Fluorescence in situ hybridization (FISH) using whole chromosome-specific probes (wcp) is an important cytogenetic tool to study the origin and evolution of sex chromosomes in several organisms [1–8]. The diversity of sex-determining mechanisms, as well as the absence of heteromorphic sex chromosomes in many fish species make this group a useful model to study the evolution of vertebrate sex chromosomes [9, 10]. However, research involving chromosome painting in fish is rarely performed because it is difficult to obtain the necessary probes. The few available studies are focused on karyotypic [11, 12] and sex chromosome evolution [2, 5, 7, 13–15]. The current literature suggests that a variety of sex-determining mechanisms and sex chromosomes may have evolved independently in different fish species.
Erythrinidae is a Neotropical fish family that is characterized by species that have a wide variety of chromosomal forms, as well as a wide range of distinct sex chromosomes. The red wolf fish Erythrinus erythrinus (EER) is karyotypically diverse among different populations, with four currently identified karyomorphs (A to D) . Karyomorph A is characterized by 2n = 54 chromosomes that have very similar karyotypic structures and the absence of heteromorphic sex chromosomes. Karyomorphs B, C and D share an X1X1X2X2/X1X2Y sex chromosome system, but they can differ in the diploid number and chromosomal morphology. Karyomorph D has 2n = 52 chromosomes in females and 2n = 51 in males; previously published data suggests that karyomorph D was derived from a karyomorph A-like system . In fact, although there are differences in the diploid number between karyomorphs A and D and only karyomorph D has a differentiated X1X1X2X2/X1X2Y sex system, they share a relatively similar karyotypic structure that is characterized by several acrocentric chromosomes and a few bi-armed chromosomes. Additionally, mapping of distinct classes of repetitive sequences (5S rDNA, Cot-1 DNA, Rex3 and telomeric repeats) in the centromeric region of the Y chromosome indicated that a centric fusion between acrocentric pair number 5 and 12 in karyomorph A led to the formation of these sex chromosomes, in addition to the unpaired X1 and X2 chromosomes in the male karyotype of karyomorph D .
Similarly, the wolf fish Hoplias malabaricus (HMA) also demonstrates significant karyotypic diversity and well-defined population differences in the diploid number, chromosome morphology and sex chromosome systems. Currently, seven easily distinguishable karyomorphs (A to G) have been identified . Three well-differentiated sex chromosome systems occur in this group, namely XX/XY in karyomorph B, X1X1X2X2/X1X2Y in karyomorph D and XX/XY1Y2 in karyomorph G; additionally, karyomorph C has an early differentiated XX/XY system [18, 19]. Karyomorph D has 2n = 40 chromosomes in females and 2n = 39 chromosomes in males; repetitive DNA chromosomal mapping suggests that karyomorph D may have derived from a karyotype similar to karyomorph C, which is characterized by 2n = 40 chromosomes in both sexes [19, 20]. A conspicuous proximal GC-rich heterochromatic/18S rDNA site, which is present on the long arms of the X and Y chromosomes in karyomorph C, is also located in the same region on the X1 chromosome and the short arm of the large Y chromosome of karyomorph D; this suggests that the X1 and Y chromosomes of karyomorph D derived from the XY chromosomes of karyomorph C. In addition, the Y chromosome of karyomorph D shares similar DNA sequences with chromosomes Y and 20 of karyomorph C. Chromosomal pair number 20 in karyomorph C and its homolog in karyomorph D (X2) have centromeric satellite 5S HindIII-DNA, as well as an exclusive interstitial site that is present on the long arms of the Y chromosome of karyomorph D, which is the only non-centromeric location of this DNA sequence in the entire karyotype. These data indicate that this interstitial site is derived from the centromere of chromosome 20, which was fused to the ancestral Y chromosome in karyomorph C and resulted in the dicentric Y chromosome currently present in karyomorph D . Moreover, additional studies have shown that this dicentric Y chromosome behaves as a stable component of the karyotype having a correct segregation during meiosis .
In the present study, we analyzed the origin of the EER and HMA X1X2Y sex chromosomes by performing chromosome painting analysis with sex-chromosome-specific probes established by microdissection. The X1 chromosome of HMA (karyomorph D) and the Y chromosome of EER (karyomorph D) were microdissected and wcp-FISH was performed on the EER (karyomorphs A and D) and HMA (karyomorphs C and D) chromosomes. The results characterized the chromosomes that gave rise to the multiple sex determination systems and that both sex systems originated from different autosomal pairs. Our data provide new insights into the origin and evolution of sex chromosomes in fish, which increases our understanding of vertebrate sex chromosome evolution.