Karyotype Structure Research Articles

High chromosomal variation in wild horn fly Haematobiairritans (Linnaeus) (Diptera, Muscidae) populations.

The horn fly, Haematobia irritans is an obligate haematophagous cosmopolitan insect pest. The first reports of attacks on livestock by Haematobia irritans in Argentina and Uruguay occurred in 1991, and since 1993 it is considered an economically important pest. Knowledge on the genetic characteristics of the horn fly increases our understanding of the phenotypes resistant to insecticides that repeatedly develop in these insects. The karyotype of Haematobia irritans, as previously described using flies from an inbred colony, shows a chromosome complement of 2n=10 without heterochromosomes (sex chromosomes). In this study, we analyze for the first time the chromosome structure and variation of four wild populations of Haematobia irritans recently established in the Southern Cone of South America, collected in Argentina and Uruguay. In these wild type populations, we confirmed and characterized the previously published “standard” karyotype of 2n=10 without sex chromosomes; however, surprisingly a supernumerary element, called B-chromosome, was found in about half of mitotic preparations. The existence of statistically significant karyotypic diversity was demonstrated through the application of orcein staining, C-banding and H-banding. This study represents the first discovery and characterization of horn fly karyotypes with 2n=11 (2n=10+B). All spermatocytes analyzed showed 5 chromosome bivalents, and therefore, 2n=10 without an extra chromosome. Study of mitotic divisions showed that some chromosomal rearrangements affecting karyotype structure are maintained as polymorphisms, and multiple correspondence analyses demonstrated that genetic variation was not associated with geographic distribution. Because it was never observed during male meiosis, we hypothesize that B-chromosome is preferentially transmitted by females and that it might be related to sex determination.

Molecular and cytogenetic analyses of cryptic species within the Synbranchus marmoratus Bloch, 1795 (Synbranchiformes: Synbranchidae) grouping: species delimitations, karyotypic evolution and intraspecific diversification

The fish species Synbranchus marmoratushas been reported to exist as a species complex due to high intraspecific karyotypic variability in spite of the difficulty or impossibility to distinguish them using morphological traits alone. The goal of this work was to use cytogenetic and molecular methods to determine the species delimitations and understand the karyoevolution ofS. marmoratususing samples collected from distinct Brazilian localities. Among the analyzed specimens, a large degree of cytogenetic variation related to diploid numbers and karyotype structure was observed, with karyotypes showing 2n=42, 44 and 46 chromosomes. In addition, using sequences of three mitochondrial genes, the phylogenetic relationships between every sample with a known karyotype were determined, which revealed significant nucleotide divergence among the karyomorphs. Also, the analyses indicate that chromosomal rearrangements occurred independently within the distinct lineages of S. marmoratuscomplex, which resulted in the appearance of distinct karyotypic variants in a non-linear fashion related to diploid numbers and in the appearance of similar non-homologous chromosomes. Finally, the integration of both molecular cytogenetic and phylogenetic approaches allowed the determination of specific chromosomes possibly involved in rearrangements and a better understanding about the evolutionary processes involved in the differentiation ofSynbranchusgenus.

Karyotype structure of Hypostomus cf. plecostomus (Linnaeus, 1758) from Tapajós River basin, Southern Amazon: occurrence of sex chromosomes (ZZ/ZW) and their evolutionary implications.

