Chromosome spreading of associated transposable elements and ribosomal DNA in the fish Erythrinus erythrinus. Implications for genome change and karyoevolution in fish

Background  

The fish, Erythrinus erythrinus, shows an interpopulation diversity, with four karyomorphs differing by chromosomal number, chromosomal morphology and heteromorphic sex chromosomes. Karyomorph A has a diploid number of 2n = 54 and does not have differentiated sex chromosomes. Karyomorph D has 2n = 52 chromosomes in females and 2n = 51 in males, and it is most likely derived from karyomorph A by the differentiation of a multiple X₁X₂Y sex chromosome system. In this study, we analyzed karyomorphs A and D by means of cytogenetic approaches to evaluate their evolutionary relationship.     

Tracking the evolutionary pathway of sex chromosomes among fishes: characterizing the unique XX/XY<Subscript>1</Subscript>Y<Subscript>2</Subscript> system in <Emphasis Type="Italic">Hoplias malabaricus</Emphasis> (Teleostei,Characiformes)

The Neotropical fish, Hoplias malabaricus, is one of the most cytogenetically studied fish taxon with seven distinct karyomorphs (A–G) comprising varying degrees of sex chromosome differentiation, ranging from homomorphic to highly differentiated simple and multiple sex chromosomes. Therefore, this fish offers a unique opportunity to track evolutionary mechanisms standing behind the sex chromosome evolution and differentiation. Here, we focused on a high-resolution cytogenetic characterization of the unique XX/XY₁Y₂ multiple sex chromosome system found in one of its karyomorphs (G). For this, we applied a suite of conventional (Giemsa-staining, C-banding) and molecular cytogenetic approaches, including fluorescence in situ hybridization FISH (with 5S and 18S rDNAs, 10 microsatellite motifs and telomeric (TTAGGG) _n sequences as probes), comparative genomic hybridization (CGH), and whole chromosome painting (WCP). In addition, we performed comparative analyses with other Erythrinidae species to discover the evolutionary origin of this unique karyomorph G-specific XY₁Y₂ multiple sex chromosome system. WCP experiments confirmed the homology between these multiple sex chromosomes and the nascent XX/XY sex system found in the karyomorph F, but disproved a homology with those of karyomorphs A–D and other closely related species. Besides, the putative origin of such XY₁Y₂ system by rearrangements of several chromosome pairs from an ancestral karyotype was also highlighted. In addition, clear identification of a male-specific region on the Y₁ chromosome suggested a differential pattern of repetitive sequences accumulation. The present data suggested the origin of this unique XY₁Y₂ sex system, revealing evidences for the high level of plasticity of sex chromosome differentiation within the Erythrinidae.     

Whole chromosome painting reveals independent origin of sex chromosomes in closely related forms of a fish species

The wolf fish Hoplias malabaricus includes well differentiated sex systems (XY and X₁X₂Y in karyomorphs B and D, respectively), a nascent XY pair (karyomorph C) and not recognized sex chromosomes (karyomorph A). We performed the evolutionary analysis of these sex chromosomes, using two X chromosome-specific probes derived by microdissection from the XY and X₁X₂Y sex systems. A putative-sex pair in karyomorph A was identified, from which the differentiated XY system was evolved, as well as the clearly evolutionary relationship between the nascent XY system and the origin of the multiple X₁X₂Y chromosomes. The lack of recognizable signals on the sex chromosomes after the reciprocal cross-FISH experiments highlighted that they evolved independently from non-homologous autosomal pairs. It is noteworthy that these distinct pathways occur inside the same nominal species, thus exposing the high plasticity of sex chromosome evolution in lower vertebrates. Possible mechanisms underlying this sex determination liability are also discussed.     

Molecular and karyotypic phylogeography in the Neotropical Hoplias malabaricus (Erythrinidae) fish in eastern Brazil

The sedentary, predatory characin Hoplias malabaricus has one of the widest distributions of freshwater fishes in South America and is characterized by seven karyomorphs (A–G) that occur in sympatric and allopatric populations. Karyotypical patterns of variation in wild populations have been interpreted as evidence of multiple lineages within this nominal species, a possibility that may limit the validity of experimental data for particular karyomorphs. This study used the phylogeographic and genealogical concordance between cytogenetic (N = 49) and molecular (mitochondrial DNA) (N = 73) data on 17 samples, collected in 12 basins from south‐eastern and north‐eastern Brazil, to assess the systematic value of cytogenetic data. Cytogenetic patterns show a sex chromosome system in the 2n = 40F karyomorph. Molecular and cytogenetic data indicate a long, independent evolutionary history of karyomorphs and a coastal origin of continental populations in south‐eastern Brazil. The lack of fit with molecular clock expectations of divergence between groups is likely to be due to strong demographic fluctuations during the evolution of this species complex. The results indicate that karyotypical identification provides a reliable baseline for placing experimental studies on Hoplias spp. in a phylogenetic context.     

