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

Larissa Bettin Pires Mariana Campaner Usso Lucia Giuliano-Caetano Ana Lúcia Dias About the authors

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.

Keywords:
Chromosome banding; fish cytogenetics; karyotype diversification; ribosomal DNA

Introduction

Cichlidae represents the largest and most diverse family among Neotropical Perciformes, with about 1700 fish species (Eschmeyer and Fong, 2018Eschmeyer WN and Fong JD (2018) Species by family/subfamily in the catalog of fishes, Eschmeyer WN and Fong JD (2018) Species by family/subfamily in the catalog of fishes, http://researcharchive.calacademy.org/research/Ichthyology/catalog/SpeciesByFamily.asp (accessed 15 January 2018).
http://researcharchive.calacademy.org/re...
). Based on morphological and molecular data, Smith et al. (2008Smith WL, Chakrabarty P and Sparks JS (2008) Phylogeny, taxonomy, and evolution of Neotropical cichlids (Teleostei: Cichlidae: Cichlinae). Cladistics 24:625-641.) 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, 2003Kullander SO (2003) Family Cichlidae. In: Reis RE, Kullander SO and Ferraris Jr CJ (eds) Check list of the freshwater fishes of South and Central America. EdiPUCRS, Porto Alegre, p 605.). Cichlasoma presents a wide distribution, occurring in almost all Neotropical regions, from Mexico to the South of South America (Rican and Kullander, 2006Rican O and Kullander SO (2006) Characterand tree-based delimitation of species in the Cichlasoma‘ facetum group (Teleostei, Cichlidae) with the description of a new genus. J Zool Syst Evol Res 44:136-152.). 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, 1988Reis RE and Malabarba LR (1988) Revision of the neotropical cichlid genus Gymnogeophagus Ribeiro, 1918, with descriptions of two new species (Pisces, Perciformes). Rev Bras Zool 4:259-305.).

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., 2003Feldberg E, Porto JIR and Bertollo LAC (2003) Chromosomal changes and adaptation of cichlid fishes during evolution. In: Val AL and Kapoor BG (eds) Fish adaptations. Science publishers, Enfield, 418 p.; Poletto et al., 2010Poletto AB, Ferreira IA, Cabral-de-Mello DC, Nakajima RT, Mazzuchelli J, Ribeiro HB, Venere PC, Nirchio M, Kocher TD and Martins C (2010) Chromosome differentiation patterns during cichlid fish evolution. BMC Genetics 11:50.). Several cytogenetic analyses with the Cichlasomatini tribe show great chromosomal variation in this tribe (Feldberg et al., 2003Feldberg E, Porto JIR and Bertollo LAC (2003) Chromosomal changes and adaptation of cichlid fishes during evolution. In: Val AL and Kapoor BG (eds) Fish adaptations. Science publishers, Enfield, 418 p.) in contrast with low ecomorphological diversity, compared with other tribes, such as Geophagini (López-Fernandes et al., 2013López-Fernández H, Arbour JH, Winemiller KO and Honeycutt RL (2013) Testing for ancient adaptive radiations in Neotropical cichlid fishes. Evolution 67:1321-1337.), with few chromosomal data (Feldberg and Bertollo, 1984Feldberg E and Bertollo LAC (1984) Discordance in chromosome number among somatic and gonadal tissue cells of Gymnogeophagus balzanii (Pices: Cichlidae). Braz J Genet 4:639-645.; Pires et al., 2010Pires LB, Giuliano-Caetano L and Dias AL (2010) Cytogenetic characterization of Geophagus brasiliensis and two species of Gymnogeophagus (Cichlidae: Geophaginae) from Guaíba Lake, RS, Brazil. Folia Biol (Krakow) 58:29-34.; Paiz et al., 2017Paiz LM, Baumgärtner L, da Graça WJ, Margarido VP and Pavanelli CS (2017) Cytogenetics of Gymnogeophagus setequedas (Cichlidae: Geophaginae), with comments on its geographical distribution. Neotrop Ichthyol 15:e160035.). 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, 1979Thompson KW (1979) Cytotaxonomy of 41 species of Neotropical Cichlidae. Copeia 4:679-691.; Feldberg and Bertollo, 1985Feldberg E and Bertollo LAC (1985) Karyotypes of 10 species of Neotropical cichlids (Pisces, Perciformes). Caryologia 38:257-268.). In recent years, different classes of repetitive DNA have been used to better understand the karyotypic structure of Neotropical cichlids (Gross et al., 2010Gross MC, Schneider CH, Valente GT, Martins C and Feldberg E (2010) Variability of 18S rDNA locus among Symphysodon fishes: Chromosomal rearrangements. J Fish Biol 76:1117-1127.; Poletto et al., 2010Poletto AB, Ferreira IA, Cabral-de-Mello DC, Nakajima RT, Mazzuchelli J, Ribeiro HB, Venere PC, Nirchio M, Kocher TD and Martins C (2010) Chromosome differentiation patterns during cichlid fish evolution. BMC Genetics 11:50.). 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). The specimens were deposited in the Museum of Zoology at the State University of Londrina (MZUEL) under the voucher numbers: 3937 (Cichlasoma paranaense - Taquari), 3479 (C. paranaense - Paranapanema), 13128 (C. dimerus), 4860 (C. portalegrense), 20102 (Gymnogeophagus rhabdotus), and 20103 (G. lacustris). For convenience, different populations of C. paranaense were called population A (Taquari) and population B (Paranapanema), as shown in Table 1. The samples were collected with the permission of the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA), protocol number 11399-1. We also obtained permission from the research ethics committee of the State University of Londrina (Animal Use Ethics number: CEUA 5579.2018.72).

