Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(1): 43-51, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-839 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: A. Lemos Costa, C. Fur- lan Lopes, M. Santos de Souza, S. Alves Barcellos, P. Giordani Vielmo, R. José Gunski, A. Del Valle Garnero (2021) Comparative cytogenetics in three species of Wood-Warblers (Aves: Pas- seriformes: Parulidae) reveal divergent banding patterns and chromatic hetero- geneity for the W chromosome. Caryo- logia 74(1): 43-51. doi: 10.36253/caryolo- gia-839 Received: January 22, 2020 Accepted: April 26, 2021 Published: July 20, 2021 Copyright: © 2021 A. Lemos Costa, C. Furlan Lopes, M. Santos de Souza, S. Alves Barcellos, P. Giordani Vielmo, R. José Gunski, A. Del Valle Garnero. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID ALC: 0000-0003-4620-2989 CFL: 0000-0002-4783-4315 MSS: 0000-0002-2130-6100 SAB: 0000-0003-2863-9976 PGV: 0000-0003-3491-2115 RJG: 0000-0002-7315-0590 ADVG: 0000-0003-4252-8228 Comparative cytogenetics in three species of Wood-Warblers (Aves: Passeriformes: Parulidae) reveal divergent banding patterns and chromatic heterogeneity for the W chromosome Alice Lemos Costa1,*, Cassiane Furlan Lopes1, Marcelo Santos de Souza1, Suziane Alves Barcellos1, Pâmela Giordani Vielmo2, Ricardo José Gunski1, Analía Del Valle Garnero1 1 Laboratório de Diversidade Genética Animal, Programa de Pós-Graduação em Ciências Biológicas, Universidade Federal do Pampa, São Gabriel, Rio Grande do Sul, Brazil 2 Laboratório de Diversidade Genética Animal, Curso de Graduação em Ciências Biológi- cas, Universidade Federal do Pampa, São Gabriel, Rio Grande do Sul, Brazil *Corresponding author. E-mail: alicelemoscosta14@hotmail.com Absract. Chromosomal rearrangements are an important process in the evolution of species. It is assumed that these rearrangements occur near repetitive sequences and heterochromatic regions. Avian karyotypes have diverse chromosomal band patterns and have been used as the parameters for phylogenetic studies. Although the group has a high diversity of species, no more than 12% has been analyzed cytogenetically, and the Parulidae family are extremely underrepresented in these studies. The aim of this study was to detect independent or simultaneous chromosomal rearrangements, and also to analyze chromosomal banding convergences and divergences of three Wood- Warblers species (Myiothlypis leucoblephara, Basileuterus culicivorus, and Setophaga pitiayumi). Our CBG-band results reveal an unusual W sex chromosome in the three studied species, containing a telomeric euchromatic region. The GTG and RBG bands identify specific regions in the macrochromosomes involved in the rearrangements. Cytogenetic data confirm the identification of speciation processes at the karyotypic of this group. Keywords: chromosomal evolution, karyotype, diploid number, chromosomal band- ing, constitutive heterochromatin. INTRODUCTION The Avian Class is characterized by a bimodal karyotype, composed of many pairs of microchromosomes and just a few macrochromosomes (Chris- tidis 1990). The Class presents several patterns of chromosomal bands. In CBG-banding, species of Passeriformes usually reveal the W chromosome 44 Alice Lemos Costa et al. heterochromatic (Kretschmer et al. 2018a). In con- trast, some Struthioniformes species show a complete- ly euchromatic chromosome (Nishida-Umehara et al. 2007). In other Orders such as Tinamiformes, this chro- mosome exhibits an intermediate CBG-banding pattern, containing euchromatic and heterochromatic blocks (Garnero et al. 2006). Some classical cytogenetic techniques provide pat- terns of positive and negative bands, exposing points of reference on the full length of the chromosome and enabling the creation of ideograms (Ladjali et al. 1999). Changes in these patterns suggest the possible types of rearrangements caused by chromosomal