Repetitive DNAs and chromosome evolution in Megaleporinus obtusidens and M. reinhardti (Characiformes: Anostomidae).

The high dynamism of repetitive DNAs is a major driver of chromosome evolution. In particular, the accumulation of repetitive DNA sequences has been reported as part of the differentiation of sex-specific chromosomes. In turn, the fish species of the genus Megaleporinus are a monophyletic clade in which the presence of differentiated ZZ/ZW sex chromosomes represents a synapomorphic condition, thus serving as a suitable model to evaluate the dynamic evolution of repetitive DNA classes. Therefore, transposable elements (TEs) and in tandem repeats were isolated and located on chromosomes of Megaleporinus obtusidens and M. reinhardti to infer their role in chromosome differentiation with emphasis on sex chromosome systems. Despite the conserved karyotype features of both species, the location of repetitive sequences - Rex 1, Rex 3, (TTAGGG)n, (GATA)n, (GA)n, (CA)n, and (A)n - varied both intra and interspecifically, being mainly accumulated in Z and W chromosomes. The physical mapping of repetitive sequences confirmed the remarkable dynamics of repetitive DNA classes on sex chromosomes that might have promoted chromosome diversification and reproductive isolation in Megaleporinus species.

Integrating morphological, molecular and cytogenetic data for F2 sea turtle hybrids diagnosis revealed balanced chromosomal sets.

Hybridization could be considered part of the evolutionary history of many species. The hybridization among sea turtle species on the Brazilian coast is atypical and occurs where nesting areas and reproductive seasons overlap. Integrated analysis of morphology and genetics is still scarce, and there is no evidence of the parental chromosome set distribution in sea turtle interspecific hybrids. In this study, chromosome markers previously established for pure sea turtle species were combined with morphological and molecular analyses aiming to recognize genetic composition and chromosome sets in possible interspecific hybrids initially identified by mixed morphology. The data showed that one hybrid could be an F2 individual among Caretta caretta × Eretmochelys imbricata × Chelonia mydas, and another is resulting from backcross between C. caretta × Lepidochelys olivacea. Native alleles of different parental lineages were reported in the hybrids, and, despite this, it was verified that the hybrid chromosome sets were still balanced. Thus, how sea turtle hybridism can affect genetic features in the long term is a concern, as the implications of the crossing-over in hybrid chromosomal sets and the effects on genetic function are still unpredictable.

U2 and U4 snDNA Comparative Chromosomal Mapping in the Neotropical Fish Genera Apareiodon and Parodon (Characiformes: Parodontidae).

Small nuclear DNA (snDNA) are valuable cytogenetic markers for comparative studies in chromosome evolution because different distribution patterns were found among species. Parodontidae, a Neotropical fish family, is known to have female heterogametic sex chromosome systems in some species. The U2 and U4 snDNA sites have been found to be involved in Z and W chromosome differentiation in Apareiodon sp., Apareiodon affinis, and Parodon hilarii. However, few studies have evaluated snDNA sites as propulsors of chromosome diversification among closely related fish species. In this study, we investigated the distribution of U2 and U4 snDNA clusters in the chromosomes of 10 populations/species belonging to Apareiodon and Parodon, aiming to identify chromosomal homeologies or diversification. In situ localization data revealed a submetacentric pair carrying the U2 snDNA site among the populations/species analyzed. Furthermore, all studied species demonstrated homeology in the location of U4 snDNA cluster in the proximal region of metacentric pair 1, besides an additional signal showing up with a divergence in Apareiodon. Comparative chromosomal mapping of U4 snDNA also helped to reinforce the proposal of the ZZ/ZW1W2 sex chromosome system origin in an A. affinis population. According to cytogenetic data, the study corroborates the diversification in Parodontidae paired species with uncertain taxonomy.

Comparative cytogenetics among Boana species (Anura, Hylidae): focus on evolutionary variability of repetitive DNA.

