Furnariidae (ovenbirds) is one of the most diversified families in the Passeriformes order and Suboscines suborder. Despite the great diversity of species, cytogenetic research is still in its early stages, restricting our knowledge of their karyotype evolution. We combined traditional and molecular cytogenetic analyses in three representative species, Synallaxis frontalis, Syndactyla rufosuperciliata, and Cranioleuca obsoleta, to examine the chromosomal structure and evolution of ovenbirds. Our findings revealed that all the species studied had the same diploid number (2n = 82). Differences in chromosomal morphology of some macrochromosomes indicate the presence of intrachromosomal rearrangements. Although the three species only had the 18S rDNA on one microchromosome pair, chromosomal mapping of six simple short repeats revealed a varied pattern of chromosome distribution among them, suggesting that each species underwent different repetitive DNA accumulation upon their divergence. The interspecific comparative genomic hybridization experiment revealed that the Furnariidae species investigated carry centromeric regions enriched in similar repetitive sequences, bolstering the Furnariidae family's karyotype conservation. Nonetheless, the outgroup species Turdus rufiventris (Turdidae) demonstrated an advanced stage of sequence divergence with hybridization signals that were almost entirely limited to a few microchromosomes. Overall, the findings imply that Furnariidae species have a high degree of chromosomal conservation, and we could also observe a differentiation of repetitive sequences in both Passeriformes suborders (Suboscines and Oscines).

Passeriformes is the largest avian order, with more than 6,000 widely distributed species that show an extraordinary morphological and ecological diversity [Ericson et al., 2014]. The order is divided into two suborders: Oscines (vocal learners), comprising 776 genera and roughly 80% of all Passeriformes species, and Suboscines (vocal non-learners), which has 284 genera, and represents the Passeriformes’ most basal lineage [Feduccia, 1999; Selvatti et al., 2015]. Among the Suboscines, Furnariidae (ovenbirds) is represented by species of small birds that are widespread in tropical forests (from Mexico to South America) and are renowned for their enormous variety in nest construction [Zyskowski and Prum, 1999].

Despite such a wide range of behavior, morphology, and ecology [Moyle et al., 2009], about 40% of all species so far show evolutionary stability of around 78–80 chromosomes [Degrandi et al., 2020a].

Most bird species studied thus far contain 80 chromosomes, with a few big macrochromosomes (20) and numerous indistinguishable microchromosomes (60) [Santos and Gunski, 2006; Griffin et al., 2007; Kretschmer et al., 2018a]. Except for woodpeckers, most birds have a low number of repetitive DNAs in comparison to other tetrapod vertebrates, as well as a compact genome that originated predominantly because of lineage-specific erosion of repetitive elements, major segmental deletions, and gene loss [Szarski, 1976; Burt, 2002; Zhang et al., 2014; de Oliveira et al., 2017]. Furthermore, avian genomes exhibit a remarkable degree of evolutionary stability at the levels of nucleotide sequence, gene synteny, and chromosomal organization [Burt, 2002; Zhang et al., 2014; Gregory, 2022]. An important feature that differentiates the karyotype of Oscines from that of Suboscines is the presence of a germline-restricted chromosome, which is a chromosome that is only present during meiosis of songbirds [Torgasheva et al., 2019]. All members of this group have a ZZ/ZW sex chromosome system, with the Z chromosome being more conserved and often bigger than the W [Schartl et al., 2016]. The W chromosome, on the other hand, displays substantial variability, although it is usually small and mostly heterochromatic, as found in Passeriformes [dos Santos et al., 2015; Barcellos et al., 2019].

Cytogenetics encompasses any kind of study concerning chromosomes, isolated or in conjunction with each other, condensed or distended, and concerns their evolution and variability as well as their morphology, function, organization, and replication [Guerra, 1988]. Since the middle of the 20th century, with the advance of cell culture techniques and chemical treatments, it has been possible to considerably improve the quality of chromosome preparations [Barcellos et al., 2022]. Most of the karyological data in birds are based on classical cytogenetic techniques, involving conventional staining and chromosomal banding methods, such as C-banding and Ag-NOR staining [Sumner, 1972; Guerra, 1988; Degrandi et al., 2020a]. Banding resolution stands as a limitation in the identification of chromosome homology and comparative studies, which limits deeper studies in chromosomes. The improvement of the FISH technique permitted and progressed towards more precise cytogenetic investigations, becoming a crucial tool for cytogenetic studies in birds, as well as clarifying phylogenies in some families. This resulted in the ability to identify and localize DNA sequences at particular locations along the chromosomes [Kretschmer et al., 2018a, 2021a].

Studies that mapped rDNA through FISH are quite common in vertebrates. Whereas, simple short repeats (SSRs) can be used to select the desirable allele when the gene of interest is known [Christiakov et al., 2006; Mazzoleni et al., 2018]. There is a classification according to the type of repetitive sequences where microsatellites can present two or more different motifs in tandem. It can be defined as perfect or imperfect, when they are composed by only perfect repetitions and when the repetitive sequences are interrupted by distinct nucleotides that are not repeated, respectively. The most common choices for molecular analysis are the sequences that contain two, three or four nucleotide repeats [Selkoe and Toonen, 2006]. Even though repetitive sequences constitute only a minor fraction of avian genomes, study of this genomic component has been useful in understanding the evolution of sex chromosomes and chromosomal differentiation [Cioffi and Bertollo, 2012; Kretschmer et al., 2018b]. Microsatellites, for example, have recently been analyzed in oscine species, where they preferentially accumulate in centromeric, pericentromeric, or telomeric areas of both macro- and microchromosomes and display a unique pattern in sex chromosomes [Barcellos et al., 2019; Kretschmer et al., 2021b]. In swallows with larger W chromosomes, a huge concentration of distinct microsatellite sequences was detected on this sex chromosome throughout its length, but the saffron finch, which has a typical small-sized W chromosome, displayed dispersed signals, similar to autosomes. In contrast, the saffron finch's Z chromosome revealed a substantial overall accumulation in its short arms [Barcellos et al., 2019; Kretschmer et al., 2021b]. Although these studies helped to improve our understanding of sex chromosome evolution and chromosome differentiation in Passeriformes species, the dynamics of repetitive DNA sequences in this group have received little attention because no similar study involving members of Suboscines has been conducted so far.

Based on the FISH technique, Kallioniemi et al. [1992] developed comparative genomic hybridization (CGH), a molecular cytogenetic method for detecting imbalanced chromosomal rearrangements (i.e., duplications, deletions, and copy number variation) between two DNA sources. After being utilized to identify chromosomal abnormalities in tumor cells compared to normal cells [Kallioniemi et al., 1992], it is now being used to detect chromosomal rearrangements between species, as previously demonstrated by de Oliveira et al. [2017] and Cioffi et al. [2019]. Recent CGH investigations suggest that the two nightingale species (Oscines) diverged in centromeric repetitive sequences in most macro- and microchromosomes [Poignet et al., 2021].

