Abstract. This paper describes a new technique for preparing mitotic fish chromosomes using short-term in vitro treatment with colchicine. The results show that a large number of good quality metaphases (many suitable for chromosome banding) can be obtained by this technique, which requires an average of 1 h and 30 min for all steps. The procedure considerably reduces the time normally required for chromosome preparations in fish.Key words. Fish cytogenetics; mitotic chromosomes; technique.Fish chromosome data have great importance in studies on evolution, systematics and mutagenesis, and in aquaculture. Although many techniques employing the cell culture technique to obtain chromosome preparations have been described 3'4, they are usually expensive and time consuming, a fact which limits their use in many laboratories. Direct preparations are less expensive and quicker to prepare but require injection of mitotic inhibitors into and killing of the animals 5-7. The air-drying technique, the most common procedure used in chromosome preparations, was initially developed for mammalian chromosome studies a and was first used in fishes by Ojima and Hitotsumachi 9. Although the basic steps remain the same, many small modifications have been added to this technique in recent years 1~ The present paper describes a modified method for obtaining fish chromosome preparations using the air-drying technique preceded by short-term colchicine treatment of the cells in vitro. The method results in a reasonable quantity and quality of chromosome spreads and is particularly useful for large fish specimens. Material and methodsMitotic chromosome preparations were obtained from cephalic kidney or gills or regenerated fin epithelium according to the following procedures: The animals were anaesthetized by immersion in a solution of 0.01% benzocaine. As soon as respiratory activity stopped, tissue was obtained either from the gills, by opening the operculum and pulling a forceps over the filaments of the branchial arches, or by cutting a piece of the fin epithelium, or by dissecting out pieces of cephalic kidney. Tissues were washed once in a Petri dish containing Hanks' saline solution (HSS) to remove any adhering fat or blood, and minced in 5-8 ml of clean HSS at room temperature. After discarding residual tissue lumps, the clean cell suspension was placed into a 20-ml conical centrifuge tube and 1 drop of 0.03% colchicine was added. The suspension was stirred lightly and the tube placed in an incubator at 37 ~ for 15 min. After colchicine treatment, the cell suspension was centrifuged at 800-1000 rpm for 7 min. The supernatant was removed by suction and the pellet resuspended by vigorous tapping of the tube, before adding 7 ml of a 0.075 M KC1 hypotonic solution. The tube was then stirred lightly and placed in an incubator at 37 ~ for 30-40 min. After hypotonization, 5 drops of a freshly prepared ice-cold fixative (3 methanol: 1 acetic acid) were added to the tube and the cell suspension stirred gently at room temperature. After 5 mi...

Major and 5S ribosomal genes have been localized in chromosomes from five fish species, genus Astyanax, using in situ hybridization (FISH) with 28S and 5S rDNA probes. In situ signals for the major rDNA co-localized with the 5S rDNA clusters in the pericentromeric region of one marker chromosome in all five species analyzed. The conserved local- ization of these two rDNA clusters in the five related Astyanax species was considered as indicative of a close relationship among them. The use of these molecular markers for elucidating evolutionary relationships among closely related taxa is discussed.

Chromosomes of Moenkhausia sanctaefilomenae (10 males and 20 females) collected from a tributary of the Tiet6 River (Botucatu, S.P. Brazil) were examined using kidney and testicular cells. Both males and females presented 2n = 50 chromosomes and 1 to 8 small supernumerary microchromosomes. C-banding demonstrated positively stained heterochromatic blocks in almost every chromosome and a pattern of interstitial bands located in the same position in relation to the centromere on the long arm of a large number of chromosomes. Two large NORs were detected in pair 19 of all the silver-stained metaphases; zero to 6 additional small NORs were detected in other chromosomes. A general survey of the occurrence of supernumerary chromosomes in fishes is presented.

