The Crenuchidae is widespread in freshwater systems of the South and Central Americas . The Characidium is the most representative group in this family, comprising 50 valid species . This is a particularly interesting Neotropical fish group for cytogenetic studies because it presents a diversified model of sex chromosomes . In the Characidium, a diploid number of 50 chromosomes is observed in all studied species and a karyotype of 32 m + 18 sm is most commonly reported [2–10].
Although the diploid number is conserved, the Characidium exhibit remarkable interspecific and interpopulation differences, such as (i) inter- and intraindividual variation of B chromosomes in some species [3–5]; (ii) a sex chromosome system with ZZ/ZW female heterogamety [2–9]; (iii) variation in the location and number of rDNA sites in distinct chromosomal pairs [2–4, 8, 10]; and (iv) occurrence of natural triploidy in C. gomesi and C. cf. zebra [6, 10].
The first cytogenetic report in Characidium was for C. zebra  and, so far, this is the only species where heteromorphic sex chromosomes are absent [4, 6, 10]. Based on morphological analysis, C. zebra exhibits several plesiomorphic traits, being placed as basal in Characidium phylogeny . The chromosomal characters of C. zebra have already been referred to as putatively plesiomorphic for the genus . Therefore, changes in the karyotypes observed in other species of Characidium, when compared with C. zebra, should be the result of secondary chromosomal rearrangements.
The karyotypes of C. lauroi and C. alipioi have been described, and while the latter was characterized by the presence of a ZZ/ZW sex chromosome system, C. lauroi was considered homomorphic because of the lack of differential heterochromatic bands . However, further comparative studies in seven species of this genus (C. zebra, C. lanei, C. pterostictum, C. lauroi, C. oiticicai, Characidium sp. and C. schubarti) showed that only C. zebra lacked differentiated sex chromosomes and the other species presented sex chromosomes in different W-heterochromatin amplification stages and had terminal NORs on both Z and W chromosomes . Characidium gomesi is another species with highly differentiated sex chromosomes, bearing entirely heterochromatic W chromosomes and NORs on autosomes [2, 3, 6].
A sex proto-chromosome pair for Characidium species was proposed, from which both Z and W chromosomes have evolved by structural rearrangements, such as duplications, deletions, and/or inversions. By contrast, the heterochromatin amplification of W was regarded as a predominant event in the differentiation of the sex chromosomal pair . Later, it was inferred that the NORs on ZW chromosomes in most species of Characidium represented an ancestor state of sex proto-chromosomes, prior to heterochromatin amplification . Heterochromatin amplification in one of the sex chromosomes driven by the accumulation of certain repetitive DNA classes in one homologous is relatively common in fish . Such a process would favor recombination suppression and thereby the independent evolution of chromosomes or chromosomal regions [14–17].
In the present study, we analyzed the origin, composition, and differentiation of sex and NOR-bearing chromosomes in populations of C. zebra, C. lauroi, and C. gomesi, using chromosomal painting with a W-specific probe from C. gomesi. We also mapped the 18S rDNA to infer the evolution, phylogeny and biogeography of the group.