Chromosomal polymorphism and speciation in ...

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Martins-Santos* Isabel Cristina, Ana Luiza De Brito Portela-Castro and Horácio. Ferreira Julio Jr. ..... tiple chromosome polymorphism in Gobius pagan-.
CARYOLOGIA

Vol. 58, no. 2: 95-101, 2005

Chromosomal polymorphism and speciation in Laetacara cf. dorsigera (Teleostei, Perciformes, Cichlidae) from the river Parana´ PR Brazil Martins-Santos* Isabel Cristina, Ana Luiza De Brito Portela-Castro and Hora´cio Ferreira Julio Jr. Department of Cell and Genetic Biology/Nupelia, State University of Maringa´; Department of Cell Biology and Genetics, Avenida Colombo, 5790; 87.020-900, Maringa´, Parana´, Brazil.

Abstract — Four sympatric and syntopic karyotype forms of Laetacara cf. dorsigera, centric fusion-originated were analyzed. Specimens with 2n=46, 2n=45, 2n=44 and 2n=43 chromosomes and differences with regard to the number of metacentric chromosomes, inversely proportional to their diploid number, were reported. Karyotypic formulae of the four cytotypes were: cytotype A=2M + 44A; cytotype B=3M + 42A; cytotype C=4M + 40A and cytotype D=5M + 38A and fundamental number constant (NF=48). Two centric fusion events are suggested: a) Robertsonian translocation, which originated the first and second metacentric chromosomal pairs; b) isochromosome formed by the fusion of acrocentric homologous chromosomes in cytotypes with odd chromosomes. Genetic balance in the four karyotype forms of Laetacara cf. dorsigera seemed to have been determinant to their reproductive isolation. The presence of individuals with different karyotypic structures living in sympatry and syntopy and the absence of “intermediate individuals” (recombinants), that would have been expected in a panmitic population, permit one to conclude that probably form 4 cryptic species exist in the population. Key words: Centric fusion, chromosomal polymorphism, Cichlidae, Laetacara cf. dorsigera, Perciformes, sympatric evolution.

INTRODUCTION Cichlids comprise a species-rich family with an extremely fast speciation rate and form highly specialized fish, according to some authors (Kornfield 1978; Thompson 1979). Whereas Africa has the highest diversity of cichlids, with South America as second, they represent only 6% of freshwater fish in Brazil and, in contrast to their endemism in African lakes, most species are widely distributed (Lowe-McConnel 1969). Since this group of fish doesn’t have a very specific reproduction period, with more than one annual generation, the fixation of new genetic recombinations is possible. Further, the fact they are territorial fishes favors the evolution process. Cytogenetic studies have shown a highly conservative karyotypic evolution for this family. The majority of the species has diploid number 2n=48 chromosomes, low karyotypic structure variability

* Corresponding author: phone: 00 55 44 32614342; fax: 00 55 44 32634839; e-mail: icmdsantos @uem.br.

and many acrocentric chromosomes. The karyotypic stability in the family may be also observed by the absence of sexual chromosome differentiation mechanisms or numerical intra- and interindividual variations. In spite of this stability, intraspecies karyotypic differences in the chromosome structure have been reported in populations of Geophagus brasiliensis (Michele and Takahashi l977; Thompson 1979; Feldberg and Bertollo 1985; Martins-Santos et al. 1995), Cichlasoma citrinellum (Ojima et al. 1976; Thompson 1979), C. facetum (Oyhernart-Perera et al. 1975; Feldberg and Bertollo 1985) and Astronotus ocellatus (Thompson 1979; Feldberg and Bertollo 1985). Variations that maintain diploid number have been attributed to structural rearrangement mechanisms, mainly pericentric inversions, while those that modify the chromosome number are attributed to centric fission and fusion. The former have been the most frequent among the cichlids and greatly responsible for intra- and interspecific variations (Brum and Galleti 1997; Feldberg et al. 2003). However, chromosomal polymor-

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phism among the Perciformes which involve Robertsonian rearrangements have been mainly reported in the gobids (Thode et al. 1985; Giles et al. 1985). In the current research, four sympatric and syntopic karyotypic forms of Laetacara cf. dorsigera, originated from centric fusion, have been analyzed. These rearrangements produce genic unbalance in the intermediate recombinants descendents and may be probably involved in the speciation process these forms.

MATERIALS AND METHODS Specimens of Laetacara cf. dorsigera were collected from three lagoons (Guarana´, Garc¸as and Pontal), from the river Parana´, Parana´, Brazil. Mitotic chromosome preparations were obtained from kidney cells by air-drying technique described by Bertollo et al. (1978). Constitutive heterochromatin pattern was characterized according to the technique by Sumner (1972), whereas silver coloration method by Howell and Black (1980) was used to identify the nucleolus organizer region (NOR). Chromosomal types were identified by arm ratio criterion proposed by Levan et al. (1964).

