Testing monophyly of the freshwater fish Leporinus (Characiformes, Anostomidae) through molecular analysis.

Monophyly of the genus Leporinus (Characiformes: Anostomidae) was tested by sequencing and analysing a total of 4732 bp, including two mitochondrial [cytochrome oxidase subunit 1 (CO1) and cytochrome b (Cytb)] and three nuclear [myosin heavy chain 6 cardiac muscle alpha (Myh6), recombination activating gene 1 (RAG1) and recombination activating gene 2 (RAG2)] loci for 22 species of Leporinus, or c. 25% of all described species in the genus. Phylogenetic tree analyses (maximum parsimony, maximum likelihood and Bayesian species tree) indicate Leporinus to be paraphyletic, with monophyly being rejected by both Kishino-Hasegawa and Shimodaira-Hasegawa tests. The sequenced species of Leporinus are distributed across five clades that are interleaved among other anostomid genera. Several taxonomic changes are suggested as being necessary to restore monophyly for the group. The clade containing the type species, Leporinus fasciatus, should be considered Leporinus sensu stricto and at least three new genera should be described for other species currently considered part of Leporinus.

Molecular systematics of the neotropical shovelnose catfish genus Pseudoplatystoma Bleeker 1862 based on nuclear and mtDNA markers.

Pseudoplatystoma is a commercially important genus of Neotropical migratory catfishes widely distributed in all major river basins of South America. Historically, only three species were recognized, but a recent revision proposed eight putative morphospecies for the genus. A molecular study based on mitochondria DNA (mtDNA) provided support for recognition of only some of the species and raised questions about species boundaries in this group. We present a more encompassing analysis based on mtDNA (cytochrome b, 818bp) and nuclear DNA-based phylogenies (Rag1 intron 1, 664bp and S7 intron 1, 635bp) for a more extensive sampling (279 individuals from 42 localities) of all putative species in all major river basins. Patterns generated by individual gene genealogies and a multispecies coalescent analysis provided evidence to suggest recognition of only four distinct species in this genus: Pseudoplatystoma magdaleniatum, Pseudoplatystoma corruscans, Pseudoplatystoma tigrimun (sensu lato) and Pseudoplatystoma fasciatum (sensu lato). The species phylogeny places P. magdaleniatum as the sister group to all the other species in the genus, but the relationships among P. fasciatum s.l, P. tigrimum s.l., and P. corruscans could not be resolved with confidence.

Genetic analysis reveals population structuring and a bottleneck in the black-faced lion tamarin (Leontopithecus caissara).

The ability of a population to evolve in a changing environment may be compromised by human-imposed barriers to gene flow. We investigated the population structure and the possible occurrence of a genetic bottleneck in two isolated populations of the black-faced lion tamarin (Leontopithecus caissara), a species with very reduced numbers (less than 400) in a very restricted range in the Atlantic Forest of southeast Brazil. We determined the genotypes of 52 individuals across 9 microsatellite loci. We found genetic divergence between the populations, each exhibiting low genetic diversity. Analysis revealed broad- and fine-scale population structuring. Both populations have evidently experienced population reduction and a genetic bottleneck without presenting any apparent detrimental effect. Anyway, measures should be taken to effectively protect the forests where L. caissara occurs in order to allow its populations to increase and counteract the eventual effects of genetic impoverishment.

Contribution of conservation genetics in assessing neotropical freshwater fish biodiversity.

Human activities have a considerable impact on hydrographic systems and fish fauna. The present review on conservation genetics of neotropical freshwater fish reveals that DNA analyses have been promoting increased knowledge on the genetic structure of fish species and their response to environmental changes. This knowledge is fundamental to the management of wild fish populations and the establishment of Evolutionary Significant Units capable of conserving genetic integrity. While population structuring can occur even in long-distance migratory fish, isolated populations can show reduced genetic variation and be at greater risk of extinction. Phylogeography and phylogeny have been powerful tools in understanding the evolution of fish populations, species and communities in distinct neotropic environments. Captive fish can be used to introduce new individuals and genes into the wild and their benefits and disadvantages can be monitored through genetic analysis. Understanding how fish biodiversity in neotropical freshwaters is generated and maintained is highly important, as these habitats are transformed by human development and fish communities are increasingly exploited as food sources to sustain a growing human population.

