Chromosomal Evolution in Aspredinidae (Teleostei, Siluriformes): Insights on Intra- and Interspecific Relationships with Related Groups.

The family Aspredinidae comprises a clade of complex systematic relationships, both from molecular and morphological approaches. In this study, conventional and molecular cytogenetic studies coupled with nucleotide sequencing were performed in 6 Aspredininae species (Amaralia hypsiura, Bunocephalus cf. aloikae, Bunocephalus amaurus, Bunocephalus aff. coracoideus, Bunocephalus verrucosus, and Platystacus cotylephorus) from different locations of the Amazon hydrographic basin. Our results showed highly divergent diploid numbers (2n) among the species, ranging from 49 to 74, including the occurrence of an XX/X0 sex chromosome system. A neighbor-joining phylogram based on the cytochrome c oxidase I (COI) showed that Bunocephalus coracoideus is not a monophyletic clade, but closely related to B. verrucosus. The karyotypic data associated with COI suggest an ancestral karyotype for Aspredinidae with a reduced 2n, composed of bi-armed chromosomes and a trend toward chromosomal fissions resulting in higher diploid number karyotypes, mainly composed of acrocentric chromosomes. Evolutionary relationships were discussed under a phylogenetic context with related species from different Siluriformes families. The karyotype features and chromosomal diversity of Aspredinidae show an amazing differentiation, making this family a remarkable model for investigating the evolutionary dynamics in siluriforms as well as in fish as a whole.

Venomous Loxosceles Species (Araneae, Haplogynae, Sicariidae) from Brazil: 2n♂ = 23 and X1X2Y Sex Chromosome System as Shared Characteristics.

The family Sicariidae comprises the genera Hexophthalma, Sicarius and Loxosceles. This latter is subdivided in eight monophyletic groups based on genitalia morphology and molecular analyses: amazonica, gaucho, laeta, and spadicea (South America); reclusa (North America); rufescens (Mediterranean); spinulosa and vonwredei (Africa). In Brazil, the genus Loxosceles is represented by 50 species. The mitotic and meiotic characteristics of eight Loxosceles species were analyzed in order to discuss the chromosome evolution, as well as the correspondence between cytogenetic data and morphological/molecular data for the delimitation of the South American groups of species belonging to this genus. All species studied in this work showed 2n♂ = 23, including a X1X2Y sex chromosome system (SCS). Despite the similarity of diploid number and SCS, the species studied here differed regarding the chromosome morphology of some autosomal pairs, presence of secondary constrictions, size of X chromosomes and localization of Ag-NOR/rDNA sites. Based on all these chromosomal data, we verified a close relationship between Loxosceles species belonging to the amazonica and gaucho groups. Transmission electron microscopy (TEM) analysis of spread pachytene cells of L. gaucho showed regular synapsis between homologous autosomal chromosomes, but asynaptic behavior of the sex chromosomes. The axial elements of the sex chromosomes undergo conspicuous morphological modifications resulting in shortening of their length.

Sex Chromosome Mosaicism in the Gonads of DSD Patients: A Karyotype/Phenotype Correlation.

Sex chromosome mosaicism results in a large clinical spectrum of disorders of sexual development (DSD). The percentage of 45,X cells in the developing gonad plays a major role in sex determination. However, few reports on the gonadal mosaic status have been published, and the phenotype is usually correlated with peripheral lymphocyte karyotypes, which makes the phenotype prediction imprecise. This study was conducted on 7 Egyptian DSD patients to demonstrate the effect of sex chromosome constitution of both blood lymphocytes and gonadal tissues on the phenotypic manifestations. Conventional cytogenetic and FISH analyses of blood lymphocytes were conducted, and laparoscopy with gonadal biopsy was performed for histopathologic examination and FISH analysis. Gonosomal mosaicism was detected in 3 patients who had a non-mosaic chromosome pattern in blood lymphocytes. Two patients showed the same type of sex chromosome mosaicism in both the blood and gonadal tissues but with different distributions. Two other patients revealed a non-mosaic pattern in both tissues. The present study elucidates the importance of examining sex chromosome mosaicism in gonadal tissues of DSD patients and highlights the critical role of 45,X mosaicism which can lead to serious effects during early gonadal organogenesis.

Extraordinary Diversity in the Origins of Sex Chromosomes in Anurans Inferred from Comparative Gene Mapping.

