Comparative chromosome painting in Spizaetus tyrannus and Gallus gallus with the use of macro- and microchromosome probes

Although most birds show karyotypes with diploid number (2n) around 80, with few macrochromosomes and many microchromosomes pairs, some groups, such as the Accipitriformes, are characterized by a large karyotypic reorganization, which resulted in complements with low diploid numbers, and a smaller number of microchromosomal pairs when compared to other birds. Among Accipitriformes, the Accipitridae family is the most diverse and includes, among other subfamilies, the subfamily Aquilinae, composed of medium to large sized species. The Black-Hawk-Eagle (Spizaetus tyrannus-STY), found in South America, is a member of this subfamily. Available chromosome data for this species includes only conventional staining. Hence, in order to provide additional information on karyotype evolution process within this group, we performed comparative chromosome painting between S. tyrannus and Gallus gallus (GGA). Our results revealed that at least 29 fission-fusion events occurred in the STY karyotype, based on homology with GGA. Fissions occurred mainly in syntenic groups homologous to GGA1-GGA5. On the other hand, the majority of the microchromosomes were found fused to other chromosomal elements in STY, indicating these rearrangements played an important role in the reduction of the 2n to 68. Comparison with hybridization pattern of the Japanese-Mountain-Eagle (Nisaetus nipalensis orientalis), the only Aquilinae analyzed by comparative chromosome painting previously, did not reveal any synapomorphy that could represent a chromosome signature to this subfamily. Therefore, conclusions about karyotype evolution in Aquilinae require additional painting studies.

