Molecular cytogenetics in artificial hybrid and highly polyploid sturgeons: An evolutionary story narrated by repetitive sequences.

We applied comparative genomic hybridization (CGH) and genomic in situ hybridization (GISH) to examine genomes of artificially produced sturgeon hybrids between sterlet, Acipenser ruthenus female (∼120 chromosomes) or Russian sturgeon, A. gueldenstaedtii female (∼240 chromosomes) and a spontaneous triploid Siberian sturgeon A. baerii male (∼360 chromosomes), respectively. The ploidy levels of progenies were analyzed by karyotyping and flow cytometry. We found that the species-specific regions were surprisingly identifiable only on some micro- and small(er) macrochromosomes in hybrid metaphases. We hypothesize that these distinguishable regions are represented by species-specific repetitive sequences driven by more dynamic molecular evolutionary mechanisms. On larger chromosomes, GISH faintly visualized only blocks of pericentromeric and telomeric repetitive sequences, remaining regions were equally shared by both parental species. We concluded that the interspecies hybridization producing viable and even fertile progeny is enabled by the fact that genomes of the species involved are likely divergent at the level of the repetitive sequences only and probably highly conserved in the coding sequences. These small differences of coding sequences are in concordance with previous estimations of relatedness of examined species producing artificial as well as natural hybrids. CGH and GISH represent a challenge in sturgeon cytogenetics as a valuable though technically not simple tool to discriminate chromosomes of parental species in hybrids. The potentials and drawbacks of CGH and GISH application in sturgeons are discussed and further experimental possibilities are proposed.

Evidence for integrity of parental genomes in the diploid hybridogenetic water frog Pelophylax esculentus by genomic in situ hybridization.

The Western Palearctic water frogs Pelophylax ridibundus and P. lessonae were identified as parental (sexual) species and P. esculentus as their interspecific, hybridogenetically reproducing hybrid with hemiclonal heredity. We used genomic in situ hybridization (GISH) to identify parental chromosomes of P.lessonae and P.ridibundus in diploid P. esculentus karyotypes (2n = 26). GISH probes were made by fluorochrome labeling of total genomic DNA extracted from the sexual progenitors. The labeled probe from one species was hybridized to chromosomes of P. esculentus in the presence of excess of unlabeled genomic DNA from the other species. Thus, the P. lessonae probe was blocked by P. ridibundus unlabeled DNA, and vice versa. We successfully discriminated each of the 13 respective parental chromosomes in metaphase complements of the hybrids according to species-specific hybridization signals. GISH enabled us to confirm additional differences between parental chromosomes in size (smaller chromosomes belong to P. lessonae) and in the presence of DAPI-positive centromeric heterochromatin (detected in chromosomes of P. ridibundus, but not in P. lessonae). The fact that no visible intergenomic exchanges were found in metaphase chromosomes of diploid P. esculentus provides important information on the genomic integrity of hemiclonal transmission and supports hybridogenesis as a reproductive mode at the chromosome level for the specimens examined.

Karyotype and chromosomal characteristics of Ag-NOR sites and 5S rDNA in European smelt, Osmerus eperlanus.

Karyotype and cytogenetic characteristics of European smelt Osmerus eperlanus were investigated using different staining techniques (sequential Ag-, CMA3 and DAPI banding) and PRINS to detect 5S rDNA and telomeric sites. The diploid chromosome number was invariably 2n = 56 and karyotype composed of 5 pairs of metacentrics, 9 pairs of subtelocentrics and 14 pairs of subtelo- to acrocentrics. The DAPI-positive heterochromatic regions were found in centromeric positions on bi-armed chromosomes and few acrocentrics. Additionally, some interstitial DAPI-positive bands were identified on three pairs of submetacentric chromosomes. The nucleolar organizer regions (NORs) were detected in the short (p) arms of the largest metacentric pair of chromosomes No. 1. Sequential banding (Giemsa-, AgNO(3) and CMA(3) stainings) revealed NOR sites corresponding to achromatic regions but not associated with CMA(3)-positive blocks of heterochromatin located on either side of NORs. Individuals from the analyzed population had this conspicuous pair of chromosomes always in heterozygous combination. A complex inversion system was hypothesized to be involved in the origin of the observed variation but analysis with telomeric PRINS and PNA-FISH did not reveal any Interstitial Telomeric Sites (ITS). Hybridization signals were confined exclusively to terminal chromosomal regions. The 5S ribosomal sites as revealed by PRINS were found to be invariably located in the short (p) arms of four pairs of subtelocentric chromosomes. Cytotaxonomic comparisons of the present results with the voluminous available cytogenetic data-set from salmoniform and esociformes fishes appear to support the recent view, based on robust molecular-based phylogeny, that salmoniform and osmeriform fishes are not as closely related as previously assumed.