Except for some Tyranni species, suboscine members have a typical avian karyotype with approximately 80 chromosomes [Degrandi et al., 2020a]. Considering only Furnariidae, there is a significant lack of knowledge about the chromosomal complement of this family, with less than 10% of the recognized species karyotyped and a smaller number of studies incorporating molecular cytogenetic methods, such as FISH, performed to date [Kretschmer et al. 2018c; de Souza et al., 2019; Degrandi et al., 2020a].

In order to get a more comprehensive understanding of the mechanisms underlying the chromosomal evolution of ovenbird species, we used a combination of conventional and molecular cytogenetic methodologies on Synallaxis frontalis, Syndactyla rufosuperciliata, and Cranioleuca obsoleta (the last two being analyzed for the first time). These methods were used to examine their karyotype structure, repetitive DNA distribution, and interspecific genomic divergences.

Samples

We collected 3 species of the Furnariidae family, S. rufosuperciliata (3 ♀), C. obsoleta (2 ♀), and S. frontalis (2 ♀, 2 ♂), and 1 species of the Turdidae family, Turdus rufiventris (4 ♀). The individuals were collected in three municipalities of Rio Grande do Sul (Brazil), São Gabriel, Porto Vera Cruz, and Pelotas. Data collection was performed according to the protocols approved by the Animal Ethics Committee (CEUA 019/2020) and Biodiversity Authorization and Information System (SISBIO 61047-3, 33860-2, and 81564-1).

Chromosomal Preparations and Classical Cytogenetics

Metaphases were obtained from distinct preparations: bone marrow direct, fibroblast or embryo cell culture [Sasaki et al., 1968; Garnero and Gunski, 2000; Barcellos et al., 2022]. C-banding analysis was performed according to Sumner [1972], with minor modifications. After incubation at 60°C for 1 h, slides were treated with 0.2 n HCl at 42°C for 10 min, then in 5% Ba(OH)2 also at 42°C for 3 min and finally treated with 2× SSC at 60°C for 1 h and 30 min. Chromosomes were counterstained using 0.7 μL propidium iodide (50 μg/mL) mounted in 20 μL of Vectashield (Vector Laboratories, Burlingame, CA, USA) [Lui et al., 2012]. The chromosome number was determined by counting 20–30 metaphases.

Repetitive DNA Mapping by FISH

For the detection of the 45S ribosomal DNA (rDNA)-bearing chromosomes, we have isolated the 18S rDNA sequence from the Ocyurus chrysurus (Perciformes: Lutjanidae) genome [White et al., 1990] and labeled it in green by nick-translation with Atto448-dUTP, following the manufacturer’s instructions (Jena Biosciences, Jena, Germany). The repetitive sequences (CA)n, (GA)n, (CGG)n, (GAA)n, (CAG)n, and (GAG)n were directly labeled with Cy-3 during the synthesis [Kubat et al., 2008]. FISH experiments were done following the methodology described in Kretschmer et al. [2022]. Chromosomes were denatured by immersion in 70% formamide (70 mL formamide and 30 mL 2× SSC) at 72°C for 1 min 20 s, and probes were denatured at 86°C for 10 min and cooled at 4°C for at least 5 min before hybridization. Chromosomes were counterstained with Vectashield with DAPI (Vector Laboratories). FISH experiments were done at least twice to confirm the hybridization signals and, in addition, 15–20 metaphases were analyzed to establish the pattern obtained for each microsatellite probe.

Comparative Genomic Hybridization

DNA from both female and male samples from all species was extracted from the liver tissue by the phenol-chloroform-isoamyl alcohol method [Sambrook and Russell, 2001]. DNA from S. frontalis female specimens was chosen as a reference since Synallaxis is the most species-rich genus in the family Furnariidae, with 33 recognized species [Remsen, 2003] and used for hybridization against metaphase chromosomes of the other species. The first approach focused on the intraspecific comparison between the female and male genomes of S. frontalis. For this purpose, the gDNAs were labeled in red and green with Atto550-dUTP and Atto488-dUTP, respectively, by nick-translation (Jena Biosciences) and hybridized against the female background of S. frontalis. To block the repetitive sequences, we used C0t-1 DNA (i.e., genomic DNA enriched for highly and moderately repetitive sequences) prepared according to Zwick et al. [1997]. The final probe mixture was composed of 500 ng of female-derived DNA, 500 ng of male-derived gDNA, and 3 μg of male-derived C0t-1 DNA. The probe was precipitated with ethanol, and the dry pellets were mixed with hybridization buffer containing 50% formamide, 2× SSC, 10% SDS, 10% dextran sulfate, and Denhardt’s buffer at pH 7.0. For the second set of experiments, we also used the female’s chromosomes of S. frontalis as background and performed an interspecific comparison, using the gDNA of Furnariidae (C. obsoleta, S. frontalis, and S. rufosuperciliata) and Turdidae (T. rufiventris) representatives. We labeled the female-derived gDNA of S. frontalis with Atto550-dUTP and the female-derived gDNAs of C. obsoleta, T. rufiventris, and S. rufosuperciliata with Atto488-dUTP with the Nick-Translation mix kit (Jena Biosciences). The final probe cocktail for each experiment was composed of 500 ng of female-derived gDNA of S. frontalis, 500 ng of female-derived DNA of the compared species, and 3 μg of male-derived C0t-1 DNA (1.5 μg of each species), diluted in 10 μL of hybridization buffer. The chosen ratio of probe versus C0t-1 DNA was based on previous experiments [de Moraes et al., 2019; Toma et al., 2019; Sassi et al., 2020]. CGH probes were denatured at 86°C for 8 min, followed by a 37°C pre-hybridization for 40 min and cooled at 4°C and the experiments followed the methodology described in Sember et al. [2018].

Microscopy and Image Processing

To confirm the diploid number, karyotype structure and FISH results in at least 30 metaphase spreads were analyzed per individual. Images were captured using an Olympus BX53 microscope (Olympus Corporation, Ishikawa, Japan) coupled with CoolSNAP and processed using ISIS software (MetaSystems). Chromosomes were arranged by size and the morphology was classified according to the centromere position [Guerra, 1988] as metacentric, submetacentric, acrocentric, and telocentric. GNU Image Manipulation Program (GIMP) software was used to assemble the karyotypes.

Karyotype Description and C-Banding

The results showed a diploid number of 2n = 82 in all ovenbirds investigated, and 2n = 78 in T. rufiventris. Both S. frontalis and T. rufiventris karyotypes have already been published, as 2n = 82 [Kretschmer et al., 2018c] and 2n = 78 [Kretschmer et al., 2014], respectively, corroborating the present results. Hence, we present the first detailed karyotype description of C. obsoleta and S. rufosuperciliata (Fig. 1). Pairs 1, 6, and 7 are acrocentric in S. rufosuperciliata, while pairs 2, 3, 4, 5, and the Z chromosome are submetacentric (Fig. 1a). Pairs 1, 6, 7, and the Z chromosome are acrocentric in C. obsoleta, but the remaining chromosomes are all submetacentric (Fig. 1b). C-positive heterochromatin regions were detected in all three ovenbird species' microchromosomes, and the W chromosomes seem to be entirely heterochromatic (Fig. 2).

Fig. 1.

Images and karyotypes of S. rufosuperciliata (a) and C. obsoleta (b).

Fig. 1.

Images and karyotypes of S. rufosuperciliata (a) and C. obsoleta (b).

