BackgroundThe megadiverse Neotropical freshwater ichthyofauna is the richest in the world with approximately 6,000 recognized species. Interestingly, they are distributed among only 17 orders, and almost 80% of them belong to only three orders: Characiformes, Siluriformes and Perciformes. Moreover, evidence based on molecular data has shown that most of the diversification of the Neotropical ichthyofauna occurred recently. These characteristics make the taxonomy and identification of this fauna a great challenge, even when using molecular approaches. In this context, the present study aimed to test the effectiveness of the barcoding methodology (COI gene) to identify the mega diverse freshwater fish fauna from the Neotropical region. For this purpose, 254 species of fishes were analyzed from the Upper Parana River basin, an area representative of the larger Neotropical region.ResultsOf the 254 species analyzed, 252 were correctly identified by their barcode sequences (99.2%). The main K2P intra- and inter-specific genetic divergence values (0.3% and 6.8%, respectively) were relatively low compared with similar values reported in the literature, reflecting the higher number of closely related species belonging to a few higher taxa and their recent radiation. Moreover, for 84 pairs of species that showed low levels of genetic divergence (<2%), application of a complementary character-based nucleotide diagnostic approach proved useful in discriminating them. Additionally, 14 species displayed high intra-specific genetic divergence (>2%), pointing to at least 23 strong candidates for new species.ConclusionsOur study is the first to examine a large number of freshwater fish species from the Neotropical area, including a large number of closely related species. The results confirmed the efficacy of the barcoding methodology to identify a recently radiated, megadiverse fauna, discriminating 99.2% of the analyzed species. The power of the barcode sequences to identify species, even with low interspecific divergence, gives us an idea of the distribution of inter-specific genetic divergence in these megadiverse fauna. The results also revealed hidden genetic divergences suggestive of reproductive isolation and putative cryptic speciation in some species (23 candidates for new species). Finally, our study constituted an important contribution to the international Barcoding of Life (iBOL.org) project, providing barcode sequences for use in identification of these species by experts and non-experts, and allowing them to be available for use in other applications.

An analysis of the nucleolar organizer region (NOR) by silver staining of five species of the order Gymnotiformes, Gymnotus carapo, Apteronotus albijrons, Sternopygus macrurus, Eigenmannia virescens, and Eigenmannia sp., is reported. The five species presented only one pair of homologs bearing NORs. In G. carapo, A. albijrons, and S. macrurus the silver-stained NORs were present as small black dots over the chromosomes. In E. virescens, the short arm of one of the largest chromosome pairs was entirely stained with silver. Eigenmannia sp. presented an intraspecific variability of the NOR-bearing pair and an increase in the length of this region, the larger one being about six times the size of the smaller. These findings suggest that an increase in the number of ribosomal genes in the NORs of certain species occurred during the evolution of this group.

SummaryThis survey was performed to characterize the embryogenesis of Prochilodus lineatus. Seven stages of embryo development were identified -zygote, cleavage, blastula, gastrula, segmentation, larval and hatching -after a period of incubation of 22 h (24• C) or 14 h (28 • C). The following cleavage pattern was identified: the first plane was vertical (2 blastomeres); the second was vertical and perpendicular to the first (4 blastomeres); the third was vertical and parallel to the first (4 × 2); the fourth cleavage was vertical and parallel to the second (4 × 4); the fifth was vertical and parallel to the first (4 × 8); and the sixth cleavage was horizontal (64 blastomeres • C); 11.0-13.0 h (28 • C)) showed a free tail, more than 25 somites, an optic vesicle and a ready-to-hatch larval shape. The blastomeres at cleavage stage had disorganized nuclei indicating high mitotic activity. At gastrula, the blastomeres and the periblast had euchromatic nuclei and a large number of mitochondria and vesicles. The yolk was organized into globose sacs, which were dispersed into small pieces prior to absorption.

The main objectives of this study are estimate a species-dense, time-calibrated molecular phylogeny of Hypoptopomatinae, Neoplecostominae, and Otothyrinae, which together comprise a group of armoured catfishes that is widely distributed across South America, to place the origin of major clades in time and space, and to demonstrate the role of river capture on patterns of diversification in these taxa. We used maximum likelihood and Bayesian methods to estimate a time-calibrated phylogeny of 115 loricariid species, using three mitochondrial and one nuclear genes to generate a matrix of 4,500 base pairs, and used parametric biogeographic analyses to estimate ancestral geographic ranges and to infer the effects of river capture events on the geographic distributions of these taxa. Our analysis recovered Hypoptopomatinae, Neoplecostominae, and Otothyrinae as monophyletic with strong statistical support, and Neoplecostominae as more closely related to Otothyrinae than to Hypoptopomatinae. Our time-calibrated phylogeny and ancestral-area estimations indicate an origin of Hypoptopomatinae, Neoplecostominae, and Otothyrinae during the Lower Eocene in the Atlantic Coastal Drainages, from which it is possible to infer several dispersal events to adjacent river basins during the Neogene. In conclusion we infer a strong influence of river capture in: (1) the accumulation of modern clade species-richness values; (2) the formation of the modern basin-wide species assemblages, and (3) the presence of many low-diversity, early-branching lineages restricted to the Atlantic Coastal Drainages. We further infer the importance of headwater stream capture and marine transgressions in shaping patterns in the distributions of Hypoptopomatinae, Neoplecostominae and Otothyrinae throughout South America.

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...

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.

A substantial fraction of the eukaryotic genome consists of repetitive DNA sequences that include satellites, minisatellites, microsatellites, and transposable elements. Although extensively studied for the past three decades, the molecular forces that generate, propagate and maintain repetitive DNAs in the genomes are still discussed. To further understand the dynamics and the mechanisms of evolution of repetitive DNAs in vertebrate genome, we searched for repetitive sequences in the genome of the fish species Hoplias malabaricus. A satellite sequence, named 5SHindIII-DNA, which has a conspicuous similarity with 5S rRNA genes and spacers was identified. FISH experiments showed that the 5S rRNA bona fide gene repeats were clustered in the interstitial position of two chromosome pairs of H. malabaricus, while the satellite 5SHindIII-DNA sequences were clustered in the centromeric position in nine chromosome pairs of the species. The presence of the 5SHindIII-DNA sequences in the centromeres of several chromosomes indicates that this satellite family probably escaped from the selective pressure that maintains the structure and organization of the 5S rDNA repeats and become disperse into the genome. Although it is not feasible to explain how this sequence has been maintained in the centromeric regions, it is possible to hypothesize that it may be involved in some structural or functional role of the centromere organization.

DNA-based studies have been one of the major interests in conservation biology of endangered species and in population genetics. As species and population genetic assessment requires a source of biological material, the sampling strategy can be overcome by non-destructive procedures for DNA isolation. An improved method for obtaining DNA from fish fins and scales with the use of an extraction buffer containing urea and further DNA purification with phenol-chloroform is described. The methodology combines the benefits of a non-destructive DNA sampling and its high efficiency. In addition, comparisons with other methodologies for isolating DNA from fish demonstrated that the present procedure also becomes a very attractive alternative to obtain large amounts of high-quality DNA for use in different molecular analyses. The DNA samples, isolated from different fish species, have been successfully used on random amplified polymorphic DNA (RAPD) experiments, as well as on amplification of specific ribosomal and mitochondrial DNA sequences. The present DNA extraction procedure represents an alternative for population approaches and genetic studies on rare or endangered taxa.