Comparative cytogenetics and chromosomal characteristics of ribosomal DNA in the fish genus Vimba (Cyprinidae).

Karyotypic and cytogenetic characteristics of Vimba vimba and V. elongata were investigated using differential staining techniques (sequential C-banding, Ag- and CMA3-staining) and fluorescent in situ hybridization (FISH) with 28S rDNA probe. The diploid chromosome number in both species was 2n = 50 with 8 pairs of metacentrics, 14 pairs of submetacentrics to subtelocentrics and 3 pairs of subtelo- to acrocentrics. The largest chromosome pair of the complements was characteristically subtelo- to acrocentric. The nucleolar organizer regions (NORs) in both species were detected in the telomeres of a single, middle-sized subtelocentric chromosome pair, a pattern common in a number of other Leuciscinae. FISH with rDNA probe produced consistently positive hybridization signals detected in the same regions indicated by Ag-staining and CMA3-fluorescence. The distribution of C-positive heterochromatin was identical in both species, including a conspicuous size polymorphism of heterochromatic blocks in the largest metacentric and subtelo- to acrocentric chromosomal pairs. No heteromorphic sex chromosomes were detected. A single analyzed individual of V. melanops possessed the same karyotype and NOR phenotype as V. vimba and V. elongata. The apparent karyotype homogeneity and chromosomal characteristics of ribosomal DNA in all three species of the genus Vimba is consistent to that found in most other representatives of the European leuciscine cyprinid fishes.

Sex-chromosome heterochromatin variation in the wood mouse, Apodemus sylvaticus.

Variation in heterochromatin content, as revealed by G- and C-banding, was studied in the sex chromosomes of the wood mouse, Apodemus sylvaticus. The sex-chromosome heterochromatin was also characterized by DAPI staining. Variation in sex chromatin was recorded in extremely large (giant) sex chromosomes in certain individuals and populations. In some individuals, the Y chromosome was the largest element of the complement. Different variants of both the X and Y chromosomes were found within a single population. The variation is therefore a type of population polymorphism and should not be used for taxonomic discrimination.

Chromosomal characteristics of ribosomal DNA in two extant species of North American mudminnows Umbra pygmaea and U. limi (Euteleostei: Umbridae).

The locations and chromosomal characteristics of ribosomal DNA (rDNA) sites in the karyotypes of two extant North American species of mudminnows, Umbra pygmaea and U. limi (2n = 22, NF = 44), were analyzed sequentially by conventional Giemsa staining, Ag staining, CMA(3) fluorescence and fluorescence in situ hybridization (FISH). The nucleolar organizer regions (NORs) were located in the fourth chromosomal pair in both species (pericentromeric region in U. pygmaea and subtelomeric in U. LIMI). These sites were strongly CMA(3)-positive suggesting that the rDNA sites in these species are associated with GC-rich DNA. FISH with a rDNA probe gave consistently positive signals in the same regions detected by Ag-staining and CMA(3)-fluorescence. However, both species also had additional CMA(3)-positive/Ag-negative heterochromatic blocks at pericentrometric regions of several chromosomal pairs (three in U. pygmaea and five in U. limi). FISH revealed additional rDNA clusters in both species. It is hypothesized that a paracentric inversion of the chromosome arm carrying the NORs might be one of the rearrangements differentiating the karyotypes of two North American species. The presence of additional rDNA sites is indicative of more complex rearrangements. The pericentromeric NOR phenotype of Umbra pygmaea is similar to that seen in U. krameri and in the distantly related genus Esox.

