Chromosomal characteristics of the temperate notothenioid fish <Emphasis Type="Italic">Eleginops </Emphasis><Emphasis Type="Italic">maclovinus</Emphasis> (Cuvier)

The Falkland’s mullet, Eleginops maclovinus, is the only modern representative of the Sub-Antarctic family Eleginopidae, suborder Notothenioidei. Based on specimens from the Falkland Islands/Islas Malvinas, the Magellan Straits, and the southern coast of Chile, we have established the specific karyotype by conventional cytogenetic methods and have mapped the chromosomal loci of the ribosomal genes by fluorescence in situ hybridization (FISH). With respect to the basal notothenioid family Bovichtidae and to the hypothetical basal condition of the suborder (diploid number = 48, fundamental number = 48), E. maclovinus displays a slightly derived karyotype (diploid number = 48, fundamental number = 54). In contrast to the bovichtids, the 45S and 5S ribosomal DNAs are co-localized to a single chromosome pair. Condensation of the ribosomal genes to a single locus is likely to represent an intermediate stage in the evolution of notothenioid karyology. Features unique to E. maclovinus (e.g., morphology of its large, rDNA-bearing chromosome pair) probably result from divergence during the long evolutionary isolation of the family.     

Ribosomal genes in notothenioid fishes: Focus on the chromosomal organisation

This mini-review makes a survey and a summary of some major issues concerning the chromosomal organisation of ribosomal genes in fish genomes, by using Notothenioidei as the model. The increasing body of information, published during the last two decades on the chromosomal mapping of the two ribosomal genes classes (45S rDNA and 5S rDNA) in notothenioids, makes it possible to recognise the main evolutionary trends across the phylogeny of the group. As one of the major features, the rDNA clusters are organised in a single chromosomal locus in most of the species. This locus is located at different positions along the chromosomes in the basal groups (non-Antarctic Clade), whereas it maintains a strongly conserved location in the cold-adapted species (Antarctic Clade). Important structural changes, leading to the co-localisation of the two ribosomal gene classes, occurred early in the notothenioid phylogeny, perhaps in the common ancestor of the Eleginopidae and Nototheniidae. The cytogenetic evidences indicate that an increased amount of ribosomal genes, organised in two large chromosomal loci, is present in the giant Antarctic fish Dissostichus mawsoni. This gain in rRNA genes is an important genomic change, having possible implications for the fitness of this notothenioid fish that combines large size, pelagic lifestyle and cold-adaptation.     

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.     

Physical mapping of 5S and 45S rDNA loci in pufferfishes (Tetraodontiformes)

Chromosomal features, location and variation of the major and minor rDNA genes cluster were studied in three pufferfish species: Sphoeroides greeleyi and Sphoeroides testudineus (Tetraodontidae) and Cyclichthys spinosus (Diodontidae). The location of the major rDNA was revealed with an 18S probe in two loci for all species. The minor rDNA loci (5S rDNA) was found in one chromosome pair in tetraodontid fishes and four sites located on two distinct chromosomal pairs in C. spinosus. A syntenical organization was not observed among the ribosomal genes. Signal homogeneity for GC/AT-DNA specific fluorochromes was observed in diodontid fish except in the NORs regions, which were CMA₃-positive. Giemsa karyotypes of tetraodontid species presents 2n = 46, having the same diploid value of other Sphoeroides species that have been investigated. On the other hand, the karyotype of C. spinosus, described for the first time, shows 2n = 50 chromosomes (4m + 18sm + 12st + 16a). The foreknowledge of the karyotypic structure of this group and also the physical mapping of certain genes could be very helpful for further DNA sequence analysis.     

Cytogenetics of the bathydraconid fish Gymnodraco acuticeps (Perciformes, Notothenioidei) from Terra Nova Bay, Ross Sea

In the framework of the I.C.E.FISH (International Collaborative Effort on Antarctic Fish Adaptive Evolution) project, during the 1998-1999 season at the Italian shore-based Terra Nova Bay Station, the broad fields of adaptation and evolution of the fish suborder Notothenioidei were tackled through the integration of many disciplines. As a representative contribution of I.C.E.FISH, a cytogenetic study of the bathydraconid fish Gymnodraco acuticeps is reported. The Bathydraconidae is a heterogeneous notothenioid taxon whose intra-family relationships are very uncertain. The conventional karyotype and the location of nuclear ribosomal genes, by means of fluorescence in situ hybridisation, contributed to species-specific characterisation and provided the basis for comparative analyses. The karyotype of G. acuticeps consists of 48 chromosomes (2 metacentric, 2 submetacentric and 44 acrocentric); major ribosomal cistrons correspond to a large region in a single chromosome pair. Mapping of the available karyotypic data on a molecular phylogenetic tree provided information on chromosomal diversification during the cladogenesis of the bathydraconids.     

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.     