Hypostomus is a group of fish with numerical and struc-tural karyotypic variability. Among them, only six species, three of which belong to the Amazon basin, show a sex chromosome. In this study, we present the karyotype structure of Hypostomus cf. plecos-tomus from the Teles Pires river basin in the municipality of Alta Flo-resta, MT. The species has 2n = 68 and the karyotype formula 14m+ 24sm+ 14st+ 16a [fundamental number (FN) = 120] in males and 15m+ 24sm+14st+15a (FN = 121) in females and sex chromosomes ZZ/ZW. Argyrophilic nucleolar organizer regions (AgNORs) were identified in two pairs of chromosomes at different positions: short arm of the pair 21and long arm of the pair 27, matching the signals displayed by 18S FISH and indicating multiple NORs. Analysis of band C detected few blocks of constitutive heterochromatin in the pericentromeric regions of most chromosomes and the telomeric regions of some pairs, includ-ing the nucleolar pair 21. However, large blocks on the long arm of the nucleolar pair 27 still stood out. GC-rich heterochromatin (CMA3) was visualized only coincidently with nucleolar sites. Mapping of 5S rDNA sites with FISH revealed markings in eight chromosomes, demonstrat-ing synteny between the 18S and 5S sites. The data obtained for H. cf. plecostomus are important for taxonomic studies of this Amazon com-plex "H. plecostomus group". The occurrence of sex chromosomes in Amazon species of Hypostomus suggests an evolutionary event that is independent of other species in the group.

Some features of cytogenetic differentiation of striped hamsters, Cricetulus barabensis and Cricetulus pseudogriseus (Rodentia, Cricetidae)

The karyotypes of C. barabensis (2n = 20, NF = 38) and C. pseudogriseus (2n = 24, NF = 38) from different geographic regions of Russia have been studied. Differences in the karyotype structure (chromosome morphology and distribution of heterochromatin) between the two species are described. Variation in heterochromatin staining of the first two autosomal pairs in C. barabensis from different parts of its range has been revealed for the first time.

Reconstruction of gross avian genome structure, organization and evolution suggests that the chicken lineage most closely resembles the dinosaur avian ancestor.

BackgroundThe availability of multiple avian genome sequence assemblies greatly improves our ability to define overall genome organization and reconstruct evolutionary changes. In birds, this has previously been impeded by a near intractable karyotype and relied almost exclusively on comparative molecular cytogenetics of only the largest chromosomes. Here, novel whole genome sequence information from 21 avian genome sequences (most newly assembled) made available on an interactive browser (Evolution Highway) was analyzed.ResultsFocusing on the six best-assembled genomes allowed us to assemble a putative karyotype of the dinosaur ancestor for each chromosome. Reconstructing evolutionary events that led to each species’ genome organization, we determined that the fastest rate of change occurred in the zebra finch and budgerigar, consistent with rapid speciation events in the Passeriformes and Psittaciformes. Intra- and interchromosomal changes were explained most parsimoniously by a series of inversions and translocations respectively, with breakpoint reuse being commonplace. Analyzing chicken and zebra finch, we found little evidence to support the hypothesis of an association of evolutionary breakpoint regions with recombination hotspots but some evidence to support the hypothesis that microchromosomes largely represent conserved blocks of synteny in the majority of the 21 species analyzed. All but one species showed the expected number of microchromosomal rearrangements predicted by the haploid chromosome count. Ostrich, however, appeared to retain an overall karyotype structure of 2n = 80 despite undergoing a large number (26) of hitherto un-described interchromosomal changes.ConclusionsResults suggest that mechanisms exist to preserve a static overall avian karyotype/genomic structure, including the microchromosomes, with widespread interchromosomal change occurring rarely (e.g., in ostrich and budgerigar lineages). Of the species analyzed, the chicken lineage appeared to have undergone the fewest changes compared to the dinosaur ancestor.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1060) contains supplementary material, which is available to authorized users.

New types of wheat chromosomal structural variations in derivatives of wheat-rye hybrids.

