Medicago murex with 2n=16 and 2n=14 chromosome complements

Contrary to the generally held view, M. murex Willd. was found to be of 2n=14 constitution in the great majority of accessions. The 2n= 16 type, though distributed sparsely over a wide area, is most likely a relic. Chromosome studies at pachytene indicate that the 2n=14 type probably arose from the 2n=16 type by translocation of the chromosomal material of the smallest chromosome in the 2n=16 complement to one of the longer chromosomes, with loss of the centromere of the small chromosome. The total chromosome lengths of the two types thus remained not significantly different, which is in line with the few small morphological differences which were detectable between the two types. Of considerably phylogenetic interest was the finding of complete inter-sterility of the two M. murex types. Neither M. aculeata 2n= 16 nor M. constricta 2n=14 could be crossed with M. murex of the same chromosome number. Inviable hybrids were obtained on crossing M. murex 2n= 16 with M. turbinata 2n=16. The spiny vs. spineless pod character was investigated in the 2n=16 type and found to be inherited on a monofactorial basis.     

黄藤染色体核型及基因组大小分析

为探究黄藤(Daemonoropsjenkinsiana)染色体核型和基因组的大小,采用体细胞染色体常规制片法与显微摄影技术相结合的方法,对黄藤染色体进行了核型分析,同时以番茄(Lycopersicon esculentum)为内标,应用流式细胞术对黄藤叶片基因组大小、DNA含量和DNA倍性进行了测定。结果表明,黄藤茎尖是理想的染色体制片材料;黄藤的染色体数为2n=24,核型公式为K(2n)=1M+17m+5sm+1st,核型类型为2C;核型不对称系数61.20%;黄藤的DNA含量为1.57 pg,基因组大小为1 539.53 Mb,黄藤的DNA倍性为二倍体(2n)。这是首次报道黄藤的核型和基因组大小,为深入开展黄藤属及其近缘属植物的核型和基因组比较分析提供了参考依据。     

Karyomorphology of Maianthemum sensu lato (Polygonatae, Ruscaceae)

We report results of karyotype analyses using nine species of Maianthemum from China. The species, except M. atropurpureum (with 2n=72), had 2n=36, and the results support the earlier suggestion that Maianthemum has x=18 with 2n=36 in most species. The species examined, however, showed marked differences in karyotype, particularly in the numbers of metacentric, submetacentric, and acrocentric chromosomes as well as in the number of satellites. In addition, we distinguished three different modes based on the number of clear gaps in chromosome length variation: unimodal, bimodal, and trimodal. The unimodal variation (with no gap) was found in M. dahuricum and M. atropurpureum, the bimodal variation (with one gap) in M. tatsienense, and the trimodal variation (with two gaps) in M. bifolium, M. forrestii, M. japonicum, M. henryi, M. purpureum, and M. lichiangense. In the trimodal variation, the positions of the two gaps may differ from species to species. In addition, the frequency of acrocentric chromosomes per complement was generally higher in the trimodal variation than in the unimodal and bimodal variations. Results of our analyses, which had not been clearly presented prior to this, may provide a better understanding of species evolution in the tribe Polygonatae.     

Comparative gene mapping in cattle,Indian muntjac,and Chinese muntjac by fluorescence in situ hybridization

The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n = 6 in the female and 2n = 7 in the male. The karyotypic evolution of Indian muntjac via extensive tandem fusions and several centric fusions are well documented by molecular cytogenetic studies mainly utilizing chromosome paints. To achieve higher resolution mapping, a set of 42 different genomic clones coding for 37 genes and the nucleolar organizer region were used to examine homologies between the cattle (2n = 60), human (2n = 46), Indian muntjac (2n = 6/7) and Chinese muntjac (2n = 46) karyotypes. These genomic clones were mapped by fluorescence in situ hybridization (FISH). Localization of genes on all three pairs of M. m. vaginalis chromosomes and on the acrocentric chromosomes of M. reevesi allowed not only the analysis of the evolution of syntenic regions within the muntjac genus but also allowed a broader comparison of synteny with more distantly related species, such as cattle and human, to shed more light onto the evolving genome organization. For convenience and to avoid confusion we added for each species a three letter abbreviation prior to the chromosomal band location discussed in this paper: BTA, Cattle chromosome; HSA, Human chromosome; MMV, M. m. vaginalis chromosome; MRE, M. reevesi chromosome.     

