Comparative cytogenetic and morphological analysis of Astyanax scabripinnis paranae (Pisces, Characidae, Tetragonopterinae)

Comparative cytogenetic and morphological analysis of Astyanax scabripinnis paranae (Pisces, Characidae, Tetragonopterinae)

Edson Luis Maistro¹, Claudio Oliveira² and Fausto Foresti²

¹ Instituto de Farmácia e Nutrição, Universidade de Alfenas, 37130-000 Alfenas, MG, Brasil.

² Departamento de Morfologia, Instituto de Biociências, UNESP, Campus de Botucatu, 18618-000 Botucatu, SP, Brasil and CAUNESP. Send correspondence to E.L.M.

ABSTRACT

Cytogenetic and morphological studies were carried out on nine local populations of Astyanax scabripinnis paranae. All populations exhibited 2n = 50 chromosomes as well as conspicuous differences involving karyotype morphology, number and position of nucleolar organizer regions (NORs) and amount and/or locations of constitutive heterochromatin blocks. A quantitative study of the cytogenetic data showed that eight populations possessed different karyotypes. Morphological analyses based on nine measurements and two meristic parameters were effective in establishing clear identification of five populations. Comparative analysis of cytogenetic and morphological traits suggests that chromosomal changes have occurred at a more rapid rate than morphological differentiation. Despite the close morphological similarity found among some populations, chromosomal differentiation was identified in all of them, even in those presenting only small morphological differences.

INTRODUCTION

An important aspect of evolutionary research is to understand the genetic changes that accompany or cause the formation of new species (Templeton, 1981). Many reports have suggested alternative hypotheses about the importance of fixation of chromosomal rearrangements and their relationship with evolution (reviewed by Sites Jr. and Moritz, 1987; King, 1993; Qumsiyeh, 1994; Sites Jr. and Reed, 1994, among others); however, according to Sites Jr. and Moritz (1987), the present knowledge of the origin and evolutionary significance of chromosome change is too fragmentary to assert that a major consequence of chromosome change is either cladogenesis or phyletic divergence. Different classes of chromosomal rearrangements may be positively or negatively selected, or approximately neutral in their fitness effects, and so become established at different rates within any given clade (Qumsiyeh, 1994).

The family Characidae is the largest group of freshwater fishes in South America, comprising about 400 described species (Nelson, 1994). The genus Astyanax, the most frequent in Brazilian rivers (Britski, 1972), includes species such as A. scabripinnis, with several interesting peculiarities for chromosomal and morphological evolutionary studies, i.e., a large number of subspecies described (Fowler, 1948), geographic distribution restricted to the headwaters of small streams (Gomes and Azevedo, 1960), phenotypic plasticity (Caramaschi, 1986; Moreira-Filho and Bertollo, 1991), and high karyotype diversity (see references in Souza et al., 1995).

In the present study nine local populations of A. s. paranae inhabiting a closely related geographical area were analyzed in order to compare the chromosomal and morphological rate of character diversification for a better understanding of the process of speciation in this group.

MATERIAL AND METHODS

The Paraná-Paraguay basin is formed by several large rivers such as the Tietê and Paranapanema Rivers. In the ridge of mountains called Serra de Botucatu there are many headwaters of small streams or rivers in a closely related area which drain in three directions: into the Tietê River, into the Paranapanema River and into the Pardo River, which is itself a large tributary of the Paranapanema River ( Figure 1).

Figure 1
- Collection sites of Astyanax scabripinnis paranae: A, Grande Stream (Pardinho, SP); B, São Pedro Stream (Itatinga, SP); C, Claro River (Botucatu, SP); D, Pardo River (Pardinho, SP); E, Pedras Stream (Itatinga, SP); F, Araquá River (Botucatu, SP); G, Capivara River (Botucatu, SP); H, Cascatinha Stream (Botucatu, SP); I, Lavapés River (Botucatu, SP).

A cytogenetic survey was performed on 106 specimens of A. s. paranae sampled from nine local populations from rivers in the Serra de Botucatu in an area of about 200 km² (Figure 1). Table I lists the samples analyzed, the collection sites and the number and sex of specimens.

Table I
- Summary of the results obtained from cytogenetic characterization of Astyanax scabripinnis paranae.

