Physical mapping of 5S rDNA in two species of Knifefishes: Gymnotus pantanal and Gymnotus paraguensis (Gymnotiformes)

Physical mapping of 5S rDNA in 2 species of knifefishes, Gymnotuspantanal and G. paraguensis (Gymnotiformes), was performed using fluorescence in situ hybridization with a 5S rDNA probe. The 5S rDNA PCR product from the genomes of both species was also sequenced and aligned to determine non-transcribed spacer sequences (NTS). Both species under study had different patterns of 5S rDNA gene cluster distribution. While in the karyotype of G. pantanal two 5S rDNA-bearing pairs were observed, the karyotype of G. paraguensis possessed as many as 19 such pairs. Such multiplication of 5S rDNA gene clusters might be caused by the involvement of transposable elements because the NTS of G. paraguensis was 400 bp long with high identity (90%) with a mobile transposable element called Tc1-like transposon, described from the cyprinid fish Labeo rohita.     

The genus Gymnotus (Gymnotiformes: Gymnotidae) in Argentina. How bad taxonomy results in poor regulations and no conservation

Four species of the genus Gymnotus are present in Argentina: G. inaequilabiatus, G. omarorum, G. pantanal, and G. sylvius, the last three species being recorded for the first time in freshwater courses. Gymnotus omarorum, G. pantanal, and G. sylvius together with others of the genus Brachyhypopomus are the group of fishes that bear the greatest impact in the trade as live bait for sport fishing in the northeastern region of Argentina. Within this large area, only the provinces of Chaco, Corrientes, and Formosa have regulations for the catch, trade, and sale of species as live bait. Unfortunately, the species covered by legal regulations are Gymnotus carapo and Brachyhypopomus brevirostris, neither of which occurs in freshwater habitats of Argentina. Comments are included as to how this bad taxonomy affects the regulations and conservation status of these species.     

Cytogenetic analysis of three sympatric Gymnotus (Gymnotiformes, Gymnotidae) species verifies invasive species in the Upper Paraná River basin, Brazil

Cytogenetic analyses were performed on three Gymnotus species, Gymnotus paraguensis , Gymnotus sylvius and Gymnotus pantanal , collected in sympatry in the region of Guaíra, Paraná State (Brazil). Gymnotus paraguensis presented 54 chromosomes (52m-sm + 2st) in both sexes, G. sylvius presented 40 chromosomes (36m-sm + 4a) in both sexes and G. pantanal presented 40 chromosomes (14m-sm + 26a) in females and 39 (15m-sm + 24a) in males, with an X₁X₁X₂X₂/X₁X₂Y multiple sex chromosome system. All three species had single nucleolar organizing regions and heterochromatin in the centromeric region of most chromosomes. Gymnotus pantanal , previously collected only in the Lower Paraná and Paraguay River basins, was confirmed for the first time in the Upper Paraná River basin. This species constitutes one of several examples of species that have colonized the region after flooding of the great Sete Quedas waterfalls by the construction of the Itaipu hydroelectric dam. The probable absence or infertility of hybrids is indicated by the high level of chromosome differentiation between these species.     

Gymnotus capanema, a new species of electric knife fish (Gymnotiformes, Gymnotidae) from eastern Amazonia, with comments on an unusual karyotype

Gymnotus capanema n. sp. is described on the basis of cytogenetic, morphometric, meristic and osteological data from nine specimens (one male and eight females) from the municipality of Capanema, Pará, in the eastern Amazon of Brazil. Later, three additional specimens were found in museums and regarded as nontypes (not cytogenetically analysed). Gymnotus capanema, which occurs in sympatry with Gymnotus cf. carapo cytotype 2n = 42 (30m/sm + 12st/a) exhibits a novel karyotype for the genus, with 2n = 34 (20m/sm + 14st/a). Gymnotus capanema can be unambiguously diagnosed from all congeners on the basis of a combination of characters from external anatomy, pigmentation and osteology. The constitutive heterochromatin, rich in adenine-thymine (A-T) base pairs [4',6 diamidino-2-phenylindole dihydrochloride (DAPI) positive], occurs in the centromeric region of all of the chromosomes, and in the pericentromeric and the entire short arm of some chromosomes. The nucleolar organizing region (NOR), stained by silver nitrate, chromomycin A(3) (CMA(3)) and 18S ribosomal (r)DNA fluorescence in situ hybridization (FISH), occurs in the short arm of pair 15. FISH, with telomeric probes did not show interstitial telomeric sequences (ITS), despite the reduced 2n in comparison to the karyotypes of other species of Gymnotus. The karyotype of G. capanema, with a reduced 2n, is strikingly different from all other previously studied congeners.     