Sex determination in frogs (anurans) is genetic and includes both male and female heterogamety. However, the origins of the sex chromosomes and their differentiation processes are poorly known. To investigate diversity in the origins of anuran sex chromosomes, we compared the chromosomal locations of sex-linked genes in 4 species: the African clawed frog (Xenopus laevis), the Western clawed frog (Silurana/X. tropicalis), the Japanese bell-ring frog (Buergeria buergeri), and the Japanese wrinkled frog (Rana rugosa). Comparative mapping data revealed that the sex chromosomes of X. laevis, X. tropicalis and R. rugosa are different chromosome pairs; however, the sex chromosomes of X. tropicalis and B. buergeri are homologous, although this may represent distinct evolutionary origins. We also examined the status of sex chromosomal differentiation in B. buergeri, which possesses heteromorphic ZW sex chromosomes, using comparative genomic hybridization and chromosome painting with DNA probes from the microdissected W chromosome. At least 3 rearrangement events have occurred in the proto-W chromosome: deletion of the nucleolus organizer region and a paracentric inversion followed by amplification of non-W-specific repetitive sequences.

Comparative chromosomal mapping of microsatellites in Leporinus species (Characiformes, Anostomidae): unequal accumulation on the W chromosomes.

Approximately 90 species in the genus Leporinus (Characiformes, Anostomidae) are known, and most of them do not present differentiated sex chromosomes. However, there is a group of 7 species that share a heteromorphic ZW sex system. In all of these species, the W chromosome is the largest one in the karyotype and is mostly heterochromatic. We investigated the distribution of several microsatellites in the genome of 4 Leporinus species that possess ZW chromosomes. Our results showed a very large accumulation of mostly microsatellites on the W chromosomes. This finding supports the presence of an interconnection between heterochromatinization and the accumulation of repetitive sequences, which has been proposed for sex chromosome evolution, and suggests that heterochromatinization is the earlier of the 2 processes. In spite of the common origin that has been proposed for W chromosomes in all of the studied species, the microsatellites followed different evolutionary trajectories in each species, which indicates a high plasticity for sex chromosome differentiation.

Karyological characterization of the endemic Iberian rock lizard, Iberolacerta monticola (Squamata, Lacertidae): insights into sex chromosome evolution.

Rock lizards of the genus Iberolacerta constitute a promising model to examine the process of sex chromosome evolution, as these closely related taxa exhibit remarkable diversity in the degree of sex chromosome differentiation with no clear phylogenetic segregation, ranging from cryptic to highly heteromorphic ZW chromosomes and even multiple chromosome systems (Z1Z1Z2Z2/Z1Z2W). To gain a deeper insight into the patterns of karyotype and sex chromosome evolution, we performed a cytogenetic analysis based on conventional staining, banding techniques and fluorescence in situ hybridization in the species I. monticola, for which previous cytogenetic investigations did not detect differentiated sex chromosomes. The karyotype is composed of 2n = 36 acrocentric chromosomes. NORs and the major ribosomal genes were located in the subtelomeric region of chromosome pair 6. Hybridization signals of the telomeric sequences (TTAGGG)n were visualized at the telomeres of all chromosomes and interstitially in 5 chromosome pairs. C-banding showed constitutive heterochromatin at the centromeres of all chromosomes, as well as clear pericentromeric and light telomeric C-bands in several chromosome pairs. These results highlight some chromosomal markers which can be useful to identify species-specific diagnostic characters, although they may not accurately reflect the phylogenetic relationships among the taxa. In addition, C-banding revealed the presence of a heteromorphic ZW sex chromosome pair, where W is smaller than Z and almost completely heterochromatic. This finding sheds light on sex chromosome evolution in the genus Iberolacerta and suggests that further comparative cytogenetic analyses are needed to understand the processes underlying the origin, differentiation and plasticity of sex chromosome systems in lacertid lizards.

Both cell-autonomous mechanisms and hormones contribute to sexual development in vertebrates and insects.