Population structuring of the endemic black-cheeked gnateater, Conopophaga melanops melanops (Vieillot, 1818) (Aves, Conopophagidae), in the Brazilian Atlantic Forest.

Randomly amplified polymorphic DNA (RAPD) markers were used to analyze genetic differentiation among three populations of the endemic Black-cheeked Gnateater (Conopophaga melanops melanops) within a larger pristine reminiscent of the Brazilian Atlantic Forest. Analyses of molecular variance (AMOVA) (phiST=0.13149, P<0.0001) and the nonparametric test for homogeneity of the molecular variance (HOMOVA) (B=0.32337; P=0.0019) showed a statistically significant genetic divergence among the three Black-cheeked Gnateater populations in a continuous transect of 250 km. Some hypothetic explanations for these results are the sedentary nature of the species and the historical isolation of the populations in refuges during the Pleistocene. The present results suggest that the local populations were naturally differentiated along the entire original range before the recent process of massive deforestation.

Genetic evidence of population structuring in the neotropical freshwater fish Brycon hilarii (Valenciennes, 1850).

Brycon hilarii is a migratory fish widely distributed throughout the Paraguay River Basin. It is appreciated in sport fishing and for its superior meat quality. It is also the main species for tourist attraction in the Bonito region (State of Mato Grosso do Sul, Brazil). Considering the lack of information on the genetic structure of the fish of this species, the aim of the present study was to detect the genetic variability of Brycon hilarii through RAPD markers. A total of eighty specimens collected in different seasons at four sites of the Miranda River sub-basin (Paraguay River Basin, Brazil) were used for analysis. The results of genetic similarity, Shannon diversity, and AMOVA revealed differences between the sampling sites. Through AMOVA, differences between populations were more evident among the animals collected during the non-reproductive season, corresponding to a time of less movement of these fish. A population structuring model in which B. hilarii appears organized into genetically differentiated reproductive units that coexist and co-migrate through the studied system was suggested, contrasting the currently accepted idea that freshwater migratory fish form large panmictic populations in a determined hydrographic system. Despite the lack of a complete picture regarding the distribution of B. hilarii in the studied region, this initial idea on its population genetic structure could be an important contribution to providing aid for management and conservation programs of these fish.

Genetic diversity of three ornamental reef fishes (Families Pomacanthidae and Chaetodontidae) from the Brazilian coast.

Reef fishes of the families Pomacanthidae (angelfish) and Chaetodontidae (butterflyfish) are popular ornamental species, intensively harvested for the aquarium trade. The impacts of such activity on intra-specific diversity and reef ecosystems are still poorly understood in the south Atlantic. In the present work, a fine-scale genetic analysis using RAPD markers was performed in distinct samples of the queen angelfish (Holacanthus ciliaris), French angelfish (Pomacanthus paru), and banded butterflyfish (Chaetodon striatus) along the Brazilian coast. Most of the genetic variation in the three species was related to intra-population diversity. However, AMOVA results demonstrated that H. ciliaris presents a subtle population structure (phi(st)=0.132, P=0.003), while P. paru and C. striatus present low genetic differentiation, especially remarkable in the latter (phi(st) = 0.090, P=0.001 and phi(st)=0.041, P=0.028, respectively). Gene flow (Nm) was also higher in C. striatus than in the angelfish species. The reported patterns of genetic differentiation contrast with the similar pelagic stage of the selected species, suggesting that larval dispersal per se is a poor predictor of population structure in these reef fishes. Ecological features coupled with biogeographic history and distinct local selective pressures might play a major role on the genetic composition of each species. Although preliminary, the present results provide a baseline for monitoring the genetic variability in these reef species. These differences in the genetic structure among co-occurring species should be taken into consideration for the conservation of eventual evolutionary units along the Brazilian Province.

