Molecular cytogenetics and characterization of a ZZ/ZW sex chromosome system in Triportheus nematurus (Characiformes, Characidae)

Chromosomes of Triportheus nematurus, a fish species from family Characidae, were analyzed in order to establish the conventional karyotype, location of C-band positive heterochromatin, Ag-NORs, GC- and AT-rich sites, and mapping of 18S and 5S rDNA with fluorescence in situ hybridization (FISH). The diploid number found was 2n = 52 chromosomes in both males and females. However, the females presented a pair of differentiated heteromorphic chromosomes, characterizing a ZZ/ZW sex chromosome system. The Z chromosome was metacentric and the largest one in the karyotype, bearing C-positive heterochromatin at pericentromeric and telomeric regions. The W chromosome was middle-sized submetacentric, appearing mostly heterochromatic after C-banding and presenting heterogeneous heterochromatin composed of GC- and AT-rich regions revealed by fluorochrome staining. Ag-NORs were also GC-rich and surrounded by heterochromatic regions, being located at the secondary constriction on the short arms of the second chromosome pair, in agreement with 18S rDNA sites detected with FISH. The 18S and 5S rDNA were aligned in tandem, representing an uncommon situation in fishes. The results obtained reinforce the basal condition of the ZZ/ZW sex system in the genus Triportheus, probably arisen prior to speciation in the group.     

Chromosome banding in amphibia. XXIII. Giant W sex chromosomes and extremely small genomes in Eleutherodactylus euphronides and Eleutherodactylus shrevei (Anura,Leptodactylidae)

Highly differentiated, heteromorphic ZZ female symbol /ZW male symbol sex chromosomes were found in the karyotypes of the neotropical leptodactylid frogs Eleutherodactylus euphronides and E. shrevei. The W chromosomes are the largest heterochromatic, female-specific chromosomes so far discovered in the class Amphibia. The analyses of the banding patterns with AT- and GC base-pair specific fluorochromes show that the constitutive heterochromatin in the giant W chromosomes consists of various categories of repetitive DNA sequences. The W chromosomes of both species are similar in size, morphology and banding patterns, whereas their Z chromosomes exhibit conspicuous differences. In the cell nuclei of female animals, the W chromosomes form very prominent chromatin bodies (W chromatin). DNA flow cytometric measurements demonstrate clear differences in the DNA content of male and female erythrocytes caused by the giant W chromosome, and also shows that these Eleutherodactylus genomes are among the smallest of all amphibian genomes. The importance of the heteromorphic ZW sex chromosomes for the study of Z-linked genes, the similarities and differences of the two karyotypes, and the significance of the exceptionally small genomes are discussed.     

An early stage of ZW/ZZ sex chromosome differentiation in Poecilia sphenops var. melanistica (Poeciliidae,Cyprinodontiformes)

The mitotic and meiotic chromosomes of the American cyprinodont fish Poecilia sphenops var. melanistica were analysed. All 46 chromosomes are telocentric. By specific staining of the constitutive heterochromatin with C-banding and various AT-specific fluorochromes, the homomorphic chromosome pair 1 could be identified as sex chromosomes of the ZW/ZZ type. All female animals exhibit a W chromosome with a large region of telomeric heterochromatin that is not present in the Z chromosome. These sex chromosomes cannot be distinguished by conventional staining; they represent the first demonstration of sex chromosomes in fishes in an early stage of morphological differentiation. The W heterochromatin and the telomeric heterochromatin in the two autosomes 18 show a very bright fluorescence when stained with AT-specific fluorochromes. This allows the direct identification of the chromosomal sex by examining the interphase nuclei: females exhibit three, males only two brightly fluorescent heterochromatic chromocenters in their nuclei. The significance of these ZW/ ZZ sex chromosomes and their specific DNA sequences, the dose compensation of the Z-linked genes, and the experimental possibilities using sex-reversed ZW males are discussed.     