Introduction 7-Page3 line3: Present an unusual (delete with) A-We deleted it. 8-Page 3 line 12: The accurate identification of the chromosomal pairs involved.… if we aim to identify synapomophies…. A-We corrected it 9-Introduction miss a clear motivation for the study. A-We improved it: "Among them, the subfamily Aquilinae includes medium and large species, distributed globally, usually known as booted eagle. Usually, ten genera are found within Aquilinae. Cytogenetically, the only information concerning Aquilinae is the definition of the diploid number of six species (four genera), ranging from 2n=66 to 82 [19]". 10-Why using GGA probes? A-Because this species is an important biological model, and because it also retained a plesiomorphic karyotype, GGA probes are used as a standard for chromosomal studies in birds. We added a small explanation in the introduction. 11-I missed some info (Figure would be better) about the phylogenetic position and relation between Chicken and Spizaetus. A-The authors agree that for this study are not necessary introduce some information about the phylogenetic position between Gallus gallus and S. tyrannus, however short information of phylogenetic relationship of the S.tyrannus within the Aquilinae Subfamily were added in the introduction aiming improve the reading of this paper. 12-Moreover, inform here as well thar STY corresponds to Spizaetus tyrannus on its first mention. A-This was corrected. Results 13-Subtitle Karyotypes before the first paragraph. A-We added it. Reviewer #2: In this study, the authors used comparative chromosome painting to reveal homology of chromosomal segments between Spizaetus tyrannus (the Black-Hawk-Eagle) and Gallus gallus. The study is conceptually and methodically motivated. The karyotype of S. tyrannus was previously studied only using conventional chromosome staining technique that showed 2n=68 (32m/sm+8st+18a+8m+ZW), and this happened for the first time that the karyotype of this bird of prey was studied by comparative chromosome painting. As a result, the study provides a novel insight into the cytogenetics of the Black-Hawk-Eagle. Both whole chromosome-specific G. gallus probes of the 1st-10th pairs and chromosome-specific G. gallus BAC probes from 11 pairs of microchromosomes were used. The study evidenced 29 evolutionary fissionfusion events that happened in the evolution of S. tyrannus and identified the particular chromosome pairs in the referenced G. gallus karyotype, which have undergone restructuring or have remained unchanged. Another sufficient result concerns the comparison between S. tyrannus and Nisaetus nipalensis orientalis, which is its only close relative studied so far by comparative chromosome painting. The comparison showed both similarities and differences between the species. The MS is well illustrated by tables and pictures of good quality. In general, the work is interesting and deserves publication in the journal. However, there are some shortcomings in the work. Main disadvantages are (1) almost complete absence of basic data on the karyotypes of the discussed species, which makes it difficult to adequately assess the results obtained, and (2) ignorance of the taxonomic component. A-We try to improve it.
All other comments that I have are mainly suggestions for improving the article (unfortunately, there is no line numbering in the MS, which makes it difficult for the reviewer to work).
3) Introduction and discussion: The manuscript is written in a manner suitable for a more specialized journal such as Cytogenetic and Genome Research or Comparative Cytogenetics, but not ideally for a general journal such as PlosOne. The introduction and discussion are rather limited to the focus of the analyses. This is a shame as I think the authors did a good job to gather nice cytogenetic results. Thus, the authors should consider redrafting these sections on a broad context.
The introduction could be more informative about the study as a whole. For example, the authors can give information about the contribution of this kind of work to karyotypic evolution and chromosomal organization of Accipitriformes. In this line, the last paragraph of introduction can be better written showing the importance of this study, and not merely comment "This study presents the cytogenetic mapping of a species…". Moreover, other intriguing topic is about the origin of microchromosomes. And maybe the authors could comment a little bit about this topic in the introduction and also discussion. Below is a reference related to this topic. A-The introduction was rewritten and several new information were added to improve the understanding of the manuscript. Also, some information from the reference recommended by the reviewer were considerate in this topic. 4) Results, paragraph 1: The authors commented that they detected "four pairs of microchromosomes" but they indicated five (29, 30, 31, 32, and 33). Considering the Figure 1B the pair 29 apparently is not a microchromosome.
A-We reviewed the Karyotype of S.tyrannus and considered only four pairs of microchromosomes ( Pairs: 30, 31, 32 and 33). Also, we corrected it throughout of the text. 5) Results, paragraph 2: "The most extreme examples are the fission of GGA1 into six pairs in STY, and GGA3 into three distinct pairs". It seems that for GGA3 are four pairs (13, 16, 19, and 20) (Figure 4), right?
A-We corrected it. 8) Discussion, paragraph 5: "These results show that they are morphologically similar species that until the last decade were part of the same genus [14].". I think the authors results did not show an association of chromosomal data with morphological similarity and this should be corrected.
A-We corrected it. 10) Methods, paragraph 1: What the name and country of the Zoos?
A-The information was added in the Methods. 11) Methods, paragraph 2: "…and labelled directly by FTIC"; "and labelled directly by fluorescein isothiocyanate (FITC)".
A-We changed it. 12) Methods: the results of this study are based on FISH with GGA probes. However, FISH procedure is not sufficiently described. I would appreciate if at least main/important steps of the FISH procedure are described. This would also avoid any doubt about the accuracy of the results.
A-We provided the main steps of the FISH procedure. 13) Figure 3: "FITC". A-We corrected it.

