Chromosomal location and distribution of As51 satellite DNA in five species of the genus Astyanax (Teleostei, Characidae, Incertae sedis)

Constitutive heterochromatin makes up a substantial portion of the genome of eukaryotes and is composed mainly of satellite DNA repeating sequences in tandem. Some satellite DNAs may have been derived from transposable elements. These repetitive sequences represent a highly dynamic component of rapid evolution in genomes. Among the genus Astyanax , the As51 satellite DNA is found in species that have large distal heterochromatic blocks, which may be considered as derived from a transposable DNA element. In the present study, As51 satellite DNA was mapped through in situ fluorescent hybridization in the chromosomes of five species of the genus. The possible roles of this type of saltatory DNA type in the genome of the species are discussed, along with its use for the phylogenetic grouping of the genus Astyanax , together with other shared chromosomal characters. However, the number of As51 clusters is presented as a homoplastic characteristic, thereby indicating evident genomic diversification of species with this type of DNA.     

Chromosome polymorphism in Astyanax fasciatus (Teleostei, Characidae). 1. Karyotype analysis, Ag-NORs and mapping of the 18S and 5S ribosomal genes in sympatric karyotypes and their possible hybrid forms

Astyanax fasciatus may be characterized as a chromosomally diversified 'species' presenting distinct cytotypes, each with its specific variants. The sympatric and syntopic occurrence of different cytotypes reinforces the hypothesis in which A. fasciatus may represent a group of species currently placed under a single common designation. Specimens from three collection points spread along the Mogi-Gua?u River in southeast Brazil were examined in the present work: (1) near its headwaters (Ouro Fino--MG), (2) in the middle region of the river (Cachoeira de Emas, Pirassunun ga--SP) and (3) close to its confluence with the Pardo River (Barrinha--SP). The 2n = 48 chromosomes cytotype was found in all sampling points, while cytotype 2n = 46 was only encountered in Barrinha and Cachoeira de Emas. In the latter locality, cytotype 2n = 46 predominated; nevertheless, other karyotype forms with 2n = 45 and 47 chromosomes also occurred, besides a structural variant of cytotype 2n = 46. One specimen with 2n = 47 chromosomes was also found in Ouro Fino. The Ag-NOR analysis, as well as the location of the 18S and 5S ribosomal genes, were conserved in all cytotypes. The data indicate that the variant karyotypes are a consequence of interbreeding between the standard cytotypes (2n = 46 and 48) and/or its descendants. This suggests a karyotype plasticity for this species, where at least a few variant karyotypes would not have deleterious effects on their bearers.     

Biogeographic patterns in the chromosomal distribution of a satellite DNA in the banded tetra Astyanax fasciatus (Teleostei: Characiformes)

The As-51 satellite DNA is a transposon-like sequence formerly described for arthropods and physically identifiable by fluorescent in situ hybridization. In the present work, we describe the occurrence of this sequence, as well the C-banding and karyotype composition, in populations of the group Astyanax fasciatus from Mogi-Guaçu (Araras-SP), Paranapanema (Angatuba and Pilar do Sul-SP), Ribeira de Iguape (Sete Barras-SP) and Tietê (Indaiatuba and Salesópolis-SP) river basins. The specimens from Sete Barras (10 M + 20SM + 12ST + 6A) and Araras (8 M + 22SM + 12ST + 6A) have 2n?=?48 chromosomes. The samples from Angatuba, Pilar do Sul and Indaiatuba presented 2n?=?46 chromosomes (12 M + 20SM + 10ST + 4A). The individuals collected in Salesópolis showed three cytotypes, bearing 2n?=?46 (12 M + 20SM + 10ST + 4A), 2n?=?48 (8 M + 22SM + 12ST + 6A) and 2n?=?50 (8 M + 16SM + 14ST + 12A). C-banding revealed large heterochromatic blocks at terminal chromosomal regions in all populations and/or cytotypes. All analyzed populations have conspicuous blocks carrying the As-51 satellite DNA, although the number of chromosomes bearing this repetitive sequence was variable among them. Such differences were not related to the diploid number of individuals, but rather to a biogeographic pattern. Aspects of the karyotype evolution and distribution of this sequence in distinct populations are discussed.     

Reaction to Alarm Substance in Cave Populations of Astyanax fasciatus (Characidae,Pisces)1

The effect of species-specific skin-extract containing alarm substance has been studied in two epigean and three hypogean populations of the Mexican-tetra Astyanax fasciatus. In all populations tested the feeding rate at the water surface decreased significantly after introducing the alarm substance and the fish avoided this surface area. Additionally epigean fish and some individuals from the Chica cave showed zigzag movements, rapid swimming and hiding. The fish did not respond to a skinextract of a species without alarm substance.     

