Comparative Cytogenetics in Tyrannidae (Aves, Passeriformes): High Genetic Diversity despite Conserved Karyotype Organization

While bird species exhibit an extensive range of variation in behavior, morphology, and ecology [1], their chromosomal history shows an intriguing degree of evolutionary stability. About 40.6% of the species that have been studied thus far have between 78 and 80 chromosomes, therefore indicating that the avian karyotypes are stable [2]. Except for a few exceptions [3], most birds have a simple ZZ/ZW sex-determination system, where the Z chromosome is relatively conserved and typically larger than the W chromosome [4, 5]. In contrast, the W chromosome shows significant variation, typically being small and heterochromatic, as observed in Passeriformes [5‒8].

With over 5,700 species, representing 60% of all bird species currently described, Passeriformes is highly diverse and includes two major suborders, named Oscines and Suboscines [9]. Among Suboscines, Tyrannidae contains about 600 species [10] and represents one of the most diverse and numerous families. For the cytogenetic information, they exhibit a high degree of stability in their diploid numbers (2n = 76–84). Nonetheless, they also show variations in chromosomal morphologies, suggesting the likelihood of various intrachromosomal rearrangements [11, 12]. However, only about 20 Tyrannidae species have been karyotyped thus far, implying a dearth of cytogenetic studies on this family [2].

A variety of repetitive elements can be found in eukaryotic genomes, and although bird genomes are generally rather repeat-poor, certain genomic areas (i.e., centromeres and telomeres) are almost exclusively made up of repetitive sequences [13]. Except for woodpeckers, most birds have a low number of repetitive DNAs compared to other vertebrates, as well as a compact genome that has predominantly originated from lineage-specific modification of repetitive elements, large segmental deletions, and gene loss [14]. Recent studies using microsatellite probes in some species of Columbidae, Picidae, Nyctibiidae, and Psittacidae have shown the importance of these elements in genome organization, as well as in sex chromosome differentiation [15‒18]. For example, a large accumulation of microsatellite sequences on the Z chromosome has been suggested as the main cause of this chromosome’s enlargement in woodpeckers and toucans [15, 17].

Nearly all the studies employing microsatellite probes on Passeriformes species were conducted on Oscines species [19, 20]. These studies evidenced an accumulation of microsatellite sequences mainly in centromeric, pericentromeric, or telomeric regions of macro- and microchromosomes. A substantial accumulation of repetitive sequences was identified along the length of the W chromosome in three species of swallows, which had a larger W chromosome than is typically found in birds [19]. On the other hand, these sequences accumulated in the short arms of the Z chromosome in the saffron finch species [17]. To date, there is only a single published study on repeated sequences in Suboscines, showing that microsatellites are scattered throughout the macro- and microchromosomes of different species of Furnariidae [21].

Other repetitive elements, such as telomeric and rDNA sequences, are also frequently mapped in bird chromosomes. Most Passeriformes species exhibit signals of 18S rDNA on a pair of microchromosomes [22], which has been postulated as the ancestral state of birds as observed in basal groups as the emu (Dromaius novaehollandiae) [23]. Although most Oscines species present several rDNA loci, including Gubernatrix cristata, Serinus canaria, and Turdus albicollis [11, 24, 25], the Suboscines demonstrate the ancestral pattern, with few exceptions as seen in Dendrocolaptes platyrostris (Furnariidae) chromosomes [26]. Regarding the telomeric sequences, it has been found on the telomeres of all chromosomes, with stronger signals on microchromosomes, which may contribute to the high rate of meiotic recombination observed in these elements [27, 28].

We used conventional and molecular cytogenetic techniques in two Tyrannidae species, Sirystes sibilator and Myiopagis viridicata, to gain an in-depth understanding of the chromosomal evolution in these species. The aforementioned methods were used to investigate their telomeric sequence organization, repetitive DNA distribution, and karyotypic structure.

Two species from the Tyrannidae, one female of S. sibilator and a male of M. viridicata, were collected in Porto Vera Cruz, State of Rio Grande do Sul, Brazil. The birds were captured in their natural habitat using mist nets. Skin biopsies were taken from each individual to establish fibroblast cell cultures, following the method described by Sasaki [29]. Chromosomal preparations were obtained using the standard methodology with colchicine (0.05%), hypotonic treatment, and cell fixation in methanol-acetic acid (3:1).

Karyotype description and diploid numbers were obtained by analyzing 50 metaphases per specimen, using conventional staining of 5% Giemsa in phosphate buffer, pH 6.8. The chromosomes were classified according to Guerra’s nomenclature [30] as metacentric, submetacentric, acrocentric, and telocentric. Blocks of constitutive heterochromatin were detected through C-banding [31].