Introduction
Usually, bird genome is organized in karyotypes consisting of few macrochromosomes and many tiny microchromosomes [1]. However, there are some exceptions. For instance, excluding the New World vultures (Cathartidae), which show similar karyotypes to the putative avian ancestral karyotype (PAK) with diploid number around 2n= 80, including 10 pairs of macrochromosomes and 30 pairs of microchromosomes [1], species belonging to the Order Accipitriformes present an interesting chromosomal diversity. They have lower diploid numbers, 2n = 54-68, and a reduction of microchromosomes to between 4 and 8 pairs, due mainly to fusions involving these small elements, occurred during their divergence [2][3][4].
In general, studies focusing on chromosome evolution in birds are based in comparative chromosome painting using chicken whole chromosome probes (Gallus gallus -GGA, 2n=78), due to the similarity of the karyotype of this species with the PAK [5]. The use of this methodology in species of birds of prey has revealed that, despite the lower diploid numbers observed in this group, the large karyotype reorganization in Accipitriformes included multiple fissions in the macrochromosome pairs homologous to GGA1-GGA5. The reduction of the chromosome number would be due to the concomitant occurrence of several fusion events involving microchromosomes [6][7][8][9][10][11].
Microchromosomes are gene rich elements, and genome comparative analyses have shown their conservation as syntenic groups among distantly related bird groups [12][13]. In fact, rearrangements involving microchromosomes were detected in few orders: Psittaciformes, Cuculiformes, Suliformes, Caprimulgiformes and the Accipitriformes [13][14][15]. Due to difficulties of the isolation of individual microchromosome pairs by flow cytometry for specific probe production, most data concerning microchromosomes were obtained by the use of pools of microchromosomes, 4 i.e., chromosome paints that recognize more than one pair. Therefore, improved identification of chromosome pairs involved in rearrangements is a priority if we are to achieve a more definitive analysis and identify synapomorphies based on chromosome characters [16][17].
Currently, the order Accipitriformes is composed of four families, of which Accipitridae is the most diverse, with approximately 230 species distributed in 14 subfamilies [18]. Among them, the subfamily Aquilinae includes medium and large species, distributed globally, usually known as booted eagle. Usually, ten genera are found within Aquilinae. Cytogenetically, the only information concerning Aquilinae is the definition of the diploid number of six species (four genera), ranging from 2n=66 to 82 [19].
The Black-Hawk-Eagle (Spizaetus tyrannus-STY) is a representative of this subfamily, found in South and Central Americas, from southern Mexico down to Argentina [18]. Considering that the only chromosomal analysis of S. tyrannus to date was based on conventional staining, revealing a karyotype within the Aquilinae standard, with 2n=68 [1], the aim of this study was to present the cytogenetic mapping of S. tyrannus by comparative painting. In addition to whole-chromosome paints of Gallus gallus (GGA), we used BAC probes from GGA clones that identified 11 individual pairs of microchromosomes. The results were compared to Nisaetus nipalensis orientalis-NNI (2n=66) [10], also from the subfamily Aquilinae, in order to identify chromosomal rearrangements related to karyotype evolution in this group. 5

Discussion
The karyotype of S. tyrannus obtained herein presented 2n=68, confirming data from a previous report [1]. We report slight differences in chromosome morphology however, due to the higher number of biarmed pairs ( Table 1).
The results of comparative chromosome painting with whole chromosome probes of G. gallus showed a similar pattern to other birds of prey in the family Accipitridae, with a large reorganization of the syntenic groups homologous to the first five pairs of G.
gallus. That is, each probe (GGA1 -GGA5) corresponded to at least two distinct pairs The closest subspecies to Spizaetus tyrannus with chromosome painting data is the Japanese-Eagle (Nisaetus nipalensis orientalis), with 2n=66 [10]. Although geographically separated, they are morphologically similar, and until the last decade were classified as part of the same genus. Despite now being separated into distinct genera, molecular data support their close phylogenetic relationship [20]. Nevertheless, the comparative chromosome painting detects many differences. For instance, GGA1-9

Conclusion
The present work is the first comparative chromosome mapping of a species in the genus S. tyrannus and has revealed substantial karyotypic reorganization common to birds of prey of the family Accipitridae. Together with G. gallus chromosome-specific probes for the larger pairs, chicken BACs were able to provide a more comprehensive result with additional information on the organization of the S. tyrannus karyotype. There are many similarities with the N. nipalensis orientalis, including numerous fissions of the first five pairs homologous to GGA with only one less in STY (21 events against 22 in NNI), and three fusions involving homologues of GGA1-GGA9 chromosomes and microchromosomes, but with breakpoints that are not shared between these two species.
For a broader analysis at the phylogenetic level, it would be necessary to have comparative mapping of other species of the genus Spizaetus so that an ancestral karyotype of this genus could be traced.

Samples and Chromosome Preparations
The experiments followed the standards approved by the Ethics Committee for Karyotype analysis was performed using conventional staining with 5% Giemsa in 0.07 M phosphate buffer (pH 6.8) for 5 minutes, slides were analyzed using a 100× objective (Leica, CO, USA) and GenASIs software (ADS Biotec, Omaha, NE, USA).

