**A.2 Methodology for obtaining cytogenetic material**

Mitotic chromosomes were obtained according to the protocol described by Bertollo et al. [67]. Genomic DNA extraction was performed using the chloroformphenol method [61]. Repetitive DNA probes were obtained using the *C*o*t*-1 DNA technique as described by Zwick et al. [45] and adapted by Vicari et al. [68]. This technique is based on re-association kinetics and enzymatic digestion by S1 nuclease. The probes were labeled by nick translation with digoxigenin 11 dUTP (Roche®) and the signals were recognized by anti-digoxigenin-rhodamin (Roche®). The hybridization techniques followed the Pinkel et al. [69] protocol, with 77% of stringency (2.5 ng/μL of each probe, 50% of deionized formamide, 10% dextran sulphate, 2X SSC at 37°C for 18 hours). The chromosomes were counterstained with DAPI in Vectashield medium (Vector®). The chromosomal preparations were analyzed by epifluorescence microscopy using an Olympus BX41® microscope fitted with a CCD Olympus DP-71® digital camera. Image capture was performed using DP controler® software (Olympus). The probes with positive hybridization signals were purified and cloned using the pMOSBlue blunt ended RPN5110 cloning kit (Amersham Biociences®) for subsequent sequencing. The samples were sequenced in an ABI-PRISM 3100 Genetic Analyzer automatic sequencer at the ACTGene laboratory (Centro de Biotecnologia, UFRGS, Porto Alegre, RS).

The sequences were aligned and edited using the CLUSTAL W program [65] using the following parameters: weights 6.66 and 10.0 for opening and extension of gaps, respectively, for pairwise alignments, and weights 10.0 and 15.0 for opening and extension of gaps in the multiple alignments respectively. Clustering analysis was performed using the parsimony method on the PAUP program v. 4.0b10 [70]. The isolated fragments of repetitive DNA sequences (*C*o*t*-1) from *G. sylvius* and *G. paraguensis* had lengths between 100 and 400 bp, respectively (**Figure 5**). These fragments were used as probes for fluorescent *in situ* hybridization (FISH) and were found to label most of the pericentromeric regions of the chromosomes of *G. sylvius* (**Figure 4a**) and all of the chromosomes of *G. paraguensis* (**Figure 4b**).

*Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material*

Molecular studies with the multigene family 5S rDNA in electric fish (Gymnotidae) have advanced a lot in recent years. The chromosomal location and distribution have been particularly interesting, since all species of the genus *Gymnotus*, analyzed so far, show great variability. Its distribution on the chromosomes of species with 2n = 54 shows to be a biogeographic marker, suggesting that the speciation of this group was recent due to migratory expansion. On the other hand, the species analyzed in the present study showed repetitive sequences composed of microsatellite replications species-specific, present around the centromeres. Although the location of the heterochromatin in *Gymnotus* was conserved, they had different constitutions and could represent important evolutionary mark-

The current study was supported in part by INCT ADAPTA II funded by CNPq - Brazilian National Research Council (465540/2014-7). The authors are grateful to Miguel Airton Carvalho for his field assistance. The author M.D.S. received a scholarship from the *Conselho Nacional de Desenvolvimento Científico e Tecnológico*

**3. Conclusions**

**Acknowledgements**

**Conflict of interest**

**A. Appendices**

(CNPq) (grant 160155/2018-5).

final extension of 5 min at 72° C.

ers for cytotaxonomic studies of this group.

The authors declare no conflict of interest.

**A.1 Methodology for obtaining cytochrome oxidase sequences**

For the Cytochrome oxidase I gene, five specimens of *G. carapo* "Catalão", four specimens of *G. carapo* "Maranhão", four specimens of *G. ucamara* and seven specimens of *G. mamiraua* were sequenced, using the primers BOL-COIfishF1 (5'TCAACYAATCAYAAAGATATYGGCAC´3′) and BOL-COI-fishR1 (5´-ACTTCYGGGTGRCCRAARAATCA- 3′) [60]. Genomic DNA extraction was performed using the chloroform-phenol method [61]. The PCR reactions were performed in a final volume of 25 μL, containing genomic DNA (100 ng), 10x buffer with 1.5 mM MgCl2, Taq DNA polymerase (5 U/μL), dNTPs (1 mM), pair of primers (5 pM) and Milli-Q water. The conditions for amplification were: 1 min 95° C, followed by 30 cycles of 1 min at 94° C, 1 min at 59° C, 1 min 30 sec at 72° C and

Sequences of *Gymnotus* species for the Clado *carapo*, available in the NCBI database, from the Paraná-Paraguay basin were added to our analysis: *G. sylvius*, *G.* 

The calculation of intra and interspecific distance was performed using the Mega 5 software [62] with the Kimura-2-parameters evolutionary model. The identification was carried out according to the protocol established by DNA barcoding through the Neighbor Joining (NJ) analysis [63], which consists of looking for the tree with the lowest total sum of branches, using Kimura-2-parameters as an evolutionary model (K2P) [64]. The topology confidence test was performed with

*inaequilabiatus*, *G*. *pantanal* and *G. carapo* "Pantanal" (**Table 1**).

**24**
