Nuclear DNA content of thirty species of Neotropical fishes

 

Margarida Lima Carvalho¹, Claudio Oliveira ² and Fausto Foresti ^2
1 Departamento de Ciências da Natureza, Universidade Federal do Acre, Rio Branco, Acre, Brasil.
² Departamento de Morfologia, Instituto de Biociências, UNESP, Campus de Botucatu, 18618-000 Botucatu, São Paulo, Brasil. Send correspondence to C.O.

 

 

ABSTRACT

The present paper reports nuclear DNA content in 30 Neotropical freshwater fish species and summarizes the data on other Neotropical species presented in the literature. Among Neotropical fishes, the nuclear DNA content ranges from 1.04 ± 0.09 pg/nucleus in Corydoras cf. simulatus (2n = 62) to 248.0 pg/nucleus in Lepidosiren paradoxa (2n = 38). A general analysis of the data obtained in the present study for each species showed that DNA measurements were practically constant at the individual level, while significant differences were observed among individuals of the same population. This observation was valid for all species analyzed and was more evident in those species that presented other karyotypic particularities such as sex chromosomes or supernumerary chromosomes. The importance of changes in nuclear DNA content in the evolutionary process of Neotropical fishes is discussed.

 

 

INTRODUCTION

Fishes are a large group of organisms comprising about 24,618 valid species, which constitute slightly more than one-half of the total number of living vertebrates (Nelson, 1994). Although the total number of living fish species from the Neotropical region is not known, this is probably the richest region of the world, with more than 5,000 species.

Cytogenetic studies of Neotropical fish have greatly expanded over the last few years, and haploid or diploid numbers are currently known for 706 species, including representatives from 207 genera in 38 families of freshwater species (Oliveira et al., 1996) and for about 39 species including representatives from 35 genera in 21 families of saltwater species (Oliveira and Foresti, 1994). In contrast, studies about nuclear DNA content were performed in only about 76 freshwater species and in two saltwater species (Table I).

 

Table I - DNA content data of Neotropical freshwater fishes.

*Diploid numbers cited by Oliveira et al. (1988).
**Mean values obtained by the authors for different lines.

 

Several evolutionary studies of Neotropical fishes have suggested the occurrence of drastic chromosomal rearrangements (e.g. Feldberg et al., 1993) and sometimes polyploidy (e.g. Oliveira et al., 1993b); thus, the determination of nuclear DNA content would be very important to better understand the overall evolutionary process in Neotropical fishes. Moreover, the wide variability of nuclear DNA content found among fishes led Ohno (1974) to state that comparative karyotype studies in this group would be very limited without information about genome size variation. The main objective of this paper was to employ a cytological technique to estimate the nuclear DNA content in fish erythrocytes from 30 Neotropical species and compare it with fishes previously studied, in order to provide new information for the study of evolutionary steps in this group of vertebrates.

 

MATERIAL AND METHODS

Thirty species of fishes were used in the present study. Their taxonomic status, collection site, and the number and sex of specimens analyzed are presented in Table II. After processing, all specimens were fixed and maintained in the fish collection of the Laboratory of Genetics and Fish Biology, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, São Paulo, Brazil.

 

Table II - Species analyzed.

 

