The complete sequence of the W chromosome, which carries feminization activity in the silkworm, is crucial for understanding the sex-determination system in Lepidoptera. However, extensive accumulation of transposons due to lack of recombination, the very rare protein-coding genes and almost no information about molecular markers has hindered full W sequencing. We report the first complete silkworm W sequence (T2T_W, 11683305 bp) obtained by combining sequencing-assembly technologies and newly developed error detection methods, evaluated with genetically mapped W-RAPD markers, W-mutants, and W-derived BAC clones. The T2T_W sequence showed that the W is composed of a massive 92% accumulation of transposons and repeat sequences, among which the main constituents are intact LTR/LINE retrotransposons indicating recent expansions. In addition to Fem clusters producing Fem piRNA (Feminizer-derived PIWI-interacting RNA), we found 26 protein-coding genes in the W sequence. These include four gene pairs encoding zinc-finger motifs designated z1:z20 and a gene encoding serine/arginine repetitive matrix protein 1-like (SRRM1-like). To identify candidate genes for female sex-determination and differentiation we also sequenced the shortest W (3.8 Mb) from a translocation mutant with feminizing activity, which harbored four conventional genes: a Fem cluster, a pair of z1:z20 isoforms, z20-S, and a SRRM1-like gene. Phylogenetic analysis revealed that z1:z20 originated from a copy of an autosomal zinc-finger gene pair, z2:z21, translocated onto the W around 2.43 Mya and subsequently amplified to yield 4 W-linked zinc-finger gene pairs. The complete W sequence revealed that large-scale deletions and amplifications played a significant role in W chromosome evolution.

Knowledge about sex determination in Lepidoptera is starting to unfold just over a decade after the discovery of the primary sex determination trigger in the silkworm Bombyx mori. The silkworm has a W-dominant sex determination mechanism with a PIWI-interacting RNA (piRNA) precursor gene called Feminizer (Fem) as the primary trigger. The emerging view is that the silkworm is unsuitable to predict primary triggers in other Lepidoptera species, despite its role as model organism. However, the Z-linked gene named Masculinizer (Masc), which is targeted by Fem piRNA in the silkworm, plays a key role in sex determination in all species studied so far. This conserved role of Masc at the beginning of the sex determination cascade differs from what is known in other insects, where the cascade is initially diverse and becomes increasingly conserved towards the end, where doublesex (dsx) is alternatively spliced into a female or male variant. Sex-specific dsx splicing is also conserved in Lepidoptera, while the other genes which make up the sex-determining cascade are yet to be revealed in full detail. The sex determination mechanisms in two species are highlighted because, unlike the silkworm, they do not rely on a primary trigger from the W chromosome. The moth Samia cynthia ricini uses the ratio of Z chromosomes to autosome sets to determine sex. The butterfly Bicyclus anynana has a sex determination more similar to the honey bee than to the silkworm, with the zygosity of a hypervariable region of Masc determining whether individuals become female or male.

Moths and butterflies (Lepidoptera) are the largest group of organisms with female heterogamety and the sex chromosome system WZ/ZZ (female/male) or exceptionally Z0/ZZ. However, the genetic basis of sex determination in Lepidoptera remained unknown for a long time until the sex-determining pathway was discovered in 2014 in the silkworm Bombyx mori. In this species, the dominant W chromosome carries a Feminizer (Fem) gene encoding a precursor of a Fem piRNA that promotes femaleness by downregulating the expression of a Z-linked gene, Masculinizer (Masc). In the W chromosome absence, Masc promotes male development and controls dosage compensation. In the 10 years since this discovery, significant progress has been made in understanding the molecular mechanisms of sex determination in Lepidoptera. Data from recent studies discussed in this review suggest a conserved role for Masc in male sex determination and dosage compensation in the clade Ditrysia, which comprises the majority of Lepidoptera. Although the primary sex-determining signals are not conserved, the presence of feminizing piRNAs of different origins in distantly related species suggests convergent evolution of a similar mechanism of female sex determination. A unique exception is zygosity-based sex determination in the butterfly Bicyclus anynana, where the primary signal is the state of the hypervariable Masc gene. In other species with a dispensable W chromosome, such as the silkmoth Samia cynthia, sex is determined by the Z:A ratio, but a molecular mechanism is not yet known. Overall, the available data suggest considerable diversity in the upstream molecular mechanisms of sex determination in Lepidoptera.