Evidence for a highly differentiated sex chromosome heteromorphism in the salamander Necturus maculosus (Rafinesque)

The mitotic chromosomes of the neotenic (sensu Gould, 1977, and Alberch et al., 1979) salamander Necturus maculosus (Rafinesque) have been examined using a C-band technique to demonstrate the distribution of heterochromatin. The C-banded mitotic chromosomes provide evidence of a highly differentiated XY male/XX female sex chromosome heteromorphism, in which the X and Y chromosomes differ greatly in size and morphology, and in the amount and distribution of C-band heterochromatin. The X chromosome represents one of the largest biarmed chromosomes in the karyotype and is indistinguishable from similar sized autosomes on the basis of C-band heterochromatin. The Y chromosome, on the other hand, is diminutive, morphologically distinct from all other chromosomes of the karyotype, and is composed almost entirely of C-band heterochromatin. The discovery of an X/Y chromosome heteromorphism in this species is consistent with the observation by King (1912) of a heteromorphic spermatogenic bivalent. Karyological and phylogenetic implications are discussed.     

Differentiation of repetitive DNA sites and sex chromosome systems reveal closely related group in Parodontidae (Actinopterygii: Characiformes)

Parodon and Apareiodon lack sufficiently consistent morphological traits to be considered a monophyletic group in Parodontidae. Species within this family are either sex-homomorphic or sex-heteromorphic (i.e., lacking a differentiated sex chromosome system, ZZ/ZW or ZZ/ZW(1)W(2)). In this study, a DNA fragment from the heterochromatin segment of the W chromosome of Apareiodon ibitiensis (named WAp) was microdissected and used for in situ mapping of nine Parodontidae species. The species were also characterized using a satellite DNA probe (pPh2004). The species were phylogenetically clustered according to 17 characters, which were examined by both classical and molecular cytogenetic techniques. Given the present results, the single ZZ/ZW sex chromosome system seems to have been derived from a paracentric inversion of a terminal WAp site onto the proximal regions of the short arms of a metacentric chromosome pair, followed by WAp site amplification. We reason that these events restrained recombination and favored differentiation of the W chromosome in some species. Moreover, co-hybridization experiments targeting the WAp and pPh2004 repetitive DNA sites of A. affinis suggest that the ZZ/ZW(1)W(2) sex chromosomes of this species may have arisen from a translocation between the proto-sex chromosome and an autosome. Our phylogenetic analysis corroborates the hypothesis of sex chromosome differentiation and establishes groups of closely related species. The phylogenetic reorganization in response to these new data supports the presence of internal monophyletic groups within Parodontidae.     

Diverse stages of sex-chromosome differentiation in tinamid birds: evidence from crossover analysis in <Emphasis Type="Italic">Eudromia elegans</Emphasis> and <Emphasis Type="Italic">Crypturellus tataupa</Emphasis>

All extant birds share the same sex-chromosome system: ZZ males and ZW females with striking differences in the stages of sex-chromosome differentiation between the primitive palaeognathus ratites and the large majority of avian species grouped within neognaths. Evolutionarily close to ratites is the neotropical order Tinamiformes that has been scarcely explored regarding their ZW pair morphology and constitution. Tinamous, when compared to ratites, constitute a large group among Palaeognathae, therefore, exploring the extent of homology between the Z and W chromosomes in this group might reveal key features on the evolution of the avian sex chromosomes. We mapped MLH1 foci that are crossover markers on pachytene bivalents to determine the size and localization of the homologous region shared by the Z and W chromosomes in two tinamous: Eudromia elegans and Crypturellus tataupa. We found that the homologous (pseudoautosomal) region differ significantly in size between these two species. They both have a single recombination event on the long arm of the acrocentric Z and W chromosomes. However, in E. elegans the pseudoautosomal region occupies one-fourth of the W chromosome, while in C. tataupa it is restricted to the tip of the long arm of the W. The W chromosomes in these two species differ in their heterochromatin content: in E. elegans it shows a terminal euchromatic segment and in C. tataupa is completely heterochromatic. These results show that tinamous have ZW pairs with more diversified stages of differentiation compared to ratites. Finally, the idea that the avian proto-sex chromosomes started to diverge from the end of the long arm towards the centromere of an acrocentric pair is discussed.     

