Phylogenetic Analysis and Karyotype Evolution in Two Species of Core Gruiformes: Aramides cajaneus and Psophia viridis

Gruiformes is a group with phylogenetic issues. Recent studies based on mitochondrial and genomic DNA have proposed the existence of a core Gruiformes, consisting of five families: Heliornithidae, Aramidae, Gruidae, Psophiidae and Rallidae. Karyotype studies on these species are still scarce, either by conventional staining or molecular cytogenetics. Due to this, this study aimed to analyze the karyotype of two species (Aramides cajaneus and Psophia viridis) belonging to families Rallidae and Psopiidae, respectively, by comparative chromosome painting. The results show that some chromosome rearrangements in this group have different origins, such as the association of GGA5/GGA7 in A. cajaneus, as well as the fission of GGA4p and association GGA6/GGA7, which place P. viridis close to Fulica atra and Gallinula chloropus. In addition, we conclude that the common ancestor of the core Gruiformes maintained the original syntenic groups found in the putative avian ancestral karyotype.


Introduction
Despite major progress in the reconstruction of phylogeny of Aves in the last decade, the classification of species within the order Gruiformes still represents one of the least stable among this class [1,2]. Nowadays, the order Gruiformes contains five modern families: Heliornithidae, Aramidae, Gruidae, Psophiidae and Rallidae, this last one greatly exceeding other Gruiformes families in species richness (144 species), geographical range and taxonomic complexity [3,4]. In addition, some gaps on the evolutionary history and interrelationships among Gruiformes species also remain unsolved. For example, the taxonomy of the Rallidae family has been the subject of debate, mainly because this group has adapted to similar environments across their geographic distribution, and consequently, they have been subject to convergence evolution, making difficult the understanding of their evolutionary origins [3][4][5]. Members of the Rallidae family inhabit a range of ecological environments, including freshwater and saltwater marshes, mangroves, sparsely vegetated atolls, cool-temperate woodlands, tropical forests and grasslands [3]. Similarly, the relationship of Psophiidae, a family of birds restricted to the Amazon basin forests, with the other five families included in the core Gruiformes is still controversial [4].
The advances in comparative chromosome mapping with the use of chromosome painting has provided important information for inferences about phylogenetic relationships in some groups of birds, clarifying some problems left by the analyses of molecular biology [9][10][11][12][13][14]. Furthermore, despite the apparent karyotypical conservation among birds observed by conventional staining, comparative chromosome painting has revealed many rearrangements, such as fusions and fissions in several macrochromosomes; this information allowed the inference of a putative ancestral karyotype (PAK) of birds, which is actually highly similar to the chromosomal complement of Gallus gallus, with the exception of pair 4, which corresponds to two distinct pairs in the proposed ancestral karyotype [9][10][11]15,16].
Up to now, comparative chromosome painting has been performed in only two species of Gruiformes-the coot (Fulica atra-FAT) and common moorhen (Gallinula chloropus-GCH), both belonging to the Rallidae family. Fulica atra (2n = 92) and Gallinula chloropus (2n = 78) share the fissions of the ancestral chromosomes GGA5 and GGA4 [17]. Moreover, these species also share chromosome associations between GGA4/5 and GGA6/7 [17]. However, in order to understand the dynamics of the karyotype evolution in this group, it is necessary to analyze other species belonging to different families of Gruiformes. Therefore, with the aim of broadening our understanding of the events occurring during the chromosomal evolution of Gruiformes, we carried out the comparative chromosome painting with chicken macrochromosome paints in two species of this order: Aramides cajaneus-ACA (Gray-necked Wood-Rail), a member of the Rallidae family and Psophia viridis-PVI (Green-winged Trumpeter), a member of the Psophiidae family. The goal of this study was to investigate (a) whether Aramides cajaneus shows a karyotype organization similar or different to Fulica atra and Gallinula chloropus and (b) whether Psophia viridis has similar or different rearrangements compared to Rallidade species. Based on the chromosome painting data for Aramides cajaneus and Psophia viridis, together with the data from the literature for other Gruiformes species, we discuss the possible process of karyotype evolution in Gruiformes.