Comparative cytogenetic analysis of Cestrum (Solanaceae) reveals different trends in heterochromatin and rDNA sites distribution

Species of Cestrum L. (Solanaceae) exhibit large variability in the accumulation of repetitive DNA, although their species possess a stable diploid number with 2n = 16. In this study, we used chromosome banding and fluorescence in situ hybridization (FISH) to characterize the karyotypes and populations of two species, Cestrum nocturnum L. and C. mariquitense Kunth. We also performed a karyotype comparison using 16 idiograms, of which 4 were developed in this study and 12 were obtained from the literature. Cestrum nocturnum displayed more bands than C. mariquitense, but the latter exhibited greater interpopulational variation in the band patterns. There was a tendency for large bands to be located at intercalary/terminal regions and for small bands to be located at intermediate/proximal regions. The idiogram comparison revealed a large variation in the amount, distribution, and size of heterochromatic bands. FISH with rDNA probes revealed stability in the number and location of 5S sites, while 45S was more variable in size and number of sites. Although 45S rDNA always appeared in the subterminal regions, this DNA family exhibited a mobility among chromosome pairs. These data highlight the dynamic of repetitive DNA families in these genomes, as well as the contribution for intra- and interspecific karyotype differentiation in Cestrum.     

Cytotaxonomical analysis of <Emphasis Type="Italic">Momordica</Emphasis> L. (Cucurbitaceae) species of Indian occurrence

Somatic chromosome number and detailed karyotype analysis were carried out in six Indian Momordica species viz. M. balsamina, M. charantia, M. cochinchinensis, M. dioica, M. sahyadrica and M. cymbalaria (syn. Luffa cymbalaria; a taxon of controversial taxonomic identity). The somatic chromosome number 2n = 22 was reconfirmed in monoecious species (M. balsamina and M. charantia). Out of four dioecious species, the chromosome number was reconfirmed in M. cochinchinensis (2n = 28), M. dioica (2n = 28) and M. subangulata subsp. renigera (2n = 56), while in M. sahyadrica (2n = 28) somatic chromosome number was reported for the first time. A new chromosome number of 2n = 18 was reported in M. cymbalaria against its previous reports of 2n = 16, 22. The karyotype analysis of all the species revealed significant numerical and structural variations of chromosomes. It was possible to distinguish chromosomes of M. cymbalaria from other Momordica species and also between monoecious and dioecious taxa of the genus. Morphology and crossability among the dioecious species was also studied. Evidence from morphology, crossability, pollen viability and chromosome synapsis suggests a segmental allopolyploid origin for M. subangulata subsp. renigera. The taxonomic status of the controversial taxon M. cymbalaria was also discussed using morphological, karyological and crossability data.     

Isozyme variation and alleged progenitor-derivative relationships in theMedicago murex complex (Fabaceae)

Chromosomal studies ofMedicago lesinsii (n = 8) and its close relativeM. murex (n = 7) have led to the competing hypotheses that the latter is derived directly from the former, or that both originated from a common ancestor. In contrast to the relatively variableM. murex, M. lesinsii proved to be almost uniform isozymically, except that most populations of Greece differed by one allele from plants of the remainder of the range. This Greek variant ofM. lesinsii was indistinguishable from one of the isozyme variants ofM. murex. The greater level of allozyme variation inM. murex was consistent with its greater ecological amplitude and competitive ability. Also, this suggests thatM. murex is unlikely to have originated directly from the less variableM. lesinsii. The data suggest that either both species originated from a common ancestor, or that the n = 8 species evolved from the n = 7 species, a mode of chromosome evolution not previously hypothesized for the genus.     

Seed-borne alfalfa mosaic virus infecting annual medics (Medicago spp.) in Western Australia