Differentiation and evolutionary relationships in Erythrinus erythrinus (Characiformes, Erythrinidae): comparative chromosome mapping of repetitive sequences

Erythrinus erythrinus presents extensive karyotypic diversity, with four karyomorphs (A–D) differing in the number of chromosomes, karyotype structure or sex chromosomes systems. Karyomorph A has 2n = 54 chromosomes in males and females without heteromorphic sex chromosomes, while karyomorph C has 2n = 52 chromosomes in females and 2n = 51 chromosomes in males, due a X₁X₁X₂X₂/X₁X₂Y sex chromosome system. Three allopatric populations of the karyomorph A and one population of the karyomorph C were now in deep investigated by molecular cytogenetic analyses, using repetitive DNAs as probes. The results reinforced the relatedness among populations of the karyomorph A, despite their large geographic distribution. Karyomorph C, however, showed a remarkably difference in the genomic constitution, especially concerning the amount and distribution of the 5S rDNA and Rex3 sequences on chromosomes. In addition, although karyomorphs C and D share several features, exclusive chromosomal markers show the derivative evolutionary pathway between them. Thus, besides the classical chromosomal rearrangements, the repetitive DNAs were useful tools to reveal the biodiversity, relatedness and differentiation of this fish group. The chromosomal set strongly corroborates that E. erythrinus corresponds to a species complex instead of a single biological entity.     

Discovery of a diploid population of theHemidactylus garnotii-vietnamensis complex (Reptilia: Gekkonidae)

A diploid member of the parthenogenetic gekkonid species complexHemidactylus garnotii-vietnamensis was discovered for the first time from Thailand. This gecko, seemingly unisexual and parthenogenetic, possesses 2n=2x=38 chromosomes, showing distinct heteromorphisms. The absence of bisexual congeneric species with a combination of karyomorphs to produce this karyotype indicates the occurrence of chromosomal rearrangements after the initial estabilishment of a diploid clonal lineage of hybrid origin. Results of karyotypic comparisons of the present sample and the three known triploid species belonging to theH. garnotii-vietnamensis complex suggest that a triploid karyomorph similar to that ofH. vietnamensis has first emerged through an insemination of the diploid parthenogen's egg by the sperm from a bisexual species having 44 chromosomes (all telocentric), and that the karyomorph subsequently experienced some minor chromosomal aberrations to produce the karyomorphs ofH. vietnamensis andH. garnotii. The origin of theH. stejnegeri karyotype still remains an open question for future studies.     

The chromosomal distribution of microsatellite repeats in the genome of the wolf fish Hoplias malabaricus, focusing on the sex chromosomes

Distribution of 12 mono-, di- and tri-nucleotide microsatellites on the chromosomes of 2 karyomorphs with 2 distinct sex chromosome systems (a simple XX/XY - karyomorph B and a multiple X(1)X(1)X(2)X(2)/X(1)X(2)Y - karyomorph D) in Hoplias malabaricus, commonly referred to as wolf fish, was studied using their physical mapping with fluorescence in situ hybridization (FISH). The distribution patterns of different microsatellites along the chromosomes varied considerably. Strong hybridization signals were observed at subtelomeric and heterochromatic regions of several autosomes, with a different accumulation on the sex chromosomes. A massive accumulation was found in the heterochromatic region of the X chromosome of karyomorph B, whereas microsatellites were gathered at centromeres of both X chromosomes as well as in corresponding regions of the neo-Y chromosome in karyomorph D. Our findings are likely in agreement with models that predict the accumulation of repetitive DNA sequences in regions with very low recombination. This process is however in contrast with what was observed in multiple systems, where such a reduction might be facilitated by the chromosomal rearrangements that are directly associated with the origin of these systems.     

