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

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 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, approximately = 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 [2-4].

In general, studies focusing on chromosome evolution in birds are based on 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 [6-11].

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: Accipitriformes, Caprimulgiformes, Cuculiformes, Psittaciformes, and the Suliformes [13-15]. 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, 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.

Results

Karyotype description

The karyotype of Spizaetus tyrannus presented 2n = 68, consisting of 21 meta-submetacentric pairs (pairs 1–4, 6–7, 9–10, 12, 14, 16–17, 19–22, 24–29 and the sex chromosomes, Z and W), seven acrocentric (pairs 5, 8, 11, 13, 15, 18 and 23), and four pairs of microchromosomes (pairs 30–33). The Z chromosome is a large metacentric, with size between pairs 3 or 4, while the W chromosome is an average submetacentric, similar in size to pairs 8 or 9 (Fig 1). In Table 1, we reported some differences in chromosome morphology of S. tyrannus described by Tagliarini et al., [1].

Fig 1

Metaphase (A) and karyotype (B) of S. tyrannus with 2n = 68, obtained with Giemsa conventional staining.

The red arrows in (A) indicate the sex chromosomes. Scale bar: 5μm.

Table 1

Karyotype of S. tyrannus described by Tagliarini et al. [1] and at this study.

Pairs	This study	[1]	Pairs	This study	[1]
Chr 1.	SM	SM	Chr 18.	AC	AC
Chr 2.	SM	SM	Chr 19.	SM	SM
Chr 3.	SM	SM	Chr 20.	SM	SM
Chr 4.	SM	SM	Chr 21.	SM	SM
Chr 5.	AC	SM	Chr 22.	SM	SM
Chr 6.	SM	ST	Chr 23.	AC	AC
Chr 7.	SM	SM	Chr 24.	SM	AC
Chr 8.	AC	ST	Chr 25.	SM	AC
Chr 9.	SM	SM	Chr 26.	SM	AC
Chr 10.	SM	ST	Chr 27.	SM	SM
Chr 11.	AC	SM	Chr 28.	SM	AC
Chr 12.	SM	SM	Chr 29.	SM	SM
Chr 13.	AC	SM	Chr 30.	Micro	Micro
Chr 14.	SM	AC	Chr 31.	Micro	Micro
Chr 15.	AC	AC	Chr 32.	Micro	Micro
Chr 16.	SM	ST	Chr 33.	Micro	Micro
Chr 17.	SM	ST	Chr ZW.	M and SM	M and SM

(Metacentric: M; Submetacentric: SM; Subtelocentric: ST; Acrocentric: AC).

Comparative chromosome painting

Gallus gallus probes used in the fluorescent in situ hybridization (FISH) experiments produced reproducible results. Hybridizations with chromosome-specific probes for the first ten pairs of GGA produced 22 signals, with the first five pairs producing multiple signals, ranging from 2 to 6 number (Fig 2). For instance, GGA1 probe painted six distinct pairs in the S. tyrannus karyotype (pairs 5, 6, 12, 14, 18, and 25), while the probes GGA6-10 pairs showed only one signal each. Table 2 details the distribution of the signals produced by GGA whole specific probes in the karyotype of S. tyrannus.

Fig 2

Representative results of FISH experiments using G. gallus chromosome-specific probes corresponding to pairs GGA1 to GGA5 in S. tyrannus karyotype.

Red and green signals represent probes labelled with Cy3 or FITC, respectively. Scale bar: 5μm.

Table 2

Results of hybridizations with G. gallus probes showing the homology between GGA probes in the karyotype of S. tyrannus (STY).

Probes	STY Chromosomes	Probes	STY Chromosomes
GGA1	(5, 6, 12, 14, 18, 25)	GGA6	9
GGA2	(1, 3q, 21)	GGA7	8
GGA3	(13, 16q, 19, 20)	GGA8	7
GGA4	(2, 17)	GGA9	11q
GGA5	(4, 15q)	GGA10	10q

(q = long arm).

