Chromosomal diversification in populations of Characidium cf. gomesi (Teleostei, Crenuchidae)

Comparative cytogenetic studies carried out in two populations of Characidium cf. gomesi from Botucatu region, SP, Brazil, showed a similar karyotypic structure in a diploid number of 50 chromosomes, 32 metacentric and 18 submetacentric chromosomes for males and 31 metacentric and 19 submetacentric chromosomes for females as well as a ZZ-ZW sex chromosome system. Differences between both populations, however, were found in relation to the occurrence of B chromosomes and the distribution of 18S and 5S ribosomal DNA (rDNA) sites. Characidium cf. gomesi from the Alambari Stream, a component of the Tietê River basin, revealed 18S rDNA on Z and W chromosomes, while this gene was located on autosomes in the sample from the Paranapanema River basin. The 5S rDNA sites were observed in a single chromosomal pair (number 25) in the populations from Paranapanema and in two pairs in the specimens from Tietê (numbers 20 and 25). Besides that, in the sample from Paranapanema, both inter and intra-individual variations were found due to the occurrence of up to four heterochromatic supernumerary chromosomes in the cells. The life mode of this fish, restricted to headwaters and subjected to frequent breakdown into sub-populations, may have contributed to the fixation of such chromosomal differences. The karyotypic similarities found in the analysed populations, however, suggest that all are descended from the same ancestor group whereas their differences indicate that they are already existing in reproductively isolated populations.     

Diversification of a ZZ/ZW sex chromosome system in Characidium fish (Crenuchidae, Characiformes)

Karyotype and other chromosomal markers of Characidium cf. gomesi were analyzed using conventional (Giemsa-staining, Ag-NOR and C-banding) and molecular (Fluorescent in situ hybridization (FISH) with 18S and 5S rDNA biotinylated probes) techniques. Both sexes had invariably diploid chromosome number 2n = 50 while karyotypes of males and females differed. That of male consisted of 32 metacentric + 18 submetacentric chromosomes and that of female consisted 31 metacentric + 18 submetacentric + 1 subtelocentric chromosomes. The Z chromosome was medium-sized metacentric, while W was highly heterochromatinized subtelocentric element. NORs as revealed by Ag-staining were situated at 2–7 telomeric regions while FISH with 18S probes showed consistently 10 signals at telomeric regions. FISH with 5S rDNA probe showed constantly signals at one metacentric pair. Distribution of centromeric heterochromatin was mostly in all chromosome pairs, besides some telomeric sites. The common origin of the sex chromosome system of ZZ/ZW type in the karyotypes of other representatives of the genus analyzed so far might be hypothesized based on biogeography and partial phylogeny of the group.     

A new species of Characidium (Characiformes: Crenuchidae) from the rio Madeira basin,Brazil

This study describes Characidium nambiquara, a new species from the upper rio Guaporé, rio Madeira basin, Brazil. The new species differs from most congeners by the presence of isthmus and area between the contralateral pectoral-fin bases completely naked. From congeners with some degree of scaleless ventral surface of the body C. nambiquara differs by having 10 circumpeduncular scales. The new species is also distinguished from congeners by the spotted colour pattern on body of the large-sized specimens and by having black dashes on all fins and conspicuous midlateral longitudinal dark stripe or conspicuous vertical bars absent. Characidium nambiquara further differs from most congeners by the presence of 34–36 pored scales on the lateral line, 3 horizontal scale rows above the lateral line and 3 horizontal scale rows from the lateral line to the midventral scale series. Remarks on intraspecific colour variation within the genus, not related to sexual dimorphism, are also provided.     

Chromosome painting of Z and W sex chromosomes in Characidium (Characiformes, Crenuchidae)