Table 1
Collection sites and hydrographic basins of Cichlidae specimens analyzed. MS = Mato Grosso do Sul; PR = Paraná; RS = Rio Grande do Sul.

Mitotic chromosomes were obtained by direct preparation removing the anterior kidney according to Bertollo et al. (1978)Bertollo LAC, Takahashi CS and Moreira-Filho O (1978) Cytotaxonomic considerations on Hoplias lacerdae (Pisces, Erythrinidae). Braz J Genet 1:103-120. 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)Levan A, Fredga K and Sandberg AA (1964) Nomenclature for centromeric position on chromosome. Hereditas 52:201-204.. 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)Howell WM and Black DA (1980) Controlled silver staining of nucleolus organizing regions with a protective colloidal developer: a one step method. Experientia 36:1014-1015.. 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, 1972Sumner AT (1972) A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 74:304-306.). GCand AT-rich sites were detected with chromomycin A3 (CMA3) and 4’,6-diamino-2-phenylindole (DAPI) according to Schweizer (1980)Schweizer D (1980) Simultaneous fluorescent staining of R bands and specific heterochromatic regions (DA/DAPI) in human chromosomes. Cytogenet Cell Genet 27:190-193.. Fluorescence in situ hybridization (FISH) was performed according to the protocol of Pinkel et al. (1986)Pinkel D, Straume T and Gray JW (1986) Cytogenetic analysis using quantitative, high sensitivity fluorescence hybridization. Proc Natl Acad Sci USA 83:2934-2938., with modifications according to Gouveia et al. (2013)Gouveia JG, Moraes VPO, Sampaio TR, Rosa R and Dias AL (2013) Considerations on karyotype evolution in the genera Imparfinis Eigenmann and Norris 1900 and Pimelodella Eigenmann and Eigenmann 1888 (Siluriformes: Heptapteridae). Rev Fish Biol Fisheries 23:215-227., using an 18S rDNA probe (Hatanaka and Galetti Jr, 2004Hatanaka T and Galetti Jr PM (2004) Mapping of the 18S and 5S ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, 1829 (Characiformes, Prochilodontidae). Genetica 122:239-244.). 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).

Results

All specimens of Cichlasoma and Gymnogeophagus presented a diploid number (2n) equal to 48; however, different karyotype formulae were found: 12m-sm + 36st-a and a fundamental number (NF) equal to 60 for Cichlasoma dimerus (Figure 1a), 14m-sm + 34st-a (NF = 62) for C. portalegrense and population A of C. paranaense (Figures 1b and 1c, respectively) and 4m-sm + 44 st-a (NF = 52) for the population B of C. paranaense (Figure 1d). Gymnogeophagus rhabdotus showed 6m-sm + 42st-a (NF = 54), and G. lacustris 8m-sm + 40st-a (NF = 56) (Figures 2a and 2b, respectively). In the latter, an interstitial secondary constriction was identified in the short arm of the largest chromosomal pair, with small heteromorphism (Figure 2b, Table 2). No differences were observed between the karyotypes of males and females.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).