differences that may have occurred during the evolution of the genome (Griffin et al. 2007). Examples of this are the chromo- somal rearrangements already reported by GTG and RBG bands in Gallus gallus (Galliformes), which iden- tified a paracentric inversion in the long arm of chro- mosome 2 (Nanda et al. 1994). Chromosomal polymor- phisms were identified by GTG bands in Synallaxis fron- talis (Passeriformes), where pericentric inversion involv- ing the first and third pairs was observed (de Souza et al. 2019), and in Treron phoenicoptera (Columbiformes) in the first and second pairs (Gupta and Kaul 2014). C h r om o s om a l r e a r r a n g e m e nt s o c c u r du r- ing the evolutionar y process at the specimen level (Kretschmer et al. 2018b). Among these chromosom- a l changes are commonly obser ved translocations, duplications, inversions, deletions, fusion, and fissions (Stock and Bunch 1982; Nascimento et al. 1994; Nanda et al. 2011). This occurs in regions involving repetitive sequences and in the proximity of heterochromatic regions (Farre et al. 2016). Less than 12% of the species of the Aves Class have been characterized by cytogenetic studies, where Pas- seriformes Order contains most of the species described (Griffin et al. 2007; Degrandi et al. 2020). Parulidae (Passeriformes) is strictly underrepresented in these studies, the family contains 119 species divided into 21 genera, but only 8% of all species have been investigat- ed cytogenetically by Giemsa staining, shown diploid variation from 76 to 80 chromosomes (Carvalho 1989; Hobart 1991). This study aimed to detect independ- ent or simultaneous chromosomal rearrangements, and it also analyzes chromosomal banding convergences and divergences of three species of the Parulidae fam- ily – Myiothlypis leucoblephara, Basileuterus culicivorus, and Setophaga pitiayumi – using techniques of classical cytogenetics such as CBG, GTG, and RBG bands. MATERIAL AND METHODS Sampling and Collecting Five specimens of Wood-Warblers were analyzed in the present study: Myiothlypis leucoblephara (1 male and 1 female), Basileuterus culicivorus (1 male and 1 female), and Setophaga pitiayumi (1 female). All specimens were collected using a mist net in São Gabriel, Rio Grande do Sul state, Brazil (latitude - 30°20’38’’S and longitude -54°20’31’’W), under license SISBIO nº 61047-3, and CEUA/UNIPAMPA nº 010/2018. Cell Culture and Chromosome Preparation Mitotic cells were obtained using a short-term bone marrow extraction technique (Garnero and Gunski 2000). Initially, biological material was extracted from femurs in 10ml of RPMI 1640 medium and incubated with 0.01 ml of colchicine solution (0.05%) at 37°C for 1 h. Cells were subsequently centrifugated and incubated for 20 min in hypotonic solution (0,075 M KCL) at 37°C. Finally, the cells were fixed with methanol and acetic acid (3:1). We analyzed approximately 40 metaphases per specimen to determine the diploid number in an optical microscope (OLYMPUS DP53). For composing karyotype figures, it was used program Corel Draw12®, and the chromosomes were classified in decrescent order according to the long arm (p), short arm (q), arm radio (r) and centromeric index (i) (Guerra 1986). CBG, GTG, and RBG Banding Regions of heterochromatic blocks were analyzed by CBG-banding (Ledesma et al. 2006). After treatment in 0.2N HCl for 15 min, the slides were incubated in Barium Hydroxide (50%) for 17 min at 37°C. Structural investigations of the GTG-banding were done accord- ing to Schnedl (1971), with modifications to the immer- sion period in saline solution, which occurred for 1 min. To obtain the RBG-banding, the protocol by Popescu (2000) was replicated with a modification of the incuba- tion period in Earl buffer (pH 5.1) saturated with Na2H- PO4, which occurred for 30 min at 87°C. Subsequently, a wash step with distilled water was performed followed by immersion for 