Comparative Karyotype Characterization And Chromosomal Evolution Of Six Groupers

The karyotypic characteristics of 6 groupers Epinephelus coioides,E. awara,E. fasciatomaculosus,E. akaara,E. bruneus and E. amblycephalus were investigated in the present paper by using Giemsa-staining, C-band, Ag-NORs, DAPI staining and Fluorescence in situ hybridization (FISH) with repetitive sequencing (5S rDNA, 18S rDNA, and (TTAGGG)_n ). The results are as following:1. All six species presented uniform diploid numbers (2n =48), yet differed in their karyotypic formulae: E. coioides is 2n=48, 2sm+46t, NF=50; E. awara is 2n=48, 48t, NF=48;E.fasciatomaculosus is 2n=48, 1st+47t, NF=48; E. akaara is 2n=48, 2sm+8st +38t, NF=50; E. bruneus is 2n=48, 2m+4sm+42t, NF=54; E. amblycephalus is 2n= 48,2m+46t,NF = 50.2. All six species showed species-specific C-band and DAPI-band patterns.Approximately two groups of C-band patterns could be obtained: (1) the constitutiveheterochromatin was observed at the centromeric region on most chromosomes, as in E. coioides and E. bruneus; 2) heterochromatin was absent at the centromeric region of most chromosomes, as in the other four species. And in the second group, still two sub-types could be observed: a) C-banding heterochromatin constantly occupied the total short arm of the bi-armed chromosomes, as in E. fasciatomaculosus and E. akaara; b) C-banding heterochromatin was occasionally observed at the centromeric region on some chromosomes, as in E. awara and E. amblycephalus.DAPI fluorochrome analyses showed that all six species of Epinephelus possess distinct DAPI fluorescent bands except E. coioides. Fluorescent band signals were observed at the centromeric regions of chromosomes in E. awara and E. bruneus, fainter in E. awara and stronger in E. bruneus; the whole short arms of biarmed chromosomes showed bright DAPI fluorescent bands in E. fasciatomaculosus and E. akaara; and fluorescent bands were observed at the centromeric regions of some chromosomes and the subtelomeric region of pair No.24 chromosome in E. amblycephalus.The concurrence of C-positive bands and DAPI fluorescent bands at the pericentromeric regions of most chromosomes in E. bruneus and on the whole short arm of bi-armed chromosomes in E. fasciatomaculosus and E. akaara indicated that at these particular locations, the chromosome structure and bases were similar and therefore could be homeologous.3. Chromosome pair No.1 of E. fasciatomaculosus was heteromorphic. Not only Giemsa-staining results showed that it is consisted of a subtelocentric chromosome and a telocentric chromosome, C-bands and DAPI fluorescent bands also occurred only on one of the homeologous chromosomes-the subtelocentric chromosome. No C-banding or DAPI fluorescent signals were detected on the telocentric chromosome. In the genus of Epinephelus, no such pair of heteromorphic homeologous chromosomes has ever been reported.4. There are inter-species variations in the number and distribution pattern of NORs in the six species of Epinephelus. In E. awara,E. fasciatomaculosus,E. akaara and E. amblycephalus, a single pair of NORs was detected at the paracentromeric region of chromosome pair No.24. In E. coioides, a single pair of NORs was also detected on chromosome pair No.24, but it was located on the telomere of the short arms of chromosome pair No.24. In E. bruneus, the number of NORs varies from 2 to 6, and they always appeared on the telomere of the short arm of biarmed chromosomes (No.2, 9 and 24) .5. Fluorescence in situ hybridization (FISH) technique was used to study the distribution patterns of 5S rDNA and 18S rDNA on chromosmoes of six species of Epinephelus. No inter- or intra- species differences were detected in the distribution pattern of 5S rDNA. In all 6 species, 5S rDNA was located at the paracentromeric region of a pair of middle-sized telocentric chromosome. This indicated that 5S rDNA distribution patterns were conservative and the 5S rDNA bearing chromosomes were homeologous in these 6 species. In five of the six species, a single pair of 18S rDNA hybridization signals could be detected only on chromosome pair No.24, corresponding to silver staining results. The exception was E. bruneus, in which strong hybridization signals were observed on three pairs of chromosomes (No.2, 9 and 24) as well as weak signals on some other chromosomes. 18S rDNA distribution patterns revealed that chromosome pair No.24 were homeologous in these 6 species. 6. The localization of the telomeric sequence (TTAGGG)_n on chromosomes of the six species was also analyzed. In all species, the telomeric sequences, detected by FISH, were restricted to telomeres, and no interstitial sites were observed. However, in E. bruneus, significant increase of hybridization signal intensity at one end of chromosome was observed in 10 pairs of chromosomes. This suggested that at these specific locations, telomeric sequences were repeated many more times than at the other locations.7. To explore the evolutionary trend and process of chromosomes in the Epinephelus during diversification, this study summarized the karyotypic characteristics of six species of groupers and built a phylogenetic tree based on the Giemsa-staining, C-band, Ag-NORs, DAPI and FISH with repetitive sequencing (5S rDNA, 18S rDNA, and (TTAGGG)_n ) experimental results. The groupings of this tree corresponded well with many other former studies in which phylogenetic trees were built based on molecular marker information. Moreover, this karyotypic phylogenetic tree further revealed that species of different clade tend to evolve through different approaches.