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.

Mechanisms of Karyotypic Diversification in Ancistrus (Siluriformes, Loricariidae): Inferences from Repetitive Sequence Analysis.

Ancistrus is a highly diverse neotropical fish genus that exhibits extensive chromosomal variability, encompassing karyotypic morphology, diploid chromosome number (2n = 34-54), and the evolution of various types of sex chromosome systems. Robertsonian rearrangements related to unstable chromosomal sites are here described. Here, the karyotypes of two Ancistrus species were comparatively analyzed using classical cytogenetic techniques, in addition to isolation, cloning, sequencing, molecular characterization, and fluorescence in situ hybridization of repetitive sequences (i.e., 18S and 5S rDNA; U1, U2, and U5 snDNA; and telomere sequences). The species analyzed here have different karyotypes: Ancistrus sp. 1 (2n = 38, XX/XY) and Ancistrus cirrhosus (2n = 34, no heteromorphic sex chromosomes). Comparative mapping showed different organizations for the analyzed repetitive sequences: 18S and U1 sequences occurred in a single site in all populations of the analyzed species, while 5S and U2 sequences could occur in single or multiple sites. A sequencing analysis confirmed the identities of the U1, U2, and U5 snDNA sequences. Additionally, a syntenic condition for U2-U5 snDNA was found in Ancistrus. In a comparative analysis, the sequences of rDNA and U snDNA showed inter- and intraspecific chromosomal diversification. The occurrence of Robertsonian rearrangements and other dispersal mechanisms of repetitive sequences are discussed.

Robertsonian Fusion Site in Rineloricaria pentamaculata (Siluriformes: Loricariidae): Involvement of 5S Ribosomal DNA and Satellite Sequences.

Cytogenetic studies demonstrated that unstable chromosomal sites in armored catfishes (Loricariidae) triggered intense karyotypic diversification, mainly derived from Robertsonian rearrangements. In Loricariinae, the presence of ribosomal DNA (rDNA) clusters and their flanking repeated regions (such as microsatellites or partial transposable element sequences) was proposed to facilitate chromosomal rearrangements. Hence, this study aimed to characterize the numerical chromosomal polymorphism observed in Rineloricaria pentamaculata and evaluate the chromosomal rearrangements which originated diploid chromosome number (2n) variation, from 56 to 54. Our data indicate a centric fusion event between acrocentric chromosomes of pairs 15 and 18, bearing 5S rDNA sites on their short (p) arms. This chromosome fusion established the numerical polymorphism, decreasing the 2n from original 56 (karyomorph A) to 55 in karyomorph B and 54 in karyomorph C. Although vestiges of telomeric sequences were evidenced at the fusion point, no 5S rDNA was detected in this region. The acrocentric chromosomes involved in the origin of the fusion were enriched with (CA)n and (GA)n microsatellites. Repetitive sequences in the acrocentric chromosomes subtelomeres have facilitated the rearrangement. Our study thus reinforces the view on the important role of particular repetitive DNA classes in promoting chromosome fusions which frequently drive Rineloricaria karyotype evolution.

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.

Evolutionary breakpoint regions and chromosomal remodeling in Harttia (Siluriformes: Loricariidae) species diversification.

The Neotropical armored catfish genus Harttia presents a wide variation of chromosomal rearrangements among its representatives. Studies indicate that translocation and Robertsonian rearrangements have triggered the karyotype evolution in the genus, including differentiation of sex chromosome systems. However, few studies used powerful tools, such as comparative whole chromosome painting, to clarify this highly diversified scenario. Here, we isolated probes from the X1 (a 5S rDNA carrier) and the X2 (a 45S rDNA carrier) chromosomes of Harttia punctata, which displays an X1X1X2X2/X1X2Y multiple sex chromosome system. Those probes were applied in other Harttia species to evidence homeologous chromosome blocks. The resulting data reinforce that translocation events played a role in the origin of the X1X2Y sex chromosome system in H. punctata. The repositioning of homologous chromosomal blocks carrying rDNA sites among ten Harttia species has also been demonstrated. Anchored to phylogenetic data it was possible to evidence some events of the karyotype diversification of the studied species and to prove an independent origin for the two types of multiple sex chromosomes, XX/XY1Y2 and X1X1X2X2/X1X2Y, that occur in Harttia species. The results point to evolutionary breakpoint regions in the genomes within or adjacent to rDNA sites that were widely reused in Harttia chromosome remodeling.

Comparative Cytogenetics of Four Sea Turtle Species (Cheloniidae): G-Banding Pattern and in situ Localization of Repetitive DNA Units.