Close modal
Fig. 2.

C-banded female chromosomes of S. frontalis (a), S. rufosuperciliata (b), and C. obsoleta (c). Sex chromosomes are indicated. Scale bar, 10 μm.

Fig. 2.

C-banded female chromosomes of S. frontalis (a), S. rufosuperciliata (b), and C. obsoleta (c). Sex chromosomes are indicated. Scale bar, 10 μm.

Close modal

Chromosomal Distribution of SSRs and 18S rDNA

Microsatellite sequences accumulated mostly on microchromosomes, resulting in a scattered pattern overall. In C. obsoleta (Fig. 3) and S. rufosuperciliata (Fig. 4), a single chromosome pair showed a strong signal with the (CA)n probe as opposed to the scattered pattern reported in S. frontalis, which also had a small accumulation on the W chromosome (Fig. 5). Despite the lack of a distinct distribution pattern in C. obsoleta chromosomes, (GA)n was found in the telomeric region of the short arm of two autosome pairs and the W chromosome of S. rufosuperciliata. The same repetitive sequence (GA)n was found dispersed in both the macrochromosomes and microchromosomes in S. frontalis, with significant signal accumulation on the Z chromosome (Fig. 3, 5). In S. rufosuperciliata and S. frontalis chromosomes, no hybridization signal was detected for either (GAG)n or (GAA)n sequences (Fig. 4, 5), however in C. obsoleta, the (GAA)n probe accumulated in a single microchromosome pair, whereas (GAG)n motifs were distributed over all chromosomes (Fig. 3). Almost all microchromosomes in C. obsoleta and S. rufosuperciliata were hybridized with (CGG)n, whereas signals accumulated in one microchromosome in S. frontalis. Furthermore, the (GA)n microsatellite was detected in an interstitial block on the q arms of the second pair of S. frontalis chromosomes. Finally, whereas few diffused signals for (CAG)n were discovered in C. obsoleta chromosomes, this pattern was found on nearly all S. rufosuperciliata and S. frontalis chromosomes (Fig. 3, 5). The sequences (GAA)n and (GAG)n showed signals only in C. obsoleta, while (CAG)n, (CGG)n, (GA)n, and (CA)n were observed in all species, presenting mostly only weak signals. It was also observed that the (CA)n and (CGG)n sequences had strong signals on the microchromosomes in all species analyzed. The (CGG)n sequence, on the other hand, showed strong signals on the microchromosomes in general, or on a specific pair, in all three Furnariidae species. The findings of microsatellite mapping in all species are summarized in Table 1. The 18S rDNA was mapped to a single microchromosome pair in all species (Fig. 6). In T. rufiventris, 18S rDNA has already been analyzed, where signals have been found in three pairs of microchromosomes [Kretschmer et al., 2014].

Fig. 3.

Distribution of microsatellite sequences on C. obsoleta female chromosomes. Sex chromosomes are indicated in the metaphase plates, and the probes used are highlighted in the upper left. Scale bar, 10 μm.

Fig. 3.

Distribution of microsatellite sequences on C. obsoleta female chromosomes. Sex chromosomes are indicated in the metaphase plates, and the probes used are highlighted in the upper left. Scale bar, 10 μm.

Close modal
Fig. 4.

FISH with microsatellite probes on S. rufosuperciliata female chromosomes. Sex chromosomes are indicated in the metaphase plates, and the probes used are highlighted in the upper left. Scale bar, 10 μm.

Fig. 4.

FISH with microsatellite probes on S. rufosuperciliata female chromosomes. Sex chromosomes are indicated in the metaphase plates, and the probes used are highlighted in the upper left. Scale bar, 10 μm.

Close modal
Fig. 5.

Microsatellite mapping on S. frontalis female chromosomes. Sex chromosomes are indicated in the metaphase plates, and the probes used are highlighted in the upper left. Scale bar, 10 μm.

Fig. 5.

Microsatellite mapping on S. frontalis female chromosomes. Sex chromosomes are indicated in the metaphase plates, and the probes used are highlighted in the upper left. Scale bar, 10 μm.

Close modal
Table 1.

Distribution of microsatellite sequences in the Furnariidae species

SSRsSpecies
Synallaxis frontalisSyndactyla rufosuperciliataCranioleuca obsoleta
(GAG)10 Scattered signals on the telomere regions of macrochromosomes and centromere of W 
(CAG)15 Scattered signals in all chromosomes but strong signals on the telomere regions of macrochromosomes Scattered signals in all chromosomes but strong signals on macro- and microchromosomes, and in the telomere of Zp and Zq Scattered and weak signals on macro- and microchromosomes, Zq 
(GAA)10 Strong signal on one pair of microchromosomes 
(CGG)10 Two pairs of microchromosomes Strong and scattered signals on all microchromosomes Strong signals on microchromosoomes, scattered on macrochromosomes, Zp 
(GA)15 Scattered signals in some macro- and microchromosomes, and strong signal on the Z Telomere of two pairs of macrochromosomes and scattered signals in some microchromosomes; strong signal on Wp Centromere of macrochromosomes 
(CA)15 Telomere of Wq Scattered signals on the microchromosomes but strong signal on one pair of microchromosomes Strong signal on one pair of microchromosomes 
SSRsSpecies
Synallaxis frontalisSyndactyla rufosuperciliataCranioleuca obsoleta
(GAG)10 Scattered signals on the telomere regions of macrochromosomes and centromere of W 
(CAG)15 Scattered signals in all chromosomes but strong signals on the telomere regions of macrochromosomes Scattered signals in all chromosomes but strong signals on macro- and microchromosomes, and in the telomere of Zp and Zq Scattered and weak signals on macro- and microchromosomes, Zq 
(GAA)10 Strong signal on one pair of microchromosomes 
(CGG)10 Two pairs of microchromosomes Strong and scattered signals on all microchromosomes Strong signals on microchromosoomes, scattered on macrochromosomes, Zp 
(GA)15 Scattered signals in some macro- and microchromosomes, and strong signal on the Z Telomere of two pairs of macrochromosomes and scattered signals in some microchromosomes; strong signal on Wp Centromere of macrochromosomes 
(CA)15 Telomere of Wq Scattered signals on the microchromosomes but strong signal on one pair of microchromosomes Strong signal on one pair of microchromosomes 

p, short arm; q, long arm.

Fig. 6.

FISH with 18S rDNA probe (green) in S. frontalis (a), S. rufosuperciliata (b), and C. obsoleta (c) chromosomes (blue). Sex chromosomes are indicated. Scale bar, 10 μm.

Fig. 6.

FISH with 18S rDNA probe (green) in S. frontalis (a), S. rufosuperciliata (b), and C. obsoleta (c) chromosomes (blue). Sex chromosomes are indicated. Scale bar, 10 μm.

Close modal

Comparative Genomic Hybridization

Intraspecific genomic hybridizations in S. frontalis revealed a conspicuous block of female-specific sequences on the whole W chromosome. Microchromosomes held the most significant accumulation of both female and male-derived genomic probes when compared to macrochromosomes (Fig. 7).

Fig. 7.

