Research Article |
Corresponding author: Rafael Kretschmer ( rafa.kretschmer@gmail.com ) Academic editor: Andrei Barabanov
© 2018 Rafael Kretschmer, Vanusa Lilian Camargo de Lima, Marcelo Santos de Souza, Alice Lemos Costa, Patricia C. M. O’Brien, Malcolm A. Ferguson-Smith, Edivaldo Herculano Corrêa de Oliveira, Ricardo José Gunski, Analía Del Valle Garnero.
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Citation:
Kretschmer R, de Lima VLC, de Souza MS, Costa AL, O’Brien PCM, Ferguson-Smith MA, de Oliveira EHC, Gunski RJ, Garnero ADV (2018) Multidirectional chromosome painting in Synallaxis frontalis (Passeriformes, Furnariidae) reveals high chromosomal reorganization, involving fissions and inversions. Comparative Cytogenetics 12(1): 97-110. https://doi.org/10.3897/CompCytogen.v12i1.22344
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In this work we performed comparative chromosome painting using probes from Gallus gallus (GGA) Linnaeus, 1758 and Leucopternis albicollis (LAL) Latham, 1790 in Synallaxis frontalis Pelzeln, 1859 (Passeriformes, Furnariidae), an exclusively Neotropical species, in order to analyze whether the complex pattern of intrachromosomal rearrangements (paracentric and pericentric inversions) proposed for Oscines and Suboscines is shared with more basal species. S. frontalis has 82 chromosomes, similar to most Avian species, with a large number of microchromosomes and a few pairs of macrochromosomes. We found polymorphisms in pairs 1 and 3, where homologues were submetacentric and acrocentric. Hybridization of GGA probes showed syntenies in the majority of ancestral macrochromosomes, except for GGA1 and GGA2, which hybridized to more than one pair of chromosomes each. LAL probes confirmed the occurrence of intrachromosomal rearrangements in the chromosomes corresponding to GGA1q, as previously proposed for species from the order Passeriformes. In addition, LAL probes suggest that pericentric inversions or centromere repositioning were responsible for variations in the morphology of the heteromorphic pairs 1 and 3. Altogether, the analysis of our data on chromosome painting and the data published in other Passeriformes highlights chromosomal changes that have occurred during the evolution of Passeriformes.
Avian cytogenetics, chromosome painting, macrochromosome syntenies, chromosome fission, intrachromosomal rearrangements
Passeriformes (passerines) are the largest and most diverse order of birds, with approximately 5,700 species, representing almost 60 % of all living birds (
Among birds, Passeriformes have the highest number of species analyzed by classical cytogenetics (
Besides information on diploid number and chromosome morphology, classical cytogenetic analyses have detected examples of chromosomal polymorphisms in some species of Passeriformes, such as Saltator similis d’Orbigny and Lafresnaye, 1837 (
Fourteen species of the suborder Oscines have been analyzed by chromosome painting (
Species belonging to the genus Synallaxis Vieillot, 1818 (Subfamily Furnariinae) show higher diversification when compared to other Furnariidae, probably due to the shift in their nesting habits and an expansion of their habitats to open areas (Irested et al. 2009), but only a few species of this family have been karyotyped. Hence, the aim of this study was to analyze the karyotype of Synallaxis frontalis Pelzeln, 1859, a species belonging to family Furnariidae, by chromosome painting using GGA and LAL probes, in order to verify if this complex pattern of intrachromosomal rearrangements is also present in more basal species for Passeriformes.
The experiments followed protocols approved by the ethics committee (CEUA-Universidade Federal do Pampa, no. 026/2012, SISBIO 33860-3 and 44173-1). Seven specimens of Synallaxis frontalis (SFR), four males and three females, were caught in São Gabriel, Rio Grande do Sul State, Brazil, within the natural area of Universidade Federal do Pampa. Skin biopsies were used for fibroblast cultures according to
Diploid number and chromosome morphology were determined by the analysis of at least 20 metaphases per individual, conventionally stained with Giemsa. C-banding (
Fluorescent in situ hybridization (FISH) experiments were performed with whole chromosome probes from two different species – Gallus gallus (pairs GGA1-GGA10) and Leucopternis albicollis (LAL), pairs homologous to GGA 1 (LAL 3, 6, 7, 15 and 18), GGA 2 (LAL 2, 4 and 20), GGA 3 (LAL 9, 13, 17 and 26), GGA 4 (LAL 1 and 16), GGA 5 (LAL 5) and GGA 6 (LAL 3) (
We found a karyotype of 2n=82 in Synallaxis frontalis, with 11 pairs of macrochromosomes, including the Z and the W chromosomes, and 30 pairs of microchromosomes (Figure
Metaphases and partial karyotype of female Synallaxis frontalis with heteromorphism in pair 1: Giemsa (A, C), C-banding (B, D). Partial karyotype of male S. frontalis with heteromorphism in pair 3 (E). Arrows indicate the Z and W chromosomes. Scale bar: 5 µm.