BackgroundChromosomal rearrangements induced by wheat-rye hybridization is a very well investigated research topic. However, the structural alterations of wheat chromosomes in wheat-rye hybrids are seldom reported.Methodology/Principal FindingsOctoploid triticale lines were derived from common wheat Triticum. aestivum L. ‘Mianyang11’×rye Secale cereale L. ‘Kustro’. Some progeny were obtained by the controlled backcrossing of triticale with ‘Mianyang11’ and common wheat T. aestivum L. ‘Chuannong27’ followed by self-fertilization. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) using Oligo-pSc119.2-1, Oligo-pTa535-1 and rye genomic DNA as probes were used to analyze the mitotic chromosomes of these progeny. Alterations of wheat chromosomes including 5A, 6A, 1B, 2B, 6B, 7B, 1D, 3D and 7D were observed. 5AL arm carrying intercalary Oligo-pSc119.2-1, Oligo-pTa535-1 or both Oligo-pSc119.2-1 and Oligo-pTa535-1 signals, 6AS, 1BS and 1DL arms containing terminal Oligo-pSc119.2-1 signal, 6BS and 3DS arms without terminal Oligo-pSc119.2-1 signal, 7BS without subtelomeric Oligo-pSc119.2-1 signal and 7DL with intercalary Oligo-pSc119.2-1 signal have been observed. However, these changed wheat chromosomes have not been detected in ‘Mianyang11’ and Chuannong 27. The altered 5A, 6A, 7B and 7D chromosomes in this study have not been reported and represent several new karyotype structures of common wheat chromosomes.Conclusions/SignificanceThese rearranged wheat chromosomes in the present study afford some new genetic variations for wheat breeding program and are valuable materials for studying the biological function of tandem repetitive DNA sequences.

The influence of climatic changes on chromosome inversion polymorphism in a population of malarial mosquito Anopheles messeae (CULICIDAE) in Tomsk

In 2013, the karyotypic structure of the population of malarial mosquito Anopheles messeae in the village of Kolarovo, Tomsk Region, was studied. A level of polymorphism close to the values of the last 20 years has been noted in the studied population. A decrease in autosome heterozygosis and rise of XL0, 2R0, 3R0 and 3L0 chromosomal variant frequencies has been shown. The results have been compared with the data obtained earlier. During 40 years of monitoring, a significant transformation of frequency characteristics of A. messeae inversion polymorphism has been detected, which occurred gradually. During the period from 1974 to 1979, the frequency stability of the concentration of chromosomal inversion has remained stable in the population of Tomsk Region (Kolarovo village), which is typical for the centre of the specific range. Furthermore, during the period until 1992, there was a serious frequency restructuring in favour of inversions that was specific to the south-western part of the range. In the period from 1993 to 2013, this restructuring has been constantly reproducing from one year to the next. Such a tendency correlates well with climate warming.

Chromosomal polymorphism in Rineloricaria lanceolata Günther, 1868 (Loricariidae: Loricariinae) of the Paraguay basin (Mato Grosso do Sul, Brazil): evidence of fusions and their consequences in the population.

Rineloricaria is the most species-rich genus of the Loricariinae (armored catfish) with 65 valid species. However, the karyotype structure is known only for eight species in this group. This study provides cytogenetic data for Rineloricaria lanceolata collected from the upper Paraguay basin (Mato Grosso do Sul). The specimens revealed extensive chromosomal polymorphism constituting 10 karyotypes, which differed in the diploid number (48 to 45 chromosomes) and fundamental number (FN) between 52 and 55. Three types of chromosome variants were observed: a medium-sized submetacentric, a large submetacentric, and a small acrocentric form. Internal telomere sequences were demonstrated by a telomeric (TTAGGG)n probe in submetacentric chromosome variants, suggesting Robertsonian and tandem fusions. Considering the karyotype 2n=48 (4m+2st+42a, FN=54) as the starting point for this polymorphism, these rearrangements contributed to the reduction in diploid number (48-45). Furthermore, a remarkable polymorphism of 18S rDNA resulted in three nucleolus organizer region phenotypes (I, II, and III) with variable frequencies. Interestingly, this polymorphism has remained in the population through interbreeding between specimens, resulting in different viable combinations. The data obtained confirm that diversification/karyotype evolution in Rineloricaria was marked by numerous chromosomal rearrangements which appear to be well tolerated in the panmitic population.

Ploidy-dependent survival of progeny arising from crosses between natural allotriploid Cobitis females and diploid C. taenia males (Pisces, Cobitidae).

