Cytogenetic and morphometric differences in populations of Astyanax "scabripinnis" (Pisces, Characidae) from Maringá region, PR, Brazil

Cytogenetic and morphometric differences in populations of Astyanax "scabripinnis" (Pisces, Characidae) from Maringá region, PR, Brazil

Sônia Maria Hiromi Nakagawa Mizoguchi and Isabel Cristina Martins-Santos

Universidade Estadual de Maringá, Departamento de Biologia Celular e Genética, Caixa Postal 331, 87020-900 Maringá, PR, Brasil. Send correspondence to S.M.H.N.M.

ABSTRACT

A cytogenetic and morphometric study of four populations of characid fish assigned to the "complex" of Astyanax scabripinnis originating from three riverine basins, namely Ivaí (populations A and B), Paranapanema (C) and Paraná (D), was carried out. Karyological analysis showed the diploid chromosome number 2n = 48 for population B and 2n = 50 for the remaining populations. All populations under study differed in the composition of karyotypes and C-banding patterns. Canonical variable analysis used to assess the morphometric data revealed that 1) the four populations under study were entirely discriminated from each other and 2) the coefficients that most contributed to this pattern were standard length, rostrodorsal distance, eye diameter and snout length. The results indicate that the differences in karyotypes were paralleled by the morphological differentiation of the populations under study and contributed to the problem of systematics of A. scabripinnis "complex".

INTRODUCTION

The family Characidae is one of the most complex groups among the Characiformes and represents the largest group of freshwater fish in South America with 170 genera and 885 species (Nelson, 1994).

Among the characids, the subfamily Tetragonopterinae comprises morphologically ill-defined genera, which are probably a non-monophyletic group (Menezes, 1992). One of them is the Astyanax genus, with 74 species and subspecies living in continental Brazilian water (Eigenmann, 1921).

Cytogenetic studies on Astyanax, in particular the species Astyanax scabripinnis, have shown broad interpopulation karyotype diversity and diploid chromosome numbers of 2n = 46, 48 or 50 (Morelli et al., 1983; Martins et al., 1984; Moreira-Filho and Bertollo, 1986; Maistro, 1991; Salvador and Moreira-Filho, 1992; Rocon-Stange and Almeida-Toledo, 1993). Different chromosome banding patterns were also observed (Moreira-Filho and Bertollo, 1991; Maistro, 1991; Souza, 1996), suggesting that this taxon may be kind of a species complex denoted by Moreira-Filho (1989) as scabripinnis "complex". These interpopulational chromosome differences have been strongly related to their populational structure (Moreira-Filho and Bertollo, 1991). This species complex is restricted to the headwaters of small tributaries (Caramaschi, 1986) and constitutes a large complex of isolate population, which are decisive for the chromosome differentiation (Galetti Jr. et al., 1994). These differences may be explained on the basis of a population model of chromosome evolution similar to that proposed by Wilson et al. (1975) and Lande (1979). The early publications have proposed that a relationship exists between social structuring of animal populations, rate of speciation and involvement of chromosomal rearrangement in those processes. Moreover, they made no distinction between those rearrangements which were polymorphic or fixed, thus casting doubt on any conclusion related to chromosome change and speciation (King, 1995).

Morphologic differentiation in the scabripinnis group was first noted in 1921 by Eigenmann, who reported the existence of several subspecies, and later by Fowler (1948), who catalogued six subspecies in this group. Effectively, integrated karyotypic and morphologic analysis used by Moreira-Filho and Bertollo (1991) and Maistro (1991) has proven to be quite an effective tool as a complementary method for the identification of distinct forms within this complex taxon.

Moreira-Filho (1989) was the first who used this type of integrated analysis, evidencing that some populations that are not chromosomally differentiated can be distinguished by morphological characters, others that showed identical morphological form can be separated by karyotype differences, while others are distinguished by both types of characteristics.

MATERIAL AND METHODS

Karyotypic studies were carried out on four different populations of Astyanax scabripinnis originated from river headwaters close to the large riverine basins of the Ivaí River (population A, Yukatan stream, and population B, Ligeiro stream), Paranapanema River (population C, Sarandi stream), and Paraná River (population D, Água do Rancho stream). All the populations were obtained from municipality of Maringá, State of Paraná, Brazil.

Mitotic chromosomes were prepared from kidney cells using the technique of Egozcue (1971) modified by Bertollo et al. (1978). They were classified by the arm ratio criterion of Levan et al. (1964). C banding patterns were obtained by the method of Sumner (1972).