Some species of the genus Characidium have heteromorphic ZZ/ZW sex chromosomes with a totally heterochromatic W chromosome. Methods for chromosome microdissection associated with chromosome painting have become important tools for cytogenetic studies in Neotropical fish. In Characidium cf. fasciatum, the Z chromosome contains a pericentromeric heterochromatin block, whereas the W chromosome is completely heterochromatic. Therefore, a probe was produced from the W chromosome through microdissection and degenerate oligonucleotide-primed polymerase chain reaction amplification. FISH was performed using the W probe on the chromosomes of specimens of this species. This revealed expressive marks in the pericentromeric region of the Z chromosome as well as a completely painted W chromosome. When applying the same probe on chromosome preparations of C. cf. gomesi and Characidium sp., a pattern similar to C. cf. fasciatum was found, while C. cf. zebra, C. cf. lagosantense and Crenuchus spilurus species showed no hybridization signals. Structural changes in the chromosomes of an ancestral sexual system in the group that includes the species C. cf. gomesi, C. cf. fasciatum and Characidium sp., could have contributed to the process of speciation and could represent a causal mechanism of chromosomal diversification in this group. The heterochromatinization process possibly began in homomorphic and homologous chromosomes of an ancestral form, and this process could have given rise to the current patterns found in the species with sex chromosome heteromorphism.     

A new species of miniature Characidium (Characiformes: Crenuchidae) from the upper Rio Paraguay basin,Mato Grosso state,Brazil

A new miniature species of Characidium is described from the upper Rio Paraguay basin, Brazil. The new species can be diagnosed from all congeners by the presence of a dark-brown humeral blotch, vertically elongated (spanning 5 to 7 horizontal scale rows), with the shape of an upside-down acute triangle. Additionally, it can be diagnosed by a short lateral line (6 to 8 perforated scales), the absence of a conspicuous peduncular blotch, a lower number of principal caudal-fin rays (14–16) and by absence of the adipose fin. The new species is the smallest species of the genus; the largest specimen was 19.3 mm standard length.     

Sex chromosome composition revealed in Characidium fishes (Characiformes: Crenuchidae) by molecular cytogenetic methods

The W chromosome of the fishes Characidium cf. fasciatum, Characidium sp. and Characidium cf. gomesi is heterochromatic, as is usually seen in most Characidium species. Samples of W-chromatin were collected by mechanical microdissection and amplified by DOP-PCR (degenerate oligonucleotide-primed polymerase chain reaction), to be used as painting probes (DCg and CgW) and for sequence analysis. FISH (fluorescence in situ hybridization) with DCg probe painted the whole W chromosome, the pericentromeric region of Z chromosomes and the terminal region of B chromosomes. DOP-PCR-generated fragments were cloned, sequenced and tested by in situ hybridization, but only CgW4 produced positive hybridization signals. Clone sequence analysis recovered seven distinct sequences, of which six did not reveal any similarity to other known sequences in the GenBank or GIRI databases. Only CgW9 clone sequence was recognized as probably derived from a Helitron-transposon similar to that found in the genome of the zebrafish Danio rerio. Our results show that the composition of Characidium’s W chromosome does seem rich in repetitive sequences as well as other W chromosomes found in several species with a ZW sex-determining mechanism.     

Cytogenetic divergence in two sympatric fish species of the genus Astyanax Baird and Girard, 1854 (Characiformes,Characidae) from northeastern Brazil

The fish genus Astyanax is widespread throughout the Neotropical region and is one of the most species-rich genera of the Characiformes. Cytogenetic studies of Astyanax have revealed marked intra- and interspecific diversity, with the identification of various species complexes. In this report, we describe the karyotypic structure of two sympatric species of Astyanax (Astyanax sp. and Astyanax aff. fasciatus) from the Middle Contas River basin in the northeastern Brazilian state of Bahia. Both species had 2n = 48 but differed in their karyotypic formulae. Small heterochromatic blocks and multiple nucleolar organizer regions (NORs) were identified in both species. Terminal CMA₃⁺/DAPI⁻ signals were observed in Astyanax sp. and A. aff. fasciatus, mostly coincident with NORs. These results show that chromosomal markers can be used to identify species in this fish complex. These markers can provide useful information for evolutionary studies and investigations on the mechanisms of chromosomal diversity in Astyanax.     