Crosses between 21 triploid hybrid Cobitis females and 19 C. taenia (2n=48) males led to viable progeny; whereas no embryonic development was observed in crosses with tetraploid males (4n=98). The ploidy status of 491 progenies randomly selected with flow cytometry (316) or chromosome analysis (175) revealed an average of 55.2% triploids and 44.8% tetraploids, but the ratio of 3n versus 4n fish did change during development. In the first 2days after hatching, approximately 65.1% of tetraploid larvae were observed. Their number decreased significantly to 30.8 and 6.2% on average during 2-5 and 10-15months of life, respectively. The karyotype of tetraploid progeny (4n=98) included 3n=74 chromosomes of the parental female and n=24 of C. taenia male. The number of tetraploid progeny indicated indirectly that about 66% of eggs from 3n females were fertilized with C. taenia. The rest of the eggs developed clonally via gynogenesis or hemiclonally via hybridogenesis into triploids of the same karyotype structure as parental females. We have documented for the first time that (at least under experimental conditions) tetraploids are commonly formed, but are less viable than triploids, and a ratio similar to what is found under natural conditions is finally attained. The current explanation concerning the ploidy and karyotype structure of the progeny confirms that the eggs of 3n Cobitis females are not only capable of maintaining all chromosomes but are also capable of incorporating the sperm genome, thus creating the potential to produce tetraploids.

Karyotype structure and chromosome fragility in the grass Phleum echinatum Host.

Phleum echinatum Host (2n = 2x = 10) is an annual Mediterranean species which differs from other representatives of the genus Phleum by reduced chromosome number, asymmetric karyotype and unusually high amount of DNA in the genome. Chromosomes of this plant were studied using conventional acetic-orcein staining and fluorescence in situ hybridization (FISH). FISH showed the major 35S ribosomal DNA (rDNA) site at the secondary constriction of satellite chromosome (3) and the minor 35S rDNA site near 5S rDNA cluster in the monobrachial chromosome 5. Telomeric repeats were detected at all chromosome ends within secondary constriction in satellited chromosome 3 and at the centromeric regions of chromosomes 1 and 2. Intrachromosomally located telomeric repeats are probably traces of chromosomal rearrangements that have shaped P.echinatum genome; they were prone to breakage which was manifested in chromosome fragmentation. The most distinct telomeric signals, suggesting massive amplification of interstitial telomeric sequences (ITRs), were observed at the nucleolar organizer region (NOR) of the third chromosome pair. Double FISH confirmed co-localization of telomeric and 35S rDNA repeats in this locus characterized by the biggest fragility in the karyotype. Fragile sites of P.echinatum, composed of amplified telomeric repeats, may bear a resemblance to metazoan rare fragile sites enriched in microsatellite repeats.Electronic supplementary materialThe online version of this article (doi:10.1007/s00709-014-0681-5) contains supplementary material, which is available to authorized users.

Sistema de Cromossomos Sexuais Múltiplos X1 X1 X2 X2 /X1 X2 Y na Mosca-das-Frutas Anastrepha sororcula (Diptera: Tephritidae)

Simple sex chromosome systems are disseminated among the Tephritidae of the Anastrepha genus. Anastrepha species are tremendously important given the impact they cause in cultivated fruit plants, mostly in the northeast of Brazil. Cytogenetic analyses initiated in A. sorocula through the examination of the karyotypic structure and C-band exposed the presence of a multiple sex chromosome system X 1 X 1 X 2 X 2 /X 1 X 2 Y in this species. Whereas the female homomorphic karyotype comprises 2n=12, the male karyotype consists of 2n=11, highlighting one large unpaired Y chromosome. The level of karyotypic divergence between the A. sorocula in the northeast - with the presence of a multiple sex chromosome system - and the southeast of Brazil, suggests the existence of reproductive impairments between specimens from the two areas and a possible birth of a new species. Keywords: Alosomes; agriculture pest; insect cytogenetics; heterochromatin.

