Mauro Nirchio. M.Sc. in Marine Sciences, Universidad de Oriente (UDO), Venezuela. Professor, UDO. Address: Escuela de Ciencias Aplicadas del Mar, UDO, Núcleo Nueva Esparta. Isla de Margarita, Venezuela. e-mail: nirchio@cantv.net

Hernán Cequea. M.Sc. and Ph.D. Texas Tech University, USA. Professor, UDO. Address: Departamento de Biología, Escuela de Ciencias, Núcleo Sucre, UDO. Cumaná, Venezuela. e-mail: hcequea@udo.edu.ve

Bruce J. Turner. M.A. and Ph.D, University of California at Los Angeles, USA. Associate Professor of Biology. Address: Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406. e-mail: fishgen@vt.edu

Conventional Giemsa and silver staining techniques were used to analyze the chromosome complement of the "pupfish" Cyprinodon dearborni, using specimens from Margarita Island, Venezuela. This species is of potential interest because of its unique geographic distribution: All other pupfish species are Nearctic or Antellian, and no other Nearctic killifish genus (e.g. Fundulus) is represented on the South American mainland. The karyotype of our specimens comprises 1 metacentric (M) pair, 5 submetacentric (SM) pairs and 18 acrocentric (A) pairs, with an arm number of NF=60. Nucleolus organizer regions (NORs) are located terminally on the short arms of a medium subtelocentric pair. Sequential staining allowed to identify a homologous pair, designated as chromosome pair 9, as NOR-bearing. There may be a few arm ratio/shape differences between the karyotype of C. dearborni and those of congeneric species reported in the literature. However, the differences appear minor and may stem from variation in chromosome contraction related to fixation rather than real cytogenetic divergence. The present data extend the observation of apparent karyological conservatism in Cyprinodon to an unusual geographic "outlier" species. The clarity with which silver staining is able to resolve the NOR-bearing chromosome pair suggests that this technique may prove informative when applied to the chromosomes of congeneric species for higher resolution chromosome comparisons within the genus.

Mediante las técnicas de tinción convencional de Giemsa y de impregnación argéntica se analizó el complemento cromosómico de la "petota" Cyprinodon dearborni, usando especímenes provenientes de la Isla de Margarita, Venezuela. Esta especie es de interés potencial debido a su distribución geográfica única; todas las otras especies de petotas son Nearcticas o Antillanas y ningún otro género Neárctico (e.g. Fundulus) está representado en el continente Suramericano. El cariotipo de los especimenes analizados está constituido por 1 par metacéntrico (M), 5 pares submetacéntricos (SM) y 18 pares acrocéntricos (A), con un número de brazos (NF) de 60. Las regiones organizadoras del nucleolo (NORs) están localizadas terminalmente en el brazo corto de un par acrocéntrico. La tinción secuencial permitió identificar un par homólogo, designado par 9, como portador de NORs. Puede haber unas pocas diferencias entre el cariotipo de C. dearborni y el de las especies congenéricas reportadas en la literatura. Sin embargo las diferencias parecen ser menores y pueden derivar de la variación en la contracción de los cromosomas debidas a la fijación de las muestras más que a una divergencia citogenética real. Existe una aparente conservación cariológica en Cyprinodon que los datos presentados amplían a una especie única por su distanciamiento geográfico de las demás especies del género. La claridad con que la tinción argéntica define el par de cromosomas portadores de NORs sugiere que esta técnica puede resultar muy informativa al aplicarla a los cromosomas de las especies congenéricas para establecer comparaciones cromosómicas de mayor resolución dentro del grupo.

Mediante as técnicas de tinção convencional de Giemsa e de impregnação argêntea se analisou o complemento cromossômico da "petota" Cyprinodon dearborni, usando espécimes provenientes da Ilha de Margarita, Venezuela. Esta espécie é de interesse potencial devido a sua distribuição geográfica única; todas as outras espécies de "petotas" são Nearcticas or Antilhanas e nenhum outro gênero Neárctico (e.g. Fundulus) está representado no continente Sul-americano. O cariótipo dos espécimes analisados está constituído por 1 par metacêntrico (M), 5 pares sub-metacêntricos (SM) e 18 pares acrocêntricos (A), com um número de braços (NF) de 60. As regiões organizadoras do nucléolo (NORs) estão localizadas terminalmente no braço curto de um par acrocêntrico. A tinção seqüencial permitiu identificar um par homólogo, designado par 9, como portador de NORs. Pode haver umas poucas diferenças entre o cariótipo de C. dearborni e o das espécies congenéricas reportadas na literatura. No entanto, as diferenças parecem ser menores e podem derivar da variação na contração dos cromossomos devido à fixação das amostras mais que a uma divergência cito genética real. Existe uma aparente conservação cariológica em Cyprinodon que os dados apresentados ampliam a uma espécie única por seu distanciamento geográfico das demais espécies do gênero. A claridade com que a tinção argêntea define o par de cromossomos portadores de NORs sugere que esta técnica pode resultar muito informativa ao aplicá-la aos cromossomos das espécies congenéricas para estabelecer comparações cromossômicos de maior resolução dentro do grupo.