Cell Cultures and Chromosome Preparations
Skin biopsies of Aramides cajaneus (one female) and Psophia viridis (one female) were collected at Museu Paraense Emilio Goeldi (Belém, PA, Brazil). The experiments were carried out according to the ethical protocols approved by an ethics committee (CEUA-Federal University of Pará) under no. 170/2013 and SISBIO 68443-1). Fibroblast cells were obtained from skin biopsies after dissociation with collagenase IV (0.0186 g in 4 mL of DMEM (Dulbecco's Modified Eagle's medium, Sigma-Aldrich, MO, USA), for 1 h at 37 • C, and maintained in DMEM medium (Sigma-Aldrich, MO, USA) supplemented with antibiotics (1%) and fetal bovine serum (15%) at 37 • C [18]. Chromosomal preparations were obtained after mitotic arrest by adding 100 µL colcemid (0.05 µg/mL) for 1 h, followed by suspension and incubation with 0.075 M KCl (10 min at 37 • C) and fixed in Carnoy's fixative (3 methanol:1 acetic acid). Chromosome preparations were kept at −20 • C until the analyses.

Microscopic Analyses
At least 30 metaphases with conventional staining (Giemsa 5% in phosphate buffer, pH 6.8) were examined to determine the diploid number and chromosome morphology for each species. Images were captured using a 100× objective, microscopy DM1000 (Leica, CO, USA) and GenASIs software (ADS Biotec, Omaha, NE, USA) and the karyotype were ordered according to their arm ratios.
At least 10 metaphase spreads per individual were analyzed to confirm the hybridizations signals. FISH results were analyzed using a Zeiss Imager 2 microscope, 63× objective and images were captured using Axiovision v.4.8 software (Zeiss, Jena, Germany). Final edition of images was made using Adobe Photoshop CS6 software. For chromosomal evolution inferences, we used chromosome painting data from Fulica atra (FAT) and Gallinula chloropus (GCH) [17].

Phylogenetic Analysis
The inference of the phylogenetic tree was made based on cytogenetic information (chromosome painting) of four Gruiformes species, three belonging to the Rallidae Family (Fulica atra, Gallinula chloropus and Aramides cajaneus) and Psophiidae family (Psophia viridis), taking into consideration the presence or absence of chromosome features in these species.

Karyotypes of Aramides cajaneus and Psophia viridis
The karyotype of Aramides cajaneus has 2n = 78. Pairs 1, 2 and 4 are submetacentric, while 5, 6 and 8 are metacentric. The remaining autosomal chromosomes are telocentric ( Figure 1A). The Z and W sex chromosomes are metacentric. Psophia viridis has a karyotype comprised of 80 chromosomes. Pair 1 is submetacentric, while pairs 2 and 5 are acrocentric, and pair 4 is metacentric and easily distinguishable from other chromosomes. The remaining autosomal chromosomes are telocentric. Among the sex chromosomes, Z is submetacentric and the W chromosome is telocentric ( Figure 1B).
Furthermore, a fission in GGA4 and an association between GGA6/GGA7 in (PVI4) were observe in this species, thereby, a total of 12 homology signals were observed between the chromosomes of P. viridis and GGA probes (Figure 3; Figure 4B).

Syntenic Blocks Shared among Gruiformes Species and Phylogentic Analyses
In order to proceed with the phylogenetic analysis, comparative chromosome painting data covering the homology of macrochromosome pairs of four species of Gruiformes-Aramides cajaneus and Psophia viridis from the present work, and Fulica atra and Gallinula chloropus previously described by [17]-were organized in a matrix ( Table 1). The phylogenetic tree obtained is shown in Figure 5. F. atra and G. chloropus are more derived in relation to the A. cajaneus, and form a clade supported by three rearrangements-the fusion GGA4/GGA5 and GGA6/GGA7, and also the fission of GGA4p-chromosome features not observed in A. cajaneus. Concerning P. viridis, it would be closer to the clade formed by F. atra and G. chloropus, with which this species shares two rearrangements: the fission of GGA4p and the association GGA6/GGA7. Genes 2019, 10 FOR PEER REVIEW 6 147     and Psophia viridis from the present work, and Fulica atra and Gallinula chloropus previously described 156 by [17]-were organized in a matrix ( Table 1). The phylogenetic tree obtained is shown in Figure 5.