Infection with alfalfa mosaic virus (AMV) was widespread in introduction, evaluation and seed increase plots of cultivars and numbered selections of annual medics (Medicago spp.) in Western Australia; the virus was detected in plots of seven species. When seed stocks from the West Australian annual medic collection harvested in 1984–1986 were sown and seedlings tested, seed-borne AMV was found in all 12 cultivars and in 44/50 numbered selections, belonging to 10 species. Seed transmission rates to seedlings ranged from 0.3–74% and exceeded 5% in 33 seed lots. By contrast, when seedlings of four species grown from seed harvested in 1971–1978 were tested, no AMV was detected; the oldest infected seed stock found was from 1980. In commercial seed stocks of two cultivars released in 1987, the levels of seedling infection with AMV found were 0–0.2% for M. polymorpha cv. Santiago and 526% for M. murex cv. Zodiac. In commercial 1986 seed of M. polymorpha cvs Serena and Circle Valley, AMV was detected in 3/13 and 6/9 stocks respectively; transmission rates to seedlings in infected stocks were 0.1–0.7%. In a survey of 47 annual medic pastures in medium and low rainfall zones of the Western Australian wheat belt in 1987, the virus was detected in leaf samples from only three sites. When inoculated mechanically, AMV systemically infected 11 cultivars and 12 selections belonging to 13 species, but did not infect one selection each of M. aculeata and M. orbicularis. Infected plants in ten species developed only faint mosaics or were symptomlessly infected, but M. littoralis, M. polymorpha and M. tornata developed distinct mottling, reduction in leaf size and, in some instances, leaf deformation and dwarfing. In pot tests, AMV infection decreased herbage and root production (dry wts) of M. polymorpha cvs Serena and Circle Valley by about 30% and 50–60% respectively, but did not decrease herbage production in M. murexcv. Zodiac. In spaced plants growing outside, AMV decreased herbage, root (dry wts) and seed production of M. polymorpha cvs Circle Valley and Santiago by about 60%.     

Speciation and chromosomal evolution of the Palearctic species of chironomids of the genus Sergentia Kieffer (Diptera, Chironomidae): Divergence of the Karyotypes of S. baueri Wülker et al., 1999, S. prima Proviz et al., 1997, S. electa Proviz et al., 1999 and their relationship with the endemic species from Baikal lake

The banding structures of chromosomes of three Palearctic species of chironomids of the genus Sergentia Kieffer, 1922 from the Irkutsk reservoir were comparatively analyzed: S. baueri Wülker et al., 1999 (2n = 8), S. prima Proviz et al., 1997 (2n = 8), and S. electa Proviz et al., 1999 (2n = 6). The S. baueri karyotype was taken as a standard in chromosome mapping. It was established that in contrast to S. baueri and S. prima the S. electa karyotype was formed due to a complex translocation: transfer of chromosome IV in the median part of chromosome III. The sequences of chromosome I bands were found to be completely homologous in all Palearctic species and in S. rara Proviz et al., 1999 (2n = 8) taken as the original species in the group of Baikalian endemics. In both groups, seven homozygous inversions were detected, of which the common one is the inversion in chromosome III. It was assumed on the basis of the results of the karyological analysis that among the Palearctic species S. baueri is most similar to the Baikalian endemics and S. electa is phylogenetically closer to S. prima. Specific features of the chromosome and morphological evolutions of the Sergentia genus species are considered.     

The Symbiotic Requirements of Different Medicago Spp. Suggest the Evolution of Sinorhizobium Meliloti and S. Medicae with Hosts Differentially Adapted to Soil pH

Nitrogen fixing rhizobia associated with the Medicago L. genus belong to two closely related species Sinorhizobium medicae and S. meliloti. To investigate the symbiotic requirements of different Medicago species for the two microsymbionts, 39 bacterial isolates from nodules of eleven Medicago species growing in their natural habitats in the Mediterranean basin plus six historical Australian commercial inocula were symbiotically characterized with Medicago hosts. The bacterial species allocation was first assigned on the basis of symbiotic proficiency with M. polymorpha. PCR primers specific for 16S rDNA were then designed to distinguish S. medicae and S. meliloti. PCR amplification results confirmed the species allocation acquired in the glasshouse. PCR fingerprints generated from ERIC, BOXA1R and nif-directed RPO1 primers revealed that the Mediterranean strains were genetically heterogenous. Moreover PCR fingerprints with ERIC and BOX primers showed that these repetitive DNA elements were specifically distributed and conserved in S. meliloti and S. medicae, clustering the strains into two divergent groups according to their species. Linking the Sinorhizobium species with the plant species of origin we have found that S. medicae was mostly associated with medics well adapted to moderately acid soils such as M. polymorpha, M. arabica and M. murex whereas S. meliloti was predominantly isolated from plants naturally growing on alkaline or neutral pH soils such as M. littoralis and M. tornata. Moreover in glasshouse experiments the S. medicae strains were able to induce well-developed nodules on M. murex whilst S. meliloti was not infective on this species. This feature provides a very distinguishing characteristic for S. medicae. Results from the symbiotic, genotypic and cultural characterization suggest that S. meliloti and S. medicae have adapted to different Medicago species according to the niches these medics usually occupy in their natural habitats.     