Figure 1
Karyotype and chromosome pairs with silver nitrate staining, FISH with 18S rDNA probe, and CMA3/DAPI in Cichlasoma dimerus (a), C. portalegrense (b), and C. paranaense, populations A (c) and B (d), respectively.

Figure 2
Karyotype and chromosome pairs with silver nitrate staining, FISH with 18S rDNA probe, and CMA3/DAPI in Gymnogeophagus rhabdotus .(a) and G. lacustris (b), respectively.

Table 2
Karyotype results for the species of Cichlasoma and Gymnogeophagus analyzed in this study: 2n = diploid number, FN = fundamental number, SC= secondary constriction, NORs = nucleolar organizer regions; CMA3 = chromomycin A3.

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 (Figure 2b). Staining with fluorochromes revealed CMA +/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).

Figure 3
Somatic metaphases after C banding in Cichlasoma dimerus (a), C. portalegrense (b), C. paranaense, populations A (c) and B (d), Gymnogeophagus rhabdotus (e) and G. lacustris (f), respectively. The arrows indicate the NORs...

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 studied populations (Martins et al., 1995Martins IC, Portella-Castro ALB and Júlio JRHF (1995) Chromosomes analysis of 5 species of the Cichlidae family (Pisces, Perciformes) from the Parana river. Cytologia 60:223-231.; Feldberg et al., 2003Feldberg E, Porto JIR and Bertollo LAC (2003) Chromosomal changes and adaptation of cichlid fishes during evolution. In: Val AL and Kapoor BG (eds) Fish adaptations. Science publishers, Enfield, 418 p.; Roncati et al., 2007Roncati HA, Pastori MC and Fenocchio AS (2007) Cytogenetic studies and evolutive considerations on fishes of the amily Cichlidae (Perciformes) from Parana River (Argentina). Cytologia 72:379-384.; Poletto et al., 2010Poletto AB, Ferreira IA, Cabral-de-Mello DC, Nakajima RT, Mazzuchelli J, Ribeiro HB, Venere PC, Nirchio M, Kocher TD and Martins C (2010) Chromosome differentiation patterns during cichlid fish evolution. BMC Genetics 11:50.). 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)Thompson KW (1979) Cytotaxonomy of 41 species of Neotropical Cichlidae. Copeia 4:679-691., Feldberg et al. (2003)Feldberg E, Porto JIR and Bertollo LAC (2003) Chromosomal changes and adaptation of cichlid fishes during evolution. In: Val AL and Kapoor BG (eds) Fish adaptations. Science publishers, Enfield, 418 p., and Poletto et al. (2010)Poletto AB, Ferreira IA, Cabral-de-Mello DC, Nakajima RT, Mazzuchelli J, Ribeiro HB, Venere PC, Nirchio M, Kocher TD and Martins C (2010) Chromosome differentiation patterns during cichlid fish evolution. BMC Genetics 11:50. 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., 2010Poletto AB, Ferreira IA, Cabral-de-Mello DC, Nakajima RT, Mazzuchelli J, Ribeiro HB, Venere PC, Nirchio M, Kocher TD and Martins C (2010) Chromosome differentiation patterns during cichlid fish evolution. BMC Genetics 11:50.), 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., 2008Mesquita DR, Porto JIR and Feldberg E (2008) Chromosomal variability in the wild ornamental species of Symphysodon (Perciformes: Cichlidae) from Amazon. Neotrop Ichthyol 6:181-190.).

