Analysis of genetic variability in three species of Pimelodidae (Ostariophysi - Siluriformes)

Fernanda S. de Almeida Leda M.K. Sodré About the authors

Abstracts

Genetic variability of three Pimelodidae species, Pimelodus maculatus, Iheringichthys labrosus, and Pinirampus pirinampu, collected at one site in the Tibagi River, was comparatively analyzed using protein data for six systems which code 15 loci in liver, muscle, and heart. The proportion of polymorphic loci (<img src="http:/img/fbpe/gmb/v21n4/21n4a14.jpg" alt="21n4a14.jpg (768 bytes)" align="middle">) for P. maculatus, I. labrosus, and P. pirinampu was 13.33, 20, and 6.67%, respectively, and mean heterozigosity was 6, 8.3, and 4.3%. The genetic identity value (I) was 0.32 between P. maculatus and I. labrosus, 0.37 between P. maculatus and P. pirinampu, and 0.58 between I. labrosus and P. pirinampu. This value suggests that these two latter species are congeneric. However, morphological characteristics place these species in distinct genera.


A variabilidade genética de 3 espécies da família Pimelodidae, Pimelodus maculatus, Iheringichthys labrosus e Pinirampus pirinampu, coletadas em um ponto do rio Tibagi, foi analisada comparativamente utilizando dados protéicos de 6 sistemas que codificam 15 locos em fígado, músculo e coração. A proporção de locos polimórficos (<img src="http:/img/fbpe/gmb/v21n4/21n4a14.jpg" alt="21n4a14.jpg (768 bytes)" align="middle">) para P. maculatus, I. labrosus e P. pirinampu foi de 13,33, 20 e 6,67%, respectivamente, e a heterozigosidade média foi de 6, 8,3 e 4,3%. A identidade genética (I) foi de 0,32 entre P. maculatus e I. labrosus, 0,37 entre P. maculatus e P. pirinampu e 0,58 entre I. labrosus e P. pirinampu. O valor de I (0,58) encontrado entre I. labrosus e P. pirinampu sugere que estas são espécies congenéricas. No entanto, as características morfológicas colocam estas espécies em gêneros distintos. Os resultados obtidos nesse estudo podem ser úteis para um melhor conhecimento de espécies de Pimelodidae. Elas também reforçam a necessidade da preservação do rio Tibagi (Paraná - Brasil) através de uma análise cuidadosa no caso de construção de hidroelétricas.


Analysis of genetic variability in three species of Pimelodidae (Ostariophysi - Siluriformes)

Fernanda S. de Almeida and Leda M.K. Sodré

Departamento de Biologia Geral, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Campus Universitário, Caixa Postal 6001, 86051-990 Londrina, PR, Brasil. Send correspondence to L.M.K.S.

ABSTRACT

Genetic variability of three Pimelodidae species, Pimelodus maculatus, Iheringichthys labrosus, and Pinirampus pirinampu, collected at one site in the Tibagi River, was comparatively analyzed using protein data for six systems which code 15 loci in liver, muscle, and heart. The proportion of polymorphic loci () for P. maculatus, I. labrosus, and P. pirinampu was 13.33, 20, and 6.67%, respectively, and mean heterozigosity was 6, 8.3, and 4.3%. The genetic identity value (I) was 0.32 between P. maculatus and I. labrosus, 0.37 between P. maculatus and P. pirinampu, and 0.58 between I. labrosus and P. pirinampu. This value suggests that these two latter species are congeneric. However, morphological characteristics place these species in distinct genera.

INTRODUCTION

Isoenzymatic markers have been utilized to study genetic variability, gene flow among populations, natural hybridization process, species dispersion, as well as to analyze plants, microorganisms, and animal phylogeny. In fish, several studies have used isoenzymatic analysis (Whitt, 1970, 1975; Van Der Bank et al., 1989; Araújo, 1990; Farias and Almeida-Val, 1992; Matthiensen et al., 1993; Verneau et al., 1994; Johansen and Naevdal, 1995; Karakousis et al., 1995; Omel'chenko et al., 1995; Zawadzki, 1996; Chiari and Lima, 1997; Revaldaves et al., 1997).