A study on sex-determination and karyotypic evolution inTetranychidae

The chromosome complements of 45 species of spider mites (Tetranychidae) were studied, making the total number of species now examined in this family 57, approximately 10% of all species known. The chromosome numbers range fromn=2 ton=7. The modal number of the family is 3 (found in 44% of the species). It is argued that the ancestral number isn=2 (21% of the species).In the more primitive subfamily of theBryobiinae both thelytokous and arrhenotokous species occur, whereas the subfamily of theTetranychinae exclusively exhibits arrhenotoky. The karyotype evolution is discussed in connection with arrhenotoky. It is stated that karyotype information is a useful tool for the spider mite taxonomist.This study was financially supported by the Netherlands Foundation for the Advancement of Tropical Research (W.O.T.R.O.) and the Uyttenboogaart-Eliasen Foundation.     

Morphological,karyological and palynological investigation of endemic Centaurea kurdica Reichardt from Turkey

The purpose of this study was to determine the morphological, morphometrical, karyological and palynological features of the endemic Centaurea kurdica Reichardt species from East Anatolian region. Some morphological features of the species like morphology of capitula, involucra, involucral leaves (phyllaries) and achene have been investigated. The chromosome number of Centaurea kurdica was found as 2n = 18 and haploid karyotype formula 6m+2sm+1M. Metaphase chromosome length ranged from 5.81 to 3.91 μm and the total haploid chromosome length was 41.09 μm. The results of the light microscope investigation of pollen revealed that it is radially symmetrical, isopolar tricolporate and has spheroid-type pollen, and exine ornamentation was also determined as scabrate.     

Reconstruction of ancestral karyotype illuminates chromosome evolution in the genus Cucumis

Karyotype dynamics driven by complex chromosome rearrangements constitute a fundamental issue in evolutionary genetics. The evolutionary events underlying karyotype diversity within plant genera, however, have rarely been reconstructed from a computed ancestral progenitor. Here, we developed a method to rapidly and accurately represent extant karyotypes with the genus, Cucumis, using highly customizable comparative oligo-painting (COP) allowing visualization of fine-scale genome structures of eight Cucumis species from both African-origin and Asian-origin clades. Based on COP data, an evolutionary framework containing a genus-level ancestral karyotype was reconstructed, allowing elucidation of the evolutionary events that account for the origin of these diverse genomes within Cucumis. Our results characterize the cryptic rearrangement hotspots on ancestral chromosomes, and demonstrate that the ancestral Cucumis karyotype (n = 12) evolved to extant Cucumis genomes by hybridizations and frequent lineage- and species-specific genome reshuffling. Relative to the African species, the Asian species, including melon (Cucumis melo, n = 12), Cucumis hystrix (n = 12) and cucumber (Cucumis sativus, n = 7), had highly shuffled genomes caused by large-scale inversions, centromere repositioning and chromothripsis-like rearrangement. The deduced reconstructed ancestral karyotype for the genus allowed us to propose evolutionary trajectories and specific events underlying the origin of these Cucumis species. Our findings highlight that the partitioned evolutionary plasticity of Cucumis karyotype is primarily located in the centromere-proximal regions marked by rearrangement hotspots, which can potentially serve as a reservoir for chromosome evolution due to their fragility.     

我国入侵植物薇甘菊(菊科)的细胞学研究

为了解入侵植物薇甘菊(Mikania micrantha)的细胞学特性,研究了薇甘菊在我国广东东南部4居群和台湾2居群的染色体数目和染色体形态。结果表明,所有居群的薇甘菊染色体数目均为2n=36,第一对染色体为近中部着丝粒染色体,其长臂中部具有次缢痕,显著大于其余染色体。广东深圳大学和深圳水库居群的核型公式为2n=20m+14sm+2st;广东汕头、阳江和台湾屏东居群为2n=22m+12sm+2st;台湾宜兰居群为2n=18m+16sm+2st。此前有报道薇甘菊深圳大学居群的染色体数目为2n=38,推断为制片过程中第一对染色体从次缢痕处断裂而导致的计数错误。因此,薇甘菊虽存在非整倍性和多倍性变化,但在广东东南部和台湾的入侵居群中目前仅发现基于x=18的二倍体(2n=36),该种在这些地区的成功入侵与多倍性无关。     