Non-optical releasers for aggressive behavior in blind and blinded Astyanax (Teleostei, Characidae)

Prior to this study, it was believed that epigean and hypogean Astyanax differ markedly in their display of agonistic behavior. Research suggested that surface-dwelling individuals were extremely aggressive whereas their blind, cave-dwelling counterparts tended to show little or no aggressive behavior. Aggression in Astyanax was thought to be triggered by visual stimuli because surface fish in a dark environment or surface fish blinded late in life did not show aggression. Here, we demonstrate that surface fish blinded early on in their embryonic development are highly aggressive as adults. We also report the first case of a population of blind cave-dwelling Astyanax that is highly aggressive. We conclude that reduced aggression is not the only evolutionary pathway for troglobitic Astyanax and that there is some degree of developmental plasticity in the releaser of aggression and in the selection of its triggering stimuli.     

Oxygen consumption of Astyanax fasciatus (Characidae,Pisces): a comparison of epigean and hypogean populations

Synopsis The standard and routine oxygen consumptions of Astyanax fasciatus from one surface population (Rio Teapao) and three cave populations (Chica, Micos and Pachon caves: sAnoptichthys jordani, the Micosfish and Anoptichthys antrobius) were determined individually over 24 hours by the use of a flow-through respirometer and polarographic oxygen electrodes. The phylogenetically oldest Pachon fish had a significantly lower standard metabolic rate (0.230 ± 0.036 mg O₂ g^-1 h^-1) than the epigean Teapao fish, the hybrid Chica fish and the phylogenetically younger Micos fish (0.314 ± 0.081 mg O₂g-^-1h^-1, 0.284 ± 0.048 mg O₂g^-1h^-1, 0.277 ± 0.063 mg O₂g^-1h^-1). No significant differences could be determined among the latter three populations. A significant difference in routine metabolic rate existed only between the Pachon fish (0.309 ± 0.0.56 mg O₂g^-1h^-1) and the Teapao fish (0.415 ± 0.071 mg O₂g^-1h^-1). The Chica fish (0.356 ± 0.084 mg O₂g^-1h^-1) and the Micos fish (0.355 ± 0.080 mg O₂ g^-1h^-1) could not be separated from either the Teapao or the Pachon fish, but a decreasing trend from the surface population through the Chica and the Micos to the Pachon population was obvious. During a starvation period of 29 days the metabolic rate of epigean Teapao and hypogean Pachon fish decreased significantly by 32.5% and 34.8% (standard oxygen consumption rate) and 27.5% and 28.2% (routine oxygen consumption rate), respectively. Body mass loss during the starvation period was 16.3% for the Teapao fish and 9.5% for the Pachon fish.     

Genetic divergence between cave and surface populations of Astyanax in Mexico (Characidae,Teleostei)

A study of genetic diversity at microsatellite loci and the mitochondrial DNA (mtDNA) cytochrome b gene was carried out to assess genetic relationships among four Mexican cave (Pachon, Sabinos, Tinaja, Chica) and four surface populations of Astyanax fasciatus (Characidae) from northeast Mexico and the Yucatan. With the exception of Chica, the cave populations were all characterized by extremely low microsatellite variability, which most likely resulted from bottleneck events. Population analyses of the microsatellite data indicated no measurable levels of gene flow between all cave and surface populations (F(ST) > 0.0707). Phylogenetic analyses of mtDNA data showed that only two cave populations - Sabinos and Tinaja - group together to the exclusion of surface populations. From the microsatellite data these cave populations cluster with the Pachon cave fish population. The mtDNA thus appears to have been replaced in Pachon because of introgressive hybridization. It is likely that these three cave populations have descended from a surface ancestor in common with current surface populations, rather than evolving recently from one of the extant surface populations. Like Pachon, the Chica population clustered with the surface populations according to mtDNA data, but was not clearly associated with either the surface or the other cave populations according to the microsatellite data. Our data indicate that the Chica population evolved recently from a surface population, and subsequently hybridized with a phylogenetically older cave population. In conclusion, both the microsatellite and mtDNA data suggest multiple origins of cave populations and the Chica and Sabinos/Tinaja/Pachon were founded after at least two independent invasions from surface populations.     

Gill‐derived glands in species of Astyanax (Teleostei: Characidae)

The presence of a gill‐derived gland is herein reported for the first time in males of species of Astyanax and related genera; they are described through histological cuts and SEM. The gill‐derived glands described for the Characidae, when fully developed, present a similar structure in different species. The main external feature of gill‐derived glands is the fusion of anteriormost gill filaments on the ventral branch of first gill arch. This fusion is caused by squamous stratified epithelial tissue that covers adjacent filaments, forming a series of chambers. In the region where the gill‐derived gland develops, the secondary lamellae of the gill filaments are much reduced or completely atrophied being characterized by the presence of glandular cells forming nests.     