A total of 19 out of 22 G. gallus BAC clones produced results. Both BACs from the GGA22 chromosome did not produce any detectable signal, as well as one of the BACs from GGA21. Among the 19 probes that gave good quality results, both proximal (BACp) and distal (BACd) referring to 8 pairs, were found in the same segment in the STY karyotype. However, BACs corresponding to GGA17 hybridized to two different pairs—BAC17p marked STY 9q, while BAC17d marked STY 24q. (Fig 3). All Chicken BACs and their respective homology in the karyotype of S. tyrannus are summarized in Table 3.

Fig 3

Representative results of hybridizations with some G. gallus BACs probes in the karyotype of S. tyrannus. (A1 and A1.1) chicken BAC17 was the only one to hybridize to different chromosomes.

Red signals represent probes labeled with Cy3, corresponding to the proximal region (px); Green signals represent probes labeled with FITC, corresponding to the distal region (d). Arrows indicate the signals. Scale bar: 5μm.

Table 3

Summary of the results of experiments using GGA BACs on the karyotype of S. tyrannus (* = BACs marking the same segment in STY karyotype; px = proximal region; d = distal region; p = short arm; q = long arm).

GGA	BAC ID	STY	GGA	BAC ID	STY
17px	CH261-113A7	9q	23d	CH261-90K11	23p*
17d	CH261-42P16	24q	24px	CH261-103F4	15p*
18px	CH261-60N6	19p*	24d	CH261-65O4	15p*
18d	CH261-72B18	19p*	25px	CH261-59C21	20p*
19px	CH261-10F1	13p*	25d	CH261-127K7	20p*
19d	CH261-50H12	13p*	26px	CH261-186M13	27p*
21px	CH261-83I20	No signal	26d	CH261-170L23	27p*
21d	CH261-122K8	4p	27px	CH261-66M16	16p*
22px	CH261-40J9	No signal	27d	CH261-28L10	16p*
22d	CH261-18G17	No signal	28px	CH261-64A15	11p*
23px	CH261-191G17	23p*	28d	CH261-72A10	11p*

Homologies obtained both by whole chromosome painting and BAC probes are shown in Fig 4.

Fig 4

Idiogram representing the homology between the S. tyrannus chromosomes and the macrochromosome chromosome-specific probes and microchromosomes BAC clones from G. gallus.

Empty boxes mean no signal detected in those chromosomes with the set of probes used. BACs corresponding to pairs 20 and 22 (*) was not used or did not produce any detectable signals, respectively.

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 (Fig 3). The most extreme examples are the fission of GGA1 into six pairs in STY, and GGA3 into four distinct pairs. These results are congruent with other birds of prey, considering that GGA1 can reveal syntenic segments in four pairs (Gypaetus barbatus, 2n = 60) to seven pairs (Nisaetus nipalensis orientalis—NNI), while GGA3 is hybridized to four pairs in all species analyzed in this family. The exception is NNI where it hybridizes to only 2 pairs [11]. On the other hand, GGA6—GGA10 are conserved syntenies, with only one signal for each pair.

All associations observed in the karyotype of STY based in its homology with G. gallus are represented in Fig 4. In general, 16 fissions and 13 fusions were detected, totalizing 29 rearrangements in the karyotype of STY when compared to G. gallus, with fissions occurring mainly in relation to the first five pairs of macrochromosomes and fusions involving mainly the microchromosomes. In the microchromosomes, chicken BAC probes showed that their syntenies were not disrupted by fission events as probes for proximal and distal regions were found hybridizing to the same pair in STY, except for GGA17, which produced signals in STY9 and STY24. However, all the identified BAC signals showed that each GGA microchromosome was fused to a STY segment homologous to either a GGA macro or microchromosome. This indicates that chromosomal fusions played an important role in reducing the diploid number in STY and other Accipitriformes. It is important to note that not all GGA microchromosomes are represented by chicken BACs, and hence other fusions must have occurred in this species to maintain 2n = 68.

