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.

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.

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.

The order Charadriiformes comprises three major clades: Lari and Scolopaci as sister group to Charadrii. Until now, only three Charadriiformes species have been studied by chromosome painting: Larus argentatus (Lari), Burhinus oedicnemus and Vanellus chilensis (Charadrii). Hence, there is a lack of information concerning the third clade, Scolapaci. Based on this, and to gain a better understanding of karyotype evolution in the order Charadriiformes, we applied conventional and molecular cytogenetic approaches in a species belonging to clade Scolopaci - the wattled jacana (Jacana jacana) - using Gallus gallus and Zenaida auriculata chromosome-specific probes. Cross-species evaluation of J. jacana chromosomes shows extensive genomic reshuffling within macrochromosomes during evolution, with multiple fission and fusion events, although the diploid number remains at high level (2n=82). Interestingly, this species does not have the GGA7-8 fusion, which was found in two representatives of Charadrii clade, reinforcing the idea that this fusion may be exclusive to the Charadrii clade. In addition, it is shown that the chromosome evolution in Charadriiformes is complex and resulted in species with typical and atypical karyotypes. The karyotypic features of Scolopaci are very different from those of Charadrii and Lari, indicating that after divergence, each suborder has undergone different chromosome rearrangements.

Although birds belonging to order Caprimulgiformes show extensive karyotype variation, data concerning their genomic organization is still scarce, as most studies have presented only results obtained from conventional staining analyses. Nevertheless, some interesting findings have been observed, such as the W chromosome of the Common Potoo, Nyctibius griseus (2n = 86), which has the same morphology and size of the Z chromosome, a rare feature in Neognathae birds. Hence, we aimed to investigate the process by which the W chromosome of this species was enlarged. For that, we analyzed comparatively the chromosome organization of the Common Potoo and the Scissor-tailed Nightjar, Hydropsalis torquata (2n = 74), which presents the regular differentiated sex chromosomes, by applying C-banding, G-banding and mapping of repetitive DNAs (microsatellite repeats and 18S rDNA). Our results showed an accumulation of constitutive heterochromatin in the W chromosome of both species. However, 9 out of 11 microsatellite sequences hybridized in the large W chromosome in the Common Potoo, while none of them hybridized in the W chromosome of the Scissor-tailed Nightjar. Therefore, we can conclude that the accumulation of microsatellite sequences, and consequent increase in constitutive heterochromatin, was responsible for the enlargement of the W chromosome in the Common Potoo. Based on these results, we conclude that even though these two species belong to the same order, their W chromosomes have gone through different evolutionary histories, with an extra step of accumulation of repetitive sequences in the Common Potoo.

Avian cell culture is widely applied for cytogenetic studies, the improvement of which increasingly allows for the production of high-quality chromosomes, essential to perform both classical and molecular cytogenetic studies. Among these approaches, there are two main types: fibroblast and bone marrow culture. Despite its high cost and complexity, fibroblast culture is considered the superior approach due to the quality of the metaphases produced. Short-term bone marrow cultivation provides more condensed chromosomes but nonetheless is quicker and easier. In the search for a quicker, cheaper way to prepare metaphases without losing quality, the present work developed a novel, widely applicable protocol for avian chromosome preparation. Twenty-one bird embryos from distinct families were sampled: Icteridae, Columbidae, Furnariidae, Estrildidae, Thraupidae, Troglodytidae and Ardeidae. The protocol was based on a combination of modified fibroblast culture and bone marrow cultivation, taking the advantages of both. The results show that all species consistently presented good mitotic indexes and high-quality chromosomes. Overall, the application of this protocol for bird cytogenetics can optimize the time, considering that most fibroblast cultures take at least 3 days and often much longer. However, our protocol can be performed in 3 h with a much-reduced cost of reagents and equipment.