Comparative cytogenetics in Tyrannidae (Aves, Passeriformes): High genetic diversity despite conserved karyotype organization.

Introduction Passeriformes has the greatest species diversity among Neoaves, and the Tyrannidae is the richest in this order with about 600 valid species. The diploid number of this family remains constant, ranging from 2n = 76 to 84, but the chromosomal morphology varies, indicating the occurrence of different chromosomal rearrangements. Cytogenetic studies of the Tyrannidae remain limited, with approximately 20 species having been karyotyped thus far. This study aimed to describe the karyotypes of two species from this family, Myiopagis viridicata and Sirystes sibilator. Methods Skin biopsies were taken from each individual to establish fibroblast cell cultures and to obtain chromosomal preparations using the standard methodology. The chromosomal distribution of constitutive heterochromatin was investigated by C-banding, while the location of simple repetitive sequences (SSRs), 18S rDNA, and telomeric sequences were found through fluorescence in situ hybridization. Results The karyotypes of both species are composed of 2n = 80. The 18S rDNA probes hybridized into two pairs of microchromosomes in M. viridicata, but only a single pair in S. sibilator. Only the telomeric portions of each chromosome in both species were hybridized by the telomere sequence probes. Most of the SSRs were found accumulated in the centromeric and telomeric regions of several macro- and microchromosomes in both species, which likely correspond to the heterochromatin-rich regions. Conclusion Although both species analyzed showed a conserved karyotype organization (2n = 80), our study revealed significant differences in their chromosomal architecture, rDNA distribution, and SSR accumulation. These findings were discussed in the context of the evolution of Tyrannidae karyotypes.

Turnover of multiple sex chromosomes in Harttia catfish (Siluriformes, Loricariidae): a glimpse from whole chromosome painting.

The remarkable fish biodiversity encompasses also great sex chromosome variability. Harttia catfish belong to Neotropical models for karyotype and sex chromosome research. Some species possess one of the three male-heterogametic sex chromosome systems, XY, X1X2Y or XY1Y2, while other members of the genus have yet uncharacterized modes of sex determination. Particularly the XY1Y2 multiple sex chromosome system shows a relatively low incidence among vertebrates, and it has not been yet thoroughly investigated. Previous research suggested two independent X-autosome fusions in Harttia which led to the emergence of XY1Y2 sex chromosome system in three of its species. In this study, we investigated evolutionary trajectories of synteny blocks involved in this XY1Y2 system by probing six Harttia species with whole chromosome painting (WCP) probes derived from the X (HCA-X) and the chromosome 9 (HCA-9) of H. carvalhoi. We found that both painting probes hybridize to two distinct chromosome pairs in Amazonian species, whereas the HCA-9 probe paints three chromosome pairs in H. guianensis, endemic to Guyanese drainages. These findings demonstrate distinct evolutionary fates of mapped synteny blocks and thereby elevated karyotype dynamics in Harttia among the three evolutionary clades.

Zebrafish (Danio rerio) Gynogenetic Production by Heat Shock: Comparison Between Mitotic and Meiotic Treatment.

Inbred species are useful resources for a variety of biomedical research applications. To create isogenic zebrafish, it is feasible to stop meiosis II (repeatedly) or mitosis (two times) in a haploid embryo by applying pressure or by delivering a heat shock, respectively. In this study, to improve the repeatability, we suggest a less complicated approach based on sperm ultraviolet-C (UV-C) exposure for a shorter period followed by heat shock at various temperatures, eliminating the use of pressure in meiotic therapy since heat shock is more accessible to laboratories. In this study, the survivability rates of meiotic (Mei) and mitotic (Mit) gynogenesis offspring produced by various combinations of irradiation (28.5, 105, and 210 mJ/cm2) and temperature (Mei: 40.40°C, 40.60°C, or 40.90°C; Mt: 41.40°C, 41.90°C, or 42.45°C) were compared with diploid (C) and haploid (H) controls. Our findings demonstrated that 40.60°C and 41.90°C were the most suitable temperatures to produce meiotic and mitotic gynogenesis, respectively, whereas 28.5 mJ/cm2 was more successful in ensuring haploid embryos. As a result, we deduced that meiotic gynogenesis produces more viable offspring than the mitotic approach and requires a lower temperature to maintain the second polar body.

Small Body, Large Chromosomes: Centric Fusions Shaped the Karyotype of the Amazonian Miniature Fish Nannostomus anduzei (Characiformes, Lebiasinidae).

