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Genet. Mol. Biol. vol. 21 n. 1 São Paulo Mar. 1998
Leuciscus (Pisces, Cyprinidae) karyotypes: Transect of Portuguese populations
Maria João Collares-Pereira, M.I. Próspero, R.I. Biléu and E.M. Rodrigues
Departamento de Zoologia e Antropologia, Centro de Biologia Ambiental, Faculdade de Ciências, Campo Grande, C2, 3º Piso, 1700 Lisboa, Portugal. Fax: 351 1 7500028. E-mail: email@example.com. Send correspondence to M.J.C.-P.
The presently described Iberian chubs - Leuciscus carolitertii and L. pyrenaicus - sampled throughout their distribution ranges in Portugal were cytogenetically analyzed. Their chromosome numbers were consistently 2n = 50, except for two specimens of L. carolitertii, which exhibited a supernumerary chromosome in some of the metaphases. The karyotypes were found to be highly typical for other Leuciscus taxa, as well as for European leuciscine cyprinids: the chromosome sets are dominated by metacentric and submetacentric elements with a reduced number of acrocentric pairs (three to four); the largest pair of the complements belongs typically to this latter category. The chubs from northern drainages, assignable to L. carolitertii, have apparently a more stable karyotype structure (12M:30S:8A) than the chubs from L. pyrenaicus, which have 12M:32S:6A, but may exhibit in the most southern river basins (Guadiana, Mira, Aljezur, Bordeira and Arade) more variable karyotypes. Besides, these data support the very recent discovery of two genetically distinct Leuciscus taxa in this region of the Iberian Peninsula, suggesting the stochastic fixation of structural chromosome rearrangements in these small and isolated drainages, which may be affected by bottlenecks due to significant variations in hydrological regimes. The NORs were apparently located in one small submetacentric pair of chromosomes and the presence of a heteromorphic sex chromosome system of the ZW/ZZ type was also evidenced for the Iberian endemic chubs.
Cyprinid fishes represent an important element in the Iberian ichthyofauna. As in other European southern peninsulas, they are a taxonomically complex group, due to the high number of endemics, in general with restricted distribution areas. Most of the controversies are related to the correct definition of both the specific and the generic limits, because only a small number of taxa were appropriately revised. Such a situation applies also to the taxonomy of the chubs, the fish of the genus Leuciscus, that have been studied mainly based on morphological and anatomical characters (e.g. Almaça, 1965; Stephanidis, 1971; Bianco and Knezevic, 1987; Doadrio, 1988), in spite of not providing fully reliable criteria at phenotypic level.
In the Iberian Peninsula, the genus Leuciscus has been considered to be represented by three species: the widely distributed European L. cephalus (Linnaeus, 1758), that occurs from the Llobragat River to the French border in Catalonia, and the endemic species, with allopatric but adjacent distribution, L. carolitertii Doadrio, 1988, in the northern part from Lopez River to Mondego River basin, and L. pyrenaicus Gunther, 1868, inhabiting the central and southern parts including the Jucar River basin in Spain (Doadrio et al., 1991).
However, the application of non-morphological methods revealed the existence of more complex patterns as regards population differentiation. Coelho et al. (1995), while studying the genetic variation at 27 presumptive loci of distinct chub populations in Portugal, found new genetically taxa of Leuciscus in the southern drainages of Mira and Arade, similarly to what happened with Tsingenopoulos and Karakousis (1996) after comparing Greek chubs.
The present paper deals with the first cytogenetical data (i.e., diploid chromosome numbers, description of karyotypes with some banding) of the two described endemic chubs, which were sampled in the main populations over both ranges. It is hoped that they will provide a framework for a correct specific definition, and valuable information for the future establishment of phylogenetic relationships among Iberian cyprinids.
MATERIAL AND METHODS
The specimens studied (N = 223) were sampled by electrofishing in 14 different drainages (in a total of 35 distinct sites) from 1989 until 1994. They were brought to the laboratory and kept alive until analysis. Sex was determined whenever possible by macroscopic observation of gonads. The list of analyzed material per drainage is given in Table I and the specific localization of collection sites in Figure 1. Specimens were deposited in the Zoology Department collections.