Sea turtles are considered flagship species for marine biodiversity conservation and are considered to be at varying risk of extinction globally. Cases of hybridism have been reported in sea turtles, but chromosomal analyses are limited to classical karyotype descriptions and a few molecular cytogenetic studies. In order to compare karyotypes and understand evolutive mechanisms related to chromosome dif-ferentiation in this group, Chelonia mydas, Caretta caretta, Eretmochelys imbricata, and Lepidochelys olivacea were cytogenetically characterized in the present study. When the obtained cytogenetic data were compared with the putative ancestral Cryptodira karyotype, the studied species showed the same diploid number (2n) of 56 chromosomes, with some variations in chromosomal morphology (karyotypic formula) and minor changes in longitudinal band locations. In situ localization using a 18S ribosomal DNA probe indicated a homeologous microchromosome pair bearing a 45S ribosomal DNA locus and size heteromorphism in all 4 species. Interstitial telomeric sites were identified in a microchromosome pair in C. mydas and C. caretta. The data showed that interspecific variations occurred in chromosomal sets among the Cheloniidae species, in addition to other Cryptodira karyotypes. These variations generated lineage-specific karyotypic diversification in sea turtles, which will have considerable implications for hybrid recognition and for the study, the biology, ecology, and evolutionary history of regional and global populations. Furthermore, we demonstrated that some chromosome rearrangements occurred in sea turtle species, which is in conflict with the hypothesis of conserved karyotypes in this group.

Heterochromatin and microsatellites detection in karyotypes of four sea turtle species: Interspecific chromosomal differences.

The wide variation in size and content of eukaryotic genomes is mainly attributed to the accumulation of repetitive DNA sequences, like microsatellites, which are tandemly repeated DNA sequences. Sea turtles share a diploid number (2n) of 56, however recent molecular cytogenetic data have shown that karyotype conservatism is not a rule in the group. In this study, the heterochromatin distribution and the chromosomal location of microsatellites (CA)n, (GA)n, (CAG)n, (GATA)n, (GAA)n, (CGC)n and (GACA)n in Chelonia mydas, Caretta caretta, Eretmochelys imbricata and Lepidochelys olivacea were comparatively investigated. The obtained data showed that just the (CA)n, (GA)n, (CAG)n and (GATA)n microsatellites were located on sea turtle chromosomes, preferentially in heterochromatic regions of the microchromosomes (mc). Variations in the location of heterochromatin and microsatellites sites, especially in some pericentromeric regions of macrochromosomes, corroborate to proposal of centromere repositioning occurrence in Cheloniidae species. Furthermore, the results obtained with the location of microsatellites corroborate with the temperature sex determination mechanism proposal and the absence of heteromorphic sex chromosomes in sea turtles. The findings are useful for understanding part of the karyotypic diversification observed in sea turtles, especially those that explain the diversification of Carettini from Chelonini species.

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.

Chromosomal Diversification in Ancistrus Species (Siluriformes: Loricariidae) Inferred From Repetitive Sequence Analysis.

The Ancistrus genus has extensive chromosomal diversity among species, including heteromorphic sex chromosomes occurrence. However, studies have been shown that chromosomal diversity may still be underestimated. Repetitive sequences represent a large part of eukaryotic genomes, associated with mechanisms of karyotypic diversification, including sex chromosomes evolution. This study analyzed the karyotype diversification of two Ancistrus species (Ancistrus sp. 1 and Ancistrus sp. 2) from the Amazon region by classical and molecular chromosomal markers. Conventional chromosome bands and fluorescence in situ hybridization using probes 18S and 5S rDNA, besides (CA)n, (CG)n, (GA)n, (CAC)n, (CAG)n, (CAT)n, (GAA)n, (GAC)n, (TAA)n, and (TTAGGG)n in tandem repeats were determined on the karyotypes. Ancistrus sp. 1 and Ancistrus sp. 2 presented karyotypes with 2n = 38 (20 m + 14sm+4st, XX/XY) and 2n = 34 (20 m + 14sm, without heteromorphic sex chromosomes), respectively. Robertsonian rearrangements can explain the diploid number difference. C-bands occurred in pericentromeric regions in some chromosomes, and a single 18S rDNA locus occurred in both species. The 5S rDNA showed variation in the number of loci between species karyotypes, suggesting the occurrence of unstable sites and rearrangements associated with these sequences in Ancistrus. The microsatellite mapping evidenced distinct patterns of organization between the two analyzed species, occurring mainly in the sex chromosomes in Ancistrus sp. 1, and in the centromeric and pericentromeric regions of chromosomes m/sm in Ancistrus sp. 2. These data shows the extensive chromosomal diversity of repetitive sequences in Ancistrus, which were involved in Robertsonian rearrangements and sex chromosomes differentiation.

Chromosomal Rearrangements and Origin of the Multiple XX/XY1Y2 Sex Chromosome System in Harttia Species (Siluriformes: Loricariidae).