Female mitotic chromosomes of S. frontalis after intraspecific CGH procedure. Female- (green) and male-derived (red) genomic probes were hybridized together. a DAPI image (blue). b Hybridization pattern of the female-derived probe (green). c Hybridization pattern of the male-derived probe (red). d Merged images of both genomic probes and DAPI staining. The common genomic regions for males and females are depicted in yellow. Sex chromosomes are indicated. Scale bar, 10 μm.

Fig. 7.

Female mitotic chromosomes of S. frontalis after intraspecific CGH procedure. Female- (green) and male-derived (red) genomic probes were hybridized together. a DAPI image (blue). b Hybridization pattern of the female-derived probe (green). c Hybridization pattern of the male-derived probe (red). d Merged images of both genomic probes and DAPI staining. The common genomic regions for males and females are depicted in yellow. Sex chromosomes are indicated. Scale bar, 10 μm.

Close modal

The interspecific comparisons using S. frontalis chromosomes showed that they exhibit divergent genomic patterns. The genomic probes of C. obsoleta, S. rufosuperciliata, and T. rufiventris successfully hybridized with the chromosomes of S. frontalis. Notably, the Furnariidae species share more repetitive sequences in their genomes than with T. rufiventris, especially at the pericentromeric regions (Fig. 8). Furthermore, all the analyzed Furnariidae species showed a similarity of signals on the terminal portion of the short arms of the W chromosome, indicating a conserved state for these species.

Fig. 8.

Female mitotic chromosomes of S. frontalis after interspecific CGH procedures. Female- (green) and male-derived (red) genomic probes were hybridized together, and the merged results are presented in the last column. First column: DAPI images (blue). Second column: hybridization pattern of the female-derived probe of S. frontalis (green). Third column: hybridization pattern of the female-derived probes (red) of C. obsoleta, S. rufosuperciliata, and T. rufiventris. Fourth column: merged images of both genomic probes and DAPI staining. The common genomic regions for both species are depicted in yellow. Sex chromosomes are indicated. Scale bar, 10 μm.

Fig. 8.

Female mitotic chromosomes of S. frontalis after interspecific CGH procedures. Female- (green) and male-derived (red) genomic probes were hybridized together, and the merged results are presented in the last column. First column: DAPI images (blue). Second column: hybridization pattern of the female-derived probe of S. frontalis (green). Third column: hybridization pattern of the female-derived probes (red) of C. obsoleta, S. rufosuperciliata, and T. rufiventris. Fourth column: merged images of both genomic probes and DAPI staining. The common genomic regions for both species are depicted in yellow. Sex chromosomes are indicated. Scale bar, 10 μm.

Close modal

Avian evolutionary history has produced an amazing group with diverse adaptations and a vast number of species [Barrowclough et al., 2016]. Despite this tremendous variety, there are currently limited cytogenetic data, particularly for ovenbirds, where the majority of studies are out of date and scarce, making understanding their karyotype evolution difficult [de Lucca and Rocha, 1992]. Here, the chromosomal and genomic organization of three representative ovenbird species (Furnariidae) were studied, with a focus on their repetitive DNA content. Furnariidae is a suborder of the Passeriformes and represents a primitive lineage of the Passeriformes [Feduccia, 1999; Selvatti et al., 2015]. Currently, the majority of cytogenetic investigations in Passeriformes have been conducted in Oscines members; hence, this work contributes to a better knowledge of chromosomal organization and evolution in Suboscines, and thus in birds as a whole.

Karyotypes of several Passeriformes have been recorded, and the majority of them have a typical karyotype structure, such as a diploid number of approximately 80 chromosomes with evident differentiation between macro- and microchromosomes [Degrandi et al., 2020a]. Indeed, our findings on the S. frontalis karyotype indicated 2n = 82, the same number as previously published [Kretschmer et al., 2018c, de Souza et al., 2019]. Similarly, both S. rufosuperciliata and C. obsoleta reported for the first time here, have 2n = 82. Although all species had the same diploid number, we noticed a few modest variations in the chromosome morphology of some autosomal macrochromosomes, indicating that intrachromosomal rearrangements may have played a significant role in ovenbird chromosome evolution. Furthermore, S. rufosuperciliata and S. frontalis have submetacentric Z chromosomes, but C. obsoleta has an acrocentric Z chromosome, indicating that intrachromosomal rearrangements have occurred, such as pericentric inversions. Previous studies have shown that the Z chromosome in birds is generally conserved in size, but not in morphology, due to intrachromosomal rearrangements [Nanda and Schmid, 2002]. From FISH analyses, comparative chromosome painting has revealed a low degree of chromosomal variation within Passeriformes, although these species share a complex pattern of paracentric and pericentric inversions, and this pattern has been observed in both Oscines and Suboscines. These rearrangements must have occurred before the separation of these two groups [Kretschmer et al., 2014; Rodrigues et al., 2018].

C-banding showed that the W chromosome and several microchromosomes preferentially accumulated constitutive heterochromatin in all the species that were investigated (Fig. 2). The centromeric region of the autosomes and Z chromosome of S. frontalis has previously been described, and blocks of constitutive heterochromatin were detected there [Kretschmer et al., 2018c], whereas the W chromosome was almost entirely heterochromatic. The role of constitutive heterochromatin in sex chromosomal differentiation has already been identified by other groups [Marchal et al., 2004; Yano et al., 2016]. It appears that its accumulation in sex chromosomes is a regular phenomenon during the chromosomal evolution of Passeriformes. Similar patterns of C-positive heterochromatin accumulation in several pairs of microchromosomes were seen in both S. rufosuperciliata and C. obsoleta, as well as in other Suboscines species [Rodrigues et al., 2018; de Souza et al., 2019]. Similar results were seen in Oscines like Saltator similis, Taeniopygia guttata, and Serinus canaria [dos Santos et al., 2015, 2017], highlighting the general tendency for conservation observed in all bird genomes.

The three species studied in this work all have one pair of microchromosomes containing 18S rDNA genes, which has been postulated as the ancestral state for birds [Nishida-Umehara et al., 2007]. In fact, the genomic organization of these sequences is dynamic, but in most bird species, including basal ones like the emu (Dromaius novaehollandiae), they are restricted to one pair of microchromosomes. On the other hand, a larger number of 18S rDNA clusters was found in several Oscines species, including four in Gubernatrix cristata, S. canaria, and Turdus albicollis [dos Santos et al., 2017; Sember et al., 2018; de Moraes et al., 2019; Bülau et al., 2021]. The repetitive nature of the clusters or their intense gene expression activity is related to the location of rDNA sites, which causes chromosomal break hotspots [Huang et al., 2008; Cazaux et al., 2011]. Breaks like this on the chromosome can lead to a variety of rearrangements, including translocations, fusions, duplications, and inversions, which can quickly affect the rDNA site distribution on the chromosome in closely related species [Degrandi et al., 2020b].