Table I - Specimens examined by drainage and their chromosomal formula and NF. (M - Metacentric; S - Submetacentric and Subtelocentric; A - Acrocentric and Telocentric).
No. of specimens examined
|Leuciscus carolitertii||Lima|| |
|50||12 M + 30 S + 8 A||92|
|50||10-12 M + 30-32 S + 8 A||92|
|50||12 M + 30 S + 8 A||92|
|50||12 M + 30 S + 8 A||92|
|50||12 M + 30 S + 8 A||92|
|Leuciscus pyrenaicus||Samarra|| |
|50||12 M + 32 S + 6 A||94|
|50||12 M + 32 S + 6 A||94|
|50||12 M + 32 S + 6 A||94|
|50||12 M + 32 S + 6 A||94|
|50||12 M + 30-32 S + 6-8 A||92-94|
|50||10-12 M + 30-32 S + 8 A||92|
|50||10 M + 32 S + 8 A||92|
|50||12 M + 32 S + 6 A||94|
|50||12-14 M + 30-32 S + 6 A||94|
(*) Most southern populations of L. pyrenaicus showing more variable karyotypes.
|Figure 1 - Localities where the fishes were sampled per drainage: Lima (1 - R. Vez, 2 - R. Azêre); Cávado (3 - R. Cávado); Douro (4 - R. Tâmega, 5 - R. Rabaçal, 6 - R. Tuela, 7 - R. Maças); Vouga (8 - R. Vouga); Mondego (9 - R. Mondego, 10 - R. Ceire, 11 - R. Alva); Tejo (12 - R. Sertã, 13 - R. Codes, 14 - R. Zêzere; 15 - R. Nabão, 16 - R. Sever, 17 - R. Sôr, 18 - R. Divor, 19 - R. Canha, 20 - R. Ulme); Samarra (21 - R. Samarra); Cheleiros (22 - R. Cheleiros); Sado (23 - Sta. Susana); Mira (24 , 25 - R. Torgal); Aljezur (26 - R. Aljezur); Arade (27 - R. Odelouca); Bordeira (28 - R. Bordeira); Guadiana (29 - R. Arronches, 30 - R. Xévora, 31 - R. Lucefecit; 32 - R. Degebe, 33 - R. Carreiras, 34 - R. Vascão, 35 - R. Beliche).|
Chromosome preparations were made initially from kidney, using the direct air-drying technique. For the most recently analyzed specimens, fibroblast fin cultures were used as described in Rodrigues and Collares-Pereira (1996). C- and G-bandings were performed following Sumner (1972) and Gold et al. (1990), respectively. Silver positive NORs were visualized according to Rufas et al. (1983) with slight modifications. Slides were also analyzed by counterstain-enhanced fluorescence banding with CMA3 using Sola et al. (1992) protocol, and by propidium iodide fluorescence staining after heat denaturation (DPI) according to Rab et al. (1996b).
At least five to ten Giemsa or banded metaphases per specimen were examined and the chromosomes classified into the three groups M (metacentric), S (submetacentric to subtelocentric) and A (acrocentric to telocentric). To estimate the NF value, the chromosomes of the groups M and S were scored as bi-armed and the chromosomes of the group A as uni-armed.
The diploid chromosome numbers of all specimens examined were 2n = 50 (Table I). The only exceptions were found in two specimens originated from Vouga and Mondego drainages, respectively, showing some metaphases with 2n = 51 due to the existence of a supernumerary chromosome (Figure 2f).
