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Chromosomal painting of the sandpiper (Actitis
macularius) detects several ssions for the
Scolopacidae family (Charadriiformes)
Melquizedec Luiz Silva Pinheiro
Universidade Federal do Para
Cleusa Yoshiko Nagamachi
Universidade Federal do Para
Talita Fernanda Augusto Ribas
Universidade Federal do Para
Cristovam Guerreiro Diniz
Instituto Federal de Educacao Ciencia e Tecnologia do Para
Patricia Caroline Mary O´Brien
University of Cambridge
Malcolm Andrew Ferguson-Smith
University of Cambridge
Fengtang Yang
Wellcome Sanger Institute
Julio Cesar Pieczarka ( juliopieczarka@gmail.com )
Universidade Federal do Para https://orcid.org/0000-0003-2951-8877
Research article
Keywords: Chromosomal evolution, Burhinus oedicnemus, classic and molecular cytogenetics, phylogeny
DOI: https://doi.org/10.21203/rs.3.rs-44925/v3
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
Background: The Scolopacidae family (Suborder Scolopaci, Charadriiformes) is composed of sandpipers
and snipes; these birds are long-distance migrants that show great diversity in their behavior and habitat
use. Cytogenetic studies in the Scolopacidae family show the highest diploid numbers for order
Charadriiformes. This work analyzes for the rst time the karyotype of
Actitis macularius
by classic
cytogenetics and chromosome painting.
Results: The species has a diploid number of 92, composed mostly of telocentric pairs. This high 2n is
greater than the proposed 80 for the avian ancestral putative karyotype (a common feature among
Scolopaci), suggesting that ssion rearrangements have formed smaller macrochromosomes and
microchromosomes. Fluorescence
In Situ
Hybridization using
Burhinus oedicnemus
whole chromosome
probes conrmed the ssions in pairs 1, 2, 3, 4 and 6 of macrochromosomes.
Conclusion: Comparative analysis with other species of Charadriiformes studied by chromosome
painting together with the molecular phylogenies for the order allowed us to raise hypotheses about the
chromosomal evolution in suborder Scolopaci. From this, we can establish a clear idea of how
chromosomal evolution occurred in this suborder.
Background
The order Charadriiformes (Aves) comprises shorebirds and is divided into three suborders: Charadrii
(plovers and allies), Scolopaci (sandpipers and allies) and Lari (gulls and allies). Although cases of
convergence have complicated efforts to establish their phylogenetic relationships based on morphology,
the molecular phylogenies of this order have proved to be quite consistent and have given rise to few
controversies [1].
The suborder Scolopaci appeared 70 million years ago and is formed by ve families: Jacanidae,
Rostratulidae, Thinocoridae, Pedionomidae and Scolopacidae; the latter is the most specious, with about
100 species [2]. Phylogenetically, this suborder is divided into two major branches: one leads to
Scolopacidae, and one leads to the other families [2-4].
The Scolopacidae family is composed of sandpipers and snipes, which exhibit a wide distribution. These
long-distance migratory birds show great diversity in their behavior and habitat use, and thus offer an
important opportunity for studying the evolutionary forces that have acted on the various species [5].
Cytogenetic studies in Charadriiformes have revealed considerable karyotypic variability, with diploid
numbers ranging from 2n = 42 in
Burhinus oedicnemus
[6] to 2n = 98 in
Gallinago gallinago
[7].
Compared to the members of other suborders, members of Scolopaci tend to have higher diploid
numbers (Table 1), ranging from 2n = 82 in
Jacana jacana
8 to the previously mentioned 2n = 98 in
Gallinago gallinago
[7]. The karyotypes diverge between the two major phylogenetic branches of
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Scolopaci; Scolopacidae present karyotypes composed mainly of telocentric chromosomes, while in the
other families most chromosomal pairs are biarmed (meta and submetacentric) [7-14].
Table 1: A review of cytogenetic information available for the Suborder Scolopaci. 2n =
Diploid number: FN = Fundamental number; CP = Chromosome Painting, species studied
using whole chromosome probes; GGA =
Gallus gallus
; ZAU =
Zenaida auriculata
; BOE =
Burhinus oedicnemus
.