Recent studies show that the centromeres can be repositioned without any chromosomal rearrangement (Rocchi et al., 2012Rocchi M, Archidiacono N, Schempp W, Capozzi O and Stanyon R (2012) Centromere repositioning in mammals. Heredity 108:59-67.). This phenomenon of centromere repositioning could explain the difference in the karyotype formulae between C. paranaense of the two localities, as also proposed by Schneider et al. (2013)Schneider CH, Gross MC, Terencio ML, Artoni RF, Vicari MR, Martins C and Feldberg E (2013) Chromosomal evolution of Neotropical cichlids: The role of repetitive DNA sequences in the organization and structure of karyotype. Rev Fish Biol Fisheries 23:201-214. 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)Feldberg E, Porto JIR and Bertollo LAC (2003) Chromosomal changes and adaptation of cichlid fishes during evolution. In: Val AL and Kapoor BG (eds) Fish adaptations. Science publishers, Enfield, 418 p.. 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, 1985Feldberg E and Bertollo LAC (1985) Karyotypes of 10 species of Neotropical cichlids (Pisces, Perciformes). Caryologia 38:257-268.; Vicari et al., 2006Vicari MR, Artoni RF, Moreira-Filho O and Bertollo LAC (2006) Basic and molecular cytogenetics in freshwater Cichlidae (Osteichthyes, Perciformes). Karyotypic conservationism and divergence. Caryologia 59:260-266.), C. paranaense (Martins et al., 1995Martins IC, Portella-Castro ALB and Júlio JRHF (1995) Chromosomes analysis of 5 species of the Cichlidae family (Pisces, Perciformes) from the Parana river. Cytologia 60:223-231.), and G. labiatus (Pires et al., 2010Pires LB, Giuliano-Caetano L and Dias AL (2010) Cytogenetic characterization of Geophagus brasiliensis and two species of Gymnogeophagus (Cichlidae: Geophaginae) from Guaíba Lake, RS, Brazil. Folia Biol (Krakow) 58:29-34.), presenting only a variation in the identification of the carrier chromosome, or in metacentric (Martins et al., 1995Martins IC, Portella-Castro ALB and Júlio JRHF (1995) Chromosomes analysis of 5 species of the Cichlidae family (Pisces, Perciformes) from the Parana river. Cytologia 60:223-231.) or subteloacrocentric chromosomes (Vicari et al., 2006Vicari MR, Artoni RF, Moreira-Filho O and Bertollo LAC (2006) Basic and molecular cytogenetics in freshwater Cichlidae (Osteichthyes, Perciformes). Karyotypic conservationism and divergence. Caryologia 59:260-266.), evidencing once again that chromosomal rearrangements are occurring in the group.