|Figure 2 - (a) C-banded metaphase from a female collected in Guadiana (Ampl. 3000X); (b) G-banded metaphase from a female collected in Tejo (Ampl. 3500X); arrows, sex chromosomes ZW; (c) G-banded metaphase from a male collected in Guadiana (Ampl. 3500X); arrows, sex chromosomes ZZ; (d) CMA3 staining metaphase from an unsexed fish collected in Mondego (Ampl. 3000X); arrows, two NORs; (e) DPI staining metaphase from a female collected in Vouga (Ampl. 3000X); arrows, two NORs; (f) Giemsa-staining metaphase with a supernumerary chromosome (arrow) from an unsexed fish collected in Mondego (Ampl. 4000X).|
Karyotype morphologies of all examined taxa were generally very identical though a certain differentiation at the population level was apparent (Table I). The chubs from northern drainages, assignable to L. carolitertii, exhibited nearly identical karyotypes (12M:30S:8A) (Figure 3). However, the limited number of examined specimens from Cávado left some doubts as to the number of chromosome pairs in the groups M and S (10M:32S vs. 12M:30S).
|Figure 3 - Giemsa-stained karyotypes (2n = 50) of Leuciscus specimens sampled at distinct drainages (all females, with the exception of Aljezur specimen, that was a male), with (M) metacentric, (S) submetacentric and (A) acrocentric chromosome groups. Arrowheads, sex chromosomes (ZW / ZZ); single arrow, Ag positive regions (Ampl. 3500X).|
The Leuciscus from the remaining drainages, generally assignable to L. pyrenaicus, exhibited more variable karyotypes. So, specimens from Samarra, Cheleiros, Tejo, and Sado river basins had complements characterized by the presence of only three pairs of acrocentrics (12M:32S:6A) (Figure 3). The analysis of the small sample of Arade did not allow an unequivocal assignment of the elements with median to submedian centromere positions (12-14M:30-32S:6A). Besides, the same applies to the karyotypes of Guadiana basin specimens, but for the elements with terminal to subterminal centromere positions (12M:30-32S:8-6A). The chubs originated from Mira and Aljezur drainages exhibited karyotypes typically with four pairs of acrocentric chromosomes 10-12M:32-30S:8A (Mira) and 10M:32S:8A (Aljezur). On the contrary, the 11 specimens from Bordeira drainage exhibited a karyotype similar to the one of L. pyrenaicus (12M:32S:6A).
Differences in karyotype morphology between males and females were evidenced in the distinct populations. In fact, in the best metaphase figures, the presence of a large, unpairable submetacentric was observed (Figures 2a,b,c, and 3). The pairing of the remaining chromosomes suggested that the homologue of this element was one of the biggest chromosome of the same group. Such large, unpairable element, was not detected in male complements (Figure 2c). This finding suggests the existence of a female heterogametic system of the ZW/ZZ type.
The examination of chromosomal location of NORs using Ag, CMA3 and DPI stainings revealed one identical NOR phenotype, present in all examined populations. The NORs were located in the telomeres of the short arm of a medium-size submetacentric to subtelocentric pair (Figures 2d,e, and 3).
The interpopulation variability of C- and G-banding patterns could not be well analyzed due to the difficulties in obtaining high resolution metaphases. However, there were faintly C-positive centromeric and telomeric blocks (Figure 2a) as well as some trypsin induced G-bands (Figure 2b,c).
The karyological pattern observed is generally consistent with the situation more commonly found in Leuciscinae: a relative low number of acrocentric chromosome pairs and a dominance of meta/submetacentrics, with one of the latters bearing the NOR sites. However, the number of elements with terminal or almost terminal centromeres seems to be even more reduced in the Iberian chubs than in the known European Leuciscus taxa (Table II).
Table II - Summarized cytogenetic data of species of the genus Leuciscus.