The family Pimelodidae is the largest and most diverse family among neotropical Siluriformes, and it is distributed throughout Central and South America (Ubeda et al., 1981). These highly diverse species always have three pairs of barbels, no scales, and usually the first ray of the dorsal and pectoral fins has a strong and pungent aculeus. A large number of economically important fishes are included in this group.

Bennemann et al. (1995), through an ichthyofaunistic study done in five sites along the Tibagi River (Paraná, Brazil) in the Sertanópolis region (river's mouth), discovered three fish species of the family Pimelodidae, Pimelodus maculatus, Iheringichthys labrosus, and Pinirampus pirinampu.

The objectives of the present study were to quantify the genetic variability and genetic distance in P. maculatus, I. labrosus and P. pirinampu, using six protein systems, and to alert about the importance of heterogeneous environment preservation for the maintenance of high levels of genetic variability.

MATERIAL AND METHODS

Specimens of Pimelodus maculatus, Iheringichthys labrosus, and Pinirampus pirinampu were collected monthly, between August 1994 and March 1995, in the Sertanópolis region (Paraná, Brazil) of the Tibagi River. Samples of liver, heart, muscle, and eye (analyzed for lactate dehydrogenase) were collected from live fish. These samples were stored at -20ºC. Individual tissue samples were homogenized using Tris-EDTA buffer, pH 7.0 (Degani and Veith, 1990), and centrifuged at 5000 rpm for 15 min at 4°C. The supernatant obtained was absorbed in Whatman number 3 paper for analysis in horizontal electrophoresis in corn starch gel (Val et al., 1981). The methodology used to prepare the buffer solutions and reaction mixtures was based on Shaw and Prasad (1970), Harris and Hopkinson (1976), and Lima and Contel (1990).

Table I summarizes the electrophoretic conditions used to study the following protein systems: malate dehydrogenase (MDH- E.C.1.1.1.42), lactate dehydrogenase (LDH- E.C.1.1.1.27), isocitrate dehydrogenase (IDHP- E.C.1.1.1.42), phosphoglucomutase (PGM- E.C.5.4.2.2), carboxylesterases (EST- E.C.3.1.1.-), and nonspecific proteins (PT).

The data were analyzed using the Fortran Biosys-1 program (Swoford and Selander, 1981). Genetic variability was estimated through polymorphic locus proportion calculation (99% criterion), intralocus and mean heterozigosity () obtained by direct counting, and expected intralocus and mean heterozygosities (), calculated according to Nei (1972). Distance and genetic identity calculations were based on Nei (1972). The distance values, which were utilized to build the dendogram, were obtained through the unweighted pair-group method arithmetic averages (UPGMA).

The zones of activity were numbered according to their electrophoretic mobilities, from the anode to cathode. Nomenclature for designation of the loci and alleles was the same as proposed by Shaklee et al. (1990).

RESULTS

After analyzing the six protein systems, 15 I. labrosus loci and 14 P. maculatus and P. pirinampu loci were detected (Figure 1). Analysis of each isoenzymic system of different tissues from one individual showed differential tissue distribution in the activity of each locus (Table II), regarding both number and color intensity of the bands. It was also observed that loci were expressed in the same manner in a given tissue among different species.

Figure 1
- Electrophoretic patterns of the six protein systems analyzed. A: Carboxylesterases (EST), nonspecific proteins (PT), phosphoglucomutase (PGM). B: Isocitrate dehydrogenase (IDHP), lactate dehydrogenase (LDH), malate dehydrogenase (MDH). Samples: 1) Pimelodus maculatus, 2) Iheringichthys labrosus, 3) Pinirampus pirinampu.

