The late replication banding patterns of chromosomes are highly conserved in the genera Rana,Hyla, and Bufo (Amphibia: Anura)

Late replication banding and C-banding analyses were performed on the metaphase chromosomes of six species and one subspecies of Palearctic water frogs, genus Rana. Although C-banding patterns showed interspecific or intersubspecific variation, late replication banding patterns of all 13 chromosome pairs of these species were homologous. Minor differences of banding patterns were observed only in chromosomes 2, 7 and 13. Close comparison of the late replication banding patterns with those of three non-water frog species of Rana, and one each of Hyla and Bufo, provided important information on interspecific and intergeneric variability. In the Rana species, the banding patterns of all 13 pairs were homologous except for those some regions of 8 pairs. In one species each of Hyla and Bufo that was examined, the six large chromosome pairs (Nos. 1-6) showed banding homologies. Furthermore, among the Rana, Hyla and Bufo species the four large chromosome pairs (Nos. 1-3, 5 of Rana and Hyla, and Nos. 1, 3–5 of Bufo) shared banding homologies. These results show that the large chromosomes have been highly conserved in the evolutionary history of the three genera.     

Cold-sensitive chromosome regions and heterochromatin inCestrum (Solanaceae):C. strigillatum,C. fasciculatum,andC. elegans

Cold-induced mitotic under-condensation of certain chromosome segments is a rare phenomenon in plants. There are about 11 genera of monocotyledons and only 3 of dicotyledons, where species are known to have such cold-sensitive regions (CSRs). The molecular causes of cold-induced undercondensation are not clear, and no consistent cytochemical characteristics of CSRs are known. Recently we have presented a chromosome banding analysis on CSRs and their relation to constitutive heterochromatin inCestrum parqui (Solanaceae), a species of sect.Cestrum. The present study is concerned with a similar analysis inC. strigillatum of sect.Cestrum, and inC. fasciculatum andC. elegans of sect.Habrothamnus. Chromomycin/DAPI fluorescent double staining, sequential C-banding, and sequential silver impregnation were applied. The species differ in detail but are similar qualitatively. Four classes of heterochromatin can be discriminated. (1) CSRs, with banding properties indicating AT-rich constitutive heterochromatin. After cold-treatment CSR heterochromatin can be silver-impregnated from interphase, as chromocentres, to metaphase, as undercondensed segments. CSRs are subject to frequent heteromorphy. (2) Nucleolar organizers. Two pairs were identified in the karyotypes. Banding properties indicate GC-rich heterochromatin. The nucleolar organizing regions are less evident and their silver-reducing capability reduces during metaphase. (3) Non-nucleolar CMA-positively fluorescing bands. These are minute, polymorphic, positively C-stained, and restricted to one or a few sites in the karyotypes. (4) Indifferently fluorescing, positively C-stained bands. They occur on centromeres, some chromosome ends, and clustered over the chromosome arms. They are mostly very delicate and do not resist harsh banding treatments. — The species investigated here andC. parqui resemble each other qualitatively in heterochromatin classes (1), (2), and (3), but differ much in banding properties of class (4). Therefore, heterochromatin characteristics in the genus are not so uniform as the present results inC. strigillatum, C. fasciculatum, andC. elegans appear to show.     

Distribution of heterochromatin in a reconstructed karyotype of Vicia faba as identified by banding- and DNA-late replication patterns