Karyotype, genetic and morphological variability in North China zokor, Myospalax psilurus (Rodentia, Spalacidae, Myospalacinae)

Both genetic and morphometric methods have been applied for studying of differentiation in North China zokor, Myospalax psilurus, which includes zokors from Russian Far East (Primorsky Krai), North China, Eastern Mongolia and Russian Transbaikalia (Zabaykalsky Krai). There are two subspecies of North China zokor which were described earlier: M. p. psilurus and M. p. epsilanus. The taxonomic statuses of these forms are debated. The diploid number of chromosomes in tested zokors was 64, but there were notable differences in the karyotype structures from Zabaykalsky Krai ('epsilanus': 9-12 M-SM, 10-13 ST, 8-9 A:) and from Primorsky Krai ('psilurus': 9 M-SM, 13-14 ST, 8-9 A). These populations are clearly differed by spectra of blood serum proteins (transferrins): all samples from Primorsky Krai were found to be monomorphic by TF-B, while the samples from Zabaykalsky Krai possessed TF-C only. Marginal popula- tions from Zabaykalsky Krai and Primorsky Krai have significant differences according to RAPD-PCR results of the study. That allows estimating these differences as corresponding to interspecies level. Genetic distance between them is high and it may be compared with the distance between another zokor species, M. aspalax and M. armandii (Pavlenko & Korablev, 2003a; Puzachenko et al., 2009; Tsvirka et al., 2011). A similar pattern of differentiation is detected by sequencing of mitochondrial markers: hypervariable region D-loop and cytochrome b (Tsvirka et al, 2009a). The main morphological differences within the North China zokor were found out in hard palate features (foramina incisive size and construction, length of maxillary bone). According to the available data, zokors from Primorsky Krai are identical to the zokors from the southern part of the species range and are considered as typical M. p. psilurus. The zokors from the Great Khingan region must be considered as M. p. epsilanus. The zokors from Zabaykalsky Krai and Eastern Mongolia are definitely close to M. p. epsilanus by the morphometric data, but have some specific features in foramina incisiva construction, the length of the M 1 and M 2 and the total length of the upper tooth-row. So, we propose to consider this form as a separate subspecies of the North China zokor provisionally. For the further revision, integrated genetic and morphological research of the zokors from the Chinese part of the species range is necessary to be done.

Evidence of incipient speciation in Astyanax scabripinnis species complex (Teleostei: Characidae)

Two populations of the Astyanax scabripinniscomplex, isolated by a waterfall with over 100 meters depth and inhabiting different altitudes of the same river (1850 m a.s.l. and 662 m a.s.l.) were compared in reproductive data, geometric morphometry, tooth morphology, anal-fin rays counts, and karyotype, in order to test the hypothesis of speciation between the two populations. The results in the geometric morphometry analysis showed differences between the populations. Discriminant function analysis (DFA) and canonical variance analysis revealed sexual dimorphism. Secondary sexual characters, such as hooks in the anal fin rays of the males are absent in the lower altitude population. Both populations had the same macro karyotype structure, except for the absence of B chromosomes in the lower altitude population. The fluorescence in situ hybridization showed differences for both markers (18S rDNA and 5S rDNA), and reproductive data suggests pre-zygotic reproductive isolation among the two populations. The data showed the absence of gene flow, indicating that an incipient speciation process has occurred, which leads the two populations to follow independent evolutionary pathways.

C-banding patterns in six taxa ofParis(Melanthiaceae)

C-banding patterns were analyzed in six taxa of Paris: P. polyphylla var. chinensis, P. marmorata, P. luquanensis, P. thibetica, P. polyphylla var. yunnanensis and P. polyphylla var. alba. All plants had a diploid chromosome number of 10. The basic banding consisted of three metacentric and two teloblastic chromosomes. All taxa showed interspecific variation in karyotype structure. P. thibetica had a unique C-banding pattern, having six bands on the long arm of the middle median centromeric chromosomes. P. polyphylla var. yunnanensis had mostly faint C-bands. Chromosome morphology and C-banding patterns have been used for chromosome identification and population divergence detection.