Family Species 2n CP References
Jacanidae
Hydrophasianus chirurgus
82 -- 9
Jacana jacana
82 GGA, ZAU 8
Actitis hypoleucos
86 -- 9
Actitis macularius
92 BOE Present study
Tringa glareola
72 -- 9
Tringa totanus
88 -- 7
Scolopacidae
Tringa flavipes
88 -- 12
Tringa nebularia
88 -- 10
Tringa erythropus
88 -- 11
Tringa semipalmatus
88 -- 13
Tringa ochropus
88 -- 11
Gallinago gallinago
98 -- 7
Scolopax rusticola
88 -- 10
Calidris alpina
88 -- 10
Calidris ruficollis
86 -- 11
Calidris temminckii
90 -- 11
Calidris acuminata
84 -- 11
Calidris canutus
90 -- 11
Calidris tenuirostris
88 -- 11
Arenaria interpres
88 -- 10
Limosa limosa limosa
90 -- 14
Limosa lapponica
94 -- 11
Numenius arquata
78 -- 7
Cytogenetic studies of class Aves using chromosomal painting started with the development of whole-
chromosome probes from the
Gallus gallus
macrochromosomes (GGA) [15]. These probes quickly
became a reference tool in birds; their use in different orders demonstrated the recurrence of a karyotype
very similar to that of GGA, leading to the proposition of a Putative Ancestral Karyotype (PAK) [16]. The
PAK differs from GGA in that GGA4 is represented by two pairs in the ancestral karyotype (Table 2): PAK4
(GGA4q) and PAK10 (GGA4p). Since its proposal, the PAK has been used as a reference in comparative
analyses [16-19].
Table 2: Chromosomal correspondence between
Gallus gallus
(GGA)
, Burhinus
oedicnemus
(BOE),
Larus argentatus
(LAR),
Actitis macularius
(AMA) and
Jacana jacana
(JJA) demonstrated by chromosome painting. The numbers of chromosome pairs are the
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ones of the karyotype of each species. Micro = microchromosome. ? = Hybridization did
not work.
PAK [16] GGA [15] BOE [6] LAR [20] AMA
(present work)
JJA [8]
1 1 1 1 1, 2, Wq 1
2 2 2 2 3, 11, 12, 13, Wq 4, 5p, 6p, 9
3 3 3 3 4, 14, 15, Wq 2q, 3p, 7q
4 4q 4 5 6, 16, W 2p, 3q
7, 8 7, 8 5 7, 8 7, 8 7p,6q
5 5 6 4 9, 10, Wq 5q, 8q
9 9, 2 micros (R3 & R6) 7 6, 7, 11 5, 2 micros, Wq 10
10 4p, 1 micro (R2) 8 9 ? 15
6 6, 1 micro 9 6, 18 8 micros, Zq, Wq 13, 14
- 2 micros (R1 & R4) 10 4, 8 17, 20 -
- 2 micros (R2 & R7) 11 10, 16 18, 2 micros -
- 2 micros (R5) 12 12, 17 19 20
- 2 micros (R6 & R9) 13 15, 25 6 micros, Wq -
- 2 micros (R5) 14 13 2 micros, Wq 21
- 3 micros 15, 16 14, 19, 23 6 micros, Wq -
- 1 micro (R9) 17, 18, 19, 20 22, 24, 26 6 micros, Wq -
Z Z Z Z Z, Wq Z
W W W W W, Zq W
In the order Charadriiformes, only four species have been studied by chromosome painting to date: 1)
Burhinus oedicnemus
(BOE, 2n = 42) has one of the lowest diploid numbers among birds (2n = 42); this is
due to the fusion of many microchromosomes giving rise to macrochromosomes and made it possible
(unlike the case of the GGA genome) for researchers to generate probes for all chromosomes of the
karyotype [6]. 2)
Larus argentatus
(LAR, 2n = 70) was hybridized with BOE probes, which demonstrated
several associations (e.g., BOE6 / BOE10 and BOE7 / BOE9) that were proposed as signatures for
suborder Lari [20]. 3)
Vanellus chilensis
(VCH, 2n = 78) was hybridized with GGA probes; this
demonstrated the fusion of GGA 7 and GGA 8, which was proposed as a possible common characteristic
in suborder Charadrii [21]. 4)
Jacana jacana
(JJA, 2n = 82), which was also hybridized with GGA probes
and presented numerous ssions and fusions, demonstrating that JJA had undergone extensive genomic
reorganization [8].
