Cytotaxonomy and karyoevolution of the genus Crenicichla ( Perciformes , Cichlidae )

Karyotypes of six cichlid species of the genus Crenicichla were investigated. The species C. cincta, C. inpa, C. reticulata, C. lugubris, and C. cf. johanna were collected from Amazon basin, and C. britskii was collected from the Paraná-Paraguai basin. All of the analysed species showed 2n = 48 chromosomes; however, C. cincta, C. lugubris, C. cf. johanna, and C. britskii had a karyotype formula of 8M-SM+40ST-A, FN = 56, while C. inpa and C. reticulata exhibited the formula 6M-SM+42ST-A, FN = 54. Analysis of active Ag-NORs revealed two NOR-bearing chromosomes in all species; however, theses cistrons were located on different chromosome pairs and/or in different chromosome locations in each species. This condition bears evolutionary significance, since it is the main chromosome marker of the process of karyotypic evolution among the species of the genus Crenicichla. In general, C-banding revealed a similar constitutive heterochromatin pattern in all species, although it was possible to detect some features that led us to infer that Crenicichla also presents a species-specific heterochromatin pattern.

In the Perciformes group, cichlids are the most species-rich non-Ostariophysan freshwater fish family in the world, and one of the largest vertebrate families, with at least 1,300 species and estimates to approach 1,900 species (Kullander, 1998).The pike cichlids of the genus Crenicichla Heckel, 1840 figure among the most speciesrich genera in the family and the genus includes about 80 species.Its geographical distribution comprises most of cis-Andean South America, from Trinidad to Argentina, including the Orinoco, Amazon, and Paraguay-Paraná basins and most coastal rivers between Guianas and the La Plata River (Kullander, 1982;Ploeg, 1991;Lucena and Kullander, 1992;Kullander, 1998Kullander, , 2003)).Based on characteristics such as body shape, the number of scale series below lateral lines, and presence or absence of a humeral blotch, the Crenicichla species are included into nine groups (lugubris, acutirostris, lacustris, missioneira, reticulata, saxatilis, scotti, wallacii, and macrophtalma) or into two more inclusive groups: large-scaled and small-scaled spe-cies (Ploeg, 1991;Lucena and Kullander, 1992;Kullander and Lucena, 2006).
The family Cichlidae is considered a group with a conservative chromosome macrostructure, where 60% of the analysed species have a diploid number of 48 chromosomes (Feldberg et al., 2003).This characteristic has been proposed by some authors as a conserved character for this family, suggesting that the ancestral karyotype consisted of 48 acrocentric chromosomes (Thompson, 1979;Kornfield et al., 1979;Martins et al., 1995;Feldberg et al., 2003).
The aim of this paper is to update the karyotypic information on Crenicichla species and to present additional cytogenetic data for six species, and to evaluate the usefulness of cytotaxonomic information for the understanding of the evolution of pike cichlids.
Chromosome preparations were obtained from a kidney cell suspension using the standard air-drying technique of Bertollo et al. (1978).Fish were anesthetized in chilled water before being sacrificed.Mitotic induction with biological yeast (Oliveira et al., 1988) was performed in order to obtain a higher number of dividing cells.Nucleolar organizer regions (NORs) were identified by silver nitrate staining according to Howell and Black (1980), and C-banding to locate constitutive heterochromatin was performed using barium hydroxide according to Sumner (1972).When pos-sible, slides were stained sequentially with Giemsa, Cbanding, and silver nitrate solution (Ag-NOR) according to Centofante et al. (2002).Chromosomes were measured and arranged in decreasing order of size into two groups: metacentric-submetacentric (M-SM) and subtelocentric-acrocentric (ST-A) according to Levan et al. (1964) and Thompson (1979).The fundamental number (FN) or chromosome arm number was determined by counting M-SM chromosomes with two arms and ST-A with only one.
Table 1 summarizes the chromosome characteristics of the Crenicichla species that were analyzed in the present study and those available in the literature.These data are hierarchically organized according to the species group division proposed by Ploeg (1991)  A single pair of NORs was observed in all six species; however, these cistrons were located on different chromosome pairs and/or different locations.In C. cincta the Ag-NORs were located on the terminal region of the short arms of the first pair (M-SM); in C. inpa they were located on the interstitial region of the long arms of the first pair (M-SM); in C. reticulata the Ag-NORs were located on the interstitial region of the short arms of the first pair (M-SM); in C. lugubris the Ag-NORs were located on the terminal region of the long arms of the third pair (M-SM); in C. cf.johanna the Ag-NORs were located on the terminal region of the short arms of the 24 th pair (ST-A); and in C. britskii the Ag-NORs were located on the terminal region of the long arms of the fourth pair (M-SM).Heteromorphism in the size of the NORs was detected in all species, more markedly in C. britskii and C. cf.johanna (Figure 1A-F).
All the six analyzed species were characterized by several C-band positive heterochromatin blocks in the pericentromeric region of all chromosomes, although some of theses blocks were more conspicuous than others.In C. cincta, C. inpa and C. reticulata, the Ag-NORs were not coincident with heterochromatic blocks, although these ribosomal cistrons were closely adjacent to clearly evident C-bands.In contrast, in C lugubris, C. cf.Johanna, and C. britskii, Ag-NORs were coincident with C-bands.Moreover, some other conspicuous heterochromatic blocks could also be observed in the six species.C. reticulata presented an interstitial heterochromatic block on the long arms of the 10 th chromosome pair of its karyotype complement.C. lugubris showed an interstitial C-band on the long arms of the second chromosome pair, and C. cf.johanna has a terminal heterochromatic block on the long arms of the 19 th chromosome pair.In C britskii, it was possible to detect a heterochromatic block that occupies the whole short arms of the first chromosome pair (Figure 1 G-L).These unique heterochromatic blocks are chromosome markers that could also be useful in future comparisons among several Crenicichla species.