The use of MES buffer in early nodulation studies with annual Medicago species

The usefulness of 2-(N-morpholino)ethanesulfonic acid (MES) to stabilize the pH of solutions in the range pH 5.5 to 7.0 during early growth and nodulation of annual Medicago (medic) species was investigated.In the first experiment, effects of MES concentrations (0, 1, 2, 5, 10 mM) on growth and nodulation of Medicago polymorpha L. and the stability of solution pH were investigated. In the second experiment we assessed the effect of MES on the growth and nodulation of three medic species (M. truncatula Gaernt., M. polymorpha and M. murex Willd.) at a range of starting pH levels (5.5, 6.0, 6.5 and 7.0) with different concentrations of MES buffer (0, 0.5, 10 mM). In a third experiment, the effects of the concentration of extra cations and the species (K-ion or Na-ion), used as hydroxide to bring solutions containing different MES concentrations to target pH were investigated.MES had no effect on growth at MES concentrations as high as 10 mM but nodule numbers were increased by concentrations of MES of 2 mM or greater. The presence of extra K-ion or Na-ion in solutions had no effect on plant growth or nodulation. With low MES concentrations (0.5 mM), solution pH stability was improved by daily titration and the use of a high nitrate to ammonium ratio (9 : 1) in solution. This combination of strategies maintained pH within a narrow range without effects on annual medic growth or nodulation.     

Chromosome diversity and evolution in Allium (Allioideae,Amaryllidaceae)

Despite extensive literature on the diversity of karyotypes in Allium is available, no attempt to analyse these data together, within a robust phylogenetic framework, has been carried out so far. Thus, we examined patterns and trends in chromosome evolution across the genus. Based on literature survey, karyo-morphometric features for 207 species belonging to 12 subgenera of Allium were obtained. Included in the data-set were basic chromosome number (x), somatic chromosome number (2n), total haploid (monoploid) chromosome length (THL) and three different measures defining karyotype structure: CV_CI, measuring how heterogeneous are centromeres positions in a karyotype, CV_CL and M_CA, quantifying interchromosomal and intrachromosomal karyotype asymmetry, respectively. Trends in karyotype evolution were analysed by phylogenetic regressions and independent contrasts. Mean karyotypes highlighted differences and similarities in karyotype structure between the 12 subgenera. Further differences were noted when the two parameters for analysing karyotype asymmetry were assessed. In addition, by examining the effects of increasing karyotype dimensions (a proxy for genome size) on karyotype structure and asymmetry, it was shown that in Allium species, the DNA was added proportionally to their arm lengths. Overall, p = 8 and somehow intermediate karyotype asymmetry levels seem to represent plesiomorphic character-states in Allium.     

Karyotypes of parasitic Hymenoptera: Diversity, evolution and taxonomic significance

Haploid chromosome numbers (n) of parasitic Hymenoptera (= traditional Parasitica + Chrysidoidea) vary from 2 to 23. However, this range can be subdivided into three intervals with n= 14–23 (less derived parasitic wasps, e.g., some Ichneumonidae and Braconidae as well as Gasteruptiidae), 8–13 (many other parasitic Hymenoptera) and 2–7 (Dryinidae, the majority of Chalcidoidea and some advanced Braconidae, e.g. Aphidiinae). The symmetric karyotype with a relatively high chromosome number (n= 14–17) and the prevalence of biarmed chromosomes must be considered as a groundplan feature of parasitic Hymenoptera. Independent reductions of chromosome numbers (n≤ 10–11) occurred in some groups of the superfamily Ichneumonoidea as well as in the common ancestor of the Proctotrupoidea sensu lato, Ceraphronoidea, Cynipoidea and Chalcidoidea. Further multiple decreases in chromosome numbers (n≤ 4–6) took place in some Braconidae, various lineages of the superfamily Chalcidoidea as well as in the family Dryinidae. Two main trends prevailed in the karyotype evolution of parasitic wasps: the reduction of chromosome numbers (mainly due to tandem fusions and less frequently due to centric ones) and karyotypic dissymmetrization (through an increase in size differentiation of chromosomes and/or in the share of acrocentrics in a chromosome set). Although karyotypic features of parasitic Hymenoptera can be used for solving taxonomic problems at various levels, this method is the most effective at the species level.     