Molecular cytogenetic analysis of Haemulidae fish (Perciformes): Evidence of evolutionary conservation

Several fish species belonging to the family Haemulidae present a karyotype consisting of 48 acrocentric chromosomes (FN = 48), and apparently similar chromosomal microstructure, especially in genus Haemulon, representing a striking example of intrafamiliar chromosomal conservation. In this study, a more detailed cytogenetic analysis of the species Conodon nobilis and Pomadasys corvinaeformis was performed using C-banding, Ag-NOR, DAPI/CMA₃ staining, in situ digestion by distinct endonucleases and double-FISH to map the 18S and 5S ribosomal genes. Both species showed a similar karyotypic macrostructure with 2n = 48 acrocentric chromosomes and active ribosomal sites at interstitial position on long arms of chromosomal pair 18 and 24 in P. corvinaeformis and C. nobilis, respectively. These sites were the only CMA₃⁺/DAPI^-regions in the karyotype. Digestion with restriction enzymes revealed a low number of digestion sites in the heterochromatic segments of both species. The data indicate some degree of interspecific evolutionary diversification At the microstructural level, incorporated in a general pattern of extensive karyotypic conservatism. Thus, the interspecific reproductive isolation leading to phyletic diversification apparently occurred without the contribution of conspicuous karyotypic changes.     

Extensive chromosome conservatism in Atlantic butterflyfishes,genus Chaetodon Linnaeus, 1758: Implications for the high hybridization success

Chaetodontidae is a family of marine butterflyfishes phylogenetically derived within the Perciformes, whose representatives are important members of coral reef ecosystems worldwide. Biological aspects of Chaetodontidae have been intensively studied, except for chromosomal analyses. Although previous reports indicate a conserved perciform-like karyotype in butterflyfishes, it remains unclear if this pattern extends to the chromosomal microstructure. New cytogenetic data are presented for two Chaetodontidae species (Chaetodon striatus and Chaetodon ocellatus) from the Western Atlantic, including karyotyping, C-banding, Ag-NOR, CMA₃/DAPI staining, and two-color-FISH for mapping of 18S and 5S ribosomal genes. All populations of both species shared a karyotype with 2n = 48 acrocentric chromosomes, with pericentromeric C-positive heterochromatin and 5S and 18S rDNA located in the same region on the long arms of pairs 10 and 21, respectively. The cytogenetic similarities within and between both Chaetodon species reinforce their remarkable stability also in the chromosome microstructure. Therefore, speciation in this genus was not followed by significant karyotypic changes. Both ecological and chromosomal properties, combined with recent diversification, might be responsible for the apparent karyotype stasis and high hybridization levels found in marine butterflyfishes.     

Comparative cytogenetics of six Indo‐Pacific moray eels (Anguilliformes: Muraenidae) by chromosomal banding and fluorescence in situ hybridization

A comparative cytogenetic analysis, using both conventional staining techniques and fluorescence in situ hybridization, of six Indo‐Pacific moray eels from three different genera (Gymnothorax fimbriatus, Gymnothorax flavimarginatus, Gymnothorax javanicus, Gymnothorax undulatus, Echidna nebulosa and Gymnomuraena zebra), was carried out to investigate the chromosomal differentiation in the family Muraenidae. Four species displayed a diploid chromosome number 2n = 42, which is common among the Muraenidae. Two other species, G. javanicus and G. flavimarginatus, were characterized by different chromosome numbers (2n = 40 and 2n = 36). For most species, a large amount of constitutive heterochromatin was detected in the chromosomes, with species‐specific C‐banding patterns that enabled pairing of the homologous chromosomes. In all species, the major ribosomal genes were localized in the guanine‐cytosine‐rich region of one chromosome pair, but in different chromosomal locations. The (TTAGGG)_n telomeric sequences were mapped onto chromosomal ends in all muraenid species studied. The comparison of the results derived from this study with those available in the literature confirms a substantial conservation of the diploid chromosome number in the Muraenidae and supports the hypothesis that rearrangements have occurred that have diversified their karyotypes. Furthermore, the finding of two species with different diploid chromosome numbers suggests that additional chromosomal rearrangements, such as Robertsonian fusions, have occurred in the karyotype evolution of the Muraenidae.     

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.     

