Chromosome comparison among five species of Neotropical cichlids of Cichlasoma and Gymnogeophagus (Perciformes)

Abstract The genera Cichlasoma and Gymnogeophagus belong to the subfamily Cichlinae, the only one in Neotropical cichlids. Cichlasoma dimerus, C. paranaense, C. portalegrense, Gymnogeophagus rhabdotus, and G. lacustris were collected at different points in the Paranapanema and Paraguay basins and the Lagoon of Patos hydrographic system. In addition to conventional analysis, CMA3 fluorochrome staining, and FISH with 18S rDNA probe were performed. All species had a diploid number equal to 48, with interand intraspecific differences in karyotype formulae. All species presented a single AgNOR site, except G. rhabdotus and the C. paranaense population of the Paranapanema River, which revealed more than one pair of nucleolar chromosomes. AgNORs were coincident to 18S rDNA and CMA3. Heterochromatin was distributed in the pericentromeric chromosomal regions and coincident with NORs. For the first time, this work shows cytogenetic data for C. portalegrense, G. lacustris, and G. rhabdotus. Although some results reinforce the idea of conservative chromosome evolution of 2n in Cichlinae, interspecific and populational variations observed confirm that chromosomal rearrangements affect the microstructural karyotype diversification in this group of fish.


Introduction
Cichlidae represents the largest and most diverse family among Neotropical Perciformes, with about 1700 fish species (Eschmeyer and Fong, 2018). Based on morphological and molecular data, Smith et al. (2008) proposed that all Neotropical cichlids belong to a single subfamily, Cichlinae, as a monophyletic group. This subfamily is subdivided into seven tribes: Astronotini, Chaetobranchini, Cichlasomatini, Cichlini, Geophagini, Heroini, and Retroculini. The genera Cichlasoma and Gymnogeophagus belong to the Cichlasomatini and Geophagini tribes, respectively (Kullander, 2003). Cichlasoma presents a wide distribution, occurring in almost all Neotropical regions, from Mexico to the South of South America (Rican and Kullander, 2006). In contrast, Gymnogeophagus has a more restricted distribution, in which the majority of species is endemic to the coastal river drainage of Uruguay and southern Brazil, in the states of Rio Grande do Sul and Santa Catarina, with exception of G. balzanii, which presents a wider distribution (Reis and Malabarba, 1988).
Most of the species of Neotropical cichlids, approximately 60%, present a karyotype with 2n = 48, but a variation from 2n = 32 to 2n = 60 is observed, and chromosomal rearrangements have already been reported in the family (Feldberg et al., 2003;Poletto et al., 2010). Several cytogenetic analyses with the Cichlasomatini tribe show great chromosomal variation in this tribe (Feldberg et al., 2003) in contrast with low ecomorphological diversity, compared with other tribes, such as Geophagini (López-Fernandes et al., 2013), with few chromosomal data (Feldberg and Bertollo, 1984;Pires et al., 2010;Paiz et al., 2017). Hence, these tribes are of interest for cytogenetic studies.
Most cytogenetic studies on Neotropical cichlids are limited to the description of the karyotypic macrostructure (Thompson, 1979;Feldberg and Bertollo, 1985). In recent years, different classes of repetitive DNA have been used to better understand the karyotypic structure of Neotropical cichlids (Gross et al., 2010;Poletto et al., 2010). However, available information is restricted to a small number of species.
This work presents a comparative karyotype analysis of five species of cichlids: Cichlasoma paranaense, C. dimerus, C. portalegrense, Gymnogeophagus rhabdotus, and G. lacustris, using techniques of conventional and molecular chromosomal banding, and provides the first cytogenetic information for the last three species. The data presented are a contribution to a better understanding of the structure and karyotype evolution in this group of fish.