Triploidy in Hochstetter's frog,Leiopelma hochstetteri,from New Zealand

Abstract

Autotriploidy is described in a female of the endemic New Zealand frog Leiopelma hochstetteri. This frog was found to have 3n=33 chromosomes plus 2 supernumerary chromosomes. All the chromosomes in the karyotype of this species contained C-band heterochromatin at the centromeres. A prominent C-band was found to be associated with a secondary constriction on chromosome no. 7. The supernumerary chromosomes in this species appear to be mitotically stable and contain C-band heterochromatin at the centromeres. From the limited data presently available, the triploid individual may have resulted from the fertilisation of a diploid egg produced when the second meiotic division had been suppressed.     

Highly differentiated and conserved sex chromosome in fish species (Aulopus japonicus: Teleostei, Aulopidae)

While highly differentiated and long-conserved sex chromosomes such as XY and ZW chromosomes are observed, respectively, in mammalian and avian species, no counterparts to such chromosomes were observed in fish until we reported in the previous study that well-conserved and highly differentiated ZW sex chromosomes existed in the family of Synodontidae. Then, the problem was if the evolutionary history of the fish ZW chromosomes was long enough to be comparable to the mammalian and avian counterparts. To tackle the problem, we had to extend our finding of the fish sex chromosomes further than a family alone. For this purpose, we chose Aulopus japonicus that belonged to one of the related families to Synodontidae.Our cytogenetic and fluorescence in situ hybridization (FISH) analyses have clearly demonstrated that A. japonicus also has ZW chromosomes. We have also found that 5S rDNA clusters are located on the Z and W chromosomes in this species. Using nontranscribed intergenic sequences in the 5S rDNA clusters as PCR primers, we successfully amplified a 6-kb-long female-specific sequence on the W chromosome. The 6-kb-long sequence contained one transposable element and two tRNA sequences. The function of the sequence remains to be studied. Our Southern blot analysis confirmed that the 6-kb sequence was located only on the W chromosome.Therefore, it is now said that highly differentiated ZW chromosomes have been conserved over two fish families. As these families were reported to have been diverged 30-60 million years ago, the fish ZW chromosomes have an evolutionary history corresponding to the history of the families. This is perhaps the first case that fish sex chromosomes are shown to have such a long evolutionary lineage.     

A Novel ZZ/ZW Sex Chromosome System for the Genus Leporinus (Pisces, Anostomidae, Characiformes)

A wide range of sex chromosome mechanisms, including simple and multiple chromosome systems is characteristic of fishes. The Leporinus genus represent a good model to study sex chromosome mechanisms, because an unambiguous ZZ/ZW sex chromosome system was previously described for seven species, while the remaining studied species of the genus do not show differentiated sex chromosomes. The occurrence of sex chromosomes in Leporinus trifasciatus and Leporinus sp2 from the Araguaia river, Amazon basin, Brazil, was here investigated. ZZ/ZW sex chromosomes were detected for both species. The Z and W chromosome morphology of L. trifasciatus is the same as described for other species of the genus Leporinus. However, the Z and W chromosomes of L. sp2 were quite different in their morphology and banding pattern suggesting that the ZW system of this species have originated independently from the ZW system previously described for other Leporinus.     

Sister chromatid exchange points in the heterochromatin and euchromatin regions of Chinese hedgehog chromosomes

Summary The Chinese hedgehog has a diploid chromosome number of 48 in which there are eleven pairs of telo- or subtelocentric autosomes, twelve pairs of meta- or submetacentric autosomes, a metacentric X chromosome and a telocentric Y chromosome. The heterochromatin is almost completely distributed in five large distal segments of chromosomes nos. 9 to 12 and no. 18. There is no positive C-band in the centromeres of the chromosomes except for the X chromosome which has a small, weakly stained C-band in the centromere. In Chinese hedgehog cells 52.1% of SCEs are found at the junction between the euchromatin and the heterochromatin, 39.5% in the heterochromatin and 8.4% in the auchromatin. The SCE number per unit C-band is double the SCE number per unit euchromatin. The SCE rate in the heterochromatin or euchromatin regions is not proportional to their chromosome length and can be quite different between different pairs of the chromosomes. Our results indicate that there is a non-uniform distribution of the SCEs in the Chinese hedgehog cells.     