The Neotropical genus Harttia comprises species with extensive chromosomal remodeling and distinct sex chromosome systems (SCSs). So far, three different SCSs with male heterogamety have been characterized in the group. In some species, the presence of the XX/XY1Y2 SCS is associated with a decrease in diploid numbers and several chromosomal rearrangements, although a direct relation to sex chromosome differentiation has not been shown yet. Here, we aimed to investigate the differentiation processes that have led to the establishment of the rare XX/XY1Y2 SCS and track its evolutionary history among other Harttia species. For that, four whole chromosome painting probes derived from chromosome 1 of H. torrenticola (HTO-1), chromosomes 9 and X of H. carvalhoi (HCA-9 and HCA-X), and chromosome X from H. intermontana (HIN-X) were applied in nine Harttia species. Homeologous chromosome blocks were located in Harttia species and demonstrated that Robertsonian (Rb) fusions originated HTO-1, HCA-9, and HCA-X chromosomes, while Rb fissions explain Y1 and Y2 sex chromosomes. Specifically, in H. intermontana, HCA-X, HCA-9, and the NOR-bearing chromosome demonstrated that homeologous blocks were used in the HIN-X and metacentric pair 2 origins. Consequently, diploid numbers changed between the studied species. Overall, the data also reinforce the existence of unstable genomic sites promoting chromosomal differentiation and remodeling within the genus Harttia.

Highly Rearranged Karyotypes and Multiple Sex Chromosome Systems in Armored Catfishes from the Genus Harttia (Teleostei, Siluriformes).

Harttia comprises an armored catfish genus endemic to the Neotropical region, including 27 valid species with low dispersion rates that are restricted to small distribution areas. Cytogenetics data point to a wide chromosomal diversity in this genus due to changes that occurred in isolated populations, with chromosomal fusions and fissions explaining the 2n number variation. In addition, different multiple sex chromosome systems and rDNA loci location are also found in some species. However, several Harttia species and populations remain to be investigated. In this study, Harttia intermontana and two still undescribed species, morphologically identified as Harttia sp. 1 and Harttia sp. 2, were cytogenetically analyzed. Harttia intermontana has 2n = 52 and 2n = 53 chromosomes, while Harttia sp. 1 has 2n = 56 and 2n = 57 chromosomes in females and males, respectively, thus highlighting the occurrence of an XX/XY1Y2 multiple sex chromosome system in both species. Harttia sp. 2 presents 2n = 62 chromosomes for both females and males, with fission events explaining its karyotype diversification. Chromosomal locations of the rDNA sites were also quite different among species, reinforcing that extensive rearrangements had occurred in their karyotype evolution. Comparative genomic hybridization (CGH) experiments among some Harttia species evidenced a shared content of the XY1Y2 sex chromosomes in three of them, thus pointing towards their common origin. Therefore, the comparative analysis among all Harttia species cytogenetically studied thus far allowed us to provide an evolutionary scenario related to the speciation process of this fish group.

Co-located hAT transposable element and 5S rDNA in an interstitial telomeric sequence suggest the formation of Robertsonian fusion in armored catfish.

Co-located 5S rDNA genes and interstitial telomeric sites (ITS) revealed the involvement of multiple 5S rDNA clusters in chromosome rearrangements of Loricariidae. Interstitial (TTAGGG)n vestiges, in addition to telomeric sites, can coincide with locations of chromosomal rearrangements, and they are considered to be hotspots for chromosome breaks. This study aimed the molecular characterization of 5S rDNA in two Rineloricaria latirostris populations and examination of roles of 5S rDNA in breakpoint sites and its in situ localization. Rineloricaria latirostris from Brazil's Das Pedras river (2n = 46 chromosomes) presented five pairs identified using a 5S rDNA probe, in addition to a pair bearing a co-located ITS/5S rDNA. Rineloricaria latirostris from the Piumhi river (2n = 48 chromosomes) revealed two pairs containing 5S rDNA, without ITS. A 702-bp amplified sequence, using 5S rDNA primers, revealed an insertion of the hAT transposable element (TE), referred to as a degenerate 5S rDNA. Double-FISH (fluorescence in situ hybridization) demonstrated co-localization of 5S rDNA/degenerate 5S rDNA, 5S rDNA/hAT and ITS/5S rDNA from the Das Pedras river population. Piumhi river isolates possessed only 5S rDNA sites. We suggest that the degenerate 5S rDNA was generated by unequal crossing over, which was driven by invasion of hAT, establishing a breakpoint region susceptible to chromosome breakage, non-homologous recombination and Robertsonian (Rb) fusion. Furthermore, the presence of clusters of 5S rDNA at fusion points in other armored catfish species suggests its re-use and that these regions represent hotspots for evolutionary rearrangements within Loricariidae genomes.