Karyotype analysis of the Russian wheat aphid, <Emphasis Type="Italic">Diuraphis noxia</Emphasis> (Kurdjumov) (Hemiptera: Aphididae) reveals a large X chromosome with rRNA and histone gene families

The Russsian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is a worldwide pest of cereals. Despite its economic importance, little is known about its genome. Here we investigated physical genomic features in RWA by karyotype analysis using differential staining with AgNO₃, CMA₃, and DAPI, by chromosomal localization of ribosomal DNA (rDNA), H3 and H4 histone genes, and the “arthropod” telomeric sequence (TTAGG) _n using fluorescence in situ hybridization (FISH), and by measuring the RWA genome size using flow cytometry. The female karyotype, 2n = 10, is composed of four autosome pairs and a pair of X chromosomes, whereas the male karyotype, 2n = 9, has a single X. The X chromosome is the largest element in the karyotype. All three molecular markers used, i.e., 18S rRNA and both H3 and H4 probes are co-localized at one end of the X chromosome. The FISH probes revealed that the AgNO₃-positive bridge between two prometaphase X chromosomes of females, which is believed to be responsible for the elimination of one X chromosome in aphid oocytes determined to undergo male development, contains clusters of both histone genes, in addition to an rDNA cluster. Interestingly, RWA lacks the (TTAGG) _n telomeric sequence in its genome, in contrast to several previously investigated aphid species. Additionally, we compared female and male genome sizes. The female genome size is 2C = 0.86 pg, whereas the male genome size is 2C = 0.70 pg. The difference between the DNA content in the two genders suggests that the RWA X chromosome occupies about 35% of the female haploid genome (1C = 0.43 pg), which makes it one of the largest sex chromosomes in the animal kingdom.     

Comparative Molecular Cytogenetic Analysis of Two Congeneric Species, <Emphasis Type="Italic">Mugil Curema</Emphasis> and <Emphasis Type="Italic">M. Liza</Emphasis> (Pisces,Mugiliformes), Characterized by Significant Karyotype Diversity

Two congeneric mullet species, Mugil liza and M. curema, respectively with an all-uniarmed and an all-biarmed karyotype, were cytogenetically studied by base-specific fluorochrome staining and FISH-mapping of 45S and 5S ribosomal RNA genes (rDNA) and the (TTAGGG)_n telomeric repeats. Whereas 45S rDNA sites might be homeologus in the two species, 5S rDNA sites are not, as they are localized on chromosome arms of different size. In both species, the (TTAGGG)_n telomeric probe hybridized to natural telomeres and was found scattered along the NORs. In metacentric chromosomes of M. curema, no pericentromeric signals of the telomeric probe were detected. Data are discussed in relation to the karyotype evolution in Mugilidae and to the mechanisms and the evolutionary implications of Robertsonian rearrangements in M. curema.     

Fissions,fusions, and translocations shaped the karyotype and multiple sex chromosome constitution of the northeast‐Asian wood white butterfly,Leptidea amurensis

Previous studies have shown a dynamic karyotype evolution and the presence of complex sex chromosome systems in three cryptic Leptidea species from the Western Palearctic. To further explore the chromosomal particularities of Leptidea butterflies, we examined the karyotype of an Eastern Palearctic species, Leptidea amurensis. We found a high number of chromosomes that differed between the sexes and slightly varied in females (i.e. 2n = 118–119 in females and 2n = 122 in males). The analysis of female meiotic chromosomes revealed multiple sex chromosomes with three W and six Z chromosomes. The curious sex chromosome constitution [i.e. W_1–3/Z_1–6 (females) and Z_1–6/Z_1–6 (males)] and the observed heterozygotes for a chromosomal fusion are together responsible for the sex‐specific and intraspecific variability in chromosome numbers. However, in contrast to the Western Palearctic Leptidea species, the single chromosomal fusion and static distribution of cytogenetic markers (18S rDNA and H3 histone genes) suggest that the karyotype of L. amurensis is stable. The data obtained for four Leptidea species suggest that the multiple sex chromosome system, although different among species, is a common feature of the genus Leptidea. Furthermore, inter‐ and intraspecific variations in chromosome numbers and the complex meiotic pairing of these multiple sex chromosomes indicate the role of chromosomal fissions, fusions, and translocations in the karyotype evolution of Leptidea butterflies.