RESULTS Analysis of metaphase chromosomes of Laetacara cf. dorsigera showed four different groups of individuals with diploid numbers 2n=46, 2n=45, 2n= 44 and 2n=43 chromosomes. Karyotype analysis revealed differences between the groups with regard to the number of metacentric chromosomes, inversely proportional to the diploid number, or rather, 2, 3, 4 and 5 metacentric chromosomes respectively (Fig.1a, b, c, d). Karyotypic formulae of the four cytotypes were: cytotype A=2M + 44A (Fig. 1a); cytotype B=3M + 42A (Fig. 1b); cytotype C= 4M + 40A (Fig. 1c) and cytotype D=5M + 38A (Fig. 1d). Fundamental number (NF=48) is constant. Table 1 shows the frequency of each karyotype in the populations of Guarana´, Garc¸as and Pontal lagoons, collected during 1992-2002. Since the three sites are intercommunicating during the flooding season, the four karyotypic structures were reported in the three collection site. However, the most frequent were those with karyotypes containing 3 and 4 metacentric chromosomes. Coloration with silver nitrate revealed only one NOR pair in the four cytotypes, in one of the

Table 1 — Frequency of each karyotype form in populations of Guarana´, Garc¸as and Pontal lagoons. Locality

43

44

45

46

Total

Guarana´ Garc¸as Pontal Total %

04 04 04 12 11.2

12 11 12 35 32.7

12 09 23 44 41.3

05 06 05 16 14.9

33 30 44 107 100

acrocentric chromosomes, probably the 7th acrocentric pair (Fig. 2). Constitutive heterochromatin pattern showed centromeric markings in all the karyotypic forms with NOR positive C band (Fig. 3a, b, c, d). Extra chromosomes, ranging 0 to 2, were reported only among the specimens with 2n=45 chromosomes (Fig. 4). Figure 5 shows the diagram of panmitic crossing of specimen of the 4 cytotypes with no intermediary individual in the population.

DISCUSSION Although scantily represented among Brazilian freshwater fishes, cichlids are widely distributed in all South American river basins. Cytogenetic studies on species of cichlids have shown variations in the diploid number, ranging from 38 to 60 chromosomes (Thompson 1979). However, species with 2n=48 chromosomes are the most frequent (65.4%) (Scheel 1973; Ojima et al. 1976; Michele and Takahashi 1977; Thompson 1979; Feldberg and Bertollo 1985; Martins et al. 1995; Brum and Galetti Jr 1997). Taking into account the hypothesis of Thompson (1979) that the cichlids’ karyotype ancestral must have had 48 acrocentric chromosomes, and considering the fact that diploid numbers 46, 45, 44 and 43 chromosomes found in Laetacara cf. dorsigera are inversely proportional to the number of metacentric chromosomes and that their fundamental number is constant, it might be suggested that the 4 karyotypes were originated from chromosomal centric fusion rearrangements characterizing a Robertsonian translocation. According by Feldberg et al. (2003) one of the evolutionary trends of the subfamily Cichlinae, Geophaginae and Cichlasomatinae (that includes genus Laetacara) is characterized by a decrease in diploid number in parallel with a large number of bi-armed chromosomes (M-SM), suggesting chromosomal fusions and pericentric inversions. According to Medrano et al. (1988), a parallelism exists between the occurrence of chromo-

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Figure 1 — Karyotype of the Laetacara cf. dorsigera a) Cytotype A with 2n=46 chromosome; b) Cytotype B with 2n=45 chromosomes; c) Cytotype C with 2n=44 chromosomes and d) Cytotype D with 2n=43 chromosomes.

some banding and the degree of compartmentalization or low DNA heterogeneity of the genome. Thus, differences in the compartmentalization by base composition when comparing the genomes of warm-blooded and cold-blooded vertebrates (Cuny et al. 1981) have been considered limiting factors to obtain multiple structural bands of fish. The presence of exclusively centromeric C bands and the difficulty of longitudinal banding production in fishes have failed to determine which chromosomes were involved in these rearrangements. NOR analysis in the four karyotypic forms showed the presence of only one nucleolar pair in one of the acrocentric chromosomes, common in

most cichlids. This fact indicates that this pair is not involved in the rearrangements occurred during formation of the cytotypes different. Results show the presence of B chromosomes in cytotype 3 M. Among the species of cichlids studied, B chromosomes had been detected in germinative cells of Gymnogeophagus balzanii (Feldberg and Bertollo l984) and mitotic cells of Cichla monoculus, Cichla sp., and Crenicichla reticulata (feldberg et al. 2004). Martins et al. (1995) reported the occurrence of “chromatin corpuscles” and stated that this structure might have been a possible loss of chromatin due to chromosomal rearrangements. However, the au-

