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.     

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.     

Evolutionary aspects of the ZZ/ZW sex chromosome system in the Characidae fish,genus Triportheus. A monophyletic state and NOR location on the W chromosome

Four species/populations of Triportheus, T. guentheri, T. cf. elongatus and T. paranense from different Brazilian hydrographic basins, were studied cytogenetically. All the species showed a similar karyotypic macrostructure, with a diploid chromosome number 2n = 52 and a ZZ/ZW sex chromosome system. Besides silver- and fluorochrome-staining, the chromosome mapping of 18S rDNA was also investigated using a biotinylated probe. In spite of some variation in the number of the NORs, a major chromosome site was always present on the short arm of an autosomal pair. In addition, a characteristic rDNA site was also observed on the telomeric region of the W chromosome in the four species/populations. In Triportheus differential reduction in size and heterochromatin accumulation appear to be the main processes associated with the evolution of the sex W chromosome. The location of rRNA genes on this chromosome may correspond to a plesiomorphic condition in the genus and, if so, predates to the sex chromosome system differentiation, with a possible influence in the initial steps of this process.     

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.     

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.     

广义烙铁头属三种烙铁头的核型及分类地位初步探讨

报道３种烙铁头蛇的核型。其中,烙铁头２ｎ＝３６＝１６Ｍ（１４Ｖ＋２ＳＶ／Ｖ）＋２０ｍ,ＺＷ型性决定,Ｚ为Ｖ型,Ｗ为ＳＩ／ＳＶ型,Ｚ明显大于Ｗ;菜花烙铁头２居群２ｎ＝３６＝１６Ｍ（１４Ｖ＋２ＳＩ）＋２０ｍ,ＺＷ型性决定,Ｚ为Ｖ型,Ｗ为ＳＩ型,Ｚ明显大于Ｗ;云南竹叶青２ｎ＝３６＝１６Ｍ（１２Ｖ／ＳＶ＋２ＳＶ／ＳＩ＋２ＳＩ）＋２ｍ,无异型性染本,对３种的核型及烙铁头属已知核型进行了比较分析,并对云南竹叶     

Evolutionary and biogeographical implications of the karyological variations in the oviparous and viviparous forms of the lizard Lacerta (Zootoca) vivipara

The lizard Lacerta ( Zootoca ) vivipara has two modes of reproduction and is variable karyologically. We describe its karyological variation from literature data and from new data on two viviparous populations from France, on two oviparous populations from the Pyrenees in south-western France and on three oviparous populations recently discovered in Slovenia. Males have 36 chromosomes, whereas females have only 35 chromosomes in all viviparous populations and in the Pyrenean oviparous populations. This karyotype has been interpreted to result from a fusion of an ancestral sexual W chromosome with an autosome from the Zl or from the Z2 pair. The karyotype formula is 32 autosomes + ZIZ2W for the female and 32 autosomes + Z1Z1Z2Z2 for the male. The karyotype of the Slovenian oviparous populations, 34 autosomes + ZW in the male and 34 autosomes +ZW in the female, represents an evolutionary stage that preceded the chromosomal fusion. There is minor karyological variation, mainly concerning the W and Z2 chromosomes, within the Pyrenean oviparous populations. This parallels the geographic variation of the W-linked alleles of the MPI enzyme and suggests that allopatric differentiation of these oviparous populations might have occurred in the vicinity of the Pyrenees during the Pleistocene.
The viviparous populations from western Europe carry a metacentric W chromosome, whereas oviparous populations from south-western Europe and eastern viviparous populations both show an acrocentrie, or a subtelocentrie. W chromosome. This suggests that the acrocentric-subtelocentric W is a primitive character and that viviparity probably arose in an eastern lineage of the species.     

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.     