Genetic diversity within and between broodstocks of the white shrimp Litopenaeus vannamei (Boone, 1931) (Decapoda, Penaeidae) and its implication for the gene pool conservation.

Genetic variation within and between fifteen closed broodstock lines of the Pacific white shrimp Litopenaeus vannamei, reared at different hatcheries in the Brazilian coast, was assessed by RAPD analysis. Fifty two polymorphic loci were identified when a set of five decamer primers was used in PCR. The genetic diversity analysis within lines evidenced genetic variation loss probably related to bottleneck effects and inbreeding. In addition, the genetic divergence values between the different samples appear to reflect the initial founder composition of such stocks, in some cases, sharing a common origin, suggesting a putative importance of interbreeding for the establishment of genetic improvement programs for these broodstocks. The genetic variation monitoring appears to be helpful to the gene pool conservation of this aquaculture species, mainly if considered its exotic status in Brazil and the current impossibility of new introduction of wild individuals.

B chromosomes in the fish Astyanax scabripinnis (Characidae, Tetragonopterinae): an overview in natural populations.

Astyanax scabripinnis, a small neotropical freshwater fish, is a headwater species living in small tributaries of many Brazilian rivers, where they form isolated populations. This species harbors a B chromosome system in several populations. Among the several kinds of Bs reported in this species, the B(M) variant, a large metacentric of a similar size to the largest A chromosome, is the most widespread in natural populations. It probably corresponds to the ancestral B type in this species and a very similar B chromosome is also found in other Astyanax species. Strong evidence suggests that this B is an isochromosome showing structural and functional homology between its two arms, as shown by satellite DNA localization and the formation of a ring B univalent during meiosis. The B(SM) and B(m) variants, a large submetacentric and a small metacentric, respectively, represent rare variants and may be derived from structural rearrangements of the B(M) chromosome. In addition, B microchromosomes (B(micro)) were found in some populations. Frequency analyses in mountain populations have shown that B chromosomes are found in populations located at high altitude, but are absent in populations at low altitude, which is consistent with their parasitic nature, given the ecological peculiarities of both kinds of populations.

A polymorphic, telomeric-like sequence microsatellite in the Neotropical fish Prochilodus.

A microsatellite locus from the Neotropical fish genus Prochilodus was isolated using PCR-based isolation of microsatellite arrays. Of 470 positive clones, 15 were sequenced, and 10 of them showed an (AATTT)(n) repeat. Primers were designed, and analysis of polymorphism revealed 11 alleles in three Prochilodus species. Fluorescence in situ hybridization analysis showed signals predominantly in the telomeric regions of several chromosomes. The description of this microsatellite may contribute to studies of the population structure of this fish group.

Molecular organization of 5S rDNA in fishes of the genus Brycon.

There are few reports on the genomic organization of 5S rDNA in fish species. To characterize the 5S rDNA nucleotide sequence and chromosomal localization in the Neotropical fishes of the genus Brycon, 5S rDNA copies from seven species were generated by PCR. The nucleotide sequences of the coding region (5S rRNA gene) and the nontranscribed spacer (NTS) were determined, revealing that the 5S rRNA genes were highly conserved, while the NTSs were widely variable among the species analyzed. Moreover, two classes of NTS were detected in each species, characterized by base substitutions and insertions-deletions. Using fluorescence in situ hybridization (FISH), two 5S rDNA chromosome loci that could be related to the two 5S rDNA NTS classes were observed in at least one of the species studied. 5S rDNA sequencing and chromosomal localization permitted the characterization of Brycon spp. and suggest a higher similarity among some of them. The data obtained indicate that the 5S rDNA can be an useful genetic marker for species identification and evolutionary studies.

Organization of 5S rDNA in species of the fish Leporinus: two different genomic locations are characterized by distinct nontranscribed spacers.

