomes were prepared according to Bertollo et al. [19] with slight modifications. Briefly, fish were injected with 0.1% colchicine solution (1 mL/100 g of body weight) 45 min before being sacrificed using an overdose of anaesthetic (clove oil). The kidneys were dissected in 0.075 M KCl at room temperature. The cell suspension free of tissue fragments was hypotonised for 30 min in 0.075 M KCl, fixed in freshly prepared fixative (methanol: acetic acid 3:1, v/v), washed twice in fixative, and finally spread onto slides. Chromosomal preparations from all individuals were stained with conventional Giemsa solution (5%, 10 min) or DAPI (4′,6-diamidino-2-phenylindole) in order to confirm number and morphology of their chromosomes. C banding, visualising blocks of constitutive heterochromatin, was performed according to Sumner [20] with slight modifications described in Pokorná et al. [21]. After C-banding, the chromosomes were counterstained by DAPI to enhance the contrast, and the images were captured in the fluorescent regime and inverted. We applied reversed fluorescent staining with chromomycin A3 (CMA3) specific for GC-rich regions, counterstained with DAPI, with a higher affinity for AT-rich regions [22].

Fluorescence in Situ Hybridisation (FISH) with Telomeric and rRNA Genes Probes
The organisation of the telomeric motif (TTAGGG)n, as well as rDNA genes, within the genomes was analysed by fluorescence in situ hybridisation (FISH). Telomeric FISH was performed using a Cy3-labelled PNA (peptide nucleic acid) probe according to the manufacturer's instructions (Telomere PNA FISH Kit/Cy3, Dako). Probes for FISH experiments were produced by PCR with primer pairs and thermal cycling conditions according to Komiya et al. [23] for 5S rDNA and Zhang et al. [24] for 28S rDNA. The PCR reactions were carried out in a final volume of 25 μL consisting of 100 ng of genomic DNA of C. rhombosomoides, 12.5 μL of PPP master mix, 0.01 mM of each primer, and PCR water to complete the volume (all reagents from TopBio, Prague, Czech Republic). Probes were indirectly labelled with biotin-16-dUTP (Roche, Mannheim, Germany) and digoxigenin-11-dUTP (Roche) through PCR reamplification of PCR products. Reamplification was carried out under 40 14 10 59 22

Cairnsichthys rhombosomoides 2
Genes 2020, 11, x FOR PEER REVIEW 3 of 12 We examined 25 individuals from five species as follows: three males and three females of Melanotaenia sp. (F1 captive bred fish from Running River, Queensland, Australia); five males and two females of I. werneri (obtained via the European aquarium trade); two males and two females from G. incisus (obtained via the Australian aquarium trade); two males and two females from R. ornatus (large captive population originally from Spring Creek, Carindale, Queensland, Australia); two males and two females from C. rhombosomoides (wild fish from Ninds Creek, Queensland, Australia). Wild samples were obtained under state fishery permits, and research was conducted with approval from the University of Canberra Ethics Committee (CEAE. . More information about the number of cells is listed in Table  1.

Chromosome Preparation and Staining
Metaphase chromosomes were prepared according to Bertollo et al. [19] with slight modifications. Briefly, fish were injected with 0.1% colchicine solution (1 mL/100 g of body weight) 45 min before being sacrificed using an overdose of anaesthetic (clove oil). The kidneys were dissected in 0.075 M KCl at room temperature. The cell suspension free of tissue fragments was hypotonised for 30 min in 0.075 M KCl, fixed in freshly prepared fixative (methanol: acetic acid 3:1, v/v), washed twice in fixative, and finally spread onto slides. Chromosomal preparations from all individuals were stained with conventional Giemsa solution (5%, 10 min) or DAPI (4′,6-diamidino-2-phenylindole) in order to confirm number and morphology of their chromosomes. C banding, visualising blocks of constitutive heterochromatin, was performed according to Sumner [20] with slight modifications described in Pokorná et al. [21]. After C-banding, the chromosomes were counterstained by DAPI to enhance the contrast, and the images were captured in the fluorescent regime and inverted. We applied reversed fluorescent staining with chromomycin A3 (CMA3) specific for GC-rich regions, counterstained with DAPI, with a higher affinity for AT-rich regions [22].