Three of the 15 loci analyzed were polymorphic, EST-4* in P. maculatus and I. labrosus, PGM-1* in the three species, and mMDH* in I. labrosus (Table III). I. labrosus showed the largest variability (= 20% and = 0.083 ± 0.048) (Table IV). Genetic distance (D) and genetic identity (I) values (Nei, 1972) are shown in Table V, and a dendogram was constructed with genetic distance values by the UPGMA method (Figure 2).

Figure 2 -
Dendogram obtained by the UPGMA method for genetic distance (Nei, 1972) data.

DISCUSSION

The number of loci detected and differential tissue distribution for the different protein systems were mostly consistent with electrophoretic patterns, genetic inferences, and molecular protein structure described for other fish species (Panepucci et al., 1984; Monteiro, 1989; Araújo, 1990; Degani and Veith, 1990; Farias and Almeida-Val, 1992; Matthiensen et al., 1993; Verneau et al., 1994; Johansen and Naedval, 1995; Karakousis et al., 1995; Zawadzki, 1996; Chiari and Lima, 1997; Revaldaves et al.,1997). Though a small number of protein systems were analyzed, a larger number of loci were detected than in many other studies due to the use of various tissues from each individual.

Almost every bony fish has the LDH-C*locus, which is believed to have originated through a duplication of the LDH-B* locus. The LDH-C* locus in bony fish shows different tissue regulation patterns in different taxons. Primitive orders of bony fish have a generalized tissue expression; in contrast, representatives of more advanced orders show a specialized tissue pattern (Kettler and Whitt, 1986; Basaglia, 1989; Rao et al., 1989). LDH-C* locus activity was not detected in the tissues analyzed (liver, muscle, eye, and heart) from the species studied. Whitt (1975), Panepucci et al. (1984), and Rao et al. (1989) observed an absence of the LDH-C* locus in the order Siluriformes (to which P. maculatus, I. labrosus, and P. pirinampu belong).

The genetic variability data showed that the proportion of polymorphic loci () observed in P. maculatus, I. labrosus, and P. pirinampu was 13.33, 20, and 6.67%, respectively. By comparing the data obtained, it was observed that the estimated values of were compatible. Nevo (1978) estimated a value of 15.2% for 51 species of Teleostei. Some studies with Brazilian fresh-water fish observed a value of 30% in Prochilodus lineatus (Revaldaves et al., 1997), while Zawadzki (1996) detected values varying from 11.54 to 19.23% in three species of Hypostomus. Chiari and Lima (1997) obtained values varying from 16.7 to 36.8% in five species of Anostomidae.

Ward et al. (1992), in a review of the literature data obtained from various fresh- and salt-water fish, estimated mean heterozigosity to be 5.1% for 150 fish species. The values of 4.3% obtained for P. pirinampu, 6.0% for P. maculatus, and 8.3% for I. labrosus do not diverge from those found in the literature. Variation in the estimated data of found in the literature can be explained in different manners. For example, according to Lewontin (1974) and Nei (1978), mean heterozigosity is highly affected by the choice and number of loci analyzed.

Bennemann et al. (1995) verified that I. labrosus and P. maculatus were detected in four of the five collection sites of the Tibagi River (Ipiranga, Sapopema, Londrina, and Sertanópolis) and P. pirinampu was found only in two sites (Londrina and Sertanópolis), which are close to one another and had few individuals.

The number of P. maculatus and I. labrosus individuals analyzed was larger than those of P. pirinampu, which had a smaller population density at the collection site (Table III). These two factors could explain the larger genetic variability (values of and ) detected for P. maculatus and I. labrosus. Greater values of heterozigosity and polymorphism are expected for species that show a large population size, since the possibility of inbreeding is lower when they occupy a more extensive niche with heterogeneous environments (Nei, 1977; Zimmerman, 1987).

Estimate of for neotropical fish vary from 1.1% for the sedentary species Hypostomus derbyi (Zawadzki, 1996) to 14.2% in Leporinus elongatus (Chiari and Lima, 1997), which was detected in three sites along the Tibagi River by Bennemann et al. (1995).