Molecular characterization and physical mapping of two classes of 5S rDNA in the genomes of Gymnotus sylvius and G. inaequilabiatus (Gymnotiformes, Gymnotidae)

The nucleotide sequences of the 5S rRNA multigene family and their distribution across the karyotypes in 2 species of Gymnotiformes, genus Gymnotus (G. sylvius and G. inaequilabiatus) were investigated by means of fluorescence in situ hybridization (FISH). The results showed the existence of 2 distinct classes of 5S rDNA sequences in both species: class I and class II. A high conservative pattern of the codifying region of the 5S rRNA gene was identified, contrasting with significant alterations detected in the nontranscribed spacer (NTS). The presence of TATA-like sequences along the NTS of both species was an expected occurrence, since such sequences have been associated with the regulation of the gene expression. FISH using 5S rDNA class I and class II probes revealed that both gene classes were collocated in the same chromosome pair in the genome of G. sylvius, while in that of G. inaequilabiatus, class II appeared more disperse than class I.     

Ultrastructure,meiotic behavior,and evolution of sex chromosomes of the genus Ellobius

The whole-mount SC preparations from males of three species of the genus Ellobius (Ellobius fuscocapillus, Ellobius lutescens), and Ellobius tancrei were studied by electron microscopy. In the males of Ellobius fuscocapillus, behavioral peculiarities of the sex bivalent (viz. the normal male heterozygosity) are characterized by early complete desynapsis of sex chromosomes (X, Y), occurring at late pachytene-early diplotene. The karyotype of species Ellobius lutescens is unique for mammals. In both sexes it is characterized by an odd number of chromosomes (2n=17). At prophase I the unpaired chromosome 9 is not involved in synapsis with other chromosomes and forms a sex body at the end of pachytene.The complete Robertsonian fan has been described for superspecies Ellobius tancrei. As shown on the basis of G-band patterns the male and female sex chromosomes are cytologically indistinguishable.Analysis of whole-mount SC preparations revealed the formation of a closed sex SC bivalent and showed some morphological differences in the axes of sex chromosomes at meiotic prophase I. A number of assumptions are made about the relationship between the behavior of sex chromosomes, their evolution and the sex determination system in the studied species of genus Ellobius.

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Non-homologous sex chromosomes in two species of the genus Eigenmannia (Teleostei: Gymnotiformes)

The Neotropical genus Eigenmannia is a fish group with unknown species diversity where representatives possess a broad range of chromosomal sex determining systems namely XY/XX, X(1)X(2)Y/X(1)X(1)X(2)X(2), ZZ/ZW as well as homomorphic sex chromosomes. To test the homology of two heteromorphic XY sex chromosome systems present in two sympatric populations, reciprocal cross-species FISH experiments were performed using probes derived by microdissection of X and Y chromosomes present in analyzed specimens of Eigenmannia virescens and Eigenmannia sp.2, respectively. While X and Y paint probes hybridized to species-specific sex chromosomes, in reciprocal cross-FISH both probes hybridized exclusively to autosomes. The result suggests multiple independent origins of the XY systems in the analyzed populations.     

Redescription of the Central American electric fish Gymnotus cylindricus (Ostariophysi: Gymnotiformes: Gymnotidae), with comments on character ambiguity within the ostariophysan clade

Gymnotus cylindricus LaMonte, a poorly studied Central American electric fish primarily observed in the Rio Motagua basin (Guatemala) and north-western Honduras, is redescribed in detail. A clear diagnosis of this species is provided, with new information on overall morphology, osteology, and colour pattern. In a separate section, comments on character ambiguity within the Ostariophysi are made, which led to a reinterpretation of seven homoplastic characters proposed by Fink & Fink (1981) in their work on interrelationships among five major lineages of the superorder. Although the topology of their cladogram remains unchanged, the present view is that alternative explanations regarding evolution of those characters are possible, and the new ones here offered agree with initial hypotheses of primary homologies.     

