BackgroundWith nearly 1,100 species, the fish family Characidae represents more than half of the species of Characiformes, and is a key component of Neotropical freshwater ecosystems. The composition, phylogeny, and classification of Characidae is currently uncertain, despite significant efforts based on analysis of morphological and molecular data. No consensus about the monophyly of this group or its position within the order Characiformes has been reached, challenged by the fact that many key studies to date have non-overlapping taxonomic representation and focus only on subsets of this diversity.ResultsIn the present study we propose a new definition of the family Characidae and a hypothesis of relationships for the Characiformes based on phylogenetic analysis of DNA sequences of two mitochondrial and three nuclear genes (4,680 base pairs). The sequences were obtained from 211 samples representing 166 genera distributed among all 18 recognized families in the order Characiformes, all 14 recognized subfamilies in the Characidae, plus 56 of the genera so far considered incertae sedis in the Characidae. The phylogeny obtained is robust, with most lineages significantly supported by posterior probabilities in Bayesian analysis, and high bootstrap values from maximum likelihood and parsimony analyses.ConclusionA monophyletic assemblage strongly supported in all our phylogenetic analysis is herein defined as the Characidae and includes the characiform species lacking a supraorbital bone and with a derived position of the emergence of the hyoid artery from the anterior ceratohyal. To recognize this and several other monophyletic groups within characiforms we propose changes in the limits of several families to facilitate future studies in the Characiformes and particularly the Characidae. This work presents a new phylogenetic framework for a speciose and morphologically diverse group of freshwater fishes of significant ecological and evolutionary importance across the Neotropics and portions of Africa.

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.

This study provides the most comprehensive Model-Based Total Evidence (MBTE) phylogenetic analyses of the clade Gymnotiformes to date, reappraising relationships using a dataset comprised of six genes (5277bp) and 223 morphological characters, and an ingroup taxon sample including 120 of 212 valid species representing 34 of the 35 extant genera. Our MBTE analyses indicate the two main gymnotiform clades are Gymnotidae and Sternopygoidei, the latter comprised of Rhamphichthyoidea (Rhamphichthyidae+Hypopomidae) and Sinusoidea (Sternopygidae+Apteronotidae). Within Gymnotidae, Electrophorus and Gymnotus are sister taxa, and Gymnotus includes the following six clades: (i) G. pantherinus clade, (ii) G. coatesi clade, (iii) G. anguillaris clade, (iv) G. tigre clade, (v) G. cylindricus clade, and (vi) G. carapo clade. Within Rhamphichthyoidea, Steatogenae (Steatogenys+Hypopygus) is a member of Rhamphichthyidae, and Hypopomidae includes the following clades: (i) Akawaio, (ii) Hypopomus, (iii) Microsternarchini, and (iv) Brachyhypopomus. Within Sternopygidae, Sternopygus and Eigenmanninae are sister groups, Rhabdolichops is the sister to other Eigenmanninae, Archolaemus+Distocyclus is the sister to Eigenmannia, and Japigny is nested within Eigenmannia. Within Apteronotidae, Sternarchorhamphinae (Sternarchorhamphus+Orthosternarchus) is the sister to Apteronotinae, Adontosternarchus is the sister group to other Apteronotinae, Sternarchorhynchini (Sternarchorhynchus+Platyurosternarchus) is the sister to Navajini, and species assigned to Apteronotus are members of two separate clades: (i) A. sensu stricto in the Apteronotini, and (ii) the "A." bonapartii clade in the Navajini.

Until recently, the study of negative and antagonistic interactions (for example, competition and predation) has dominated our understanding of community structure, maintenance and assembly. Nevertheless, a recent theoretical model suggests that positive interactions (for example, mutualisms) may counterbalance competition, facilitating long-term coexistence even among ecologically undifferentiated species. Müllerian mimics are mutualists that share the costs of predator education and are therefore ideally suited for the investigation of positive and negative interactions in community dynamics. The sole empirical test of this model in a Müllerian mimetic community supports the prediction that positive interactions outweigh the negative effects of spatial overlap (without quantifying resource acquisition). Understanding the role of trophic niche partitioning in facilitating the evolution and stability of Müllerian mimetic communities is now of critical importance, but has yet to be formally investigated. Here we show that resource partitioning and phylogeny determine community structure and outweigh the positive effects of Müllerian mimicry in a species-rich group of neotropical catfishes. From multiple, independent reproductively isolated allopatric communities displaying convergently evolved colour patterns, 92% consist of species that do not compete for resources. Significant differences in phylogenetically conserved traits (snout morphology and body size) were consistently linked to trait-specific resource acquisition. Thus, we report the first evidence, to our knowledge, that competition for trophic resources and phylogeny are pivotal factors in the stable evolution of Müllerian mimicry rings. More generally, our work demonstrates that competition for resources is likely to have a dominant role in the structuring of communities that are simultaneously subject to the effects of both positive and negative interactions.

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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.