30 min in Earl buffer (pH 6.4), without the addition of NaHCO3, at 87°C. In all banding proto- cols, metaphases were stained with Giemsa (5% in 0.07 M phosphate buffer, pH 6.8). The GTG and RBG bands position were classified according to the International System of Standardized 45Comparative cytogenetics in three species of Wood-Warblers Avian Karyotypes (ISSAK). Band patterns were inter- preted by comparison among the three species of this study, and the types of rearrangements were detected with the inferences by homology in model species Gallus gallus (Ladjali et al. 1999). RESULTS Wood-Warblers analyzed in this study showed dif- ferences in karyotypes. We identified a chromosome number of 2n=76 for Myiothlypis leucoblephara, con- comitant with the described by Carvalho (1989) in a male specimen. Basileuterus culicivorus presented a dip- loid number of 2n=78, and Setophaga pitiayumi 2n=80 (Figure 1). In the three species, the karyotypes exhibited 14 pairs of autosomal macrochromosomes and 1 pair of sex chromosomes ZZ or ZW. The remaining pairs were composed of microchromosomes. Autosomal macro- chromosomes and sex chromosomes were morphomet- rically described, presenting only morphological diver- gences occurring among chromosomes 5, 6, and 7 in the three species (Table 1). CBG-banding analysis identified constitutive het- erochromatin in the centromeric regions of the macro- chromosomes and revealed the W chromosome. This chromosome was positioned between the 6th and 7th pair and showed chromatic heterogeneity in CBG-banding in the three species. It was formed by a block of hetero- chromatin in the short arm and partial in the long arm, containing a telomeric euchromatic region in the long arm (Figure 2). In all analyzed species, the Z chromo- some was euchromatic, with positive staining observed near the centromere and morphometrically positioned between the 4th and 5th pair of macrochromosomes. In this study, we describe by GTG-banding the first 10 autosomes macrochromosomes, and ZW sex chromo- somes (Figure 3). M. leucoblephara presented 137 GTG- bands distributed along the chromosomes, where the negatives integrated into terminal regions of the short and long arms of chromosomes 2, 4, 5, 6 and 7. Other chromosomes contained positive bands in their terminal regions. B. culicivorus had a total set of 139 GTG-bands, of which the negatives were also distributed in the ter- minal regions of the short and long arms of chromo- somes 1, 2, 4 and 5, and in the terminal region of the long arm of chromosomes 3, 6 and 7. Other terminal regions of chromosomes contained positive bands. S. pitiayumi showed 137 GTG-bands along their chromo- somes, with negatives forming the terminal regions of the short and long arms of the chromosomes 1, 3 and 4, and the terminal region of the long arm of chromo- somes 2, 5, 6, 7 and 8. The terminal regions of other chromosomes consisted of positive bands. The reverse pattern was identified by RBG-banding, performed with the first 10 pairs of autosomal chro- mosomes and ZW. This data was shown to be compat- ible with the results obtained by GTG-banding (Figure Figure 1. Species complete karyotype. Chromosomes arranged in descending order with Giemsa staining, followed by sexual chro- mosomes Z and W. Myiothlypis leucoblephara (A), Basileuterus culi- civorus (B), and Setophaga pitiayumi (C). 