For the detection of 45S DNA (rDNA), we isolated the 18S rDNA gene sequence from the Ocyurus chrysurus genome (Perciformes: Lutjanidae) [32] and labeled it in green using nick-translation with Atto448-dUTP, following the manufacturer’s instructions (Jena Biosciences, Jena, Germany). The repetitive sequences (GA)_n, (GC)_n, (TA)_n, (CAT)_n, (GAC)_n, and (GAG)_n were directly labeled with Cy-3 during synthesis [33]. These repetitive sequences were chosen because it is the most abundant sequences in birds’ chromosomes according to previous studies (15–21). Biotinylated telomere repeat probes (TTAGGG)n were generated by polymerase chain reaction in the absence of templates, according to Ijdo et al. [34], and detected by streptavidin Cy-3. The FISH experiments were performed following the methodology described by Kretschmer et al. [35]. The chromosomes were denatured by immersion in 70% formamide (70 mL formamide and 30 mL 2xSSC) at 72°C for 1 min 20 s, and the probes were denatured at 86°C for 10 min and then cooled to 4°C until the hybridization. The chromosomes were counterstained with VECTASHIELD with DAPI (Vector Laboratories, Burlingame, CA, USA). The FISH experiment was performed at least twice to confirm the hybridization signals, and in addition, the number of metaphases analyzed to establish the pattern obtained for each microsatellite probe ranged from 15 to 20.

The images were captured using an Olympus B ×53 microscope (Olympus Corporation, Ishikawa, Japan) coupled with CoolSNAP and processed using ISIS software (MetaSystems). The GNU Image Manipulation Program (GIMP) software was used to assemble the karyotypes.

Karyotype of S. sibilator is composed of 2n = 80 (Fig. 1a), with chromosome pair 1 as submetacentric, pairs 2–7 as acrocentric, and the remaining autosomal pairs are telocentric. The Z is a medium-sized acrocentric, whereas the W is a small-sized metacentric chromosome. M. viridicata also present 2n = 80 (Fig. 1b), but the pair 8 is metacentric, instated of telocentric as in S. sibilator. The Z is a medium-sized acrocentric. The W chromosome of M. viridicata could not be investigated in this study as only a male individual was obtained for analysis. C-positive heterochromatin-rich regions were detected in several microchromosome pairs and in the telomeric and centromeric regions of most macrochromosomes in both species (Fig. 2). The W chromosome of S. sibilator was almost entirely heterochromatic (Fig. 2a).

The simple repetitive sequences (SSRs) are primarily hybridized with both microchromosomes and macrochromosomes, while no labeling was observed in the W sex chromosomes of S. sibilator (Fig. 3). Table 1 provides a summary of the SSR data. Among the five sequences used, (TA)_n, (CAT)_n, and (GAG)_n did not hybridize in either species; additionally, (GC)_n also did not produce FISH signals in S. sibilator (data not shown).

The 18S rDNA probes hybridized in only one pair of microchromosomes in S. sibilator, while in M. viridicata it was found in two pairs of microchromosomes (Fig. 4). Regarding the telomeric FISH results, no interstitial telomeric sequences (ITSs) were observed in either of the two species. The probes containing telomeric sequences produced signals only in the distal region of the chromosome arms and tended to be more prominent in the microchromosomes than in the macrochromosomes (Fig. 4).

Despite the extensive diversity of birds, there is currently a lack of available cytogenetic data. This is particularly the case for the Tyrannidae, where most of the research has relied solely on conventional methods [12, 36‒38]. This study examined the chromosomal and genomic organization of two Tyrannidae species with an emphasis on their repetitive DNA content. To gain insight into their karyotypic and evolutionary aspects, we analyzed the distribution of constitutive heterochromatin regions, as well as the localization of microsatellites, 18S rDNA, and telomeric sequences.

The vast majority of cytogenetically examined Passeriformes species have a typical karyotype with around 80 chromosomes, split into macro- and microchromosomes [2, 39, 40]. Similarly, both species investigated in this study exhibited 2n = 80, with 39 pairs of autosomes and one pair of sex chromosomes. Constitutive heterochromatin is evidenced on the q and p arms, as well as in the centromeric regions of nearly all the autosomes in both species. In addition, M. viridicata showed a heterochromatin block on the Z chromosome, while the W chromosome of S. sibilator is nearly heterochromatic. On the other hand, constitutive heterochromatin is only present in the centromeres of most macro- and microchromosomes in the Tyrannidae species Pitangus sulphuratus, Serpophaga subcristata, and Satrapa icterophrys [38]. Indeed, interspecific variations of heterochromatin have also been observed for other bird lineages [41]. These results imply that constitutive heterochromatin accumulation actively influences the karyotype evolution in Tyrannidae lineage.