Cytogenetic characterization of a supernumerary chromosome segment and of B-chromosomes in Astyanax scabripinnis (Teleostei, Characidae)

The chromosome complement of a local population of Astyanax scabripinnis in Brazil was investigated with emphasis on the study of the heterochromatin attached to the A-chromosomes and present in the macro B-chromosome. Analysis after C-banding, silver and CMA₃ staining, incorporation of 5-bromo-2′-deoxyuridine and chromosome digestion with nine restriction endonucleases revealed that the heterochromatin in the B-chromosomes was different from that found in the A-chromosomes. A polymorphism due to the presence of a supernumerary heterochromatic chromosome segment was observed in the population investigated. Some aspects related to the origin of the heterochromatin polymorphism in Astyanax scabripinnis are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Population Genetic Structure of Astyanax scabripinnis (Teleostei, Characidae) from an Urban Stream

Despite the great anthropogenic interference on urban streams, information is still scarce about the genetic variability and structure of native fish populations inhabiting such streams. In the present study, random amplified polymorphic DNA (RAPD) markers were used to analyze genetic variability and structure of populations assigned to the Neotropical fish species Astyanax scabripinnis from an urban stream located in Londrina, Paraná State, southern Brazil. Thirty individuals of this species were collected from three sites throughout the upper Cambé stream. A total of 10 primers amplified 159 loci, of which 128 (80.5%) were polymorphic. Each of the three populations showed very similar proportions of polymorphic loci, which ranged from 63.5 to 64.8%. Unbiased genetic distances varied from 0.0612 to 0.0646. Theta_p-test values indicated moderate to high genetic differentiation among individuals from different localities. The number of migrants varied from 1.34 to 1.46, suggesting a low gene flow between populations. The genetic similarity among all individuals studied ranged from 0.424 to 0.848. The results suggest that populations of A. scabripinnis in Cambé stream are undergoing genetic differentiation.     

The species complex Astyanax fasciatus Cuvier (Teleostei, Characiformes) – a multidisciplinary approach

Cytogenetic data have provided important clues that the Astyanax fasciatus populations from the Upper Paraná River basin could be a part of a more diverse fish group, usually included on the same taxa. Samples collected in Cachoeira de Emas, SP, in Mogi-Guaçu River basin, show two major cytotypes presenting 2 n = 46 and 2 n = 48 chromosomes, with distinct karyotypic formula, despite the fact that the molecular data suggested some degree of gene flow between these cytotypes. Cytogenetic and morphometric analyses were performed in this species, aiming to contribute to the understanding of the natural history from such fish group. Two allopatric populations with distinct standard cytotypes were analysed, and the data obtained suggest the separation into two groups.     

Delimiting the Origin of a B Chromosome by FISH Mapping,Chromosome Painting and DNA Sequence Analysis in Astyanax paranae (Teleostei,Characiformes)

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.     

Population genetic patterns revealed by microsatellite data challenge the mitochondrial DNA based taxonomy of Astyanax in Mexico (Characidae, Teleostei)

Astyanax has become an important model system for evolutionary studies of cave animals. We investigated correlations of population genetic patterns revealed by microsatellite data and phylogeographic patterns shown by mitochondrial DNA sequences in Mexican cave and surface fish of the genus Astyanax (Characidae, Teleostei) to improve the understanding of the colonization history of this neotropical fish in Central and North America and to assess a recent taxonomic classification. The distribution of nuclear genotypes is not congruent with that of the mitochondrial clades. Admixture analyses suggest there has been nuclear gene flow between populations defined by different mitochondrial clades. The microsatellite data indicate that there was mitochondrial capture of a cave population from adjacent populations. Furthermore, gene flow also occurred between populations belonging to different nuclear genotypic clusters. This indicates that neither the nuclear genotypic clusters nor the mitochondrial clades represent independent evolutionary units, although the mitochondrial divergences are high and in a range usually characteristic for different fish species. This conclusion is supported by the presence of morphologically intermediate forms. Our analyses show that the Trans-Mexican Volcanic Belt limited gene flow, but has been crossed by Astyanax several times. In Yucatán, where obvious geographic barriers are missing, the incongruence between the distribution of nuclear and mitochondrial markers reflects random colonization events caused by inundations or marine transgressions resulting in random phylogeographic breaks. Thus, conclusions about the phylogeographic history and even more about the delimitation of species should not be based on single genetic markers.     

