Biological materials and sampling areas
A total of 15 individuals were collected for each one of the following species in different areas: Ch. interruptus (Estero Marga-Marga, Viña del Mar, V region, Chile, coordinates 19H 0263800.90 UTM E–6342166.09 UTM S), Ch. pisciculus (Río Angostura, Hospital, Metropolitan region, Chile, coordinates 19H 0338122.17 UTM E–6250729.69 UTM S), Ch. galusdae (Río Andalién, Concepción, VIII região, Chile, coordinates 18H 0682055.00 UTM E–5925386.00 UTM S), Ch. kiliani (Rio Calle-Calle, Antilhue, XIV region, Chile, coordinates 18H 0674363.00 UTM E–5592270.00 UTM S) and Ch. australe (Lagoa La Poza, desembocadura Lago Llanquihue, Puerto Varas, X região, Chile, coordinates 18G 0679651.18 UTM E–5428179.56 UTM S). The capture of the specimens was authorised by the Subsecretaría de Pesca y Acuicultura del Gobierno de Chile (SUBPESCA) and by the Servicio Nacional de Pesca y Acuicultura del Gobierno de Chile (SERNAPESCA), Authorization No. 740.
Representative individuals of different morphological types (two males and two females for each specie) were anesthetised, labelled, and preserved in absolute ethanol for taxonomic identification. The specimens were photographed using a Leica M205C Stereomicroscope to observe the ventral protrusive teeth and cusps. The analysed material was deposited in the Department of Zoology of the Universidade Federal de Rio Grande do Sul, Brazil, under vouchers UFRGS 22295 (Ch. interruptus), UFRGS 22296 (Ch. galusdae), UFRGS 22297 (Ch. pisciculus), UFRGS 22298 (Ch. kiliani), and UFRGS 22299 and UFRGS 22300 (Ch. australe).
Chromosome preparations were obtained from fin tissue regenerated for three days for animals acclimated at 20 °C in aquarium. The regeneration occurs in situ, therefore, does not require special incubation conditions (20 °C), more than those that do the fish to remain activefollowing the protocol of . This technique was employed here due to the small size of the animals (< 3.0 cm). To obtain the number, size, and morphology of chromosomes, the slides were stained with Giemsa diluted in 5% phosphate buffer (KH2 PO4 + Na2 HPO4) pH 6.8 for 10 min and then washed in running water and air dried. To visualize heterochromatic regions, we used the C-band technique by Sumner , adaptated by Lui et al. . The nucleolus organizer regions (Ag-NORs) were visualised according to .
Fluorescence in situ hybridisation (FISH) was utilised to detect chromosomal location of rDNA (18S and 5S) sites and telomeric sequences, according to the protocol of Pinkel et al. , under high stringency (2.5 ng/μL probe, 50% formamide, 2× SSC, and 10% dextran sulphate). The 18S rDNA probe was amplified from the DNA of Prochilodus argenteus (Spix & Agassiz, 1829) , using the primers NS1 5′-GTAGT CATATGCTTGTCTC-3′ and NS8 5’-TCCGCAGGTTC ACCTACGGA-3′ . The 5S probe was obtained from DNA amplification of Leporinus elongatus (Valenciennes, 1850), using the primers A 5’-TACGCCCGATCTCGTCCGATC-3′ and B 5’-GCTGGTATGGCCGTAGC-3′ . The telomere probe (TTAGGG) n was amplified using the primers (TTAGGG)5 and (CCCTAA)5, in the absence of a DNA template, according to . The chromosome mapping of the 18S probe was achieved using the Kit Biotin Nick Translation (Roche), and the 5S and telomeric mappings were performed using Kit Dig Nick Translation (Roche), following the manufacturer’s recommendations. Signal detection was performed with digoxigenin conjugated to rhodamine (18S probe) and streptavidin conjugated to FITC (5S and Telomeric probes) (Roche Applied Science).
All analyses used mitotic chromosomes subjected to sequential treatment to optimize the use of the chromosome material. The treatments were applied in order of increasing invasiveness: Giemsa stain, double-color FISH with 18S and 5S rDNA probes, C-bands and silver staining of nucleoli organizing regions (Ag-NORs).
Conventional chromosome preparations were observed under optical microscope, Olympus® BX41. FISH metaphases were analysed using an epifluorescence microscope with appropriate filters. Images were captured using a camera, CCD DP71 12 mp, with the software DP controller (Olympus). At least 10 specimens, five from males and five from females, were analysed for each species. Adobe Photoshop v. CC (2015) software was used to karyotype arrange and Easy Idio v. 1.0 software was used to construct ideograms. Chromosomes were classified following Levan et al.  and karyotypes organised in decreasing order of size.