46 Alice Lemos Costa et al. 3). Homologous and non-homologous regions among the three species were identified and compared with the homologous regions of model species Gallus gallus (Lad- jali et al. 1999). In chromosome 1, a fission in region 2 of the short arm of B. culicivorus, and a paracentric inver- sion in region 1 of this same arm in S. pitiayumi were detected. In the long arm of this same chromosome, in region 4, a paracentric inversion was found in B. culi- civorus. For chromosome 3, an inversion followed by deletion in region 1 of the long arm was detected in B. culicivorus. In the 5th pair, B. culicivorus also presented a fusion in region 1 of the short arm. A break followed by pericentric inversion was found in the 6 pair of the spe- cies M. leucoblephara and S. pitiayumi. In chromosome 7, S. pitiayumi also presented a fission in region 1 of the short arm. M. leucoblephara and S. pitiayumi showed a fusion in region 1 of the long arm in chromosome 8 (Figure 4). Table 1. Measurements and morphology of autosomal macrochromosomes and sex chromosomes of the species studied. Chromo- some Myiothlypis leucoblephara   Basileuterus culicivorus   Setophaga pitiayumi Short arma Long arma Rb CIc Morpho- logyd Short arma Long arma Rb CIc Morpho- logyd Short arma Long arma Rb CIc Morpho- logyd 1 6.3 10.6 1.68 37.28 SM 6.1 11.1 1.82 35.47 SM 6.2 10.9 1.76 36.26 SM 2 4.1 9.3 2.27 30.60 SM 4.3 9.5 2.21 31.16 SM 3.9 9.6 2.46 28.89 SM 3 2.3 8.5 3.70 21.30 A 2.2 9.8 4.45 18.33 A 2.8 9.6 3.43 22.58 A 4 2.1 8.2 3.90 20.39 A 2.1 8.9 4.24 19.09 A 2.1 8.7 4.14 19.44 A 5 1.9 7.3 3.84 20.65 A 1.7 7.4 4.35 18.68 A 3.1 6.3 2.03 32.98 SM 6 1.5 6.7 9.70 23.15 A 0 9.8 9.80 9.80 T 2.1 6.5 3.10 24.42 A 7 2.1 4.7 2.24 30.88 SM 2.1 6.2 2.95 25.30 SM 1.2 5.4 4.50 18.18 A 8 0 6.3 6.30 6.30 T 0 7.2 7.20 7.20 T 0 6.3 6.30 6,30 T 9 0 5.8 5.80 5.80 T 0 6.3 6.30 6.30 T 0 6.1 6.10 6.10 T 10 0 5.3 5.30 5.30 T 0 5.9 5.90 5.90 T 0 5.7 5.70 5.70 T 11 0 4.9 4.90 4.90 T 0 5.1 5.10 5.10 T 0 5.2 5.20 5.20 T 12 0 4.1 4.10 4.10 T 0 4.5 4.50 4.50 T 0 4.8 4.80 4.80 T 13 0 3.7 3.70 3.70 T 0 3.9 3.90 3.90 T 0 4.1 4.10 4.10 T 14 0 3.5 3.50 3.50 T 0 3.6 3.60 3.60 T 0 3.6 3.60 3.60 T Z 3.2 7.1 2.22 31.07 SM 3.3 7.4 2.24 30.84 SM 3.1 7.2 2.32 30.10 SM W 1.9 4.2 2.21 31.15 SM   1.8 4.3 2.39 29.51 SM   1.5 3.9 2.60 27.78 SM aLength in micrometer (µm) q-long arm, p-short arm. bRelationship between p/q. cCentromeric index. dChromosomal morphology: T-telo- centric, A-acrocentric, SM-submetacentric. Figure 2. CBG-Banding metaphases with emphasis on the patterns of banding of sex chromosomes. Myiothlypis leucoblephara (A), Basileu- terus culicivorus (B), and Setophaga pitiayumi (C). 47Comparative cytogenetics in three species of Wood-Warblers DISCUSSION The karyotypic structure of the three analyzed spe- cies in this study is similar to the typical avian karyo- type (Figure 1 and Table 1), containing few pairs of macrochromosomes, many microchromosomes, a ZW heterogametic sexual system for females and ZZ homo- gametic for males (Christidis 1990). In the species of the family that has been previously studied, the frequency of the diploid number was within the standard, rang- ing from 76 to 80 chromosomes (Carvalho 1989; Hobart 1991). Karyotypically, the three species presented the first pair of submetacentric chromosomes, supporting the theory that Passeriformes retain this morphology among its Oscines birds (Guttenbach et al. 2003). During the evolutionary changes of this chromosome, a break fol- lowed by fusion with a microchromosome forming this biarmed chromosome has been historically suggested in Galliformes (Stock and Bunch 1982). In Passeriformes, it was shown by fluorescent in situ hybridization (FISH) results that all species studied shared a fission of GGA1 (Kretschmer et al. 2018b). CBG-banding identified a preferential accumula- tion of constitutive heterochromatin in the centromeric regions (Figure 2). The W chromosome showed a dis- tinct banding pattern identified in Passeriformes, which is generally heterochromatic (Kretschmer et al. 2018a). In all three species, this chromosome has an euchromat- ic telomeric region in the long arm. We can infer that this chromosome has an intermediate CBG-banding pat- tern, it was seen in other Orders such as Tinamiformes in the Crypturellus tataupa species, where euchro- matic and heterochromatic blocks occur simultane- ously (Garnero et al. 2006). A similar pattern occurred in Charadriiformes