The closest subspecies to Spizaetus tyrannus with chromosome painting data is the NNI, 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 probes produced signals in 21 pairs in STY, and 22 in NNI; the difference was due to an extra fission of GGA1 in NNI. Despite both species presenting three fusions involving the first nine pairs with microchromosomes (STY: pairs 4, 7 and 9; NNI: pairs 2, 4 and 9), none of them share the same GGA syntenic groups, and microchromosomes involved in NNI were not identified. Additionally, in both species GGA3 hybridizes to 4 pairs, however in STY all these segments are fused with microchromosomes (GGA: pairs 18, 19, 24 and 25), whereas in NNI only one segment of GGA3 is fused with a microchromosome (unidentified pair) [10].

Regarding the phylogenetic relationship of Aquilinae with other subfamilies within Accipitridae, although STY and NNI present some karyotypic similarities common to diurnal birds of prey, such as recurrent breakpoints mainly in relation to the GGA1-GGA5 pairs [10, 11], we did not identify any synapomorphic associations which could represent ancestral characteristics for the Aquilinae [21, 22]. Hence, while other subfamilies, such as Buteoninae and Harpiinae present well-established chromosomal signatures that allow the elaboration of their putative ancestral karyotypes [7], the available chromosome data indicate the absence of chromosomal signatures between STY and NNI, which can be explained by their significant geographic isolation, inhabiting opposite regions in the globe.

Conclusion

The present work is the first comparative chromosome mapping of a species in the genus Spizaetus, 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 suggested.

Methods

Samples and chromosome preparations

The experiments followed the standards approved by the Ethics Committee for the Use of Animals in Research (CEPAE-UFPA under number 170–13). We performed fibroblast cell cultures from skin biopsies and feather pulp of Spizaetus tyrannus (STY) obtained from two female individuals maintained in Zoos (Criadouro Gavião Real, Capitão Poço, Brazil), following the protocol of Sasaki et al. [23] with modifications. After tissue cleavage in Petri dishes, the samples were incubated with 1% type 1 collagenase (GIBCO) for 1 hour at 37°C for tissue dissociation. Metaphase chromosomes were obtained after incubation for one hour with Colcemid (0.05 μg/mL), hypotonic solution (KCl at 0.075 M) for 20 minutes and fixation in methanol/acetic acid (3:1). 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 Cambridge Resource Center for Comparative Genomics (Cambridge, UK) using the Fluorescent Activated Cell Sorting (FACS) technique and labeled with biotin, fluorescein and/or digoxigenin (Roche Diagnostics, Mannheim, Germany), and detected with the addition of avidin-Cy3 (or Cy5) or anti-digoxigenin-rhodamin (Vector Laboratories, Burlingame, CA, USA). BAC clones ranged from 150,000 kb to 210,000 kb in size were selected from the CHORI-261 Chicken BAC library (Children’s Hospital Oakland Research Institute, Oakland-USA), corresponding to sequences from the proximal and distal regions of the microchromosomes (each pair represented by two BACs, in a total of 22 BACs covering pairs 17 to 28, except for pair 20). Clones were produced following the protocol of the mini prep kit (Qiagen, Hilden, Germany) and labelled directly by fluorescein isothiocyanate (FITC) (green) or Texas Red (red) through Nick Translation (Roche, Mannheim, Germany).

Hybridization experiments followed standard procedures [7, 12]. Probes (1 μL labelled probe in 14 μL hybridization buffer) were denatured at 70ºC for 10 min and preannealed for 30 min at 37ºC. Hybridization mix was added on slides with chromosome preparations previously denatured at 70% formamide for 1 min and 20 s and dehydrated by serial ethanol dehydration (70%, 90% and 100%). Detection was performed using Avidin-Cy5 or anti-digoxygenin (Vector Laboratories, Burlingame, CA, USA). Slides were analyzed with an Olympus BX-61 epifluorescence microscope equipped with a cooled CCD camera and appropriate filters. Images were captured using SmartCapture3 (Digital Scientific UK).