The differentiation of male and female characteristics in vertebrates and insects has long been thought to proceed via different mechanisms. Traditionally, vertebrate sexual development was thought to occur in two phases: a primary and a secondary phase, the primary phase involving the differentiation of the gonads, and the secondary phase involving the differentiation of other sexual traits via the influence of sex hormones secreted by the gonads. In contrast, insect sexual development was thought to depend exclusively on cell-autonomous expression of sex-specific genes. Recently, however, new evidence indicates that both vertebrates and insects rely on sex hormones as well as cell-autonomous mechanisms to develop sexual traits. Collectively, these new data challenge the traditional vertebrate definitions of primary and secondary sexual development, call for a redefinition of these terms, and indicate the need for research aimed at explaining the relative dependence on cell-autonomous versus hormonally guided sexual development in animals.

Analysis of chromosome and karyotype in Bali cattle and Simmental-Bali (Simbal) crossbreed cattle.

Chromosome analysis is very important part for the initial analysis of genetics. Some genetics abnormalities can be detected at the chromosome level and are usually associated with the inherited diseases. Accurate and prompt information need to be obtained for the purposes of the prevention of the genetics abnormalities genetics risk. This study was aimed to find out and to analyze the different size and morphological chromosome of Bali cattle and its crosses with Simmental cattle (Simbal cattle). Ten Simbal cattle (5 male and 5 female) and 5 female Bali cattle were used in this study. Five milliliter blood was collected using venous puncture through the jugular vein of each cattle. Chromosome was derived from white blood cells (lymphocyte) of peripheral blood. The Bali cattle and Simbal cattle have diploid chromosome (2n) of 60, with 29 pair of autosomes and one pair of sex chromosome. All autosomes are acrocentric with centromere index of 25.13 to 29.52% for Simbal cattle and 21.61 to 24.84% for Bali cattle. Sex chromosomes ware sub-metacentric in Simbal cattle either male or female and metacentric in female Bali cattle. Average length of chromosomes of female Simbal, male Simbal and female Bali cattle were 0.29 +/- 0.04 micron, 0.30 +/- 0.05 micron and 0.24 +/- 0.02 micron, respectively. Chromosome size of female Bali cattle was smaller than Simbal cattle.

Samia cynthia versus Bombyx mori: comparative gene mapping between a species with a low-number karyotype and the model species of Lepidoptera.

We performed gene-based comparative FISH mapping between a wild silkmoth, Samia cynthia ssp. with a low number of chromosomes (2n=25-28) and the model species, Bombyx mori (2n=56), in order to identify the genomic components that make up the chromosomes in a low-number karyotype. Mapping of 64 fosmid probes containing orthologs of B. mori genes revealed that the homologues of either two or four B. mori chromosomes constitute the S. c. ricini (Vietnam population, 2n=27♀/28♂, Z0/ZZ) autosomes. Where tested, even the gene order was conserved between S. c. ricini and B. mori. This was also true for the originally autosomal parts of the neo-sex chromosomes in S. c. walkeri (Sapporo population, 2n=26♀/26♂, neo-Wneo-Z/neo-Zneo-Z) and S. cynthia subsp. indet. (Nagano population, 2n=25♀/26♂, neo-WZ1Z2/Z1Z1Z2Z2). The results are evidence for an internal stability of lepidopteran chromosomes even when all autosomes had undergone fusion processes to form a low-number karyotype.

Insights into the meiotic behavior and evolution of multiple sex chromosome systems in spiders.

A characteristic feature of spider karyotypes is the predominance of unusual multiple X chromosomes. To elucidate the evolution of spider sex chromosomes, their meiotic behavior was analyzed in 2 major clades of opisthothele spiders, namely, the entelegyne araneomorphs and the mygalomorphs. Our data support the predominance of X(1)X(2)0 systems in entelegynes, while rare X(1)X(2)X(3)X(4)0 systems were revealed in the tuberculote mygalomorphs. The spider species studied exhibited a considerable diversity of achiasmate sex chromosome pairing in male meiosis. The end-to-end pairing of sex chromosomes found in mygalomorphs was gradually replaced by the parallel attachment of sex chromosomes in entelegynes. The observed association of male X univalents with a centrosome at the first meiotic division may ensure the univalents' segregation. Spider meiotic sex chromosomes also showed other unique traits, namely, association with a chromosome pair in males and inactivation in females. Analysis of these traits supports the hypothesis that the multiple X chromosomes of spiders originated by duplications. In contrast to the homogametic sex of other animals, the homologous sex chromosomes of spider females were already paired at premeiotic interphase and were inactivated until prophase I. Furthermore, the sex chromosome pairs exhibited an end-to-end association during these stages. We suggest that the specific behavior of the female sex chromosomes may have evolved to avoid the negative effects of duplicated X chromosomes on female meiosis. The chromosome ends that ensure the association of sex chromosome pairs during meiosis may contain information for discriminating between homologous and homeologous X chromosomes and thus act to promote homologous pairing. The meiotic behavior of 4 X chromosome pairs in mygalomorph females, namely, the formation of 2 associations, each composed of 2 pairs with similar structure, suggests that the mygalomorph X(1)X(2)X(3)X(4)0 system originated by the duplication of the X(1)X(2)0 system via nondisjunctions or polyploidization.