1) The distribution pattern of heterochromatin characterized by Giemsa-banding, Quinacrine-banding and DNA-late replication has been studied in a reconstructed karyotype of Vicia faba with all chromosome pairs interdistinguishable. 2) By means of two Giemsa-banding methods both an interstitial and a centromeric Giemsa-banding pattern are described. The former one comprehends 14 marker and 18 additional bands of lower but characteristic visualization frequencies. The centromeric Giemsa-banding pattern consists of 7 bands, located in the centromeric and in the secondary constrictions of the metaphase chromosomes. Chromosomes with banding patterns intermediate between the interstitial and the centromeric Giemsa-banding have also been observed. 3) Quinacrine-banding revealed 10–12 brightly fluorescent bands and 1–2 regions of dim fluorescence. Most Q-bands occupy chromosomal positions also characterized by interstitial Giemsa bands. 4) The DNA-late replication pattern, analyzed both by autoradiography and by FPG-technique, revealed 9 late replicating chromosome regions; all of these correspond positionally to the sites of interstitial Giemsa bands. 5) The results are discussed with respect to (a) the relationships between the banding- and the DNA-late replication pattern; (b) banding and heterochromatin characteristics; (c) the correlations between the distribution of chromatid aberrations and special types of heterochromatin. — The patterns of heterochromatin distribution found are in basic conformity with the corresponding patterns reported for the standard karyotype of Vicia faba. The heterochromatin type characterized by both Giemsabanding and late replication is characteristic of all those chromosome regions which after mutagen treatments show up as aberration hot spots. Positional correlations between interstitial Giemsa marker bands and chemically induced isochromatid breaks are indicative of preferential aberration clustering in heterochromatin/euchromatin junctions.     

Constitutive heterochromatin, 5S and 18S rDNA genes in Apareiodon sp. (Characiformes, Parodontidae) with a ZZ/ZW sex chromosome system

Karyotype, sex chromosome system and cytogenetics characteristics of an unidentified species of the genus Apareiodon originating from Piquiri River (Paraná State, Brazil) were investigated using differential staining techniques (C-banding and Ag-staining) and fluorescent in situ hybridization (FISH) with 5S and 18S rDNA probes. The diploid chromosome number was 2n = 54 with 25 pairs of meta- (m) to submetacentric (sm) and 2 pairs of subtelocentric (st) chromosomes. The major ribosomal rDNA sites as revealed by Ag-staining and FISH with 18S rDNA probe were found in distal region of longer arm of st chromosome pair 26, while minor 5S sites were observed in the interstitial sites on chromosome pairs 2 (smaller cluster) and 7 (larger one). The C-positive heterochromatin had pericentromeric and telomeric distribution. The heteromorphic sex chromosome system consisted of male ZZ (pair 21) and female middle-sized m/st Z/W chromosomes. The pericentric inversion of heterochromatinized short arm of ancestral Z followed by multiplication of heterochromatin segments is hypothesized for origin of W chromosome. The observed karyotype and chromosomal markers corresponded to those found in other species of the genus.     

Chromosome banding and essential oils composition of Brazilian accessions of <Emphasis Type="Italic">Lippia alba</Emphasis> (Verbenaceae)

The essential oil components and a karyotypic analysis of five Lippia alba (Verbenaceae) accessions from Brazil were performed with the objective of investigating the variation among different populations. The chemistry analysis allowed the grouping of the accessions in two main chemotypes: neral chemotype (LaCat, LaJF and LaRJ) and linalool chemotype (LaGua and LaVC). However, large karyotypic differences, verified by different chromosome banding techniques, were not detected. The results presented the same chromosome number for all accessions (2n = 30) with 10 metacentric chromosomes and 5 submetacentric. The chromosome banding showed great blocks of constitutive heterochromatin (C-bands) around the centromeric region, which was rich in AT bases (DAPI₊), while the CMA bands were observed only in terminal regions of six chromosomes. Through Ag-NOR techniques, only two active pairs of NORs were detected on the three pairs of secondary constrictions (the NOR activity is discussed). This work relates the pattern of heterochromatin for Lippia alba for the first time.     

Banded karyotype of spined loach Cobitis taenia and triploid Cobitis from Poland

The present work provides new data on the banding pattern of diploid Cobitis taenia and its triploid hybrid females, which belong to the diploid–polyploid complex in the Vistula River tributary. C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ techniques were used to describe the diploid and triploid karyotype. The karyotype of Cobitis taenia of 2n=48 was characterised by one pair of NOR-bearing subtelocentric chromosomes and at least four chromosomes with CMA₃-positive sites. The C-positive heterochromatin was present in the centromeres of almost all chromosomes and the pericentromeric regions of several metacentric and submetacentric chromosomes. The triploid females of 3n=74 had two pairs of chromosomes with active NORs. The NORs-sites were located terminally on two biarmed and two uniarmed chromosomes. The CMA₃-staining revealed at least six A₃-positive sites. The C-banded and A₃-stained triploid karyotype was composed of haploid set of Cobitis taenia and diploid set of unidentified species, so heterochromatin pattern confirmed the possibility of their hybrid origin. The characteristics of banded diploid and triploid karyotype, and the hypothetical karyotype of an unknown species of 2n=50 is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Replication banding in the chromosomes of the European eel (Anguilla anguilla)