in the Burhinus oedicnemus species, where a euchromatic band was found in the long arm of W chromosome (Nie et al. 2009). Neognathae birds tend to have a reduction in the size of the W chromosome. Suggesting that this occurs due to loss of accumulated repetitive sequences and non-recombining regions. However, there are significant morphological differences in this chromosome, refer- ring to loss and gain, followed by the accumulation of these sequences (Furo et al. 2017). In some species such as Neochmia faeton (Passeriformes), Ardeola grayii (Pele- caniformes), Gallinula melanops (Gruiformes), Amazona aestiva (Psittaciformes), and Crotophaga ani (Cucu- liformes), this chromosome is considered the largest or one of the largest among chromosomal complement (Christidis 1989; Mohanty and Bhunya 1990; Furo et al. 2017; Gunski et al. 2019; Kretschmer et al. 2021). The number of GTG and RBG bands obtained for the species was distinct (Figure 3), collaborating with the observed diploid number. It is possible to suggest the occurrence of interchromosomal and intrachromo- Figure 3. Description of GTG and RBG banding patterns and their respective ideograms. Light bands: negative GTG and RBG positive. Dark bands: positive GTG and Negative RBG. 48 Alice Lemos Costa et al. somal rearrangements for these species, since fission, fusion, inversion, and deletion processes can be detected by banding patterns, which could be used as a reference point of the genomic organization (Ladjali et al. 1999; Nanda et al. 2011). However, we suggest that results should be analyzed in future studies by fluorescent in situ hybridization (FISH), giving additional information about this issue. Comparisons of GTG and RBG band patterns among the three species showed distinct convergences and divergences (Figure 4). The macrochromosome pairs 1, 2, and 3 have similar morphology, but the banding patterns were not the same in these chromosomes for the parulids. In this context, Takagi (1974) found the same pattern in nine other Orders of the Aves Class, for example in Strigiformes, Columbiformes, and Grui- formes. Some studies have shown that chromosomes 1, 2, and 3 are actively linked to intrachromosomal rear- rangement processes in the Passeriformes (Nanda et al. 1994). Our results indicate a sharing of number of chro- mosomal bands in M. leucoblephara and B. culicivorus species, in region 1 of the first pair in short arm com- pared to the G. gallus (Ladjali et al. 1999). S. pitiayumi has a divergent pattern in this region, where possibly a paracentric inversion has caused this differentiation. B. culicivorus has rearrangement in region 2 of the short arm, where fission occurred in the telomeric region. Also, in region 4 of the long arm, there is a higher num- ber of bands compared the other parulids, where a break followed by paracentric inversion could have caused this pattern. B. culicivorus showed a reduction in the number of bands in region 2 of the long arm in the third pair compared to the other two parulids, which have simi- larities with G. gallus (Ladjali et al. 1999) in this region. For this differentiation, a possible paracentric inversion and deletion may have occurred. In Synallaxis frontalis species, there is a pericentric inversion involving the first and third pairs (de Souza et al. 2019). Nevertheless, this diversity of rearrangements involving chromosomes 1, 2, and 3 is not restricted to Passeriformes. In Columbi- dae, the Treron phoenicoptera species has a chromosomic Figure 4. Ideograms of Parulidae with compiled data obtained by GTG and RBG bands, demonstrating the type of chromosomal rearrange- ments with divergences and convergences among bands pattern. Region of bands compared with Gallus gallus available in Ladjali et al. (1999). Chr-Chromosome. 