Satellitome analysis on the pale-breasted thrush Turdus leucomelas (Passeriformes; Turdidae) uncovers the putative co-evolution of sex chromosomes and satellite DNAs.

Do all birds' sex chromosomes follow the same canonical one-way direction of evolution? We combined cytogenetic and genomic approaches to analyze the process of the W chromosomal differentiation in two selected Passeriform species, named the Pale-breasted Thrush Turdus leucomelas and the Rufous-bellied thrush T. rufiventris. We characterized the full catalog of satellite DNAs (satellitome) of T. leucomelas, and the 10 TleSatDNA classes obtained together with 16 microsatellite motifs were in situ mapped in both species. Additionally, using Comparative Genomic Hybridization (CGH) assays, we investigated their intragenomic variations. The W chromosomes of both species did not accumulate higher amounts of both heterochromatin and repetitive sequences. However, while T. leucomelas showed a heterochromatin-poor W chromosome with a very complex evolutionary history, T. rufiventris showed a small and partially heterochromatic W chromosome that represents a differentiated version of its original autosomal complement (Z chromosome). The combined approach of CGH and sequential satDNA mapping suggest the occurrence of a former W-autosomal translocation event in T. leucomelas, which had an impact on the W chromosome in terms of sequence gains and losses. At the same time, an autosome, which is present in both males and females in a polymorphic state, lost sequences and integrated previously W-specific ones. This putative W-autosomal translocation, however, did not result in the emergence of a multiple-sex chromosome system. Instead, the generation of a neo-W chromosome suggests an unexpected evolutionary trajectory that deviates from the standard canonical model of sex chromosome evolution.

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 was 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.

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.

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.

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.

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.

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.

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.

Comparative analyses of three swallow species (Aves, Passeriformes, Hirundinidae): Insights on karyotype evolution and genomic organization.

Despite the richness of species in the Hirudinidae family, little is known about the genome organization of swallows. The Progne tapera species presents genetic and morphological difference when compared to other members of the same genus. Hence, the aims of this study were to analyze the chromosomal evolution of three species Progne tapera, Progne chalybea and Pygochelidon cyanoleuca - by comparative chromosome painting using two sets of probes, Gallus gallus and Zenaida auriculata, in order to determine chromosome homologies and the relationship between these species. All karyotypes exhibited 76 chromosomes with similar morphology, except for the 5th, 6th and 7th chromosome pairs in P. cyanoleuca. Additionally, comparative chromosome painting demonstrated the same hybridization pattern in the two Progne, which was similar to the putative avian ancestral karyotype, except for the centric fission in the first pair, as found in other Passeriformes. Thus, these data display a close relationship between the Progne species. Although P. cyanoleuca demonstrated the same fission in the first pair of the ancestral syntenic (GGA1), it also showed an additional chromosomal rearrangement for this species, namely a fusion with a microchromosome in the seventh pair.

Novel insights into chromosome evolution of Charadriiformes: extensive genomic reshuffling in the wattled jacana (Jacana jacana, Charadriiformes, Jacanidae).

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.

Comparative chromosome painting in hummingbirds (Trochilidae).

Hummingbirds (Trochilidae) are one of the most enigmatic avian groups, and also among the most diverse, with approximately 360 recognized species in 106 genera, of which 43 are monotypic. This fact has generated considerable interest in the evolutionary biology of the hummingbirds, which is reflected in a number of DNA-based studies. However, only a few of them explored chromosomal data. Given this, the present study provides an analysis of the karyotypes of three species of Neotropical hummingbirds, Anthracothorax nigricollis (ANI), Campylopterus largipennis (CLA), and Hylocharis chrysura (HCH), in order to analyze the chromosomal processes associated with the evolution of the Trochilidae. The diploid number of ANI is 2n=80 chromosomes, while CLA and HCH have identical karyotypes, with 2n=78. Chromosome painting with Gallus gallus probes (GGA1-12) shows that the hummingbirds have a karyotype close to the proposed ancestral bird karyotype. Despite this, an informative rearrangement was detected: an in-tandem fusion between GGA7 and GGA9 found in CLA and HCH, but absent in ANI. A comparative analysis with the tree of life of the hummingbirds indicated that this fusion must have arisen following the divergence of a number of hummingbird species.

Polymorphism of Sooty-fronted Spinetail (Synallaxis frontalis Aves: Furnariidae): Evidence of chromosomal rearrangements by pericentric inversion in autosomal macrochromosomes.

The Passeriformes is the most diverse and cytogenetically well-known clade of birds, comprising approximately 5,000 species. The sooty-fronted spinetail (Synallaxis frontalis Aves: Furnariidae) species, which belongs to the order Passeriformes, is typically found in South America, where it is widely distributed. Polymorphisms provide genetic variability, important for several evolutionary processes, including speciation and adaptation to the environment. The aim of this work was to analyze the possible cytotypes and systemic events involved in the species polymorphism. Of the sampled 19 individuals, two thirds were polymorphic, an event supposedly linked to mutations resulting from genomic evolution that can be transmitted hereditarily. A chromosomal polymorphism was detected between the 1st and 3rdpairs of autosomal macrochromosomes. This type of polymorphism is related to a pericentric inversion in regions involving chromosomal rearrangements. Differently from other polymorphism studies that report a link between polymorphic chromosomes and phenotypic changes, S. frontalis did not present any morphological variation in the sampled individuals.

Distribution of CR1-like transposable element in woodpeckers (Aves Piciformes): Z sex chromosomes can act as a refuge for transposable elements.

Birds have relatively few repetitive sequences compared to other groups of vertebrates; however, the members of order Piciformes (woodpeckers) have more of these sequences, composed mainly of transposable elements (TE). The TE most often found in birds is a retrotransposon chicken repeat 1 (CR1). Piciformes lineages were subjected to an expansion of the CR1 elements, carrying a larger fraction of transposable elements. This study compared patterns of chromosome distribution among five bird species, through chromosome mapping of the CR1 sequence and reconstructed their phylogenetic tree. We analyzed several members of Piciformes (Colaptes campestris, Colaptes melanochloros, Melanerpes candidus, and Veniliornis spilogaster), as well as Galliformes (Gallus gallus). Gallus gallus is the species with which most genomic and hence cytogenetic studies have been performed. The results showed that CR1 sequences are a monophyletic group and do not depend on their hosts. All species analyzed showed a hybridization signal by fluorescence in situ hybridization (FISH). In all species, the chromosomal distribution of CR1 was not restricted to heterochromatin regions in the macrochromosomes, principally pair 1 and the Z sex chromosome. Accumulation in the Z sex chromosomes can serve as a refuge for transposable elements. These results highlight the importance of transposable elements in host genomes and karyotype evolution.