Collection sites	Number of specimens female/male		Number of cells female/male		2n	FN	Chromosome types				Number of NOR pairs
							M	SM	ST	A
Paranapanema River basin
Grande Stream	3	7	116	340	50	82	6	26	8	10	5
São Pedro Stream	7	3	253	93	50	80	6	24	8	12	5
Pardo River basin
Claro River	6	4	176	109	50	84	6	28	6	10	2
Pardo River	3	5	93	145	50	84	6	26	6	12	2
Pedras Stream	5	3	99	127	50	84	6	28	4	12	3
Tietê River basin
Araquá River	7	12	227	358	50	84	4	30	4	12	2
Capivara River	8	6	285	226	50	80	8	22	10	10	3
Cascatinha Stream	9	6	267	183	50	80	8	22	10	10	2
Lavapés River	7	5	231	154	50	82	6	26	8	10	2

FN = Fundamental number; M = metacentric; SM = submetacentric; ST = subtelocentric; A = acrocentric; NOR = nucleolus organizer region.

Chromosome spreads, silver staining of nucleolus organizer regions (NORs) and C-banding were performed as described by Foresti et al. (1993). Chromosome preparations were obtained from gill and kidney tissues and about 30 metaphases were examined for each specimen (Table I). Chromosome morphology was determined on the basis of arm ratios (AR) as proposed by Levan et al. (1964) and the chromosomes were classified as metacentric (M, AR = 1.00 to 1.70), submetacentric (SM, AR = 1.71 to 3.00), subtelocentric (ST, AR = 3.01 to 7.00) and acrocentric (A, AR > 7.01). The fundamental number (FN) was determined considering M/SM chromosomes to have two arms and ST/A chromosomes to have one arm. The idiograms were prepared on the basis of direct measurements of chromosomes in the karyotypes. For quantitative analysis, cytogenetic data for each population were grouped into 12 categories divided into two subgroups (Table II) and subjected to canonical variable analysis (CVA) using the 6.03 version of the Statistical Analysis System (SAS) software.

Table II
- Cytogenetical data from the nine populations of Astyanax scabripinnis paranae subjected to canonical variable analysis (CVA).

Collection sites	Number of chromosome pairs presenting
	M	SM	ST	A	NORs	NORs in short arm	NORs in long arm	C-band in M	C-band in SM	C-band in ST short arm	C-band in ST long arm	C-band in A
Paranapanema River basin
Grande Stream	3	13	4	5	5	3	2	0	0	1	2	3
São Pedro Stream	3	12	4	6	5	2	3	0	0	0	1	4
Pardo River basin
Claro River	3	14	3	5	2	1	1	1	1	1	1	1
Pardo River	3	13	3	6	2	2	0	0	0	0	1	2
Pedras Stream	3	14	2	6	3	3	0	0	0	0	0	0
Tietê River basin
Araquá River	2	15	2	12	2	1	1	0	0	1	1	3
Capivara River	4	11	5	5	3	1	2	0	0	1	1	0
Cascatinha Stream	4	11	5	5	2	1	0	1	0	0	0	4
Lavapés River	3	13	4	5	2	2	0	0	0	0	1	3
	Group 1				Group 2

A total of 212 specimens from the nine populations studied cytogenetically were used for morphometric analyses (Table III). The measurements were performed using a pachymeter as described by Britski et al. (1988). The modal score of each of the 11 parameters described in Table III was calculated for each population and the parameters were grouped into two blocks and subjected to CVA analysis. To permit a better interpretation of the quantitative analyses of cytogenetic and morphologic data the values were plotted on a single graph (Figure 3).

Table III
- Morphometric parameters of the nine populations of Astyanax scabripinnis paranae subjected to canonical variable analysis (CVA).

Morphometric analyses realized in all populations	Collection sites	Number of specimens analyzed (female/male)
Group 1 - measurement parameters	Paranapanema River basin
Standard length	Grande Stream	14/14
Rostrodorsal distance	São Pedro Stream	11/11
Rostro-anal distance	Pardo River basin
Body height	Claro River	8/8
Head length	Pardo River	15/15
Snout length	Pedras Stream	12/12
Eye diameter	Tietê River basin
Interorbital width	Araquá River	10/10
Third infraorbital width	Capivara River	12/12
Group 2 - meristic parameters	Cascatinha Stream	11/11
Number of scales on the lateral line	Lavapés River	13/13
Number of rays of the anal fin

RESULTS AND DISCUSSION

Chromosomal analysis

All local populations analyzed in the present study showed the same diploid number of 2n = 50 chromosomes, with no difference between sexes (Table I and Figure 2). Including the present investigation, cytogenetic studies have been carried out thus far on thirty-five local populations of A. scabripinnis from different Brazilian regions. In this studies, three diploid numbers have been already detected: 2n = 46 for one population, 2n = 48 for four populations and 2n = 50 for 30 populations (Souza et al., 1995). In this species, such karyotypic differences seem not to be related to the geographic logic distribution of the populations since fishes with different diploid numbers can be found nearby each other.

