Understanding microchromosomal organization and evolution in four representative woodpeckers (Picidae, Piciformes) through BAC-FISH analysis.

The genome organization of woodpeckers has several distinctive features e.g., an uncommon accumulation of repetitive sequences, enlarged Z chromosomes, and atypical diploid numbers. Despite the large diversity of species, there is a paucity of detailed cytogenomic studies for this group and we thus aimed to rectify this. Genome organization patterns and hence evolutionary change in the microchromosome formation of four species (Colaptes campestris, Veniliornis spilogaster, Melanerpes candidus, and Picumnus nebulosus) was established through fluorescence in situ hybridization using bacterial artificial chromosomes originally derived from Gallus gallus and Taeniopygia guttata. Findings suggest that P. nebulosus (2n = 110), which was described for the first time, had the most basal karyotype among species of Picidae studied here, and probably arose as a result of fissions of avian ancestral macrochromosomes. We defined a new chromosomal number for V. spilogaster (2n = 88) and demonstrated microchromosomal rearrangements involving C. campestris plus a single, unique hitherto undescribed rearrangement in V. spilogaster. This comprised an inversion after a fusion involving the ancestral microchromosome 12 (homologous to chicken microchromosome 12). We also determined that the low diploid number of M. candidus is related to microchromosome fusions. Woodpeckers thus exhibit significantly rearranged karyotypes compared to the putative ancestral karyotype.

Chromosome mapping of retrotransposon AviRTE in a neotropical bird species: Trogon surrucura (Trogoniformes; Trogonidae).

Avian genomes are characterized as being more compact than other amniotes, with less diversity and density of transposable elements (TEs). In addition, birds usually show bimodal karyotypes, exhibiting a great variation in diploid numbers. Some species present unusually large sex chromosomes, possibly due to the accumulation of repetitive sequences. Avian retrotransposon-like element (AviRTE) is a long interspersed nuclear element (LINE) recently discovered in the genomes of birds and nematodes, and it is still poorly characterized in terms of chromosomal mapping and phylogenetic relationships. In this study, we mapped AviRTE isolated from the Trogon surrucura genome into the T. surrucura (TSU) karyotype. Furthermore, we analyzed the phylogenetic relationships of this LINE in birds and other vertebrates. Our results showed that the distribution pattern of AviRTE is not restricted to heterochromatic regions, with accumulation on the W chromosome of TSU, yet another species with an atypical sex chromosome and TE hybridization. The phylogenetic analysis of AviRTE sequences in birds agreed with the proposed phylogeny of species in most clades, and allowed the detection of this sequence in other species, expanding the distribution of the element.

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.

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.

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.

Understanding the chromosomal evolution in cuckoos (Aves, Cuculiformes): a journey through unusual rearrangements.

The Cuculiformes are a family of over 150 species that live in a range of habitats, such as forests, savannas, and deserts. Here, bacterial artificial chromosome (BAC) probes (75 from chicken and 14 from zebra finch macrochromosomes 1-10 +ZW and for microchromosomes 11-28 (except 16)) were used to investigate chromosome homologies between chicken and the squirrel cuckoo (Piaya cayana). In addition, repetitive DNA probes were applied to characterize the chromosome organization and to explore the role of these sequences in the karyotype evolution of P. cayana. We also applied BAC probes for chicken chromosome 17 and Z to the guira cuckoo (Guira guira) to test whether this species has an unusual Robertsonian translocation between a microchromosome and the Z chromosome, recently described in the smooth-billed ani (Crotophaga ani). Our results revealed extensive chromosome reorganization with inter- and intrachromosomal rearrangements in P. cayana, including a conspicuous chromosome size and heterochromatin polymorphism on chromosome pair 20. Furthermore, we confirmed that the Z-autosome Robertsonian translocation found in C. ani is also found in G. guira, not P. cayana. These findings suggest that this translocation occurred prior to the divergence between C. ani and G. guira, but after the divergence with P. cayana.

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.

Comparative chromosome painting in three Pelecaniformes species (Aves): Exploring the role of macro and microchromosome fusions in karyotypic evolution.