19 Aug 2021

PONE-D-21-20639

Comparative Chromosome Painting in the Black-Hawk-Eagle (Spizaetus tyrannus) and Gallus gallus with the use of Macro and Microchromosome Probes.

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Reviewer #1: The work from Carvalho et al brings and interesting analysis in the in the Black-Hawk-Eagle (Spizaetus tyrannus) using BAC and WCP experiments, both derived from Gallus gallus. The results are very well represented in high-quality figures and the and brings new pieces of information for the big puzzle that avian karyotype evolution represents. Below I indicate some points to be adjusted and put special attention to one suggestion for discussion: The work would profit too much if the analyses were done under a phylogenetic context. An updated phylogeny with the special focus on the phylogenetic position and relation between Chicken and Spizaetus would bring more light to the general results found.

Abstract

I missed some introduction about BIRDs cytogenetic in the beginning of Abstract. The authors go too direct about Accipitriformes and most readers do not have such phylogenetic information to which group we are dealing with.

Why are the authors aiming to investigate homologies with chicken? (Please see comments below). Abstract also brings some previous data on NNI that were not originated from this work. I suggest removing. The terms STY and NNI are used without any previous information/explanation about, causing misunderstandings.

A final conclusion is missing in the abstract.

Introduction

Page3 line3: Present an unusual (delete with)

Page 3 line 12: The accurate identification of the chromosomal pairs involved.… if we aim to identify synapomophies….

Introduction miss a clear motivation for the study. Why using GGA probes? I missed some info (Figure would be better) about the phylogenetic position and relation between Chicken and Spizaetus. Moreover, inform here as well thar STY corresponds to Spizaetus tyrannus on its first mention.

Results

Subtitle Karyotypes before the first paragraph

Figures

Figure 3: I did not understand why using the metaphases present in right column? The signals and chromosomal morphology are clearly visible in the FISH images.

Scale bars are missing in all figures.

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 fission-fusion 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.

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).

1. Running title. It is too long, should be shorter, e.g., “Comparative Chromosome Painting in Spizaetus tyrannus and Gallus gallus“.

2. Abstract. Please, decipher GGA, should be Gallus gallus (GGA), as done above for Spizaetus tyrannus (STY)

3. Introduction.

Page 3. Paragraph 1. Please, provide a putative avian ancestral karyotype, with 2n and the number of microchromosomes.

Page 3. Paragraph 1. Here (or further, in Discussion), provide the G. gallus karyotype to make further reasoning clearer.

Page 3. Paragraph 3. Unify way of citing – in other places you use numbering.

Page 4. Paragraph 4. Please, specify the species studied, Spizaetus tyrannus

4. Discussion.

Page 6, Paragraph 1. Please, expand the statement “We report slight differences in chromosome morphology….” by clarifying what Tagliarini et al. (2007) have reported. Describe the differences, give for comparison one and the other karyotypes (otherwise, you only have a declaration).

Page 7. Paragraph 3. The Japanese mountain hawk-eagle Nisaetus nipalensis orientalis (Temminck & Schlegel, 1844), not the Hodgson's hawk-eagle Nisaetus nipalensis Hodgson, 1836.

Other remarks can be found in the MS attached.

Reviewer #3: This study provides a cytogenetic mapping of the Black-Hawk-Eagle (Spizaetus tyrannus) using whole-chromosome paints and BAC probes of Gallus gallus. Using this cytogenetic mapping, the authors investigate the chromosome homologies between Spizaetus tyrannus and Gallus gallus. Overall the manuscript presents an advance in cytogenetic of Accipitriformes. The manuscript is written in understandable English, but some procedures require more details. Please see below my

comments that could help to further enhance the quality of the study.

1) This version is without line numbers to make it easier for reviewers to comment on the text.

2) Abstract: “chicken (Gallus gallus – GGA)”.

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.

- Waters, P. D., Patel, H. R., Ruíz-Herrera, A., Álvarez-González, L., Lister, N. C., Simakov, O., ... & Graves, J. A. M. (2021). Microchromosomes are building blocks of bird, reptile and mammal chromosomes. bioRxiv.