49Comparative cytogenetics in three species of Wood-Warblers rearrangement of inversion in first and second pairs (Gupta and Kaul 2014). In parulids, chromosomes 4 and 5 showed the same number of regions, containing differences only in mor- phology the 5 pair and number of bands. In region 1 of the short arm of chromosome 5, species B. culicivorus has three bands, while M. leucoblephara and S. pitiayu- mi contain two bands. The corresponding region of the G. gallus (Ladjali et al. 1999) is similar, inferring that a possible fusion is related to the increase in bands in B. culicivorus. Passeriformes have a unique evolutionary history for the 5th chromosome pair, where it is assumed to have occurred by fission of the short arm of chro- mosome 1 in the putative ancestral karyotype (PAK) (Kretschmer et al. 2018b). Using G. gallus (GGA) probes in Passeriformes, GGA1 usually hybridize two distinct chromosome pairs, for example, in Saltator aurantiiro- stris (Thraupidae) the second and fifth pairs (dos Santos et al. 2015). The B. culicivorus chromosome 6 shows numerical conservation of positive and negative bands compared to the corresponding chromosome in G. gallus (Ladjali et al. 1999), which contains 7 bands in region 1 of the long arm. M. leucoblepharus and S. pitiayumi showed 5 bands for the same chromosome in this region. A pos- sible break followed by pericentric inversion may have occurred, resulting in the changes found in biarmed chromosome, which has 2 bands in region 1 of the short arm in the two species. This is a rearrangement type that has been previously found in Treron phoenicoptera and Synallaxis frontalis by GTG-bands (Gupta and Kaul 2014; de Souza et al. 2019). Morphology and number of bands in chromosome 7 found were similar in M. leucoblephara and B. culi- civorus, which contained the same number of bands in the corresponding chromosome of G. gallus (Ladjali et al. 1999). However, S. pitiayumi shown a reduction in the number of bands in region 1 and morphological difference in this chromosome. Possibly, a fission in the terminal region of the short arm might have caused this reduction of bands and morphological differentiation in S. pitiayumi. In this perspective, multiple fragments of sites interstitial were found in non-telomeric regions in Turdus merula (Passeriformes), implying how active these regions are in relation to chromosomal rearrange- ments (Nanda et al. 2002). In chromosome 8, region 1 of long arm, B. culi- civorus showed similar patterns of bands number the G. gallus (Ladjali et al. 1999). M. leucoblephara and S. pitiayumi had an increase in bands, thus inferring the occurrence of a fusion in the telomeric region. Neverthe- less, chromosomes 9 and 10 of the three species main- tained morphological and numerical band similarities. The difference between M. leucoblephara and S. pitiay- umi in chromosome 8 is a chromosomal rearrangement caused by fusion in the terminal region of the long arm, considering that B. culicivorus species has a similar pat- tern found in G. gallus (Ladjali et al. 1999) in the cor- respondent chromosome. The telomeric region is an area rich in repetitive sequences which have been reported as hotspots of chromosomal fusion and fission (Nanda et al. 2011). The similarities of chromosomes 9 and 10 of the three species suggest conservation. In the three species, Z chromosome presented high evolutionary stability in terms of morphology and band patterns. In many Passeriformes, the Z chromosome has the same submetacentric morphology (Kretschmer et al. 2018b). Furthermore, some studies have shown that there is a high syntenic degree of this chromosome among several families of this group (Griffin et al. 2007). It is important to emphasize that GTG and RBG bands analyses have already identified a paracentric inversion in the terminal region of the long arm of the Z chromo- some in Alectoris chukar (Galliformes) (Ouchia and Lad- jali 2018). Signals of hybridization in this chromosome also demonstrated that the accumulation