The relationships among Neotropical cichlids have been the object of evolutionary studies using morphological (Kullander, 1998), molecular (Farias et al., 2000), and karyological data (Feldberg et al., 2003).All these data agree that Neotropical cichlids form a monophyletic group.
According to Kullander (1998), Neotropical cichlids are organized in five major lineages, where the subfamily Cichlinae occupies a basal position and encompass only two genera: Cichla and Crenicichla.In a recent review, Feldberg et al. (2003) showed that Crenicichla and Cichla appear to fall into different karyoevolution directions.Cichla species exhibit only acrocentric chromosomes and seem to be the most primitive (Thompson, 1979;Alves-Brinn et al., 2004), while Crenicichla exhibits chromosomal variability in characteristics like karyotypic formula and fundamental chromosome number (Thompson, 1979;Feldberg and Bertollo, 1985a,b;Martins et al., 1995;Loureiro et al., 2000;present paper).According to Thompson (1979), the presence of a greater number of meta-submetacentric chromosomes characterizes a derived state, since the karyotype considered ancestral for this group has only acrocentric chromosomes.
Whereas Farias et al. (2000) proposed that Crenicichla might be related with Apistogramma forming the subfamily Geophaginae, Feldberg et al. (2003) showed that Crenicichla and Apistogramma fall into different karyoevolution directions with Apistogramma presenting 46 chromosomes.However, a closer look on the chromosomal data presented by the authors reveal that the gross karyotype macrostructure of Crenicichla and the remaining geophagines is quite similar.
In fact, Crenicichla species exhibit a diploid number of 48 chromosomes, except for Crenicichla sp. from the Paraná-Paraguay basin which supposedly possess 2n = 46 chromosomes (nome dos autores, unpublished data).Often, chromosomal variability can be attributed to problems in analysis, as well as to the interpretation of chromosome measurements.Special care must be taken when these comparisons are made among cichlids, since there are cases where cytogenetic differences indicated the existence of different species (Feldberg et al., 2003;Alves-Brinn et al., 2004).
Data regarding the prevalence of chromosomal rearrangements in Neotropical cichlids have been obtained mainly through conventional cytogenetic studies (Feldberg et al., 2003).Apparently, non-Robertsonian rearrangements, such as paracentric/pericentric inversions and translocation, might be postulated as the source of differentiation among Crenicichla species, taking into account the NORs carriers as a hallmark.Thus, the phenetic NOR character seems to be a good tool for differentiating Crenicichla species (Figure 1, Table 1).Of the 17 nominal Crenicichla species that have already been studied (Table 1), 13 had their NOR pattern determined.All 13 species exhibit a single NOR pattern, with the exception of a population of C. lepidota from the Paraná River.This population exhibits four NOR-bearing chromosome markers, characterizing a multiple NOR system (Martins et al., 1995).
According to Feldberg et al. (2003), the general trend in the family Cichlidae is to exhibit a single NOR system, with only one pair of NORs located on the largest chromosome of the complement.This chromosomal character seems to be plesiomorphic for cichlids.This is particularly true for the majority of the Crenicichla species.Every time that a large M-SM chromosome pair is observed, it usually bears Ag-NORs (except for Crenicichla britskii).When such a large M-SM chromosome pair is not so evident, then the Ag-NORs position is not on the first chromosome pair.In this way, C. reticulata, C. inpa, and C. cincta might be considered more primitive than C. cf.joahna, C. lugubris, and C. britskii, although the last species also presents a large M-SM chromosome pair in the complement.Feldberg et al. (2003) also reported that constitutive heterochromatin, in the few Neotropical cichlids analyzed so far, is present in the pericentromeric region of all chromosomes or in most of them.However, interspecific differences are clearly observed in Crenicichla, where interstitial and/or telomeric blocks, as well as regions at or near the NORs, reflect part of the chromosomal differentiation of the species.
The heterochromatin patterns of the species analyzed here are very similar; however, each species exhibits at Several studies in fish shave shown that the association of heterochromatin and NORs is an important element in chromosome differentiation.For example, Galetti et al. (1991) postulated for anostomids that heterochromatin associated with NORs may have favored breaks and consequent rearrangements during the chromosomal evolution.Furthermore, Ruiz et al. (1981) and Clark and Wall (1996) claimed that constitutive heterochromatin is often associated with ribosomal cistrons in the NORs of eukaryotic chromosomes.Thus, we are assuming for Crenicichla species that the association of heterochromatin and NORs would have played an important role in their chromosomal differentiation.
Our comparisons of cytogenetic patterns of the genera Cichla and Crenicichla make it clear that Crenicichla presents a more derived condition.It is interesting to note that Crenicichla possesses a much wider natural distribution than Cichla.Species of the C. lacustris group, which are restricted to the southern portion of the genus distribution, exhibit less-conserved characteristics than other members of this taxon.This may indicate that the dispersion and speciation of the genus Crenicichla involved non-Robertsonian rearrangements, principally inversions, and that these have occurred from North to South on the South American continent.

Table 1 -
and Lucena and Kullander Chromosome characteristics in the genus Crenicichla (2n = diploid number; KF = karyotypic formulaAll species analyzed in the present study exhibited 2n = 48 chromosomes.Although the overall karyotypes are uniform (Figure 1A-F), they could be separated into two groups, based on their chromosomal formulae: C. cincta, C. lugubris, C. cf.johanna, and C. britskii exhibit 8M-SM+40ST-A and FN = 56, and C. inpa and C. reticulata exhibit 6M-SM+42ST-A and FN = 54.No sex heteromorphism was detected in species where it was possible to analyze males and females.