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.

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?

6) Discussion, paragraph 3: “…karyotype of STY when compared to Gallus”; “…karyotype of STY when compared to Gallus gallus”.

7) Discussion, paragraph 4: “…microchromosomes (STY4, 7 and 9; NNI2, 4 and 9), none of them”; “microchromosomes (STY: pairs 4, 7 and 9; NNI: pairs 2, 4 and 9), none of them”.

“…with microchromosomes (GGA: pairs 18, 19, 24 and 25)”.

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.

9) Conclusion: “…of S.tyrannus should be…”; “…of S. tyrannus should be…”.

10) Methods, paragraph 1: What the name and country of the Zoos?

11) Methods, paragraph 2: “…and labelled directly by FTIC”; “and labelled directly by fluorescein isothiocyanate (FITC)”.

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.

13) Figure 3: “FITC”.

14) Please, give a scale bar information for all figures. It is very important, specially considering the presence of macrochromosomes and microchromosomes.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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Submitted filename: PONE-D-21-20639_reviewer.pdf

Click here for additional data file.

17 Sep 2021

Dear Editor and reviewer

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.

Sincerely,

Reviewer #1:

1- The work from Carvalho et al brings and interesting analysis in the in the Black-Hawk-Eagle (Spizaetus tyrannus) using BAC and WCP experiments, both derived from Gallus gallus. The results are very well represented in high-quality figures and the and brings new pieces of information for the big puzzle that avian karyotype evolution represents. Below I indicate some points to be adjusted and put special attention to one suggestion for discussion: The work would profit too much if the analyses were done under a phylogenetic context. An updated phylogeny with the special focus on the phylogenetic position and relation between Chicken and Spizaetus would bring more light to the general results found.

A- From the cytotaxonomic point of view, Gallus gallus has a basal karyotype, very similar to the avian putative ancestor. As 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. However, except for the use of other probes, which could reveal intrachromosomal rearrangements, a phylogenetic analysis of GGA and birds of prey is not very informative, except as outgroup

Abstract

2- I missed some introduction about BIRDs cytogenetic in the beginning of Abstract. The authors go too direct about Accipitriformes and most readers do not have such phylogenetic information to which group we are dealing with.

A- We agree, and added an introduction to the abstract “Although most birds show karyotypes with diploid number around 2n=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”

3- Why are the authors aiming to investigate homologies with chicken?

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 “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].”

4- Abstract also brings some previous data on NNI that were not originated from this work. I suggest removing.

A- We removed it

5- The terms STY and NNI are used without any previous information/explanation about, causing misunderstandings.

A- We corrected it

6- A final conclusion is missing in the abstract.

A- We added a conclusion in the abstract: “Comparison with hybridization pattern of the Japanese-Mountain-Eagle, 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.

Figures

14- Figure 3: I did not understand why using the metaphases present in right column? The signals and chromosomal morphology are clearly visible in the FISH images. Scale bars are missing in all figures.

A- We removed the right column and added the scale bars in all figures.

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 fission-fusion 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).

1-Running title. It is too long, should be shorter, e.g., “Comparative Chromosome Painting in Spizaetus tyrannus and Gallus gallus“.

A- W corrected it.

2. Abstract. Please, decipher GGA, should be Gallus gallus (GGA), as done above for Spizaetus tyrannus (STY)

A- We corrected it.

Introduction.

3- Page 3. Paragraph 1. Please, provide a putative avian ancestral karyotype, with 2n and the number of microchromosomes.

A- We provided this information in the introduction of the paper.

4- Page 3. Paragraph 1. Here (or further, in Discussion), provide the G. gallus karyotype to make further reasoning clearer.

A- We did it.

5- Page 3. Paragraph 3. Unify way of citing – in other places you use numbering.

A- We corrected it.

6- Page 4. Paragraph 4. Please, specify the species studied, Spizaetus tyrannus

A- We corrected it.

Discussion.