GGA Probes and FISH Experiments
Two types of Gallus gallus probes were used: whole-chromosome-specific probes of the first 10 pairs, and bacterial artificial chromosomes (BACs) probes from 11 microchromosome pairs. Whole chromosome paints were developed and provided by the    in fusion events .
The mMicrochromosomes in birds karyotype are gene rich elements, and and highly conserved, some genome comparative analysies have shown considerabletheir conservation as syntenic groups among distantly related bird groups [12][13]. In fact, rearrangements involving microchromosomes were detected only in in fewsome order: , such as Psittaciformes, Cuculiformes and the Accipitriformes were detected rearrangements involving these elements [13][14][15].
Due to difficulties involving of the isolation of individual microchromosome pairs by flow cytometry for specific probe production, most data concerning microchromosomes were obtained have been obtained usingby the use of pools of microchromosomes, i.e., chromosome paints that recognize more than one pair.
Currently, the order Accipitriformes is composed of four families, of which the Accipitridae family beingis the most diverse, with approximately 230 species distributed in 14 subfamilies in quantity of species, within this family is the subfamily Aquilinae [18]. Among them, the subfamily Aquilinae includes medium and large species, distributed globally, usually known as booted eagle. Usually, ten genera are found within whole-chromosome paints of Gallus gallus (GGA), we used BAC probes from GGA clones that identified 11 individual pairs of microchromosomes. The results were compared to Nisaetus nipalensis orientalis-NNI (2n=66) [10], also from the subfamily Aquilinae, in order do identify chromosomal rearrangements related to karyotype evolution in this group.
Thus,is study aimed understanding the karyotype evolution within of the subfamily Aquilinae, we presenteds the cytogenetic mapping of S. tyrannusa species of the genus Spizaetus by comparative painting, we also compared this species to the Nisaetus nipalensis orientalis-NNI (2n=66) [10], the only close relative. In addition Additionally, to whole-chromosome paints of Gallus gallus (GGA), we used bacterial artificial chromosomes (BACs) BAC probes from GGA G. gallus clones that identified corresponded to 11 individual pairs of microchromosomes were used aiming identify possible rearrangements that could help to understand the role of microchromosomes in the karyotype evolution of S. tyrannus..

Karyotype description
The karyotype of Spizaetus tyrannus presenteds a diploid number 2n=68, submetacentric, similar in size to pairs 8 or 9 (Figure 1). In table 1, we We reported at this study some differences in chromosome morphology of S.tyrannus described by Tagliarini et al., [1] (Table 1 ).
All Chicken BACs and their respective homology in the karyotype of Spizaetus S.
tyrannus are summarized in Table 32.

GGA BAC ID
Homologies obtained both by whole chromosome painting and BAC probes are shown in figure 4.

Discussion
The karyotype of Spizaetus S. tyrannus obtained herein presented 2n=68, confirming data from a previous report [1]. We report slight differences in chromosome morphology however, due to the higher number of biarmed pairs (Table 1).
Formatted Table   Formatted Table   Formatted Table   Formatted Table   Formatted Table  The results [109]. These results show that they are morphologically similar species that until the last decade were part of the same genus [14].
Moreover, despite STY and NNI presenting some karyotypic similarities common to diurnal birds of prey such as recurrent breakpoints mainly in relation to the GGA1-GGA5 pairs [109,110], we did not identify any synapomorphic associations which could represent ancestral characteristics for the Aquilinae [15,. Hence, while other subfamilies, such as Buteoninae and Harpiinae present well-established chromosomal signatures that allow the elaboration of their putative ancestral karyotypes [76], the available chromosome data indicate an absence of chromosomal signatures between STY and NNI, which can be explained by their significant geographic isolation, inhabiting opposite regions in the globe.

Samples and Chromosome Preparations
The experiments followed the standards approved by the Ethics Committee for

GGA Probes and FISH Experiments
Two types of Gallus gallus probes were used: whole-chromosome-specific probes of the first 10 pairs, and chromosome-specific bacterial artificial chromosomes            Dear Reviewers,

Figure Legends
We are thankful for the constructive reviews that we received; they certainly helped us to improve the manuscript. Please find below our responses to each of your comments.
Please find below our responses to each of your comments.