BACKGROUND:The primary signals of sex determination in insects are diverse and evolve erratically. This also appears to be the case with moths and butterflies. In the silkworm Bombyx mori, female development is triggered by a W chromosome-derived Feminizer (Fem) piRNA that degrades the mRNA of the Z-linked Masculinizer (Masc) gene, which controls male development. We investigated whether this mechanism is conserved in another group of Lepidoptera. RESULTS:We identified a putative feminizing piRNA and many partial copies of the EkMasc gene on the W chromosome of Ephestia kuehniella. The piRNA is generated by a repetitive W-linked sequence named E. kuehniella Moth-overruler-of-masculinization (EkMom). EkMom piRNA shows high similarity to a region of Z-linked EkMasc and is expressed at the onset of female development, but has no relationship to the B. mori Fem piRNA. We then mapped small RNA-seq data from embryos of the related Plodia interpunctella to the PiMasc gene and identified a single small RNA, a PiMom piRNA, able to target PiMasc and with high sequence identity to the EkMom piRNA. Both the PiMom and EkMom repeats are present in high copy number and form a single cluster on the W chromosome. In both species, the Mom piRNA is responsible for Masc mRNA cleavage, clearly demonstrating that the Mom piRNA triggers female development. CONCLUSIONS:Our study provides multiple lines of evidence that Mom piRNA is the primary sex-determining signal in two pyralid moths and highlights a possible pathway for the origin of feminizing piRNAs in Lepidoptera. The similarity in female sex determination between the phylogenetically distant species suggests convergent evolution of feminizing piRNAs in Lepidoptera.

Houseflies provide a good experimental model to study the initial evolutionary stages of a primary sex-determining locus because they possess different recently evolved proto-Y chromosomes that contain male-determining loci (M) with the same male-determining gene, Mdmd. We investigate M-loci genomically and cytogenetically revealing distinct molecular architectures among M-loci. M on chromosome V (MV) has two intact Mdmd copies in a palindrome. M on chromosome III (MIII) has tandem duplications containing 88 Mdmd copies (only one intact) and various repeats, including repeats that are XY-prevalent. M on chromosome II (MII) and the Y (MY) share MIII-like architecture, but with fewer repeats. MY additionally shares MV-specific sequence arrangements. Based on these data and karyograms using two probes, one derives from MIII and one Mdmd-specific, we infer evolutionary histories of polymorphic M-loci, which have arisen from unique translocations of Mdmd, embedded in larger DNA fragments, and diverged independently into regions of varying complexity.

Satellite DNAs (satDNAs) are abundant components of eukaryotic genomes, playing pivotal roles in chromosomal organization, genome stability, and evolution. Here, we combined cytogenetic and genomic methods to characterize the satDNAs in the genomes of Leptidea butterflies. Leptidea is characterized by the presence of a high heterochromatin content, large genomes, and extensive chromosomal reshuffling as well as the occurrence of cryptic species. We show that, in contrast to other Lepidoptera, satDNAs constitute a considerable proportion of Leptidea genomes, ranging between 4.11% and 11.05%. This amplification of satDNAs, together with the hyperactivity of transposable elements, contributes to the substantial genome expansion in Leptidea. Using chromosomal mapping, we show that, particularly LepSat01-100 and LepSat03-167 satDNAs, are preferentially localized in heterochromatin exhibiting variable distribution that may have contributed to the highly diverse karyotypes within the genus. The satDNAs also exhibit W-chromosome accumulation, suggesting their involvement in sex chromosome evolution. Our results provide insights into the dynamics of satDNAs in Lepidoptera genomes and highlight their role in genome expansion and chromosomal organization, which could influence the speciation process. The high proportion of repetitive DNAs in the genomes of Leptidea underscores the complex evolutionary dynamics revealing the interplay between repetitive DNAs and genomic architecture in the genus.