Differentiation of European freshwater bucephalids (Digenea: Bucephalidae) based on karyotypes and DNA sequences

Three species of bucephalid digeneans are known in European freshwater habitats. In this study parthenitae of Rhipidocotyle campanula (Dujardin, 1845) and R. fennica Gibson, Taskinen & Valtonen, 1992, infecting unionid bivalves, and adult Bucephalus polymorphus von Baer, 1827 from perch (Perca fluviatilis L.) were investigated using karyological analysis and DNA sequencing. Our previously published data on genetic characteristics of parthenitae of B. polymorphus from Dreissena polymorpha Pallas were used for comparative analysis. Ribosomal DNA sequences (ITS2 and 28S rDNA) were used to estimate the phylogenetic relationships of the three bucephalid species. Very close phylogenetic affinity between investigated species was revealed; the sequence difference between the two species of Rhipidocotyle Diesing, 1858 (3.78% based on 28S) was comparable with intergeneric differences observed in comparisons of B. polymorphus with R. campanula and R. fennica (3.43% and 4.49% based on 28S, respectively). A high degree of similarity was noted in karyotype structure of the two species of Rhipidocotyle. The diploid chromosome sets consist of 14 bi-armed chromosomes with the first pair of metacentric elements markedly larger than the remaining chromosomes. This chromosome set structure is also specific to B. polymorphus. One specimen of Anodonta anatina L. was infected with tetraploid R. fennica (4n=28). On the basis of karyotype characters and molecular data, species of the genus Rhipidocotyle cannot be recognised as more closely related to each other than to B. polymorphus. Our findings of Lithuanian and Ukrainian populations of unionid mussels infected with R. fennica provide evidence that this species occurs not only in Finland but also in Central and Eastern Europe. Previous reports of B. polymorphus in unionids in these regions are equivocal because of possible confusion with R. fennica.

Differentiation and evolutionary relationships in Erythrinus erythrinus (Characiformes, Erythrinidae): occurrence and distribution of B chromosomes

Erythrinus erythrinus, a Neotropical fish species of the Erythrinidae family, has a wide distribution in South America. Previous cytogenetic analysis showed that this species presents extensive karyotype diversity, with 4 karyomorphs (A-D) described herein. This study investigated the karyotypic structure of 2 new populations of E. erythrinus from the Brazilian Pantanal region, in order to improve the knowledge of the chromosomal diversity in this species. Both populations showed typical characteristics of karyomorph A, with 2n=54 chromosomes (6m+2st+46a), without differentiation between males and females. In addition, identical supernumerary B chromosomes, appearing as double-minute chromosomes, were also found in both populations. These findings suggest the presence of mitotic instability in view of their high intra- and inter-individual numerical variation. The presence of these chromosomes is likely a basal characteristic for this group, since the same kind of Bs also occurs in some other populations and karyomorphs of E. erythrinus. As such, they are important markers of biodiversity found in this nominal species, which probably corresponds to a species complex.

Chromosomal Mapping of Repetitive DNA and Cytochrome C Oxidase I Sequence Analysis Reveal Differentiation among Sympatric Samples of Astyanaxfasciatus (Characiformes, Characidae)

The genus Astyanax is a specious Neotropical fish group, occurring from the south area of the United States to Argentina. During the past few years, intensive studies on representatives of this group have been performed, broadening the genetic, taxonomic and biogeographical knowledge considerably. However, phylogenetic relationships among many species remain uncertain. Significant karyotypic variations occur in A. bimaculatus, A. scabripinnis and A. fasciatus, suggesting the existence of species complexes. Samples with different diploid numbers may be found in A. fasciatus and, in some cases, morphological differentiation among them is not easily verified due to the absence of clear diagnostic characteristics. In the present study, the organization of the A. fasciatus genome from the Tietê River, Mogi das Cruzes, SP, Brazil, was analyzed, describing the karyotype structure and the chromosomal mapping of 18S and 5S ribosomal DNA (rDNA), H1 and H3 histones genes, telomeric DNA, and Rex3 transposable element. Sequencing of the cytochrome c oxidase I (COI) gene was also performed. Chromosomal analyses highlighted the occurrence of 2 distinct karyomorphs in the referred population, one with 2n = 46 and the other with 2n = 48 chromosomes. Furthermore, physical mapping of the repetitive sequences and nucleotide analysis of the COI gene reinforce the genomic differentiation between those karyomorphs and corroborate the existence of 2 different lineages morphologically similar to A. fasciatus living in sympatry.