Thorpe (1982), in a literature review of genetic identity (I) data in vertebrates, invertebrates and plants, concluded that about 85% of I values among species of the same genera exceed 0.35 (76% over 0.4 and 90% over 0.3), while among genera, 77% are under 0.35 (87% under 0.4 and 93% under 0.45). A value of 0.316 was found between P. maculatus and I. labrosus, and of 0.370 between P. maculatus and P. pirinampu, which shows that they are distinct genera (Table V). However, the I value of 0.575 (Thorpe's index) found between I. labrosus and P. pirinampu suggests that these species are congeneric. According to Britski (1972), morphological characteristics such as lips, aculeus, barbels, fins, etc., put these species in distinct genera; monetheless, it is shown that they share a large number of loci at the biochemical genetic level.

This study is part of an integrated project which aims to recuperate the Tibagi River Basin (Aspects of Fauna and Flora of Tibagi River Basin). However, there are governmental projects to build five dams inside the Tibagi River. The dams themselves may be formidable barriers to the dispersal of many fresh-water organisms, especially migratory ones. Beyond the impacts caused by the flux control, they compromise the survival, mating success, and gene flow that can alter the gene frequencies of the species. Therefore, the genetic variability indexes detected for these three fish species may be reduced by the dams construction, and in the future, they can be lesser than the ones detected for P. pirinampu.

ACKNOWLEDGMENTS

The authors would like to thank Prof. Dr. Oscar Akio Shibatta and the biologist Mário Luís Orsi from the Departamento de Biologia Animal e Vegetal of Universidade Estadual de Londrina for identification and collection of the specimens. We also wish to thank Consórcio Intermunicipal para a Recuperação da Bacia do Rio Tibagi (Intermunicipal Partnership for the Recuperation of the Tibagi River Basin), Klabin Fabricadora de Papel e Celulose (Klabin paper and cellulose factory), CNPq, and Universidade Estadual de Londrina for financial support received for the execution of this study.

RESUMO

A variabilidade genética de 3 espécies da família Pimelodidae, Pimelodus maculatus, Iheringichthys labrosus e Pinirampus pirinampu, coletadas em um ponto do rio Tibagi, foi analisada comparativamente utilizando dados protéicos de 6 sistemas que codificam 15 locos em fígado, músculo e coração. A proporção de locos polimórficos () para P. maculatus, I. labrosus e P. pirinampu foi de 13,33, 20 e 6,67%, respectivamente, e a heterozigosidade média foi de 6, 8,3 e 4,3%. A identidade genética (I) foi de 0,32 entre P. maculatus e I. labrosus, 0,37 entre P. maculatus e P. pirinampu e 0,58 entre I. labrosus e P. pirinampu. O valor de I (0,58) encontrado entre I. labrosus e P. pirinampu sugere que estas são espécies congenéricas. No entanto, as características morfológicas colocam estas espécies em gêneros distintos. Os resultados obtidos nesse estudo podem ser úteis para um melhor conhecimento de espécies de Pimelodidae. Elas também reforçam a necessidade da preservação do rio Tibagi (Paraná - Brasil) através de uma análise cuidadosa no caso de construção de hidroelétricas.

(Received November 7, 1997)