The morphologic analyses were carried out on 106 specimens of the four Astyanax scabripinnis populations. The following measurements (mm) were made with the help of the pachymeter: standard length (SL), rostrodorsal distance (RDD), rostroanal distance (RAnD), body height (BH), head length (HL), snout length (StL), eye diameter (ED), interorbital width (IOW), and third infraorbital width (IFOW). Two meristic determinations were also made, i.e., number of scales along the lateral line (LL) and number of rays on the anal fin (RA).

The data for the nine morphometric characters were treated by means of canonical variable analysis (CANOVA) according to Manly (1994).

RESULTS

Counts carried out on 348 metaphases for population A (Yukatan stream), 450 for population B (Ligeiro stream), 421 for population C (Sarandi stream), and 551 for population D (Água do Rancho stream) showed a diploid number of 2n = 50 for populations A, C and D (Figures 1A and 2A,B) and 2n = 48 for population B (Figure 1B). The analysis did not reveal the presence of heteromorphic sex chromosomes.

Figure 1
- Karyotype of the Astyanax scabripinnis from Yukatan stream (A) and Ligeiro stream (B). (X1000).

Figure 2
- Karyotype of the Astyanax scabripinnis from Sarandi stream (A) and Água do Rancho stream (B). (X1000).

The karyotypes of the four populations (Figures 1A,B and 2A,B) showed differences in terms of diploid number, composition of karyotype and fundamental number (Table I).

Populations Basin Stream Karyotype 2n FN M SM ST A A Ivaí Yukatan 50 90 3 15 2 5 B Ivaí Ligeiro 48 82 5 11 1 7 C Paranapanema Sarandi 50 86 3 13 2 7 D Paraná Água do Rancho 50 84 3 14 - 8

Populations	Basin	Stream	Karyotype
			2n	FN	M	SM	ST	A
A	Ivaí	Yukatan	50	90	3	15	2	5
B	Ivaí	Ligeiro	48	82	5	11	1	7
C	Paranapanema	Sarandi	50	86	3	13	2	7
D	Paraná	Água do Rancho	50	84	3	14	-	8

Table I - Karyotype and diploid and fundamental numbers of the different populations of the Astyanax scabripinnis studied.

FN, Fundamental numbers; M, metacentric; SM, submetacentric; ST, subtelocentric; A, acrocentric.

In addition to these differences, the individuals in the population A exhibited an extra chromosome of the large metacentric type in 100% of the cells (2n = 51) in 31% of the females examined. The population D exhibited a microchromosome with intraindividual variation in 32% of the females examined (Mizoguchi and Martins-Santos, in press).

The C-banding pattern was characteristic for each population under study, with differences in the distribution pattern being observed in each of them (Figures 3A,B and 4A,B). Figure 5 shows the C band idiogram for the four populations.

Figure 4
- Karyotype of the A. scabripinnis arranged from C-banded chromosomes from Sarandi stream (A) and Água do Rancho stream (B). (X1000).

Figure 5 - Idiogram showing the C-banding patterns (in black) in Astyanax scabripinnis from Yukatan stream (A), Ligeiro stream (B), Sarandi stream (C) and Água do Rancho stream (D).

Analysis of the morphometric data showed a high canonical correlation. Of the 25 individuals of the population A from Yukatan stream, 23 were normally grouped. The same occurred for 19 of 21 individuals of the population B from Ligeiro stream, for 28 of 30 individuals of the population C from Sarandi stream, and for 29 of 30 individuals of the population D from Água do Rancho stream (Table II).

Populations A B C D Total A 23 0 1 1 25 B 0 19 1 1 21 C 1 0 28 1 30 D 0 0 1 29 30

Populations	A	B	C	D	Total
A	23	0	1	1	25
B	0	19	1	1	21
C	1	0	28	1	30
D	0	0	1	29	30

Table II - Grouping of the Astyanax scabripinnis complex specimens according to canonical correlation from the different populations studied.

Figure 6 shows that the four populations were fully discriminated on the basis of the first two canonical variables. Table III indicates that the highest coefficients produced on the basis of the first canonical variable were SL, RDD and ED. For the second canonical variable, the highest coefficients were SL, RDD and StL.

Figure 6
- Discrimination of four population of the Astyanax scabripinnis, on the basis of the first canonical variable (x). A= Yukatan stream; B = Ligeiro stream; C = Sarandi stream, and D = Água do Rancho stream.