In sum, there is relatively little cytogenetic information described so far for Charadriiformes, but there is
considerable chromosomal variation between the different taxonomic groups. Thus, further studies are
needed to improve our understanding of the karyotypic evolution of these families. In the present study,
we analyzed the karyotype of
Actitis macularius
(AMA, Scolopacidae) using BOE probes [6] and
compared it with published data for
Gallus gallus
(GGA) and Charadriiformes. We also revised the
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cytogenetic information available for the suborder Scolopaci, with special emphasis in families
Jacanidae and Scolopacidae (Table 1), and used a published molecular phylogeny [2] to determine the
direction of chromosomal rearrangements.
Results
Karyotypic description and chromosome painting in
Actitis macularius
Actitis macularius
has 2n = 92, where the rst two pairs are acrocentric and the others are telocentric
(Figure 2). This karyotype has 14 pairs of autosomal macrochromosomes and the others are
microchromosomes. For the sex chromosomes, the Z and the W are submetacentric.
FISH with BOE whole-chromosome probes in
Actitis macularius
(AMA) demonstrated the
correspondences observed in Figure 2 and Table 2. Examples of these hybridizations are found in Figure
3. These results were extrapolated to GGA and PAK using the previous reports [6,16], respectively (Table
2).
Discussion
The karyotype of
Actitis macularius
(AMA)
The karyotype of AMA, which was studied herein for the rst time, has 2n = 92, and thus exhibits a 2n
greater than the proposed 2n=80 for the PAK [16]. This difference reects the occurrence of ssion
rearrangements in all macrochromosomes. As this is a common feature among Scolopaci (Table 10, this
is not a distinctive feature specic of
Actitis macularius
.
Chromosomal rearrangements among
Actitis macularius
(AMA),
Burhinus oedicnemus
(BOE) and
Gallus
gallus
(GGA)
Using BOE probes to paint the karyotype of a species of Scolopacidae allowed us to detect the
rearrangements that occurred in the phylogenetic branch leading to the AMA karyotype. Unlike the
conserved state observed for the rst pairs of many avian species [16, 19, 22, 23], including
Burhinus
oedicnemus
[6], pairs 1, 2, 3, 4 and 6 of PAK are ssioned in AMA. Possibly other Scolopacidae with high
2n and similar chromosomes may have undergone the same rearrangements.
Many BOE probes hybridized on the long arm of the W in AMA, as observed in
Larus argentatus
[20]. This
suggests that the W carries numerous variable copies (repetitive regions) homologous to the autosomes
of species in order Charadriiformes. A similar arrangement was found in the Passeriform,
Glyphorynchus
spirurus
[24]. An experiment to test the possibility of repetitive DNA sequences spread in autosomes and
W would be the isolation of this sequence and its use as a DNA probe for FISH in AMA karyotype.
Despite BOE belonging to the same order as AMA, we observed no conservation of the
macrochromosome pairs, except for the Z and W. The ssion of submetacentric BOE1 is clear in the rst
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two AMA acrocentric pairs. Pericentric inversion may have occurred after this ssion, leading to the
formation of two pairs with small short arms (Figure 4A). Alternatively, the short arm may be the result of
telomeric amplication [25] or centromeric repositioning [26]. The submetacentric BOE2 is divided into
four telocentric pairs in AMA (pairs 3 and 11-13) due to multiple ssions; we were not able to dene which
segment of BOE2 was found in each AMA pair (Figure 4B). BOE3 experienced ssion, giving rise to pairs
AMA4, 14 and 15 (Figure 4C). Fission of BOE4 gave rise to AMA6 and AMA16 (Figure 4D). BOE5 was
divided into two pairs, AMA7 and AMA8, but this was not by ssion. Cytogenetic studies demonstrated
that the fusion of PAK7 and PAK8 is a characteristic of suborder Charadrii [21]; the presence of the
separate chromosomal pairs in
Actitis
is the ancestral form, and BOE5 is the derived form (Figure 4E).
