Evolution of bird sex chromosomes: a cytogenomic approach in Palaeognathae species.

BACKGROUND: Different patterns of sex chromosome differentiation are seen in Palaeognathae birds, a lineage that includes the ratites (Struthioniformes, Rheiformes, Apterygiformes, Casuariiformes, and the sister group Tinamiformes). While some Tinamiform species have well-differentiated W chromosomes, both Z and W of all the flightless ratites are still morphologically undifferentiated. Here, we conducted a comprehensive analysis of the ZW differentiation in birds using a combination of cytogenetic, genomic, and bioinformatic approaches. The whole set of satDNAs from the emu (Dromaius novaehollandiae) was described and characterized. Furthermore, we examined the in situ locations of these satDNAs alongside several microsatellite repeats and carried out Comparative Genomic Hybridizations in two related species: the greater rhea (Rhea americana) and the tataupa tinamou (Crypturellus tataupa). RESULTS: From the 24 satDNA families identified (which represent the greatest diversity of satDNAs ever uncovered in any bird species), only three of them were found to accumulate on the emu's sex chromosomes, with no discernible accumulation observed on the W chromosome. The W chromosomes of both the greater rhea and the emu did not exhibit a significant buildup of either C-positive heterochromatin or repetitive DNAs, indicating their large undifferentiation both at morphological and molecular levels. In contrast, the tataupa tinamou has a highly differentiated W chromosome that accumulates several DNA repeats. CONCLUSION: The findings provide new information on the architecture of the avian genome and an inside look at the starting points of sex chromosome differentiation in birds.

Satellite DNAs, heterochromatin, and sex chromosomes of the wattled jacana (Charadriiformes; Jacanidae): a species with highly rearranged karyotype.

Charadriiformes, which comprises shorebirds and their relatives, is one of the most diverse avian orders, with over 390 species showing a wide range of karyotypes. Here, we isolated and characterized the whole collection of satellite DNAs (satDNAs) at both molecular and cytogenetic levels of one of its representative species, named the wattled jacana (Jacana jacana), a species that contains a typical ZZ/ZW sex chromosome system and a highly rearranged karyotype. In addition, we also investigate the in situ location of telomeric and microsatellite repeats. A small catalog of 11 satDNAs was identified that typically accumulated on microchromosomes and on the W chromosome. The latter also showed a significant accumulation of telomeric signals, being (GA)10 the only microsatellite with positive hybridization signals among all the 16 tested ones. These current findings contribute to our understanding of the genomic organization of repetitive DNAs in a bird species with high degree of chromosomal reorganization contrary to the majority of bird species that have stable karyotypes.

Satellitome Analysis in the Southern Lapwing (Vanellus chilensis) Genome: Implications for SatDNA Evolution in Charadriiform Birds.

Vanellus (Charadriidae; Charadriiformes) comprises around 20 species commonly referred to as lapwings. In this study, by integrating cytogenetic and genomic approaches, we assessed the satellite DNA (satDNA) composition of one typical species, Vanellus chilensis, with a highly conserved karyotype. We additionally underlined its role in the evolution, structure, and differentiation process of the present ZW sex chromosome system. Seven distinct satellite DNA families were identified within its genome, accumulating on the centromeres, microchromosomes, and the W chromosome. However, these identified satellite DNA families were not found in two other Charadriiformes members, namely Jacana jacana and Calidris canutus. The hybridization of microsatellite sequences revealed the presence of a few repetitive sequences in V. chilensis, with only two out of sixteen displaying positive hybridization signals. Overall, our results contribute to understanding the genomic organization and satDNA evolution in Charadriiform birds.

Evolution of ancient satellite DNAs in extant alligators and caimans (Crocodylia, Reptilia).