Fluorescence in Situ Hybridisation (FISH) with Telomeric and rRNA Genes Probes
The organisation of the telomeric motif (TTAGGG)n, as well as rDNA genes, within the genomes was analysed by fluorescence in situ hybridisation (FISH). Telomeric FISH was performed using a Cy3-labelled PNA (peptide nucleic acid) probe according to the manufacturer's instructions (Telomere PNA FISH Kit/Cy3, Dako). Probes for FISH experiments were produced by PCR with primer pairs and thermal cycling conditions according to Komiya et al. [23] for 5S rDNA and Zhang et al. [24] for 28S rDNA. The PCR reactions were carried out in a final volume of 25 μL consisting of 100 ng of genomic DNA of C. rhombosomoides, 12.5 μL of PPP master mix, 0.01 mM of each primer, and PCR water to complete the volume (all reagents from TopBio, Prague, Czech Republic). Probes were indirectly labelled with biotin-16-dUTP (Roche, Mannheim, Germany) and digoxigenin-11-dUTP (Roche) through PCR reamplification of PCR products. Reamplification was carried out under ; 2 Genes 2020, 11, x FOR PEER REVIEW 3 of 12 We examined 25 individuals from five species as follows: three males and three females of Melanotaenia sp. (F1 captive bred fish from Running River, Queensland, Australia); five males and two females of I. werneri (obtained via the European aquarium trade); two males and two females from G. incisus (obtained via the Australian aquarium trade); two males and two females from R. ornatus (large captive population originally from Spring Creek, Carindale, Queensland, Australia); two males and two females from C. rhombosomoides (wild fish from Ninds Creek, Queensland, Australia). Wild samples were obtained under state fishery permits, and research was conducted with approval from the University of Canberra Ethics Committee (CEAE. . More information about the number of cells is listed in Table  1.

Chromosome Preparation and Staining
Metaphase chromosomes were prepared according to Bertollo et al. [19] with slight modifications. Briefly, fish were injected with 0.1% colchicine solution (1 mL/100 g of body weight) 45 min before being sacrificed using an overdose of anaesthetic (clove oil). The kidneys were dissected in 0.075 M KCl at room temperature. The cell suspension free of tissue fragments was hypotonised for 30 min in 0.075 M KCl, fixed in freshly prepared fixative (methanol: acetic acid 3:1, v/v), washed twice in fixative, and finally spread onto slides. Chromosomal preparations from all individuals were stained with conventional Giemsa solution (5%, 10 min) or DAPI (4′,6-diamidino-2-phenylindole) in order to confirm number and morphology of their chromosomes. C banding, visualising blocks of constitutive heterochromatin, was performed according to Sumner [20] with slight modifications described in Pokorná et al. [21]. After C-banding, the chromosomes were counterstained by DAPI to enhance the contrast, and the images were captured in the fluorescent regime and inverted. We applied reversed fluorescent staining with chromomycin A3 (CMA3) specific for GC-rich regions, counterstained with DAPI, with a higher affinity for AT-rich regions [22].

Fluorescence in Situ Hybridisation (FISH) with Telomeric and rRNA Genes Probes
The organisation of the telomeric motif (TTAGGG)n, as well as rDNA genes, within the genomes was analysed by fluorescence in situ hybridisation (FISH). Telomeric FISH was performed using a Cy3-labelled PNA (peptide nucleic acid) probe according to the manufacturer's instructions (Telomere PNA FISH Kit/Cy3, Dako). Probes for FISH experiments were produced by PCR with primer pairs and thermal cycling conditions according to Komiya et al. [23] for 5S rDNA and Zhang et al. [24] for 28S rDNA. The PCR reactions were carried out in a final volume of 25 μL consisting of 100 ng of genomic DNA of C. rhombosomoides, 12.5 μL of PPP master mix, 0.01 mM of each primer, and PCR water to complete the volume (all reagents from TopBio, Prague, Czech Republic). Probes were indirectly labelled with biotin-16-dUTP (Roche, Mannheim, Germany) and digoxigenin-11-dUTP (Roche) through PCR reamplification of PCR products. Reamplification was carried out under 80 40 15 14 28 CMA3-chromomycin A3, FISH-fluorescence in situ hybridisation.