Karyotype of an avian schistosome Trichobilharzia szidati (Digenea: Schistosomatidae)

Somatic chromosomes of Trichobilharzia szidati Neuhaus, 1952 are described from larval stages dissected from snoils, air-dried on slides and stained with Giemsa and C-banding technique, used for the first time in Trichobilharzia sp. The karyotype consisted of 7 autosomal pairs and 1 pair of sex chromosomes, ZZ in the male and ZW in the female, where Z and W chromosomes are of different sizes and both are classified as submetaceatric. C-banding aided in identification of chromosomes Nos 4, satellited 6 and 8. No heterochromatin was observed in the W chromosome. The results were not in agreement with those previously reported and represent new findings. The possible explanation for this fact is given.     

Equilocality of heterochromatin distribution and heterochromatin heterogeneity in acridid grasshoppers

Comparative fluorescence studies on the chromosome of ten species of acridid grasshoppers, with varying amounts and locations of C-band positive heterochromatin, indicate that the only regions to fluoresce differentially are those that C-band. Within a given species there is a marked tendency for groups of chromosomes to accumulate heterochromatin with similar fluorescence behaviour at similar sites. This applies to all three major categories of heterochromatin — centric, interstitial and telomeric. Different sites within the same complement, however, tend to have different fluorescence properties. In particular, centric C-bands within a given species are regularly distinguishable in their behaviour from telomeric C-bands. Different species, on the other hand, may show distinct forms of differential fluorescence at equilocal sites. These varying patterns of heterochromatin heterogeneity, both within and between species, indicate that whatever determines the differential response to fluorochromes has tended to operate both on an equilocal basis and in a concerted fashion. This is reinforced by the fact that structural rearrangements that lead to the relocation of centric C-bands, either within or between species, may also be accompanied by a change in fluorescence behaviour.We dedicate this paper, with affection, to Professor Hans Bauer on the occasion of his 80th birthday, and in appreciation of his singular contribution to the study of chromosomes     

The contrasting role of heterochromatin in the differentiation of sex chromosomes: an overview from Neotropical fishes

During the evolutionary process of the sex chromosomes, a general principle that arises is that cessation or a partial restriction of recombination between the sex chromosome pair is necessary. Data from phylogenetically distinct organisms reveal that this phenomenon is frequently associated with the accumulation of heterochromatin in the sex chromosomes. Fish species emerge as excellent models to study this phenomenon because they have much younger sex chromosomes compared to higher vertebrates and many other organisms making it possible to follow their steps of differentiation. In several Neotropical fish species, the heterochromatinization, accompanied by amplification of tandem repeats, represents an important step in the morphological differentiation of simple sex chromosome systems, especially in the ZZ/ZW sex systems. In contrast, multiple sex chromosome systems have no additional increase of heterochromatin in the chromosomes. Thus, the initial stage of differentiation of the multiple sex chromosome systems seems to be associated with proper chromosomal rearrangements, whereas the simple sex chromosome systems have an accumulation of heterochromatin. In this review, attention has been drawn to this contrasting role of heterochromatin in the differentiation of simple and multiple sex chromosomes of Neotropical fishes, highlighting their surprising evolutionary dynamism.     

C-band polymorphisms in exotic inbred strains of mice: a method for mapping centromeric ends of chromosomes.

The major satellite DNA of Mus musculus appears as a pericentromeric heterochromatin block in all chromosomes but the Y. While C-banding readily reveals the presence of this heterochromatin block, there is considerable polymorphism in C-band size among the chromosomes and among different subspecies. We have studied the distribution of C-band size differences in the chromosomes of 15 exotic inbred laboratory strains and substrains derived from wild populations of different subspecies of M. musculus. The variation in C-band size among these inbred strains can serve as a useful codominant cytological marker for estimating recombinational distances between the centromere and proximal genes in linkage crosses.     