  • Araújo, M.E. (1990). Análise filogenética do gęnero Achirus (Pleronectiformes: Soleidae) do litoral da Paraíba e do rio Tocantins (PA) baseado no estudo de isoenzimas. Master's thesis, Universidade Federal da Paraíba, Joăo Pessoa.
  • Basaglia, F. (1989). Some aspects of isozymes of lactate dehydrogenase, malate dehydrogenase and glucosephosphate isomerase in fish. Com. Biochem. Physiol. 92: 213-226.
  • Bennemann, S.T., Silva-Souza, A.T. and Rocha, C.R.A. (1995). Composicion ictiofaunística en cinco localidades de la cuenca del rio Tibagi. Intercięncia 20: 7-13.
  • Britski, H.A. (1972). Peixes de água doce do Estado de Săo Paulo: Sistemática. In: "Poluiçăo e Piscicultura - Comissăo Interestadual da Bacia do Paraná-Uruguai". Faculdade de Saúde Pública, USP/Instituto de Pesca - CPRN, Săo Paulo, pp. 79-108.
  • Chiari, L. and Lima, L.M.K.S. (1997). Análise da variabilidade genética em cinco espécies da família Anostomidae (Ostariophysi - Characiformes) do rio Tibagi - Sertanópolis (PR). Rev. Bras. Genet. 20 (Suppl.): 330.
  • Degani, G. and Veith, M. (1990). Electrophoretic variation systems in the muscle and liver of Anabantidae fish. Isr. J. Aquacult. 42: 67-76.
  • Farias, I.P. and Almeida-Val, V.M.F. (1992). Malate dehydrogenase (s-MDH) in Amazon cichlid fishes: evolutionary features. Biochem. Physiol. 103: 939-943.
  • Harris, H. and Hopkinson, D.A. (1976). Handbook of Isozyme Electrophoresis in Human Genetics North Holand Publishing Company, North Holand.
  • Johansen, T. and Naevdal, G. (1995). Genetic analysis of population structure of tusk in the North Atlantic. J. Fish Biol. 47 (Suppl. A): 226-242.
  • Karakousis, Y., Machordom, A., Doadrio, I. and Economidis, P.S. (1995). Phylogenetic relationships of Barbus peloponnesius Valenciennes, 1842 (Osteichyties: Cyprinidae) from Greece and other species of Barbus as revealed by allozyme electrophoresis. Biochem. System. Ecol. 23: 365-375.
  • Kettler, M.K. and Whitt, G.S. (1986). An apparent progressive and recurrent evolutionary restriction in tissue expression of a gene, the lactate dehydrogenase-C gene, within a family of bony fish (Salmoniformes: Umbridae). J. Mol. Evol. 23: 95-107.
  • Lewontin, R.C. (1974). The Genetic Basis of Evolutionary Change. Columbia University Press, New York. 
  • Lima, L.M.K.S. and Contel, E.P.B. (1990). Electrophoretic analysis of 12 proteins in natural populations of Spodoptera frugiperda (Lepidoptera: Noctuidae). Rev. Bras. Genet. 13: 711-729.
  • Matthiensen, A., Tellechea, E. and Levy, J.A. (1993). Biochemical characterization for the genetic interpretation of esterase isozyme in Micropogonias furnieri (Pisces, Sciaenidae) in south Brazil. Comp. Biochem. Physiol. 104B: 349-352.
  • Monteiro, M.C. (1989). Locos que codificam a malato desidrogenase solúvel (sMDH) em 22 espécies de peixes das ordens Characiformes, Siluriformes e Perciformes: aspectos evolutivos. Master's thesis, Universidade Federal de Săo Carlos, Săo Paulo.
  • Nei, M. (1972). Genetic distance between populations. Am. Nat. 106: 283-292.
  • Nei, M. (1977). F-statistics and analysis of gene diversity in subdivided populations. Ann. Hum. Genet. 41: 225-233.
  • Nei, M. (1978). Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89: 583-590.
  • Nevo, E. (1978). Genetic variation in natural populations. Evol. Biol. 8: 79-119.