Comparative chromosome mapping of 5S rDNA and 5SHindIII repetitive sequences in Erythrinidae fishes (Characiformes) with emphasis on the Hoplias malabaricus 'species complex'

Chromosomal localization of 5S rDNA and 5SHindIII repetitive sequences was carried out in several representatives of the Erythrinidae family, namely in karyomorphs A, D, and F of Hoplias malabaricus, and in H. lacerdae, Hoplerythrinusunitaeniatus and Erythrinus erythrinus. The 5S rDNA mapped interstitially in two chromosome pairs in karyomorph A and in one chromosome pair in karyomorphs D and F and in H. lacerdae. The 5SHindIII repetitive DNA mapped to the centromeric region of several chromosomes (18 to 22 chromosomes) with variations related to the different karyomorphs of H. malabaricus. On the other hand, no signal was detected in the chromosomes of H. lacerdae, H. unitaeniatus and E. erythrinus, suggesting that the 5SHindIII-DNA sequences have originated or were lost after the divergence of H. malabaricus from the other erythrinid species. The chromosome distribution of 5S rDNA and 5SHindIII-DNA sequences contributes to a better understanding of the mechanisms of karyotype differentiation among the Erythrinidae members.     

New Ctenomys karyotypes (Rodentia,Octodontidae) from north-eastern Argentina and from Paraguay confirm the extreme chromosomal multiformity of the genus

Bone-marrow karyotypes of 68 specimens of the subterranean octodontid rodent genus Ctenomys from 16 different populations of north east Argentina and one from Paraguay have been studied. A surprising variety of chromosome numbers was found, ranging from 2n=42 to 2n=70. Some of the karyomorphs are clearly assigned to named species by topotypy: C. conoveris 2n=50, FN=56; C. argentinus, 2n=44, FN=54; C. perrensi, 2n=50, FN=84; C. dorbignyi, 2n=70, FN=84; C. roigi, 2n=48, FN=80; C. yolandae, 2n=50, FN=78. Four populations of Corrientes Province similar in morphology to C. perrensi were found to be polymorphic and polytypic; they maintain the same FN=84, but diploid numbers increase from 2n=54 to 2n=58 from SW to the NE, thus suggesting Robertsonian rearrangements. In the middle of this cline, a stable karyomorph of 2n=62, FN=84 was found in two different populations, suggesting to belong to an undescribed species. Another karyomorph of 2n=42, FN=76 found in Curuzú Laurel, Corrientes, may also prove to represent another undescribed species. One karyomorph of 2n=52, FN=74, and another of 2n=56, FN=78 from Paraná and Ubajaý (Entre Ríos Province, Argentina) respectively are close to C. rionegrensis. The relationships among these karyomorphs is considered in light of data on sperm morphology. The hypothesis is advanced that karyotypic rearrangements among the FN=84 group may be the result of Robertsonian repatterning from a 2n=70 original widespread form. Fixation of chromosomal variants is correlated with patchy distribution and small size of unstable demes, and may or may not have resulted in reproductive isolation.     

Different rate of intrapopulation chromosome polymorphism in two species of Black Sea Blennies (Blenniidae,Pisces)

Somatic karyotypes in seven specimens of Blennius sanguinolentus include 22 subtelocentric and 26 acrocentric chromosomes, whereas one male has 2n=47=1M+22ST+24A: polymorphism is evidently a result of centric fusion of two acrocentrics. Blennius tentacularis is characterized by the availability of four karyomorphs out of which three coincide with karyotypes described earlier (Carbone et al., 1987). Karyttype-I consists of a 48 small uni-armed chromosome, but both karyotypes II and III with 2n=48 and 2n=47 respectively include one large acrocentric chromosome, and karyotype-IV has one large submetacentric out of the 47 chromosomes. Karyotypic variability of B. tentacularis is attributed either to polymorphism by 1–3 chromosome rearrangements or to availability of sex-determining mechanism, including the Y-autosome translocation. This diverse series of male karyomorphs may reflect the complicated behavioural structure.     

A De Novo Case of Floating Chromosomal Polymorphisms by Translocation in Quasipaa boulengeri (Anura,Dicroglossidae)

Very few natural polymorphisms involving interchromosomal reciprocal translocations are known in amphibians even in vertebrates. In this study, thirty three populations, including 471 individuals of the spiny frog Quasipaa boulengeri, were karyotypically examined using Giemsa stain or FISH. Five different karyomorphs were observed. The observed heteromorphism was autosomal but not sex-related, as the same heteromorphic chromosomes were found both in males and females. Our results indicated that the variant karyotypes resulted from a mutual interchange occurring between chromosomes 1 and 6. The occurrence of a nearly whole-arm translocation between chromosome no. 1 and no. 6 gave rise to a high frequency of alternate segregation and probably resulted in the maintenance of the translocation polymorphisms in a few populations. The translocation polymorphism is explained by different frequencies of segregation modes of the translocation heterozygote during meiosis. Theoretically, nine karyomorphs should be investigated, however, four expected karyotypes were not found. The absent karyomorphs may result from recessive lethal mutations, position effects, duplications and deficiencies. The phylogenetic inference proved that all populations of Q. boulengeri grouped into a monophyletic clade. The mutual translocation likely evolved just once in this species and the dispersal of the one karyomorph (type IV) can explain the chromosomal variations among populations.     