Mitotic chromosome preparations were obtained from kidney and gill cells using the air-drying technique described by Foresti et al. (1993). Relative DNA content of each fish was determined according to the technique described by Gold and Price (1985) with some minor modifications. Blood was collected by caudal puncture and smeared near the frosted end of three slides. Blood smears from chicken, common carp, and rainbow trout served as standard references for DNA quantification. Absorbance values of fish nuclei from each slide were standardized as a percentage of the mean absorbance value of the three controls: chicken erythrocytes, common carp erythrocytes, and rainbow trout erythrocytes. For conversion to picograms (pg) of DNA, the standardized data were multiplied by known values for these species (2.5 pg, 3.4 pg, and 5.5 pg, respectively, according to Tiersch et al., 1989b). Chicken blood was obtained from a Hamphisare strain male; rainbow trout and common carp blood was obtained from domestic stocks. The slides were fixed for 20 min in 9:1 methanol-formaldehyde (37%), rinsed twice (10 min each) in distilled water, dehydrated in 70% ethanol (2 min) and 95% ethanol (2 min), and stored overnight under desiccative conditions at 4°C. The following day, individual batches of 20 randomized slides were hydrolyzed for 15 min in 1.0 N HCl at 60°C, rinsed briefly in distilled water, and stained for 2 h in Schiff’s reagent (Feulgen stain). Hydrolysis time was determined empirically as the point of maximum absorbance in a hydrolysis curve. Following staining, the slides were rinsed twice (10 min each) in SO₂ water and once (10 min) in  distilled water,  air dried  in the  dark and   analyzed.

Microdensitometry analysis was performed under a Zeiss microscope using a 100X oil-immersion objective. Analyses were done using OPTIMAS software version 4.1. For each fish, 15 nuclei were measured from two slides each (30 nuclei per individual). The third slide prepared from each specimen served as a backup in case of breakage. Only nuclei which were roughly spherical, homogeneously Feulgen-stained and from clear slide areas were chosen for measurement. The decision to measure 30 nuclei per individual was based on preliminary experiments (measurements of 10-300 cells per slide and over 20 slides) that showed an average coefficient of variation (per slide and per individual) of 1-3%. This means that measuring 30 nuclei per fish should distinguish a 1-3% difference in mean genome size at a- and b-probability levels of 0.05 (Gold et al., 1975).

 

RESULTS AND DISCUSSION

Including the present data, nuclear DNA content is known for 106 freshwater fish species from the Neotropical region and ranges from 1.04 ± 0.09 pg/nucleus in Corydoras cf. simulatus (2n = 62) to 248.0 pg/nucleus in Lepidosiren paradoxa (2n = 38) (Table I). However, a general analysis of Table I shows that only the families Callichthyidae (26 species) and Cyprinodontidae (11 species) were relatively well studied, while for all other groups only a few isolated data are known.

A general analysis of the data obtained in the present study for each species showed that at the individual level the coefficient of variation for DNA content was about 1%, while the coefficient of variation at the species level was about 4-6%. These data showed that while for each specimen the nuclear DNA content is practically constant, at the species or local population level there is a wider range of variation. This observation was valid for all species analyzed and was more evident in those species which presented other karyotypic particularities such as sex chromosomes or supernumerary chromosomes. Furthermore, it agrees with other studies, which also observed large differences in genome size among local populations and significant differences among specimens of the same population (Gold and Price, 1985; Gold and Amemiya, 1987; Johnson et al., 1987; Ragland and Gold, 1989; Tiersch et al., 1989a; Oliveira et al., 1992).

DNA content analysis of Characidium cf. fasciatus, which has a ZZ/ZW sex chromosome system (Maistro et al., 1996), showed that six males had less DNA than two females. On the other hand, one male presented more DNA than the females. In this species the W chromosome is larger than the Z chromosome. Considering that a variable number of medium-sized supernumerary chromosomes was found in the cells of the individuals analyzed, we suggest that they could be responsible for the additional DNA content in the cells of this male.

The presence of supernumerary chromosomes produced a large coefficient of variation in the values obtained for Moenkhausia sanctaefilomenae (9.15%), Steindachnerina insculpta (7.71%), and Prochilodus lineatus (9.42%). Since these species presented a variable number of supernumerary microchromosomes among different specimens of each population, the occurrence of such genomic elements could cause individual differences in DNA content in the populations. In the future we intend to analyze specifically the relationship between the number of supernumerary chromosomes and DNA content variation. The objective would be to estimate the size and real participation of these additional chromosomes in the genomic configuration of each individual.