The genus Eigenmannia comprises several species groups that display a surprising variety of diploid chromosome numbers and sex-determining systems. In this study, hypotheses regarding phylogenetic relationships and karyotype evolution were investigated using a combination of molecular and cytogenetic methods. Phylogenetic relationships were analyzed for 11 cytotypes based on sequences from five mitochondrial DNA regions. Parsimony-based character mapping of sex chromosomes confirms previous suggestions of multiple origins of sex chromosomes. Molecular cytogenetic analyses involved chromosome painting using probes derived from whole sex chromosomes from two taxa that were hybridized to metaphases of their respective sister cytotypes. These analyses showed that a multiple XY system evolved recently (o7 mya) by fusion. Furthermore, one of the chromosomes that fused to form the neo-Y chromosome is fused independently to another chromosome in the sister cytotype. This may constitute an efficient post-mating barrier and might imply a direct function of sex chromosomes in the speciation processes in Eigenmannia. The other chromosomal sexdetermination system investigated is shown to have differentiated by an accumulation of heterochromatin on the X chromosome. This has occurred in the past 0.6 my, and is the most recent chromosomal sex-determining system described to date. These results show that the evolution of sexdetermining systems can proceed very rapidly.

Karyotypes and nuclear DNA content were studied in 11 species of the genus Corydoras from rivers in South America: C. sp. from Caripi river 2n = 60, C. cf. simulatus 2n = 62, C. simulatus 2n = 62, C. reticulafus 2n = 74, C. sp. from Galheiro river 2n = 84, C. aff. punctatus from Negro river 2n = 102, C.Javeolus 2n = 58, C. arcuatus 2n = 46, C . trilineatus 2n =46, C. schwartzi 2n = 46, and C. metae 2n=92. Extensive chromosome diversity and differences in DNA content were detected among species. The high variability in chromosome counts was not exclusively related to chromosomal structural rearrangements, but also to large changes in DNA content. Species could be grouped using their shared cytogenetic characteristics, suggesting that within the genus Corydoras different groups of species followed distinct evolutionary trends. Chromosomal rearrangements in Corydoras are, apparently, more frequent that morphological modifications, so cytogenetic data may be very useful for species delimitation and for the understanding of interrelationships among species.

Chromosomes of a species of Eigenmannia presenting a X1X1X2X2:X1X2Y sex chromosome system, resulting from a Y-autosome Robertsonian translocation, were analyzed using the C-banding technique, chromomycin A3 (CMA3) and mithramycin (MM) staining and in situ digestion by the restriction endonuclease AluI. A comparison of the metacentric Y chromosome of males with the corresponding acrocentrics in females indicated that a C-band-positive, CMA3/MM-fluorescent and AluI digestion-resistant region had been lost during the process of translocation, resulting in a diminution of heterochromatin in the males. It is hypothesized that the presence of a smaller amount of G + C-rich heterochromatin in the sex chromosomes of the heteromorphic sex when compared with the homomorphic sex may be associated with the sex determination mechanism in this species and may be a more widely occurring phenomenon in fish with differentiated sex chromosomes than was initially thought.

SUMMARY -Cytogenetic studies involving conventional Giemsa stammg, Cbanding analysis and silver staining of NORs were performed on nine species belonging to six genera of the family Callichthyidae. The diploid number ranged from 2n = 44 to 2n = 100, the number of chromosomal pairs with NORs ranged from 1 to 4 and constitutive heterochromatin was mainly distributed in the centromeric and/or pericentromeric position of the chromosomes. The DNA content of erythrocytes from six species studied ranged from 1.18±0.07 to 2.77±0.22 pg/nucleus. The extensive variability in karyotypes and in nuclear DNA content detected are in accordance with the initial hypothesis that chromosome rearrangements and polyploidy have played a significant role in the evolutionary history of Callichthyidae.

Here we describe a new species of Gymnotus, G. pantanal n. sp., from the Pantanal Matogrossense of Brazil, using morphological, cytogenetic, and molecular data. Specimens ascribed to the new species are also known from areas downstream in Paraguay, and from the adjacent Guaporé basin of Bolivia. The new species most closely resembles G. anguillaris in possessing an elongate body, slender profile, long body cavity, and shorter head than other congeners. The new species also resembles G. anguillaris in the presence of pale narrow bands restricted to the area below the lateral line on the anterior half of the body. The new taxon differs from G. anguillaris in possessing more narrowly set eyes, a wider and deeper head, a larger branchial opening, longer pectoral fins with more fin rays, and fewer pored posterior lateral-line scales. The new species inhabits rooted grasses and floating macrophytes in small creeks and along the banks of larger blackwater rivers. Populations are found syntoptically with G. inaequilabiatus and G. sylvius. Compared with these species, the new species exhibits a distinct combination of microsatellite DNA amplification patterns, and chromosomal and external features. These results confirm earlier studies showing the power of a multidisciplinary approach to characterizing the enormous and often cryptic diversity of Neotropical fishes.