Chromosomal Evolution and Evolutionary Relationships of Lebiasina Species (Characiformes, Lebiasinidae)

Abstract

We present the first cytogenetic data for Lebiasina bimaculata and L. melanoguttata with the aim of (1) investigating evolutionary events within Lebiasina and their relationships with other Lebiasinidae genera and (2) checking the evolutionary relationships between Lebiasinidae and Ctenoluciidae. Both species have a diploid number 2n = 36 with similar karyotypes and microsatellite distribution patterns but present contrasting C-positive heterochromatin and CMA₃⁺ banding patterns. The remarkable interstitial series of C-positive heterochromatin occurring in L. melanoguttata is absent in L. bimaculata. Accordingly, L. bimaculata shows the ribosomal DNA sites as the only GC-rich (CMA₃⁺) regions, while L. melanoguttata shows evidence of a clear intercalated CMA₃⁺ banding pattern. In addition, the multiple 5S and 18S rDNA sites in L. melanogutatta contrast with single sites present in L. bimaculata. Comparative genomic hybridization (CGH) experiments also revealed a high level of genomic differentiation between both species. A polymorphic state of a conspicuous C-positive, CMA₃⁺, and (CGG)n band was found only to occur in L. bimaculata females, and its possible relationship with a nascent sex chromosome system is discussed. Whole chromosome painting (WCP) and CGH experiments indicate that the Lebiasina species examined and Boulengerella maculata share similar chromosomal sequences, thus supporting the relatedness between them and the evolutionary relationships between the Lebiasinidae and Ctenoluciidae families.

Keywords: comparative genomic hybridization; fish; karyotype evolution; whole chromosome painting.

Figure 1

Female karyotypes of Lebiasina bimaculata (a–c) and Lebiasina melanoguttata (d–f) arranged after different cytogenetic procedures. Giemsa staining (a,d), C-banding (b,e), and dual-color fluorescence in situ hybridization (FISH) with 18S (red) and 5S (green) ribosomal DNA probes (c,f). Chromosomes were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) in blue. The inserts highlight the homomorphic condition related to pair 3 in the males of L. bimaculata. Scale bar = 5 μm.

Figure 2

Metaphase plates of male (a) and female (b) Lebiasina bimaculata and male (c) and female (d) Lebiasina melanoguttata after DAPI-CMA₃ staining. The arrows indicate the unique CMA₃⁺ site and its polymorphic state between male and females of L. bimaculata. In L. melanoguttata, males and females display a set of CMA₃⁺ (GC-rich) and DAPI⁺ (AT-rich) regions on the chromosomes. Scale bar = 5 µm.

Figure 3

Metaphase plates of Lebiasina bimaculata (upper line) and Lebiasina melanoguttata hybridized with the microsatellite probes (CA)n, (GA)n, (CGG)n, and (CAT)n, respectively, showing the general distribution pattern of these repetitive DNAs in the chromosomes. Bar = 5 μm.

Figure 4

Female metaphase plate of Lebiasina bimaculata (a) and Lebiasina melanoguttata (b) showing the distribution of the telomeric (TTAGGG)n repeats. Bar = 5 µm.

Figure 5

Comparative genomic hybridization (CGH) for intra- and interspecific comparison in the female metaphase plates of Lebiasina bimaculata (a–d and m–p) and L. melanoguttata (e–h and i–l). Male- and female-derived genomic probes from L. bimaculata mapped against female chromosomes of L. bimaculata (a–d); Male- and female-derived genomic probes from L. melanoguttata mapped against female chromosomes of L. melanoguttata (e–h); female-derived genomic probes from both L. bimaculata and L. melanoguttata hybridized together against female chromosomes of L. melanoguttata (i–l); and female-derived genomic probes from both L. bimaculata and Boulengerella lateristriga (Ctenolucidae) hybridized together against female chromosomes of L. bimaculata (m–p). First column (a,e,i,m): DAPI images (blue); second column (b,f,j,n): hybridization patterns using male gDNA of L. bimaculata (b), male gDNA of L. melanoguttata (f), female gDNA of L. melanoguttata (j), and female gDNA of B. lateristriga probes (red); third column (c,g,k,o): hybridization patterns using female gDNA of L. bimaculata (c,o) and female gDNA of L. melanoguttata (g,k) probes (green); fourth column (d,h,l,p): merged images of both genomic probes and DAPI staining. The common genomic regions are depicted in yellow. Scale bar = 5 µm.

Figure 6

WCP with the LEB-1 (red) and BOU-1 (green) probes derived from pair 1 of Lebiasina bimaculata and Boulengerella lateristriga, respectively, hybridized against female metaphase chromosomes of Lebiasina bimaculata and Lebiasina melanoguttata. No differences between the sexes were observed. Bar = 5 μm.

Figure 7

Map of South America highlighting the collection sites of Lebiasina bimaculata (1—red circle) and L. melanoguttata (2—purple and 3—green circles). The maps were created using the following software: QGis 3.4.3, Inkscape 0.92, and Photoshop 7.0.