The molecular cytogenetic characterization of Conopophaga lineata indicates a common chromosome rearrangement in the Parvorder Furnariida (Aves, Passeriformes)

Abstract Cytogenetic analyses of the Suboscines species are still scarce, and so far, there is no karyotype description of any species belonging to the family Conopophagidae. Thus, the aim of this study is to describe and analyze the karyotype of Conopophaga lineata by chromosome painting using Gallus gallus (GGA) probes and to identify the location of the 18/28S rDNA cluster. Metaphases were obtained from fibroblast culture from two individuals of C. lineata. We observed a diploid number of 2n=78. GGA probes showed that most ancestral syntenies are conserved, except for the fission of GGA1 and GGA2, into two distinct pairs each. We identified the location of 18S rDNA genes in a pair of microchromosomes. The fission of the syntenic group corresponding to GGA2 was observed in other Furnariida, and hence may correspond to a chromosomal synapomorphy for the species of Parvorder Furnariida.

The order Passeriformes encompasses approximately 5700 species, equivalent to 60% of existing birds, and besides their large diversity, they can also be considered a cosmopolitan group, thus becoming the focus of different studies. The order encompasses two large Suborders -Oscines and Suboscines (Ericson et al., 2003(Ericson et al., , 2014. The process of song learning is the main distinction between Oscines and Suboscines: Oscines are characterized by complex vocalizations, which are often learned through imitation. On the other hand, Suboscines have less complex vocal organs, and their songs do not seem to be learned by imitation (Raikow and Bledsoe, 2000). Suboscines are traditionally divided into two infraorders -Tyrannides (272 genera), endemic to the New World, and Eurylaimides (12 genera), which are widely distributed in the Old World (Selvatti et al., 2015).
Tyrannides are divided into the Parvorders -Furnariida and Tyrannida (Selvatti et al., 2015). The species Conopophaga lineata (CLI), the focus of this study, belongs to the Conopophagidae family, which is included in the Parvorder Furnariida.
Despite these data, information on events occurring during the karyotype evolution of Passeriformes is still fragmentary, as observed in most groups of birds. In this sense, studies involving species from basal clades are important to reconstruct the sequence of rearrangements arising during Passeriformes diversification. Considering that Conopophagidae represents one of the most basal lineages of passerines (Selvatti et al., 2015), a detailed study of one species of this family may shed some light on the chromosome evolution of Passeriformes. Hence, we describe here for the first time the karyotype of a species belonging to this family, the rufous gnateater (Conopophaga lineata).
The protocols were approved by the Committee of Ethics on the use of Animals (CEUA-Universidade Federal do Pampa, 026/2012), and SISBIO (Permission Number: 101 33860-4). Skin biopsies were collected from two females of C. lineata in Porto Vera Cruz and São Gabriel (Rio Grande do Sul, Brazil), and used for cell culture, following Sasaki et al. (1968), with modifications. In this process, cells were dissociated with collagenase type IV (Sigma) and grown in DMEM medium supplemented with fetal bovine serum (20%). Chromosome preparations were obtained after exposure to colcemid (1 h, 37 ºC), hypotonic treatment (0.075M KCl, 15 min, 37 ºC) and methanol/acetic acid (3:1). fixative Fluorescence in situ hybridization (FISH) experiments were performed using whole chromosome probes from Gallus gallus (GGA 1-10), obtained by flow cytometry at the Cambridge Resource Centre for Comparative Cytogenetics, (Cambridge, UK), amplified and labeled with biotin by DOP-PCR. Hybridizations were carried out according to Oliveira et al. (2010). Detection was performed with the use of Streptavidin-CY3 (Invitrogen). 18S rDNA probe fragments were labeled with digoxigenin by Nick Translation (Nick Translation Kit, Roche) and detected with Anti-Digoxigenin-Rhodamine, following the manufacturer's instructions, slide preparation, hybridization and washing were performed according to Daniels and Delany (2003).
Approximately 30 mitotic metaphases from each specimen were analyzed in order to determine the diploid number, chromosome morphology and confirm FISH experiments. Metaphases were analyzed in an epifluorescence light microscope (Imager Z2, Zeiss, Germany), and the images were acquired with the software Axiovision 4.8 (Zeiss, Germany).
The diploid number of C. lineata is 78. Pairs 1 to 7 are acrocentric, except for pair 4, which is submetacentric. The other autosomal chromosomes are telocentric, while the Z sex chromosome is submetacentric and W sex chromosome possibly is a telocentric microchromosome (Figure 1).
GGA probes 1-10 produced 13 different signals, revealing chromosome rearrangements. Most of the ancestral macrochromosomes are conserved in C. lineata, except for GGA1 and GGA2, which are fissioned in two pairs each. GGA 4 probe hybridized to two chromosome pairs, as in the putative bird ancestral karyotype. GGA3 and 5-10 hybridized to only a single pair each, revealing conserved syntenies. In addition, CLI 5 is the result of a fusion between a segment of GGA2 and an unidentified chromosome, possibly a microchromosome (Figures 2A,B and 3).
The diploid number observed, 2n = 78, is found in most bird species and is similar to the hypothetical bird ancestor (80 chromosomes) . It was possible to observe that the first and second pairs have a similar size, differently from most of Passerines studied so far (Kretschmer et al., 2014;Santos et al., 2017), indicating an additional fission in Conopophaga lineata.
Molecular cytogenetic in C. lineata 3  In addition to the fission of GGA2, we have identified that pair 5 of C. lineata was formed from a fusion between one of the segments originated from the GGA2 fission and a microchromosome (Figure 3).
Despite the fact that these rearrangements have been observed in a species belonging to the basal family Conopophagidae, the localization of ribosomal clusters in a pair of microchromosomes, corresponds to a plesiomorphic characteristic, usually observed in the order Passeriformes and in other avian orders, demonstrating the conservation of the ancestral state ( Figure 2F) (Nishida-Umehara et al., 2007;Oliveira et al., 2017;Santos et al., 2017).
In conclusion, we demonstrate that the morphology of macrochromosomes in C. lineata is significantly different from other Passeriformes species. Furthermore, we found a fission in GGA2, which appears to be a common chromosome rearrangement in Furnariidae and possibly other Parvorder Furnariida species that have minimal size difference between the first chromosomal pairs, in addition to the fissions that are typically found in Passeriformes (GGA1). However, since passerines present a high degree of chromosomal rearrangement, subsequent mapping and sequencing studies allowing the investigation of intrachromosomal rearrangements may elucidate these events.