Materials and Methods
The species of Cichlasoma and Gymnogeophagus were collected from different localities of the Paranapanema (PR/SP) and Paraguay/MS hydrographic basins and the hydrographic system Lagoon of Patos/RS ( Table  1) Mitotic chromosomes were obtained by direct preparation removing the anterior kidney according to Bertollo et al. (1978) and then stained with 5% Giemsa in phosphate buffer (pH 6.8). The morphology of the chromosomes was determined based on the ratio of arms, as proposed by Levan et al. (1964). For determination of the fundamental number (FN), the meta-submetacentric (m-sm) chromosomes were considered biarmed and the subtelo-acrocentric (st-a) uniarmed.
Silver nitrate staining revealed active nucleolus organizer regions (AgNORs) and was performed according to Howell and Black (1980). The distribution of constitutive heterochromatin was analyzed by Giemsa C-banding after treatments with 0.1 M HCl, Ba(OH) 2 , and 2 X SSC (Sumner, 1972). GC-and AT-rich sites were detected with chromomycin A 3 (CMA 3 ) and 4',6-diamino-2-phenylindole (DAPI) according to Schweizer (1980). Fluorescence in situ hybridization (FISH) was performed according to the protocol of Pinkel et al. (1986), with modifications according to Gouveia et al. (2013), using an 18S rDNA probe (Hatanaka and Galetti Jr, 2004). Finally, the slides were analyzed on an epifluorescence microscope (Leica DM2000), equipped with a digital camera. Metaphase images were captured using the Leica Application Suite version 3.1.0. (Leica Microsystems).
AgNORs were located on a pair of chromosomes for all species, except for the population B of C. paranaense and G. rhabdotus, which showed three to four chromosomes bearing these regions (Figures 1 and 2, boxes). In the population B of C. paranaense, it was possible to observe a variation of two to three AgNORs in the terminal regions of the short arm of a submetacentric pair (pair 1) and the long arm of a subtelo-acrocentric chromosome (chromosome 11) (Figure 1d, box). In Gymnogeophagus rhabdotus, the AgNORs were located on st-a chromosomes: long arm of pair 5 and short arm of pair 12 (Figure 2a).
The other species of Cichlasoma, including population A of C. paranaense, presented terminal AgNOR on the short arm of one pair of meta-submetacentric chromosomes (Figures 1a-c, boxes); in G. lacustris AgNOR was located interstitially on the short arm of the largest metacentric pair 2 Pires et al.  ( Figure 2b). Staining with fluorochromes revealed CMA 3 + /DAPIcoincident with NORs in all species (Figures 1 and 2).
FISH with 18S rDNA probe demonstrated that C. dimerus, C. portalegrense, C. paranaense (population A), and G. lacustris, present two ribosomal cistrons corresponding to AgNORs (Figures 1a-c, and 2b, boxes). In the other two species, four ribosomal cistrons were observed: in pairs 5 and 12 in the terminal region of G. rhabdotus (Figure 2a, box), and in C. paranaense (population B) in the short arm of pair 1, in the long arm of chromosomes 5 and 11, and in interstitial and terminal regions, respectively (Figure 1d, box).
Heterochromatic regions were observed in the pericentromeric regions of the majority of chromosomes and associated with NORs in all species (Figure 3); C. paranaense also showed an interstitial marking on the long arm of a subtelo-acrocentric chromosome of pair 5 ( Figure  3d) corresponding to NOR, and in G. rhabdotus terminal heterochromatic blocks were observed in some chromosomes (Figure 3e).

Discussion
Despite conservation in diploid number, variations were found in the karyotype formulae of C. dimerus and C. paranaense (population B) in comparison to previously 4 Pires et al.  studied populations (Martins et al., 1995;Feldberg et al., 2003;Roncati et al., 2007;Poletto et al., 2010). Pericentric inversions seem to be the mechanism that predominantly contributed to these variations, since the diploid number was not altered, as observed by Thompson (1979), Feldberg et al. (2003), and Poletto et al. (2010) in other cichlid species. However, other rearrangement events cannot be ruled out in the family, as in Tilapia mariae, in which chromosomal fusion processes would explain the reduction of 2n to 40 chromosomes (Poletto et al., 2010), and in Symphysodon species, where successive translocation events, fissions, and/or fusions would have contributed to the formation of the most highly derived karyotype in the Cichlidae family (2n = 60) (Mesquita et al., 2008).
Recent studies show that the centromeres can be repositioned without any chromosomal rearrangement (Rocchi et al., 2012). This phenomenon of centromere repositioning could explain the difference in the karyotype formulae be-tween C. paranaense of the two localities, as also proposed by Schneider et al. (2013) for some species of cichlids.
Except for population B of C. paranaense and G. rhabdotus, which presented multiple NORs, all cichlids analyzed in the present study had only one nucleolar chromosomal pair, characterizing a single NOR system and confirming the ancestral condition proposed by Feldberg et al. (2003). However, differences in chromosome types and location of these sites were observed. These results are similar to those found in other species of Cichlasoma and Gymnogeophagus, such as C. facetum (Feldberg and Bertollo, 1985;Vicari et al., 2006), C. paranaense (Martins et al., 1995), and G. labiatus (Pires et al., 2010), presenting only a variation in the identification of the carrier chromosome, or in metacentric (Martins et al., 1995) or subteloacrocentric chromosomes (Vicari et al., 2006), evidencing once again that chromosomal rearrangements are occurring in the group.