of the repeti- tive sequences are responsible for the main cause of its enlargement, as in Myiopsitta monachus (Psittaciformes) (Furo et al. 2017) and Nyctibius griseus (Caprimulgi- formes) (de Souza et al. 2020). In conclusion, the cytogenetic analyses performed in this study in the three parulids species provided an accurate description of the karyotypic structuring. Through CBG, GTG, and RBG bands, the information was obtained on chromatic patterns and chromosomal rearrangements which should be analyzed by molecular cytogenetic techniques in the future. Our results support the identification of speciation processes at the karyo- typic of this group. ACKNOWLEDGEMENTS The authors would like to thank all colleagues from the Grupo de Pesquisa Diversidade Genética Animal from the Universidade Federal do Pampa and Dr. Rafael Kretschmer for comments on the manuscript. STATEMENT OF ETHICS The protocols used in t his experiment were approved by the Ethics Committee on the use of animals (CEUA – Universidade Federal do Pampa, 010/2018). 50 Alice Lemos Costa et al. FUNDING SOURCES This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Bra- sil (CAPES) – Finance Code 001. REFERENCES Carvalho MVP. 1989. Estudos Citogenéticos na Família Fringillidae (Passeriformes-Aves). UFRGS, Porto Alegre. Christidis L. 1989. Chromosomal evolution within the family Estrildidae (Aves) I. The Poephilae. Genetica 71:81–97. Christidis L. 1990. Animal Cytogenetics 4: Chordata 3 B: Aves, pp 55–57. Gebrüder Borntraeger, Berlin. Degrandi TM, Barcellos SA, Costa AL, Garnero AD, Hass I. et al. 2020. Introducing the Bird Chromosome Database: An Overview of Cytogenetic Studies in Birds. Cytogenetic and Genome Research, 160: 199- 205. de Souza MS, Kretschmer R, Barcellos S, Costa AL, Cioffi MB, et al. 2020. Repeat Sequence Mapping Shows Different W Chromosome Evolutionary Pathways in Two Caprimulgiformes Families. Birds 1:19-34. de Souza MS, Barcellos SA, Costa AL, Kretschmer R, Garnero ADV, et al. 2019. Polymorphism of Sooty- fronted Spinetail (Synallaxis frontalis Aves: Furnarii- dae): Evidence of chromosomal rearrangements by pericentric inversion in autosomal macrochromo- somes. Genetics and Molecular Biology 42:62-67. dos Santos MDS, Kretschmer R, Silva FAO, Ledesma MA, O’Brien PC, et al. 2015. Intrachromosomal rear- rangements in two representatives of the genus Salta- tor (Thraupidae, Passeriformes) and the occurrence of heteromorphic Z chromosomes. Genetica, 143: 535-543. Farre M, Narayan J, Slavov GT, Damas J, Auvil L. et al. 2016. Novel insights into chromosome evolution in birds, archosaurs, and reptiles. Genome biology and evolution, 8:2442-2451. Furo IO, Kretschmer R, dos Santos MS, Carvalho CAL, Gunski RJ, et al. 2017. Chromosomal mapping of repetitive DNAs in Myiopsitta monachus and Ama- zona aestiva (Psittaciformes, Psittacidae: Psittaci- formes), with emphasis on the sex chromosomes. Cytogenetic and Genome Research 151:151–160. Garnero AD, Ledesma MA, Gunski RJ. 2006. Alta homeologia cariotípica na família Tinamidae (Aves: Tinamiformes). Revista Brasileira de Ornitologia 14:53-58. Garnero AV, Gunski RJ. 2000. Comparative analysis of the karyotypes of Nothura maculosa and Rynchotus rufescens (Aves: Tinamidae). A case of chromosom- al polymorphism. Nucleus-Calcutta - International Journal of Cytology 43:64-70. Griffin DK, Robertson LB, Tempest HG, Skinner BM. 2007. The evolution of the avian genome as revealed by comparative molecular cytogenetics. Cytogenetic and Genome Research 117: 64–77. Guerra MS. 1986. Reviewing the chromosome nomen- clature of Levan et al. Brazilian Journal of Genetics 9:741-743. Gunski RJ, Kretschmer R, de Souza MS, de Oliveira FI, Barcellos SA, et al. 2019. Evolution of Bird Sex Chro- mosomes Narrated by Repetitive Sequences: Unu- sual W Chromosome Enlargement in Gallinula mel- anops (Aves: Gruiformes: Rallidae). Cytogenetic and Genome Research 