Variable organization of repeats and hidden diversity of XY sex chromosomes in Pentatomidae true Bugs (Hemiptera) revealed through comparative genomic hybridization.

Relationships between Vertebrate ZW and XY Sex Chromosome Systems

Background: Species with ‘young’ or nascent sex chromosomes provide unique opportunities to understand early evolutionary mechanisms (e.g. accumulation of repetitive sequences, cessation of recombination and gene loss) that drive the evolution of sex chromosomes. Among vertebrates, fishes exhibit highly diverse and a wide spectrum of sex-determining mechanisms and sex chromosomes, ranging from cryptic to highly differentiated ones, as well as, from simple to multiple sex chromosome systems. Such variability in sex chromosome morphology and composition not only exists within closely related taxa, but often within races/populations of the same species. Inside this context, the wolf fish Hoplias malabaricus offers opportunity to investigate the evolution of morphologically variable sex chromosomes within a species complex, as homomorphic to highly differentiated sex chromosome systems occur among its different karyomorphs.Materials & Methods: To discover various evolutionary stages of sex chromosomes and to compare their sequence composition among the wolf fish´s karyomorphs, we applied multipronged molecular cytogenetic approaches, including C-banding, repetitive DNAs mapping, Comparative Genomic Hybridization (CGH) and Whole Chromosomal Painting (WCP). Our study was able to characterize a cryptically differentiated XX/XY sex chromosome system in the karyomorph F of this species.Conclusion: The Y chromosome was clearly identified by an interstitial heterochromatic block on the short arms, primarily composed of microsatellite motifs and retrotransposons. Additionally, CGH also identified a male specific chromosome region in the same chromosomal location, implying that the accumulation of these repeats may have initiated the Y chromosome differentiation, as well as played a critical role towards the evolution and differentiation of sex chromosomes in various karyomorphs of this species.

BackgroundThe Characidium (a Neotropical fish group) have a conserved diploid number (2n = 50), but show remarkable differences among species and populations in relation to sex chromosome systems and location of nucleolus organizer regions (NOR). In this study, we isolated a W-specific probe for the Characidium and characterized six Characidium species/populations using cytogenetic procedures. We analyzed the origin and differentiation of sex and NOR-bearing chromosomes by chromosome painting in populations of Characidium to reveal their evolution, phylogeny, and biogeography.ResultsA W-specific probe for efficient chromosome painting was isolated by microdissection and degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) amplification of W chromosomes from C. gomesi. The W probe generated weak signals dispersed on the proto sex chromosomes in C. zebra, dispersed signals in both W and Z chromosomes in C. lauroi and, in C. gomesi populations revealed a proximal site on the long arms of the Z chromosome and the entire W chromosome. All populations showed small terminal W probe sites in some autosomes. The 18S rDNA revealed distinctive patterns for each analyzed species/population with regard to proto sex chromosome, sex chromosome pair, and autosome location.ConclusionsThe results from dual-color fluorescence in situ hybridization (dual-color FISH) using W and 18S rDNA probes allowed us to infer the putative evolutionary pathways for the differentiation of sex chromosomes and NORs, from structural rearrangements in a sex proto-chromosome, followed by gene erosion and heterochromatin amplification, morphological differentiation of the sex chromosomal pair, and NOR transposition, giving rise to the distinctive patterns observed among species/populations of Characidium. Biogeographic isolation and differentiation of sex chromosomes seem to have played a major role in the speciation process in this group of fish.

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

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.

The South American fruit fly Anastrepha fraterculus sp. 1 belongs to a cryptic species complex with only a single morphotype present in Argentina, which possess polymorphisms in the sex chromosomes, identified by two X (X1, X2) and two Y (Y5, Y6) variants. Our aim was to explore the molecular composition of the sex chromosomes identified in the laboratory strain Af-Y-short due to it possesses the most frequent sex chromosomes variants, and the smallest Y chromosome found in wild populations and laboratory colonies of A. fraterculus sp. 1. Whole Comparative Genomic Hybridization (W-CGH) was applied to the Af-Y-short laboratory strain to identify potential differences in the size and composition of highly repetitive DNA blocks between individuals, morphs or sexes. Our results showed that the X1 and X2 share complete homology in euchromatic regions, also sharing similar sequences with the Y5 chromosome in the centromeric region. Females X1X1 and X1X2 exhibited repetitive DNA in telomeric regions which contributed to their morphological differentiation, while the Y chromosome evidenced the accumulation of specific and differential repetitive sequences. In contrast, autosomes showed balanced signal with no sex-chromosome-specific sequence accumulation. Our findings highlight the role of repetitive DNA in the differentiation and evolution of sex chromosomes in A. fraterculus sp. 1, shedding new light on the origin and diversification of Anastrepha species in South America and providing valuable cytogenetic insights for future research.