Boana comprises a diverse genus of Neotropical treefrogs, currently rearranged into seven taxonomic species groups. Although cytogenetic studies have demonstrated diversity in its representatives, the chromosomal mapping of repetitive DNA sequences is still scarce. In this study, Boana albopunctata, Boana faber, and Boana prasina were subjected to in situ localization of different repetitive DNA units to evaluate trends of chromosomal evolution in this genus. Boana faber and B. prasina had 2n=24 chromosomes, while B. albopunctata has 2n=22 and an intra-individual variation related to the presence/absence of one B chromosome. The location of 45S rDNA sites was different in the analyzed karyotypes, corroborating with what was found in the distinct phylogenetic groups of Boana. We presented the first description of 5S rDNA in a Boana species, which showed markings resulting from transposition/translocation mechanisms. In situ localization of microsatellite loci proved to be a helpful marker for karyotype comparison in Boana, commonly with cis accumulation in the heterochromatin. On the other hand, genomic dispersion of microsatellites may be associated with hitchhiking effects during the spreading of transposable elements. The obtained results corroborated the independent diversification of these lineages of species from three distinct phylogenetic groups of Boana.

Comparative Cytogenetic and Sequence Analysis of U Small Nuclear RNA Genes in Three Ancistrus Species (Siluriformes: Loricariidae).

Ancistrus presents a wide karyotypic diversity, resulting from numeric and structural chromosomal rearrangements. It has been proposed that some genome-specific regions containing repetitive units could organize prone-to-break DNA sites in Loricariidae, triggering chromosomal rearrangements such as Robertsonian fusions (Rb fusions), centric fissions, translocations, and inversions. The tandemly repeats of the small nuclear RNAs (snRNAs) gene families are considered good cytogenetic markers for understanding chromosomal remodeling events among closely related species, but these snRNAs have been scarcely analyzed in Ancistrus. This study presented the nucleotide sequencing and comparative in situ location of U snRNA sequences from Ancistrus aguaboensis, Ancistrus cf. multispinis, and Ancistrus sp. (2n = 50, 52, and 50, respectively), aiming to provide information about snRNA clusters in the genome and chromosome evolution in Ancistrus. U snRNA nucleotide sequences of Ancistrus presented identity to orthologous copies and folded their secondary structures correctly. In situ localization and karyotyping of the three Ancistrus species revealed clustered copies of U2 and U5 snRNA gene families to a single chromosome site, one chromosome pair bearing U1 snRNA sequence, and one main locus of U4 snRNA sequence, besides scattered signals along the chromosomes. Previous studies related the participation of the rRNA gene families in centric fusion events, contributing to chromosome rearrangements and karyotype plasticity present in Loricariidae. In this study, homeologies in U snRNA loci chromosomal locations were detected, indicating the occurrence of conserved sites of these gene families in these three Ancistrus species with 2n = 50 or 52 chromosomes.

Enriched tandem repeats in chromosomal fusion points of Rineloricaria latirostris (Boulenger, 1900) (Siluriformes: Loricariidae).

Cytogenetic data showed the enrichment of repetitive DNAs in chromosomal rearrangement points between closely related species in armored catfishes. Still, few studies integrated cytogenetic and genomic data aiming to identify their prone-to-break DNA sites. Here, we aimed to obtain the repetitive fraction in Rineloricaria latirostris to recognize the microsatellite and homopolymers flanking the regions previously described as chromosomal fusion points. The results indicated that repetitive DNAs in R. latirostris are predominantly DNA transposons, and considering the microsatellite and homopolymers, A/T-rich expansions were the most abundant. The in situ localization demonstrated the A/T-rich repetitive sequences were scattered on the chromosomes, while A/G-rich microsatellite units were accumulated in some regions. The DNA transposon hAT, the 5S rDNA, and 45S rDNA (previously identified in Robertsonian fusion points in R. latirostris) were clusterized with some microsatellites, especially (CA)n, (GA)n, and poly-A, which were also enriched in regions of chromosomal fusions. Our findings demonstrated that repetitive sequences such as rDNAs, hAT transposons, and microsatellite units flank probable evolutionary breakpoint regions in R. latirostris. However, due to the sequence unit homologies in different chromosomal sites, these repeat DNAs only may facilitate chromosome fusion events in R. latirostris rather than working as a double-strand breakpoint site.

Major and minor U small nuclear RNAs genes characterization in a neotropical fish genome: Chromosomal remodeling and repeat units dispersion in Parodontidae.