Highly Differentiated ZW Sex Microchromosomes in the Australian Varanus Species Evolved through Rapid Amplification of Repetitive Sequences

Transitions between sex determination systems have occurred in many lineages of squamates and it follows that novel sex chromosomes will also have arisen multiple times. The formation of sex chromosomes may be reinforced by inhibition of recombination and the accumulation of repetitive DNA sequences. The karyotypes of monitor lizards are known to be highly conserved yet the sex chromosomes in this family have not been fully investigated. Here, we compare male and female karyotypes of three Australian monitor lizards, Varanus acanthurus, V. gouldii and V. rosenbergi, from two different clades. V. acanthurus belongs to the acanthurus clade and the other two belong to the gouldii clade. We applied C-banding and comparative genomic hybridization to reveal that these species have ZZ/ZW sex micro-chromosomes in which the W chromosome is highly differentiated from the Z chromosome. In combination with previous reports, all six Varanus species in which sex chromosomes have been identified have ZZ/ZW sex chromosomes, spanning several clades on the varanid phylogeny, making it likely that the ZZ/ZW sex chromosome is ancestral for this family. However, repetitive sequences of these ZW chromosome pairs differed among species. In particular, an (AAT)n microsatellite repeat motif mapped by fluorescence in situ hybridization on part of W chromosome in V. acanthurus only, whereas a (CGG)n motif mapped onto the W chromosomes of V. gouldii and V. rosenbergi. Furthermore, the W chromosome probe for V. acanthurus produced hybridization signals only on the centromeric regions of W chromosomes of the other two species. These results suggest that the W chromosome sequences were not conserved between gouldii and acanthurus clades and that these repetitive sequences have been amplified rapidly and independently on the W chromosome of the two clades after their divergence.     

Satellite DNA and evolution of sex chromosomes

The satellite DNA (satellite III) which is mainly represented in the female of Elaphe radiata (Ophidia, Colubridae) has been isolated and its buoyant density has been determined (=1.700 g cm^–3). In situ hybridisation of radioactive complementary RNA of this satellite DNA with the chromosomes of different species has revealed that it is mainly concentrated on the W sex chromosome and its sequences are conserved throughout the sub-order Ophidia. From hybridisation studies these sequences are absent from the primitive family Boidae which represents a primitive state of differentiation of sex chromosomes. Chromosome analysis and C-banding have also revealed the absence of heteromorphism and of an entirely heterochromatic chromosome in the species belonging to the primitive family and their presence in the species of highly evolved families. It is suggested that the origin of satellite DNA (satellite III) in the W chromosome is the first step in differentiation of W from the Z in snakes by generating asynchrony in the DNA replication pattern of Z and W chromosomes and thus conceivably reducing the frequency of crossing-over between them which is the prerequisite of differentiation of sex chromosomes. Presence of similar sex chromosome associated satellite DNA in domestic chicken suggests its existence in a wider range of vertebrates than just the snakes.     

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.     

Sex and death in birds: a model of dosage compensation that predicts lethality of sex chromosome aneuploids

Birds show female heterogamety, with ZZ males and ZW females. It is still not clear whether the W is female-determining, or whether two doses of the Z chromosomes are male-determining, or both. This question could easily be settled by the sexual phenotypes of ZZW and ZO birds, in the same way that the sexual phenotypes of XXY and XO showed that the Y is male determining in humans, but that the dosage of an X-borne gene determines sex in Drosophila. However, despite extensive searches, no ZZW or ZO diploid birds have been satisfactorily documented, so we must assume that these genotypes are embryonic lethals. Given that ZW and ZZ are viable and the W contains few genes it is not clear why this should be so. Here I propose that sex chromosome aneuploids are lethal in chicken because, to achieve dosage compensation, a locus on the W chromosome controls the upregulation of genes on the Z in ZW females. ZO birds would therefore have only half the normal dose of Z-linked gene product and ZZW would have twice the amount, both of which would undoubtedly be incompatible with life. Reports of other aneuploids and triploids are also consistent with this hypothesis.     

Origin and Differentiation of a Sex Chromosome System in Parodon hilarii (Pisces, Parodontidae). Satellite DNA, G- and C-banding

A satellite DNA sequence of Parodon hilarii (named pPh2004) was isolated, cloned and sequenced. This satellite DNA is composed of 200 bp, 60% AT rich. In situ hybridization (FISH) results revealed that the satellite DNA pPh2004 is located in the terminal regions of several chromosomes, forming highly evident blocks in some and punctual marks in others. The comparison between the FISH and C-banding results showed that the location of this satellite DNA coincides with that of most terminal heterochromatins. However, some regions are only marked by FISH whereas other regions are only marked by C-banding. The possible existence of more than one satellite DNA family could explain these partial differences. The in situ hybridization with the satellite DNA and the G- and C-bandings confirmed the presence of a sex chromosome system of the ZZ/ZW type in P. hilarii, as well as the correct identification of the Z chromosome in the karyotype. This chromosome displays a segment of terminal heterochromatin in the long arm, similar to the segment observed in the short arm of the W chromosome, also showing a G-banding pattern similar to that of the short arm and part of the long arm of the W chromosome. A hypothesis on the origin of the W chromosome from an ancestral chromosome similar to the Z chromosome is presented.     

