Luciana Sola

New developments in vertebrate cytotaxonomy III. Karyology of bony fishes: A review

Several years after the introduction of modern cytological techniques in the karyological study of the bony fishes, an attempt may be made to take stock of the results obtained, the problems raised and the difficulties encountered by workers engaged in this type of investigation. The first limitation is set by the value of the karyotype itself. Because it is purely morphological and its immediate adaptive value is unknown, karyotype transformations are difficult to interpret in analyses of populations of the same species or of related species. When related species are being compared, similar karyotypes are considered indicative of a relatively recent separation, whereas widely differing karyotypes are believed to indicate an earlier separation.However, this assumption is not always valid. In the bony fishes a karyotype composed of 48 acrocentric chromosomes occurs not only in related species but also in species that are phylogenetically further apart. In some taxa the karyotype therefore appears ‘neutral’ with respect to both speciation and phyletic evolution (Fig. 1) and it is therefore often impossible to establish a definite correlation between karyotype and karyotype transformations and to identify the processes involved. Although this situation diminishes the immediate value of the karyological evidence, it raises a series of questions that, when set in the right problematical context, make karyological analyses extremely interesting .

Allozyme and microsatellite loci provide discordant estimates of population differentiation in the endangered dusky grouper (Epinephelus marginatus) within the Mediterranean Sea.

The dusky grouper, Epinephelus marginatus, inhabits coastal reefs in the Mediterranean Sea and Atlantic Ocean. A decline in the abundance of this long‐lived protogynous hermaphrodite has led to its listing as an endangered species in the Mediterranean, and heightened management concerns regarding its genetic variability and population substructure. To address these concerns, we analysed genetic variation at seven microsatellite and 28 allozyme loci in dusky groupers sampled from seven areas (for microsatellites) and three areas (for allozymes) in the west‐central Mediterranean. Levels of genetic variability were higher for microsatellites than for allozymes (mean HE = 0.78 and 0.07, respectively), but similar to those observed in other marine fishes with comparable markers. Both microsatellites and allozymes revealed significant genetic differentiation among all areas analysed with each class of marker, but the magnitude of differentiation revealed by allozymes over three locales (FST = 0.214) was greater than that detected with microsatellites over seven areas, or over the three areas shared with the allozyme analysis (FST = 0.018 and ~0, respectively). A large proportion of the allozyme differentiation was due to a single locus (ADA*) possibly influenced by selection, but allozyme differentiation over the three areas was still highly significant (FST = 0.06, P < 0.0001), and the 95% confidence intervals for allozyme and microsatellite FST did not overlap when this locus was excluded. There was no evidence of isolation by distance with either class of markers. Our results lead us to conclude that dusky groupers are not panmictic in the Mediterranean Sea and suggest that they should be managed on a local basis. However, more work is needed to elucidate genetic relationships among populations.

Classical and molecular cytogenetics of the zebrafish, Danio rerio (Cyprinidae, Cypriniformes): an overview.

The zebrafish, Danio rerio, has recently become the model system for the genetic analysis of vertebrate development. This paper reviews the advances in zebrafish cytogenetics, obtained through classical and molecular techniques, which will lead to the assignment of specific linkage groups to specific chromosome pairs in the zebrafish genome project. Several chromosome pairs of the 50-chromosome karyotype of D. rerio were differentially stained by classical staining techniques and additional information has been obtained by molecular cytogenetics. Indeed, the analysis of constitutive heterochromatin by C-banding and base-specific fluorochrome staining had suggested a differential composition of peri- and paracentromeric constitutive heterochromatin. The chromosome mapping of distinct AT- and GC-rich zebrafish satellite DNAs by means of PRINS (Primed in situ) and multicolor FISH (Fluorescence in situ Hybridization) has confirmed this hypothesis, which therefore provided the chromosome localization of 10% of the zebrafish genome. The analysis of nucleolus organizer regions (NORs) by silver staining and by FISH with 18S rDNA has also revealed the existence of variable and inactive NORs, in addition to those on the terminal regions of the long arms of the three NOR-bearing chromosome pairs. Other multicopy genes, such as minor ribosomal genes, or multicopy repeats, such as telomere specific sequences, have now been mapped on zebrafish chromosomes. The latest advancement in zebrafish molecular cytogenetics is the chromosome mapping of single locus genes. Single-copy genes from each of the 25 genetic linkage groups are now being mapped on zebrafish chromosomes by using PAC clones.

CMA3-banding pattern and fluorescence in situ hybridiz ation with 18S rRNA genes in zebrafish chromosomes

This study provides new data on zebrafish chromosomes, obtained from the chromomycin A3-banding pattern and mapping of18S rRNA genes by fluorescence in situ hybridization (FISH). C-banding and Ag-staining were also performed to analyse whether variation in heterochromatin and Ag-nucleolus organizer regions (NORs) exists among various commercially purchased strains. The results provide information on heterochromatin composition and on the existence of inter individual NOR polymorphism and contribute to the construction of an idiogram suitable for gene mapping.

Cytogenetic analysis of global populations of Mugil cephalus (striped mullet) by different staining techniques and fluorescent in situ hybridization

The present paper reports the results of cytogenetic analysis carried out on several scattered populations of the striped mullet, Mugil cephalus, the most widespead among mugilid species. The karyotype was investigated through Ag-staining, C-banding, fluorochrome-staining (chromomycin A3/DAPI) and fluorescent in situ hybridization with rDNA genes. All populations showed the same chromosome number and morphology and no changes were detected in heterochromatin and NORs. Therefore, neither population- nor sex-specific marker chromosomes were identified. In some of the specimens, NOR size heteromorphism was detected. Results are discussed with respect to karyotype and ribosomal cistrons organization and to cytotaxonomic implications.