Ribosomal genes in Coregonid fishes (Coregonus lavaretus, C. albula and C. peled) (Salmonidae): single and multiple nucleolus organizer regions.

Major rDNA loci, i.e. nucleolus-organizing regions (NORs), were assigned using chromomycin-A3 (CMA3) staining followed by sequential silver (Ag) staining and in situ hybridization (ISH) with a rDNA probe to the chromosomes of the European whitefish (Coregonus lavaretus), the peled (Coregonus peled) and the vendace (Coregonus albula), three closely related coregonine salmonid fishes. One pair of NOR-bearing chromosomes was found in the peled karyotype. Multichromosomal, but stable, locations of rDNA sites on three pairs of chromosomes were observed in the European whitefish karyotype. Multichromosomal polymorphic locations, both in site and number, were observed in the karyotype of the vendace. Several Ag-, CMA3- and ISH-positive regions were found which defined up to seven cytotypes of five NOR-bearing chromosomes. All positive Ag-NORs detected corresponded both to rDNA-ISH- and CMA3-positive signals, which suggests extensive structural polymorphism in the locations of rDNA sites. Stable NOR sites were found at the same location on both homologous elements of the chromosome no. 9 in all individuals, while the remaining NORs were quite variable between individuals, and often present in heterozygous condition. The apparently similar and parallel evolutionary rDNA differentiation patterns in the subfamilies Coregoninae and Salmoninae (family Salmonidae) are observed and discussed.

Extensive polymorphism and chromosomal characteristics of ribosomal DNA in a loach fish, Cobitis vardarensis (Ostariophysi, Cobitidae) detected by different banding techniques and fluorescence in situ hybridization (FISH).

When surveying the karyotype diversity of European loaches of the genus Cobitis to identify species involved in hybrid polyploid complexes, an extensive polymorphism in number and location of NORs was discovered in C. vardarensis using Ag-staining, C-banding, CMA3-fluorescence and fluorescence in situ hybridization (FISH). This species had 2n = 50, the karyotype contained 13 pairs of metacentric, 10 pairs of submetacentric and two pairs of subtelocentric chromosomes. The NOR-bearing chromosomes included one medium-sized metacentric pair with a large CMA3-positive heterochromatic pericentromeric block, one small metacentric as well as one large submetacentric pairs. Ribosomal sites were always located in telomeres of these chromosomes. Each of the pair of NOR-bearing chromosomes occurred in three variants - (1) presence and/or (2) absence of NORs on both homologues and (3) heterozygous combination where only one of the homologues bears NORs. Altogether, 10 different NOR cytotypes from 27 theoretically possible ones were discovered among 20 indviduals examined. The number of NORs ranged from two to five per specimen. The results regarding the number and locations of NORs as revealed by banding techniques were confirmed using FISH with rDNA probe. NOR sites were of CMA3-positive, suggesting that ribosomal sites are associated with GC-rich DNA. Very similar structural polymorphism with multiple NORs is expressed in the Danubian loach C. elongatoides indicating a close relationship between both species.

Chromosomal characteristics of ribosomal DNA in the primitive semionotiform fish, longnose gar Lepisosteus osseus.

The chromosomes of longnose gar, Lepisosteus osseus, an extant representative of early radiation of actinopterygian fishes, were studied using conventional Giemsa-staining, Ag-staining, CMA3-fluorescence and fluorescence in-situ hybridization (FISH). The diploid chromosome number was 2n = 56 and the karyotype contained 11 pairs of metacentric, 6 pairs of submetacentric, 3 pairs of subtelocentric macrochromosomes and 16 microchromosomes. Nearly all macrochromosomes showed large CMA3-positive regions resembling the R-bands of higher vertebrates, indicating extensive distribution of GC-rich DNA along chromosomes. The nucleolar organizer regions (NORs) were located on the end of the short arm of a single small metacentric macrochromosomal pair. These sites were strongly CMA3-positive, suggesting that ribosomal sites are associated with GC-rich DNA. In-situ hybridization (FISH) with a rDNA probe gave consistently positive signals in the same regions detected by Ag-staining and CMA3-fluorescence. The evolutionary conservation of positive CMA3-fluorescence of ribosomal sites in 'holostean' and teleostean fishes is discussed.