Population differentiation and speciation in the genus Characidium (Characiformes: Crenuchidae): effects of reproductive and chromosomal barriers

Both time and low gene flow are the key factors by which different biological species arise. The divergence process among lineages and the development of pre‐ or postzygotic isolation occur when gene flow events are lacking. The separation among species of the genus Characidium was analysed in relation to the geomorphological mechanisms in river courses, events of captured adjacent upland drainages in south‐eastern Brazil, and sex chromosome differences. The ZZ/ZW sex chromosomes of Characidium vary in size, morphology, degree of heterochromatinization, and presence/absence of ribosomal DNA. The goal of this study was to understand the mechanism of sex chromosome differentiation, its close association with the geological history of cladogenetic events among drainages, and reproductive isolation leading to Characidium speciation. The W‐specific probe from Characidium gomesi generated a highlighted signal on the entire W chromosome of C. gomesi, Characidium heirmostigmata, Characidium pterostictum, and Characidium sp., instead of karyotypes of three Characidium aff. zebra populations, which showed scattered signals. An evolutionary and biogeographic landscape arose by analysis of ribosomal DNA site location and differentiation of the sex chromosomes, which established mechanisms of reproductive isolation leading to meiotic barriers, keeping the biological unit distinct even if the contact among species was restored. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 541–553.     

Unusual occurrence of a ZZ/ZW sex-chromosome system and supernumerary chromosomes in Characidium cf. fasciatum (Pisces, Characiformes, Characidiinae)

Individuals of two populations of the fish Characidium cf. fasciatum were cytogenetically studied and showed a basic diploid number of 50 chromosomes. Some fishes were found to have 51 to 54 chromosomes due to the presence of one to four small subtelocentric/acrocentric supernumerary chromosomes. When analyzed by conventional Giemsa staining, male and female specimens of C. cf. fasciatum from the Quinta stream and Pardo River presented the same basic karyotypic macro- and microstructure, consisting of 32 metacentric and 18 submetacentric chromosomes. Ag-NORs were terminally located on the long arms of two submetacentric chromosome pairs. Constitutive heterochromatin was identified by C-banding as small pericentromeric blocks in the majority of the chromosomes, and B-chromosomes were found to be heterochromatic. The occurrence of one totally heterochromatic submetacentric chromosome restricted to females and considered as an unusual feature in fish karyotypes led to the identification of a ZZ/ZW sex-chromosome system. The implications of chromosomic differentiation observed in the genus Characidium are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

An unusual ZZ/ZW sex chromosome system in Characidium fishes (Crenuchidae, Characiformes) with the presence of rDNA sites

Characidium fishes with a sex chromosome system form a monophyletic group. This work presents data of Characidium lanei from the South Atlantic basin (Brazil), including an unknown type of ZW sex chromosome system for the groups including the presence of rDNA sites on sex chromosomes.     

Karyotypic conservatism in samples of Characidium cf. zebra (Teleostei, Characiformes, Crenuchidae): Physical mapping of ribosomal genes and natural triploidy

Basic and molecular cytogenetic analyses were performed in specimens of Characidium cf. zebra from five collection sites located throughout the Tietê, Paranapanema and Paraguay river basins. The diploid number in specimens from all samples was 2n = 50 with a karyotype composed of 32 metacentric and 18 submetacentric chromosomes in both males and females. Constitutive heterochromatin was present at the centromeric regions of all chromosomes and pair 23, had additional interstitial heterochromatic blocks on its long arms. The nucleolar organizer regions (NORs) were located on the long arms of pair 23, while the 5S rDNA sites were detected in different chromosomes among the studied samples. One specimen from the Alambari river was a natural triploid and had two extra chromosomes, resulting in 2n = 77. The remarkable karyotypic similarity among the specimens of C. cf. zebra suggests a close evolutionary relationship. On the other hand, the distinct patterns of 5S rDNA distribution may be the result of gene flow constraints during their evolutionary history.     

Genetics of male nuptial colour divergence between sympatric sister species of a Lake Victoria cichlid fish

The hypothesis of sympatric speciation by sexual selection has been contentious. Several recent theoretical models of sympatric speciation by disruptive sexual selection were tailored to apply to African cichlids. Most of this work concludes that the genetic architecture of female preference and male trait is a key determinant of the likelihood of disruptive sexual selection to result in speciation. We investigated the genetic architecture controlling male nuptial colouration in a sympatric sibling species pair of cichlid fish from Lake Victoria, which differ conspicuously in male colouration and female mating preferences for these. We estimated that the difference between the species in male nuptial red colouration is controlled by a minimum number of two to four genes with significant epistasis and dominance effects. Yellow colouration appears to be controlled by one gene with complete dominance. The two colours appear to be epistatically linked. Knowledge on how male colouration segregates in hybrid generations and on the number of genes controlling differences between species can help us assess whether assumptions made in simulation models of sympatric speciation by sexual selection are realistic. In the particular case of the two sister species that we studied a small number of genes causing major differences in male colouration may have facilitated the divergence in male colouration associated with speciation.     