|L. aula||50||15 M + 23 Sm, St + 12 A (2 unpaired)||88||1 ? (*)||Fontana et al., 1970a|
|L. bergi||50||18 M + 22 Sm + 10 A||90||18 M, 23 F, 5 ? (*)||L. Issyk-Kul||Mazik et al., 1986|
|L. borysthenicus||50||16 M + 28 Sm, St + 6 A||94||1 M||R. Strymon||Rab et al., 1996a|
|L. carolitertii||50||12 M + 30 S + 8 A (ZW/ZZ)||92||11 M, 11 F, 58 (*)||B.Lima, Cáv.,Douro, Vouga, Mond.||Present study|
|L. cephalus||50||38 M, Sm + 12 St, A||88||Freiburg||Wolf et al., 1969|
|50||11 M + 29 Sm, St + 10 A (2 unpaired)||3 ? (*)||Fontana et al., 1970a|
|50||20 M, Sm + 30 A||70||R. Fojnica||Berberovic and Sofradzija, 1972|
|L. cephalus cephalus||50||36 M, Sm + 14 St, A||86||20 M, 25 F||L. Turano, L. Piediluco, R. Stura, R. Zeljeznica||Sofradzija, 1977|
|50||18 M + 20 Sm, St + 12 A||88||R. Garonne||Hafez et al., 1978|
|50||10 M + 16 Sm + 14 St + 10 A (ZW/ZZ)||90||15 M, 6 F||R. Morava, R. Moravica||Vujosevic et al., 1983|
|50||34 M, Sm + 16 St, A||84||9 ? (*)||R. Kupa||Al-Sabti 1986, 1987|
|L. cephalus albus||50||36 M, Sm + 14 St, A||86||20 M, 12 F||L. Jablanicko||Sofradzija, 1977|
|L. cephalus cabeda||50||16 M + 12 Sm + 12 St + 10 A||78||4 M, 5 F||R. Savuto, R. Tiber||Cataudella et al., 1977|
|L. idus||52||Lueken and Foerster, 1969|
|(25)||Hinegardner and Rosen, 1972|
|50||38 M, Sm + 12 St, A||88||6 M, 8 F||R. Ukrina, R. Bardaca||Sofradzija, 1977|
|L. keadicus||50||14 M + 12 Sm + 24 St, A||3 ? (*)||R. Evrotas||Tsingenopoulos and Karakousis, 1996|
|L. leuciscus||50||40 M, Sm + 10 St, A||90||9 M, 16 F||R. Ukrina, R. Matura||Sofradzija, 1977|
|50||16 M + 22 Sm, St + 12 A||88||R. Garonne||Hafez et al., 1978|
|L. leuciscus kirgisorum||50||18 M + 22 Sm + 10 A||90||6 ? (*)||R. Chu, R. Turgay, R. Nura, R. Ulja Kojak||Mazik et al., 1986|
|L. pyrenaicus||50||12 M + 32 S + 6 A (ZW/ZZ)||94||8M, 10F, 78 (*)||B. Samarra, Cheleiros, Tejo, Sado||Present study|
|L. schmidti||50||18 M + 22 Sm + 10 A||90||10 M, 5 F, 1 ? (*)||L. Issyk-Kul||Mazik et al., 1986|
|L. souffia muticellus||50||15 M + 23 Sm + 12 A (2 unpaired)||2 ? (*)||Fontana et al., 1970a,b|
|50||20 M + 12 Sm + 8 St + 10 A||82||2 M, 2 juv.||R. Mignone, R. Stura||Cataudella et al., 1977|
|L. souffia agassizi||50||42 M, Sm + 8 St, A||92||12 M, 14 F||R. Drina||Sofradzija, 1977|
|L. svallize||50||24 M, Sm + 26 St, A||74||R. Neretva||Berberovic and Sofradzija, 1972|
|L. svallize svallize||50||32 M, Sm + 18 St, A||82||26 M, 16 F||L. Jablanicko||Sofradzija, 1977|
|L. turskyii||50||68||R. Plavuca||Berberovic and Sofradzija, 1972|
|L. turskyii tenellus||50||34 M, Sm + 16 St, A||84||21 M, 14 F||R. Plavuca, R. Struba||Sofradzija, 1977|
M - Male; F - female; (*) not reported or unsexed.
Although the detection of sex heteromorphic systems is still problematical and demands a careful confirmation, especially in groups with small chromosomes as the cyprinids (Rab and Collares-Pereira, 1995), the apparent evidence of a female heterogamety in Iberian chubs is very interesting. Besides, it was already suggested by Vujosevic et al. (1983) for L. cephalus from lower Danube, but the W chromosome in the Iberian taxa seems to be the biggest S chromosome and not the small A element as in the previous case. Moreover, Fontana et al. (1970a) found unpaired chromosomes in the karyotypes of L. aula, L. cephalus and L. souffia muticellus unfortunately without having the possibility to reference them to sex differentiation.