Fission of BOE6 gave rise to AMA9 and 10 (Figure 4F). BOE8 did not hybridize in the AMA genome,
possibly for technical reasons. So, this is rst conrmation of the ssions in pairs BOE2, 3, 4 and 6 and
also the rst demonstration of ssion in pair BOE1 in Scolopaci.
Fissions in BOE1, 2 and 5, were observed in
Glyphorynchus spirurus
[24], Strigiformes, Passeriformes,
Columbiformes and Falconiformes also have the ssion of GGA1 (BOE1) [20, 27-29]. For Scolopaci, in
contrast, a ssion of PAK1 (= GGA1, BOE1) seems to be a character shared only among members of the
Scolopacidae family. Its presence in other orders would therefore be an example of homoplasy.
The correspondences among the AMA, BOE, LAR, GGA and PAK chromosomes are shown in Table 2.
Chromosome evolution in suborder Scolopaci
It is accepted that the ancestral putative karyotype (PAK) with 2n = 80, which is commonly found in
several orders of birds, remained conserved for about 100 million years, with few variations for Neoaves
[16]. However, order Charadriiformes presents a high level of karyotypic diversity [14]. An interesting point
is that the three suborders originated in the late Cretaceous between 79 and 102 Mya [3], which indicates
that little time has passed from the origin of PAK to the ancestral Charadriiformes karyotype.
Suborder Scolopaci has a high diploid number, ranging from 78 to 98 chromosomes [9]. In addition to
Actitis macularius
(described here), chromosome painting was previously used to examine the karyotype
of
Jacana jacana
[8]. Our comparative analysis between these two species (Table 2) shows that both
share the following ssions: PAK2 (GGA2, BOE2) in four segments; PAK3 (GGA3, BOE3) in three
segments; and PAK4 (GGA4q, BOE4) and PAK6 (GGA6, BOE9) in two segments. After these ssions, a
series of fusions occurred between several chromosome pairs in the evolutionary branch that gave rise to
JJA [8]. Although the literature lacks any additional chromosome painting study of the Jacanidae, the
karyotype described from the other genus of this family,
Hydrophasianus
, has the same diploid number
and appears similar to JJA on conventional staining [9]. This suggests that the chromosomal
characteristics found in JJA are not restricted to this species and may be chromosome signatures for the
Jacanidae family (Figure 5).
An interesting feature is seen for chromosome PAK1 (= BOE1, GGA1): It is split into two pairs in AMA but
remains whole in JJA. This suggests that PAK1 underwent ssion in the branch that led to AMA but
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remained in its ancestral form in JJA. Since the morphology of the full-length PAK1 chromosome is quite
different from that of its split version, information about the timing of this ssion can be obtained by
analyzing other karyotypes along the branch that leads to AMA (family Scolopacidae), even using
conventional staining data. The karyotypes of genera
Tringa
,
Calidris
,
Arenaria
and
Limosa
[7, 9-11, 13,
14] show the rst chromosomal pair as an acrocentric that is similar in size to the long arm of PAK1,
according to the measurements performed by Hammar [7]. This suggests that ssion occurred in the
branches that lead to these genera (Figure 5). Two branches cast some doubt on this proposition,
however. The
Gallinago
and
Scolopax
genera have similar karyotypes, in which the rst pair is a
submetacentric chromosome [7, 10, 13]. Hammar [7] measured the chromosomes of several species of
Charadriiformes and demonstrated that the rst whole-chromosome pair (PAK1) is equivalent to 14% of
the karyotype. In contrast, that of
Tringa
(long arm of PAK1) corresponds to 10%. The rst pair of the
Gallinago
karyotype corresponds to 9.3% of the karyotype, suggesting that it is similar to
Tringa
, but with
an inversion. Chromosomal painting studies are needed in these species to conrm if the rst pair of
Tringa
and
Gallinago
are homeologues. The second branch that generates doubt is the one that leads to
Numenius
and
Bartramia
(Figure 5). The second chromosomal pair of the karyotype of
Numenius
arquata
is an acrocentric corresponding to 9.8% of the karyotype [7]; it may be equivalent to the rst pair
of the other species of Scolopaci (the rst pair of the karyotype of
Numenius
is a metacentric of similar
size, possibly the result of a fusion). Studies with chromosome painting in
Numenius
and/or
Bartramia
are needed to test this possible correspondence. Thus, it is not clear whether the ssion break in PAK1
occurred at the base of the branch that gave rise to the Scolopacidae family or after the separation of the
branch that gave rise to
Numenius
and
Bartramia
(Figure 5). If additional studies conrm that
Numenius
arquata
pair 2 is equivalent to the rst chromosome of the other species of Scolopacidae, the ssion of
PAK1 would be a chromosomal signature for this family.
