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Abstract The W chromosome of Lepidoptera is typically gene-poor, repeat-rich and composed of heterochromatin. Pioneering studies investigating this chromosome reported an abundance of mobile elements. However, the actual composition of the W chromosome varies greatly between species, as repeatedly demonstrated by comparative genomic hybridization (CGH) or fluorescence in situ hybridization (FISH). Here we present an analysis of repeats on the W chromosome in the willow beauty, Peribatodes rhomboidaria (Geometridae), a species in which CGH predicted an abundance of W-enriched or W-specific sequences. Indeed, comparative analysis of male and female genomes using RepeatExplorer identified ten putative W chromosome-enriched repeats, most of which are LTR or LINE mobile elements. We analysed the two most abundant: PRW LINE-like and PRW Bel-Pao. The results of FISH mapping and bioinformatic analysis confirmed their enrichment on the W chromosome, supporting the hypothesis that mobile elements are the driving force of W chromosome differentiation in Lepidoptera. As the W chromosome is highly underrepresented in chromosome-level genome assemblies of Lepidoptera, this recently introduced approach, combining bioinformatic comparative genome analysis with molecular cytogenetics, provides an elegant tool for studying this elusive and rapidly evolving part of the genome.

Abstract Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution.

Abstract Background Despite progress in genomic analysis of spiders, their chromosome evolution is not satisfactorily understood. Most information on spider chromosomes concerns the most diversified clade, entelegyne araneomorphs. Other clades are far less studied. Our study focused on haplogyne araneomorphs, which are remarkable for their unusual sex chromosome systems and for the co-evolution of sex chromosomes and nucleolus organizer regions (NORs); some haplogynes exhibit holokinetic chromosomes. To trace the karyotype evolution of haplogynes on the family level, we analysed the number and morphology of chromosomes, sex chromosomes, NORs, and meiosis in pholcids, which are among the most diverse haplogyne families. The evolution of spider NORs is largely unknown. Results Our study is based on an extensive set of species representing all major pholcid clades. Pholcids exhibit a low 2n and predominance of biarmed chromosomes, which are typical haplogyne features. Sex chromosomes and NOR patterns of pholcids are diversified. We revealed six sex chromosome systems in pholcids (X0, XY, X1X20, X1X2X30, X1X2Y, and X1X2X3X4Y). The number of NOR loci ranges from one to nine. In some clades, NORs are also found on sex chromosomes. Conclusions The evolution of cytogenetic characters was largely derived from character mapping on a recently published molecular phylogeny of the family. Based on an extensive set of species and mapping of their characters, numerous conclusions regarding the karyotype evolution of pholcids and spiders can be drawn. Our results suggest frequent autosome–autosome and autosome–sex chromosome rearrangements during pholcid evolution. Such events have previously been attributed to the reproductive isolation of species. The peculiar X1X2Y system is probably ancestral for haplogynes. Chromosomes of the X1X2Y system differ considerably in their pattern of evolution. In some pholcid clades, the X1X2Y system has transformed into the X1X20 or XY systems, and subsequently into the X0 system. The X1X2X30 system of Smeringopus pallidus probably arose from the X1X20 system by an X chromosome fission. The X1X2X3X4Y system of Kambiwa probably evolved from the X1X2Y system by integration of a chromosome pair. Nucleolus organizer regions have frequently expanded on sex chromosomes, most probably by ectopic recombination. Our data suggest the involvement of sex chromosome-linked NORs in achiasmatic pairing.