Mechanisms of Chromosomal Diversification in Species of Rineloricaria (Actinopterygii: Siluriformes: Loricariidae).

Cytogenetic studies in fish of the Rineloricaria genus have already shown a high variation in diploid number (2n). Along with fusion/fission events for 2n alteration, inversions contribute to the diversification of chromosome formulae within this group. The present study assessed different populations/species of the Rineloricaria aiming to describe the karyotype organization of its members and understand the mechanisms that lead to the variation of chromosome numbers. Cytogenetic data showed distinct karyotype organization among Rineloricaria populations/species studied, ranging in diploid number from 46 to 64 chromosomes, syntopic species and two karyomorphs in Rineloricaria pentamaculata. Using ribosomal DNAs (rDNAs) and TTAGGGn probes by fluorescence in situ hybridization in species with low diploid numbers, we detected sites of 18S rDNA, 5S rDNA, and TTAGGGn in centromeric regions of metacentric chromosomes, which participated in chromosome rearrangements like centric fusions. In species with high 2n, centric fissions probably occurred in karyotypic diversification. In this study, we assessed the telomeric instability, chromosomal breaks, and rearrangements due to interstitial telomeric site vestiges detection, in addition to the probable role of rDNAs in chromosome fusions in karyotypic diversification of this group.

Comparative cytogenetic analyses in Ancistrus species (Siluriformes: Loricariidae)

Ancistrus is a specious genus of armored catfishes that has been extensively used for cytogenetic studies in the last 17 years. A comparison of the extensive karyotypic plasticity within this genus is presented with new cytogenetic analysis for Ancistrus cf. multispinis and Ancistrus aguaboensis. This study aims to improve our understanding of chromosomal evolution associated with changes in the diploid number (2n) and the dispersion of ribosomal DNAs (rDNAs) within Ancistrus. Ancistrus cf. multispinis and A. aguaboensis exhibit 2n of 52 and 50 chromosomes, respectively. Given that A. cf. multispinis shares a 2n = 52 also found in Pterygoplichthyini, the sister group for Ancistrini, a Robertsonian (Rb) fusion event is proposed for the 2n reduction in A. aguaboensis. 5S rDNAs pseudogenes sites have already been associated with Rb fusion in Ancistrus and our analysis suggests that the 2n reduction in A. aguaboensis was triggered by double strand breaks (DSBs) and chromosomal rearrangements at 5S rDNA sites. The presence of evolutionary breakpoint regions (EBRs) into rDNA cluster is proposed to explain part of the Rb fusion in Ancistrus. Cytogenetic data presented extends the diversity already documented in Ancistrus to further understand the role of chromosomal rearrangements in the diversification of Ancistrini.(AU)

Ancistrus é um gênero rico em espécies de peixes conhecidos como cascudos e tem sido alvo de estudos citogenéticos nos últimos 17 anos. Uma comparação da plasticidade presente no gênero é apresentada com novas análises citogenéticas para Ancistrus cf. multispinis e Ancistrus aguaboensis. Este estudo visa melhorar nossa compreensão da evolução cromossômica associada as alterações do número diploide (2n) e a dispersão de DNAs ribossômicos (rDNAs) em Ancistrus. Ancistrus cf. multispinis e A. aguaboensis apresentaram 2n de 52 e 50 cromossomos, respectivamente. Visto que A. cf. multispinis compartilha 2n = 52 também encontrado em Pterygoplichthyini, o grupo irmão para Ancistrini, um evento de fusão Robertsoniana (Rb) é proposto para a redução do 2n em A. aguaboensis. Sítios de pseudogenes de rDNA 5S já foram associados a eventos de fusão Rb em Ancistrus e nossas análises sugerem que a redução do 2n em A. aguaboensis foi desencadeada por quebras na dupla fita e rearranjos cromossômicos em sítios de rDNA 5S. A presença de evolutionary breakpoint regions (EBRs) em clusters de rDNA foi proposta para explicar parte da fusão Rb em Ancistrus. Os dados citogenéticos apresentados ampliam a diversidade já documentada em Ancistrus visando melhor entender o papel dos rearranjos cromossômicos na diversificação de Ancistrini.(AU)

Chromosome Mapping and Molecular Characterization of the Tc1/Mariner Element in Rineloricaria (Siluriformes: Loricariidae)

ABSTRACT The Tc1/Mariner sequence was isolated and mapped on chromosomes aiming to verify the association of this transposable element (TE) and chromosomal rearrangements in Rineloricaria. Cytogenetic analysis showed that Tc1/Mariner does not co-localize with chromosomal fusion points, in addition the analysis revealed intense molecular degeneration in its DNA sequence.