Nature has devised many ways of producing males and females. Here, we report on a previously undescribed mechanism for Lepidoptera that functions without a female-specific gene. The number of alleles or allele heterozygosity in a single Z-linked gene ( BaMasc ) is the primary sex-determining switch in Bicyclus anynana butterflies. Embryos carrying a single BaMasc allele develop into WZ (or Z0) females, those carrying two distinct alleles develop into ZZ males, while (ZZ) homozygotes initiate female development, have mismatched dosage compensation, and die as embryos. Consequently, selection against homozygotes has favored the evolution of spectacular allelic diversity: 205 different coding sequences of BaMasc were detected in a sample of 246 females. The structural similarity of a hypervariable region (HVR) in BaMasc to the HVR in Apis mellifera csd suggests molecular convergence between deeply diverged insect lineages. Our discovery of this primary switch highlights the fascinating diversity of sex-determining mechanisms and underlying evolutionary drivers.

In this study, we performed a cytogenetic analysis of Acanthocephalus lucii specimens from three sites with different levels of environmental pollution. Standard and fluorochrome staining (CMA3/DAPI), fluorescence in situ hybridization (FISH) with 18S rDNA and histone H3 probes, and silver impregnation were performed. Chromosome complements of 2n = 7/8 (male/female), n = 1m + 2sm + 1a (X), and CMA3-positive bands in all chromosomes were found in all three populations. FISH revealed one 18S rDNA locus on the X chromosome and one locus of H3 histone genes on the first chromosome pair. At the intraspecific level, the populations differed in the presence of supernumerary B chromosomes, which were found in all specimens from Zemplínska Šírava and in 89.4% of specimens from the Laborec River, but not at the reference site. The first two sites are considered to be water bodies with high toxin contamination. Based on this fact, we assume an increased frequency of chromosome breaks leading to the formation of DNA fragments that have the potential to form B chromosomes. The present results add to the very limited data on the organization of multigene families in the genome of Acanthocephala and suggest a possible causal link between water pollution and the occurrence of B chromosomes in fish parasites.

Genetic sexing strains (GSS), such as the Ceratitis capitata (medfly) VIENNA 8 strain, facilitate male-only releases and improve the efficiency and cost-effectiveness of sterile insect technique (SIT) applications. Laboratory domestication may reduce their genetic diversity and mating behaviour and hence, refreshment with wild genetic material is frequently needed. As wild males do not carry the T(Y;A) translocation, and wild females do not easily conform to artificial oviposition, the genetic refreshment of this GSS is a challenging and time-consuming process. In the present study, we report the development of a novel medfly GSS, which is based on a viable homozygous T(XX;AA) translocation using the same selectable markers, the white pupae and temperature-sensitive lethal genes. This allows the en masse cross of T(XX;AA) females with wild males, and the backcrossing of F1 males with the T(XX;AA) females thus facilitating the re-establishment of the GSS as well as its genetic refreshment. The rearing efficiency and mating competitiveness of the novel GSS are similar to those of the T(Y;A)-based VIENNA 8 GSS. However, its advantage to easily allow the genetic refreshment is of great importance as it can ensure the mass production of high-quality males and enhanced efficacy of operational SIT programs.

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.