The chromosomes of the European eel Anguilla anguilla have been analyzed with a replication banding technique from lymphocyte cultures treated with 5-BrdU. This technique allows us to identify with high resolution the individual chromosome pairs and to differentiate classes of chromatin by the order of replication. The replication banding obtained on the chromosomes of European eel can be related with the structural bands described in this species.     

Chromosome banding in the genusPinus

The chromosomes (2n=24) ofPinus densiflora Sieb. et Zucc. andP. thunbergii Parl. collected from several localities were analyzed on their fluorescent banding patterns by sequential staining with the base specifically binding fluorochromes, CMA and DAPI. In both species, the CMA-bands were localized at the proximal and/or interstitial regions of most of the chromosomes. The CMA-banding pattern was constant among the cells in a plant and was specific to respective species with a few variations. After the CMA and DAPI stainings each chromosome was identified individually. The fluorescent banding patterns of the two species were somewhat similar, but were diferent with respect to in some characters.Pinus thunbergii had two pairs of metacentric chromosomes without CMA-band and two pairs of metacentric chromosomes with an additional thin CMA-band at the interstitial region. The 10th and 11th pairs of chromosomes of both species, which showed similarity in interstitial CMA and DAPI banding and chromosome shape, had the proximal CMA-bands inP. densiflora and DAPI-band inP. thunbergii. The interspecific F₁ hybrid between the two species could easily be identified by the fluorescent banding method.     

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.     

Comparative cytogenetics between the species Passiflora edulis and Passiflora cacaoensis

Passiflora edulis Sims is the most economically important species of the genus Passiflora. A new species was described recently, Passiflora cacaoensis Bernacci & Souza, which displayed morphologic characteristics very similar to P. edulis. Due to the need for delimitation of the two species, karyomorphological and banding analyses were carried out. Both species have 2n = 18, with the same karyotype formula 16 m + 2sm. There was variation between the species regarding the location of satellites and the width of chromosome pairs 2, 4 and 8. C banding revealed the presence of constitutive heterochromatin in the centromeric and telomeric regions of all chromosomes in both species. However, only in P. cacaoensis did chromosomes 3 and 9 have a large quantity of heterochromatin. Fluorochrome banding revealed CMA⁺ bands only in the satellites, but no DAPI⁺ bands. Fluorescence in situ hybridisation (FISH) showed that in P. cacaoensis the rDNA 5S probe is located in a single site in the subterminal position of the long arm of chromosome 5. However, for the rDNA 45S probe, two sites were detected in terminal positions of the long arms of chromosome 7, with a bigger and stronger signal, and of chromosome 9. According to the asymmetry index and the quantity of heterochromatin, P. cacaoensis is a more basal species than P. edulis. The cytogenetic data indicate that P. cacaoensis is closely related to P. edulis, but is a different species.     

Chromosome studies of Saguinus midas niger (Callithricidae,Primates) from Tucurui,Para, Brazil: Comparison with the karyotype of Callithrix jacchus

The karotype of Saguinus midas niger was studied by G-, C-, and nuclear organizer region (NOR)-banding techniques. Variations in C-banding patterns were observed in some chromosomes. The banding patterns obtained were compared with those previously described for Callithrix jacchus. The two species differ by a reciprocal translocation involving pairs 9 and 16; by a paracentric inversion in chromosomes 1, 13, 14, 18, and 22; and by a pericentric inversion in at least four subtelocentric pairs (chromosomes 19, 20, 21, and 22), dislocating the nucleolar organizer region from the small short arm in C. jacchus to the proximal segment of the long arm in S. m. niger (or vice versa). The amount of constitutive heterochromatin is greater in S. m. niger than in C. jacchus, especially in chromosomes 4, 7, and 14. The Y chromosome is smaller in C. jacchus than in S. m. niger.     