Pelecaniformes is an order of waterbirds that exhibit diverse and distinct morphologies. Ibis, heron, pelican, hammerkop, and shoebill are included within the order. Despite their fascinating features, the phylogenetic relationships among the families within Pelecaniformes remain uncertain and pose challenges due to their complex evolutionary history. Their karyotypic evolution is another little-known aspect. Therefore, to shed light on the chromosomal rearrangements that have occurred during the evolution of Pelecaniformes, we have used whole macrochromosome probes from Gallus gallus (GGA) to show homologies on three species with different diploid numbers, namely Cochlearius cochlearius (2n = 74), Eudocimus ruber (2n = 66), and Syrigma sibilatrix (2n = 62). A fusion between GGA6 and GGA7 was found in C. cochlearius and S. sibilatrix. In S. sibilatrix the GGA8, GGA9 and GGA10 hybridized to the long arms of biarmed macrochromosomes, indicating fusions with microchromosomes. In E. ruber the GGA7 and GGA8 hybridized to the same chromosome pair. After comparing our painting results with previously published data, we show that distinct chromosomal rearrangements have occurred in different Pelecaniformes lineages. Our study provides new insight into the evolutionary history of Pelecaniformes and the chromosomal changes involving their macrochromosomes and microchromosomes that have taken place in different species within this order.

Microchromosome BAC-FISH Reveals Different Patterns of Genome Organization in Three Charadriiformes Species.

Microchromosomes, once considered unimportant elements of the genome, represent fundamental building blocks of bird karyotypes. Shorebirds (Charadriiformes) comprise a wide variety of approximately 390 species and are considered a valuable model group for biological studies. Despite this variety, cytogenetic analysis is still very scarce in this bird order. Thus, the aim of this study was to provide insight into the Charadriiformes karyotype, with emphasis on microchromosome evolution in three species of shorebirds-Calidris canutus, Jacana jacana, and Vanellus chilensis-combining classical and molecular approaches. Cross-species FISH mapping applied two BAC probes for each microchromosome, GGA10-28 (except GGA16). The experiments revealed different patterns of microchromosome organization in the species investigated. Hence, while in C. canutus, we found two microchromosomes involved in chromosome fusions, they were present as single pairs in V. chilensis. We also described a new chromosome number for C. canutus (2n = 92). Hence, this study contributed to the understanding of genome organization and evolution of three shorebird species.

Chromosomal distribution of major rDNA and genome size variation in Belostoma angustum Lauck, B. nessimiani Ribeiro & Alecrim, and B. sanctulum Montandon (Insecta, Heteroptera, Belostomatidae).

Known as "electric-light bugs", belostomatids potentially act as agents of biological control. The Belostoma genus has holokinetic chromosomes, interspecific variation in diploid number, sex chromosome system and DNA content. Thus, the chromosomal complement, the accumulation of constitutive heterochromatin and the distribution of rDNA clusters by fluorescence in situ hybridization (FISH) in Belostoma angustum (BAN), Belostoma sanctulum (BSA), and Belostoma nessimiani (BNE) were evaluated. In addition, a comparative analysis of the DNA content of these species and B. estevezae (BES) was performed. BES has the highest Belostoma DNA content, while BSA has the lowest. BAN showed 2n = 29 + X1X2Y, while BSA and BNE had 2n = 14 + XY. BSA showed 18S rDNA markings on sex chromosomes, while BNE and BAN did on autosomes. The difference between BSA and BNE occurs because of the possible movement of the rDNA cluster in BNE. We suggest the occurrence of fusion in the autosomes of BSA and BNE, and fragmentation in the sex chromosomes in BAN. Also, the genome size of 1-2 pg represents a haploid DNA content of a common ancestor, from which the genomes of BES and BAN had evolved by gene duplication and heterochromatinization events.

Cytogenetic Evidence Clarifies the Phylogeny of the Family Rhynchocyclidae (Aves: Passeriformes).