Differentiation of sex chromosomes and karyotypic evolution in the eye-lid geckos (Squamata: Gekkota: Eublepharidae), a group with different modes of sex determination.

The eyelid geckos (family Eublepharidae) include both species with temperature-dependent sex determination and species where genotypic sex determination (GSD) was suggested based on the observation of equal sex ratios at several incubation temperatures. In this study, we present data on karyotypes and chromosomal characteristics in 12 species (Aeluroscalabotes felinus, Coleonyx brevis, Coleonyx elegans, Coleonyx variegatus, Eublepharis angramainyu, Eublepharis macularius, Goniurosaurus araneus, Goniurosaurus lichtenfelderi, Goniurosaurus luii, Goniurosaurus splendens, Hemitheconyx caudicinctus, and Holodactylus africanus) covering all genera of the family, and search for the presence of heteromorphic sex chromosomes. Phylogenetic mapping of chromosomal changes showed a long evolutionary stasis of karyotypes with all acrocentric chromosomes followed by numerous chromosomal rearrangements in the ancestors of two lineages. We have found heteromorphic sex chromosomes in only one species, which suggests that sex chromosomes in most GSD species of the eyelid geckos are not morphologically differentiated. The sexual difference in karyotype was detected only in C. elegans which has a multiple sex chromosome system (X(1)X(2)Y). The metacentric Y chromosome evolved most likely via centric fusion of two acrocentric chromosomes involving loss of interstitial telomeric sequences. We conclude that the eyelid geckos exhibit diversity in sex determination ranging from the absence of any sexual differences to heteromorphic sex chromosomes, which makes them an interesting system for exploring the evolutionary origin of sexually dimorphic genomes.

A W-linked palindrome and gene conversion in New World sparrows and blackbirds.

A hallmark feature of the male-specific region of the human Y chromosome is the presence of large and near-identical palindromes. These palindromes are maintained in a state of near identity via gene conversion between the arms of the palindrome, and both neutral and selection-based theories have been proposed to explain their enrichment on the human Y and X chromosomes. While those proposed theories would be applicable to sex chromosomes in other species, it has not been established whether near-identical palindromes are a common feature of sex chromosomes in a broader range of taxa, including other tetrapods. Here, we report the genomic sequencing and features of a 279-kb region of the non-recombining portion of the W chromosome spanning the CHD1W locus in a New World sparrow, the white-throated sparrow (Zonotrichia albicollis), and the corresponding region on the Z chromosome. As has been observed for other Y and W chromosomes, we detected a high repetitive element content (51%) and low gene content on the white-throated sparrow W chromosome. In addition, we identified a 22-kb near-identical (>99%) palindrome on the W chromosome that flanks the 5' end of the CHD1W gene. Signatures of gene conversion were readily detected between the arms of this palindrome, as was the presence of this palindrome in other New World sparrows and blackbirds. Near-identical palindromes are therefore present on the avian W chromosome and may persist due to the same forces proposed for the enrichment of these elements on the human sex chromosomes.

miRNA and piRNA localization in the male mammalian meiotic nucleus.

During mammalian meiosis, transcriptional silencing of the XY bivalent is a necessary event where defects may lead to infertility in males. While not well understood, the mechanism of meiotic gene silencing is believed to be RNA-dependent. In this study, we investigated the types and localization of non-coding RNAs in the meiotic nucleus of the male mouse using a microarray screen with different cell isolates as well as FISH. We report that the dense body, a component of the murine spermatocyte sex body similar to that of a dense body in Chinese hamster spermatocytes, is DNA-negative but rich in proteins and RNA including miRNAs (micro RNAs) and piRNAs (PIWI associated small RNAs), or their precursors. Selective miRNAs and piRNAs localize to chromosome cores, telomeres and the sex body of spermatocytes. These RNAs have not previously been detected in meiotic nuclei. These RNAs appear to associate with the nucleolus of the Sertoli cells as well as with the dense body. While in MIWI-null male mice the nucleolar signal from miRNA and piRNA probes in Sertoli cells is largely diminished, a differential regulation must exist in meiotic nuclei since the localization of these two components appears to be unaffected in the null animal.