Molecular cytogenetic analysis of Brassica rapa-Brassica oleracea var. alboglabra monosomic addition lines

Interspecific alien chromosome addition lines can be very useful for gene mapping and studying chromosome homoeology between closely related species. In this study we demonstrate a simple but robust manner of identifying individual C-genome chromosomes (C5, C8 and C9) in the A-genome background through the simultaneous use of 5S and 25S ribosomal probes on mitotic and meiotic chromosomes of three different Brassica rapa-B. oleracea var. alboglabra monosomic addition lines. Sequential silver staining and fluorescence in situ hybridisation indicated that 18S-5.8S-25S rRNA genes on the additional chromosome C9 are expressed in the A-genome background. Meiotic behaviour of the additional chromosomes was studied in pollen mother cells at diakinesis and metaphase I. In all of the addition lines the alien chromosome was most frequently observed as a univalent. The alien chromosome C5, which carries an intercalary 5S rDNA locus, occasionally formed trivalents that involved either rDNA- or non rDNA-carrying chromosomes from the A genome. In the case of chromosomes C8 and C9, the most frequently observed intergenomic associations involved the regions occupied by 18S-5.8S-25S ribosomal RNA genes. It is possible that not all such associations represent true pairing but are remnants of nucleolar associations from the preceding interphase. Variations in the numbers and distribution of 5S and 25S rDNA sites between cultivars of B. oleracea, B. oleracea var. alboglabra and B. rapa are discussed.This revised version was published online in April 2005 with corrections to Fig. 2.     

Chromosomal localization of rDNA genes and genomic organization of 5S rDNA in <Emphasis Type="Italic">Oreochromis mossambicus,O. urolepis hornorum</Emphasis> and their hybrid

In this study, classical and molecular cytogenetic analyses were performed in tilapia fishes, Oreochromis mossambicus (XX/XY sex determination system), O. urolepis hornorum (WZ/ZZ sex determination system) and their hybrid by crossing O. mossambicus female × O. u. hornorum male. An identical karyotype ((2n = 44, NF (total number of chromosomal arms) = 50) was obtained from three examined tilapia samples. Genomic organization analysis of 5S rDNA revealed two different types of 5S rDNA sequences, 5S type I and 5S type II. Moreover, fluorescence in situ hybridization (FISH) with 5S rDNA probes showed six positive fluorescence signals on six chromosomes of all the analysed metaphases from the three tilapia samples. Subsequently, 45S rDNA probes were also prepared, and six positive fluorescence signals were observed on three chromosome pairs in all analysed metaphases of the three tilapia samples. The correlation between 45 rDNA localization and nucleolar organizer regions (NORs) was confirmed by silver nitrate staining in tilapia fishes. Further, different chromosomal localizations of 5S rDNA and 45S rDNA were verified by two different colour FISH probes. Briefly, the current data provide an insights for hybridization projects and breeding improvement of tilapias.     

Species relations among wild Arachis species with the A genome as revealed by FISH mapping of rDNA loci and heterochromatin detection

Section Arachis of the homonymous genus includes 29 wild diploid species and two allotetraploids (A. monticola and the domesticated peanut, A. hypogaea L.). Although, three different genomes (A, B and D) have been proposed for diploid species with x = 10, they are still not well characterized. Moreover, neither the relationships among species within each genome group nor between diploids and tetraploids (AABB) are completely resolved. To tackle these issues, particularly within the A genome, in this study the rRNA genes (5S and 18S–26S) and heterochromatic bands were physically mapped using fluorescent in situ hybridization (FISH) in 13 species of Arachis. These molecular cytogenetic landmarks have allowed individual identification of a set of chromosomes and were used to construct detailed FISH-based karyotypes for each species. The bulk of the chromosome markers mapped revealed that, although the A genome species have a common karyotype structure, the species can be arranged in three groups (La Plata River Basin, Chiquitano, and Pantanal) on the basis of the variability observed in the heterochromatin and 18S–26S rRNA loci. Notably, these groups are consistent with the geographical co-distribution of the species. This coincidence is discussed on the basis of the particular reproductive traits of the species such as autogamy and geocarpy. Combined with geographic distribution of the taxa, the cytogenetic data provide evidence that A. duranensis is the most probable A genome ancestor of tetraploid species. It is expected that the groups of diploid species established, and their relation with the cultigen, may aid to rationally select wild species with agronomic traits desirable for peanut breeding programs.     

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.     

基于荧光原位杂交技术的紫薇属植物核型分析

以紫薇(Lagerstroemia indica)、尾叶紫薇(L.caudata)、屋久岛紫薇(L.fauriei)和福建紫薇(L.limii)4种紫薇属植物为材料,利用染色体荧光原位杂交技术(FISH)获得了4种紫薇属植物的有丝分裂中期染色体FISH图及核型参数,分析了45SrDNA在紫薇属植物染色体上的数量和分布特点。结果表明,4种紫薇属植物染色体上均具有1对45SrDNA杂交位点,位于较长染色体短臂的近端部,紫薇、尾叶紫薇、屋久岛紫薇和福建紫薇的核型公式分别为2n=48=2M+24m+22sm、2n=48=30m+18sm、2n=48=2M+20m+26sm和2n=48=2M+32m+14sm,均为2A型。该研究首次获得了紫薇属植物45SrDNA荧光原位杂交核型,为紫薇属植物亲缘关系研究和细胞生物学研究提供了分子细胞学依据。     