BACKGROUND: Crocodilians are one of the oldest extant vertebrate lineages, exhibiting a combination of evolutionary success and morphological resilience that has persisted throughout the history of life on Earth. This ability to endure over such a long geological time span is of great evolutionary importance. Here, we have utilized the combination of genomic and chromosomal data to identify and compare the full catalogs of satellite DNA families (satDNAs, i.e., the satellitomes) of 5 out of the 8 extant Alligatoridae species. As crocodilian genomes reveal ancestral patterns of evolution, by employing this multispecies data collection, we can investigate and assess how satDNA families evolve over time. RESULTS: Alligators and caimans displayed a small number of satDNA families, ranging from 3 to 13 satDNAs in A. sinensis and C. latirostris, respectively. Together with little variation both within and between species it highlighted long-term conservation of satDNA elements throughout evolution. Furthermore, we traced the origin of the ancestral forms of all satDNAs belonging to the common ancestor of Caimaninae and Alligatorinae. Fluorescence in situ experiments showed distinct hybridization patterns for identical orthologous satDNAs, indicating their dynamic genomic placement. CONCLUSIONS: Alligators and caimans possess one of the smallest satDNA libraries ever reported, comprising only four sets of satDNAs that are shared by all species. Besides, our findings indicated limited intraspecific variation in satellite DNA, suggesting that the majority of new satellite sequences likely evolved from pre-existing ones.

Chromosomal Rearrangements and Satellite DNAs: Extensive Chromosome Reshuffling and the Evolution of Neo-Sex Chromosomes in the Genus Pyrrhulina (Teleostei; Characiformes).

Chromosomal rearrangements play a significant role in the evolution of fish genomes, being important forces in the rise of multiple sex chromosomes and in speciation events. Repetitive DNAs constitute a major component of the genome and are frequently found in heterochromatic regions, where satellite DNA sequences (satDNAs) usually represent their main components. In this work, we investigated the association of satDNAs with chromosome-shuffling events, as well as their potential relevance in both sex and karyotype evolution, using the well-known Pyrrhulina fish model. Pyrrhulina species have a conserved karyotype dominated by acrocentric chromosomes present in all examined species up to date. However, two species, namely P. marilynae and P. semifasciata, stand out for exhibiting unique traits that distinguish them from others in this group. The first shows a reduced diploid number (with 2n = 32), while the latter has a well-differentiated multiple X1X2Y sex chromosome system. In addition to isolating and characterizing the full collection of satDNAs (satellitomes) of both species, we also in situ mapped these sequences in the chromosomes of both species. Moreover, the satDNAs that displayed signals on the sex chromosomes of P. semifasciata were also mapped in some phylogenetically related species to estimate their potential accumulation on proto-sex chromosomes. Thus, a large collection of satDNAs for both species, with several classes being shared between them, was characterized for the first time. In addition, the possible involvement of these satellites in the karyotype evolution of P. marilynae and P. semifasciata, especially sex-chromosome formation and karyotype reduction in P. marilynae, could be shown.

Following the Pathway of W Chromosome Differentiation in Triportheus (Teleostei: Characiformes).

In this work, we trace the dynamics of satellite DNAs (SatDNAs) accumulation and elimination along the pathway of W chromosome differentiation using the well-known Triportheus fish model. Triportheus stands out due to a conserved ZZ/ZW sex chromosome system present in all examined species. While the Z chromosome is conserved in all species, the W chromosome is invariably smaller and exhibits differences in size and morphology. The presumed ancestral W chromosome is comparable to that of T. auritus, and contains 19 different SatDNA families. Here, by examining five additional Triportheus species, we showed that the majority of these repetitive sequences were eliminated as speciation was taking place. The W chromosomes continued degeneration, while the Z chromosomes of some species began to accumulate some TauSatDNAs. Additional species-specific SatDNAs that made up the heterochromatic region of both Z and W chromosomes were most likely amplified in each species. Therefore, the W chromosomes of the various Triportheus species have undergone significant evolutionary changes in a short period of time (15-25 Myr) after their divergence.

Cross-species chromosome painting and repetitive DNA mapping illuminate the karyotype evolution in true crocodiles (Crocodylidae).

Crocodilians have maintained very similar karyotype structures and diploid chromosome numbers for around 100 million years, with only minor variations in collinearity. Why this karyotype structure has largely stayed unaltered for so long is unclear. In this study, we analyzed the karyotypes of six species belonging to the genera Crocodylus and Osteolaemus (Crocodylidae, true crocodiles), among which the Congolian endemic O. osborni was included and investigated. We utilized various techniques (differential staining, fluorescence in situ hybridization with repetitive DNA and rDNA probes, whole chromosome painting, and comparative genomic hybridization) to better understand how crocodile chromosomes evolved. We studied representatives of three of the four main diploid chromosome numbers found in crocodiles (2n = 30/32/38). Our data provided new information about the species studied, including the identification of four major chromosomal rearrangements that occurred during the karyotype diversification process in crocodiles. These changes led to the current diploid chromosome numbers of 2n = 30 (fusion) and 2n = 38 (fissions), derived from the ancestral state of 2n = 32. The conserved cytogenetic tendency in crocodilians, where extant species keep near-ancestral state, contrasts with the more dynamic karyotype evolution seen in other major reptile groups.