The rearrangements described here are restricted to suborder Scolopaci, since chromosomal painting in
Larus argentatus
[20], a species of suborder Lari (a sister group of Scolopaci) [1, 4], revealed a karyotype
similar to the ancestral birds in pairs PAK1-4, with fusions of microchromosomes with
macrochromosomes (LAR4 and 7-9) and none of the ssions observed in Scolopaci (Table 2).
The data analyzed here allow us to propose an ancestral karyotype for suborder Scolopaci using PAK as
an outgroup, in which chromosome PAK1 is preserved, PAK2 is broken into four pairs, PAK3 is fragmented
into three segments and PAK4 and PAK6 are divided into two segments each (Scolopaci Putative
Ancestral Karyotype, SPAK, Figure 5).
Conclusions
This study examined for the rst time a species of family Scolopacidae by classic and molecular
cytogenetics.
Actitis macularius
has a high diploid number with several ssions. This work suggests that
ssions and possible pericentric inversions occurred in suborder Scolopaci, leading to a karyotype formed
almost exclusively of telocentric pairs. Only ve species have been examined to date with
Burhinus
oedicnemus
probes, but the data from the previous and present studies enable us to establish a clear
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idea of how chromosomal evolution occurred in this suborder. Studies of more species are needed to test
the hypotheses raised here and further clarify the evolutionary history of this group of birds.
Methods
Ethics
The specimens were kept stress-free with full access to food and water until their necessary euthanasia,
which was performed by intraperitoneal injection of buffered and diluted barbiturates (86 mg/kg) after
anesthesia with ketamine (40 mg/kg), following The American Veterinary Medical Association Guidelines
for the Euthanasia of Animals, in accordance with animal welfare guidelines established by Brazilian
resolution CFMV n.1000/2012, and with animal welfare guidelines established by the Animal Ethics
Committee (Comitê de Ética Animal) from Universidade Federal do Pará (UFPA), which authorized the
present study (Permit 68-2015). JCP has a permanent eld permit, number 13248 from “Instituto Chico
Mendes de Conservação da Biodiversidade”. The Cytogenetics Laboratory from UFPA has a special
permit number 19/2003 from the Ministry of Environment for samples transport and 52/2003 for using
the samples for research.
Sampling
Three
Actitis macularius
adult females were collected during eld research for the Molecular Biology and
Neuroecology Laboratory of the Federal Institute of Pará (Laboratório de Biologia Molecular e
Neuroecologia do Instituto Federal do Pará), Campus Bragança, and so they provided all technical
support. Collections occurred at Praia do Pilão (0º47'46.08" S and 46°40'29.64" W) in the state of Pará,
Brazil (Figure 1). Nets of 12 m x 2 m in size and made with a 36-mm mesh were extended, and then
visited every 30 minutes for sample retrieval [30]. Voucher specimens (BCAM108, BCAM109 and
BCAM126) were deposited in the collection of the Laboratório de Biologia Molecular e Neuroecologia,
Instituto Federal do Pará (Bragança, Para, Brazil).
Cytogenetic methods
Chromosome preparation and classic cytogenetics
As the sample would be euthanatized for other research purposes not related to cytogenetics, bone
marrow preparations were performed in the eld after colchicine treatment according to the literature [31].
Mitotic chromosomes were classied in decreasing sizes according to the proposed nomenclature [32].