Molecular phylogeny and karyotype differentiation in <Emphasis Type="Italic">Paratelmatobius</Emphasis> and <Emphasis Type="Italic">Scythrophrys</Emphasis> (Anura,Leptodactylidae)

Paratelmatobius and Scythrophrys are leptodactylid frogs endemic to the Brazilian Atlantic forest and their close phylogenetic relationship was recently inferred in an analysis that included Paratelmatobius sp. and S. sawayae. To investigate the interspecific relationships among Paratelmatobius and Scythrophrys species, we analyzed a mitochondrial region (approximately 2.4 kb) that included the ribosomal genes 12S and 16S and the tRNAval in representatives of all known localities of these genera and in 54 other species. Maximum parsimony inferences were done using PAUP* and support for the clades was evaluated by bootstrapping. A cytogenetic analysis using Giemsa staining, C-banding and silver staining was also done for those populations of Paratelmatobius not included in previous cytogenetic studies of this genus in order to assess their karyotype differentiation. Our results suggested Paratelmatobius and Scythrophrys formed a clade strongly supported by bootstrapping, which corroborated their very close phylogenetic relationship. Among the Paratelmatobius species, two clades were identified and corroborated the groups P. mantiqueira and P. cardosoi previously proposed based on morphological characters. The karyotypes of Paratelmatobius sp. 2 and Paratelmatobius sp. 3 described here had diploid chromosome number 2n = 24 and showed many similarities with karyotypes of other Paratelmatobius representatives. The cytogenetic data and the phylogenetic analysis allowed the proposal/corroboration of several hypotheses for the karyotype differentiation within Paratelmatobius and Scythrophrys. Namely the telocentric pair No. 4 represented a synapomorphy of P. cardosoi and Paratelmatobius sp. 2, while chromosome pair No. 5 with interstitial C-bands could be interpreted as a synapomorphy of the P. cardosoi group. The NOR-bearing chromosome No. 10 in the karyotype of P. poecilogaster was considered homeologous to chromosome No. 10 in the karyotype of Scythrophrys sp., chromosome No. 9 in the karyotype of Paratelmatobius sp. 1, chromosome No. 8 in the karyotypes of Paratelmatobius sp. 2 and of Paratelmatobius sp. 3, and chromosome No. 7 in the karyotype of P. cardosoi. A hypothesis for the evolutionary divergence of these NOR-bearing chromosomes, which probably involved events like gain in heteochromatin, was proposed.     

Chromosome studies in Orchidaceae: karyotype divergence in Neotropical genera in subtribe Maxillariinae

Here, we study karyotype divergence in the closely related genera Brasiliorchis, Christensonella and Trigonidium belonging to subtribe Maxillariinae of subfamily Epidendroideae (Orchidaceae). We compare karyotypes in 15 species by (1) measuring 1C genome sizes, (2) mapping the distribution of 4′,6‐diamidino‐2‐phenylindole and chromomycin A3 chromosome bands and (3) localizing 5S and 45S nuclear ribosomal DNA (rDNA) sequences using fluorescent in situ hybridization. Recently, phylogenetic studies have been conducted to resolve species and genera relationships in subtribe Maxillariinae. We used these phylogenetic trees to map the cytogenetic characters in an evolutionary framework. This has enabled a better understanding of the patterns of genomic divergence in the group. Genome sizes range from 1C = 1.85 to 4.1 pg. The largest, B. schunkeana, shows evidence of genome upsizing, probably through the acquisition of tandem repeats that now form large 4′,6‐diamidino‐2‐phenylindole‐positive blocks of heterochromatin. Our cytogenetic data are consistent with a base chromosome number of 2n = 40, although Christensonella is characterized by a dysploid reduction in chromosome number to 2n = 36. The number of 5S and 45S rDNA sites is variable between species, consistent with high rates of karyotype divergence. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 29–39.     

Physical mapping of rDNA genes establishes the karyotype of almond

The localisation of ribosomal RNA genes on chromosomes of almond (Prunus amygdalus, 2n = 16) was studied by fluorescence in situ hybridisation. Simultaneous double-colour hybridisation with both 18S–5.8S–25S and 5S rDNA probes demonstrated that all chromosomes can be identified. In spite of the small size, differences in length between chromosomes that hybridised with the same rDNA probe as well as between chromosomes without hybridisation signal are apparent. Chromosomes were ordered in the karyotype according to their length. The 18S-5.8S-25S rDNA genes were detected in subdistal positions of chromosomes 2, 3, and 8. Sites located on chromosomes 2 and 3 carry a higher number of repeats than the site of chromosome 8. The 5S rDNA genes were found proximally located on chromosomes 5 and 7, the signal on chromosome 5 showing higher intensity than the signal on chromosome 7. Chromosomes 1, 4, and 6 show no hybridisation signal.