Each Furnariidae species has a unique pattern of repetitive DNA hybridization, encompassing the macro- and microchromosomes as well as the sex chromosomes, which indicates species-specific patterns. So far, only four Passeriformes species (Progne tapera, Progne chalybea, Pygochelidon cyanoleuca, and Sicalis flaveola) have been studied using chromosomal mapping of SSR sequences [Barcellos et al., 2019; Kretschmer et al., 2021b]. The (GAA)n and (GAG)n sequences did not show signals in any of the chromosomes in S. frontalis and S. rufosuperciliata, but when we analyze these sequences in other bird species, there is a different pattern, where in S. flaveola it shows very strong and visible signals. The (GAA)n sequence, for example, was present in all bird species that have been studied by microsatellites to date (S. flaveola, P. tapera, P. chalybea, and P. cyanoleuca) and also shows a pattern of strong markings in all these species, unlike S. frontalis and S. rufosuperciliata, which showed no signal [Barcellos et al., 2019; Kretschmer et al., 2021b]. We can deduce that C. obsoleta, the most derived species among the three Furnariidae species here analyzed, may yet maintain an apomorphic trait because (GAG)n and (GAA)n are only present in this species.

Despite the presence of interstitial blocks of repetitive DNA, microsatellite accumulations were detected in the centromeric and telomeric regions of several macrochromosomes. According to Cella and Ferreira [1991] and Bueno et al. [2013], the interstitial signals in S. frontalis were thought to be driven on by the accumulation of repetitive DNAs in the long arms of the second chromosomal pair. Although the W chromosomes in Oscines are small and comparable to a microchromosome [Chen et al., 2012; Zhang et al., 2014], there are other species that deviate significantly from this trend, such as swallows, where the W chromosome reaches nearly the size of the Z chromosome [Barcellos et al., 2019]. The size of the W is conserved in Suboscines, as it is in other birds, and both species karyotyped in this study show the same pattern. However, there was no evidence of sex-specific accumulation of these sequences in our study. Because avian genomes contain fewer repetitive sequences than those of other vertebrates [Zhang et al., 2014], there is a huge gap in information relating to avian repetitive DNA fraction of the genome. Except in Piciformes [Kretschmer et al., 2020], cytogenetic studies show that microsatellites accumulate preferentially on W chromosomes [de Oliveira Furo et al., 2017; Barcellos et al., 2019; Gunski et al., 2019]. Furthermore, these motifs are preferentially linked with heterochromatic regions, lending support to the theory that repetitive DNAs are present in condensed and inactive parts of the genome [Ellegren, 2004]. The (CA)n and (GA)n sequences are found in small amounts on the telomere of the W chromosome in S. frontalis and S. rufosuperciliata, respectively. Microsatellite probes provided insights into the sex chromosome evolution in the Furnariidae family. For example, the sequences (CGG)n, (GAA)n, and (GAG)n were not detected in the W of the two Furnariidae species, confirming that there was no amplification of these sequences, while a distinct scenario was observed in C. obsoleta, which had minor markings of the (CGG)n and (GAG)n sequences in the W.

Despite the fact that the three species studied here are from distinct genera, our CGH data indicated that their genomes share considerable portions of their repetitive DNA fractions. The presence of repeated DNAs in the centromeric heterochromatic blocks of several chromosomes was revealed in S. frontalis when the gDNA was hybridized against its own chromosomal complement. CGH investigations with C. obsoleta and S. rufosuperciliata gDNA probes revealed multiple overlapping signals in the centromeric regions, indicating that these species' centromeres are enriched in homologous repetitive sequences (Fig. 8). Incompatibilities between quickly evolving centromeric components might be responsible for both centromeric region organization and reproductive isolation of emerging species [Henikoff et al., 2001]. Centromeric repeats are the most quickly changing DNA sequences in eukaryotic genomes, varying even within populations of the same species [Murphy and Karpen, 1998] and between species that naturally hybridize [Poignet et al., 2021]. The interspecific CGH experiment in Oscines indicated that the two nightingale species studied (Luscinia megarhynchos and Luscinia luscinia) differed in their centromeric repetitive sequences in all chromosomes. In fact, certain chromosomes demonstrated differences in the copy number of centromeric repeats between species [Poignet et al., 2021]. Concerning the sex chromosomes, the results demonstrated (1) that sequences are still shared in the terminal portion of the short arms of the W chromosomes (as previously proposed by the whole avian order) [Mank and Ellegren, 2007] and (2) the accumulation of species-specific sequences in the long arms of the W chromosome, evidencing a varying degree of W chromosome differentiation between species, accompanied by species-specific patterns of repetitive DNA, as observed in other ZW species from different vertebrate groups [Mank and Ellegren, 2007; Fraïsse et al., 2017; Barcelllos et al., 2019; de Souza et al., 2020; Viana et al., 2020]. The majority of the chromosomes in S. frontalis were hybridized by the gDNA probes during intraspecific analysis, highlighting repetitive DNA content (indicated by yellowish signals) and produced strong signals on the W chromosome. That is, both male and female probes hybridized to chromosome W, but the female probe stained roughly half of chromosome W (Wq) more intensively, indicating that the W displayed preferential binding of the corresponding female probe (Fig. 9). Taking all of this into consideration, we propose that each species followed different trajectories in these chromosomes.

Here we conducted the most comprehensive cytogenetic investigation for any ovenbird species to date, providing an understanding of their chromosomal organization and evolution. The described karyotypes (2n = 82) showed a typical avian structure. Our chromosomal mapping of SSRs revealed a reasonable differentiation between these sequences in both suborders (Suboscines and Oscines). In terms of the W chromosome, oscine species accumulated more repetitive sequences preferentially over suboscine ones. To assess significant evolutionary elements of the genomic architecture and composition of other Furnariidae species, as well as to determine if the other members of this family have a common pattern of sequence organization, more studies are still required.

The authors are grateful to all colleagues at the Laboratório de Diversidade Genética Animal from the Universidade Federal do Pampa (RS, Brazil) and Laboratório de Citogenética de Peixes of the Universidade Federal de São Carlos (SP, Brazil) for their technical support. We are also grateful to Guilherme Castro Franco de Lima for the illustration of the S. rufosuperciliata and C. obsoleta used in Figure 1.

All protocols were approved by the Ethics Committee on the use of animals (CEUA 019/2020) and IBAMA (SISBIO 61047-1).

The authors have no conflicts of interest to declare.

This research was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES, Finance Code 001) and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Proc. 407285/2021-0). M.B.C. was supported by CNPq (Proc. 302928/2021-9) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Proc. 2020/1172-8).