Differentiated ZZ/ZW sex chromosomes in Apareiodon ibitiensis (Teleostei,Parodontidae): cytotaxonomy and biogeography

Conventional and molecular chromosomal analyses were carried out on three populations of Apareiodon ibitiensis sampled from the hydrographic basins of the São Francisco River and Upper Paraná River (Brazil). The results reveal a conserved diploid number (2n = 54 chromosomes), a karyotype formula consisting of 50 m‐sm + 4st and a ZZ/ZW sex chromosome system that has not been previously identified for the species. C‐banding analysis with propidium iodide staining revealed centromeric and terminal bands located in the chromosomes of the specimens from the three populations and allowed the identification of heteromorphism of heterochromatin regions in the Z and W chromosomes. The number of 18S sites located through fluorescent in situ hybridization (FISH) varied between the populations of the São Francisco and Upper Paraná Rivers. The location of 5S rDNA sites proved comparable in one pair of metacentric chromosomes. Thus, the present study proposes a ZZ/ZW sex chromosome system for A. ibitiensis among the Parodontidae, and a hypothesis is presented regarding possible W chromosome differentiation stages in this species through DNA accumulation, showing geographical variations for this characteristic, possibly as a consequence of geographical reproductive isolation.     

Heterochromatin organization in the nucleus of Indian muntjac (Muntiacus muntjak)

The heterochromatin in Indian muntjac (Muntiacus muntjak) is located at the periphery of primary constrictions of all the chromosomes. The X chromosome contains significantly larger amounts of heterochromatin than the rest of the complement by C-banding technique. However, the small portion of C-band region was found to be resistant by restriction endonuclease HaeIII (5'...GG decreases CC...3') and was clearly visible on the nucleus. Therefore, the position of this large heterochromatic segment is examined at somatic metaphases. The distribution of the heterochromatin of the X chromosome observed in Indian muntjac is contrary to the general pattern observed in other species, i.e., the chromosomes consisting greater amount of heterochromatin are located more peripherally than those with lesser amount. However, the smaller Y chromosome (Y1) is frequently found at the periphery. The present findings suggest that the role of heterochromatin organization in the nucleus vary between different heterochromatic segments of the same species and vary from species to species.     

In situ nick translation distinguishes between C-band positive regions on mouse chromosomes

In situ nick translation of mouse metaphase chromosomes by non-radioactive detection means and DNase I digestion followed by Giemsa staining were used to analyse the DNase I resistance of two different C-band positive regions. These were the centromeric heterochromatin of aero- and metacentric chromosomes and an interstitial C- band on chromosome 1 of wild mice, IS(HSR;1C5D)1Lub. Whereas the centromeric heterochromatin was clearly resistant to DNase I, the interstitial C-band showed very high DNase I sensitivity. Among centromeric C-bands, the heterochromatin in Robertsonian fusion biarmed chromosomes was more resistant to DNase I action than was the centromeric heterochromatin of the acrocentric chromosomes.     

A new approach in recognition of heterochromatic regions of human chromosomes by means of restriction endonucleases.

The heteromorphisms of C-band regions of human chromosomes are evaluated by means of restriction endonucleases AluI, DdeI, MboI, and RsaI. Every chromosome exhibits heteromorphic markers of the C-band regions except chromosome 8. Each enzyme was found to be highly characteristic in its staining profile, a result that clearly suggests the diversity of heterochromatin. The inherent C-band-region heterochromatin variability that is revealed by these enzymes provides a valuable tool in identifying markers as compared with other previously described techniques.     

Length of human C-bands in relation to the degree of chromosome condensation

Summary The C-band length of human chromosome 1 in prophase and prematurely condensed interphase chromosomes is relatively shorter than in metaphase chromosomes. However, even in chromosomes with the same degree of contraction the absolute length of the C-band varies considerably. This allocyclic behaviour of human constitutive heterochromatin has to be kept in mind if C-bands of different individuals are compared.Sponsored by the Deutsche Forschungsgemeinschaft (Sp 144)