Cytotaxonomy of Gallinula melanops (Gruiformes, Rallidae): Karyotype evolution and phylogenetic inference

Abstract Although Rallidae is the most diverse family within Gruiformes, there is little information concerning the karyotype of the species in this group. In fact, Gallinula melanops, a species of Rallidae found in Brazil, is among the few species studied cytogenetically, but only with conventional staining and repetitive DNA mapping, showing 2n=80. Thus, in order to understand the karyotypic evolution and phylogeny of this group, the present study aimed to analyze the karyotype of G. melanops by classical and molecular cytogenetics, comparing the results with other species of Gruiformes. The results show that G. melanops has the same chromosome rearrangements as described in Gallinula chloropus (Clade Fulica), including fission of ancestral chromosomes 4 and 5 of Gallus gallus (GGA), beyond the fusion between two of segments resultants of the GGA4/GGA5, also fusions between the chromosomes GGA6/GGA7. Thus, despite the fact that some authors have suggested the inclusion of G. melanops in genus Porphyriops, our molecular cytogenetic results confirm its place in the Gallinula genus.


Introduction
Gruiformes is an avian order showing great heterogeneity of habits, habitats and morphology and a wide geographic distribution (Del Hoyo et al., 1996;Garcia et al., 2014).Because of their great diversity, phylogenetic relationships among the different families in this order are still controversial, despite the number of phylogenetic studies performed so far.One of the first proposals based on morphological characters classified Gruiformes into 12 families (Wetmore, 1960).However, with the introduction of new methods, such as molecular studies using mitochondrial-nuclear DNA or genome sequencing, it has been possible to reach a consensus that there is a monophyletic core of five families known as "Core Gruiformes":Rallidae, Heliornithidae, Psophiidae, Aramidae, and Gruidae (Fain et al., 2007;Hackett et al., 2008;Jarvis et al., 2014;Prum et al., 2015).
Within the core Gruiformes, Rallidae is the family with the highest number of species, around 152, distributed in 33 to 40 genera, comprising 85% of the order diversity (Garcia et al., 2014;Gill et al., 2020).The phylogenetic relationships within Rallidae still present many inconsistencies, due mainly to the small numbers of species that have been sampled in the different approaches (Garcia et al., 2014).
There is an urgent need to use these new techniques to clarify the problems concerning avian karyotypes and phylogenetic relationships in a greater number of species (Dobigny et al., 2004;Furo et al., 2015Furo et al., , 2018;;Nie et al., 2015;Seligmann et al., 2019).The main aim of this study was to characterize the karyotype of G. melanops by classic cytogenetics, GGA chromosome painting probes and FISH with BACs selected from the genome library from microchromosomes of G. gallus in order to contribute to the phylogeny and karyotype evolution of the Rallidae family.

Chromosome preparation
Fibroblast cultures obtained from wing skin biopsies of five female specimens of Gallinula melanops were collected in São Gabriel, Rio Grande do Sul State, (RS, Brazil), following Sasaki et al. (1968) with modifications.The samples were first mechanically fractionated in a Petri dish after incubation in type IV collagenase for tissue dissociation.The cells were cultured in DMEM (GIBCO) supplement with calf bovine serum 20%, Aminiomax TM -II 5% and penicillin (PNS) 1% then incubated at 37 ºC.Afterwards, metaphase arrest was obtained by adding colcemid (Gibco, 100 μl for 5 ml of complete medium) followed by incubation for 1 hour at 37 ºC, and hypotonic solution treatment (KCl 0,075 M) for 15 minutes.Finally, the suspensions were fixed using Carnoy's fixative methanol: acetic acid (3:1 v/v).The experiments followed ethical protocols approved by the Ethics Committee nº018/2014 (UNIPAMPA) and SISBIO: 44173-3.

Fluorescence in situ Hybridization (FISH)
Gallus gallus (GGA) chromosome probes from 1 to 14 obtained by flow-sorting and labeled with biotin-16-dUTP or digoxigenin-11-dUTP (Roche Diagnostics, Mannheim, Germany) by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) (Telenius et al., 1992) were hybridized to metaphase chromosomes of G. melanops, following standard protocols, as described previously by de Oliveira et al. (2010).In this study ZW chromosome probes of G. gallus were not used.The FISH results were analyzed using a Zeiss Imager 2 microscope, 63x objective and images were captured using Axiovision 4.8 software (Zeiss, Germany).At least 10 metaphases were analyzed to confirm the hybridizations signals.Final editing of images was performed using Adobe Photoshop CC software.For chromosomal evolution inferences, we used chromosome painting data from Fulica atra, Gallinula chloropus and Aramides cajaneus (Nanda et al., 2011;Furo et al., 2020), also these data were plotted in a phylogenetic tree proposed by Garcia et al., (2014), to clarify the phylogenetic position of some Rallidae species.
The bacterial artificial chromosomes (BACs) were selected from the genome library of G. gallus or Taeniopygia guttata (Zebra finch) for the microchromosomes GGA17-28, following O' Connor et al. (2019).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).