7- Page 6, Paragraph 1. Please, expand the statement “We report slight differences in chromosome morphology….” by clarifying what Tagliarini et al. (2007) have reported. Describe the differences, give for comparison one and the other karyotypes (otherwise, you only have a declaration).

A- In order to clarify the difference in chromosome morphology described between this study and the Tagliarini et al., (2007), we made a comparative table that was introduce in the results.

8- Page 7. Paragraph 3. The Japanese mountain hawk-eagle Nisaetus nipalensis orientalis (Temminck & Schlegel, 1844), not the Hodgson's hawk-eagle Nisaetus nipalensis Hodgson, 1836.

A- We corrected it.

9- Other remarks can be found in the MS attached.

A- We followed all the instructions of attached.

Reviewer #3:

This study provides a cytogenetic mapping of the Black-Hawk-Eagle (Spizaetus tyrannus) using whole-chromosome paints and BAC probes of Gallus gallus. Using this cytogenetic mapping, the authors investigate the chromosome homologies between Spizaetus tyrannus and Gallus gallus. Overall the manuscript presents an advance in cytogenetic of Accipitriformes. The manuscript is written in understandable English, but some procedures require more details. Please see below my

comments that could help to further enhance the quality of the study.

2) Abstract: “chicken (Gallus gallus – GGA)”.

A- We corrected it.

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.

- Waters, P. D., Patel, H. R., Ruíz-Herrera, A., Álvarez-González, L., Lister, N. C., Simakov, O., ... & Graves, J. A. M. (2021). Microchromosomes are building blocks of bird, reptile and mammal chromosomes. bioRxiv.

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- The reviewer is right. The GGA3 probe correspond to four pairs (13,16,19 and 20).

We changed it.

6) Discussion, paragraph 3: “…karyotype of STY when compared to Gallus”; “…karyotype of STY when compared to Gallus gallus”.

A- We corrected it.

7) Discussion, paragraph 4: “…microchromosomes (STY4, 7 and 9; NNI2, 4 and 9), none of them”; “microchromosomes (STY: pairs 4, 7 and 9; NNI: pairs 2, 4 and 9), none of them”.

“…with microchromosomes (GGA: pairs 18, 19, 24 and 25)”.

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 removed it.

9) Conclusion: “…of S.tyrannus should be…”; “…of S. tyrannus should be…”.

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.

14) Please, give a scale bar information for all figures. It is very important, specially considering the presence of macrochromosomes and microchromosomes.

A- We added the scale bar in all figures.

Submitted filename: Carvalho et al-Response to the reviewers-Plos One.pdf

Click here for additional data file.

27 Oct 2021

PONE-D-21-20639R1Comparative Chromosome Painting in the Black-Hawk-Eagle (Spizaetus tyrannus) and Gallus gallus with the use of Macro and Microchromosome Probes.PLOS ONE

Dear Dr. de Oliveira,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Academic Editor

PLOS ONE

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Additional Editor Comments:

I return with another round of review. Two reviewers (2 and 3) make small suggestions to improve the quality of the manuscript. Also, the Plos one does not submit proof before publication, for this reason, I suggest special care in this review.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: N/A

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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Reviewer #2: Yes

Reviewer #3: Yes

**********

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The manuscript has been extensively revised. Generally, all my comments were addressed and I am satisfied with the revision. However, minor typos remain (listed below and marked in the attached PDF), which can be corrected while editorial processing and preparing the manuscript for publication.

Title

1. Comparative Chromosome Painting in the Black-Hawk-Eagle

(Spizaetus tyrannus) and Gallus gallus with the use of Macro

and Microchromosome Probes.

It is better to use only Latin names in both cases, e.g.

Comparative Chromosome Painting in Spizaetus tyrannus and Gallus gallus with the use of Macro-and Microchromosome Probes.

Then, Macro-and Microchromosome Probes should be written with a hyphen.

