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.

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.

Investigating the evolutionary dynamics of diploid number variation in Ctenomys (Ctenomyidae, Rodentia).

Contrary to predictions from classical hybrid sterility models of chromosomal speciation, some organisms display high rates of karyotype variation. Ctenomys are the current mammals with the greatest interspecific and intraspecific chromosomal variation. A large number of species have been studied cytogenetically. The diploid numbers of chromosomes range from 2n = 10 to 2n = 70. Here, we analyzed karyotype evolution in Ctenomys using comparative phylogenetic methods. We found a strong phylogenetic signal with chromosome number. This refutes the chromosomal megaevolution model, which proposes the independent accumulation of multiple chromosomal rearrangements in each closely related species. We found that Brownian motion (BM) described the observed characteristic changes more thoroughly than the Ornstein-Uhlenbeck and Early-Burst models. This suggests that the evolution of chromosome numbers occurs by a random walk along phylogenetic clades. However, our data indicate that the BM model alone does not fully characterize the chromosomal evolution of Ctenomys.

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.

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.

New karyotype information for Ctenomys (Rodentia: Ctenomyidae) from Midwest and Northern Brazil.

Its wide karyotypic variation characterizes the genus Ctenomys, and in Brazil, the genus is distributed in the country's southern, Midwest, and Northern regions. Recently, populations of Ctenomys have been found in the Midwest and northern Brazil, with two new lineages named C. sp. "xingu" and C. sp. "central". This work combines classical cytogenetic and molecular analyses to provide new chromosomal information on the boliviensis group distributed in Northern and Midwestern Brazil. This includes the validation of the karyotype of C. bicolor and C. nattereri, and the description of the karyotype of C. sp. "xingu" and C. sp. "central". We found three different karyotypes: 2n=40 for C. bicolor; 2n= 36 for C. nattereri, and specimens from a locality belonging to C. sp. "central"; 2n=34 for the lineage C. sp. "xingu" and specimens from a locality belonging to C. sp. "central". Furthermore, GTG-banding revealed homologous chromosomes between species/lineages and allowed the identification of the rearrangements that occurred, which proved the occurrence of fissions. Considering our results on the variation of 2n in the boliviensis group, we found two possibilities: the first, deduced by parsimony, is that 2n=36 appeared initially, and two fissions produced gave rise to 2n=40, and an independent fusion gave rise to 2n=34 from 2n=36; moreover, the second explanation is that all karyotypes arose independently.

Phylogeography, genetic diversity, and intraspecific genetic structure of the black-horned capuchin (Sapajus nigritus).

Recent molecular studies have clarified the overarching taxonomy of capuchin monkeys, but intraspecific genetic diversity remains unexplored for most capuchin species. One example is Sapajus nigritus, the southernmost capuchin monkey, found in Brazil and Argentina; its phenotypic diversity has been recognized as two geographic subspecies, but the intraspecific genetic structure of this taxon is poorly known. Here, we sampled across most of this species' geographic distribution, producing a newly sequenced data set for genetic analyses that included 78 individuals from 14 populations. We investigated the intraspecific diversity, genetic structure, and evolutionary history using three mitochondrial markers. Our results indicated that S. nigritus populations exhibited high levels of genetic structure. We found strong support for two monophyletic clades within this species with a deep phylogenetic split, and clear separation from other related taxa. Vicariance events seem to have played a prevalent role in shaping S. nigritus genetic differentiation. The Paraíba do Sul River may have driven the deep divergence between southern and northern clades, whereas the Tietê River may have had a weaker, more recent effect on the divergence of populations within the southern clade.

Cranial morphological variation of Ctenomys lami (Rodentia: Ctenomyidae) in a restricted geographical distribution.