A common placement for most sex chromosomes that is involved in their evolutionary histories is the accumulation of distinct classes of repetitive DNAs. Here, with the aim of understanding the poorly studied repetitive DNA organization in crickets and its possible role in sex chromosome differentiation, we characterized the chromosomes of the cricket species Cycloptiloides americanus, a species with the remarkable presence of the unusual sex chromosome system X1X20♂/X1X1X2X2♀. For these proposes, we used C-banding and mapping through the fluorescence in situ hybridization of some repetitive DNAs. The C-banding and distribution of highly and moderately repetitive DNAs (C 0t-1 DNA) varied depending of the chromosome. The greater accumulation of repetitive DNAs in the X2 chromosome was evidenced. The microsatellites were spread along entire chromosomes, but (AG)10 and (TAA)10 were less enriched, mainly in the centromeric areas. Among the multigene families, the 18S rDNA was spread throughout almost all of the chromosomes, except for pair 5 and X2, while the U2 snDNA was placed exclusively in the largest chromosome. Finally, the 5S rDNA was exclusively located in the short arms of the sex chromosomes. The obtained data reinforce the importance of chromosomal dissociation and inversion as a primary evolutionary mechanism to generate neo-sex chromosomes in the species studied, followed by the repetitive DNAs accumulation. Moreover the exclusive placement of 5S rDNA in the sex chromosomes suggests the involvement of this sequence in sex chromosome recognition throughout meiosis and, consequently, their maintenance, in addition to their avoiding degeneration.

We performed a detailed karyotype analysis in the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), the key pest of pome fruit in the temperate regions of the world. The codling moth karyotype consisted of 2n = 56 chromosomes of a holokinetic type. The chromosomes were classified into 5 groups according to their sizes: extra large (3 pairs), large (3 pairs), medium (15 pairs), small (5 pairs), and dot-like (2 pairs). In pachytene nuclei of both sexes, a curious NOR (nucleolar organizer region) bivalent was observed. It carried 2 nucleoli, each associated with one end of the bivalent. FISH with an 18S ribosomal DNA probe confirmed the presence of 2 clusters of rRNA genes at the opposite ends of the bivalent. In accordance with this finding, 2 homologous NOR chromosomes were identified in mitotic metaphase, each showing hybridization signals at both ends. In highly polyploid somatic nuclei, females showed a large heterochromatin body, the so-called sex chromatin or W chromatin. The heterochromatin body was absent in male nuclei, indicating a WZ/ZZ (female/male) sex chromosome system. In keeping with the sex chromatin status, pachytene oocytes showed a sex chromosome bivalent (WZ) that was easily discernible by its heterochromatic W thread. To study molecular differentiation of the sex chromosomes, we employed genomic in situ hybridization (GISH) and comparative genomic hybridization (CGH). GISH detected the W chromosome by strong binding of the Cy3-labelled, female-derived DNA probe. With CGH, both the Cy3-labelled female-derived probe and Fluor-X labelled male-derived probe evenly bound to the W chromosome. This suggested that the W chromosome is predominantly composed of repetitive DNA sequences occurring scattered in other chromosomes but accumulated in the W chromosome. The demonstrated ways of W chromosome identification will facilitate the development of genetic sexing strains desirable for pest control using the sterile insect technique.