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Figure 2 — Mitotic metaphase showing NOR (arrow) and chromosomes involved in the Robertsonian translocation (arrowhead) in the four Laetacara cf. dorsigera cytotypes.

thors do not discard the existence of supranumerary chromosomes. According to Volobujev (1981), the origin of B chromosomes may be explained as the remaining portions of structural rearrangements or the results of chromosomal nondisjunction. In this manner, the B chromosomes of Laetacara cf. dorsigera seem to have originated from the loss of segments evolving the centromeric region during the fusion process of two acrocentric chromosomes. The fact that these karyotype forms have been maintained in the population during several generations indicates a stable genetic balance and suggests that two centric fusion events must have occurred: a) Robertsonian translocation has given rise to the first chromosomal pair in the four cytotypes (Fig. 1) and the second chromosomal pair in the cytotypes C and D (Fig. 1 c, d); b) isochromosome formed by the fusion of acrocentric homologous chromosomes in odd chromosomes cytotypes (2n=45 and 43 chromosomes, Fig. 1b, d). The genetic balance reported in the four karyo-

type forms of Laetacara cf. dorsigera seems to have determined their reproductive isolation. Therefore, in spite of the great number of specimens analyzed, the intermediate forms expected from random crossings have not been observed in the population (Table 1 and Fig. 5). Specimens with different karyotypic structures living in sympatry and syntopy and the absence of “intermediate individuals” (recombinants), normally expected in a panmitic population (Fig. 5), permit one to conclude that probably 4 cryptic species exist in this population. Therefore, only descendants of crossings between individuals of the same cytotypes are viable in this population, which, in turn, also indicates their reproductive isolation. Reproductive isolation probably occurred because of the production of unbalanced gametes that carry loss and/or addition of the chromosomes involved in the original rearrangements. However, high frequency of karyotypic forms with 3 and 4 metacentric chromosomes (Table 1)

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Figure 3 — C-banding patterns of Laetacara cf. dorsigera a) Cytotype A with 2n=46 chromosome; b) Cytotype B with 2n=45 chromosomes; c) Cytotype C with 2n=44 chromosomes and d) Cytotype D with 2n=43 chromosomes.

may be due to some balanced recombinants, which, because of similarity between individuals with one only 2nd metacentric translocated chromosome and individuals with isochromosome in odd forms, are computed as such. Thus the intermediary recombinant 3M + 42A (Fig. 5, arrow) from crossing of cytotypes 2M + 44A x 4M + 40A is balanced and viable, since it showed one 2nd metacentric chromosome originating from Robertsonian translocation and its respective homologous chromosomes are present free as acrocentric ones. The same occurs with the intermediary recombinant 4M + 40A (Fig. 5, arrow), descendant

of crossing between 5M + 38A and 3M +42 A. According to Futuyma (1992), the best criterion for the definition of species is not the degree of morphologic differences but the non-intercrossing of different forms in nature. The evidence of reproductive isolation of two populations is frequently indirect and may be inferred through the absence of individuals with presumed or recognized inherited features. Further, according to this author, a discrete difference in a single characteristic may indicate merely species polymorphism. However, when two or more alleles or characters distinguish two or more forms, the absence of recombinants

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Figure 4 — Mitotic metaphase of Laetacara cf. dorsigera with one (a) and two (b) B chromosomes (arrow).

Figure 5 — Diagram of random crossing of individual of the 4 cytotypes. Dark box indicates the specimen found in the population. Black chromosome = 1st metacentric chromosome pair; gray chromosome = 2nd metacentric chromosome pair; white chromosome = isochromosome; M = metacentric chromosome A = acrocentric chromosome. Arrow indicates balanced recombinant descendents.

polymorphism and speciation in laetacara cf. dorsigera

probably indicates that these forms are not intercrossing. This seems to be the case in Laetacara cf. dorsigera in the four cytotypes, if the involvement of three pairs of chromosomes (1st and 2nd metacentric and isochromosome) at the origin of the 4 cytotypes is considered. Nevertheless, more morphometric and molecular studies must be undertaken for a better understanding of cryptic species in this population. Acknowledgements — The authors are grateful to the CNPq for its financial support and to Nupelia for supplying the specimens analyzed.

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