Molecular Genetics Study On Hybirdization Between Paralichthys Olivaceus (♀) And P. Dentatus (♂)and Phylogenetic Relationships Of 18 Pleuronectiformes Fishes

Mitochondrial DNA and ribosomal DNA have long been considered useful marker for comparative evolutionary and phylogenetic studies. In this study, Partial 16S rRNA gene sequences, Internal transcribed spacer one(ITS1)and Internal transcribed spacer two(ITS2)regions were analysed using female Paralichthys olivaceus , male P. dentatus and their hybrids to exploring the phylogenetic utility of these regions for Pleuronectiformes fishes. Previous studies of phylogenetic relationships of Pleuronectiformes fishes have relied mostly on morphological data.But there are many controversial problems with morphologic studies.Their phylogeny and classification are still far from being firmly established.More recently,molecular approaches have combined with morphological data expected to provide interesting results on such interrelationships of Pleuronectiformes taxa.In this study,the first ribosomal internal transcribed spacers (ITS1) of nuclear ribosomal DNA from eighteen species of Pleuronectiformes fishes were used for phylogenetic analysis.The major results in this paper were presented as follows: 1. ITS1 and ITS2 regions of mitochondrial DNA were obtained from female Paralichthys olivaceus , male P. dentatus and their hybrids. Intraindividual, intraspecific and interspecific sequence variation were analysed. The results were partially congruent between ITS1 and ITS2. Using molecular approaches, it is confirmed that these hybrids are true filial generation of these two flounders, not the gynogenesis of p. olivaceus was induced by heterogeneous sperm of P. dentatus Two divergent ITS1 and ITS2 repeat types were sampled in P. olivaceus, One repeat type was the same as P. dentatus These types of ITS1 and ITS2 maybe represent the ancient paralogs. This paper also demonstrate that in artifical flounder gybrids , the expected additive pattern was observed and two repeat types are maintained but undergo various degrees of recombination.2. Partial 16S rRNA gene sequences of mitochondrial DNA were obtained from female Paralichthys olivaceus , male P. dentatus and their hybrids. The alignment length consists of 590 sites.The variable sites were 28 and all the sites were parsimony informative. One conserved primer pair was used to amplify partial 16S rRNA gene of all samples containing hybrid flounders and their parents. The sequence was identical in all the specimens of hybridization and three P. olivaceus samples analysed. Male-transmitted mitochondrial DNA was not detected in hybrid flounders. The results indicate that mitochondrial DNA is inherited maternally in hybridization between Paralichthys olivaceus and P. dentatus.3. The ITS1 region were sequenced from 9 flatfish species of family Psettodidae: Pseudopleuronectes yokohamae, P. herzensteini, Limanda limanda, Pleuronichthys cornutus, Cleisthenes herzensteini, Kareius bicoloratus, Verasper variegates, Microstomus achne, Platichthys stellatus. The ITS1 region provided limited insight within this family. Phylogenetic analysis show that Pleuronichthys cornutus arose early. Congruence with morphological data, Pseudopleuronectes yokohamae, P. herzensteini and Limanda limanda grouped together. Intraindividual, intraspecific and interspecific sequence variation in family Psettodidae were analysed. Significant interspecific length variation of ITS1 region was observed, the spacers ranged in length from 585bp to 777bp. Two repeats types were exhibited in Platichthys stellatus. This paper shows that phylogenetic resolution of Psettodidae fishes will require additional data and new or combined methodologies.4. In this study, besides C.purpureomaculatus , Cynoglossus oligolepis, C.sinicus, C.abbreviatus, C.lighti and Paraplagusia japonica all five species exhibit two divergent ITS1 repeat types. One type is also observed in Platichthys stellatus and Psettodes erumei. The other ITS1 repeat type from six species of the subfamily Cynoglossinae were used for phylogenetic analysis.Significant length variation of ITS1 region was observed in Cynoglossinae species,the spacers ranged in length from 404bp to 744bp.Regarding the length of the whole ITS1 region,a clade-specific pattern was revealed with three well-defined groups.The first group had a length of 404-405bp (containing C. purpureomaculatus and C. abbreviatus),C. sinicus and P. japonica belong to the second group (having a length of 462-463bp),and the last group corresponded to C. oligolepis and C. lighti(741-744bp).The alignment length of ITS1 consists of 756 sites.The variable sites were 286 and 285 sites were parsimony informative.High variability was found in the ITS1 region and conserved motifs were very short except one motif (ACCCAACTCTCC) which was shared by all sequences.Nucleotide frequencies of the sequences showed a relatively high GC content ranging from 66.2% to 69.5%,with an average of 67.2%.The mean distances within groups were 0.00248,0.00217and 0.00169,and the mean distances between groups were 0.38453(between groupâ… and groupâ…¡),0.90879(between groupâ…¡and groupâ…¢)and 0.86845(between groupâ… and groupâ…¢)respectively.The distances within groups were very low compared with those between groups,suggesting that the species in the same group have very close relationships.DNA data were analysed using neighbour-joining (NJ) and maximum parsimony (MP) methods to infer phylogenetic relationships within the subfamily.Combined morphological,nuclear DNA and mitochondrial DNA data to analyse,C. abbreviatus and C. purpureomaculatus should be the same species with different morphs.Incongruence with morphological perspective,C. sinicus of genus Cynoglossus and P. japonica (the only one species under genus Paraplagusia) formed a clade with high bootstrap values.C. oligolepis and C. lighti grouped together.5. Historical relationships of 17 Pleuronectiformes fishes and 4 relatived species containing Engraulis japonicus, Auxis rochei, Siniperca chuatsi, Oncorhynchus mykiss were analysed based on ITS1 sequences. The results show that the fishes of Perciformes have closer relationships with Pleuronectiformes fishes. The order Pleuronectiformes is monophyletic, based on ITS1 region.