To address understanding the organization of the 5S rRNA multigene family in the fish genome, the nucleotide sequence and organization array of 5S rDNA were investigated in the genus Leporinus, a representative freshwater fish group of South American fauna. PCR, subgenomic library screening, genomic blotting, fluorescence in situ hybridization, and DNA sequencing were employed in this study. Two arrays of 5S rDNA were identified for all species investigated, one consisting of monomeric repeat units of around 200 bp and another one with monomers of 900 bp. These 5S rDNA arrays were characterized by distinct NTS sequences (designated NTS-I and NTS-II for the 200- and 900-bp monomers, respectively); however, their coding sequences were nearly identical. The 5S rRNA genes were clustered in two chromosome loci, a major one corresponding to the NTS-I sites and a minor one corresponding to the NTS-II sites. The NTS-I sequence was variable among Leporinus spp., whereas the NTS-II was conserved among them and even in the related genus Schizodon. The distinct 5S rDNA arrays might characterize two 5S rRNA gene subfamilies that have been evolving independently in the genome.

Two 5S rDNA arrays in neotropical fish species: is it a general rule for fishes?

In this paper we describe Southern blot hybridization results probed with 5S rRNA genes for several Neotropical fish species representing different taxonomic groups. All the studied species showed a general trend with the 5S rDNA tandem repeats organized in two distinct size-classes. At the same time, data on 5S rDNA organization in fish genome were summarized. Previous information on the organization and evolution of 5S rRNA gene arrays in the genome of this vertebrate group are in agreement with the Southern results here presented. Sequences obtained for several fish species have revealed the occurrence of two distinct 5S rDNA classes characterized by distinct nontranscribed spacer sequences, which are clustered in different chromosomes in some species. Moreover, the 5S rDNA loci are generally distributed in an interstitial position in the chromosomes and they are usually not syntenic to the 45S rDNA. The presence of two classes of 5S rDNA in several non-related fish species suggests that this could be a common condition for the 5S rRNA gene organization in the fish genome.

Structural and functional evidence that a B chromosome in the characid fish Astyanax scabripinnis is an isochromosome.

Astyanax scabripinnis possesses a widespread polymorphism for metacentric B chromosomes as large as the largest chromosome pair in the A complement. On the basis of C-banding pattern, it was hypothesized that these B chromosomes are isochromosomes that have arisen by means of centromere misdivision and chromatid nondisjunction. In the present paper we test this hypothesis by analysing (i) the localization of a repetitive DNA sequence on both B chromosome arms, and (ii) synaptonemal complex formation, in order to test the functional homology of both arms. Genomic DNA digested with KpnI and analysed by gel electrophoresis showed fragments in a ladder-like pattern typical of tandemly repetitive DNA. These fragments were cloned and their tandem organization in the genome was confirmed. A 51-bp long consensus sequence, which was AT-rich (59%) and contained a variable region and two imperfect reverse sequences, was obtained. Fluorescence in situ hybridization (FISH) localized this repetitive DNA into noncentromeric constitutive heterochromatin which encompasses the terminal region of some acrocentric chromosomes, the NOR region, and interstitial polymorphic heterochromatin in chromosome 24. Most remarkably, tandem repeats were almost symmetrically placed in the two arms of the B chromosome, with the exception of two additional small clusters proximally located on the slightly longer arm. Synaptonemal complex (SC) analysis showed 26 completely paired SCs in males with 1B. The ring configuration of the B univalent persisting until metaphase I suggests that the two arms formed chiasmata. All these data provided strong support for the hypothesis that the B chromosome is an isochromosome.

Chromosomal localization of 5S rDNA genes in Leporinus fish (Anostomidae, Characiformes).

The large 45S rDNA chromosome sites have often been analyzed in fish. In contrast, little is known about the 5S genes in this animal group. In the genus Leporinus, the NOR chromosomal location has been shown to be very diverse. In the present work, chromosome mapping of 5S rDNA in three anostomids, Leporinus elongatus, L. obtusidens and L. friderici, is investigated using fluorescence in-situ hybridization (FISH) with PCR-obtained 5S probes and primed in-situ labeling (PRINS). Major 5S rDNA chromosomal sites were found to be subterminally located in a small metacentric pair, while minor ones were detected near the centromeric region of a medium-sized submetacentric pair in all studied species. The 5S rDNA genes were not associated with the NORs or sex chromosomes. A highly conserved chromosomal location of these genes appears to characterize the karyotype evolution of this fish group.