Discussion
The diploid chromosome numbers of both species analyzed are very similar, A. cajaneus 2n = 78 and P. viridis 2n = 80 (Figure 1). Additionally, both species are characterized by a typical avian karyotype, since the mode of the chromosome number in birds is 2n = 80, with a high number of microchromosomes [16,20].
In general, rearrangements found in Gruiformes involved chromosome pairs homologous to GGA4, GGA5, GGA6 and GGA7. The fission of ancestral syntenies GGA4p and GGA5 followed by fusion have been proposed as ancestral events in Gruiformes, since these rearrangements are present in F. atra and G. chloropus [17]. However, in P. viridis, only the fission in GGA4p has been observed, without the fusion between GGA4q/GGA5 ( Figure 4A). Furthermore, A. cajaneus does not share these rearrangements ( Figure 5).
The apparent multiple independent origins of associations between GGA6/GGA7 in avian species suggest that there are specific sites in these chromosomes that are susceptible to rearrangement processes (hotspots) [27][28][29]. This fact highlights the potential for the occurrence of rearrangements, such as chromosomal fusions, inversions and centromere shifts [30].

Phylogenetic Analysis
The results obtained from the experiments in this study, together with data previously published concerning two other Gruiformes (Table 1; Table 2), were plotted using the phylogenetic tree proposed by [4] and [5] to improve understanding of chromosomal evolution in this order ( Figure 5). Gallinula chloropus * * * [17] The presence of the rearrangement is indicated by *.
In respect of family Rallidae, some phylogenetic relationships, such as F. atra and G. chloropus as sister-groups, are well supported [31,32]. According to [5], A. cajaneus was included in the "Aramides clade", as a sister-group of "Fulica clade". The chromosome data of these species corroborate this relationship, since F. atra and G. chloropus share the same chromosome rearrangements, such as associations GGA6/GGA7, GGA4/GGA5 and fissions of GGA4p and GGA5, not found in A. cajaneus ( Figure 4B and Tables 1 and 2). In this way A. cajaneus, which shares less chromosome syntenies with the other two species (F. atra and G. chloropus), would be more basal than the 'Fulica clade'.
In our previous study with Eurypyga helias (Eurypygidae), formerly included in Gruiformes, but now regarded as belonging to a different order (Eurypygiformes) with only two families (Rynochetidae and Eurypygidae), we had proposed that the putative common ancestral karyotype of core Gruiformes would have a fission of GGA4p, since it had been found in the three species studied (P. viridis, F. atra and G. chloropus) ( Table 1; Table 2) [10,17]. If this hypothesis was correct, A. cajaneus would have regained the ancestral character. Alternatively, the absence of this fission in A. cajaneus indicates that it does not represent a synapomorphy of the Gruiformes.
According to [29], there are regions of bird genomes that are prone to breakage, facilitating chromosomal rearrangements, and this could explain the fission of GGA4p in some species but not in A. cajaneus. As more bird genomes are sequenced, the reasons for the conservation of some syntenic groups, while others were disrupted and reorganized, will become clearer.
Despite the fact that the phylogenetic position of the Psophiidae family is not well resolved within the Core Gruiformes [4], our results show that P. viridis shares some rearrangements with F. atra and G. chloropus, such as fission of GGA4p and association of GGA6/GGA7 (Table 1; Table 2); these rearrangements place P. viridis close to F. atra and G. chloropus (Rallidae family) ( Figure 5).
Accordingly, the last common ancestor from the Core Gruiformes would have a karyotype similar to the putative ancestral avian karyotype (PAK) [16], because some rearrangements that were found in this group are the result of independent events, such as the association between GGA5/GGA7 in A. cajaneus, despite the fact that some association and fission events were not found in all the species analyzed ( Figure 5).
Thus, molecular cytogenetic analysis confirms earlier studies on the relationships between F. atra and G. chloropus. Furthermore, they support the fact that the chromosome rearrangements accumulated in P. viridis are similar to the Rallidae species, although A. cajaneus shows a karyotype organization different from F. atra and G. chloropus. Nevertheless, this study has improved our understanding of the process of karyotype evolution in the Core Gruiformes.