Cyprinodon dearborni (Meek, 1909; Pisces: Cyprinodontidae), is an endemic "pupfish" species known from the Caribbean coast of Venezuela, Aruba, Curação, and Bonaire, where it is often locally abundant in hypersaline littoral lagoons (Cervigón, 1991). The precise extent of its geographic distribution has not yet been firmly established. For example, Wildekamp (1995) notes that there are unconfirmed records of the species from Colombia and Guyana, and his distribution map, without additional comment, shows a record from Trinidad. On morphological grounds, C. dearborni is a component of a loosely defined "Cyprinodon variegatus complex," otherwise known from the Atlantic coast of North America, the Greater Antilles, Bahamas, Yucatan/Belize, Jamaica and the Caymans, and it has sometimes been considered a subspecies of C. variegatus itself. However, zoogeographically, C. dearborni must be considered a unique "outlier" species, for the distribution of no other essentially Nearctic cyprinodontoid genus (e.g., Fundulus, which is otherwise widespread) reaches the coast of northern South America. At present there is no readily apparent historical explanation for this unusual distribution. Its closest congeneric neighbors are C. v. artifrons (Yucatan-Belize, southern range limit unknown) and C. v. "riverendi" (Cuba, Jamaica and Grand Cayman).

Studies of C. dearborni have thus far included descriptions of feeding habits, growth rates and maturation, embryonic and larval development, reproductive behavior (Cardona and Gómez, 2001), temperature acclimatization (Chung, 1980) and genetic variation estimates by enzyme electrophoresis (Nirchio, 1996; González and Nirchio, 1997). In this article we report on the chromosome complement of C. dearborni, as represented by specimens from Margarita Island, Venezuela. Cyprinodon species from the North American mainland are known to be karyologically conservative to the point of being essentially invariant (see Klinkhardt et al., 1995). We asked if this conservatism extended to this geographically unusual species. We also used silver staining to visualize the nucleolar organizer regions of the C. dearborni genome in order to assess the potential of this technique for future comparative studies of the Cyprinodon genus.

Chromosome data are from ten adult specimens (5 males and 5 females), collected from La Restinga Lagoon, Margarita Island, Venezuela (10º59'7''N, 64º07'58''W). Voucher specimens were deposited at the Ichthyology Collection of the Escuela de Ciencias Aplicadas del Mar, Universidad de Oriente.

Mitotic chromosomes were prepared from the gill epithelium of specimens injected intraperitoneally with 0.1% colchicine (1ml/100g fish weight) employing an insulin syringe. The fish were maintained for 4h in a well-aerated aquarium. Then, their gills were extracted and placed in a hypotonic solution of 0.4% KCl. Tissue was minced with fine forceps and thereafter with a glass syringe by repeated aspiration and forced release, until a fine cellular suspension was obtained. After 30min in the hypotonic solution, the cellular suspension was centrifuged at 1000rpm for 3min. After discarding the supernatant, the cellular button was suspended and washed 3 times in a methanol-acetic acid mixture 3:1 (v:v). One droplet of the cellular suspension was dropped on a clean microscope slide, previously chilled in a freezer, from a height of 45cm. The slides were briefly put over a flame and then allowed to air-dry. To corroborate the karyotype of mitotic cells (2n), meiotic analysis from samples of testes from mature individuals were performed. Testicular tissues were dissected and fixed in methanol-acetic acid mixture (3:1) for 12h. For the conventional karyotypes, the preparations were stained for 20min with 10% Giemsa in phosphate buffer, pH 6.88. Metaphase I configurations were analyzed by squashing a small sample of testicular tissue stained with acetic orcein. To identify correspondences among NOR bearing chromosomes with chromosomes previously stained with Giemsa, slides were then destained in methanol-acetic acid (3:1) for 10min and treated according to the controlled silver nitrate one step method (Howell and Black, 1980) for the characterization of nucleolar organizer regions (NORs).