As a general remark, it was observed that the variation of DNA content inside each family or sub-family analyzed was equal or similar to that observed for its respective order. Thus, among Anostomidae the nuclear DNA content ranged from 2.57 ± 0.14 pg for Leporinus friderici to 3.48 ± 0.15 pg for Leporinus octofasciatus (Table I). Considering that both species have 2n = 54 chromosomes and similar karyotypes with only metacentric and submetacentric chromosomes (Galetti Jr. et al., 1981), the difference in nuclear DNA content may be distributed among the chromosomes. Among the Tetragonopterinae, the nuclear DNA content ranged from 2.09 ± 0.15 for Astyanax bimaculatus to 3.74 ± 0.13 for A. scabripinnis (Table I). In this case, both species have 2n = 50 chromosomes; however, C- banding showed that A. scabripinnis presents large heterochromatic segments which are not observed in A. bimaculatus (Daniel-Silva, 1996). The occurrence of differences in nuclear DNA content among species of the same group was observed previously in different situations (Thode et al., 1985; Majumdar and McAndrew, 1986; Oliveira et al., 1992, 1993a; among others).

In a study of nuclear DNA content of 275 teleost species, Hinegardner and Rosen (1972) found that the modal value observed was 2.0 pg. Analysis of data available for the order Characiformes showed that species of this group present a mean value of 3.0 pg, which represents 1.0 pg above the mean value observed for fishes by Hinegardner and Rosen (1972). Analysis of the data available for the order Siluriformes showed that species of this group present a mean value of 3.4 pg, which represents 1.4 pg above the mean value observed for fishes by Hinegardner and Rosen (1972).

The more specialized or evolutionarily advanced fishes usually have less DNA than the more generalized forms; however, there are several exceptions, which may represent fish groups that are now in the process of evolutionary radiation (Hinegardner and Rosen, 1972). Although several exceptions to this rule are known, the data obtained for the orders Siluriformes and Characiformes are in accordance with it since their superorder Ostariophysi is a primitive group of fishes (Nelson, 1994).

Although the data obtained in the present investigation have significantly increased the number of species whose nuclear DNA content is known, these data are still insufficient when compared to the known number of fish species in the Neotropical region. Considering the wide variation of nuclear DNA content in the species already studied, future studies are necessary to better evaluate the relationship between changes in DNA content and the evolutionary process in Neotropical fishes.

 

ACKNOWLEDGMENTS

The authors are grateful to Dr. F. Langeani Filho for taxonomic identification of the specimens, R. Devidé, R.T.C. Dellevedove, M.R. Vieira, and P.C. Vissotto for technical assistance, and Dr. V. Dal Pai for the use of the micro-densitometer. The chicken blood used was provided by Mr. Heraldo Emílio from the Biotério Central of UNESP, Botucatu, SP, Brazil; common carp blood was provided by Mr. João Batista from CAUNESP, Jaboticabal, SP, and rainbow trout blood was provided by MsC. Marcos Guilherme Rigolino from Estação Experimental de Salmonicultura, Campos do Jordão, SP. Research supported by FAPESP, CAPES, CNPq, and FUNDUNESP. Publication supported by FAPESP.

 

 

RESUMO

O presente trabalho descreve os dados obtidos da análise de conteúdo de DNA nuclear de 30 espécies de peixes Neotropicais de água doce e apresenta uma revisão dos dados disponíveis sobre o conteúdo de DNA nuclear para as espécies Neotropicais. Entre os peixes Neotropicais, o conteúdo nuclear de DNA varia de 1,04 ± 0,09 pg/núcleo em Corydoras cf. simulatus (2n = 62) a 248,0 pg/núcleo em Lepidosiren paradoxa (2n = 38). Uma análise geral dos dados obtidos no presente estudo para cada espécie mostrou que a nível de indivíduo as medidas de DNA foram praticamente constantes, enquanto diferenças significativas foram observadas entre indivíduos da mesma população. Essa observação foi válida para todas as espécies analisadas e foi mais evidente naquelas espécies que apresentaram outras particularidades cariotípicas como a presença de cromossomos sexuais ou supranumerários. A importância das alterações no conteúdo de DNA nuclear no processo evolutivo dos peixes Neotropicais é discutida.