Chromosome Preparation and Staining
Metaphase chromosomes were prepared according to Bertollo et al. [19] with slight modifications. Briefly, fish were injected with 0.1% colchicine solution (1 mL/100 g of body weight) 45 min before being sacrificed using an overdose of anaesthetic (clove oil). The kidneys were dissected in 0.075 M KCl at room temperature. The cell suspension free of tissue fragments was hypotonised for 30 min in 0.075 M KCl, fixed in freshly prepared fixative (methanol: acetic acid 3:1, v/v), washed twice in fixative, and finally spread onto slides. Chromosomal preparations from all individuals were stained with conventional Giemsa solution (5%, 10 min) or DAPI (4 ,6-diamidino-2-phenylindole) in order to confirm number and morphology of their chromosomes. C banding, visualising blocks of constitutive heterochromatin, was performed according to Sumner [20] with slight modifications described in Pokorná et al. [21]. After C-banding, the chromosomes were counterstained by DAPI to enhance the contrast, and the images were captured in the fluorescent regime and inverted. We applied reversed fluorescent staining with chromomycin A 3 (CMA 3 ) specific for GC-rich regions, counterstained with DAPI, with a higher affinity for AT-rich regions [22].

Fluorescence In Situ Hybridisation (FISH) with Telomeric and rRNA Genes Probes
The organisation of the telomeric motif (TTAGGG) n , as well as rDNA genes, within the genomes was analysed by fluorescence in situ hybridisation (FISH). Telomeric FISH was performed using a Cy3-labelled PNA (peptide nucleic acid) probe according to the manufacturer's instructions (Telomere PNA FISH Kit/Cy3, Dako). Probes for FISH experiments were produced by PCR with primer pairs and thermal cycling conditions according to Komiya et al. [23] for 5S rDNA and Zhang et al. [24] for 28S rDNA. The PCR reactions were carried out in a final volume of 25 µL consisting of 100 ng of genomic DNA of C. rhombosomoides, 12.5 µL of PPP master mix, 0.01 mM of each primer, and PCR water to complete the volume (all reagents from TopBio, Prague, Czech Republic). Probes were indirectly labelled with biotin-16-dUTP (Roche, Mannheim, Germany) and digoxigenin-11-dUTP (Roche) through PCR reamplification of PCR products. Reamplification was carried out under the same conditions as the previous PCR reaction. Labelled PCR products were precipitated. The hybridisation mixture consisted of hybridisation buffer [25] and differently labelled PCR products of both genes.
The hybridisation and detection procedures were carried out under conditions described in Symonová et al. [25]. The biotin-dUTP-labelled probes were detected by the Invitrogen Cy™3-Streptavidin (Invitrogen, San Diego, CA, USA; cat. no. , while the digoxigenin-dUTP-labelled probes were detected by the Roche Anti-Digoxigenin-Fluorescein (cat. no. 11207741910). Finally, the slides were mounted with Vectashield DAPI anti-fade medium (Vector Laboratories, Burlingame, CA, USA).

Microscopy and Image Analyses
Chromosomal preparations were examined using an Olympus Provis AX 70 epifluorescence microscope (Olympus, Tokyo, Japan). Images of metaphase chromosomes were recorded with a cooled Olympus DP30BW CCD camera. The IKAROS and ISIS imaging programs (Metasystems, Altlussheim, Germany) were used to analyse grey-scale images. The captured digital images from FISH experiments were pseudocoloured (red for Anti-Digoxigenin-Rhodamine, green for Invitrogen FITC-Streptavidin) and superimposed using Adobe Photoshop software, version CS5. In the case of CMA 3 /DAPI staining, the CMA 3 signal was inverted into the red and the DAPI signal into the green channel to enhance the contrast between these two types of signals. Chromosomes were ordered within each group (sm = submetacentric, st = subtelocentric, a = acrocentric) in decreasing size order.

Karyotypes
Karyotypes of all five species examined possessed the diploid chromosome number 2n = 48 ( Figure 1, Table 2). However, species differed in chromosome morphology and fundamental number (NF). Melanotaenia sp., G. incisus, and I. werneri possessed the same karyotype composed of exclusively 48 subtelo/acrocentric chromosomes and NF = 48, while the karyotype of R. ornatus was composed of six pairs of submetacentric and 18 pairs of subtelo/acrocentric chromosomes with NF = 60. C. rhombosomoides karyotype was composed of four submetacentric and 20 subtelo/acrocentric pairs of chromosomes with NF = 56. No intraspecific numerical or structural polymorphisms between males and females were observed in any of the study species ( Figure S1, Supplementary Materials). We noticed differences in size of the first chromosome pair between C. rhombosomoides male and female, possibly associated with different accumulation of 28S rDNA copies in the p-arms of these chromosomes ( Figure S1, Supplementary Materials).