In association with many proteins, small nuclear RNAs (snRNAs) organize the spliceosomes that play a significant role in processing precursor mRNAs during gene expression. According to snRNAs genic arrangements, two kinds of spliceosomes (major and minor) can be organized into eukaryotic cells. Although in situ localization of U1 and U2 snDNAs have been performed in fish karyotypes, studies with genomic characterization and functionality of U snRNAs integrated into chromosomal changes on Teleostei are still scarce. This study aimed to achieve a genomic characterization of the U snRNAs genes in Apareiodon sp. (2n = 54, ZZ/ZW), apply these data to recognize functional/defective copies, and map chromosomal changes involving snDNAs in Parodontidae species karyotype diversification. Nine snRNA multigene families (U1, U2, U4, U5, U6, U11, U12, U4atac and U6atac) arranged in putatively functional copies in the genome were analyzed. Proximal Sequence Elements (PSE) and TATA-box promoters occurrence, besides an entire transcribed region and conserved secondary structures, qualify them for spliceosome activity. In addition, several defective copies or pseudogenes were identified for the snRNAs that make up the major spliceosome. In situ localization of snDNAs in five species of Parodontidae demonstrated that U1, U2, and U4 snDNAs were involved in chromosomal location changes or units dispersion. The U snRNAs defective/pseudogenes units dispersion could be favored by the probable occurrence of active retrotransposition enzymes in the Apareiodon genome. The U2 and U4 snDNAs sites were involved in independent events in the differentiation of sex chromosomes among Parodontidae lineages. The study characterized U snRNA genes that compose major and minor spliceosomes in the Apareiodon sp. genome and proposes that their defective copies trigger chromosome differentiation and diversification events in Parodontidae.

Population structuration and chromosomal features homogeneity in Parodon nasus (Characiformes: Parodontidae): A comparison between Lower and Upper Paraná River representatives

The ichthyofauna of the La Plata hydrographic basin is divided into Upper and Lower Paraná River systems due to the geographic isolation of the Sete Quedas waterfalls, currently flooded by the lake of the Itaipu dam. In Parodontidae, pairs of species, or groups of cryptic species were described between these systems. Although genetic isolation and speciation have already been proposed in other species in the group, Parodon nasus has been maintained as a valid species and distributed throughout the La Plata river basin. In this perspective, specimens of P. nasus from four different sampling sites in the Upper and Lower Paraná River systems were compared regarding the karyotypes, molecular analyzes of population biology and species delimitation to investigate their genetic and population isolation in the La Plata river basin. Despite a geographic barrier and the immense geographic distance separating the specimens sampled from the Lower Paraná River system compared to those from the Upper Paraná River, the data obtained showed P. nasus as a unique taxon. Thus, unlike other species of Parodontidae that showed diversification when comparing the groups residing in the Lower versus Upper Paraná River, P. nasus showed a population structure and a karyotypic homogeneity.(AU)

A ictiofauna do sistema hidrográfico La Plata é dividida em alto e baixo rio Paraná devido ao isolamento geográfico dos Saltos das Sete Quedas há 22 milhões de anos, atualmente inundado pelo lago da represa da Usina de Itaipu. Em Parodontidae, espécies pares ou grupos de espécies crípticas foram descritos entre esses sistemas. Contudo, embora o isolamento genético e especiação já tenham sido propostos em outras espécies do grupo, Parodon nasus tem sido mantido como espécie válida e distribuída em toda a bacia do rio La Plata. Nessa perspectiva, exemplares de P. nasus de quatro diferentes pontos de amostragem nos sistemas do alto e baixo rio Paraná foram comparados quanto ao arranjo dos cariótipos, análises moleculares de biologia populacional e delimitação de espécies, afim de investigar seu isolamento genético e populacional na bacia do rio La Plata. Apesar da barreira geográfica e imensa distância geográfica separando os exemplares amostrados no sistema baixo rio Paraná em comparação àqueles do alto rio Paraná, os dados obtidos demonstraram P. nasus como único táxon válido. Dessa forma, diferentemente de outras espécies de Parodontidae que demonstraram diversificação quando comparados grupos pares residentes no baixo e alto rio Paraná, P. nasus demonstrou estruturação populacional e homogeneidade cariotípica.(AU)

Karyotypic Diversification in Two Megaleporinus Species (Characiformes, Anostomidae) Inferred from In Situ Localization of Repetitive DNA Sequences.