Cytogenetics Meets Genomics: Cytotaxonomy and Genomic Relationships among Color Variants of the Asian Arowana Scleropages formosus.

Scleropages formosus (Osteoglossiformes, Teleostei) represents one of the most valued ornamental fishes, yet it is critically endangered due to overexploitation and habitat destruction. This species encompasses three major color groups that naturally occur in allopatric populations, but the evolutionary and taxonomic relationships of S. formosus color varieties remain uncertain. Here, we utilized a range of molecular cytogenetic techniques to characterize the karyotypes of five S. formosus color phenotypes, which correspond to naturally occurring variants: the red ones (Super Red); the golden ones (Golden Crossback and Highback Golden); the green ones (Asian Green and Yellow Tail Silver). Additionally, we describe the satellitome of S. formosus (Highback Golden) by applying a high-throughput sequencing technology. All color phenotypes possessed the same karyotype structure 2n = 50 (8m/sm + 42st/a) and distribution of SatDNAs, but different chromosomal locations of rDNAs, which were involved in a chromosome size polymorphism. Our results show indications of population genetic structure and microstructure differences in karyotypes of the color phenotypes. However, the findings do not clearly back up the hypothesis that there are discrete lineages or evolutionary units among the color phenotypes of S. formosus, but another case of interspecific chromosome stasis cannot be excluded.

B Chromosomes in Psalidodon scabripinnis (Characiformes, Characidae) Species Complex.

B chromosomes are extra-genomic components of cells found in individuals and in populations of some eukaryotic organisms. They have been described since the first observations of chromosomes, but several aspects of their biology remain enigmatic. Despite being present in hundreds of fungi, plants, and animal species, only a small number of B chromosomes have been investigated through high-throughput analyses, revealing the remarkable mechanisms employed by these elements to ensure their maintenance. Populations of the Psalidodon scabripinnis species complex exhibit great B chromosome diversity, making them a useful material for various analyses. In recent years, important aspects of their biology have been revealed. Here, we review these studies presenting a comprehensive view of the B chromosomes in the P. scabripinnis complex and a new hypothesis regarding the role of the B chromosome in the speciation process.

Revealing the Satellite DNA History in Psalidodon and Astyanax Characid Fish by Comparative Satellitomics.

Eukaryotic genomes are usually enriched in repetitive DNA sequences, which can be classified as dispersed or tandemly repeated elements. Satellite DNAs are noncoding monomeric sequences organized in a head-to-tail fashion that are generally located on the subtelomeric and/or pericentromeric heterochromatin. In general, a single species incorporates a diverse group of satellite DNA families, which collection is called satellitome. Here, we characterized three new satellitomes from distinct characid fish (Psalidodon fasciatus, P. bockmanni, and Astyanax lacustris) using a combination of genomic, cytogenetic, and bioinformatic protocols. We also compared our data with the available satellitome of P. paranae. We described 57 satellite DNA (satDNA) families of P. fasciatus (80 variants), 50 of P. bockmanni (77 variants), and 33 of A. lacustris (54 variants). Our analyses demonstrated that several sequences were shared among the analyzed species, while some were restricted to two or three species. In total, we isolated 104 distinctive satDNA families present in the four species, of which 10 were shared among all four. Chromosome mapping revealed that the clustered satDNA was mainly located in the subtelomeric and pericentromeric areas. Although all Psalidodon species demonstrated the same pattern of clusterization of satDNA, the number of clusters per genome was variable, indicating a high dynamism of these sequences. In addition, our results expand the knowledge of the As51 satellite DNA family, revealing that P. bockmanni and P. paranae exhibited an abundant variant of 39 bp, while P. fasciatus showed a variant of 43 bp. The majority of satDNAs in the satellitomes analyzed here presented a common library repetitive sequence in Psalidodon and Astyanax, with abundance variations in each species, as expected for closely related groups. In addition, we concluded that the most abundant satDNA in Psalidodon (As51) passed through a diversification process in this group, resulting in new variants exclusive of Psalidodon.