Cytogenetic studies in the speciesCercopithecus pogonias (Bennet 1833) andCercopithecus nictitans (Linnaeus 1966)

We describe the banding patterns of the chromosomes of Cercopithecus pogonias(2n = 72) and Cercopithecus nictitans nictitans(2n = 70), the two species which exhibit the highest diploid numbers among the Cercopithecidae, using G-banding, C-banding, and nucleolar organizing region (NOR)-staining techniques. The karyotypes of these two species show a large number of morphological homologies, but several chromosome pairs cannot be matched. It is suggested that translocations and insertions may have been important in the chromosomal evolution of this group.     

Chromosome banding in Amphibia

High-resolution replication banding patterns were induced in prometaphase and prophase chromosomes of Xenopus laevis by treating kidney cell lines with 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT) in succession. Up to 650 early and late replicating bands per haploid karyotype were demonstrated in the very long prophase chromosomes. This permits an exact identification of all chromosome pairs of X. laevis. Late replicating heterochromatin was located by analysing the time sequence of replication throughout the second half of S-phase. Neither heteromorphic sex chromosomes nor sex chromosome-specific replication bands were demonstrated in the heterogametic ZW females of X. laevis. A detailed examination of the BrdU/dT-labelled prometaphases and prophases revealed that the X. laevis chromosomes can be arranged in groups of four (quartets), most of which show conspicuous similarities in length, centromere position, and replication pattern. This is interpreted as further evidence for an ancient allotetraploid origin of X. laevis.by H.C. MacgregorThis paper is dedicated to Prof. Wolfgang Engel on the occasion of his 50th birthday     

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-, AgNO₃ and CMA₃ stainings) revealed NOR sites corresponding to achromatic regions but not associated with CMA₃-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.     

Gene mapping of 28S and 5S rDNA sites in the spined loach Cobitis taenia (Pisces, Cobitidae) from a diploid population and a diploid–tetraploid population

We compare the chromosomal 28S and 5S rDNA patterns of the spined loach C. taenia (2n = 48) from an exclusively diploid population and from a diploid–polyploid population using 28S and 5S rDNA probe preparation and labelling, and fluorescence in situ hybridization (FISH). The 5S rDNA was located in two to three chromosome pairs, and separated from the 28S loci for the males and one female (F1) from the diploid population. Loaches from a diploid–polyploid population, and one female (F2) from the diploid population were characterized by at least one chromosome pair with 5S and 28S overlapping signals. The fishes differed mainly in their number of 28S rDNA loci, located on 3–6 chromosomes. All individuals from both populations were characterized by one acrocentric chromosome bearing a 28S rDNA signal on the telomeres of its long arm. The number of major ribosomal DNA in the karyotype of C. taenia by FISH was always higher than the number of Ag-NORs. Our data confirm the extensive polymorphism of NORs in both populations, as already has been observed in closely related Cobitis species, and less polymorphic 5S rDNA pattern. However, this preliminary result highlights the need for a wider scale study.     

Heterochromatin and chromosome variation in cultivated species ofTulipa subg.Eriostemones (Liliaceae)

The chromosomes of several cultivatedTulipa species belonging to the subg.Eriostemones were examined using conventional staining and C-banding techniques. Most of the species have lightly banded chromosomes with heterochromatin content varying from nil to about 15%. The banding patterns of several taxa are described and discussed in regard to species relationships.     

Giemsa C-banded karyotypes of some diploidArtemisia species

Detailed C-banded karyotypes of eight diploidArtemisia species from three different sections are reported together with preliminary observations on three additional related diploid species. In the majority, the overall amount of banding is relatively low. Bands are mostly confined to distal chromosome regions; intercalary banding is virtually absent and centromeric heterochromatin is also scarce. With the exception ofA. judaica there is in general great uniformity in karyotype structure but considerable interspecific variation in total karyotype length (and hence DNA content) ranging from 44 µm inA. capillaris (2n = 18) to 99 µm inA. atrata (2n = 18).A. judaica (2n = 16; total karyotype length 97 µm) was distinguished by its karyomorphology, with one large non-banded metacentric chromosome pair and 7 pairs of smaller terminally banded meta- or submetacentric chromosomes.     