The relationship between chromosomal and morphological variation in mammals is poorly understood. We analyzed the cranial size and shape variation in Ctenomys lami concerning to the geographic variation in their chromosome numbers. This subterranean rodent occurs in a narrow range of sand-dunes in the Coastal Plain of southern Brazil. This species presents a high karyotypic variation with diploid numbers varying from 2n = 54 to 2n = 58, involving the fission and fusion of chromosome pairs 1 and 2. Due to different chromosome rearrangement frequencies along their geographic distribution, four karyotypic blocks were proposed. This study, explored cranium shape and size variation in geographical, chromosomal polymorphism, and chromosome rearrangements contexts to test whether the four karyotypic blocks reflect morphologically distinct units. For this, we measured 89 craniums using geometric morphometrics and used uni and multivariate statistics to discriminate the predicted groups and test for an association among chromosomal and morphological variation. Our results show the size and shape of sexual dimorphism, with males larger than females, and support the existence of four karyotypic blocks for Ctenomys lami based on morphological variation. However, our results do not support a direct relationship between chromosomal and cranial morphological variation in C. lami.

Genomic Divergence and the Evolution of Ecotypes in Bottlenose Dolphins (Genus Tursiops).

Climatic changes have caused major environmental restructuring throughout the world's oceans. Marine organisms have responded to novel conditions through various biological systems, including genomic adaptation. Growing accessibility of next-generation DNA sequencing methods to study nonmodel species has recently allowed genomic changes underlying environmental adaptations to be investigated. This study used double-digest restriction-site associated DNA (ddRAD) sequence data to investigate the genomic basis of ecotype formation across currently recognized species and subspecies of bottlenose dolphins (genus Tursiops) in the Southern Hemisphere. Subspecies-level genomic divergence was confirmed between the offshore common bottlenose dolphin (T. truncatus truncatus) and the inshore Lahille's bottlenose dolphin (T. t. gephyreus) from the southwestern Atlantic Ocean (SWAO). Similarly, subspecies-level divergence is suggested between inshore (eastern Australia) Indo-Pacific bottlenose dolphin (T. aduncus) and the proposed Burrunan dolphin (T. australis) from southern Australia. Inshore bottlenose dolphin lineages generally had lower genomic diversity than offshore lineages, a pattern particularly evident for T. t. gephyreus, which showed exceptionally low diversity. Genomic regions associated with cardiovascular, musculoskeletal, and energy production systems appear to have undergone repeated adaptive evolution in inshore lineages across the Southern Hemisphere. We hypothesize that comparable selective pressures in the inshore environment drove similar adaptive responses in each lineage, supporting parallel evolution of inshore bottlenose dolphins. With climate change altering marine ecosystems worldwide, it is crucial to gain an understanding of the adaptive capacity of local species and populations. Our study provides insights into key adaptive pathways that may be important for the long-term survival of cetaceans and other organisms in a changing marine environment.

Hybridization in Canids-A Case Study of Pampas Fox (Lycalopex gymnocercus) and Domestic Dog (Canis lupus familiaris) Hybrid.

Hybridization between species with different evolutionary trajectories can be a powerful threat to wildlife conservation. Anthropogenic activities, such as agriculture and livestock, have led to the degradation and loss of natural habitats for wildlife. Consequently, the incidence of interspecific hybridization between wild and domestic species has increased, although cases involving species of different genera are rare. In Vacaria, a Southern city in Brazil, a female canid with a strange phenotype, which had characteristics between the phenotype of the domestic dog (Canis familiaris) and that of the pampas fox (Lycalopex gymnocercus), was found. Our analysis suggests that the animal is a hybrid between a domestic dog and a pampas fox, but future studies are necessary to investigate additional cases of this hybridization in nature. This finding worries for the conservation of wild canids in South America, especially concerning Lycalopex species. Hybridization with the domestic dog may have harmful effects on pampas fox populations due to the potential for introgression and disease transmission by the domestic dog. Therefore, future studies to explore the consequences of hybridization on genetics, ecology, and behavior of wild populations will be essential to improve the conservation of this species.

Oxidative status of Ctenomys flamarioni (Rodentia: Ctenomyidae) in natural areas with different levels of anthropic activity in southern Brazil.