Figure 2
- Idiograms showing the chromosome types, the C-banding patterns (in black), and Ag-NOR position (asterisks) found in local populations of Astyanax scabripinnis paranae. A, Grande Stream; B, São Pedro Stream; C, Claro River; D, Pardo River; E, Pedras Stream; F, Araquá River; G, Capivara River; H, Cascatinha Stream; I, Lavapés River; M, metacentric; SM, submetacentric; ST, subtelocentric; A, acrocentric.

Figure 3
- Discrimination of populations of Astyanax scabripinnis paranae from the different sites studied on the basis of canonical variable analysis (CVA). The numbers refer to the morphological data and the letters to cytogenetic data: 1-A, Grande Stream; 2-B, São Pedro Stream; 3-C, Claro River; 4-D, Pardo River; 5-E, Pedras Stream; 6-F, Araquá River; 7-G, Capivara River; 8-H, Cascatinha Stream; 9-I, Lavapés River.

Almost all local populations examined in the present study showed different numbers of metacentric, submetacentric, subtelocentric and acrocentric chromosomes (Tables I and II, Figure 2). All combinations of karyotypes found were inside the limits previously described for populations of A. s. paranae, with 2n = 50 chromosomes ranging from 36m,sm + 14st,a (FN = 86) to 14m,sm + 36st,a (FN = 64) (Souza et al., 1995).

Five populations showed two chromosome pairs with Ag-NORs, two showed three chromosome pairs with Ag-NORs, and two showed five chromosome pairs with Ag-NORs (Table I and Figure 2). Only one chromosome pair (a small subtelocentric) was present in almost all samples (Figure 2). Differences in NOR position in A. scabripinnis were also reported by Moreira-Filho (1989) who found one to seven chromosome pairs stained by the Ag-NOR technique, among seven local populations studied. In A. scabripinnis, the maximum number of NOR-bearing chromosomes was described for a sample from the Jucu River with 13 labelled chromosome pairs (Rocon-Stange and Almeida-Toledo, 1993).

The pattern of heterochromatin distribution was the best discriminating feature for the local populations of A. s. paranae. Although all samples had chromosomes with small pericentromeric blocks, the amount of heterochromatin on the long arm of subtelocentric and acrocentric chromosomes was characteristic for each population, permitting its identification (Figure 2). Moreira-Filho and Bertollo (1991) were also able to differentiate six of seven populations of A. scabripinnis on the basis of heterochromatin distribution on the chromosomes.

Although some controversy exists about heterochromatin function, it has been long accepted that the highly repetitive sequences of DNA also play a structural role in conserving important sites of chromosomes (telomeres, centromeres) and/or intergene spaces (e.g., rDNA sites) (Eberl et al., 1993). Thus, we may assume that the most important gene changes that have occurred during the chromosome differentiation process in A. scabripinnis are related to modifications in heterochromatic chromosome segments. Differences involving heterochromatin have been frequently found among related species of Neotropical fish (Andreata et al., 1993, among others) and several other species of plants and animals (King, 1993).

The CVA results permitted us to determine that eight of nine populations studied can be quantitatively differentiated. Although the population from Lavapés River had a unique karyotype, it could not be clearly discriminated by the CVA method on the basis of quantitative parameters. Analysis of Figure 3 shows that despite the high level of chromosomal diversification, nearby populations do not necessarily have similar karyotypes, and distant populations do not always have different karyotypes.

Morphological analysis

Morphological analysis of nine populations of A. scabripinnis by canonical variable analysis permitted the unambiguous identification of five of them (Grande Stream, São Pedro Stream, Claro River, Pardo River, and Pedras Stream) (Figure 3). The remaining four populations could not be individually identified by the application of the CVA method. The populations from Paranapanema basin and Pardo basin could be discriminated from one another while the populations from Tietê basin could not.

The application of CVA analysis to morphological studies of six local populations of A. scabripinnis from different hydrographic basins in Brazil permitted Moreira-Filho and Bertollo (1991) to identify four of them. The better discrimination of populations obtained by Moreira-Filho and Bertollo (1991) could be ascribed to the fact that their specimens were collected from different and distant Brazilian hydrographic basins (some sampling sites were up to 650 km distant from each other). However, morphological studies with A. scabripinnis have been limited to the analysis of only a few external body parts.