BackgroundThe accumulation of repetitive DNA during sex chromosome differentiation is a common feature of many eukaryotes and becomes more evident after recombination has been restricted or abolished. The accumulated repetitive sequences include multigene families, microsatellites, satellite DNAs and mobile elements, all of which are important for the structural remodeling of heterochromatin. In grasshoppers, derived sex chromosome systems, such as neo-XY♂/XX♀ and neo-X1X2Y♂/X1X1X2X2♀, are frequently observed in the Melanoplinae subfamily. However, no studies concerning the evolution of sex chromosomes in Melanoplinae have addressed the role of the repetitive DNA sequences. To further investigate the evolution of sex chromosomes in grasshoppers, we used classical cytogenetic and FISH analyses to examine the repetitive DNA sequences in six phylogenetically related Melanoplinae species with X0♂/XX♀, neo-XY♂/XX♀ and neo-X1X2Y♂/X1X1X2X2♀ sex chromosome systems.ResultsOur data indicate a non-spreading of heterochromatic blocks and pool of repetitive DNAs (C0t-1 DNA) in the sex chromosomes; however, the spreading of multigene families among the neo-sex chromosomes of Eurotettix and Dichromatos was remarkable, particularly for 5S rDNA. In autosomes, FISH mapping of multigene families revealed distinct patterns of chromosomal organization at the intra- and intergenomic levels.ConclusionsThese results suggest a common origin and subsequent differential accumulation of repetitive DNAs in the sex chromosomes of Dichromatos and an independent origin of the sex chromosomes of the neo-XY and neo-X1X2Y systems. Our data indicate a possible role for repetitive DNAs in the diversification of sex chromosome systems in grasshoppers.

Among amniote vertebrates, geckos represent a clade with exceptional variability in sex determination; however, only a minority of species of this highly diverse group has been studied in this respect. Here, we describe for the first time a female heterogamety in the genus Paroedura, the group radiated in Madagascar and adjacent islands. We identified homomorphic ZZ/ZW sex chromosomes with a highly heterochromatic W chromosome in Paroedura masobe, Paroedura oviceps, Paroedura karstophila, Paroedura stumpffi, and Paroedura lohatsara. Comparative genomic hybridization (CGH) revealed that female-specific sequences are greatly amplified in the W chromosome of P. lohatsara and that P. gracilis seems to possess a derived system of multiple sex chromosomes. Contrastingly, neither CGH nor heterochromatin visualization revealed differentiated sex chromosomes in the members of the Paroedura picta-Paroedura bastardi-Paroedura ibityensis clade, which is phylogenetically nested within lineages with a heterochromatic W chromosome. As a sex ratio consistent with genotypic sex determination has been reported in P. picta, it appears that the members of the P. picta-P. bastardi-P. ibityensis clade possess homomorphic, poorly differentiated sex chromosomes and may represent a rare example of evolutionary loss of highly differentiated sex chromosomes. Fluorescent in situ hybridization (FISH) with a telomeric probe revealed a telomere-typical pattern in all species and an accumulation of telomeric sequences in the centromeric region of autosomes in P. stumpffi and P. bastardi. Our study adds important information for the greater understanding of the variability and evolution of sex determination in geckos and demonstrates how the geckos of the genus Paroedura provide an interesting model for studying the evolution of the sex chromosomes.

Characterizing the abundance and genomic distribution of repetitive DNAs provides information on genome evolution, especially regarding the origin and differentiation of sex chromosomes. Triportheus fishes offer a useful model to explore the evolution of sex chromosomes, since they represent a monophyletic group in which all species share a ZZ/ZW sex chromosome system. In this study, we analyzed the distribution of 13 classes of repetitive DNA sequences by FISH, including microsatellites, rDNAs, and transposable elements in 6 Triportheus species, in order to investigate the fate of the sex-specific chromosome among them. These findings show the dynamic differentiation process of the W chromosome concerning changes in the repetitive DNA fraction of the heterochromatin. The differential accumulation of the same class of repeats on this chromosome, in both nearby and distant species, reflects the inherent dynamism of the microsatellites, as well as the plasticity that shapes the evolutionary history of the sex chromosomes, even among closely related species sharing a same sex chromosome system.