The influence of a substrate including extracellular matrix proteins on karyotypic variability in two cell lines of Indian muntjac skin fibroblasts

The influence of cell-culture conditions on numerical and structural karyotypic variability has been investigated in two Indian muntjac skin fibroblast “markerless” cell lines, M and MT. The cells were cultivated on a substrate consisting of extracellular matrix proteins (ECMs) synthesized by human mesenchymal stem cells (SC5-MSC). The character of cell distribution for the chromosome number of the cell line M changed after cultivation for 1 and 4 days as compared to control cells, which were cultured on a hydrophilic surface without ECM coating. These changes involve a significant decrease in frequency of cells with the modal numbers of chromosomes and an increase in frequency of cells with lower chromosome numbers. Many new types of additional structural variants of the karyotype (SVK) appear. The MT cell line, differing from M line in the number of homologous chromosomes, exhibited a character of cell distribution similar to that of the M line for the chromosome number for only 1 day after cultivation on the ECM substrate, but not after 4 days under the same culture conditions, when no difference from the control cells was observed. Further cultivation of MT cells for 8 days did not change the character of cell distribution for the chromosome number relative to the control variant. The observed alterations seem to be due to disturbances in the correct chromosome-segregation process, which were caused by an abrupt shift in the cell-culture conditions. Analysis of the structural karyotypic variability revealed a significant increase in frequency of chromosomal aberrations in the M cell line for 1 and 4 days in culture on the ECM substrate as compared to the control cells. The frequency of dicentric chromosomes (telomeric associations) was increased and constituted more than 50% of all chromosome aberrations. No increase in frequency of chromosome aberrations was observed for MT cells cultured under the same conditions. It can be suggested that the differing by the karyotypic structure, but the genetically identical cell lines have different response to the substrate. In contrast to the M line, in the MT line, a fast normalization of numerical karyotypic characteristics and no enhancement of structural karyotypic variability takes place. This provides a possibility to cultivate an MT cell on the given protein substrate while maintaining a balanced karyotypic structure characteristic of MT cell line.

Karyotypes of four species of chironomids (Diptera, Chironomidae) from Northern Italy

Karyotypes of 4 chironomid species were studied: Cryptochironomus obreptans Walker, Criptochironomus sp., Chironomus plumosus Linnaeus and Stictochironomus rosenscholdi Zetterstedt. All these species belong to the subfamily Chironominae. Each species is characterized by the specific karyotype structure. The first species in the list has 2n = 4, while the other 3 species have 2n = 8.

Karyological and taxonomical considerations on Epipactis cupaniana sp. nov. (Orchidaceae) from Sicily

A new species of Epipactis from the Madonie massif (Sicily), E. cupaniana C. Brullo, D’Emerico and Pulv. sp. nov., is described and illustrated. It is a nemoral species, occurring in Holm oak woods, and is characterized by a diploid chromosome complement of 2n = 2x = 38. In the metaphase I, the mother cells of the embryo sac contain 19 bivalents. With respect to morphology and karyotype structure, the new species shows a close relationships with E. helleborine and other allied species, such as E. meridionalis and E. schubertiorum, both occurring in southern Italy and Sicily. However, several morphological features, chromosome size, and differences in the heterochromatin distribution allow for a clear distinction between E. cupaniana and the above‐mentioned species.