 

 

REFERENCES

Daniel-Silva, M.F.Z. (1996). Estudos citogenéticos comparativos em quatro espécies do gênero Astyanax (Pisces, Characidae). Master’s thesis, Instituto de Biociências, Universidade de São Paulo, IB/USP, SP.         [ Links ]

Feldberg, E., Porto, J.I.R., Nakayama, C. and Bertollo, L.A.C. (1993). Karyotype evolution in Curimatidae (Teleostei, Characiformes) from the Amazon region. II. Centric fissions in the genus Potamorhina. Genome 36: 372-376.         [ Links ]

Foresti, F., Oliveira, C. and Almeida-Toledo, L.F. (1993). A method for chromosome preparations from large specimens of fishes using in vitro short treatment with colchicine. Experientia 49: 810-813.         [ Links ]

Galetti Jr., P.M., Foresti, F., Bertollo, L.A.C. and Moreira- Filho, O. (1981). Heteromorphic sex chromosomes in three species of the genus Leporinus (Pisces, Anostomidae). Cytogenet. Cell Genet. 29: 138-142.         [ Links ]

Gold, J.R. and Amemiya, C.T. (1987). Genome size variation in North American minnows (Cyprinidae). II. Variation among 20 species. Genome 29: 481-489.         [ Links ]

Gold, J.R. and Price, H.J. (1985). Genome size variation among North American minnows (Cyprinidae). I. Distribution of the variation in five species. Heredity 54: 297-305.         [ Links ]

Gold, J.R., Bennett, L.F. and Gall, G.A.E. (1975). A Set of Tables for Determining Minimum Sample Sizes Necessary to Show Statistically Significant Differences Among Treatment Groups Using Analysis of Variance. Division of Agricultural Sciences, University of California, Davis, California,         [ Links ]

Gomes, V., Vazzoler, A.E.A.M. and Phan, V.N. (1983a). Estudos cariotípicos de peixes da família Scianidae (Teleostei, Perciformes) da região de Cananéia, SP, Brasil. 1. Sobre o cariótipo de Micropogonias furnieri (Desmarest, 1823). Bol. Inst. Oceanogr. 32: 137-142.         [ Links ]

Gomes, V., Vazzoler, A.E.A.M. and Phan, V.N. (1983b). Estudos cariotípicos de peixes da família Scianidae (Teleostei, Perciformes) da região de Cananéia, SP, Brasil. 2. Sobre o cariótipo de Menticirrhus americanus (Linnaeus, 1758). Bol. Inst. Oceanogr. 32: 187-191.         [ Links ]

Hinegardner, R. and Rosen, D.E. (1972). Cellular DNA content and evolution of teleostean fishes. Am. Nat. 106: 621-644.         [ Links ]

Johnson, O.W., Utter, F.M. and Rabinovitch, P.S. (1987). Interspecies differences in salmonid cellular DNA identified by flow cytometry. Copeia 1987: 1001-1009.         [ Links ]

Maistro, E.L., Mata, E.P., Oliveira, C. and Foresti, F. (1996). Cromossomos sexuais e supranumerários em Characidium gomesi (Pisces, Characiformes, Characdiinae). Rev. Bras. Genet. 19 (Suppl.): 115 (Abstract).         [ Links ]

Majumdar, K.C. and McAndrew, B.J. (1986). Relative DNA content of somatic nuclei and chromosomal studies in three genera, Tilapia, Sarotherodon, and Oreochromis of the Tribe Tilapiini (Pisces, Cichlidae). Genetica 68: 175-188.         [ Links ]