Phylogeny,biogeography, and electric signal evolution of Neotropical knifefishes of the genus Gymnotus (Osteichthyes: Gymnotidae)

The Neotropical knifefish genus Gymnotus is the most broadly distributed and the most diverse (34 + species) gymnotiform genus. Its wide range includes both Central and South American drainages, including the Amazon, Orinoco, and La Plata Basins. Like all gymnotiforms, Gymnotus species produce weak electric fields for both navigation and communication, and these fields exhibit interspecific variation in electric waveform characteristics. Both biogeography and electric signal evolution can profitably be analyzed in a phylogenetic context. Here, we present a total evidence phylogeny for 19 Gymnotus species based on data from the mitochondrial cytochrome b and 16S genes (1558 bp), the nuclear RAG2 gene (1223 bp), and 113 morphological characters. Our phylogenetic hypothesis resolves five distinct Gymnotus lineages. In a previous morphology-based analysis, the Central American Gymnotus cylindricus lineage was hypothesized as the sister group to all other Gymnotus species. In our analysis, the G. cylindricus lineage is nested within South American species, and molecular age estimates support a relatively recent origin for the clade in Central America. Phylogenetic optimization of electric signal waveforms indicate that the ancestral state in Gymnotus is a multiphasic (4 + phases of alternating polarity) condition, and independent phase loss has occurred in multiple lineages. Gymnotus is a model group for understanding Neotropical diversification and the evolution of communication at a continental scale.     

Innervation pattern and electric organ discharge waveform in Gymnotus carapo (Teleostei; Gymnotiformes)

The electrogenic organ (EO) of Gymnotus carapo has two main portions: a posterior region consisting of four bilaterally arranged electrocyte rows; and an anterior portion composed of only two. The lateral row (LR) of the anterior portion contains doubly innervated electrocytes with axon terminals from different nerves on their rostral and caudal faces. The LR is continuous with the most dorsal row of the caudal region. This row also contains doubly innervated electrocytes. The medial row (MR) electrocytes of the anterior region and ventral rows of the caudal region are exclusively caudally innervated. All caudal faces of the anterior or abdominal region are supplied by two nerves which originate from spinal roots VIII to XXI. Roots I to VII give origin to pure rostral nerves whose electromotor axons terminate on the rostral surfaces of the first seven LR electrocytes. A given doubly innervated electrocyte is supplied on its caudal face by a nerve originating several segments (usually seven) posterior to the spinal root supplying its rostral face. Transections of the spinal cord at the level of root VIII isolate the activity of the rostral surfaces of the first electrocytes. The EO discharge (EOD) then appears as a head negative deflection which arises from abdominally located electrocytes. Its monophasic character reveals that the activity remains restricted to the rostral electrocyte surfaces. Damage of the abdominal portion of the EO abolishes the first negative deflection of the normal pulse. Transections of the spinal cord at the level of root XXI isolate the activity of the whole abdominal portion of the EO. Since both doubly and singly innervated electrocytes remain active, the EOD appears biphasic. Comparative studies have shown that the EOD of Hypopomus sp. lacks any early negative wave and correspondingly all its electrocytes are exclusively caudally innervated.     

Phylogenetic systematics and historical biogeography of the Neotropical electric fish Gymnotus (Teleostei: Gymnotidae)

Phylogenetic interrelationships of the Neotropical electric fish genus Gymnotus are documented from comparative study of phenotypic data. A data matrix was compiled of 113 phenotypic characters for 40 taxa, including 31 recognized Gymnotus species, six allopatric populations of G. carapo, two allopatric populations of G. coropinae, and three gymno‐tiform outgroups. MP analysis yielded 15 trees of equal length, the strict consensus of which is presented as a working hypothesis of Gymnotus interrelationships. Diagnoses are presented for 26 clades, including three species groups; the G. cylindricus group with two species restricted to Middle America, the G. pantherinus group with 12 species in South America, and the G. carapo group with 16 species in South America. The basal division of Gymnotus is between clades endemic to Middle and South America. Both the G. pantherinus and G. carapo groups include trans‐Andean sister‐taxon pairs, suggesting a minimum date for the origins of these groups in the late Middle Miocene (c. 12 Ma.). The geographically widespread species G. carapo is paraphyletic. Analysis of character state evolution shows characters of external morphology are more phylogenetically plastic and provide more phylogenetic information in recent branches than do characters of internal morphology, which themselves provide the more information in deeper branches. Nine regional species assemblages of Gymnotus are recognized, none of which is monophyletic. There are at least two independent origins of Gymnotus species in sediment rich, high conductivity, perennially hypoxic whitewater floodplains (varzea´) derived from an ancestral condition of being restricted to low conductivity non‐floodplain (terra firme) black and clearwater rivers and streams. These phylogenetic, biogeographic and ecological patterns suggest a lengthy and complex history involving numerous instances of speciation, extinction, migration and coexistence in sympatry. Evolution in Gymnotus has been a continent‐wide phenomenon; i.e. Amazonian species richness is not a consequence of strictly Amazonian processes. These patterns are similar to those of other highly diverse groups of Neotropical fishes and do not resemble those of monophyletic, rapidly generated species flocks.     