Miniature refers to species with extraordinarily small adult body size when adult and can be found within all major metazoan groups. It is considered that miniature species have experienced severe alteration of numerous morphological traits during evolution. For a variety of reasons, including severe labor concerns during collecting, chromosomal acquisition, and taxonomic issues, miniature fishes are neglected and understudied. Since some available studies indicate possible relationship between diploid chromosome number (2n) and body size in fishes, we aimed to study one of the smallest Neotropical fish Nannostomus anduzei (Teleostei, Characiformes, Lebiasinidae), using both conventional (Giemsa staining, C-banding) and molecular cytogenetic methods (FISH mapping of rDNAs, microsatellites, and telomeric sequences). Our research revealed that N. anduzei possesses one of the lowest diploid chromosome numbers (2n = 22) among teleost fishes, and its karyotype is entirely composed of large metacentric chromosomes. All chromosomes, except for pair number 11, showed an 18S rDNA signal in the pericentromeric region. 5S rDNA signals were detected in the pericentromeric regions of chromosome pair number 1 and 6, displaying synteny to 18S rDNA signals. Interstitial telomeric sites (ITS) were identified in the centromeric region of pairs 6 and 8, indicating that centric fusions played a significant role in karyotype evolution of studied species. Our study provides further evidence supporting the trend of diploid chromosome number reduction along with miniaturization of adult body size in fishes.

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.

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.

Chromosomal Evolution of Suboscines: Karyotype Diversity and Evolutionary Trends in Ovenbirds (Passeriformes, Furnariidae).

Furnariidae (ovenbirds) is one of the most diversified families in the Passeriformes order and Suboscines suborder. Despite the great diversity of species, cytogenetic research is still in its early stages, restricting our knowledge of their karyotype evolution. We combined traditional and molecular cytogenetic analyses in three representative species, Synallaxis frontalis, Syndactyla rufosuperciliata, and Cranioleuca obsoleta, to examine the chromosomal structure and evolution of ovenbirds. Our findings revealed that all the species studied had the same diploid number (2n = 82). Differences in chromosomal morphology of some macrochromosomes indicate the presence of intrachromosomal rearrangements. Although the three species only had the 18S rDNA on one microchromosome pair, chromosomal mapping of six simple short repeats revealed a varied pattern of chromosome distribution among them, suggesting that each species underwent different repetitive DNA accumulation upon their divergence. The interspecific comparative genomic hybridization experiment revealed that the Furnariidae species investigated carry centromeric regions enriched in similar repetitive sequences, bolstering the Furnariidae family's karyotype conservation. Nonetheless, the outgroup species Turdus rufiventris (Turdidae) demonstrated an advanced stage of sequence divergence with hybridization signals that were almost entirely limited to a few microchromosomes. Overall, the findings imply that Furnariidae species have a high degree of chromosomal conservation, and we could also observe a differentiation of repetitive sequences in both Passeriformes suborders (Suboscines and Oscines).

Tracking the evolutionary pathways among Brazilian Lebiasina species (Teleostei: Lebiasinidae): a chromosomal and genomic comparative investigation

Despite several difficulties in chromosomal analyses of small-sized fishes, the cytogenetics of the Lebiasinidae was largely improved in the last years, showing differential patterns in the chromosomal evolution inside the family. In this context, it has been shown that genus Lebiasina preserves its karyotypic macrostructure, composed of 2n = 36 chromosomes, whereas the other genera generally present higher 2n. This study focused on the comparative cytogenetics of three Lebiasina species, one of them analyzed here for the first time, using conventional and molecular procedures. The results reinforced the differentiated evolutionary path of the genus Lebiasina while, at the same time, highlighted the genomic particularities that have accompanied the evolution of each species. In this sense, the repetitive components of the genome played a significant role in the differentiation of each species. It is also notable that L. minuta and L. melanoguttata, the two species that occur exclusively in the Brazilian territory, show greater chromosomal similarities to each other than to the trans-Andean sister species, L. bimaculata.(AU)

Apesar das dificuldades encontradas em se realizar análises cromossômicas em peixes de pequeno porte, os estudos citogenéticos em Lebiasinidae vêm crescendo nos últimos anos e demonstrando padrões diferenciados na evolução cromossômica entre os membros da família. Nesse contexto, o gênero Lebiasina tem mostrado preservar sua macroestrutura cariotípica, composta por 2n = 36 cromossomos, enquanto os demais gêneros geralmente apresentam 2n maiores. Este estudo tem como foco a citogenética comparativa de três espécies de Lebiasina, sendo uma delas analisada pela primeira vez aqui, através do emprego de técnicas convencionais e moleculares. Os resultados obtidos reforçam a trajetória evolutiva diferenciada do gênero Lebiasina, ao mesmo tempo em que evidenciam as particularidades genômicas que acompanham a evolução de cada uma das espécies. Neste contexto, os componentes repetitivos do genoma tiveram um papel importante na caracterização particular de cada uma das espécies. Também, é notável que L. minuta e L. melanoguttata, duas espécies que ocorrem exclusivamente no território brasileiro, apresentam maior proximidade citogenética entre elas do que com a espécie irmã transandina, L. bimaculata.(AU)

Tracking the Evolutionary Trends Among Small-Size Fishes of the Genus Pyrrhulina (Characiforme, Lebiasinidae): New Insights From a Molecular Cytogenetic Perspective.