Evolution of sex-chromosomes and formation of W-chromatin in snakes

The analysis of sex-chromosome complexes and formation of W-chromatin in 16 species of snakes of the families Boidae, Colubridae, Elapidae, and Hydrophiidae, reveal three very pertinent facts. First, the snakes exhibit various states of the differentiation of the Z and W chromosomes, apparently according to the evolutionary status of the families, being homomorphic in primitive families and well differentiated in highly evolved ones. Second, the demonstration of a heteropycnotic body in the interphase nuclei of the families of a large number of species of snakes has definitely shown that the nuclear sexing is possible not only in those species of snakes where the W chromosome is morphologically distinguishable from the Z, but also in those species where it is not so, but shows an asynchrony in the replicating pattern of W. It is suggested that development of allocycly rather than establishment of structural changes is the first step in the differentiation of the W from the Z in snakes. Third, the absence of coexistence of nucleolus and W-chromatin in a condensed state in the interphase nuclei of different tissues in a few species of snakes reported in this paper suggests that the W-chromatin is responsible for the synthesis of the nucleolus in these snakes.Paper presented at the Third Oxford Chromosome Conference, September, 1970.     

Heterosynapsis and axial equalization of the sex chromosomes of the northern bobwhite quail.

The pairing behavior of the Z and W chromosomes in the female northern bobwhite quail (Colinus virginianus) was analyzed by electron microscopy of silver-stained synaptonemal complexes (SCs). After autosomal pairing was completed, synapsis of the sex chromosomes initiated at the short-arm end of the W chromosome and one end of the Z chromosome. Synapsis then progressed unidirectionally, producing a sex bivalent in which the entire length of the W axis was paired with an equivalent length of the Z axis. Progressive contraction and asymmetrical twisting of the Z axis ultimately resulted in a fully paired configuration with aligned axial ends. Further contraction of the Z axis reduced the extent of asymmetrical twisting such that only the nonaligned centromeric regions distinguished the SC of the ZW bivalent from SCs of similar-sized autosomes in late-pachytene nuclei. Quantitative analyses indicated that the length of the Z axis shortened significantly during the adjustment process, whereas no significant difference occurred in the length of the W axis. The nonalignment of the centromeric regions during transitional stages of ZW synapsis indicates that direct heterosynapsis of nonhomologous segments, followed by axial equalization of the length inequality, is responsible for the length adjustment during synapsis in the sex chromosomes of the bobwhite quail.     

Cytogenetics of the Javan file snake (Acrochordus javanicus) and the evolution of snake sex chromosomes

Advanced snakes (Caenophidia) are an important group including around 90% of the recent species of snakes. The basal splitting of the clade is still rather controversial, and it is not fully understood when the differentiation of sex chromosomes started in snake evolution. To help resolve these questions, we performed cytogenetic analysis on the Javan file snake, also known as the elephant trunk snake (Acrochordus javanicus) from the family Acrochordidae, which occupies an informative phylogenetic position. For the first time for acrochordids, we identified heteromorphic ZZ/ZW sex chromosomes with a highly heterochromatic W chromosome. These traits are likely synapomorphies of advanced snakes. In contrast to other caenophidian snakes, the Javan file snake lacks an accumulation of Bkm repeats and interstitial telomeric repeats on the W chromosome. This observation supports the sister group relationship between acrochordids and all other caenophidian snakes including the family Xenodermatidae and questions the suggested role of Bkm repeats in the formation of sex heterochromatin in snakes. The revealed partial gene content of the Z chromosome in acrochordids supports the hypothesis that the progressive degeneration of the W chromosome commenced in snakes before the basal split of Caenophidia, albeit its evolutionary rate in file snakes might be slower than in their sister lineage.