rDNA Chromosome Mapping
The FISH (fluorescence in situ hybridisation) experiments with 5S and 28S rDNA probes showed specific signals on non-homologous chromosomal pairs among the studied species. The 28S rDNA probe hybridised at the pericentromeric regions of one subtelo/acrocentric chromosomal pair in all species. The number of the 5S rDNA loci differed among studied species. Melanotaenia sp., G. incisus, I. werneri, and R. ornatus possessed a total four hybridisation signals of 5S rDNA, two on the telomeric region of one subtelo/acrocentric chromosomal pair, and two signals in the centromeric regions of the another subtelo/acrocentric chromosomal pair. Cairnsichthys rhombosomoides possessed the highest number of 5S rDNA copies (eight signals in total), with two signals located in the telomeric region of one subtelo/acrocentric chromosomal pair and six interstitial (between the telomeres and centromeres) signals on three other subtelo/acrocentric chromosomal pairs (Figures 2 and 3).

Telomere Mapping
In order to document interstitial telomeric sequences (ITSs) as remnants of chromosomal rearrangements, we performed FISH with the conserved vertebrate telomeric repeat (TTAGGG) n . We detected signals at the termini of all chromosomes but did not detect any ITSs in any species examined (Figures 2 and 3).

CMA 3 /DAPI Staining
Reversed fluorescence staining (CMA 3 /DAPI) revealed homogeneous staining patterns across chromosomes with a moderately GC-rich centromeric and telomeric regions. Extremely GC-rich signals were found in the centromeric region of one subtelo/acrocentric chromosomal pair in all but R. ornatus. In R. ornatus, strong GC-rich signals were located in the telomeric region of one subtelo/acrocentric chromosomal pair, corresponding to the signals revealed also by C banding (Figure 3). In addition to signals in telomeric regions, several CG-rich signals were observed in pericentromeric regions in both male and female R. ornatus (Figure 3).

C Banding
C banding revealed interspecies differences of constitutive heterochromatin among species. In Melanotaenia sp., we observed approximately 32 C-positive bands in centromeric regions, while, in G. incisus, there were 14 C-positive bands in centromeric regions. I. werneri displayed 36 C-positive bands in pericentromeric regions and four interstitial C-positive bands (Figure 2). In R. ornatus 28 C-positive bands in centromeric regions, in addition to two C-positive bands in telomeric regions of subtelo/acrocentric chromosomes, were observed. In C. rhombosomoides C banding showed eight C-positive bands in centromeric regions together with four interstitial C-positive bands and two subtelo/acrocentric chromosomes with large heterochromatin blocks (Figure 3). No sex-specific C banding pattern was observed in any species.