Considering the potential application of the cytological approach to cyprinid systematic (see Buth et al., 1991), this study provides also data for a correct specific definition, due to the relatively high conservative character of karyotypes in the family (Rab and Collares-Pereira, 1995). So, as regards the interpopulation variability observed, L. carolitertii seems to be more homogeneous in terms of karyological formula, conversely to L. pyrenaicus inhabiting the most southern isolated drainages. Among these latter, the karyotypic data strongly suggest the occurrence of new Leuciscus forms.
This is congruent with the very recent results of Brito et al. (1997), who confirmed the existence of two new taxa respectively in the Mira and Arade drainages by sequencing the cytochrome b. Although our sample from Arade was very small, and Bordeira specimens presented an identical diploid formula to L. pyrenaicus, at least the karyotypes observed in fishes captured in Mira and Aljezur basins seemed to be in fact distinct from the remaining southern samples. Such a chromosome variability suggests the occurrence of a speciation process in those small and isolated southern drainages, which have typically drastic changes in their hydrological regimes. The bottleneck effect due to sporadic mass reductions of population size may effectively favor the stochastic fixation of some chromosome rearrangements and explain the observed chromosome formulae, even in such an apparently conservative family from the karyological standpoint.
In this genus, as well as in the previously genera analyzed cytogenetically and inhabiting Iberian freshwaters (see Collares-Pereira, 1985), there is an apparent tendency for the occurrence of the same type of chromosome structural rearrangements (namely small pericentric inversions, duplications and deletions). However, only the very recent improvement of cell culture techniques allowing to obtain high quality metaphases will give the possibility of acquisition of valuable information through the application of sequencial banding and of new molecular markers. Such data, coupled with research on nuclear DNA molecules, will certainly allow a deep insight into Cyprinidae differentiation processes and evolution.
We are specially grateful to P. Rab for his critical revision of the manuscript, and also to M.M. Coelho, R.M. Brito, M.J. Alves, J.A. Rodrigues, A.M. Pires, T.R. Pacheco and D. Figueiredo for providing material from northern drainages. This project was partially funded by the EU "Environment Program" (contract No. EV5V-CT92-0097) and by the "Centro de Biologia Ambiental" (JNICT - FCUL). M.I. Próspero and R.I. Biléu were recipients of Praxis XXI (BTL/4619/95 and BTL/6370/95 fellowships, respectively).
A análise citogenética dos dois endemismos de Leuciscus atualmente descritos na Península Ibérica foi efetuada em amostras obtidas ao longo da sua área de distribuição. Apresentaram um valor diplóide de 2n = 50, com exceção de dois exemplares de L. carolitertii, os quais exibiam um cromossomo supranumerário em algumas metáfases. Caracterizaram-se por um padrão cariológico idêntico ao dos restantes táxons do mesmo gênero, igualmente partilhado pelos Leuciscinae europeus: um elevado número de cromossomos metacêntricos e submetacêntricos e um número reduzido de acrocêntricos (três a quatro pares), incluindo-se neste último grupo o maior par do complemento. As NORs localizaram-se tipicamente num par de submetacêntricos de reduzidas dimensões, existindo também evidências de um sistema cromossômico de determinação sexual do tipo ZW/ZZ. Porém, os espécimes do norte, L. carolitertii, aparentaram possuir um cariótipo mais estável em termos estruturais (12M:30S:8A) do que os do sul, pertencentes a L. pyrenaicus (12M:32S:6A). Esta espécie exibiu, nas suas populações mais meridionais que se localizam nas bacias do Mira, Aljezur, Bordeira e Arade, alterações da referida fórmula cromossômica, fato que vem em apoio da recente descoberta de dois taxóns de Leuciscus geneticamente diferenciados no sul da Península Ibérica, sugerindo a fixação de rearranjos cromossômicos estruturais nestas pequenas e isoladas bacias hidrográficas, com grandes variações dos seus regimes hidrológicos produzindo os chamados "efeitos de gargalo".
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(Received May 9, 1997)