Moths and butterflies (Lepidoptera) have sex chromosome systems with female heterogamety, and 2 models, W-dominance and Z-counting, have been proposed to determine sex. The W-dominant mechanism is well known in Bombyx mori. However, little is known about the Z-counting mechanism in Z0/ZZ species. We investigated whether ploidy changes affect sexual development and gene expression in the eri silkmoth, Samia cynthia ricini (2n = 27♀/28♂, Z0♀/ZZ♂). Tetraploid males (4n = 56, ZZZZ) and females (4n = 54, ZZ) were induced by heat and cold shock, and then, triploid embryos were produced by crosses between diploids and tetraploids. Two karyotypes (3n = 42, ZZZ and 3n = 41, ZZ) were identified in triploid embryos. Triploid embryos with 3 Z chromosomes showed male-specific splicing of the S. cynthia doublesex (Scdsx) gene, whereas 2-Z triploid embryos showed both male- and female-specific splicing. From larva to adult, 3-Z triploids showed a normal male phenotype, except for defects in spermatogenesis. However, abnormal gonads were observed in 2-Z triploids, which showed both male- and female-specific Scdsx transcripts not only in the gonads but also in somatic tissues. Two-Z triploids were thus obviously intersexes, suggesting that sexual development in S. c. ricini depends on the Z:A ratio and not only on the Z number. Moreover, mRNA-seq analyses in embryos showed that relative levels of gene expression are similar between samples with different doses of Z chromosomes and autosome sets. Our results provide the first evidence that ploidy changes disrupt sexual development but have no effect on the general mode of dosage compensation in Lepidoptera.

High-quality haploid genomes corroborate 29 chromosomes and 1 highly conserved synteny of genes in Hyles hawkmoths 2 (Lepidoptera: Sphingidae) 3 4 Anna K. Hundsdoerfer , Tilman Schell , Franziska Patzold , Charlotte J. Wright , 5 Atsuo Yoshido , František Marec , Hana Daneck , Sylke Winkler , Carola Greve , 6 Lars Podsiadlowski , Michael Hiller 2 & Martin Pippel 5, 7 7 8 1 Senckenberg Natural History Collections Dresden, Königsbrücker Landstr. 159, 9 01109 Dresden, Germany 10 2 LOEWE-Centre for Translational Biodiversity Genomics (LOEWE-TBG), Frankfurt 11 am Main, Germany. 12 3 Tree of Life, Wellcome Sanger Institute, Cambridge, CB10 1SA, UK 13 4 Biology Centre of the Czech Academy of Sciences, Institute of Entomology, 14 Branišovská 31, 370 05 České Budějovice, Czech Republic 15 5 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstraße 16 108, 01307 Dresden, Germany 17 6 Centre for Molecular Biodiversity Research, Leibniz Institute for the Analysis of 18 Biodiversity Change, Adenauerallee 127, 53113 Bonn, Germany 19 7 Center for Systems Biology Dresden, Pfotenhauerstr. 108, 01307 Dresden, 20 Germany 21 22 23 24 25 # Corresponding author: anna.hundsdoerfer@senckenberg.de 26 27

Sex chromosome-autosome fusions give rise to neo-sex chromosomes, which provide an insight into early evolution of sex chromosomes and drivers of chromosomal fusions. While sex chromosome-autosome fusions are scarce in vertebrates with female heterogamety (♀ZW/♂ZZ), they are common in moths and butterflies (Lepidoptera), the most species rich group with heterogametic females. This contradicts theoretical model that assumes chromosome fusions to be random and predicts them to be rare in taxa with high chromosome number such as Lepidoptera. In the present study we analyzed sex chromosomes in nine ermine moths of the genus Yponomeuta (Yponomeutidae) and their two outgroups, Teinoptila gutella (Yponomeutidae) and Plutella xylostella (Plutellidae). We employed genomic in situ hybridization to identify sex chromosomes and used a custom designed microarray to identify Z-linked genes. Our results confirmed a multiple sex chromosome system Z1Z2W to be present in T. gutella and all Yponomeuta spp. except for Y. tokyonella . The multiple sex chromosome system resulted from a fusion between the W chromosome and autosome homeologous to the Bombyx mori chromosome 2 (BmChr2). The BmChr2 bears a cluster of genes with ovary-specific expression which suggests that sexually antagonistic selection could have driven fixation of the fusion in a common ancestor of Yponomeuta and Teinoptila genera. We hypothesize that sex chromosome turnover in Lepidoptera could be driven by sexual antagonism.