Cytogenetics of the genus Leporinus (Pisces, Anostomidae). 1. Karyotype analysis, heterochromatin distribution and sex chromosomes.

Cytogenetic analyses (Giemsa staining, C-banding, AgNO3 labelling of nucleolus organizer regions (NORs) and staining with base-specific fluorochromes) were performed on the South American fish species Leporinus friderici, L. obtusidens and L. elongatus. The overall karyotypic structure, position of NORs, as well as the amount, distribution and composition of constitutive heterochromatin were determined. Particular attention was given to the highly differentiated ZZ/ZW sex chromosome system of L. obtusidens and L. elongatus. Sharing the apparently ancient macroscopic karyotype of Anostomidae, all three species have 2n = 54 meta- or submetacentric chromosomes. NORs were found exclusively on chromosome pair 2, which may represent the ancestral NOR-bearing chromosome of the anostomid karyotype. Observed differences in the relative position of NORs along chromosome 2 and variations in the amount and distribution of constitutive heterochromatin throughout the karyotype were most probably caused by heterochromatin-mediated chromosome rearrangements. Detailed analysis of the morphologically similar heteromorphic ZZ/ZW sex chromosomes of L. obtusidens and L. elongatus allowed detection of differences in the DNA composition of the largely heterochromatic W chromosomes. However, since these and the W chromosomes of three other Leporinus species exhibit homologies with respect to their relative size, centromere position and amount and distribution of heterochromatin, it is concluded that they evolved from the same ancestral W chromosome.

Post-zygotic modifications and intra- and inter-individual nucleolar organizing region variations in fish: report of a case involving Leporinus friderici.

Silver nitrate staining, a rapid and efficient method, has proven to be excellent for nucleolar organizing region (NOR) studies in fish. Some fish appear to have only two NOR-bearing chromosomes in their karyotype, whereas others probably have several. In the present study we analyzed the NORs of Leporinus friderici, a species that, on the basis of previous studies, has been considered as representative of species with NORs carried by a single chromosome pair. The analyses were performed by a combination of three methods, i.e. silver nitrate staining, staining with the GC-specific fluorochrome chromomycin A3, and in situ hybridization with digoxigenin-labeled probes. The results showed that, although more frequent and conspicuous in a single chromosome pair, the NORs of this species are present in multiple chromosomes. Intra- and inter-individual variations observed by the three methods strongly suggest the occurrence of post-zygotic modifications involving NORs. NOR identification in fish, almost exclusively performed by the silver nitrate method, is currently being re-evaluated by methods such as chromomycin A3 staining and in situ hybridization, which may provide important information leading to a better understanding of chromosome evolution in these animals.

Organization and molecular cytogenetics of a satellite DNA family from Hoplias malabaricus (Pisces, Erythrinidae).

The chromosomes of the primitive South American teleost fish Hoplias malabaricus have been analyzed by classical cytogenetic (C-, AgNOR-, Hoechst 33258-, and Q-banding) techniques. A highly repetitive DNA family has been cloned and sequenced. It is a tandemly repeated sequence of about 355 bp, yielding an overall base pair composition of 67% AT with long runs of > 50% As and 70% Ts. Analysis of sequence variation has allowed the further categorization of Hoplias satellite DNA into two evolutionary related subfamilies A and B, distinguishable by characteristic insertions and deletions within this 355-bp monomer. Subfamily A satellite is found (in diverged form) at the centromeres of most H. malabaricus chromosomes. Sequence variants are clustered on specific chromosomal subsets. Subfamily B satellite is highly specific for the paracentromeric heterochromatin on one particular chromosome pair by fluorescence in situ hybridization. These results indicate that the Hoplias satellite DNA family has evolved in a concerted manner predominantly via recombination events involving homologous, rather than non-homologous chromosome regions. The clones isolated here may be useful for the molecular, genetic, and cytological analysis of the genus Hoplias.