  • Omel'chenko, V.T., Salmenkova, E.A., Ivanov, A.N., Afanas'ev, K.I., Savvushkina, N.E. and Roslyy, Yu. S. (1995). Genetic identification of the origin of the chum salmon Oncohynchus keta, taken by the marine fishery along northwest Sakhalin. J. Ichthyol. 35: 146-157.
  • Panepucci, L.L.L., Schwantes, M.L. and Schwantes, A.R. (1984). Loci that encode the lactate dehydrogenase in 23 species of fish belonging to the orders Cypriniformes, Siluriformes and Perciformes: adaptive features. Comp. Biochem. Physiol. 77B: 867-876.
  • Rao, M.R.K., Padhi, B.K. and Khuda-Bunkhsh, A.R. (1989). Lactate dehydrogenase isozymes in fifty-two species of teleostean fish: taxonomic significance of LDH-C gene expression. Biochem. Syst. Ecol. 17M: 69-76.
  • Revaldaves, E., Renesto, E. and Machado, M.F.P.S. (1997). Genetic variability of Prochilodus lineatus (Characiformes, Prochilodontidae) in the upper Paraná River. Rev. Bras. Genet. 20: 381-388.
  • Shaklee, J.B., Allendorf, F.W., Morizot, F.W. and Whitt, G.S. (1990). Gene nomenclature for protein-coding loci in fish. Trans. Am. Fish Soc. 119: 2-15.
  • Shaw, C.R. and Prasad, R. (1970). Starch gel electrophoresis of enzymes - A compilation of recipes. Biochem. Genet. 4: 297-320.
  • Swoford, D.L. and Selander, R.B. (1981). BIOSYS-1: a fortran program for the comprehensive analysis of electrophoretic data in population genetics and systematics. J. Hered. 72: 281-283.
  • Thorpe, J.P. (1982). The molecular clock hypothesis: biochemical evolution, genetic differentiation and systematics. Ann. Rev. Ecol. Syst. 13: 139-168.
  • Ubeda, C.A., Vignes, I.E. and Drago, L.P. (1981). Contribuicion para el conocimiento del bagre amarillo (Pimelodus maculatus, Lacépede 1803) (Pisces, Pimelodidae) del rio de La Plata: redescripcion, biologia, distribucion geográfica, importância econômica. Physis. Secc. B Aguas Cont. Org. 40: 63-76.
  • Val, A.L., Schwantes, A.R., Schwantes, M.L.B. and Luca, P.H. den (1981). Amido hidrolisado em milho como suporte eletroforético. Cięnc. Cult. 33: 992-996.
  • Van Der Bank, F.H., Grant, W.S. and Ferreira, J.T. (1989). Electrophoretically detectable genetic data for fifteen southern African cichlids. J. Fish Biol. 34: 463-483.
  • Verneau, O., Moreau, C., Catzeflis, F.M. and Renaud, F. (1994). Phylogeny of flatfishes (Pleuronectiformes): Comparisons and contradictions of molecular and morphoanatomical data. J. Fish Biol. 45: 685-696.
  • Ward, R.D., Skibinski, O.F. and Woodwark, M. (1992). Protein heterozygosity, protein structure, and taxonomic differentiation. Evol. Biol. 26: 73-159.
  • Whitt, G.S. (1970). Developmental genetics of the lactate dehydrogenase isozyme of fish. J. Exp. Zool. 175: 1-3.
  • Whitt, G.S. (1975). A unique lactate dehydrogenase isozyme in the teleost retina. In: Vision in Fishes (Ali, M.A., eds.). Plenum Publishing Corporation, New York, pp. 459-470.
  • Zawadzki, C.H. (1996). Análise genética e morfométrica de tręs espécies do gęnero Hypostomus Lacépede, 1803 (Osteichtyes: Loricariidae) da bacia do rio Iguaçu. Master's thesis, Universidade Estadual de Maringá, Paraná.
  • Zimmerman, E.G. (1987). Relationships between genetic parameters and life-history characteristics of stream fish. In: Community and Evolutionary Ecology of American Stream Fishes (Mathews, W.J. and Heins, D.C., eds.). University of Oklahoma Press, Norman and London Copyright, Oklahoma, pp. 239-244.

Publication Dates

  • Publication in this collection
    01 Mar 1999
  • Date of issue
    Dec 1998

History

  • Received
    07 Nov 1997
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