Background Morphological and traditional genetic studies of the young Pliocene genus Hyles have led to the understanding that despite its importance for taxonomy, phenotypic similarity of wing patterns does not correlate with phylogenetic relationship. To gain insights into various aspects of speciation in the Spurge Hawkmoth ( Hyles euphorbiae ), we assembled a chromosome-level genome and investigated some of its characteristics. Results The genome of a male H. euphorbiae was sequenced using PacBio and Hi-C data, yielding a 504 Mb assembly (scaffold N50 of 18.2 Mb) with 99.9% of data represented by the 29 largest scaffolds forming the haploid chromosome set. Consistent with this, FISH analysis of the karyotype revealed n = 29 chromosomes and a WZ/ZZ (female/male) sex chromosome system. Estimates of chromosome length based on the karyotype image provided an additional quality metric of assembled chromosome size. Rescaffolding the published male H. vespertilio genome resulted in a high-quality assembly (651 Mb, scaffold N50 of 22 Mb) with 98% of sequence data in the 29 chromosomes. The larger genome size of H. vespertilio (average 1C DNA value of 562 Mb) was accompanied by a proportional increase in repeats from 45% in H. euphorbiae (measured as 472 Mb) to almost 55% in H. vespertilio . Several wing pattern genes were found on the same chromosomes in the two species, with varying amounts and positions of repetitive elements and inversions possibly corrupting their function. Conclusions Our two-fold comparative genomics approach revealed high gene synteny of the Hyles genomes to other Sphingidae and high correspondence to intact Merian elements, the ancestral linkage groups of Lepidoptera, with the exception of three simple fusion events. We propose a standardized approach for genome taxonomy using nucleotide homology via scaffold chaining as the primary tool combined with Oxford plots based on Merian elements to infer and visualize directionality of chromosomal rearrangements. The identification of wing pattern genes promises future understanding of the evolution of forewing patterns in the genus Hyles , although further sequencing data from more individuals are needed. The genomic data obtained provide additional reliable references for further comparative studies in hawkmoths (Sphingidae).

ABSTRACT The classical model of sex chromosome evolution has been recently challenged in moths and butterflies (Lepidoptera). According to the current hypothesis, the adoption of a supernumerary chromosome may have driven the transition from the Z0 to the WZ sex chromosome system in females. However, the evolutionary history of the W chromosome remains enigmatic, especially in the early-diverging lepidopteran lineages. In ghost moths of the family Hepialidae, one of the most basal lepidopteran clades, there is conflicting evidence regarding their sex chromosomes. In this study, we aimed to clarify the status of the hepialid W chromosome. Using cytogenetics and genomics, we investigated the karyotype, sex chromosomes, genome size and repeatome of multiple ghost moth species and reconstructed basic phylogenetic relationships in the group. Our data show that Hepialidae have unusually large genomes (reaching up to 1C = 3 Gb) and are the oldest known lepidopteran clade with a W chromosome. However, the W does not form a typical heterochromatin body in polyploid nuclei, known as sex chromatin, previously employed to detect the presence of W chromosomes across Lepidoptera. Moreover, in some species, the W does not exhibit distinct repeat content and can escape detection via methods that rely on W-specific sequences. Analysis of the Z chromosome confirmed highly conserved gene content, arguing for a possible origin of the hepialid W chromosome from a B chromosome. We hypothesize that the mechanism underlying the formation of sex chromatin could be used in future research to study the origin of the W chromosome.