Chromosomal and morphological comparative analysis

Morphological differentiation may be concurrent with karyotype divergence and also may be a consequence of geographical barriers (Wilson et al., 1974 and 1975; Lamborot and Eaton, 1992) or, conversely, morphological differentiation may occur independently of chromosome divergence (Baker et al., 1979; Sites Jr., 1982; Larson et al., 1984). Thus, chromosomal rearrangements cannot be pointed out as the major cause of morphological change (Lande, 1979; Schwenk et al., 1982).

The geological events which gave origin to the Paraná River basin began during the Paleozoic period and were completed during the Cretaceous period, about 135 millions of years ago (Penteado, M., personal communication); therefore, the local populations found today may have been maintained isolated for a considerable period of time, with consequent fixation of chromosome rearrangements and of morphological differentiation.

The lack of obvious morphological characters separating each of the nine cytotypes of A. s. paranae studied here indicates that external morphological differentiation is not directly related to karyotype differentiation. These different rates of evolution are not unexpected since A. s. paranae lives only in the headwaters of small streams and rivers in mountain regions (Gomes and Azevedo, 1960); thus, the body morphologies are adaptive for life in these particular regions. The better discrimination of the populations obtained by using cytogenetic data suggests that karyotypic structure changed more rapidly than external body morphology in A. s. paranae. Cytogenetic and morphologic analyses conducted on a shrew species complex of the genus Sorex also showed that morphological differentiation was not necessarily related to karyotype differentiation (Ivanitskaya, 1994; Halka et al., 1994, among others).

The frequency of chromosomal rearrangements (including constitutive heterochromatin additions) varies considerably as a function of many interrelated internal (e.g., molecular, cytomechanical, meiotic) and external factors (e.g., effective population size, gene flow) (Spirito, 1992; Sites Jr. and Reed, 1994). A. scabripinnis populations are characterized by their small size and low vagility. These characteristics are favorable to rapid chromosomal change and speciation, as occurs in several other vertebrate genera (Sites Jr. and Moritz, 1987; King, 1993; Sites Jr. and Reed, 1994).

The conclusions reached in the present study are quite different from those presented by Moreira-Filho and Bertollo (1991) who suggested that karyotypic and morphological diversification occurred at approximately the same rate in A. scabripinnis. This divergence may be due to the fact that in the present study the fish samples were obtained from streams belonging to closely related river basins at distances ranging from four to 40 km (in an area of about 200 km²) and in which the fish fauna probably originated from a single ancestral stock. Conversely, in the study by Moreira-Filho and Bertollo (1991) the samples analyzed were captured in different large river basins, at distances ranging from 50 km to 650 km, and the fish populations probably originated from different ancestral stocks.

The karyotypic differentiation could prevent effective reproduction between populations. To test this hypothesis we intend to perform artificial crosses between specimens from different populations and examine their viability.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Francisco Langeani Neto for taxonomic identification of specimens, to Dr. Valdener Garutti for a critical reading of the manuscript, to MSc. Luzia Aparecida Trinca and MSc. Carlos Tadeu dos Santos Dias for helping with the canonical variable analysis, and to Mr. Renato Devidé for technical assistance. Thanks are also due to two anonymous referees for their perceptive criticism. Funds supporting this study were provided by FUNDUNESP, CNPq, and FAPESP. Publication supported by FAPESP.

RESUMO

Foram realizados estudos citogenéticos e morfológicos em nove populações locais de Astyanax scabripinnis paranae. Todas as populações apresentaram 2n = 50 cromossomos e conspícuas diferenças envolvendo a morfologia dos cromossomos, número e posição das NORs e quantidade e localização dos blocos de heterocromatina constitutiva. O estudo quantitativo dos dados citogenéticos, usando a análise das variáveis canônicas (CVA), mostraram que oito populações apresentaram diferentes constituições cariotípicas. Análises morfológicas baseadas em nove caracteres morfométricos e dois merísticos foram eficazes no estabelecimento de uma clara identificação de cinco populações. Análises comparativas dos dados citogenéticos e morfológicos sugerem que as modificações cromossômicas e morfológicas ocorreram em diferentes taxas, sendo as cromossômicas mais rápidas do que as morfológicas. A despeito da similaridade morfológica encontrada entre algumas populações, diferenças cromossômicas foram detectadas entre todas as populações estudadas.

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(Received November 25, 1996)

Publication Dates

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

History

• Received
  25 Nov 1996