Conceptualization: Victoria Tura, Rafael Kretschmer, and Francisco de Menezes Cavalcante Sassi. Data curation: Victoria Tura and Marcelo de Bello Cioffi. Formal analysis: Victoria Tura, Rafael Kretschmer, Francisco de Menezes Cavalcante Sassi, Renata Luiza Rosa de Moraes, Suziane Alves Barcellos, and Marcelo de Bello Cioffi. Funding acquisition: Marcelo de Bello Cioffi, Ricardo José Gunski, and Analía del Valle Garnero. Investigation: Victoria Tura, Rafael Kretschmer, Francisco de Menezes Cavalcante Sassi, and Marcelo de Bello Cioffi. Methodology: Victoria Tura, Francisco de Menezes Cavalcante Sassi, Renata Luiza Rosa de Moraes, Suziane Alves Barcellos, and Marcelo Santos de Souza. Project administration: Marcelo de Bello Cioffi and Ricardo José Gunski. Supervision: Rafael Kretschmer, Francisco de Menezes Cavalcante Sassi, Marcelo de Bello Cioffi, Ricardo José Gunski, and Analía del Valle Garnero. Validation: Victoria Tura and Rafael Kretschmer. Visualization: Victoria Tura, Francisco de Menezes Cavalcante Sassi, and Marcelo de Bello Cioffi. Writing – original draft: Victoria Tura, Francisco de Menezes Cavalcante Sassi, Suziane Alves Barcellos, and Vitor Oliveira de Rosso. Writing – review and editing: Rafael Kretschmer and Francisco de Menezes Cavalcante Sassi. Data curation: Victoria Tura, Renata Luiza Rosa de Moraes, Marcelo de Bello Cioffi, Rafael Kretschmer, Ricardo José Gunski, and Analía del Valle Garnero. All authors have read and agreed to the published version of the manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