The data obtained for the meristic parameters showed that lateral line scales (LL) ranged from 31 to 34 for populations A and B, from 30 to 34 for population C, and from 33 to 35 for population D, whereas number of rays on the anal fin ranged from 20 to 24 for populations A and D, from 18 to 21 for population B and from 18 to 24 for population C.

DISCUSSION

The diploid number of 2n = 50 found in three of the four populations was the most common one observed in the Astyanax scabripinnis populations analyzed thus far (Moreira-Filho and Bertollo, 1991; Maistro, 1991; Maistro et al., 1992; Souza and Moreira- Filho, 1995; Rocon-Stange and Almeida-Toledo, 1993). Despite the identical diploid number, differences in the structure of karyotypes (Table I) and C-banding patterns were observed showing that they are cytogenetically distinct.

Population D differed from the other populations in that it was the only one showing absence of subtelocentric chromosomes (3M, 14SM, 8A). This was also observed in the Jucu River population (3M, 4SM, 18A), investigated by Rocon-Stange and Almeida-Toledo (1993). However, these two populations differed in number of submetacentric and acrocentric chromosomes.

Populations A and B, belonging to the same riverine basin (Ivaí basin), differed in both diploid number and fundamental number. Furthermore, population B (Ligeiro stream) differed from all other populations by the presence of five metacentric chromosomes. Differences in the number of metacentrics were also observed by Maistro (1991) who, in a study of 10 A. scabripinnis populations, detected a different number of three metacentric pairs in only three populations. Souza (1996) and Centofante and Vênere (1995) also observed populations with variations in number of metacentric chromosomes of two and seven pairs, respectively. However, populations with three pairs of these chromosomes are the most frequent, indicating that these pairs are more stable from an evolutionary point of view than the remaining chromosome types which vary from population to population within the scabripinnis "complex". Moreira-Filho and Bertollo (1991) suggest that the metacentric chromosome group represents a conservative characteristic in relation to the variation detected in the submetacentric, subtelocentric and acrocentric chromosomes within the scabripinnis group. Considering the most common principle, the conditions of 2n = 50 and three metacentric pairs among A. scabripinnis populations may be considered to be a pleiomorphic characteristic and their variations may be considered to be derived conditions.

Population B was characterized by a diploid number 2n = 48, the same as for the populations from the Marrecos (Moreira-Filho and Bertollo, 1991), Canta Galo (Souza, 1996) and Tamanduá (Maistro, 1991) and São Domingos streams (Alves and Martins-Santos, personal communication), although the latter populations differed in fundamental number and C-banding patterns.

Despite the occurrence of extensive diversification in karyotype morfology among the Astyanax scabripinnis populations, the majority has unchanged diploid numbers. In these cases, rearrangements that modify the centromere position such as pericentric inversions appear to be predominant in chromosome diversifications. For instance, those populations which show chromosome number 2n = 48 and 2n = 46 may represent derived evolutionary states and the main rearrangement responsible for the redution in diploid number appears to have been the centric fusions. The fact that these populations occur exclusively in the headwater of small streams and represent isolated reproductive populations favors the notion of chromosome rearrangements and fits the model of population evolution proposed by Wilson et al. (1975).

With respect to the heterochromatin pattern, the four populations could also be distinguished by the presence of marker chromosomes characteristic for each one. Population A showed an extremely variable pattern with marked interindividual variation, with the most frequent heterochromatin blocks being an interstitial and terminal one on the long arm of the first metacentric and a telomeric one on the short arm of one of the homologues of the second metacentric pair. The pattern was more stable in the remaining populations, with population B presenting telomeric heterochromatin blocks on the long arm of the subtelocentric chromosome pair 17 and of the acrocentric pairs 18 to 22, as well as an interstitial band in pair 21 and a telomeric band on the short arm of one of the homologues of pair 8. The characteristic markers of population C were chromosome pair 20 with a strong heterochromatin block in one of the homologues and weak telomeric marks on the short arm of the third metacentric pair. In contrast, population D showed distal interstitial band on the short arm of the first metacentric pair and strong heterochromatin blocks in the telomeric region of the long arm of the acrocentric pairs 18 to 23 (Figure 5). The Astyanax scabripinnis populations studied by Maistro (1991) also showed karyotypic distinctions involving chromosome morphology and mainly related to constitutive heterochromatin distribution.

Thus, karyotypic analysis showed differences between the populations of these species both at the level of karyotype macrostructure and at the level of chromosome structure. These differences occurred both between populations from different riverine basins and between populations belonging to the same basin. This appears to occur due in part to the fact that these populations living at headwater of small streams form a large complex of isolated populations, which, according to Galetti Jr. et al. (1994), is decisive for chromosome diversification.