An intraspecific comparative analysis of character divergence between sympatric species

Although sympatric character divergence between closely related species has been described in a wide variety of taxa, the evolutionary processes responsible for generating these patterns are difficult to identify. One hypothesis that can explain sympatric differences is ecological character displacement: the sympatric origin of morphologically divergent phenotypes in response to selection caused by interspecific competition. Alternatively, populations may adapt to different conditions in allopatry, with sympatric distributions evolving through selective colonization and proliferation of ecologically compatible phenotypes. In this study, I characterize geographic variation within two sibling species of rocky-shore gastropods that have partially overlapping distributions in central California. In sympatry, both Nucella emarginata and N. ostrina show significant differences in shell shape and shell ornamentation that together suggest that where the two species co-exist, divergent phenotypes arose as an evolutionary consequence of competition. To examine the evolutionary origins of divergent characters in sympatry, I used a comparative method based on spatial autocorrelation to remove the portion of the phenotypic variance among populations that is explained by genetic distance (using mitochondrial DNA sequences and allozyme frequency data). Because the remaining portion of the phenotypic variance represents the independent divergence of individual populations, a significant sympatric difference in the corrected dataset provides evidence of true character displacement: significant sympatric character evolution that is independent of population history. After removal of genetic distance effects in Nucella, shell shape differences remain statistically significant in N. emarginata, providing evidence of significant sympatric character divergence. However, for external shell ornamentation in both species and shell shape in N. ostrina, the significance of sympatric differences is lost in the corrected dataset, indicating that colonization events and gene flow have played important roles in the evolutionary history of character divergence in sympatry. Although the absence of a widely dispersing planktonic larva in the life cycle of Nucella will promote local adaptation, the results here indicate that once advantageous traits arise, demographic processes, such as recurrent gene flow between established populations and extinction and recolonization, are important factors contributing to the geographic pattern of sympatric character divergence.     

Genetic divergence in adaptive characters between sympatric species of stickleback

This study explored the genetic basis of phenotypic differences between two sympatric species of ecologically and morphologically divergent sticklebacks (Gasterosteus aculeatus complex). The aim was to understand how many loci determine the differences and to what extent the differences are due to additive or nonadditive gene action. I reared the two parental species, F1 and F2 hybrids, and both backcrosses in the laboratory and measured the following quantitative characters: gill raker number and length (both involved in feeding), lateral plate number and pelvic spine length (both involved in predator defense), and growth (a fitness component). I then applied joint-scaling regression models to estimate composite additive, dominance and epistatic effects, and their contribution to divergence of parental lines. A simple additive model was sufficient for gill raker number and growth; additive and dominance effects contributed significantly to divergence in plate number and pelvic spine length; and additive, dominance, and epistatic effects contributed significantly to divergence in gill raker length. Wright's estimator for the number of loci for the four morphological characters ranged from 1 to 50. My results suggest that adaptive divergence between limnetic and benthic sticklebacks has taken place through a variety of genetic mechanisms specific to different traits. Though interspecific hybrids are completely fertile and viable in the laboratory, they are selected against in the wild. The pattern of inheritance for the traits examined here directly influences how well hybrids can exploit the two major resource environments in the wild.     

Trophic specialization and morphological divergence between two sympatric species in Lake Catemaco,Mexico

The association of morphological divergence with ecological segregation among closely related species could be considered as a signal of divergent selection in ecological speciation processes. Environmental signals such as diet can trigger phenotypic evolution, making polymorphic species valuable systems for studying the evolution of trophic‐related traits. The main goal of this study was to analyze the association between morphological differences in trophic‐related traits and ecological divergence in two sympatric species, Astyanax aeneus and A. caballeroi, inhabiting Lake Catemaco, Mexico. The trophic differences of a total of 70 individuals (35 A. aeneus and 35 A. caballeroi) were examined using stable isotopes and gut content analysis; a subset of the sample was used to characterize six trophic and six ecomorphological variables. In our results, we recovered significant differences between both species in the values of stable isotopes, with higher values of δ¹⁵N for A. caballeroi than for A. aeneus. Gut content results were consistent with the stable isotope data, with a higher proportion of invertebrates in A. caballeroi (a consumption of invertebrates ten times higher than that of A. aeneus, which in turn consumed three times more vegetal material than A. caballeroi). Finally, we found significant relationship between ecomorphology and stable isotopes (r = .24, p < .01), hence, head length, preorbital length, eye diameter, and δ¹⁵N were all positively correlated; these characteristics correspond to A. caballeroi. While longer gut and gill rakers, deeper bodies, and vegetal material consumption were positively correlated and corresponded to A. aeneus. Our results are consistent with the hypothesis that morphological divergence in trophic‐related traits could be associated with niche partitioning, allowing the coexistence of closely related species and reducing interspecific competition.     