Anostomidae species have conserved diploid numbers (2n = 54), although comparative cytogenetic studies have demonstrated chromosomal rearrangements occurrence among them, especially in repetitive DNA rich regions. The location and distribution of ribosomal DNA (rDNA) and small nuclear RNAs (snRNAs) multigene families are highly dynamic in the genomes of several organisms. In this study, we in situ located the rDNA and snRNA sites in two populations of Megaleporinus obtusidens and a sample of Megaleporinus reinhardti to infer their chromosomal changes in the evolutionary lineages. Both species of Megaleporinus shared 2n = 54 chromosomes with the presence of ZZ/ZW sex chromosome system, but they diverged in relationship to the location of 5S and 45S rDNAs as well as the distribution of snRNAs sites. The characterization of the analyzed sequences revealed the presence of complete rDNA and snRNAs sequences as well as snRNAs containing transposable elements (TEs) and microsatellite repeats. After chromosomal mapping, the sequences encompassing TEs proved to be dispersed through autosomes and accumulated on sex chromosomes. The data demonstrate that intra- and interspecific chromosomal changes occurred involving the multigene family's sites in Megaleporinus karyotypes. Furthermore, we detected TE-like sequences in the differentiation of sex chromosome systems in M. obtusidens and M. reinhardti.

Chromosomal Diversification in Two Species of Pimelodus (Siluriformes: Pimelodidae): Comparative Cytogenetic Mapping of Multigene Families.

Pimelodidae has a high number of species, but cytogenetic studies are generally restricted to classical chromosomal characterization and in situ localization of ribosomal DNA (rDNA) genes. This study was developed to compare Pimelodus microstoma and Pimelodus pohli focusing on chromosomal diversification provided by the transposition of DNA sequences containing multigene families. Both species share 56 chromosomes, with centromeric and terminal heterochromatic blocks. The silver nucleolus organizer regions (Ag-NORs)/45S rDNA was located in the chromosome pair 24 for both species. The 5S rDNA sites were evidenced in the pair 8 of P. microstoma, and in the pairs 1, 17, and 18 in P. pohli. The U1 small nuclear RNA (snRNA) was located at terminal site in the first subtelocentric pair in both species. The U2 snRNA site was syntenic to 5S rDNA in non-homeologue chromosomes between analyzed species. The histones H3 and H4 were clustered in chromosome pairs 19 and 23 in P. microstoma, and 21 and 22 in P. pohli. Our study proposes that the movement of DNA sequences carrying multigene families has been driven on the chromosomal diversification of Pimelodidae. These multigene location in the genomes can explain most of the visualized chromosomal rearrangements in Pimelodidae and it is useful to understand the chromosomal changes and their distinctive karyotype formulae.

Cytogenetic and Molecular Characterization of Three Mimetic Species of the Genus Alagoasa Bechyné 1955 (Coleoptera: Alticinae) from the Neotropical Region.

Coleoptera is a mega-diverse order, but only about 1% of its species have been analyzed cytogenetically. In this order, the subfamily Alticinae presents many identification problems, mainly due to the occurrence of mimicry. The objective of this work was to cytogenetically characterize 3 very similar species of the genus Alagoasa (A. pantina, A.areata, and A.scissa). We used classical and molecular cytogenetic as well as molecular genetic techniques. All 3 species showed a diploid chromosome number of 2n = 22 (20+X+y), but differences in the morphology of the chromosomes. All had a meiotic formula of 2n = 10II+X+y and an X+y sex determination system with giant, fully asynaptic sex chromosomes, concordant characteristics observed in the subtribe Oedionychina. FISH demonstrated the presence of 18S and 5S rDNA clusters in 1 pair of autosomes, syntenic and colocalizing in the 3 analyzed species. However, in A. areata, heteromorphism between the cistrons was observed. The telomeric (TTAGG)n probe showed signals in all 3 species, with proximal signals in the X and dispersed signals in the y chromosome of A. areata, and 2 proximal signals in the X chromosome of A. scissa. Molecular analysis of the COI gene indicated that they are 3 distinct species, corroborating the observed cytogenetic characteristics.