Satellitome analysis illuminates the evolution of ZW sex chromosomes of Triportheidae fishes (Teleostei: Characiformes).

Satellites are an abundant source of repetitive DNAs that play an essential role in the chromosomal organization and are tightly linked with the evolution of sex chromosomes. Among fishes, Triportheidae stands out as the only family where almost all species have a homeologous ZZ/ZW sex chromosomes system. While the Z chromosome is typically conserved, the W is always smaller, with variations in size and morphology between species. Here, we report an analysis of the satellitome of Triportheus auritus (TauSat) by integrating genomic and chromosomal data, with a special focus on the highly abundant and female-biased satDNAs. In addition, we investigated the evolutionary trajectories of the ZW sex chromosomes in the Triportheidae family by mapping satDNAs in selected representative species of this family. The satellitome of T. auritus comprised 53 satDNA families of which 24 were also hybridized by FISH. Most satDNAs differed significantly between sexes, with 19 out of 24 being enriched on the W chromosome of T. auritus. The number of satDNAs hybridized into the W chromosomes of T. signatus and T. albus decreased to six and four, respectively, in accordance with the size of their W chromosomes. No TauSat probes produced FISH signals on the chromosomes of Agoniates halecinus. Despite its apparent conservation, our results indicate that each species differs in the satDNA accumulation on the Z chromosome. Minimum spanning trees (MSTs), generated for three satDNA families with different patterns of FISH mapping data, revealed different homogenization rates between the Z and W chromosomes. These results were linked to different levels of recombination between them. The most abundant satDNA family (TauSat01) was exclusively hybridized in the centromeres of all 52 chromosomes of T. auritus, and its putative role in the centromere evolution was also highlighted. Our results identified a high differentiation of both ZW chromosomes regarding satellites composition, highlighting their dynamic role in the sex chromosomes evolution.

The Satellite DNA Catalogues of Two Serrasalmidae (Teleostei, Characiformes): Conservation of General satDNA Features over 30 Million Years.

Satellite DNAs (satDNAs) are tandemly repeated sequences that are usually located on the heterochromatin, and the entire collection of satDNAs within a genome is called satellitome. Primarily, these sequences are not under selective pressure and evolve by concerted evolution, resulting in elevated rates of divergence between the satDNA profiles of reproductive isolated species/populations. Here, we characterized two additional satellitomes of Characiformes fish (Colossoma macropomum and Piaractus mesopotamicus) that diverged approximately 30 million years ago, while still retaining conserved karyotype features. The results we obtained indicated that several satDNAs (50% of satellite sequences in P. mesopotamicus and 43% in C. macropomum) show levels of conservation between the analyzed species, in the nucleotide and chromosomal levels. We propose that long-life cycles and few genomic changes could slow down rates of satDNA differentiation.

Sex-Dependent Inheritance of B Chromosomes in Psalidodon paranae (Teleostei, Characiformes) Revealed by Directed Crossings.

B chromosomes are additional dispensable elements to the standard chromosomal set of an organism. In most cases, their transmission differs from Mendelian patterns, leading to their accumulation or extinction. The present study aimed to describe, for the first time, the transmission pattern of B chromosome in a population of Psalidodon paranae through directed crosses, as well as to analyze the populational dynamics of B chromosome. Our results revealed the possible elimination of B chromosome in crossings where only females were B-carriers, with a mean transmission rate (kB) of 0.149; however, kB was significantly higher in crossings involving male B-carriers (kB = 0.328-0.450). Moreover, we observed an increase in the frequency of B chromosomes in the natural population of P. paranae in the last two decades. These apparently contradictory results can make sense if the B chromosome provides adaptive advantages to their carriers. Here, we observed a differential transmission of B chromosomes in each sex of parental individuals, with higher transmission rates in crossing involving males B-carriers, in addition to describe the temporal changes of B chromosome frequency in P. paranae.

Chromosomal Instability and Origin of B Chromosomes in the Amazonian Glass Tetra Moenkhausia oligolepis (Günther, 1864) (Characiformes, Characidae).