Teleost fishes exhibit a breath-taking diversity of sex determination and differentiation mechanisms. They encompass at least nine sex chromosome systems with often low degree of differentiation, high rate of inter- and intra-specific variability, and frequent turnovers. Nevertheless, several mainly female heterogametic systems at an advanced stage of genetic differentiation and high evolutionary stability have been also found across teleosts, especially among Neotropical characiforms. In this study, we aim to characterize the ZZ/ZW sex chromosome system in representatives of the Triportheidae family (Triportheus auritus, Agoniates halecinus, and the basal-most species Lignobrycon myersi) and its sister clade Gasteropelecidae (Carnegiella strigata, Gasteropelecus levis, and Thoracocharax stellatus). We applied both conventional and molecular cytogenetic approaches including chromosomal mapping of 5S and 18S ribosomal DNA clusters, cross-species chromosome painting (Zoo-FISH) with sex chromosome-derived probes and comparative genomic hybridization (CGH). We identified the ZW sex chromosome system for the first time in A. halecinus and G. levis and also in C. strigata formerly reported to lack sex chromosomes. We also brought evidence for possible mechanisms underlying the sex chromosome differentiation, including inversions, repetitive DNA accumulation, and exchange of genetic material. Our Zoo-FISH experiments further strongly indicated that the ZW sex chromosomes of Triportheidae and Gasteropelecidae are homeologous, suggesting their origin before the split of these lineages (approx. 40-70 million years ago). Such extent of sex chromosome stability is almost exceptional in teleosts, and hence, these lineages afford a special opportunity to scrutinize unique evolutionary forces and pressures shaping sex chromosome evolution in fishes and vertebrates in general.

Spiders are an intriguing model to analyse sex chromosome evolution because of their peculiar multiple X chromosome systems. Y chromosomes were considered rare in this group, arising after neo-sex chromosome formation by X chromosome-autosome rearrangements. However, recent findings suggest that Y chromosomes are more common in spiders than previously thought. Besides neo-sex chromosomes, they are also involved in the ancient X1X2Y system of haplogyne spiders, whose origin is unknown. Furthermore, spiders seem to exhibit obligatorily one or two pairs of cryptic homomorphic XY chromosomes (further cryptic sex chromosome pairs, CSCPs), which could represent the ancestral spider sex chromosomes. Here, we analyse the molecular differentiation of particular types of spider Y chromosomes in a representative set of ten species by comparative genomic hybridisation (CGH). We found a high Y chromosome differentiation in haplogyne species with X1X2Y system except for Loxosceles spp. CSCP chromosomes exhibited generally low differentiation. Possible mechanisms and factors behind the observed patterns are discussed. The presence of autosomal regions marked predominantly or exclusively with the male or female probe was also recorded. We attribute this pattern to intraspecific variability in the copy number and distribution of certain repetitive DNAs in spider genomes, pointing thus to the limits of CGH in this arachnid group. In addition, we confirmed nonrandom association of chromosomes belonging to particular CSCPs at spermatogonial mitosis and spermatocyte meiosis and their association with multiple Xs throughout meiosis. Taken together, our data suggest diverse evolutionary pathways of molecular differentiation in different types of spider Y chromosomes.

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.

The wolf fish Hoplias malabaricus (Erythrinidae) presents a high karyotypic diversity, with 7 karyomorphs identified. Karyomorph A is characterized by 2n = 42 chromosomes, without morphologically differentiated sex chromosomes. Karyomorph B also has 2n = 42 chromosomes for both sexes, but differs by a distinct heteromorphic XX/XY sex chromosome system. The cytogenetic mapping of 5 classes of repetitive DNA indicated similarities between both karyomorphs and the probable derivation of the XY chromosomes from pair No. 21 of karyomorph A. These chromosomes appear to be homeologous since the distribution of (GATA)_n sequences, 18S rDNA and 5SHindIII-DNA sites supports their potential relatedness. Our data indicate that the differentiation of the long arms of the X chromosome occurred by accumulation of heterochromatin and 18S rDNA cistrons from the ancestral homomorphic pair No. 21 present in karyomorph A. These findings are further supported by the distribution of the Cot-1 DNA fraction. In addition, while the 18S rDNA cistrons were maintained and amplified on the X chromosomes, they were lost in the Y chromosome. The X chromosome was a clearly preferred site for the accumulation of DNA repeats, representing an unusual example of an X clustering more repetitive sequences than the Y during sex chromosome differentiation in fish.