The metaphases were captured and the karyotypes were assembled using the Adobe Photoshop CS6
software.
Chromosome painting
Page 9/18
The utilized whole-chromosome probes from
Burhinus oedicnemus
(Charadriiformes) were obtained by
ow cytometry [6]. The uorescence
in situ
hybridization (FISH) experiments were carried out as
described by Yang [33]. Metaphase chromosome preparations were made and aged at the same day for 1
h at 65°C, followed by incubation in 1% pepsin for 5 min. These slides preparations were denatured in
70% formamide, 2XSSC solution at 65°C for 1 min, rapidly cooled in ice-cold 70% ethanol and dehydrated
through a 70%, 90%, and 100% ethanol series. The probes (1 ml) were diluted into 15 ml of hybridization
buffer (50% deionized formamide, 10% dextran sulphate, 2XSSC, 0.5 M phosphate buffer, pH 7.3),
denatured at 65°C for 10 min, and applied onto slides, followed by a three days hybridization at 42°C.
After hybridization the preparations were washed twice in formamide 50%, 2xSSC, and once in
4xSSC/Tween at 40˚C. For visualization of the biotin-labelled probes a layer of Cy3-a or Cy5-avidin
(1:1000 dilution; Amersham) was used. For FITC-labelled probes we used a layer of rabbit anti-FITC
(1:200; DAKO). Slides were mounted in a mounting medium with DAPI called Vectashield (Vector
Laboratories). For the rst mapping single experiments (one probe by slide) were made. For the
determination of the limits of two different probes into a chromosome pair, double experiments (two
probes using different colors) were made.
Slides were analyzed in a Nikon H550S microscope, with a DS-Qi1Mc digital camera controlled by the
Nis-Elements software. The images were captured in black and white and subsequently pseudo-colored
based on the utilized uorochromes. Images were edited with the Adobe Photoshop CS6 software.
Phylogenetic inferences
To determine the direction of chromosomal rearrangements in Scolopaci, the known karyotypes were
plotted on a molecular phylogeny [2]. The phylogeny was built by those authors based on the sequences
of ve genes (RAG1, CYT B, 12S, ND2 and COI) and estimated with partitioned Bayesian analysis (Figure
5). To make the chromosomal evolution clear, we redesigned the phylogeny, where branches with genera
without cytogenetic information were removed, but at least one representative from each family
remained. We also take into account the PAK as an outgroup to dene the direction of the rearrangements
(fusion or ssion).
Abbreviations
GGA:
Gallus gallus
; PAK: Putative Ancestral Karyotype; BOE:
Burhinus oedicnemus
; 2n = diploid number;
LAR:
Larus argentatus
; VCH:
Vanellus chilensis
; JJA:
Jacana jacana
; AMA:
Actitis macularius
; FISH:
Fluorescence
in situ
hybridization.
Declarations
Ethics approval and consent to participate
The specimens were kept stress-free with full access to food and water until their necessary euthanasia
was performed in accordance with animal welfare guidelines established by Brazilian resolution CFMV
Page 10/18
n.1000/2012. The necessary euthanasia was performed by intraperitoneal injection of buffered and
diluted barbiturates after local anesthesia, in accordance with animal welfare guidelines established by
the Animal Ethics Committee (Comitê de Ética Animal) from Universidade Federal do Pará (UFPA), which
authorized the present study (Permit 68-2015). JCP has a permanent eld permit, number 13248 from
“Instituto Chico Mendes de Conservação da Biodiversidade”. The Cytogenetics Laboratory from UFPA has
a special permit number 19/2003 from the Ministry of Environment for samples transport and 52/2003
for using the samples for research.
Consent for publication
Not applicable.
Availability of data and materials
All data supporting the results reported in this article can be foundat the article itself. No additional
dataset is available.
Competing interests
The authors declare that they have no competing interests.