Miniature fishes have always been a challenge for cytogenetic studies due to the difficulty in obtaining chromosomal preparations, making them virtually unexplored. An example of this scenario relies on members of the family Lebiasinidae which include miniature to medium-sized, poorly known species, until very recently. The present study is part of undergoing major cytogenetic advances seeking to elucidate the evolutionary history of lebiasinids. Aiming to examine the karyotype diversification more deeply in Pyrrhulina, here we combined classical and molecular cytogenetic analyses, including Giemsa staining, C-banding, repetitive DNA mapping, comparative genomic hybridization (CGH), and whole chromosome painting (WCP) to perform the first analyses in five Pyrrhulina species (Pyrrhulina aff. marilynae, Pyrrhulina sp., P. obermulleri, P. marilynae and Pyrrhulina cf. laeta). The diploid number (2n) ranged from 40 to 42 chromosomes among all analyzed species, but P. marilynae is strikingly differentiated by having 2n = 32 chromosomes and a karyotype composed of large meta/submetacentric chromosomes, whose plesiomorphic status is discussed. The distribution of microsatellites does not markedly differ among species, but the number and position of the rDNA sites underwent significant changes among them. Interspecific comparative genome hybridization (CGH) found a moderate divergence in the repetitive DNA content among the species' genomes. Noteworthy, the WCP reinforced our previous hypothesis on the origin of the X1X2Y multiple sex chromosome system in P. semifasciata. In summary, our data suggest that the karyotype differentiation in Pyrrhulina has been driven by major structural rearrangements, accompanied by high dynamics of repetitive DNAs.

Comparative chromosomal mapping of microsatellite repeats reveals divergent patterns of accumulation in 12 Siluridae (Teleostei: Siluriformes) species.

The freshwater family Siluridae occurs in Eurasia and is especially speciose in South and Southeast Asia, representing an important aquaculture and fishery targets. However, despite the restricted cytogenetic data, a high diploid number variation (from 2n=40 to 92) characterizes this fish group. Considering the large genomic divergence among its species, silurid genomes have experienced an enormous diversification throughout their evolutionary history. Here, we aim to investigate the chromosomal distribution of several microsatellite repeats in 12 Siluridae species and infer about their possible roles in the karyotype evolution that occurred in this group. Our results indicate divergent patterns of microsatellite distribution and accumulation among the analyzed species. Indeed, they are especially present in significant chromosome locations, such as the centromeric and telomeric regions, precisely the ones associated with several kinds of chromosomal rearrangements. Our data provide pieces of evidence that repetitive DNAs played a direct role in fostering the chromosomal differentiation and biodiversity in this fish family.

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.

Cytogenetics of the small-sized fish, Copeina guttata (Characiformes, Lebiasinidae): Novel insights into the karyotype differentiation of the family.

Lebiasinidae is a small fish family composed by miniature to small-sized fishes with few cytogenetic data (most of them limited to descriptions of diploid chromosome numbers), thus preventing any evolutionary comparative studies at the chromosomal level. In the present study, we are providing, the first cytogenetic data for the red spotted tetra, Copeina guttata, including the standard karyotype, C-banding, repetitive DNA mapping by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH), providing chromosomal patterns and novel insights into the karyotype differentiation of the family. Males and females share diploid chromosome number 2n = 42 and karyotype composed of 2 metacentric (m), 4 submetacentric (sm) and 36 subtelocentric to acrocentric (st-a) chromosomes. Blocks of constitutive heterochromatin were observed in the centromeric and interstitial regions of several chromosomes, in addition to a remarkably large distal block, heteromorphic in size, which fully corresponded with the 18S rDNA sites in the fourth chromosomal pair. This overlap was confirmed by 5S/18S rDNA dual-color FISH. On the other hand, 5S rDNA clusters were situated in the long and short arms of the 2nd and 15th pairs, respectively. No sex-linked karyotype differences were revealed by male/female CGH experiments. The genomic probes from other two lebiasinid species, Lebiasina melanoguttata and Pyrrhulina brevis, showed positive hybridization signals only in the NOR region in the genome of C. guttata. We demonstrated that karyotype diversification in lebiasinids was accompanied by a series of structural and numeric chromosome rearrangements of different types, including particularly fusions and fissions.

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

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.