Discussion
Rainbowfishes represent one of the most widespread and abundant freshwater families in Australia and New Guinea. They became very popular among aquarium hobbyists such that they are now available in pet stores around the world [11]. Similarly, they attract the attention of the scientific world with wide-ranging studies focused on their ecology, biology, phylogeny, and conservation (e.g., McGuigan et al. [26], Page et al. [27], Colléter and Brown [28]). Despite the fact that cytogenetics provides a valuable source for understanding genome evolution using information undetectable by molecular genetics (e.g., Dion-Côté et al. [29]), chromosome studies of rainbowfishes are scarce. In five studies published so far, the chromosome number of nine species was reported, namely, M. duboulayi (2n = 48a) [17], M. maccullochi (2n = 46 or 2n = 48) [15,18], M. fluviatilis (2n = 48) [14], M. goldiei (2n = 48a) [14], M. cf. splendida (2n = 48) [16], M. bosemani (2n = 48) [18], M. lacustris (2n = 46) [18], M. praecox (2n = 48) [18], and G. incisus (2n = 48) [18]. Unfortunately, none of these studies included any images of karyotypes or detailed information on other chromosomal characteristics. Thus, it was not possible to compare these data with our results in more detail. Here, we described the chromosome number of five species representing the five major lineages of this family [7]. The Running River rainbowfish (Melanotaenia sp.) is representative of the "Australis" phylogenetic lineage which was until recently included within the M. splendida (eastern rainbowfish) complex [30]. Nevertheless, recent genetic analyses (P. Unmack unpub. data) using single-nucleotide polymorphisms (SNPs) determined that the Running River rainbowfish is an undescribed species. Glossolepis incisus, representing the northern lineage, is the only species in this study with known chromosome number as reported by Said [18]. However, this research was not published in English and provides only a statement about the chromosome number with no images of karyotypes depicting detailed chromosomal characteristics. I. werneri, R. ornatus, and C. rhombosomoides represent early branching lineages within the phylogeny of the family [7].
Previous cytogenetic analyses revealed uniform chromosome numbers across rainbowfish species (2n = 48) as the most parsimonious ancestral state for major teleostean clades [31,32]. The diploidy in rainbowfishes was probably maintained over long evolutionary time. In addition to the stable 2n, similarities in other chromosomal characteristics are presented in our study. The most closely related species, Melanotaenia sp. and G. incisus, with an estimated divergence from each other between 23.6 and 37.3 Mya [7] possessed similar karyotype characteristics, as did I. werneri, one of the early branching lineages with estimated divergence that pre-dates~40 Mya [7]. These three species possessed the same karyotypes ( Figure 1 and Figure S1, Supplementary Materials) and the same numbers of clusters of 5S and 28 rDNA genes ( Figure 2). Moreover, other chromosomal markers displayed similar patterns, i.e., constitutive heterochromatin regions were located mostly in the centromeric regions and interstitial positions of chromosomes, mainly those associated with NOR (nucleolar organizer region) signals ( Figure 2, Table 2). These phylogenetically shared chromosomal features can indicate similar levels and patterns of chromosomal evolution within a clade [33]. The hybridisation of species in the genera Melanotaenia and Glossolepis in captivity, and a lack of significant barriers to introgressive hybridisation [34] suggest that hybridisation may also be linked with karyotype stasis among these clades. There are examples of active hybridisation of representatives from these clades in nature where different widespread species are coming into reproductive contact at their boundaries with a mix of parental species, F1 hybrids, and backcrosses present [35]. Moreover, there are a number of examples where mitochondrial DNA (mtDNA) introgression was demonstrated without evidence of nuclear introgression. This usually occurs between sympatric species from different rainbowfish lineages [7]. The two other species under this study, R. ornatus and C. rhombosomoides, displayed species-specific differences at both karyotype and chromosomal marker levels, which could represent evidence of the chromosomal background of speciation. Specifically, they differ in the chromosomal morphology or in the number of rDNA sites; both species possess two pairs of 28S rDNA but four (R. ornatus) and eight signals of 5S rDNA (C. rhombosomoides) (Figure 3, Table 2). It is commonly accepted that major 28S rDNA clusters of teleostean fishes are GC-rich and, thus, can also be identified by CMA 3 staining (e.g., Mayr et al. [36], Amemiya and Gold [37], Schmid and Guttenbach [38]). The same situation was observed in all species under this study with the exception of R. ornatus. In this species, the 28S rDNA clusters were only moderately GC-rich in addition to GC-rich signals located on non-homologous chromosomes. The lack of correspondence between CMA 3 + regions and 28S rDNA loci may be explained by the lower copy number of rRNA genes, as already reported in fishes (e.g., Gromicho et al. [39], Sola et al. [40]). We observed no intraspecific numerical or structural polymorphisms between males and females, suggesting a lack of heteromorphic sex chromosomes (Figure 2, Figure 3 and Figure S1, Supplementary Materials). The only polymorphism was observed in the size of p-arms in the first chromosome pair in C. rhombosomoides, where unequal accumulation of 28S rDNA copies possibly led to slightly different size of chromosomes ( Figure S1, Supplementary Materials). In all studied species, no ITSs were observed. ITSs sites are usually considered as relicts of the ancient chromosomal rearrangements, namely, centric fusions and tandem fusions [41]. Such rearrangements were previously detected in various teleost species (reviewed in Ocalewicz [42]). However, many cases of ancestral chromosome fusions may not have the expected ITS, probably due to loss or drastic reduction of the telomeric DNA during the rearrangements that followed these events [43]. The absence of ITSs and the same 2n in studied species may indicate the absence of structural rearrangements involving terminal regions in Melanotaeniidae karyotype evolution.
In conclusion, our study provides the first cytogenetic analyses of representatives of all five lineages of the family Melanotaeniidae by combining conventional and molecular cytogenetic approaches. Despite the conservatism in chromosome number, cytogenetic differences in the topology of chromosome markers (constitutive heterochromatin regions, rDNA sites, GC-rich regions) were found among different genera. The observed differences correlate with the degree of species divergence and might be associated with chromosomal rearrangements, known to play a fundamental role in speciation [44,45]. Nevertheless, additional detailed cytogenetic studies on a wider taxonomic scale are still needed for detailed description of the karyotype evolution in the family Melanotaeniidae.