Neotropical Monogenoidea. 52. Diechodactylus joaberi n. g., n. sp. from the banded knifefish Gymnotus carapo (Gymnotiformes: Gymnotidae) in southeastern Brazil

Diechodactylus joaberi n. g., n. sp. is described from the body surface of the banded knifefish Gymnotus carapo L. (Gymnotiformes: Gymnotidae) from southeastern Brazil. The new genus is proposed to accommodate species with five pairs of hooks in anterior bilateral clusters on the haptor, three pairs of hooks in a single cluster on the posterior margin of the haptor, sclerites R1 associated with the superficial bar, and confluent intestinal caeca. The presence of five pairs of hooks in two bilateral clusters anterior in the haptor permits the differentiation of species of Diechodactylus from species of all known genera of the Gyrodactylidae. The genus is likely a member of a clade of the Gyrodactylidae comprising genera with a similar hook distribution.     

Life history of <Emphasis Type="Italic">Gymnotus refugio</Emphasis> (Gymnotiformes; Gymnotidae): an endangered species of weakly electric fish

The present study describes the life history of Gymnotus refugio, a species classified as Endangered in the last published list of threatened species of the Brazilian fauna. The study was conducted at a conservation unity that protect one of the last remaining semideciduous forests in the region. The reproductive period was estimated as occurring from the end of winter to the last summer months. Gymnotus refugio exibited fractional spawning, the lowest relative fecundity registered among the Gymnotifomes species studied at the present, and male parental care behavior. The analyses showed a seasonal pattern on the species diet, associating different food categories to winter, autumn, and spring. According to food items analysis and estimated intestinal quotient, G. refugio was classified as invertivorous, feeding mainly on autochthonous insects. The results obtained herein suggest that the position of G. refugio as an Endangered species might be influenced by its territoriality, habitat specificity, parental care behavior, and low fecundity, reinforcing the importance of swampy forest environment conservation as the only means of the species maintenance.     

Repetitive DNAs and differentiation of sex chromosomes in neotropical fishes

The processes working on sex chromosome differentiation are still not completely understood. However, the accumulation of repetitive DNA sequences has been shown to be one of the first steps in the early stages of such differentiation. In addition, regions with suppressed or no recombination have a potential to accumulate these DNA sequences and, for this reason, the absence of recombination between the sex chromosomes favors, by itself, the accumulation of repetitive sequences on these chromosomes during evolution. The diversity of sex-determining mechanisms in fish, alongside with the absence of heteromorphic sex chromosomes in many species, makes this group a useful model to better understand evolutionary processes of sex chromosomes in vertebrates, considering that fish occupy the basal position in the phylogeny of this group. In this review we draw attention to a preferential accumulation and enrichment in repetitive DNAs in sex chromosomes of many neotropical fish species in comparison with autosomes. This phenomenon has been observed between both morphologically differentiated and nascent sex chromosome systems, which highlight the potential role of these sequences in the differentiation of fish sex chromosomes generating differences in morphology and size between them.     

Cytogenetic studies in Eigenmannia virescens (Sternopygidae, Gymnotiformes) and new inferences on the origin of sex chromosomes in the Eigenmannia genus

Background

Previous studies suggested that multiple domestication events in South and South-East Asia (Yunnan and surrounding areas) and India have led to the genesis of modern domestic chickens. Ha Giang province is a northern Vietnamese region, where local chickens, such as the H'mong breed, and wild junglefowl coexist. The assumption was made that hybridisation between wild junglefowl and Ha Giang chickens may have occurred and led to the high genetic diversity previously observed. The objectives of this study were i) to clarify the genetic structure of the chicken population within the Ha Giang province and ii) to give evidence of admixture with G. gallus. A large survey of the molecular polymorphism for 18 microsatellite markers was conducted on 1082 chickens from 30 communes of the Ha Giang province (HG chickens). This dataset was combined with a previous dataset of Asian breeds, commercial lines and samples of Red junglefowl from Thailand and Vietnam (Ha Noï). Measurements of genetic diversity were estimated both within-population and between populations, and a step-by-step Bayesian approach was performed on the global data set.