The chromosomes of 11 species of cyprinidae and one cobitidae from Italy, with some remarks on the problem of polyploidy in the cypriniformes

The present paper gives a detailed description of the morphology of the karyotype of 11 Cyprinidae and 1 Cobitidae indigenous to Italian inland waters. The data relating to Chondrostoma toxostoma, C. soetta, Phoxinus phoxinus, Rutilus rubilio, Barbus meridionalis and Cobitis taenia are new from a karyological point of view; as regards Leuciscus cephalus, L. souffia, Alburnus alburnus, Scardinius erythrophtalmus, Tinca tinca and Barbus barbus the already known diploid numbers are confirmed and the morphology of the karyotypes is specified. The data on the morphology of the karyotype are used in discussing the problem of polyploidization of the chromosomal complement in both the Cyprinidae and the whole order Cypriniformes.

Phylogenetic Analysis of Mediterranean Mugilids by Allozymes and 16S mt-rRNA Genes Investigation: Are the Mediterranean Species of Liza Monophyletic?

The family Mugilidae (Pisces, Mugiliformes) includes species which are present in all tropical and temperate regions. Six species, Chelon labrosus, Mugil cephalus, Liza aurata, L. ramada, L. saliens, Oedalechilus labeo, are commonly found in the Mediterranean. These species have been widely studied through morphological, biochemical, and molecular markers. However, their phylogenetic relationships, and therefore the assumed monophyly of Liza species, still remain unclear. To further investigate this topic, gene–enzyme systems and sequences of the partial 16S rRNA mitochondrial gene were analyzed in Italian samples of all six Mediterranean species. The phylogenetic reconstructions indicated M. cephalus as being the most divergent species and the existence of a main cluster including all the Mediterranean species of Liza and C. labrosus. The parametric bootstrap approach adopted to test alternative phylogenetic hypotheses indicated that the Mediterranean species of Liza do not form a monophyletic group exclusive of Chelon.

Geographic variability in the grey mullet Mugil cephalus: preliminary results of mtDNA and chromosome analyses

Abstract The grey mullet, Mugil cephalus , plays an important role in the fisheries and aquaculture of tropical and subtropical regions of the world. This species is considered cosmopolitan, but its distribution appears peculiar with regard to the coastal ecology of the species. A multidisciplinary study of the geographic variability of this species, through a cytogenetic, molecular and morphometric characterization, was undertaken to detect whether genetically distinct populations occur. The preliminary results from analyses of mitochondrial DNA and of chromosomes of seven different populations around the world are reported. The different populations analysed are well discriminated by mtDNA analyses: samples are clustered in four groups, Mediterranean, East Atlantic, Central Pacific and East Pacific, with a maximum sequence divergence of 3.3%. The karyotype of all the populations studied is uniformly composed of 48 acrocentric chromosomes, and neither heterochromatin distribution nor nucleolus organizer regions allow the identification of chromosomal markers useful in distinguishing these genetically differentiated groups of populations.

FISH-mapping of 18S ribosomal RNA genes and telomeric sequences in the Japanese bitterlings Rhodeus ocellatus kurumeus and Tanakia limbata (Pisces, Cyprinidae) reveals significant cytogenetic differences in morphologically similar karyotypes.

The Japanese rose bitterling, Rhodeus ocellatus kurumeus, and the oily bitterling, Tanakia limbata, were cytogenetically studied by silver (Ag)- and chromomycin A3 (CMA3)-staining, by C-banding and by mapping of the 18S ribosomal genes and of the (TTAGGG)n telomeric sequence. These two representative species of related genera of the subfamily Acheilognathinae show very similar chromosome complements. Nevertheless, significant differences in the chromosomal distribution of nucleolus organizer regions (NORs) and interstitial telomeric sequences were observed. Whereas R. ocellatus kurumeus shows a single NOR-bearing chromosome pair, T. limbata is characterized by a higher number of variable NORs. Multiple telomeric sequence sites were found at the pericentromeric regions of several chromosomes in the rose bitterling. No telomeric sequence sites were detected near centromeres, but they were found to be scattered along the NORs in the oily bitterling. Two karyoevolutive trends might have been identified in the subfamily.

Zebrafish 5S rRNA genes map to the long arms of chromosome 3.

The zebra¢sh, Danio rerio, is a model species in genetic screening for embryonic mutations. In this study, we have localised the 5S ribosomal RNA (5S rDNA) genes. The 5S rDNA was ampli¢ed by polymerase chain reaction using the sequences from the rainbow trout 5S coding region as primers [1]. Two PCR ampli¢cation products of approximately 180 and 500 bp were obtained from four individuals. Two representative clones of different size, pDR-5S19-2 and pDR-5S17-1, were sequenced (Genbank AF213516-213517). Each clone contains two separate regions which form an almost complete 5S coding sequence, joined by a non-transcribed spacer (NTS). Fluorescence in-situ hybridization (FISH) with either puri¢ed PCR ampli¢cation products or the two cloned genes as probes localized the 5S rDNA scattered along most of the long arms of the largest submetacentric chromosome pair (Figure 1), classi¢ed as number 3 [2,3]. The long arms of this chromosome are slightly C [4] and slightly chromomycin A3 (CMA3)-positive [3], as well as late replicating [2]. Present data suggest that the NTS variants, averaging 56% AT, are not responsible for the CMA3-staining pattern. Instead, this could be due to the slight prevalence of GC (around 60%) in the 5S rRNA coding regions and to their high redundancy and interspersion. The 5S rRNA genes constitute the ¢rst singlechromosome marker DNA sequences mapped in the zebra¢sh karyotype. We are grateful to Dr. Alberto Penda© s and Dr. Giovanni Destro-Bisol for their advice in setting up experiments.