The molecular mechanisms of sex determination in moths and butterflies (Lepidoptera) with female heterogamety (WZ/ZZ) are poorly understood, except in the silkworm Bombyx mori. However, the Masculinizer (Masc) gene that controls male development and dosage compensation in B. mori, appears to be conserved in Lepidoptera, as its masculinizing function was recently confirmed in several moth species. In this work, we investigated the role of the Masc gene in sex determination of the codling moth Cydia pomonella (Tortricidae), a globally important pest of pome fruits and walnuts. The gene structure of the C. pomonella Masc ortholog, CpMasc, is similar to B. mori Masc. However, unlike B. mori, we identified 14 splice variants of CpMasc in the available transcriptomes. Subsequent screening for sex specificity and genetic variation using publicly available data and RT-PCR revealed three male-specific splice variants. Then qPCR analysis of these variants revealed sex-biased expression showing a peak only in early male embryos. Knockdown of CpMasc by RNAi during early embryogenesis resulted in a shift from male-to female-specific splicing of the C. pomonella doublesex (Cpdsx) gene, its downstream effector, in ZZ embryos, leading to a strongly female-biased sex ratio. These data clearly demonstrate that CpMasc functions as a masculinizing gene in the sex-determining cascade of C. pomonella. Our study also showed that CpMasc transcripts are provided maternally, as they were detected in unfertilized eggs after oviposition and in mature eggs dissected from virgin females. This finding is unique, as maternal provision of mRNA has rarely been studied in Lepidoptera.

EDITORIAL article Front. Bioeng. Biotechnol., 05 May 2023Sec. Biosafety and Biosecurity Volume 11 - 2023 | https://doi.org/10.3389/fbioe.2023.1208677

The cytogenetics of Acanthocephala is a neglected area in the study of this group of endoparasites. Chromosome number and/or karyotypes are known for only 12 of the 1,270 described species, and molecular cytogenetic data are limited to rDNA mapping in two species. The standard karyological technique and mapping of 18S rRNA and H3 histone genes on the chromosomes of Acanthocephalus anguillae individuals from three populations, one of which originated from the unfavorable environmental conditions of the Zemplínska Šírava reservoir in eastern Slovakia, were applied for the first time. All specimens had 2n = 7/8 (male/female); n = 1m + 1m-sm + 1a + 1a (X). Fluorescence in situ hybridization (FISH) revealed three loci of 18S rDNA on two autosomes and dispersion of H3 histone genes on all autosomes and the X chromosome. In addition to the standard A chromosome set, 34% of specimens from Zemplínska Šírava possessed a small acrocentric B chromosome, which was always found to be univalent, with no pairing observed between the B chromosome and the A complement. The B chromosome had a small amount of heterochromatin in the centromeric and telomeric regions of the chromosomal arms and showed two clusters of H3 genes. It is well known that an environment permanently polluted with chemicals leads to an increased incidence of chromosomal rearrangements. As a possible scenario for the B chromosome origin, we propose chromosomal breaks due to the mutagenic effect of pollutants in the aquatic environment. The results are discussed in comparison with previous chromosome data from Echinorhynchida species.

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.

Caryophyllideans are intestinal parasites of freshwater fishes, occupying a basal position among the ‘true’ tapeworms. We performed detailed cytogenetic analyses of the well-known caryophyllidean species Caryophyllaeus laticeps . For comparison, we also examined for the first time the chromosomes of Paracaryophyllaeus gotoi , a specific parasite of loaches in China. Both species showed a diploid chromosome number of 2 n = 20, n = 10 m . Chromomycin A 3 (CMA 3 )/diamidino-2-phenylindole (DAPI) staining performed for the first time in the class Cestoda revealed CMA 3 + /DAPI − bands in the pericentromeric regions of the short arms of chromosome pair no. 7 in the karyotype of C. laticeps . Fluorescence in situ hybridization with the 18S rDNA probe confirmed the presence of a single cluster of major rDNA near the centromere on a pair of small chromosomes in both species. These findings support the hypothesis that the ancestral state in the family Caryophyllaeidae is a single interstitial cluster of major rDNA genes and thus one nucleolar organizer region per haploid genome. Our results, which we presented together with literature data plotted on a phylogenetic tree, show stability of caryophyllidean karyotypes at the genus level, but showed differences between genera without a clear phylogenetic signal. The data allowed us to at least formulate a hypothesis about the ancestral haploid chromosome number of n = 10 for the family Caryophyllaeidae and possibly for the sister family Capingentidae. In addition, we compared two populations of C. laticeps from water bodies with different levels of polychlorinated biphenyl contamination, showing a slightly increased incidence of chromosomal abnormalities at the contaminated site.