The phylogenetic position and taxonomic status of Rhynchocyclidae (Aves: Passeriformes) have been the subject of debate since their first description. In most models, Rhynchocyclidae represents a subfamily-level taxon placed within the Tyrant Flycatchers (Tyrannidae). Considering that this classification does not include cytotaxonomic characters, we tested the hypothesis that the chromosome organization of Rhynchocyclidae members differs from that of Tyrannidae. Hence, we selected two species, Tolmomyias sulphurescens, and Pitangus sulphuratus, representing Rhynchocyclidae and Tyrannidae, respectively. Results revealed a diploid number (2n) of 60 in T. sulphurescens and 2n = 80 in P. sulphuratus, indicating significant chromosomal differences. Chromosome mapping of Gallus gallus (GGA) and Taeniopygia guttata bacterial artificial chromosome (BAC) corresponding to chromosomes GGA1-28 (except 16) revealed that the genome evolution of T. sulphurescens involved extensive chromosome fusions of macrochromosomes and microchromosomes. On the other hand, P. sulphuratus retained the ancestral pattern of organization of macrochromosomes (except the centric fission involving GGA1) and microchromosomes. In conclusion, comparing our results with previous studies in Tyrant Flycatchers and allies indicates that P. sulphuratus has similar karyotypes to other Tyrannidae members. However, T. sulphurescens does not resemble the Tyrannidae family, reinforcing family status to the clade named Rhynchocyclidae.

Karyotype Evolution and Genomic Organization of Repetitive DNAs in the Saffron Finch, Sicalis flaveola (Passeriformes, Aves).

The Saffron finch (Sicalis flaveola), a semi-domestic species, is tolerant of human proximity and nesting in roof spaces. Considering the importance of cytogenomic approaches in revealing different aspects of genomic organization and evolution, we provide detailed cytogenetic data for S. flaveola, including the standard Giemsa karyotype, C- and G-banding, repetitive DNA mapping, and bacterial artificial chromosome (BAC) FISH. We also compared our results with the sister groups, Passeriformes and Psittaciformes, bringing new insights into the chromosome and genome evolution of birds. The results revealed contrasting rates of intrachromosomal changes, highlighting the role of SSR (simple short repetition probes) accumulation in the karyotype reorganization. The SSRs showed scattered hybridization, but brighter signals were observed in the microchromosomes and the short arms of Z chromosome in S. flaveola. BACs probes showed conservation of ancestral syntenies of macrochromosomes (except GGA1), as well as the tested microchromosomes. The comparison of our results with previous studies indicates that the great biological diversity observed in Passeriformes was not likely accompanied by interchromosomal changes. In addition, although repetitive sequences often act as hotspots of genome rearrangements, Passeriformes species showed a higher number of signals when compared with the sister group Psittaciformes, indicating that these sequences were not involved in the extensive karyotype reorganization seen in the latter.

Cytotaxonomy of Gallinula melanops (Gruiformes, Rallidae): Karyotype evolution and phylogenetic inference.

Although Rallidae is the most diverse family within Gruiformes, there is little information concerning the karyotype of the species in this group. In fact, Gallinula melanops, a species of Rallidae found in Brazil, is among the few species studied cytogenetically, but only with conventional staining and repetitive DNA mapping, showing 2n=80. Thus, in order to understand the karyotypic evolution and phylogeny of this group, the present study aimed to analyze the karyotype of G. melanops by classical and molecular cytogenetics, comparing the results with other species of Gruiformes. The results show that G. melanops has the same chromosome rearrangements as described in Gallinula chloropus (Clade Fulica), including fission of ancestral chromosomes 4 and 5 of Gallus gallus (GGA), beyond the fusion between two of segments resultants of the GGA4/GGA5, also fusions between the chromosomes GGA6/GGA7. Thus, despite the fact that some authors have suggested the inclusion of G. melanops in genus Porphyriops, our molecular cytogenetic results confirm its place in the Gallinula genus.

Interspecies Chromosome Mapping in Caprimulgiformes, Piciformes, Suliformes, and Trogoniformes (Aves): Cytogenomic Insight into Microchromosome Organization and Karyotype Evolution in Birds.