Rampant urbanization in coastal environments turns areas close to the seafront into a highly impacted ecosystem, possibly affecting the health and well-being of resident animal species. The tuco-tuco Ctenomys flamarioni is an endemic and endangered subterranean mammal from southern Brazil, and the anthropic influence was considered one of the main threats to the species. In this context, the present study aimed to investigate the patterns of oxidative status of the species in natural areas with different levels of anthropogenic disturbance. We evaluated two populations of C. flamarioni, one located in an area with intense anthropogenic impact due to urbanization and tourist activity, and the other in a non-impacted area. Oxidative injury parameters (lipid peroxidation and carbonylated protein levels), and the activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glucose 6-phosphate dehydrogenase, glutathione S-transferase and carboxylesterase were measured. Individuals inhabiting the impacted area had lower G6PDH activity and higher levels of carbonylated proteins. This combination of higher level of oxidative damage and lower antioxidant activity is an indication that the oxidative status of animals in the impacted population is possibly being affected as a consequence of anthropogenic activities in this environment. The values of the parameters obtained in the current study can be used as a reference of the oxidative state of C. flamarioni in future studies with tuco-tucos.

The challenge of implementing environmental DNA metabarcoding to detect elasmobranchs in a resource-limited marine protected area.

Elasmobranchs are threatened and eDNA metabarcoding is a powerful tool that can help efforts to better understand and conserve them. Nevertheless, the inter-calibration between optimal methodological practices and its implementation in resource-limited situations is still an issue. Based on promising results from recent studies, the authors applied a cost-effective protocol with parameters that could be easily replicated by any conservationist. Nonetheless, the results with fewer elasmobranchs detected than expected reveal that endorsed primers and sampling strategies still require further optimization, especially for applications in resource-limited conservation programmes.

Saint Peter and Saint Paul Archipelago barcoded: Fish diversity in the remoteness and DNA barcodes reference library for metabarcoding monitoring.

In order to monitor the effects of anthropogenic pressures in ecosystems, molecular techniques can be used to characterize species composition. Among molecular markers capable of identifying species, the cytochrome c oxidase I (COI) is the most used. However, new possibilities of biodiversity profiling have become possible, in which molecular fragments of medium and short-length can now be analyzed in metabarcoding studies. Here, a survey of fishes from the Saint Peter and Saint Paul Archipelago was barcoded using the COI marker, which allowed the identification of 21 species. This paved the way to further investigate the fish biodiversity of the archipelago, transitioning from barcoding to metabarcoding analysis. As preparatory steps for future metabarcoding studies, the first extensive COI library of fishes listed for these islands was constructed and includes new data generated in this survey as well as previously available data, resulting in a final database with 9,183 sequences from 169 species and 63 families of fish. A new primer specifically designed for those fishes was tested in silico to amplify a region of 262 bp. The new approach should guarantee a reliable surveillance of the archipelago and can be used to generate policies that will enhance the archipelago's protection.

Genomic Organization of Microsatellites and LINE-1-like Retrotransposons: Evolutionary Implications for Ctenomys minutus (Rodentia: Ctenomyidae) Cytotypes.

The Neotropical underground rodents of the genus Ctenomys (Rodentia: Ctenomyidae) comprise about 65 species, which harbor the most significant chromosomal variation among mammals (2n = 10 to 2n = 70). Among them, C. minutus stands out with 45 different cytotypes already identified, among which, seven parental ones, named A to G, are parapatrically distributed in the coastal plains of Southern Brazil. Looking for possible causes that led to such extensive karyotype diversification, we performed chromosomal mapping of different repetitive DNAs, including microsatellites and long interspersed element-1 (LINE-1) retrotransposons in the seven parental cytotypes. Although microsatellites were found mainly in the centromeric and telomeric regions of the chromosomes, different patterns occur for each cytotype, thus revealing specific features. Likewise, the LINE-1-like retrotransposons also showed a differential distribution for each cytotype, which may be linked to stochastic loss of LINE-1 in some populations. Here, microsatellite motifs (A)30, (C)30, (CA)15, (CAC)10, (CAG)10, (CGG)10, (GA)15, and (GAG)10 could be mapped to fusion of chromosomes 20/17, fission and inversion in the short arm of chromosome 2, fusion of chromosomes 23/19, and different combinations of centric and tandem fusions of chromosomes 22/24/16. These data provide evidence for a correlation between repetitive genomic content and localization of evolutionary breakpoints and highlight their direct impact in promoting chromosomal rearrangements.

Bridging macroecology and macroevolution in the radiation of sigmodontine rodents.