B chromosomes occur in different species of the small characid fishes of the genus Moenkhausia. These supernumerary elements, that do not recombine with chromosomes of the standard A complement and follow their own evolutionary mechanism vary in number, morphology, and distribution. Here, we show karyotypic data of individuals of 2 populations of Moenkhausia oligolepis of the Brazilian Amazon (Pedro Correia and Taboquinha streams, Tocantins river basin), both with a diploid number of 50 chromosomes and karyotypic formula of 10m + 32sm + 8a. In addition to the normal complement, we also observed the occurrence of B chromosomes in the 2 populations with intra- and interindividual variation ranging from 0 to 10 Bs, independent of sex. The C-banding pattern evidenced heterochromatic blocks located mainly in the pericentromeric region of the chromosomes, while the B chromosomes appeared euchromatic. Silver-stained nucleolus organizer regions were identified in multiples sites, and some of these blocks were positive when stained with chromomycin A3. The karyotype analysis and the application of whole-chromosome painting in populations of M. oligolepis reinforce the conservation of the basal diploid number for the genus, as well as the evolutionary tendency in these fishes to carry B chromosomes. Both populations turned out to be in different stages of stability and expansion of their B chromosomes. We further suggest that the origin of these chromosomes is due to the formation of isochromosomes. Here, we identified a pair of complement A chromosomes involved in this process.

The B Chromosomes of Prochilodus lineatus (Teleostei, Characiformes) Are Highly Enriched in Satellite DNAs.

B or supernumerary chromosomes are dispensable elements that are widely present in numerous eukaryotes. Due to their non-recombining nature, there is an evident tendency for repetitive DNA accumulation in these elements. Thus, satellite DNA plays an important role in the evolution and diversification of B chromosomes and can provide clues regarding their origin. The characiform Prochilodus lineatus was one of the first discovered fish species bearing B chromosomes, with all populations analyzed so far showing one to nine micro-B chromosomes and exhibiting at least three morphological variants (Ba, Bsm, and Bm). To date, a single satellite DNA is known to be located on the B chromosomes of this species, but no information regarding the differentiation of the proposed B-types is available. Here, we characterized the satellitome of P. lineatus and mapped 35 satellite DNAs against the chromosomes of P. lineatus, of which six were equally located on all B-types and this indicates a similar genomic content. In addition, we describe, for the first time, an entire population without B chromosomes.

Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish.

BACKGROUND: Eukaryote genomes frequently harbor supernumerary B chromosomes in addition to the "standard" A chromosome set. B chromosomes are thought to arise as byproducts of genome rearrangements and have mostly been considered intraspecific oddities. However, their evolutionary transcendence beyond species level has remained untested. RESULTS: Here we reveal that the large metacentric B chromosomes reported in several fish species of the genus Astyanax arose in a common ancestor at least 4 million years ago. We generated transcriptomes of A. scabripinnis and A. paranae 0B and 1B individuals and used these assemblies as a reference for mapping all gDNA and RNA libraries to quantify coverage differences between B-lacking and B-carrying genomes. We show that the B chromosomes of A. scabripinnis and A. paranae share 19 protein-coding genes, of which 14 and 11 were also present in the B chromosomes of A. bockmanni and A. fasciatus, respectively. Our search for B-specific single-nucleotide polymorphisms (SNPs) identified the presence of B-derived transcripts in B-carrying ovaries, 80% of which belonged to nobox, a gene involved in oogenesis regulation. Importantly, the B chromosome nobox paralog is expressed > 30× more than the A chromosome paralog. This indicates that the normal regulation of this gene is altered in B-carrying females, which could potentially facilitate B inheritance at higher rates than Mendelian law prediction. CONCLUSIONS: Taken together, our results demonstrate the long-term survival of B chromosomes despite their lack of regular pairing and segregation during meiosis and that they can endure episodes of population divergence leading to species formation.

Establishment of rapid and non-invasive protocols to identify B-carrying individuals of Psalidodon paranae.