Interchromosomal rearrangements involving microchromosomes are rare events in birds. To date, they have been found mostly in Psittaciformes, Falconiformes, and Cuculiformes, although only a few orders have been analyzed. Hence, cytogenomic studies focusing on microchromosomes in species belonging to different bird orders are essential to shed more light on the avian chromosome and karyotype evolution. Based on this, we performed a comparative chromosome mapping for chicken microchromosomes 10 to 28 using interspecies BAC-based FISH hybridization in five species, representing four Neoaves orders (Caprimulgiformes, Piciformes, Suliformes, and Trogoniformes). Our results suggest that the ancestral microchromosomal syntenies are conserved in Pteroglossus inscriptus (Piciformes), Ramphastos tucanus tucanus (Piciformes), and Trogon surrucura surrucura (Trogoniformes). On the other hand, chromosome reorganization in Phalacrocorax brasilianus (Suliformes) and Hydropsalis torquata (Caprimulgiformes) included fusions involving both macro- and microchromosomes. Fissions in macrochromosomes were observed in P. brasilianus and H. torquata. Relevant hypothetical Neognathae and Neoaves ancestral karyotypes were reconstructed to trace these rearrangements. We found no interchromosomal rearrangement involving microchromosomes to be shared between avian orders where rearrangements were detected. Our findings suggest that convergent evolution involving microchromosomal change is a rare event in birds and may be appropriate in cytotaxonomic inferences in orders where these rearrangements occurred.

Chromosomal Analysis in Crotophaga ani (Aves, Cuculiformes) Reveals Extensive Genomic Reorganization and an Unusual Z-Autosome Robertsonian Translocation.

Although cytogenetics studies in cuckoos (Aves, Cuculiformes) have demonstrated an interesting karyotype variation, such as variations in the chromosome morphology and diploid number, their chromosome organization and evolution, and relation with other birds are poorly understood. Hence, we combined conventional and molecular cytogenetic approaches to investigate chromosome homologies between chicken and the smooth-billed ani (Crotophaga ani). Our results demonstrate extensive chromosome reorganization in C. ani, with interchromosomal rearrangements involving macro and microchromosomes. Intrachromosomal rearrangements were observed in some macrochromosomes, including the Z chromosome. The most evolutionary notable finding was a Robertsonian translocation between the microchromosome 17 and the Z chromosome, a rare event in birds. Additionally, the simple short repeats (SSRs) tested here were preferentially accumulated in the microchromosomes and in the Z and W chromosomes, showing no relationship with the constitutive heterochromatin regions, except in the W chromosome. Taken together, our results suggest that the avian sex chromosome is more complex than previously postulated and revealed the role of microchromosomes in the avian sex chromosome evolution, especially cuckoos.

Chromosomal Evolution in the Phylogenetic Context: A Remarkable Karyotype Reorganization in Neotropical Parrot Myiopsitta monachus (Psittacidae).

Myiopsitta monachus is a small Neotropical parrot (Psittaciformes: Arini Tribe) from subtropical and temperate regions of South America. It has a diploid chromosome number 2n = 48, different from other members of the Arini Tribe that have usually 70 chromosomes. The species has the lowest 2n within the Arini Tribe. In this study, we combined comparative chromosome painting with probes generated from chromosomes of Gallus gallus and Leucopternis albicollis, and FISH with bacterial artificial chromosomes (BACs) selected from the genome library of G. gallus with the aim to shed light on the dynamics of genome reorganization in M. monachus in the phylogenetic context. The homology maps showed a great number of fissions in macrochromosomes, and many fusions between microchromosomes and fragments of macrochromosomes. Our phylogenetic analysis by Maximum Parsimony agree with molecular data, placing M. monachus in a basal position within the Arini Tribe, together with Amazona aestiva (short tailed species). In M. monachus many chromosome rearrangements were found to represent autopomorphic characters, indicating that after this species split as an independent branch, an intensive karyotype reorganization took place. In addition, our results show that M. monachus probes generated by flow cytometry provide novel cytogenetic tools for the detection of avian chromosome rearrangements, since this species presents breakpoints that have not been described in other species.

A Comprehensive Cytogenetic Analysis of Several Members of the Family Columbidae (Aves, Columbiformes).