Investigations of phenotypic disparity across geography often ignore macroevolutionary processes. As a corollary, the random null expectations to which disparity is compared and interpreted may be unrealistic. We tackle this issue by representing, in geographical space, distinct processes of phenotypic evolution underlying ecological disparity. Under divergent natural selection, assemblages in a given region should have empirical disparity higher than expected under an evolutionarily oriented null model, whereas the opposite may indicate constraints on phenotypic evolution. We gathered phylogenies, biogeographic distributions, and data on the skull morphology of sigmodontine rodents to discover which regions of the Neotropics were more influenced by divergent, neutral, or constrained phenotypic evolution. We found that regions with higher disparity than expected by the evolutionary-oriented null model, in terms of both size and shape, were concentrated in the Atlantic Forest, suggesting a larger role for divergent natural selection there. Phenotypic disparity in the rest of South America, mainly the Amazon basin, northeastern Brazil, and Southern Andes, was constrained-lower than predicted by the evolutionary model. We also demonstrated equivalence between the disparity produced by randomization-based null models and constrained-evolution null models. Therefore, including evolutionary simulations into the null modeling framework used in ecophylogenetics can strengthen inferences on the processes underlying phenotypic evolution.

Expert range maps of global mammal distributions harmonised to three taxonomic authorities.

Aim: Comprehensive, global information on species' occurrences is an essential biodiversity variable and central to a range of applications in ecology, evolution, biogeography and conservation. Expert range maps often represent a species' only available distributional information and play an increasing role in conservation assessments and macroecology. We provide global range maps for the native ranges of all extant mammal species harmonised to the taxonomy of the Mammal Diversity Database (MDD) mobilised from two sources, the Handbook of the Mammals of the World (HMW) and the Illustrated Checklist of the Mammals of the World (CMW). Location: Global. Taxon: All extant mammal species. Methods: Range maps were digitally interpreted, georeferenced, error-checked and subsequently taxonomically aligned between the HMW (6253 species), the CMW (6431 species) and the MDD taxonomies (6362 species). Results: Range maps can be evaluated and visualised in an online map browser at Map of Life (mol.org) and accessed for individual or batch download for non-commercial use. Main conclusion: Expert maps of species' global distributions are limited in their spatial detail and temporal specificity, but form a useful basis for broad-scale characterizations and model-based integration with other data. We provide georeferenced range maps for the native ranges of all extant mammal species as shapefiles, with species-level metadata and source information packaged together in geodatabase format. Across the three taxonomic sources our maps entail, there are 1784 taxonomic name differences compared to the maps currently available on the IUCN Red List website. The expert maps provided here are harmonised to the MDD taxonomic authority and linked to a community of online tools that will enable transparent future updates and version control.

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.

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.

The role of the environment in the spatial dynamics of an extensive hybrid zone between two neotropical cats.

Identifying factors that create and maintain a hybrid zone is of great interest to ecology, evolution and, more recently, conservation biology. Here, we investigated the role of environmental features in shaping the spatial dynamics of a hybrid zone between the southern tigrina, Leopardus guttulus, and Geoffroy's cat, L. geoffroyi, testing for exogenous selection as the main force acting on its maintenance. These Neotropical felid species are mainly allopatric, with a restricted area of sympatry in the ecotone between the Atlantic Forest and Pampa biomes. As both biomes have experienced high rates of anthropogenic habitat alteration, we also analysed the influence of habitat conversion on the hybrid zone structure. To do this, we used 13 microsatellite loci to identify potential hybrids and generated ecological niche models for them and their parental species. We compared the influence of variables on parental species and hybrid occurrence and calculated the amount of niche overlap among them. Parental species showed different habitat requirements and predicted co-occurrence was restricted to the forest-grassland mosaic of the ecotone. However, hybrids were found beyond this area, mainly in the range of L. geoffroyi. Hybrids demonstrated higher tolerance to habitat alteration than parental types, with a probability of occurrence that was positively related with mosaics of cropland areas and remnants of natural vegetation. These results indicate that exogenous selection alone does not drive the dynamics of the hybrid zone, and that habitat conversion influences its structure, potentially favouring hybrids over parental species.

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.