Supernumerary, or B, chromosomes are present in several eukaryotes, including characid fish of the genus Psalidodon. Notably, Psalidodon paranae carries the most studied B chromosome variant, a macro-B chromosome. The origin of this element was determined to be an isochromosome; however, data regarding its inheritance remain unavailable due to methodological barriers such as the lack of an efficient, non-invasive, and rapid protocol for identifying B-carrying individuals that would enable the design of efficient crossing experiments. Thus, in this study, we primarily aimed was to develop two non-invasive and fast (approximately 2 h) methods to identify the presence of B chromosomes in live specimens of P. paranae based on satellite DNA (satDNA) sequences known to be present in this element. The methods include fluorescence in situ hybridization in interphase nuclei and relative gene quantification of satDNAs using quantitative polymerase chain reaction. Our results reveal the efficiency of quick-fluorescence in situ hybridization and quantitative polymerase chain reaction for identifying B-carrying individuals using the proposed satDNA sequences and open up new possibilities to study B chromosomes.

A Long-Term Conserved Satellite DNA That Remains Unexpanded in Several Genomes of Characiformes Fish Is Actively Transcribed.

Eukaryotic genomes contain large amounts of repetitive DNA sequences, such as tandemly repeated satellite DNAs (satDNAs). These sequences are highly dynamic and tend to be genus- or species-specific due to their particular evolutionary pathways, although there are few unusual cases of conserved satDNAs over long periods of time. Here, we used multiple approaches to reveal that an satDNA named CharSat01-52 originated in the last common ancestor of Characoidei fish, a superfamily within the Characiformes order, ∼140-78 Ma, whereas its nucleotide composition has remained considerably conserved in several taxa. We show that 14 distantly related species within Characoidei share the presence of this satDNA, which is highly amplified and clustered in subtelomeric regions in a single species (Characidium gomesi), while remained organized as small clusters in all the other species. Defying predictions of the molecular drive of satellite evolution, CharSat01-52 shows similar values of intra- and interspecific divergence. Although we did not provide evidence for a specific functional role of CharSat01-52, its transcriptional activity was demonstrated in different species. In addition, we identified short tandem arrays of CharSat01-52 embedded within single-molecule real-time long reads of Astyanax paranae (536 bp-3.1 kb) and A. mexicanus (501 bp-3.9 kb). Such arrays consisted of head-to-tail repeats and could be found interspersed with other sequences, inverted sequences, or neighbored by other satellites. Our results provide a detailed characterization of an old and conserved satDNA, challenging general predictions of satDNA evolution.

Development of a SNP linkage map and genome-wide association study for resistance to Aeromonas hydrophila in pacu (Piaractus mesopotamicus).

BACKGROUND: Pacu (Piaractus mesopotamicus) is one of the most important Neotropical aquaculture species from South America. Disease outbreaks caused by Aeromonas hydrophila infection have been considered significant contributors to the declining levels of pacu production. The current implementation of genomic selection for disease resistance has been adopted as a powerful strategy for improvement in fish species. This study aimed to investigate the genetic architecture of resistance to A. hydrophila in pacu via Genome-Wide Association Study (GWAS), the identification of suggestive Quantitative Trait Loci (QTLs) and putative genes associated with this trait. The genetic data were obtained from 381 juvenile individuals belonging to 14 full-sibling families. An experimental challenge was performed to gain access to the levels of genetic variation for resistance against the bacteria using the following trait definitions: binary test survival (TS) and time of death (TD). RESULTS: The analyses of genetic parameters estimated moderate heritability (h2) for both resistance traits: 0.20 (± 0.09) for TS and 0.35 (± 0.15) for TD. A linkage map for pacu was developed to enable the GWAS, resulting in 27 linkage groups (LGs) with 17,453 mapped Single Nucleotide Polymorphisms (SNPs). The length of the LGs varied from 79.95 (LG14) to 137.01 (LG1) cM, with a total map length of 2755.60 cM. GWAS identified 22 putative QTLs associated to A. hydrophila resistance. They were distributed into 17 LGs, and were considered suggestive genomic regions explaining > 1% of the additive genetic variance (AGV) for the trait. Several candidate genes related to immune response were located close to the suggestive QTLs, such as tbk1, trim16, Il12rb2 and lyz2. CONCLUSION: This study describes the development of the first medium density linkage map for pacu, which will be used as a framework to study relevant traits to the production of this species. In addition, the resistance to A. hydrophila was found to be moderately heritable but with a polygenic architecture suggesting that genomic selection, instead of marker assisted selection, might be useful for efficiently improving resistance to one of the most problematic diseases that affects the South American aquaculture.