Gymnogeophagus rhabdotus presented two chromosomal pairs bearing ribosomal cistrons, an unusual pattern in the Geophaginae tribe, even though only few species were analyzed. However, there are reports of single NORs in Geophagus brasiliensis, Gymnogeophagus gymnogenys, and Satanoperca acuticeps (Brum et al., 1998Brum MJI, Oliveira CC, Voigt N and Côrrea MMO (1998) Karyotypic discrepancy between populations of Geophagus brasiliensis (Perciformes: Cichlidae), including the topotypical population, with possible taxonomic implications. J Comp Biol 3:177-184.; Feldberg et al., 2003Feldberg E, Porto JIR and Bertollo LAC (2003) Chromosomal changes and adaptation of cichlid fishes during evolution. In: Val AL and Kapoor BG (eds) Fish adaptations. Science publishers, Enfield, 418 p.; Pires et al., 2010Pires LB, Giuliano-Caetano L and Dias AL (2010) Cytogenetic characterization of Geophagus brasiliensis and two species of Gymnogeophagus (Cichlidae: Geophaginae) from Guaíba Lake, RS, Brazil. Folia Biol (Krakow) 58:29-34.), and multiple NORs only in Gymnogeophagus setequedas (Paiz et al., 2017Paiz LM, Baumgärtner L, da Graça WJ, Margarido VP and Pavanelli CS (2017) Cytogenetics of Gymnogeophagus setequedas (Cichlidae: Geophaginae), with comments on its geographical distribution. Neotrop Ichthyol 15:e160035.). In population B of C. paranaense, a chromosomal pair and two non-homologous chromosomes (chromosomes 5 and 11) with ribosomal cistrons were observed; chromosome 5 had an interstitial signal, coincident with the heterochromatin, but not corresponding to AgNOR sites. The occurrence of 18S rDNA sites in non-homologous chromosomes and the location of these genes in the long arm are uncommon in C. paranaense, and may indicate a particular characteristic of this species and population. According to the literature, most sites are located on the short arm of the chromosomes, and can be of the m-sm group (Poletto et al., 2010Poletto AB, Ferreira IA, Cabral-de-Mello DC, Nakajima RT, Mazzuchelli J, Ribeiro HB, Venere PC, Nirchio M, Kocher TD and Martins C (2010) Chromosome differentiation patterns during cichlid fish evolution. BMC Genetics 11:50.; Perazzo et al., 2011Perazzo G, Noleto RB, Vicari MR, Machado PC, Gava A and Cestari MM (2011) Chromosomal studies in Crenicichla lepidota and Australoheros facetus (Cichlidae, Perciformes) from extreme southern Brazil. Rev Fish Biol Fisheries 21:509-515.), or the st-a group (Vicari et al., 2006Vicari MR, Artoni RF, Moreira-Filho O and Bertollo LAC (2006) Basic and molecular cytogenetics in freshwater Cichlidae (Osteichthyes, Perciformes). Karyotypic conservationism and divergence. Caryologia 59:260-266.; Pires et al., 2008Pires LB, Giuliano-Caetano L and Dias AL (2008) Karyotype similarities among two populations of Geophagus brasiliensis (Perciformes, Cichlidae) from the Tibagi river basin/PR/Brazil. Caryologia 61:135-138.; Gross et al., 2010Gross MC, Schneider CH, Valente GT, Martins C and Feldberg E (2010) Variability of 18S rDNA locus among Symphysodon fishes: Chromosomal rearrangements. J Fish Biol 76:1117-1127.; Poletto et al., 2010Poletto AB, Ferreira IA, Cabral-de-Mello DC, Nakajima RT, Mazzuchelli J, Ribeiro HB, Venere PC, Nirchio M, Kocher TD and Martins C (2010) Chromosome differentiation patterns during cichlid fish evolution. BMC Genetics 11:50.).

In the Geophagini and Cichlasomatini tribes, as in Cichlidae in general, the pattern of single NORs is the most common one (Poletto et al., 2010Poletto AB, Ferreira IA, Cabral-de-Mello DC, Nakajima RT, Mazzuchelli J, Ribeiro HB, Venere PC, Nirchio M, Kocher TD and Martins C (2010) Chromosome differentiation patterns during cichlid fish evolution. BMC Genetics 11:50.), indicating that this characteristic can be considered plesiomorphic. Reports of multiple NORs, confirmed by FISH in cichlids, are scarce, and were reported in only seven species, including those described in this study: Mesonauta festivus (Poletto et al., 2010Poletto AB, Ferreira IA, Cabral-de-Mello DC, Nakajima RT, Mazzuchelli J, Ribeiro HB, Venere PC, Nirchio M, Kocher TD and Martins C (2010) Chromosome differentiation patterns during cichlid fish evolution. BMC Genetics 11:50.), Symphysodon aequifasciatus S. discus and S. haraldi (Gross et al., 2010Gross MC, Schneider CH, Valente GT, Martins C and Feldberg E (2010) Variability of 18S rDNA locus among Symphysodon fishes: Chromosomal rearrangements. J Fish Biol 76:1117-1127.), and Gymnogeophagus setequedas (Paiz et al., 2017Paiz LM, Baumgärtner L, da Graça WJ, Margarido VP and Pavanelli CS (2017) Cytogenetics of Gymnogeophagus setequedas (Cichlidae: Geophaginae), with comments on its geographical distribution. Neotrop Ichthyol 15:e160035.). It is worthy of note that four of these species of the genera Mesonauta and Symphysodon belong to the Heroini tribe, considered as derived within the subfamily Cichlinae. The NORs were CMA3 positive, rich in GC base pairs, as already shown in other species of Geophaginae and Cichlasomatinae by Loureiro et al. (2000Loureiro MA, Giuliano-Caetano L and Dias AL (2000) Cytogenetic characterization of two species of the genus Crenicichla (Pisces, Cichlidae). Cytologia 65:57-63.), Vicari et al. (2006Vicari MR, Artoni RF, Moreira-Filho O and Bertollo LAC (2006) Basic and molecular cytogenetics in freshwater Cichlidae (Osteichthyes, Perciformes). Karyotypic conservationism and divergence. Caryologia 59:260-266.), and Pires et al. (2010Pires LB, Giuliano-Caetano L and Dias AL (2010) Cytogenetic characterization of Geophagus brasiliensis and two species of Gymnogeophagus (Cichlidae: Geophaginae) from Guaíba Lake, RS, Brazil. Folia Biol (Krakow) 58:29-34.).