Nelson, J.S. (1994). Fishes of the World. 3rd edn. John Wiley and Sons, Inc., New York, pp. 600.         [ Links ]

Ohno, S. (1974). Animal Cytogenetics. In: Chordata 1 - Protochordata, Cyclostomata and Pisces. 4 (Bernard J., ed.). Gebrüder Borntraeger, Berlin, pp. 92.         [ Links ]

Ohno, S. and Atkin, N.B. (1966). Comparative DNA values and chromosome complements of eight species of fishes. Chromosoma 18: 455-466.         [ Links ]

Oliveira, C. and Foresti, F. (1994). Revisão dos estudos citogenéticos em peixes Neotropicais marinhos. Resumos do V Simpósio de Citogenética Evolutiva e Aplicada de Peixes Neotropicais, pp. 7.         [ Links ]

Oliveira, C., Almeida Toledo, L.F., Foresti, F., Britski, H.A. and Toledo Filho, S.A. (1988). Chromosome formulae of Neotropical freshwater fishes. Rev. Bras. Genet. 11: 577-624.         [ Links ]

Oliveira, C., Almeida-Toledo, L.F., Mori, L. and Toledo Filho, S.A. (1992). Extensive chromosomal rearrangements and nuclear DNA content changes in the evolution of the armoured catfishes of the genus Corydoras (Pisces, Siluriformes, Callichthyidae). J. Fish Biol. 40: 419-431.         [ Links ]

Oliveira, C., Almeida-Toledo, L.F., Mori, L. and Toledo Filho, S.A. (1993a). Cytogenetic and DNA content studies on armoured catfishes of the genus Corydoras (Pisces, Siluriformes, Callichthyidae) from the southeast coast of Brazil. Rev. Bras. Gen. 16: 617-629.         [ Links ]

Oliveira, C., Almeida-Toledo, L.F., Mori, L. and Toledo Filho, S.A. (1993b). Cytogenetic and DNA content in six genera of the family Callichthyidae (Pisces, Siluriformes). Caryologia 46: 171-188.         [ Links ]

Oliveira, C., Almeida-Toledo, L.F. and Foresti, F. (1996). Revisão dos estudos citogenéticos em peixes Neotropicais de águas continentais. Resumos do VI Simpósio de Citogenética Evolutiva e Aplicada de Peixes Neotropicais, pp. 104.         [ Links ]

Ragland, C.J. and Gold, J.R. (1989). Genome size variation in the North American sunfish genus Lepomis (Pisces, Centrarchidae). Genet. Res. 53: 173-182.         [ Links ]

Sola, L. Iaselli, V., Rossi, A.R., Rasch, E.M. and Monaco, P.J. (1992). Cytogenetics of bisexual/unisexual species of Poecilia. III. The karyotype of Poecilia formosa, a gynogenetic species of hybrid origin. Cytogenet. Cell Genet. 60: 236-240.         [ Links ]

Thode, G., Alvarez, M.C., Garcia, E. and Giles, V. (1985). Variations in C-banding patterns and DNA values in two scorpion-fishes (Scorpaena porcus and S. nottata, Teleostei). Genetica 68: 69-74.         [ Links ]

Tiersch, T.R., Chandler, R.W., Kallman, K.D. and Wachtel, S.S. (1989a). Estimation of nuclear DNA content by flow cytometry in fishes of the genus Xiphophorus. Comp. Biochem. Physiol. 94B: 465-468.         [ Links ]

Tiersch, T.R., Chandler, R.W., Wachtel, S.S. and Elias, S. (1989b). Reference standards for flow cytometry and application in comparative studies of nuclear DNA content. Cytometry 10: 706-710.         [ Links ]

Turner, B.J., Diffoot, N. and Rasch, E.M. (1992). The callichthyid catfish Corydoras aeneus is an unresolved diploid-tetraploid sibling species complex. Ichthyol. Explor. Freshwaters 3: 17-23.         [ Links ]

 

(Received March 6, 1997)