Odonata have holokinetic chromosomes. About 95% of species have an XX/X0 sex chromosome system, with heterogametic males. There are species with neo-XX/neo-XY sex chromosomes resulting from an X chromosome/autosome fusion. The genus Rhionaeschna includes 42 species found in the Americas. We analyzed the distribution of the nucleolar organizer region (NOR) using FISH with rDNA probes in Rhionaeschna bonariensis (n = 12 + neo-XY), R. planaltica (n = 7 + neo-XY), and Aeshna cyanea (n = 13 + X0). In R. bonariensis and A. cyanea, the NOR is located on a large pair of autosomes, which have a secondary constriction in the latter species. In R. planaltica, the NOR is located on the ancestral part of the neo-X chromosome. Meiotic analysis and FISH results in R. planaltica led to the conclusion that the neo-XY system arose by insertion of the ancestral X chromosome into an autosome. Genomic in situ hybridization, performed for the first time in Odonata, highlighted the entire neo-Y chromosome in meiosis of R. bonariensis, suggesting that it consists mainly of repetitive DNA. This feature and the terminal chiasma localization suggest an ancient origin of the neo-XY system. Our study provides new information on the origin and evolution of neo-sex chromosomes in Odonata, including new types of chromosomal rearrangements, NOR transposition, and heterochromatin accumulation.

Tandem repeats are important parts of eukaryotic genomes being crucial e.g., for centromere and telomere function and chromatin modulation. In Lepidoptera, knowledge of tandem repeats is very limited despite the growing number of sequenced genomes. Here we introduce seven new satellite DNAs (satDNAs), which more than doubles the number of currently known lepidopteran satDNAs. The satDNAs were identified in genomes of three species of Crambidae moths, namely Ostrinia nubilalis, Cydalima perspectalis, and Diatraea postlineella, using graph-based computational pipeline RepeatExplorer. These repeats varied in their abundance and showed high variability within and between species, although some degree of conservation was noted. The satDNAs showed a scattered distribution, often on both autosomes and sex chromosomes, with the exception of both satellites in D. postlineella, in which the satDNAs were located at a single autosomal locus. Three satDNAs were abundant on the W chromosomes of O. nubilalis and C. perspectalis, thus contributing to their differentiation from the Z chromosomes. To provide background for the in situ localization of the satDNAs, we performed a detailed cytogenetic analysis of the karyotypes of all three species. This comparative analysis revealed differences in chromosome number, number and location of rDNA clusters, and molecular differentiation of sex chromosomes.

Sex chromatin is a conspicuous body that occurs in polyploid nuclei of most lepidopteran females and consists of numerous copies of the W sex chromosome. It is also a cytogenetic tool used to rapidly assess the W chromosome presence in Lepidoptera. However, certain chromosomal features could disrupt the formation of sex chromatin and lead to the false conclusion that the W chromosome is absent in the respective species. Here we tested the sex chromatin presence in 50 species of Geometridae. In eight selected species with either missing, atypical, or normal sex chromatin patterns, we performed a detailed karyotype analysis by means of comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). The results showed a high diversity of W chromosomes and clarified the reasons for atypical sex chromatin, including the absence or poor differentiation of W, rearrangements leading to the neo-W emergence, possible association with the nucleolus, and the existence of multiple W chromosomes. In two species, we detected intraspecific variability in the sex chromatin status and sex chromosome constitution. We show that the sex chromatin is not a sufficient marker of the W chromosome presence, but it may be an excellent tool to pinpoint species with atypical sex chromosomes.

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 magpie moth, Abraxas grossulariata, is an iconic species in which female heterogamety was discovered at the beginning of the 20th century. However, the sex chromosomes of this species have not yet been cytologically identified. We describe the sex chromosomes of A. grossulariata and its congener, A. sylvata. Although these species split only around 9.5 million years ago, and both species have the expected WZ/ZZ chromosomal system of sex determination and their sex chromosomes share the major ribosomal DNA (rDNA) representing the nucleolar organizer region (NOR), we found major differences between their karyotypes, including between their sex chromosomes. The species differ in chromosome number, which is 2n = 56 in A. grossularita and 2n = 58 in A. sylvata. In addition, A. grossularita autosomes exhibit massive autosomal blocks of heterochromatin, which is a very rare phenomenon in Lepidoptera, whereas the autosomes of A. sylvata are completely devoid of distinct heterochromatin. Their W chromosomes differ greatly. Although they are largely composed of female-specific DNA sequences, as shown by comparative genomic hybridization, cross-species W-chromosome painting revealed considerable sequence differences between them. The results suggest a relatively rapid molecular divergence of Abraxas W chromosomes by the independent spreading of female-specific repetitive sequences.