The morphologic diversity of A. scabripinnis has been reported by several authors (Eigenmann, 1921; Fowler, 1948; Britsky et al., 1984; Caramaschi, 1986). However, the morphological differences were very small and required maximization by analysis of canonical variables for group differentiation. Thus, the application of this method showed that the four populations analyzed can be perfectly diagnosed from one another (Figure 6). The characters that most contributed to their discrimination were standard length, rostrodorsal distance, eye diameter and snout length (Table III).

Morphometric parameters Axes CVI CVII Standard length -4.426* -3.776* Rostrodorsal distance 1.017* 3.331* Rostroanal distance 0.549 0.278 Body height 0.783 0.185 Head length 0.438 0.562 Snout length 0.522 -0.765* Eye diameter 0.785* -0.372 Interorbital width -0.184 0.730 Third infraorbital width 0.211 0.089

Morphometric parameters	Axes
	CVI	CVII
Standard length	-4.426*	-3.776*
Rostrodorsal distance	1.017*	3.331*
Rostroanal distance	0.549	0.278
Body height	0.783	0.185
Head length	0.438	0.562
Snout length	0.522	-0.765*
Eye diameter	0.785*	-0.372
Interorbital width	-0.184	0.730
Third infraorbital width	0.211	0.089

Table III - Canonical variable coefficient for the two axes CVI and CVII, considering the parameters studied in different populations analyzed.

*Coefficients with the highest values, i.e., indicating the most important traits for discrimination among populations.

CVI, CVII - Canonical variables I and II, respectively.

Population B (Ligeiro stream) showed the best differentiation from the remaining ones, especially compared to population A (Yukatan stream) belonging to the same riverine basin. This type of analysis revealed the existence of morphological differences among the four populations.

The meristic data did not contribute in an effective manner to the discrimination of the populations studied here, and therefore do not represent decisive parameters for this type of analysis. Similar results were observed in populations studied by Moreira-Filho and Bertollo (1991).

Comparative analysis carried out to correlate the karyotypic and morphological studies showed that populations A and B, belonging to the same riverine basin (Ivaí River), were those that could be best differentiated both by morphological (Figure 6) and cytogenetic (Table I and Figures 1, 2, and 5) data.

Using canonical variable analysis, Moreira-Filho and Bertollo (1991) discriminated four of seven A. scabripinnis populations studied, whereas Maistro (1991) reported that the use of this method did not permit a clear distinction of nine populations under his study. The populations that could not be discriminated by morphological analysis in these two studies were differentiated on the basis of cytogenetic data. The populations investigated in the present study revealed both morphological and cytogenetic differentiation, being the two methods in agreement. Thus, the combination of morphological and chromosomal studies has been useful not only in terms of differentiation of populations found to be similar when analyzed by one of the methods, but also by showing that in some cases karyotypic diversity is accompanied by morphological diversity.

ACKNOWLEDGMENTS

This work was supported by CNPq and CAPES. We thank Prof. Luis Maurício Bini for help in the morphometric analysis of this paper.

RESUMO

Foram realizados estudos citogenéticos e morfométricos de quatro populações de peixes characídeos, designados de "complexo" de Astyanax scabripinnis, pertencentes a três bacias hidrográficas diferentes: bacia do Ivaí (populações A e B), do Paranapanema (população C) e do Paraná (população D). A análise cariotípica mostrou um número diplóide de 2n = 48 cromossomos para a população B e de 2n = 50 cromossomos para as demais populações. Todas as populações analisadas diferiram na composição cariotípica e no padrão de banda C. A análise de variável canônica, aplicada aos dados morfométricos, revelou que: 1) as quatro populações estudadas foram totalmente discriminadas entre si, e 2) os coeficientes que mais contribuíram para isto foram o comprimento padrão, distância rostro-dorsal, diâmetro do olho e comprimento do focinho. Portanto, estes resultados indicam que as diferenças cariotípicas foram acompanhadas pelas diferenças morfológicas nas populações e contribuíram para o problema da sistemática do "complexo" A. scabripinnis.

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(Received March 12, 1997)

Figure3 - Karyotype of the Astyanax scabripinnis arranged from C-banded chromosomes from Yukatan stream (A) and Ligeiro stream (B). (X1000).

Publication Dates

• Publication in this collection
  06 Jan 1999
• Date of issue
  Mar 1998

History

• Received
  12 Mar 1997