The heterochromatin in the species of this study maintains the typical general distribution pattern found in cichlids, in pericentromeric and terminal regions, as observed in different species of Cichlasoma (Martins et al., 1995Martins IC, Portella-Castro ALB and Júlio JRHF (1995) Chromosomes analysis of 5 species of the Cichlidae family (Pisces, Perciformes) from the Parana river. Cytologia 60:223-231.; Vicari et al., 2006Vicari MR, Artoni RF, Moreira-Filho O and Bertollo LAC (2006) Basic and molecular cytogenetics in freshwater Cichlidae (Osteichthyes, Perciformes). Karyotypic conservationism and divergence. Caryologia 59:260-266.; Roncati et al., 2007Roncati HA, Pastori MC and Fenocchio AS (2007) Cytogenetic studies and evolutive considerations on fishes of the amily Cichlidae (Perciformes) from Parana River (Argentina). Cytologia 72:379-384.) and Gymnogeophagus (Roncati et al., 2007Roncati HA, Pastori MC and Fenocchio AS (2007) Cytogenetic studies and evolutive considerations on fishes of the amily Cichlidae (Perciformes) from Parana River (Argentina). Cytologia 72:379-384.; Pires et al., 2010Pires LB, Giuliano-Caetano L and Dias AL (2010) Cytogenetic characterization of Geophagus brasiliensis and two species of Gymnogeophagus (Cichlidae: Geophaginae) from Guaíba Lake, RS, Brazil. Folia Biol (Krakow) 58:29-34.), except for the population B of C. paranaense, which also pre sented a chromosome with interstitial marking.

The location of NORs in terminal regions may be the factor that facilitates the transposition of these sequences to other chromosomes through translocation events, as observed by Gross et al. (2010Gross MC, Schneider CH, Valente GT, Martins C and Feldberg E (2010) Variability of 18S rDNA locus among Symphysodon fishes: Chromosomal rearrangements. J Fish Biol 76:1117-1127.) in some species of Symphysodon, which could explain the origin of the interstitial ribosomal cistron found in only a large subtelo-acrocentric chromosome (chromosome 5). In addition, the association of heterochromatin and ribosomal sites may be related to the variability in location and number of the active NORs, a pattern commonly observed in Neotropical cichlids (Schneider et al., 2013Schneider CH, Gross MC, Terencio ML, Artoni RF, Vicari MR, Martins C and Feldberg E (2013) Chromosomal evolution of Neotropical cichlids: The role of repetitive DNA sequences in the organization and structure of karyotype. Rev Fish Biol Fisheries 23:201-214.). Besides that, the differences between the populations may be due to their geographical isolation, so that this could facilitate the fixation of chromosomal rearrangements in the populations (Oliveira et al., 1988Oliveira CLF, Almeida-Toledo LM, Foresti F, Britski HA and Toledo-Filho SA (1988) Chromosome formulae of Neotropical freshwater fishes. Braz J Genet 11:577-624.), and possibly C. paranaense is a cryptic species.

The karyotype pattern observed in the species of this study reinforces the idea of a conservative diploid number in this group of fish. However, variations in karyotype formulae and location of NORs among the species and populations of C. paranaense confirm that chromosomal rearrangements are acting in the diversification of this group of fish.

Acknowledgments

The authors thank Prof. Dr. Luiz Roberto Malabarba of the Zoology Laboratory at the Universidade Federal do Rio Grande do Sul (UFRGS) for the identification of specimens. This research was supported by Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Finance Code 001 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The research received permission from the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA) to collect fish specimens.

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Internet Resources

Publication Dates

  • Publication in this collection
    22 Apr 2020
  • Date of issue
    2020

History

  • Received
    20 Dec 2018
  • Accepted
    22 Apr 2019
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