Physical chromosome mapping of repetitive DNA sequences in Nile tilapia Oreochromis niloticus: evidences for a differential distribution of repetitive elements in the sex chromosomes.

Repetitive DNAs have been extensively applied as physical chromosome markers on comparative studies, identification of chromosome rearrangements and sex chromosomes, chromosome evolution analysis, and applied genetics. Here we report the characterization of repetitive DNA sequences from the Nile tilapia (Oreochromis niloticus) genome by construction and screening of plasmid library enriched with repetitive DNAs, analysis of a BAC-based physical map, and hybridization to chromosomes. The physical mapping of BACs enriched with repetitive sequences and C(o)t-1 DNA (DNA enriched for highly and moderately repetitive DNA sequences) to chromosomes using FISH showed a predominant distribution of repetitive elements in the centromeric and telomeric regions and along the entire length of the largest chromosome pair (X and Y sex chromosomes) of the species. The distribution of repetitive DNAs differed significantly between the p arm of X and Y chromosomes. These findings suggest that repetitive DNAs have had an important role in the differentiation of sex chromosomes.

The first karyotype study in palpigrades, a primitive order of arachnids (Arachnida: Palpigradi).

Chromosomes of palpigrades (Arachnida: Palpigradi), a rare arachnid order with numerous primitive characters, were studied for the first time. We analysed two species of the genus Eukoenenia, namely E. spelaea and E. mirabilis. Their karyotypes are uniform, consisting of a low number of tiny chromosomes that decrease gradually in size. Study of the palpigrade karyotype did not reveal morphologically differentiated sex chromosomes. Analysis of E. spelaea showed that constitutive heterochromatin is scarce, GC-rich, and restricted mostly to presumed centromeric regions. Meiosis is remarkable for the presence of a short diffuse stage and prominent nucleolar activity. During prophase I, nuclei contain a large nucleolus. Prominent knob at the end of one bivalent formed by constitutive heterochromatin is associated to the nucleolus by an adjacent NOR. Presence of a nucleolus-like body at male prophase II suggests activity of NOR also during beginning of the second meiotic division. The data suggest acrocentric morphology of palpigrade chromosomes. Palpigrades do not display holocentric chromosomes which appear to be apomorphic features of a number of arachnid groups. These are: acariform mites, buthid scorpions, and spiders of the superfamily Dysderoidea. Therefore, cytogenetic data do not support a close relationship of palpigrades and acariform mites as suggested previously.

Molecular divergence of the W chromosomes in pyralid moths (Lepidoptera).

Most Lepidoptera have a WZ/ZZ sex chromosome system. We compared structure of W chromosomes in four representatives of the family Pyralidae--Ephestia kuehniella, Cadra cautella, Plodia interpunctella, and Galleria mellonella--tracing pachytene bivalents which provide much higher resolution than metaphase chromosomes. In each species, we prepared a W-chromosome painting probe from laser-microdissected W-chromatin of female polyploid nuclei. The Ephestia W-probe was cross-hybridized to chromosomes of the other pyralids to detect common parts of their W chromosomes, while the species-specific W-probes identified the respective W chromosome. This so-called Zoo-FISH revealed a partial homology of W-chromosome regions between E. kuehniella and two other pyralids, C. cautella and P. interpunctella, but almost no homology with G. mellonella. The results were consistent with phylogenetic relationships between the species. We also performed comparative genomic hybridization, which indicated that the W chromosome of C. cautella is composed mainly of repetitive DNA common to both sexes but accumulated in the W chromosome, whereas E. kuehniella, P. interpunctella, and G. mellonella W chromosomes also possess a large amount of female specific DNA sequences, but differently organized. Our results support the hypothesis of the accelerated molecular divergence of the lepidopteran W chromosomes in the absence of meiotic recombination.

Cytogenetic analysis and description of the sexual chromosome determination system ZZ/ZW of species of the fish genus Serrapinnus (Characidae, Cheirodontinae).