157:01-08. Gupta N, Kaul D. 2014. G-Band Polymorphism in Natu- ral Populations of Yellow-Legged Green Pigeon, Tre- ron phoenicoptera Rallied from Northern India. Pro- ceedings of the National Academy of Sciences, India Section B: Biological Sciences, 84: 917-926. Guttenbach M, Nanda I, Feichtinger W, Masabanda JS, Griffin DK, et al. 2003. Comparative chromo- some painting of chicken autosomal paints 1–9 in nine different bird species. Cytogenetic and Genome Research, 103: 173-184. Hobart HH. 1991. Comparative karyology in nine-prima- ried oscines (Aves). UMI, Arizona. Kretschmer R, Ferguson-Smith M, de Oliveira E: Karyo- type Evolution in birds. 2018b. From conventional staining to chromosome painting. Genes 9:181. Kretschmer R, Gunski RJ, Garnero ADV, de Freitas TRO, Toma GA, et al. 2021. Chromosomal Analysis in Cro- tophaga ani (Aves, Cuculiformes) Reveals Extensive Genomic Reorganization and an Unusual Z-Auto- some Robertsonian Translocation. Cells, 10: 4-17. Kretschmer, R, de Lima VLC, de Souza MS, Costa AL, O’Brien PC, et al 2018a. Multidirectional chromo- some painting in Synallaxis frontalis (Passeriformes, Furnariidae) reveals high chromosomal reorganiza- tion, involving fissions and inversions. Comparative cytogenetics, 12: 97-110. Ladjali-Mohammedi K, Bitgood JJ, Tixier-Boichard M, De Leon FP. 1999. International system for standard- ized avian karyotypes (ISSAK): standardized banded karyotypes of the domestic fowl (Gallus domesticus). Cytogenetic and Genome Research, 86: 271-276. Ledesma MA, Martinez PA, Calderón OS, Boeris JM, Meriles JM. 2006. Descrição do cariótipo e padrões de bandas C e NOR em Pheucticus aureoventris 51Comparative cytogenetics in three species of Wood-Warblers (Emberizidae: Cardinalinae). Revista Brasileira de Ornitologia 14:59-62. Mohanty MK, Bhunya SP. 1990. Karyological studies in four species of ardeid birds (Ardeldae, Ciconii- formes). Genetica 81:211-214. Nanda I, Benisch P, Fetting D, Haaf T, Schmid M. 2011. Synteny Conservation of Chicken Macrochromo- somes 1–10 in Different Avian Lineages Revealed by Cross-Species Chromosome Painting. Cytogenetic and Genome Research, 132:165–181. Nanda I, Schmid M. 1994. Localization of the telomeric (TTAGGG)n sequence in chicken (Gallus domesticus) chromosomes. Cytogenet Cell Gene 65:190–193. Nanda I, Schrama D, Feichtinger W, Haaf T, Schartl M, et al. 2002. Distribution of telomeric (TTAGGG) n sequences in avian chromosomes. Chromosoma, 111: 215-227. Nascimento JAD, Carvalho FIFD, Barbosa NJF, Federizzi LC. 1994. Agentes mutagênicos e a intensidade de variabilidade genética no caráter estatura de plantas de aveia (Avena sativa L.). Ciência Rural 24:291-297. Nie W, O’Brien PC, Ng BL, Fu B, Volobouev V, et al. 2009. Avian comparative genomics: reciprocal chro- mosome painting between domestic chicken (Gallus gallus) and the stone curlew (Burhinus oedicnemus, Charadriiformes) - An atypical species with low dip- loid number. Chromosome research, 17:99-113. Nishida-Umehara C, Tsuda Y, Ishijima J, Ando J, Fuji- wara A. 2007. The molecular basis of chromosome orthologies and sex chromosomal differentiation in palaeognathous birds. Chromosome Research 15:721-734. Ouchia SB, Ladjali KM. 2018. Banding cytogenetics of the Barbary partridge Alectoris barbara and the Chu- kar partridge Alectoris chukar (Phasianidae): a large conservation with Domestic fowl Gallus domesticus revealed by high resolution chromosomes. Compara- tive Cytogenetics 12:171. Popescu P, Hayes H. 2000. Techniques in animal cytoge- netics. INRA, France. Schnedl W. 1971. Analysis of the human karyotype using a reassociation technique. Chromosoma 34:448-454. Stock A D, Bunch TD. 1982. The evolutionary implica- tions of chromosome banding pattern homologies in the bird order Galliformes. Cytogenetic and Genome Research 34:136–148. Takagi N, Sasaki M. 1974. A phylogenetic study of bird karyotypes. Chromosoma 46:91-120.