The 18S rDNA gene was identified within a single and in two pairs of microchromosomes in S. sibilator and M. viridicata, respectively. Given that both species have the same diploid number, M. viridicata may have duplicated and translocated this sequence. It is noteworthy to point out that all species of Tyranninae and Fluvicolinae subfamilies examined, including S. sibilator, have consistently shown one microchromosome with 18S clusters, suggesting that the ancestral state has been conserved (Fig. 5). However, species in the subfamily Elaeniinae, among which M. viridicata is a notable example, have shown a unique pattern, with two pairs of microchromosomes bearing 18S rDNA clusters, indicating an apomorphic condition (Fig. 5). Although the genomic arrangement of these sequences is dynamic, they are limited to a pair of microchromosomes in most bird species, particularly primitive ones like D. novaehollandiae [23]. This points to a plausible scenario where the duplication and translocation of the 18S rDNA sequence likely occurred within the last common ancestor of the Elaeniinae, considering its prevalence across all investigated species within this subgroup (Fig. 5).

While certain bird species, such as Passeriformes like Turdus iliacus and Fringilla coelebs, have ITSs [43], the two species under investigation here only displayed telomeric FISH signals in terminal regions of the chromosomes. ITS has been reported in several bird species, particularly in more basal groups like Palaeognathae, as a result of chromosomal fusions. Its gradual elimination has been noted during the divergence of Palaeognathae and Neognathae [28, 44]. Therefore, ITSs have been interpreted as an indication of the occurrence of interchromosomal rearrangements. According to chromosomal painting research, chromosome fusions did not occur in any Tyrannidae species up to now analyzed [12, 38]. Our results showing the lack of ITS further support this conclusion. However, it is noteworthy that certain species have shown no ITS in chromosomes resulting from chromosome fusions [44‒46]. Likewise, in all bird species that have been investigated up to this point, we noticed strong telomeric FISH signals on most microchromosomes. This discovery implies a possible connection to the remarkably high meiotic recombination rates found in the microchromosomes of birds [28].

Our findings indicate that each Tyrannidae species exhibits a distinct pattern of SSR hybridization. Generally, these sequences were amplified in heterochromatic regions, in either pericentromeric or telomeric regions, present in both macrochromosomes and microchromosomes. Repetitive DNA is thought to be located in less active and condensed portions of the genome, which is consistent with the aforementioned pattern [47, 48]. In contrast to Suboscines species from the Furnariidae family, the W chromosome of S. sibilator did not accumulate SSR signals [21]. On the other hand, Oscines species displayed a large accumulation of these sequences on the W chromosome [19]. Although these chromosomes display a common origin in birds, these observations point to their different evolutionary paths, even among species that belonged to the same order (e.g., Passeriformes), highlighting the significant variability in sex chromosome differentiation within this group of birds.

To summarize, we described and characterized the karyotypes of two distinct species belonging to the Tyrannidae family: S. sibilator (Tyranninae) and M. viridicata (Elaeniinae). Both species have 2n = 80, a diploid number that is in line with the usual range of passerines. Our research showed significant differences in their chromosomal architecture, rDNA distribution, and SSR accumulation. We examined these findings in the context of the evolution of Tyrannidae karyotypes. These results highlight the various evolutionary histories across the members of the Tyrannidae family and throw insight into unique evolutionary paths within each subfamily.

Samples were collected according to licenses SISBIO 61047-3, 33860-2, and 81564-1, and the animal research ethics committee (CEUA 019/2020).

The authors have no conflicts of interest to declare.

This research was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), finance Code 001 (D.M.S., M.S.d.S., V.T., and V.O.R.) and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Grant No. 302928/2021-9 (M.B.C.), 407285/2021-0 and 306717/2022-0 (A.D.V.G.), and 302835/2021-0 (E.Z).

D.M.S. and R.K. contributed to the study conception and design and wrote the original draft. Methodology was performed by D.M.S., M.S.S., V.T., V.O.R., F.M.C.S., and R.K. Formal analysis, data curation, and investigation were performed by D.M.S., M.S.S., V.T., V.O.R., and F.M.C.S. Visualization was performed by D.M.S., M.S.S., and R.K. Writing – review and editing – was performed by D.M.S., R.K., E.Z., A.V.G., R.J.G., and M.B.C. Funding was obtained by R.K., E.Z., A.V.G., R.J.G., and M.B.C. All authors read and approved the final manuscript.

All data generated or analyzed during this study are included in this article.