Funding
This study is part of the Mastership Dissertation of MLSP in Neurosciences and Cellular Biology. CYN
(305880/2017-9) and JCP (305876/2017-1) are grateful to CNPq for Productivity Grants. Funding:
Conselho Nacional de Desenvolvimento Cientíco e Tecnológico (CNPq), the Fundação Amazônia
Paraense de Amparo à Pesquisa (FAPESPA) and the Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES) on projects coordinated by CY Nagamachi (Edital BIONORTE-CNPq, Proc
552032/2010-7; Edital BIONORTE-FAPESPA, ICAAF 007/2011; Edital Pró-Amazônia Proc 047/2012); the
FAPESPA (Edital Vale – Proc 2010/110447) and Banco Nacional de Desenvolvimento Econômico e
Social – BNDES (Operação 2.318.697.0001) on a project coordinated by JC Pieczarka. The funding
bodies did not have any role in the design of the study, collection, analysis and interpretation of data, or in
writing the manuscript.
Authors contributions
MLSP: conception of the work; acquisition, analysis, and interpretation of cytogenetic data; rst version
of the manuscript. CGD: eld collection of biological samples; critical review of the manuscript for
important intellectual content. CYN: participated in the draft of the work and revised it critically for
important intellectual content; analysis, and interpretation of cytogenetic data. TFAR: acquisition,
analysis, and interpretation of cytogenetic data. PCMO’B: generation of whole chromosome probes;
revised the work critically for important intellectual content. FY: generation of whole chromosome probes;
acquisition and interpretation of cytogenetic data. MAF-S: generation of whole chromosome probes;
Page 11/18
revised the work critically for important intellectual content. JCP: conception of the work; analysis and
interpretation of cytogenetic data; participated in the draft of the work and revised it critically for
important intellectual content. All authors have read and approved the manuscript.
Acknowledgements
We would like to thank the staff of the Molecular Biology and Neuroecology Laboratory of the Federal
Institute of Pará, Campus Bragança, which provided all technical support. We also thank the technical
team of the Cytogenetics Laboratory from the CEABIO, UFPa for their constant support.
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Figures
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Figure 1
Geographic distribution map showing the collection locality (Bragança - PA) and place from which the
Actitis macularius specimens were collected. Specimens were collected at Praia do Pilão (black circle).
The map was prepared using the QUANTUM-GIS software, v. 2.10.1. The database was obtained through
IBGE and REDLIST. Note: The designations employed and the presentation of the material on this map do
not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal
status of any country, territory, city or area or of its authorities, or concerning the delimitation of its
frontiers or boundaries. This map has been provided by the authors.
Page 15/18
Figure 2
Actitis macularius karyotype with genomic mapping performed using Burhinus oedicnemus (BOE) whole-
chromosome probes, with the correspondence shown on the right. The microchromosomes were
organized by size, as the correct homologies could not be detected due to the lack of reliable markers.
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Figure 3
Chromosome painting with whole-chromosome probes from Burhinus oedicnemus (BOE) in Actitis
macularius (AMA). A) BOE1 (pairs 1, 2 and W); B) BOE2 (pairs 3, 11, 12, 13 and W); C) BOE3 (pairs 3 and
14, 2 microchromosomes and W); D) BOE4 (pair 6, 2 microchromosomes and W); E) BOE5 (pairs 7 and 8
and W); F) BOE6 (pairs 9 and 10 and W); G) BOE10 (pairs 17 and 20); H) BOE11 (pair 18 and a pair of
microchromosomes); I) BOE12 (pair 19). The probes were visualized with avidin-Cy3 (red) and the
chromosomes were counterstained with DAPI (blue).
Page 17/18
Figure 5
A simplied version of the phylogeny from Gibson & Baker [2], which was based on the sequences of ve
genes (RAG1, CYT B, 12S, ND2 and COI) and estimated with partitioned Bayesian analysis for suborder
Scolopaci. According to the authors, “all nodes received a posterior probability of 1.00 unless otherwise
labeled”. In the partial phylogeny here shown, the only node that has a posterior probability lower than
1.00 is the one that split Actitis from Tringa (0.71). Diploid numbers from literature (Table 1); karyotypes
Page 18/18
analyzed by FISH are shown in red. PAK fusions* = (4+3), (3+4), (2+5), (2+8), (7+3), (5+micros), according
to Kretschmer et al. [8]. PAK = avian putative ancestral karyotype, Grin et al. [16]. SPAK = Scolopaci PAK.