Gonadal morphology and difference in reproductive development of two isolated populations of Astyanax rivularis (Teleostei,Characidae)

Individuals of the same species may present different reproductive tactics depending on the environment in which they develop and mature. The present study aimed to define the gonadal development phases of males and females of Astyanax rivularis and to carry out a comparative analysis of the reproductive development of specimens captured in two isolated environments of the São Francisco River basin in Serra da Canastra, Brazil (Point 1: low vegetation and river showing calm and crystalline waters with small well formations; Point 2: current waters, and well-established areas of arboreal vegetation). Thus, the gonads of A. rivularis specimens were collected, fixed and processed with techniques for light microscopy. Five maturation phases of the females' reproductive cycle were established: immature, developing, spawning capable, regressing and regenerating. Three maturation phases of the males' reproductive cycle were observed: spawning capable, regressing, and regenerating. There are differences in the phases of gonadal development of A. rivularis between the two sampling points so that, possibly, animals upstream of the waterfall demonstrate a delay in the reproductive cycle in relation to animals downstream.     

Convergent evolution of the cavefish Astyanax (Characidae, Teleostei): genetic evidence from reduced eye-size and pigmentation

More than 20 populations of the cave-dwelling characid Astyanax occur within a restricted karst area in Mexico. The fish possess reduced eyes without lenses and visual cells. It is still an open question as to whether this condition evolved convergently after multiple entries of the surface ancestor into the different caves or whether a single cave ancestor, already characterized by reduced eyes, spread secondarily within them. In the crosses between specific populations, specimens appear that deviate considerably from those of the parents. They possess larger and better-developed eyes with histologically intact lenses and visual cells; they thus have the structural potential for vision. This indicates that in different cave populations, different mutations in the eye gene system have occurred. In cases where these non-functional rudimentary genes are recombined in hybrid specimens, gene expression may be restored. This is the result of separate evolution of several Astyanax cave populations.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80 , 545−554.     

Chromosome polymorphism in Astyanax fasciatus (Teleostei, Characidae), Part 3: Analysis of the RAPD and ISSR molecular markers

The Randomly Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSR) molecular markers were used to complement the study of chromosomal polymorphism in Astyanax fasciatus (Teleostei, Characidae) from the Mogi-Guaçu River (Southeastern Brazil), analyzed in three collection sites along the river (Ouro Fino – MG, Cachoeira de Emas – SP and Barrinha – SP). Two cytotypes (or karyotypic types), denominated standard cytotypes, were previously characterized, one including 2n = 46 chromosomes and the other 2n = 48 chromosomes, where all the chromosomes of the complement form homologous pairs. Additionally, variant karyotypic forms with 2n = 45, 46 and 47 chromosomes were also detected, although with a lower frequency in relation to the standard cytotypes. RAPD turned out little informative in the analysis of the observed situation, indicating a high value of migrants per generation among the cytotypes. On the other hand, ISSR showed a small structure, especially among the standard cytotypes from the Barrinha region where the Nm was 0.4301 with a genetic identity of 0.6862 and genetic distance of 0.3765. However, the general results obtained do not discard the possibility of interbreeding between both standard cytotypes and/or their descendants as a source of chromosome variation. The association between the cytogenetic and molecular markers viabilized putative explanatory scenery for the origin and evolution of the forms seen in A. fasciatus.     

Description of Dendrorchis retrobiloba n. sp. (Digenea: Gorgoderidae) from Astyanax fasciatus (Teleostei: Characidae) from Southern Uruguay, with an emended diagnosis of the genus Dendrorchis

To date, the monospecific Dendrorchis is represented by D. neivai, reported from Brazil and parasitizing Brycon lundi. Dendrorchis retrobiloba n. sp. is described from the swim bladder of Astyanax fasciatus from Ca?ada del Dragón stream in Montevideo, southern Uruguay (34 degrees 47'S, 56 degrees 14'W), and the emended diagnosis of Dendrorchis is given. The diagnosis of Dendrorchis has been modified to include the following characters: Phyllodistominae with flattened body, wider in the region posterior to acetabulum. Intestinal ceca sinuous or not. Testes branched or with superficial lobes. Ovary with deep or superficial lobes. Seminal receptacle present or absent. Vitellaria ellongate or pyriform. Uterus extends to posterior region and to portion of body anterior to acetabulum. Eggs oval, variable size, thin-shelled, without opercula. Dendrorchis retrobiloba n. sp. differs from D. neivai by having pyriform vitellaria, ovary larger than the testes and not deeply lobed, wider distribution of the uterus, smaller egg size, testes with superficial lobes, oral sucker wider than the acetabulum, shorter esophagus, 2 terminal lobes at the posterior end of the body, and much smaller body length. Despite being found in different hosts, both species of Dendrorchis parasitize the swim bladder of freshwater Characidae.