1.
Barcellos
SA
,
Kretschmer
R
,
de Souza
MS
,
Costa
AL
,
Degrandi
TM
,
dos Santos
MS
.
Karyotype evolution and distinct evolutionary history of the W chromosomes in swallows (Aves, Passeriformes)
.
Cytogenet Genome Res
.
2019
;
158
(
2
):
98
105
.
2.
Barcellos
SA
,
de Souza
MS
,
Tura
V
,
Pereira
LR
,
Kretschmer
R
,
Gunski
RJ
.
Direct chromosome preparation method in avian embryos for cytogenetic studies: quick, easy and cheap
.
DNA
.
2022
;
2
(
1
):
22
9
.
3.
Barrowclough
GF
,
Cracraft
J
,
Klicka
J
,
Zink
RM
.
How many kinds of birds are there and why does it matter
.
PLoS One
.
2016
;
11
:
e0166307
.
4.
Bueno
D
,
Palacios-Gimenez
OM
,
Cabral-de-Mello
DC
.
Chromosomal mapping of repetitive DNAs in the grasshopper Abracris flavolineata reveal possible ancestry of the B chromosome and H3 histone spreading
.
PLoS One
.
2013
;
8
(
6
):
e66532
.
5.
Bülau
SE
,
Kretschmer
R
,
Furo
IO
,
de Oliveira
EHC
,
de Freitas
TRO
.
Karyotype organization of the endangered species yellow cardinal (Gubernatrix cristata)
.
DNA
.
2021
;
1
(
2
):
77
83
.
6.
Burt
DW
.
Origin and evolution of avian microchromosomes
.
Cytogenet Genome Res
.
2002
96
1-4
97
112
.
7.
Cazaux
B
,
Catalan
J
,
Veyrunes
F
,
Douzery
EJ
,
Britton-Davidian
J
.
Are ribosomal DNA clusters rearrangement hotspots? A case study in the genus Mus (Rodentia, Muridae)
.
BMC Evol Biol
.
2011
;
11
:
124
.
8.
Cella
DM
,
Ferreira
A
.
The cytogenetics of Abracris flavolineata (Orthoptera, Caelifera, Ommatolampinae, Abracrini)
.
Brazil J Genet
.
1991
;
14
:
315
29
.
9.
Chen
N
,
Bellott
DW
,
Page
DC
,
Clark
AG
.
Identification of avian W-linked contigs by short-read sequencing
.
BMC Genomics
.
2012
;
13
:
183
.
10.
Christiakov
DA
,
Bart
H
,
Filip
AMV
.
Microsatellite and their genome distribution, evolution, function and applications; a review with special reference to fish genetics
.
Aquaculture
.
2006
255
1-4
1
29
.
11.
Cioffi
MB
,
Bertollo
LAC
.
Chromosomal distribution and evolution of repetitive DNAs in fish
.
Genome Dyn
.
2012
;
7
:
197
221
.
12.
Cioffi
MB
,
Ráb
P
,
Ezaz
T
,
Bertollo
LAC
,
Lavoué
S
,
Oliveira EA
.
Deciphering the evolutionary history of Arowana fishes (Teleostei, Osteoglossiformes, Osteoglossidae): insight from comparative cytogenomics
.
Int J Mol Sci
.
2019
20(17)
4296
.
13.
Degrandi
TM
,
Barcellos
SA
,
Costa
AL
,
Garnero
AVG
,
Hass
I
,
Gunski
RJ
.
Introducing the bird chromosome database: an overview of cytogenetic studies in birds
.
Cytogenet Genome Res
.
2020a
160
4
199
205
.
14.
Degrandi
TM
,
Gunski
RJ
,
Garnero
ADV
,
Oliveira
EHC
,
Kretschmer
R
,
Souza
MS
.
The distribution of 45S rDNA sites in bird chromosomes suggests multiple evolutionary histories
.
Genet Mol Biol
.
2020b
43
2
e20180331
.
15.
de Lucca
EJ
,
Rocha
GT
.
Citogenética de Aves
.
Bol Mus Para Emílio Goeldi
.
1992
;
8
:
33
68
.
16.
de Moraes
RLR
,
Sember
A
,
Bertollo
LAC
,
de Oliveira
EA
,
Ráb
P
,
Hatanaka
T
.
Comparative cytogenetics and neo-Y formation in small-sized fish species of the genus Pyrrhulina (Characiformes, Lebiasinidae)
.
Front Genet
.
2019
;
10
:
678
.
17.
de Oliveira
TD
,
Kretschmer
R
,
Bertocchi
NA
,
Degrandi
TM
,
de Oliveira
EHC
,
Cioffi
MB
.
Genomic organization of repetitive DNA in woodpeckers (Aves, Piciformes): implications for karyotype and ZW sex chromosome differentiation
.
PLoS One
.
2017
;
12
(
1
):
e0169987
.
18.
de Oliveira Furo
I
,
Kretschmer
R
,
dos Santos
MS
,
de Lima Carvalho
CA
,
Gunski
RJ
,
O’Brien
PCM
.
Chromosomal mapping of repetitive DNAs in Myiopsitta monachus and Amazona aestiva (Psittaciformes, Psittacidae), with emphasis on the sex chromosomes
.
Cytogenet Genome Res
.
2017
;
151
(
3
):
151
60
.
19.
de Souza
MS
,
Barcellos
S
,
Costa
AL
,
Kretschmer
R
,
Garnero
ADV
,
Gunski
RJ
.
Polymorphism of sooty-fronted spinetail (Synallaxis frontalis Aves: Furnariidae): evidence of chromosomal rearrangements by pericentric inversion in autosomal macrochromosomes
.
Genet Mol Biol
.
2019
;
42
(
1
):
62
7
.
20.
de Souza
MS
,
Kretschmer
R
,
Barcellos
SA
,
Costa
AL
,
Cioffi
MB
,
de Oliveira
EHC
.
Repeat sequence mapping shows different W chromosome evolutionary pathways in two Caprimulgiformes families
.
Birds
.
2020
;
1
:
19
34
.
21.
dos Santos
MS
,
Kretschmer
R
,
Silva
FAO
,
Ledesma
MA
,
O’Brien
PCM
,
Ferguson-Smith
MA
.
Intrachromosomal rearrangements in two representatives of the genus Saltator (Thraupidae, Passeriformes) and the occurrence of heteromorphic Z chromosomes
.
Genetica
.
2015
;
143
(
5
):
535
43
.
22.
dos Santos
MS
,
Kretschmer
R
,
Frankl-Vilches
C
,
Bakker
A
,
Gahr
M
,
O´Brien
PCM
.
Comparative cytogenetics between two important songbird, models: the zebra finch and the canary
.
PLoS One
.
2017
;
12
(
1
):
e0170997
.
23.
Ellegren
H
.
Microsatellites: simple sequences with complex evolution
.
Nat Rev Genet
.
2004
;
5
(
6
):
435
45
.
24.
Ericson
PG
,
Klopfstein
S
,
Irestedt
M
,
Nguyen
JMT
,
Nylander
JA
.
Dating the diversification of the major lineages of Passeriformes (Aves)
.
BMC Evol Biol
.
2014
;
14
:
8
.
25.
Feduccia
A
The origin and evolution of birds
New Haven
Yale University Press
1999
. p.
287
300
.
26.
Fraïsse
C
,
Picard
MAL
,
Vicoso
B
.
The deep conservation of the Lepidoptera Z chromosome suggests a non-canonical origin of the W
.
Nat Commun
.
2017
;
8
(
1
):
1486
.
27.
Garnero
AV
,
Gunski
RJ
.
Comparative analysis of the karyotype of Nothura maculosa and Rynchotus rufescens (Aves: Tinamidae). A case of chromosomal polymorphism
.
Nucleus
.
2000
;
243
:
64
70
.
28.
Gregory
TR
Animal Genome Size Database
.
2022
. http://www.genomesize.com
29.
Griffin
DK
,
Robertson
LBW
,
Tempest
HG
,
Skinner
BM
.
The evolution of the avian genome as revealed by comparative molecular cytogenetics
.
Cytogenet Genome Res
.
2007
117
1-4
64
77
.
30.
Guerra
M
.
Introdução à citogenética geral
.
Guanabara Koogan
.
1988
1
1st
142
.
31.
Gunski
RJ
,
Kretschmer
R
,
Santos de Souza
M
,
de Oliveira Furo
I
,
Barcellos
SA
,
Costa
AL
.
Evolution of bird sex chromosomes narrated by repetitive sequences: unusual W chromosome enlargement in Gallinula melanops (Aves: Gruiformes: Rallidae)
.
Cytogenet Genome Res
.
2019
;
158
(
3
):
152
9
.
32.
Henikoff
S
,
Ahmad
K
,
Malik
H
.
The centromere paradox: stable inheritance with rapidly evolving DNA
.
Science
.
2001
;
293
(
5532
):
1098
102
.
33.
Huang
J
,
Ma
L
,
Yang
F
,
Fei
SZ
,
Li
L
.
45S rDNA regions are chromosome fragile sites expressed as gaps in vitro on metaphase chromosomes of root-tip meristematic cells in Lolium spp
.
PLoS One
.
2008
;
3
(
5
):
e2167
.
34.
Kallioniemi
A
,
Kallioniemi
OP
,
Sudar
D
,
Rutovitz
D
,
Gray
JW
,
Waldman
F
.
Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors
.
Science
.
1992
;
258
(
5083
):
818
21
.
35.
Kretschmer
R
,
Gunski
RJ
,
Garnero
ADV
,
Furo
IO
,
O’Brien
PCM
,
Ferguson-Smith
MA
.
Molecular cytogenetic characterization of multiple intrachromosomal rearrangements in two representatives of the genus Turdus (Turdidae, Passeriformes)
.
PLoS One
.
2014
;
9
:
e103338
.
36.
Kretschmer
R
,
Ferguson-Smith
MA
,
de Oliveira
EHC
.
Karyotype evolution in birds: from conventional staining to chromosome painting
.
Genes
.
2018a
9
4
181
.
37.
Kretschmer
R
,
de Oliveira
TD
,
de Oliveira Furo
I
,