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.     

Chromosomal diversity in three species of electric fish (Apteronotidae,Gymnotiformes) from the Amazon Basin

Cytogenetic studies were carried out on samples of Parapteronotus hasemani, Sternarchogiton preto and Sternarchorhamphus muelleri (Apteronotidae, Gymnotiformes) from the Amazon basin. The first two species exhibited both a 2n = 52 karyotype, but differed in their karyotypic formulae, distribution of constitutive heterochromatin, and chromosomal location of the NOR. The third species, Sternarchorhamphus muelleri, was found to have a 2n = 32 karyotype. In all three species the DAPI and chromomycin A3 staining results were consistent with the C-banding results and nucleolar organizer region (NOR) localization. The 18S rDNA probe confirmed that there was only one pair of ribosomal DNA cistron bearers per species. The telomeric probe did not reveal interstitial telomeric sequences (ITS). The karyotypic differences among these species can be used for taxonomic identification. These data will be useful in future studies of these fishes and help understanding the phylogenetic relationships and chromosomal evolution of the Apteronotidae.     

Chromosomal location of 18S and 5S rDNA sites in Triportheus fish species (Characiformes, Characidae)

The location of 18S and 5S rDNA sites was determined in eight species and populations of the fish genus Triportheus by using fluorescent in situ hybridization (FISH). The males and females of all species had 2n = 52 chromosomes and a ZZ/ZW sex chromosome system. A single 18S rDNA site that was roughly equivalent to an Ag-NOR was detected on the short arms of a submetacentric pair in nearly all species, and up to two additional sites were also observed in some species. In addition, another 18S rDNA cluster was identified in a distal region on the long arms of the W chromosome; this finding corroborated previous evidence that this cluster would be a shared feature amongst Triportheus species. In T. angulatus, a heterozygotic paracentric inversion involving the short arms of one homolog of a metacentric pair was associated with NORs. The 5S rDNA sites were located on the short arms of a single submetacentric chromosomal pair, close to the centromeres, except in T. auritus, which had up to ten 5S rDNA sites. The 18S and 5S rDNA sites were co-localized and adjacent on the short arms of a chromosomal pair in two populations of T. nematurus. Although all Triportheus species have a similar karyotypic macrostructure, the results of this work show that in some species ribosomal genes may serve as species-specific markers when used in conjunction with other putatively synapomorphic features.     

Maintenance of species boundaries in three sympatric Ligularia (Senecioneae,Asteraceae) species

The key process in speciation concerns the formation and maintenance of reproductive isolating barriers between diverging lineages. Although species boundaries are frequently investigated between two species across many taxa, reproductive isolating barriers among multiple species (>2) that would represent the most common phenomenon in nature, remain to be clarified. Here, we use double digest restriction‐site associated DNA (ddRAD) sequencing to examine patterns of hybridization at a sympatric site where three Ligularia species grow together and verify whether those patterns contribute to the maintenance of boundaries among species. The results based on the RAD SNP datasets indicated hybridization Ligularia cyathiceps × L. duciformis and L. duciformis × L. yunnanensis were both restricted to F₁s plus a few first‐generation backcrosses and no gene introgression were identified, giving rise to strong reproductive isolation among hybridizing species. Moreover, hybrid swarm simulation, using HYBRIDLAB, indicated the RAD SNP datasets had sufficient discriminatory power for accurate hybrid detection. We conclude that parental species show strong reproductive isolation and they still maintain species boundaries, which may be the key mechanism to maintain species diversity of Ligularia in the eastern Qinghai‐Tibetan Plateau and adjacent areas. Moreover, this study highlights the effectiveness of RAD sequencing in hybridization studies.