Abstract

1. Here and elsewhere (Introduction, Results), should not be duplicated diploid number and 2n, as for example in the case “diploid number around 2n=80”. Should be either diploid number around 80 or 2n around 80

2. The family Accipitridae contains 14 subfamilies, not only Aquilinae, then, the phrase “the Accipitridae family is the most diverse and includes the subfamily Aquilinae…” needs to be revised (see my suggestion in the PDF text).

Results

1. Table 3. Please, explain in the title the abbreviation “q”

-

Reviewer #3: Carvalho et al. 2021 – Resubmission “Comparative Chromosome Painting in the Black-Hawk-Eagle (Spizaetus tyrannus) and Gallus gallus with the use of Macro and Microchromosome Probes”. This is a re-review of the manuscript for PlosOne. The new version of the manuscript is improved, and I do think the results overall are publishable. I only have a few suggestions that can be considered during the production process.

- Please alphabetic order: “…In fact, rearrangements involving microchromosomes were detected in few orders: Psittaciformes, Cuculiformes, Suliformes, Caprimulgiformes and the Accipitriformes [13-15].”

-“Gallus gallus probes used in the fluorescent in situ hybridization (FISH) experiments produced…”.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PONE-D-21-20639_R1_rev.pdf

Click here for additional data file.

27 Oct 2021

Reviewer #2: The manuscript has been extensively revised. Generally, all my comments were addressed and I am satisfied with the revision. However, minor typos remain (listed below and marked in the attached PDF), which can be corrected while editorial processing and preparing the manuscript for publication.

Answer: Thank you very much for the positive feedback, we accepted all the suggestion in the PDF file.

Title

1. Comparative Chromosome Painting in the Black-Hawk-Eagle (Spizaetus tyrannus) and Gallus gallus with the use of Macro and Microchromosome Probes. It is better to use only Latin names in both cases, e.g. Comparative Chromosome Painting in Spizaetus tyrannus and Gallus gallus with the use of Macro-and Microchromosome Probes. Then, Macro-and Microchromosome Probes should be written with a hyphen.

Answer: Thank you, we accepted the suggestion.

Abstract

1. Here and elsewhere (Introduction, Results), should not be duplicated diploid number and 2n, as for example in the case “diploid number around 2n=80”. Should be either diploid number around 80 or 2n around 80

Answer: We corrected it.

2. The family Accipitridae contains 14 subfamilies, not only Aquilinae, then, the phrase “the Accipitridae family is the most diverse and includes the subfamily Aquilinae…” needs to be revised (see my suggestion in the PDF text).

Answer: We corrected it.

Results

1. Table 3. Please, explain in the title the abbreviation “q”.

Answer: We explained it.

Reviewer #3: Carvalho et al. 2021 – Resubmission “Comparative Chromosome Painting in the Black-Hawk-Eagle (Spizaetus tyrannus) and Gallus gallus with the use of Macro and Microchromosome Probes”. This is a re-review of the manuscript for PlosOne. The new version of the manuscript is improved, and I do think the results overall are publishable. I only have a few suggestions that can be considered during the production process.

Answer: Thank you very much for the positive feedback.

- Please alphabetic order: “…In fact, rearrangements involving microchromosomes were detected in few orders: Psittaciformes, Cuculiformes, Suliformes, Caprimulgiformes and the Accipitriformes [13-15].”

Answer: We corrected it.

-“Gallus gallus probes used in the fluorescent in situ hybridization (FISH) experiments produced…”.

Answer: We corrected it.

Submitted filename: Carvalho et al-Response to the reviewers -2-Plos One.docx

Click here for additional data file.

29 Oct 2021

Comparative Chromosome Painting in Spizaetus tyrannus) and Gallus gallus with the use of Macro and Microchromosome Probes.

PONE-D-21-20639R2

Dear Dr. de Oliveira,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Maykon Passos Cristiano, D. Sc.

Academic Editor

PLOS ONE

5 Nov 2021

PONE-D-21-20639R2

Comparative Chromosome Painting in Spizaetus tyrannus and Gallus gallus with the use of Macro- and Microchromosome Probes

Dear Dr. de Oliveira:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Academic Editor

PLOS ONE