Oliveira Silva
FA
,
Gunski
RJ
,
Del Valle Garnero
A
.
Repetitive DNAs and shrink genomes: a chromosomal analysis in nine Columbidae species (Aves, Columbiformes)
.
Genet Mol Biol
.
2018b
41
1
98
106
.
38.
Kretschmer
R
,
de Lima
VLC
,
de Souza
MS
,
Costa
AL
,
O’Brien
PCM
,
Ferguson-Smith
MA
.
Multidirectional chromosome painting in Synallaxis frontalis (Passeriformes, Furnariidae) reveals high chromosomal reorganization, involving fissions and inversions
.
Comp Cytogenet
.
2018c
12
1
97
110
.
39.
Kretschmer
R
,
Furo
IDO
,
Cioffi
MB
,
Gunski
RJ
,
Garnero
ADV
,
O’Brien
PCM
.
Extensive chromosomal fissions and repetitive DNA accumulation shaped the atypical karyotypes of two Ramphastidae (Aves: Piciformes) species
.
Biol J Linn Soc
.
2020
;
130
(
4
):
839
49
.
40.
Kretschmer
R
,
Franz
I
,
de Souza
MS
,
Garnero
ADV
,
Gunski
RJ
,
de Oliveira
EHC
.
Cytogenetic evidence clarifies the phylogeny of the family Rhynchocyclidae (Aves: Passeriformes)
.
Cells
.
2021a
10
2650
.
41.
Kretschmer
R
,
Rodrigues
BS
,
Barcellos
SA
,
Costa
AL
,
Cioffi
MB
,
Garnero
ADV
.
Karyotype evolution and genomic organization of repetitive DNAs in the saffron finch, Sicalis flaveola (Passeriformes, Aves)
.
Animals
.
2021b
11
5
1456
.
42.
Kretschmer
R
,
Santos
MS
,
Furo
IO
,
Oliveira
EHC
,
Cioffi
MB
.
FISH–in birds
. In:
Liehr
T
, editor.
Cytogenetics and Molecular Cytogenetics
1st ed
Boca Raton
CRC Press
2022
. p.
263
76
.
43.
Kubat
Z
,
Hobza
R
,
Vyskot
B
,
Kejnovsky
E
.
Microsatellite accumulation on the Y chromosome in Silene latifolia
.
Genome
.
2008
;
51
(
5
):
350
6
.
44.
Lui
RL
,
Blanco
DR
,
Moreira-Filho
O
,
Margarido
VP
.
Propidium iodide for making heterochromatin more evident in the C-banding technique
.
Biotech Histochem
.
2012
;
87
(
7
):
433
8
.
45.
Mank
JE
,
Ellegren
H
.
Parallel divergence and degradation of the avian W sex chromosome
.
Trends Ecol Evol
.
2007
;
22
(
8
):
389
91
.
46.
Marchal
JA
,
Acosta
MJ
,
Nietzel
H
,
Sperling
K
,
Bullejos
M
,
Díaz de la Guardia
R
.
X chromosome painting in Microtus: origin and evolution of the giant sex chromosomes
.
Chromosome Res
.
2004
;
12
(
8
):
767
76
.
47.
Mazzoleni
S
,
Rovatsos
M
,
Schillaci
O
,
Dumas
F
.
Evolutionary insight on localization of 18S, 28S rDNA genes on homologous chromosomes in Primates genomes
.
Comp Cytogenet
.
2018
;
12
(
1
):
27
40
.
48.
Moyle
RG
,
Chesser
RT
,
Brumfield
RT
,
Tello
JG
,
Marchese
DJ
,
Cracraft
J
.
Phylogeny and phylogenetic classification of the antbirds, ovenbirds, woodcreepers, and allies (Aves: Passeriformes: infraorder Furnariides)
.
Cladistics
.
2009
;
25
(
4
):
386
405
.
49.
Murphy
TD
,
Karpen
GH
.
Centromeres take flight: alpha satellite and the quest for the human centromere
.
Cell
.
1998
;
93
(
3
):
317
20
.
50.
Nanda
I
,
Schmid
M
.
Conservation of avian Z chromosomes as revealed by comparative mapping of the Z-linked aldolase B gene
.
Cytogenet Genome Res
.
2002
96
1-4
176
8
.
51.
Nishida-Umehara
C
,
Tsuda
Y
,
Ishijima
J
,
Ando
J
,
Fujiwara
A
,
Matsuda
Y
.
The molecular basis of chromosome orthologies and sex chromosomal differentiation in palaeognathous birds
.
Chromosome Res
.
2007
;
15
(
6
):
721
34
.
52.
Poignet
M
,
Johnson
MP
,
Altmanová
M
,
Majtánová
Z
,
Dedukh
D
,
Albrecht T
.
Comparison of karyotypes in two hybridizing passerine species: conserved chromosomal structure but divergence in centromeric repeats
.
Front Genet
.
2021
;
12
:
768987
.
53.
Remsen
JV
.
Family Furnariidae (ovenbirds)
. In:
del Hoyo
J
,
Elliott
A
,
Christie
DA
, editors.
Handbook of the Birds of the World
Barcelona
Lynx Edicions
2003
. p.
162
357
.
54.
Rodrigues
BS
,
Kretschmer
R
,
Gunski
RJ
,
Garnero
ADV
,
O’Brien
PCM
,
Ferguson-Smith
M
.
Chromosome painting in tyrant flycatchers confirms a set of inversions shared by Oscines and Suboscines (Aves, Passeriformes)
.
Cytogenet Genome Res
.
2017
;
153
(
4
):
205
12
.
55.
Sambrook
J
,
Russell
DW
Molecular Cloning: A Laboratory Manual
3rd ed
New York
Cold Spring Harbor Laboratory Press
2001
.
56.
Santos
LP
,
Gunski
RJ
.
Revisão de dados citogenéticos sobre a avifauna brasileira
.
Rev Brasil Ornitol
.
2006
;
14
:
35
45
.
57.
Sasaki
M
,
Ikeuchi
T
,
Maino
SA
.
Feather pulp culture for avian chromosomes with notes on the chromosomes of the peafowl and the ostrich
.
Experientia
.
1968
;
24
:
1923
9
.
58.
Sassi
FMC
,
Hatanaka
T
,
Moraes
RLR
,
Toma
GA
,
Oliveira
EA
,
Liehr
T
.
An insight into the chromosomal evolution of Lebiasinidae (Teleostei, Characiformes)
.
Genes
.
2020
;
11
(
4
):
365
.
59.
Schartl
M
,
Schmid
M
,
Nanda
I
.
Dynamics of vertebrate sex chromosome evolution: from equal size to giants and dwarfs
.
Chromosoma
.
2016
;
125
(
3
):
553
71
.
60.
Selkoe
KA
,
Toonen
RJ
.
Microsatellites for ecologists: a practical guide to using and evaluating microsatellite markers
.
Ecol Lett
.
2006
;
9
(
5
):
615
29
.
61.
Selvatti
AP
,
Gonzaga
LP
,
Russo
CA
.
A Paleogene origin for crown passerines and the diversification of the Oscines in the New World
.
Mol Phylogenet Evol
.
2015
;
88
:
1
15
.
62.
Sember
A
,
Bertollo
LAC
,
Ráb
P
,
Yano
CF
,
Hatanaka
T
,
de Oliveira
EA
.
Sex chromosome evolution and genomic divergence in the fish Hoplias malabaricus (Characiformes, Erythrinidae)
.
Front Genet
.
2018
;
9
:
71
.
63.
Sumner
AT
.
A simple technique for demonstrating centromeric heterochromatin
.
Exp Cell Res
.
1972
;
75
(
1
):
304
6
.
64.
Szarski
H
.
Cell size and nuclear DNA content in vertebrates
.
Int Rev Cytol
.
1976
;
44
:
93
111
.
65.
Toma
GA
,
de Moraes
RLR
,
Sassi
FMC
,
Bertollo
LAC
,
de Oliveira
EA
,
Rab
P
.
Cytogenetics of the small-sized fish, Copeina guttata (Characiformes, Lebiasinidae): novel insights into the karyotype differentiation of the family
.
PLoS One
.
2019
;
14
(
12
):
e0226746
.
66.
Torgasheva
AA
,
Malinovskaya
LP
,
Zadesenets
KS
,
Karamysheva
TV
,
Kizilova
EA
,
Akberdina
EA
.
Germline-restricted chromosome (GRC) is widespread among songbirds
.
Proc Natl Acad Sci U S A
.
2019;
116
24
11845
50
.
67.
Viana
PF
,
Ezaz
T
,
de Bello Cioffi
M
,
Liehr
T
,
Al-Rikabi
ABH
,
Goll
LG
.
Landscape of snake’ sex chromosomes evolution spanning 85 MYR reveals ancestry of sequences despite distinct evolutionary trajectories
.
Sci Rep
.
2020
;
10
(
1
):
12499
.
68.
White
TJ
,
Bruns
T
,
Lee
S
,
Taylor
J
.
Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics
. In:
Innis
MA
,
Gelfand
DH
,
Shinsky
JJ
,
White
TJ
, editors.
PCR Protocols: A Guide to Methods and Applications
San Diego
Academic Press
1990
. p.
315
22
.
69.
Yano
CF
,
Bertollo
LAC
,
Liehr
T
,
Troy
WP
,
Cioffi
MB
.
W chromosome dynamics in Triportheus species (Characiformes, Triportheidae)—an ongoing process narrated by repetitive sequences
.
J Hered
.
2016
;
107
(
4
):
342
8
.
70.
Zhang
G
,
Li
C
,
Li
Q
,
Li
B
,
Larkin
DM
,
Lee
C
.
Comparative genomics reveals insights into avian genome evolution and adaptation
.
Science
.
2014
;
346
(
6215
):
1311
20
.
71.
Zwick
MS
,
Hanson
RE
,
Islam-Faridi
MN
,
Stelly
DM
,
Wing
RA
,
Price
HJ
.
A rapid procedure for the isolation of C0t-1 DNA from plants
.
Genome
.
1997
;
40
(
1
):
138
42
.
72.
Zyskowski
K
,
Prum
RO
.
Phylogenetic analysis of the nest architecture of neotropical ovenbirds (Furnariidae)
.
The Auk
.
1999
;
116
(
4
):
891
911
.