The Columbidae species (Aves, Columbiformes) show considerable variation in their diploid numbers (2n = 68-86), but there is limited understanding of the events that shaped the extant karyotypes. Hence, we performed whole chromosome painting (wcp) for paints GGA1-10 and bacterial artificial chromosome (BAC) probes for chromosomes GGA11-28 for Columbina passerina, Columbina talpacoti, Patagioenas cayennensis, Geotrygon violacea and Geotrygon montana. Streptopelia decaocto was only investigated with paints because BACs for GGA10-28 had been previously analyzed. We also performed phylogenetic analyses in order to trace the evolutionary history of this family in light of chromosomal changes using our wcp data with chicken probes and from Zenaida auriculata, Columbina picui, Columba livia and Leptotila verreauxi, previously published. G-banding was performed on all these species. Comparative chromosome paint and G-banding results suggested that at least one interchromosomal and many intrachromosomal rearrangements had occurred in the diversification of Columbidae species. On the other hand, a high degree of conservation of microchromosome organization was observed in these species. Our cladistic analysis, considering all the chromosome rearrangements detected, provided strong support for L. verreauxi and P. cayennensis, G. montana and G. violacea, C. passerina and C. talpacoti having sister taxa relationships, as well as for all Columbidae species analyzed herein. Additionally, the chromosome characters were mapped in a consensus phylogenetic topology previously proposed, revealing a pericentric inversion in the chromosome homologous to GGA4 in a chromosomal signature unique to small New World ground doves.

The molecular cytogenetic characterization of Conopophaga lineata indicates a common chromosome rearrangement in the Parvorder Furnariida (Aves, Passeriformes).

Cytogenetic analyses of the Suboscines species are still scarce, and so far, there is no karyotype description of any species belonging to the family Conopophagidae. Thus, the aim of this study is to describe and analyze the karyotype of Conopophaga lineata by chromosome painting using Gallus gallus (GGA) probes and to identify the location of the 18/28S rDNA cluster. Metaphases were obtained from fibroblast culture from two individuals of C. lineata. We observed a diploid number of 2n=78. GGA probes showed that most ancestral syntenies are conserved, except for the fission of GGA1 and GGA2, into two distinct pairs each. We identified the location of 18S rDNA genes in a pair of microchromosomes. The fission of the syntenic group corresponding to GGA2 was observed in other Furnariida, and hence may correspond to a chromosomal synapomorphy for the species of Parvorder Furnariida.

Chromosomal evolution and phylogenetic considerations in cuckoos (Aves, Cuculiformes, Cuculidae).

The Cuckoos have a long history of difficult classification. The species of this order have been the subject of several studies based on osteology, behavior, ecology, morphology and molecular data. Despite this, the relationship between Cuculiformes and species of other orders remains controversial. In this work, two species of Cuculidae, Guira guira (Gmelin, 1788) and Piaya cayana (Linnaeus, 1766), were analyzed by means of comparative chromosome painting in order to study the chromosome evolution of this group and to undertake the first chromosome mapping of these species. Our results demonstrate high chromosomal diversity, with 2n = 76 in G. guira, with fission and fusion events involving ancestral syntenies, while P. cayana presented only fissions, which were responsible for the high diploid number of 2n = 90. Interestingly, there were no chromosomal rearrangements in common between these species. Our results, based on Giemsa staining, were compared with previous data for other cuckoos and also with taxa proposed as sister-groups of Cuculiformes (Otidiformes, Musophagiformes and Opisthocomiformes). Cytogenetic comparisons demonstrated that cuckoo species can be divided into at least three major groups. In addition, we found no evidence to place Cuculiformes close to the groups proposed previously as sister-groups.

The distribution of 45S rDNA sites in bird chromosomes suggests multiple evolutionary histories.

The distribution of 45S rDNA cluster in avian karyotypes varies in different aspects, such as position, number of bearer chromosomes, and bearers being macro- or microchromosomes. The present study investigated the patterns of variation in the 45S rDNA-bearer chromosomes of birds in order to understand the evolutionary dynamics of the cluster configuration and its contribution to the evolution of bird karyotypes. A total of 73 bird species were analyzed, including both published data and species for which rDNA-FISH was conducted for the first time. In most birds, the 45S rDNA clusters were located in a single pair of microchromosomes. Hence, the location of 45S rDNA in macrochromosomes, observed only in Neognathae species, seems to be a derived state, probably the result of chromosomal fusion between microchromosomes and distinct macrochromosomes. Additionally, the 45S rDNA was observed in multiple microchromosomes in different branches of the bird phylogeny, suggesting recurrence of dispersion processeses, such as duplications and translocations. Overall, this study indicated that the redistribution of the 45S rDNA sites in bird chromosomes followed different evolutionary trajectories with respect to each lineage of the class Aves.