Mitotic and meiotic chromosomes were photographed using a Nikon COOLPIX® 995 Digital Camera following the microscopy settings recommended by the manufacturer. (http://www.nikonusa.com/pdf/CoolpixSeriesMtngInstructions). Images were stored as *.tif files and digitally processed with ADOBE PHOTOSHOP v. 7.0. This software was also used for measuring long arm (L), short arm (S) and whole chromosome lengths, and constructing karyograms based on arm length ratios (Levan et al., 1964).

Counts of a total of 61 spreads gave 2n=48 as the modal diploid number of C. dearborni (Figure 1a). The modal complement was observed in 83% of all recorded cells, i.e. 17% of the counts were hypomodal or hypermodal. Normal meioses in metaphase I contained 24 elements, with 20 circle and 4 chain bivalents and with a chiasmata number per cell of 44. The counts of meiotic bivalents corroborate the diploid number in mitotic cells (Figure 1e). The karyogram obtained by arranging the chromosomes in order of decreasing size and centromere position is shown on Figure 1c. It consists of one metacentric (M; pair 1), 5 submetacentric (SM; pairs 2 to 6) and 18 acrocentric (A; pairs 7 to 24). Arm number (Nombre Fondamental) was 60, calculated by assigning of a value of 2 for M/SM chromosomes and 1 for the A chromosomes.

The pair of metacentric elements (pair 1) was easily identifiable as homologous by its morphology and size, but the rest of the chromosomes could not be characterized in the same way because it was impossible to establish clear differences among adjacent pairs in the size-graded series. A detail to be noticed is that there was an apparent heteromorphy of pair 1 (Figure 1d), that initially led to suspect polymorphism or heterogamety. This possibility was discarded after it was noted that the presumptive heteromorphism occurred both among and within individual spreads from the same organism, and at the same frequency in both sexes. Thus, apparent differences in arm lengths of pair 1 can be attributed to a technical artifact.

Ag-stained metaphases in C. dearborni exhibited positive signals in only one small subtelocentric chromosome pair, with the black dot located terminally on the short arm of the elements. Sequential staining allowed to identify pair 9 as the NOR bearing chromosomes and as homologous (Figures 1b and 1c).

A single pair of NOR-bearing chromosomes seems to represent the "primitive" karyotypical condition in most vertebrate species (Hsu et al., 1975; Schmidt, 1978) and this number is also characteristic of most teleostean fishes thus far studied (Vitturi et al., 1995). However, there appear to be no reports of the number or position of NOR-bearing chromosomes in other species of Cyprinodon. Consequently, the question of the relative "primitiveness" of the C. dearborni karyotype, as well as that of possible NOR-related divergence among other pupfish species, remains open.

The chromosome numbers of C. dearborni presented here agree with those of 12 taxa previously reported: C. atrorus, C. bifasciatus, C. eximius, C. macularis, C. nevadensis nevadensis, C. nevadensis mionectes, C. radiosus, C. rubrofluviatilis, C. salinus, C. variegatus variegatus, C. variegatus ovinus. In these species, the karyotype formula is essentially a 2M+14SM+32A (see Klinkhardt et al., 1995). For C. dearbornii, we found 2M+10SM+36A, suggesting the possibility of karyological divergence between it and its congeners. However, the differences appear to be minor, and may stem simply from slight differences in chromosome condensation and/or fixation, or in interpretation of images by individual observers. Comparisons of the karyotypes of different species prepared in the same laboratory are needed to evaluate this question.

Our results extend the observation of apparent chromosomal conservatism of pupfishes (Miller and Echelle, 1975) at least at the level of conventional cytological preparations, to a geographical outlier species. Given the extensive chromosomal variation seen in other cyprinodontoid groups (e.g., the West African rivuline species or goodeids in general), this conservatism is difficult to explain, especially since there is no reason to believe that pupfishes are significantly younger than other killifish groups. These observations form a specific example of the more general question of the biological significance of chromosome divergence as a whole. The cyprinodontoid fishes, with sharply contrasting patterns of chromosomal and organismal divergences, might well be a group in which this question can be profitably explored.

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