Four populations of Serrapinnus notomelas and one population of Serrapinnus sp.1, both belonging to the subfamily Cheirodontinae, were analyzed by Giemsa and silver nitrate impregnation techniques. We found 2n = 52 chromosomes for all populations, with interspecific differences in the karyotype formula; S. notomelas showed 16 m + 22 sm + 10 st + 4a, with fundamental number (FN) = 100 for males, and 16 m + 23 sm + 10 st + 3a, with FN = 101 for females. Serrapinnus sp.1 had 8m + 16 sm + 4 st + 24 a, with FN = 80 for males, and 8m + 15 sm + 4 st + 25 a, with FN = 79 for females. The difference in FN for the two sexes is due to a pair of heteromorphic chromosomes in the females of both species, which characterizes a ZZ/ZW-type mechanism of chromosome sexual determination. Interspecies differences were also found in nucleolus organizer regions (NORs). A simple NOR system was detected in three of four S. notomelas populations, while Serrapinnus sp.1 had two chromosome pairs with NOR. Although S. notomelas and Serrapinnus sp.1 have the same diploid number, differences in the karyotype structure indicate that these are different species. Apparently there was pericentric inversion during the karyotype evolution of these species.

Patient gender is associated with distinct patterns of chromosomal abnormalities and sex chromosome linked gene-expression profiles in meningiomas.

The female predominance of meningiomas has been established, but how this is affected by hormones is still under discussion. We analyzed the characteristics of meningiomas from male (n = 53) and female (n = 111) patients by interphase fluorescence in situ hybridization (iFISH). In addition, in a subgroup of 45 (12 male and 33 female) patients, tumors were hybridized with the Affymetrix U133A chip. We show a higher frequency of larger tumors (p = .01) and intracranial meningiomas (p = .04) together with a higher relapse rate (p = .03) in male than in female patients. Male patients had a higher percentage of del(1p36) (p < .001), while loss of an X chromosome was restricted to tumors from female patients (p = .008). In turn, iFISH studies showed a higher frequency of chromosome losses, other than monosomy 22 alone, in meningiomas from male patients (p = .002), while female patients displayed a higher frequency of chromosome gains (p = .04) or monosomy 22 alone (p = .03) in the ancestral tumor clone. Interestingly, individual chromosomal abnormalities had a distinct impact on the recurrence-free survival rate of male versus female patients. In turn, gene expression showed that eight genes (RPS4Y1, DDX3Y, JARID1D, DDX3X, EIF1AY, XIST, USP9Y, and CYorf15B) had significantly different expression patterns (R(2) > 0.80; p < .05) in tumors from male and female patients. In summary, we show the existence of different patterns of chromosome abnormalities and gene-expression profiles associated with patient gender, which could help to explain the slightly different clinical behavior of these two patient groups.

Evolution of multiple sex chromosomes in the spider genus Malthonica (Araneae: Agelenidae) indicates unique structure of the spider sex chromosome systems.

Most spiders exhibit a multiple sex chromosome system, X(1)X(2)0, whose origin has not been satisfactorily explained. Examination of the sex chromosome systems in the spider genus Malthonica (Agelenidae) revealed considerable diversity in sex chromosome constitution within this group. Besides modes X(1)X(2)0 (M. silvestris) and X(1)X(2)X(3)0 (M. campestris), a neo-X(1)X(2)X(3)X(4)X(5)Y system in M. ferruginea was found. Ultrastructural analysis of spread pachytene spermatocytes revealed that the X(1)X(2)0 and X(1)X(2)X(3)0 systems include a pair of homomorphic sex chromosomes. Multiple X chromosomes and the pair exhibit an end-to-end pairing, being connected by attachment plaques. The X(1)X(2)X(3)X(4)X(5)Y system of M. ferruginea arose by rearrangement between the homomorphic sex chromosome pair and an autosome. Multiple X chromosomes and the sex chromosome pair do not differ from autosomes in a pattern of constitutive heterochromatin. Ultrastructural data on sex chromosome pairing in other spiders indicate that the homomorphic sex chromosome pair forms an integral part of the spider sex chromosome systems. It is suggested that this pair represents ancestral sex chromosomes of spiders, which generated multiple X chromosomes by non-disjunctions. Structural differentiation of newly formed X chromosomes has been facilitated by heterochromatinization of sex chromosome bivalents observed in prophase I of spider females.