Analysis of different banding patterns and late replicating regions in chromosomes of Allium cepa,A. sativum and A. nigrum

Different banding procedures and preferential Giemsa staining of late replicating DNA-rich regions were carried out in metaphase chromosomes of three species belonging to different sections of the genus Allium (A. cepa, A. sativum and A. nigrum). The banding, as well as the late replicating patterns were species-specific. The late replicating pattern proved to be, in all cases, the more detailed, and represented the highest percentage of the karyotype differentially stained. Lower percents of the karyotype positively stained were accounted for by C-banding, by modified C-banding and by N-banding. In A. cepa interphase nuclei the pattern of constitutive heterochromatin fitted well with that of late replicating DNA-rich regions, but the coincidence with that revealed by C-banding was only partial. This supports the suggestion that late replicating regions may be considered to be a special category of heterochromatin. On the other hand, it seems that not all C-banded material replicates at the end of the S phase. By the modified C-banding, stained centromere dots or small bands, as well as bands at the NORs are observed.     

Using chromosomal data in the phylogenetic and molecular dating framework: karyotype evolution and diversification in Nierembergia (Solanaceae) influenced by historical changes in sea level

Karyotype data within a phylogenetic framework and molecular dating were used to examine chromosome evolution in Nierembergia and to infer how geological or climatic processes have influenced in the diversification of this solanaceous genus native to South America and Mexico. Despite the numerous studies comparing karyotype features across species, including the use of molecular phylogenies, to date relatively few studies have used formal comparative methods to elucidate chromosomal evolution, especially to reconstruct the whole ancestral karyotypes. Here, we mapped on the Nierembergia phylogeny one complete set of chromosomal data obtained by conventional staining, AgNOR‐, C‐ and fluorescent chromosome banding, and fluorescent in situ hybridisation. In addition, we used a Bayesian molecular relaxed clock to estimate divergence times between species. Nierembergia showed two major divergent clades: a mountainous species group with symmetrical karyotypes, large chromosomes, only one nucleolar organising region (NOR) and without centromeric heterochromatin, and a lowland species group with asymmetrical karyotypes, small chromosomes, two chromosomes pairs with NORs and centromeric heterochromatin bands. Molecular dating on the DNA phylogeny revealed that both groups diverged during Late Miocene, when Atlantic marine ingressions, called the ‘Paranense Sea’, probably forced the ancestors of these species to find refuge in unflooded areas for about 2 Myr. This split agrees with an increased asymmetry and heterochromatin amount, and decrease in karyotype length and chromosome size. Thus, when the two Nierembergia ancestral lineages were isolated, major divergences occurred in chromosomal evolution, and then each lineage underwent speciation separately, with relatively minor changes in chromosomal characteristics.     

Fluorescent chromosome banding in the cultivated species ofCapsicum (Solanaceae)

Fluorochrome chromosome banding is applied for the first time to 15 samples of five cultivatedCapsicum species, all with 2n = 24, and allows a detailed analysis of the karyotypes (Tables 2–3, Fig. 8). Banding patterns differ between cytotypes, species and groups, reflecting the dynamics of chromosomal differentiation and evolutionary divergence. Taxa have from 1 to 4 NOR-bearing satellited chromosome pairs and exhibit increasing numbers of terminal (rarely intercalary and indistinct centromeric) heterochromatic fluorescent bands. Amounts of heterochromatin (expressed in % of karyotype length) increase from the group withC. annuum (1.80–2.88),C. chinense (3.91–5.52), andC. frutescens (5.55) toC. baccatum (7.30–7.56), and finally toC. pubescens (18.95). In all taxa CMA+DAPI—(GC-rich) constitutive heterochromatin dominates, onlyC. pubescens has an additional CMAo DAPI+ (AT-rich) band. The fluorochrome bands generally (but not completely) correspond to the Giemsa C-bands. Structural heterozygosity can be demonstrated but is not prominent. The independent origin of at least three evolutionary lines leading to the cultivated taxa ofCapsicum is supported.Chromosome studies inCapsicum (Solanaceae), V. For the fourth part seeMoscone & al. 1995.