Investigation and documentation of hybridization betweenParkinsonia aculeata andCercidium praecox (Leguminosae:Caesalpinioideae)

Morphometric, cytogenetic, geographical and ecological evidence for hybridization betweenParkinsonia aculeata andCercidium praecox is presented. Morphometric investigation using the character count procedure and cytogenetic observations confirm hybrid status. All diagnostic morphometric characters were intermediate in the hybrid. Both parents (2n = 28) show regular tetrad formation and pollen fertility greater than 94%. Hybrids have a chromosome number of 2n = 28 or 2n = 30, and display meiotic abnormalities including lagging chromosomes and micronucleus formation; less than 21% of hybrid pollen was fertile. Ecological and geographical information suggests that hybridization is occurring at increasing frequency due to the expanding range ofP. aculeata associated with cultivation as an ornamental, coupled with ecological disturbance and weediness, and the cultivation ofC. praecox and hybrids as fodder, ornamental and shade trees. Hybrid fertility and phenological observations, in conjunction with F-weighted principal component analysis, suggest that the progeny of F₁ hybrids are established. The hybrid is formally described asP. ×carterae.     

Molecular cytogenetic evidence of hybridization in the “purple corolla clade of the genus Capsicum” (C. eximium × C. cardenasii)

Abstract

A hybrid individual between two taxa from “the Purple Corolla clade of the genus Capsicum”, C. eximium × C. cardenasii (2n = 24) was found during a cytogenetic study of a population belonging to C. cardenasii cytotype 1, from Bolivia. 5S and 45S rDNA probes were located on mitotic chromosomes by fluorescent in situ hybridization. The hybrid haploid karyotype length was an average value of the two taxa; the hybrid presented 24 45S loci in the diploid complement, 18 45S sites belonged to C. cardenasii cytotype 2, and six came from C. eximium cytotype 2, although this cytotype of C. eximium has been mentioned only for Argentina. Whereas hybrids were previously reported in the purple flower group, their existence has not been cytologically corroborated. This finding constitutes the first molecular cytogenetic evidence of hybridization between two taxa from this group.     

Genomic characterisation and the detection of raspberry chromatin in polyploid Rubus

 This paper reports genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) data for chromosomes of raspberry (Rubus idaeus 2n=2x=14), blackberry (Rubus aggregate, subgenus Eubatus. 2n=2–12x=14–84) and their allopolyploid derivatives used in fruit breeding programmes. GISH was used to discriminate labelled chromosomes of raspberry origin from those of blackberry origin in allopolyploid hybrid plants. The raspberry chromosomes were labelled by GISH at their centromeres, and 1 chromosome was also labelled over the short arm. In one allopentaploid plant a chromosome carried a terminal signal. Karyotype analysis indicated that this is a blackberry chromosome carrying a raspberry translocation. GISH analysis of an aneuoctaploid blackberry cv ‘Aurora’ (2n=8x=58) showed that both whole and translocated raspberry chromosomes were present. The basic Rubus genome has one ribosomal DNA (rDNA) locus, and in all but one case all levels of ploidy had the expected multiples of rDNA loci. Interestingly, in the blackberry cv ‘Aurora’, there were only six sites, two less than might be predicted from its aneuoctaploid chromosome number. Our results highlight the potential of GISH and FISH for genomic designation, physical mapping and introgression studies in Rosaceous fruit crops. Received: 20 February 1998 / Accepted: 12 May 1998     

An improved technique for obtaining well-spread metaphases from plants with numerous large chromosomes

Preparations that contain well-spread metaphase chromosomes are critical for plant cytogenetic analyses including chromosome counts, banding procedures, in situ hybridization, karyotyping and construction of ideograms. Chromosome spreading is difficult for plants with large and numerous chromosomes. We report here a technique for obtaining cytoplasm-free, well-spread metaphases from two Amaryllidaceae species: Sprekelia formosissima (2n = 120) and Hymenocallis howardii (2n = 96). The technique has three main steps: 1) pretreatment to cause chromosome condensation, 2) dripping onto tilted slides coated with a thin layer of pure acetic acid and 3) application of steam and acetic acid to produce cytoplasmic hydrolysis, which spreads the chromosomes.     

