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Additional file 1. Supplementary Note 1.

Additional file 3: Figure S1. A workflow of steps applied in the present study during the procedure of genomics analyses of B chromosomes in different species. Figure S2. Coverage plots of B-blocks of A. correntinus with remarkable difference in the reads coverage between 0B and 1B samples. Figure S3. Coverage plots of B-blocks of A. flavolineata with remarkable difference in the reads coverage between 0B and 2B samples. Figure S4. Identification of B chromosome genomic blocks (A) and their repeats contents (B) in A. flavolineata. Figure S5. Identification of protein-coding genes located in B chromosomes of the A. mexicanus, using the number of mapped reads that map to the CDSs found in the transcriptome, in the 0B (X axis) and 2B (Y axis). Each dot represents a coding sequence with only those labeled that recorded the log2 greater than 1.5. The plot is limited for 800 mapped reads to optimize the visualizations. Figure S6. Identification of protein-coding genes located in B chromosomes of the A. correntinus, using the number of mapped reads that map to the CDSs found in the transcriptome, in the 0B (X axis) and 1B (Y axis). Each dot represents a coding sequence with only those labeled that recorded the log2 greater than 1. The plot is limited for 800 mapped reads to optimize the visualizations. Figure S7. Coverage plots of (representative) coding sequences detected on the B chromosome of A. mexicanus using Log base 2 ratio. Each plot compares the reads depth of the transcript between 0B and 2B. Figure S8. Coverage plots of (representative) coding sequences detected on the B chromosome of A. correntinus using Log base 2 ratio. Each plot compares the reads depth of the transcript between 0B and 1B. Figure S9. Coverage plots of (representative) coding sequences detected on the B chromosome of A. flavolineata using Log base 2 ratio. Each plot compares the reads depth of the transcripts between 0B and 2B. Figure S10. Double FISH mapping of candidate blocks in A. flavolineata. Each panel represents mapping of two distinct blocks (7 and 14 – See Supplementary Table S2) labeled with digoxigenin (block 7 in red) and biotin (block 14 in green). A scattered pattern of markings of block 7 can be observed for the B chromosomes (white arrows), whereas telomeric and centromeric associated signals of blocks 7 and 14 are obvious for different A and X chromosomes. Figure S11. The methylation profile of microB blocks in A. mexicanus. (A) Graph shows the number of Bisulphite reads analyzed with B blocks and alignments. (B) Pie chart represents percentage of B block methylated Cs in different contexts with the highest percent in CpGs regions. (C) Pie chart highlights the number of hypomethylated (less than 50% methylated Cs), unmethylated, highly methylated and hypermethylated (more than 90% methylated Cs) B chromosome blocks. (D) Bisuphite coverage plots of hypermethylated blocks are shown as examples. Refer to Supplementary datasets (excel) for a complete list of methylated blocks with methylation level. Figure S12. FISH mapping of 45S rDNA (red labeling) in A. mexicanus. The micro B is indicated with an arrow showing no sign of 45S rDNA. Figure S13. Synteny dotplot of whole genome aligments between de novo B+ assembly and reference masked (coding) genome of A. mexicanus. Figure S14. Violin plots show the distribution of length for various types of genomic rearrangements from 0 to 5000 bp range. Figure S15. Synteny dotplot (synmap version, unfiltered) of self alignments of B blocks (A. mexicanus). Figure S16. Synteny dotplot (synmap version, unfiltered) of self alignments of B blocks (A. correntinus). Figure S17. Synteny dotplot (synmap version, unfiltered) of self alignments of B blocks (A. flavolineata).

Pimelodus paranaensis. The diploid number of this species was also 56 chromosomes (22 m + 22 sm + 4 st + 8 a) (Fig. 1g). The AgNORs were located in the terminal region of the long arm of a pair of subtelocentric chromosomes 24 (Fig. 1g, box). C-banding highlighted pale heterochromatin in the region of the telomere in the most of chromosomes, with more conspicuous heterochromatin being found in the telomeric region of long arm (pairs 4, 13, 19, 20, 24 and 26), in both telomeres (pairs 7 and 23), in the pericentromeric region of the long arm (pairs 13, 18 and 26) and short arm (pairs 2 and 12) (Fig. 2g). The 18S rDNA was located on the subtelocentric chromosome pair (24), which corresponds to the AgNORs, while the 5S rDNA was found at multiple sites, located in the pericentromeric region of the long arm of pairs 13, 18 and 26 (Fig. 3g)., Published as part of Girardi, Simone C., Pavanelli, Carla S. & Margarido, Vladimir P., 2018, Contributions to the systematic of Pimelodidae (Osteichthyes, Siluriformes): basic and molecular cytogenetics on seven species of Pimelodus from three Brazilian hydrographic systems, pp. 1-16 in Neotropical Ichthyology 16 (2) on page 10, DOI: 10.1590/1982-0224-20170148, http://zenodo.org/record/3709713