Karyotype and chromosome painting with G. gallus probes
The karyotype of Gallinula melanops (2n=80) is composed of 11 pairs of macrochromosomes, including the Z and W, and 29 pairs of microchromosomes, which corroborates previous findings (Gunski et al., 2019).The first and second chromosome pairs are submetacentrics, the third, fourth and fifth pairs are metacentrics and the other chromosome are telocentrics.The sex chromosomes ZW are submetacentrics, and the W is larger than the Z chromosome due to the accumulation of repetitive DNA, as described by Gunski et al. (2019) (Figure 1).
A comparison of chromosome morphology available for this family shows that generally the first six pairs are biarmed, while the remaining macrochromosomes are telocentrics, except in P. albicollis, which has a karyotype of only biarmed macrochromosomes (Table 3).Compared to other Gruiformes, which usually follow a chromosome pattern common to each family, there is great diversity in chromosomal morphology in Rallidae species, due to inversions, fusions and fissions, which play an important role in the karyotype evolution within this family (Furo et al., 2015).
Furthermore, it was possible to confirm that each chicken or Zebra finch BAC was conserved as a distinct element in each microchromosome of G. melanops (Figure 3).Microchromosomes are highly conserved in bird karyotypes, with rearrangements involving these elements detected only is some orders, such as Psittaciformes and Falconiformes (O'Connor et al., 2019).Despite the conservation of microchromosomes in the karyotype of G. melanops, the increase in diploid number in F. atra (2n=92) can be explained by extensive fission of microchromosomes.
According to Sangster et al. (2015), G. melanops should be included in the genus Porphyiro.However, the chromosome morphology data of P. porphyrio, the only species from this genus with a known karyotype, do not show many similarities with the karyotype of G. melanops.For example, in G. melanops the macrochromosomes 1-5 are biarmed and 6-10 are telocentrics, whereas in P. porphyrio chromosomes 1-7 are biarmed and 8-10 are acrocentrics (Table 3).
The phylogenetic relationships within the 'Clade Fulica' (genera Fulica, Gallinula and Porzana), based on mitochondrial DNA (mtDNA), suggest that this group is paraphyletic (Sangster et al., 2015).In the analysis based on mitochondrial and nuclear genes (Cytb, COI, 16S, FGB-7, RAG-1), performed by Garcia et al. (2014), G. melanops (2n=80) was recovered as the sister clade to G. chloropus (2n=78).However, these species share the same chromosome rearrangements, which could indicate that their common ancestor would contain the fission into GGA4 and GGA5, aside from the association between GGA4/GGA5 and GGA6/GGA7.Furthermore, other phylogenetic analyses using mtDNA recovered G. melanops in a more basal position within Clade Fulica (species of genera Fulica and Gallinula) (Garcia et al., 2014;Sangster et al., 2015), consistent with the chromosome painting data that indicate the karyotypic similarity between G. melanops and G. chloropus (Figure 5).
Additionally, the clade Aramides would be sister group to clade Fulica, despite the species A. cajaneus (Clade Aramides) not showing the fission into GGA4 and GGA5, or the fusions between GGA4/GGA5 and GGA6/GGA7 (Furo et al., 2020).Thus, the last common ancestor of these clades would have a karyotype similar to the putative avian ancestral karyotype (Furo et al., 2020).
In conclusion, the comparative chromosome painting reveals that G. melanops has a similar karyotype to G. chloropus and does not support the separation of these species into different genera.They are supported as sister species.Additionally, as in most birds studied so far, the microchromosomes are conserved as distinct pairs and do not take part in interchromosomal rearrangements (fusions or fissions).The results illustrate the value of comparative chromosome painting and BAC mapping in phylogenetic studies.

Figure 4 -Figure 5 -
Figure 4 -Homology map between chromosomes of Gallus gallus and Gallinula melanops determined by FISH experiments with GGA painting probes (GGA1-14) and GGA BAC clones of microchromosomes from 17-28.A) reporting the colour guide to GGA painting probes and BAC clones, and B) reporting the homology between the chromosomes of these two species.Segments not hybridized are indicated in white.Chicken probe 11 did not work and BACs 15 and 16 were not used in this study.The BAC 20 of Zebra finch is represented by the BAC 20 of chicken.