Results

The highest value for expected heterozygosity (> 0.60) was found in HG chickens and in the wild junglefowl populations from Thailand. HG chickens exhibited the highest allelic richness (mean A = 2.9). No significant genetic subdivisions of the chicken population within the Ha Giang province were found. As compared to other breeds, HG chickens clustered with wild populations. Furthermore, the neighbornet tree and the Bayesian clustering analysis showed that chickens from 4 communes were closely related to the wild ones and showed an admixture pattern.

Conclusion

In the absence of any population structuring within the province, the H'mong chicken, identified from its black phenotype, shared a common gene pool with other chickens from the Ha Giang population. The large number of alleles shared exclusively between Ha Giang chickens and junglefowl, as well as the results of a Bayesian clustering analysis, suggest that gene flow has been taking place from junglefowl to Ha Giang chickens.     

Two new species of Nomimoscolex (Cestoda: Proteocephalidea,Monticelliidae) from Gymnotus carapo (Pisces: Gymnotiformes) in Argentina

Nomimoscolex guillermoi n. sp. and N. dechambrieri n. sp. are described from the gymnotiform fish Gymnotus carapo from Argentina. The new species are placed into Nomimoscolex based on the cortical position of the vitelline follicles, and medullary position of the testes, ovary, and uterus. Both species were compared to the 13 species considered valid in the genus. The combination of features distinguishing N. guillermoi from N. dechambrieri is (1) the position of the vagina to cirrus pouch (anterior or posterior vs always anterior respectively), (2) the total number of testes (41-85 vs 108-130 respectively), (3) the distribution of the vitelline follicles (arranged in dorso-lateral and ventro-lateral bands vs lateral bands respectively), (4) the length of the uteroduct (ending 58% vs 35% from posterior margin of mature proglottis respectively), and (5) the presence of gland cells in the scolex (unicellular glands in the apical region and the external margin of suckers vs the presence of unicellular glands in the apex and other grouped in a cluster medially to the suckers respectively).     

Complex sex chromosome system in Eigenmannia sp. (Pisces,Gymnotiformes)

Chromosomes of Eigenmannia sp. (7 males and 15 females) collected from the Tietê River in Botucatu (SP, Brazil) were examined from gill, kidney and testicular cells. The diploid chromosome number in males was 2n=31 and in females, 2n=32. In both sexes the number of chromosomal arms was 40. The difference in diploid number was due to the fusion of two acrocentrics. Mitotic and meiotic studies suggested that one of the fused acrocentrics was the Y chromosome. The sex-determining mechanism in Eigenmannia sp. could therefore be XX, AA in the female and X, \-YA A in the males. One of the males presented 2n=30 chromosomes due to the occurrence of another fusion of acrocentrics. C-banding analysis of the mitotic chromosomes revealed constitutive heterochromatin in the centromeric regions of all acrocentrics. However, small metacentrics were C-band negative. The YA chromosome is C-band negative except for a small amount of heterochromatin in the centromeric region. The nucleolar organizer region as identified by Ag-staining is present in the interstitial region of chromosome pair No. 10.     

Chromosomal and electric signal diversity in three sympatric electric knifefish species (Gymnotus, Gymnotidae) from the Central Amazon Floodplain

We describe chromosomal and electric signal diversity in three sympatric species of Gymnotus (Gymnotidae) fish from the Central Amazon Floodplain. Gymnotus arapaima presents a karyotype of 2n = 44 (24 m-sm + 20st-a), G. mamiraua 2n = 54 (42 m-sm + 12st-a), and G. jonasi 2n = 52 (12 m-sm + 40st-a). No evidence for a chromosomal sexual system was observed in two species for which both males and females were analyzed (G. mamiraua and G. arapaima). In all three species the constitutive heterochromatin is located primarily in pericentromeric regions, but also at some other sites. G. arapaima and G. mamiraua exhibit simple nucleolar organizing regions (NORs) on short arms of chromosome pairs 19 and 24, respectively. Gymnotus jonasi exhibits a multiple interstitial NOR on the long arm of pairs 9 and 10, and on the short arm of pair 11. G. arapaima and G. mamiraua exhibit several additional similarities in their karyotypic formulas—reflecting the phylogenetic proximity of these species within a G. carapo group clade (based on molecular phylogenetic evidence). The chromosomal differences among these three sympatric species imply complete post-zygotic reproductive isolation. A prominent pattern of partitioning of the peak power frequency of the electric organ discharge of these three species indicates pre-zygotic reproductive isolation of mate attraction signals. We conclude by discussing the evolutionary events that may have promoted signal divergence and reproductive isolation in Gymnotus of the Central Amazon, and the role that chromosomal rearrangements may place in diversification.