Pimelodus absconditus. We found a diploid number of 56 chromosomes (24 m + 18 sm + 8 st + 6 a) (Fig. 1a). The AgNORs were located in the terminal region of the long arm of a pair of subtelocentric chromosomes (24) (Fig. 1a, box). C-banding revealed the existence of pale heterochromatin in the most of the centromeres of chromosomes, with some pairs showing heterochromatin conspicuous in the telomeric region of the short arm (pars 17 and 18) and the long arm (pair 24), in both telomeres (pairs 8 and 20), in the pericentromeric region of the long arm of pair 18 and in subterminal region of the long arm of pair 17 (Fig. 2a). The 18S rDNA was located on the subtelocentric chromosome pair (24), which corresponds to the AgNORs, while the 5S rDNA was found in the pericentromeric region of the long arm of pair 18 (Fig. 3a)., Published as part of Girardi, Simone C., Pavanelli, Carla S. & Margarido, Vladimir P., 2018, Contributions to the systematic of Pimelodidae (Osteichthyes, Siluriformes): basic and molecular cytogenetics on seven species of Pimelodus from three Brazilian hydrographic systems, pp. 1-16 in Neotropical Ichthyology 16 (2) on page 6, DOI: 10.1590/1982-0224-20170148, http://zenodo.org/record/3709713

Pimelodus britskii. The diploid number of this species was also 56 chromosomes (24 m + 18 sm + 8 st + 6 a) (Fig. 1b). The AgNORs were located in the terminal region of the long arm of a pair of subtelocentric chromosomes 23 (Fig. 1b, box). C-banding revealed the existence of pale heterochromatin in the region of the telomeres of some chromosomes, with more conspicuous heterochromatin being found in the telomeric region of the long arm (pairs 3, 6 and 23), in the subterminal region of the long arm (pair 15), pericentromeric region of the short arm (pair 2) and in long arm in pair 17 (Fig. 2b). The 18S rDNA was located on the subtelocentric chromosome pair (23), which corresponds to the AgNORs, while the 5S rDNA was found at multiple sites, located in the pericentromeric region of the long arm of pair 17 and terminal region of the long arm of pairs 23 and 28 (Fig. 3b)., Published as part of Girardi, Simone C., Pavanelli, Carla S. & Margarido, Vladimir P., 2018, Contributions to the systematic of Pimelodidae (Osteichthyes, Siluriformes): basic and molecular cytogenetics on seven species of Pimelodus from three Brazilian hydrographic systems, pp. 1-16 in Neotropical Ichthyology 16 (2) on page 6, DOI: 10.1590/1982-0224-20170148, http://zenodo.org/record/3709713

Pimelodus ortmanni. The diploid number was also 56 chromosomes (24 m + 18 sm + 8 st + 6 a) (Fig. 1f). Among one to four B chromosomes (micro-chromosomes and acrocentric) (Fig. 1f) were observed in all specimens, with intra- and inter-individual variation (Tab. 4). The AgNORs were located in the terminal region of the long arm of a pair of subtelocentric chromosomes 24 (Fig. 1f, box). C-banding revealed the existence of pale heterochromatin in the region of the telomeres and centromeres of some chromosomes, with more conspicuous heterochromatin being found in the telomeric region of short arm (pair 16) and long arm (pairs 10, 24 and 27), in both telomere (pair 5) and in the pericentromeric region of the long arm (pairs 17 and 18), while the B chromosomes are entirely heterochromatic (Figs. 2f, 4). The 18S rDNA was located on the subtelocentric chromosome pair (24), which corresponds to the AgNORs, while the 5S rDNA was found in the pericentromeric region of the long arm of pair 18 (Fig. 3f)., Published as part of Girardi, Simone C., Pavanelli, Carla S. & Margarido, Vladimir P., 2018, Contributions to the systematic of Pimelodidae (Osteichthyes, Siluriformes): basic and molecular cytogenetics on seven species of Pimelodus from three Brazilian hydrographic systems, pp. 1-16 in Neotropical Ichthyology 16 (2) on page 10, DOI: 10.1590/1982-0224-20170148, http://zenodo.org/record/3709713

Pimelodus maculatus. The diploid number of this species was also 56 chromosomes (24 m + 20 sm + 6 st + 6 a) (Fig. 1c). The AgNORs were located in the terminal region of the long arm of a pair of subtelocentric chromosomes 23 (Fig. 1c, box). C-banding revealed the existence of pale heterochromatin in the region of the telomeres of some chromosomes, with more conspicuous heterochromatin being found in the telomeric region of the short arm (pair 6) and long arm (pairs 17, 22, 23 and 24), in both telomeres (pair 8) and the pericentromeric region of the short arm (pairs 1 and 25) and in long arm in pair 21 (Fig. 2c). The 18S rDNA and 5S rDNA showed syntenic sites in the terminal region of the long arm of a pair of subtelocentric chromosomes (23), which corresponds to the AgNORs (Fig. 3c)., Published as part of Girardi, Simone C., Pavanelli, Carla S. & Margarido, Vladimir P., 2018, Contributions to the systematic of Pimelodidae (Osteichthyes, Siluriformes): basic and molecular cytogenetics on seven species of Pimelodus from three Brazilian hydrographic systems, pp. 1-16 in Neotropical Ichthyology 16 (2) on page 6, DOI: 10.1590/1982-0224-20170148, http://zenodo.org/record/3709713