Loss of nucleolar organizer regions during chromosomal evolution in the South American cricetid Graomys griseoflavus

Graomys griseoflavus is a South American phyllotine rodent having a remarkable Robertsonian polymorphism which may have produced reproductive isolation between 2n=42–41 and 2n=38–34 karyomorphs. Analysis of nucleolar organizer region (NOR) locations both by silver staining (Ag-NOR) and in situ hybridization revealed that 2n=42 individuals exhibit highly variable Ag-NOR patterns, while specimens of the 2n=38–34 karyomorphic group showed a single Ag-NOR pattern. The latter animals underwent two NOR deletions in reference to the 2n=42 karyomorphs, one of which would be the consequence of a Robertsonian fusion and the other would be produced by the unequal crossing-over mechanism. The differential NOR homogenization supports the hypothesis that G. griseoflavus karyomorphs are evolving separately towards the acquisition of separate species status.     

Chromosome study of hybrid and gynogenetic offspring of artificial crosses between members of the catfish families Clariidae and Pangasiidae

Synopsis Artificial hybridization between species of the catfish families Clariidae (Clarias macrocephalus) and Pangasiidae (Pangasius sutchi) resulted in first generation offspring comprising two intermediate morphotypes and one morphotype indistinguishable from its clariid parent. The two intermediate morphotypes apparently correspond to hybrid morphotypes 2 and 4 resulting from earlier hybridization experiments between the same parent species (Tarnchalanukit 1986). We did not obtain morphotypes 1 and 3. Chromosome spreads of the relatively pangasiid-like morphotype 2 reveal a diploid number of 57, presumably comprising one set of Clarias (n=27) and one of Pangasius (n=30) chromosomes. The relatively clariid-like morphotype 4 is a triploid with 84 chromosomes, presumably comprising two sets from Clarias (2n=54) and one from Pangasius (n=30). Finally, the morphotype indistinguishable from Clarias, is a diploid with 54 chromosomes, apparently arising from gynogenesis.     

Characterisation of the double genome structure of modern sugarcane cultivars (Saccharum spp.) by molecular cytogenetics

Cultivated sugarcane clones (Saccharum spp., 2n=100 to 130) are derived from complex interspecific hybridizations between the speciesS. officinarum andS. spontaneum. Using comparative genomic DNA in situ hybridization, we demonstrated that it is possible to distinguish the chromosomes contributed by these two species in an interspecific F1 hybrid and a cultivated clone, R570. In the interspecific F1 studied, we observed n+n transmission of the parental chromosomes instead of the peculiar 2n+n transmission usually described in such crosses. Among the chromosomes of cultivar R570 (2n=107–115) about 10% were identified as originating fromS. spontaneum and about 10% were identified as recombinant chromosomes between the two speciesS. officinarum andS. spontaneum. This demonstrated for the first time the occurrence of recombination between the chromosomes of these two species. The rDNA sites were located by in situ hybridization in these two species and the cultivar R570. This supported different basic chromosome numbers and chromosome structural differences between the two species and provided a first bridge between physical and genetical mapping in sugarcane.     

Comparative cytogenetics of six Indo‐Pacific moray eels (Anguilliformes: Muraenidae) by chromosomal banding and fluorescence in situ hybridization

A comparative cytogenetic analysis, using both conventional staining techniques and fluorescence in situ hybridization, of six Indo‐Pacific moray eels from three different genera (Gymnothorax fimbriatus, Gymnothorax flavimarginatus, Gymnothorax javanicus, Gymnothorax undulatus, Echidna nebulosa and Gymnomuraena zebra), was carried out to investigate the chromosomal differentiation in the family Muraenidae. Four species displayed a diploid chromosome number 2n = 42, which is common among the Muraenidae. Two other species, G. javanicus and G. flavimarginatus, were characterized by different chromosome numbers (2n = 40 and 2n = 36). For most species, a large amount of constitutive heterochromatin was detected in the chromosomes, with species‐specific C‐banding patterns that enabled pairing of the homologous chromosomes. In all species, the major ribosomal genes were localized in the guanine‐cytosine‐rich region of one chromosome pair, but in different chromosomal locations. The (TTAGGG)_n telomeric sequences were mapped onto chromosomal ends in all muraenid species studied. The comparison of the results derived from this study with those available in the literature confirms a substantial conservation of the diploid chromosome number in the Muraenidae and supports the hypothesis that rearrangements have occurred that have diversified their karyotypes. Furthermore, the finding of two species with different diploid chromosome numbers suggests that additional chromosomal rearrangements, such as Robertsonian fusions, have occurred in the karyotype evolution of the Muraenidae.