C-banding showed that blocks of constitutive heterochromatin were located in the centromeric region of the autosomes and Z chromosome, while the W chromosome was almost completely heterochromatic (Figure
GGA whole chromosome probes showed that most syntenic groups found in the putative avian ancestral karyotype (PAK) were conserved in SFR, except for GGA 1 and GGA 2, which were fissioned into two pairs each - SFR1/SFR5 and SFR3/SFR7, respectively (Fig.
Representative FISH experiments using Gallus gallus GGA (A–D) probes on metaphase chromosomes of Synallaxis frontalis (SFR). Chromosomes were counterstained with DAPI (blue), and probes detected with Cy3 (red). Probes used are indicated in the lower left corner of the images. Scale bar: 5 µm.
Representative FISH experiments using Leucopternis albicollis LAL (A–F) probes on metaphase chromosomes of Synallaxis frontalis (SFR). Chromosomes were counterstained with DAPI (blue), and probes detected with Cy3 (red). Probes used are indicated in the lower left corner of the images. Scale bar: 5 µm.
The genome of S. frontalis shows a chromosomal organization typical for Class Aves and order Passeriformes (
Most of the ancestral macrochromosomes are conserved as whole chromosomes in S. frontalis, as shown by the hybridizations of G. gallus macrochromosomes. Only the first two pairs (GGA1 and GGA2) are not conserved, due to the occurrence of fissions, and correspond to SFR1 and SFR5, SFR3 and SFR7 pairs, respectively. The fission of the ancestral chromosome 1 has been found in all species of the order Passeriformes studied to date (19 species, including S. frontalis) (
Unlike the fission of the GGA1 chromosome, the fission of the GGA2 chromosome has been described previously in only one species of the order Passeriformes, Satrapa icterophrys Vieillot, 1818 (
Hybridizations with LAL probes was not enough to identify the mechanism responsible for the heteromorphisms observed in the first and third chromosomes pairs in some SFR individuals. Both heteromorphisms may have originated either by pericentric inversions or centromere repositioning. Pericentric and paracentric inversions have been reported in several species of Passeriformes (
Homology map (13 first autosomal pairs) comparing the syntenic groups of Synallaxis frontalis to Gallus gallus (bottom) and Leucopternis albicollis (colors) (A). Schematic diagram showing the hypothetical pericentric inversion responsible for the heteromorphism observed in pair 1 from two individuals of Synallaxis frontalis (SFR1) (B). Hypothetical rearrangements observed in Synallaxis frontalis (SFR) PAK 2 (GGA2) that would have given rise to SFR3 and SFR7 (C–E). First, a centric fission in the ancestral synteny homologous to GGA2, created two distinct chromosome pairs, homologous to GGA2p (SFR7) and GGA2q (SFR3) (C). A pericentric inversion in SFR3 changed its morphology to acrocentric (D). A second pericentric inversion gave rise to the heteromorphic element in pair 3, which corresponds to a submetacentric chromosome (E).
In addition to the in silico analysis demonstrating several intrachromosomal rearrangements, chromosome painting studies with L. albicollis probes have also identified some of these rearrangements, especially in the GGA1q chromosome in Passeriformes (
Future studies on this species could use other probes such as BACs clones (
We are grateful to members of the Research Group Diversidade Genética Animal of the Universidade Federal do Pampa, SISBIO for the license to sampling of specimens studied in this paper and for the Pró-Reitoria de Pesquisa (PROPESQ-UNIPAMPA) for the financial contribution through notices of Support to Research Groups. We are also grateful to members of the Laboratory of Tissue Culture and Cytogenetics of the Evandro Chagas Institute (Ananindeua, PA, Brazil) for technical and financial support. Kretschmer R and de Lima VLC received student fellowships from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).