Phylogenetic Study Of Scombridae Fishesand Molecular Phylogeography Of K.pelamis Inferred From Mitochondrial DNA

The family Scombridae is an epipelagic and generally migratory marine fishbelongs to Perciformes, Scombroidei, which iswidespread in tropical, subtropical andtemperate marine areas. It contains15genera and about54species, and most of themwith a high economic value for commercial fisheries. Currently, molecular maker,also known as the basic method, is widely used in the molecular phylogenetics,genetic diversity and the mechanism of the speciesformation.In this study, firstly, we amplified the mitochondrial genomes and preliminarilyestablished the amplification method of Rastrelliger kanagurta. Some analyses likethe genome organization and gene arrangement of the mitogenomes, phylogeneticanalysis with mtDNA complete sequences and protein-coding sequences to exploringthe the phylogenetic relationship among20Scombridae fishes, would be representedin this paper. What’s more, the mitochondrial Cytb gene and control region ofKatsuwonus pelamis also be sequenced for the genetic diversity and populationstructure and evolution history of eight K.pelamis populations, and providing somescientific data for stock management and the development and utilization ofK.pelamis resources. The main comment are as follows:1. Mitochondrial genomes of R.kanagurtaWe designed10sets of primers to amplifysegments of the completemitochondrial genome of R.kanagurta. The amplified DNA products directlysequenced with an ABI3730automated DNA sequencer (Applied Biosystems, FosterCity, CA, USA) following the manufacturer’s protocol. The R.kanagurtamitochondrial genomes were reported for the first time and there wase16,537bp inlength with the accession numbers of JX524134. The organization of R.kanagurtamitochondrial genomes is similar toother reported bony fishes, whichcontains37genes, including13protein-coding genes,2ribosomal RNAs, and22transfer RNAs, and a major non-coding control region. Some overlapping and intervalwere found in R.kanagurta mitochondrial genomes, which maybe the secret why themitochondrial genomes in fishes with a high coding efficiency. There has lowerproportion of G nucleotide in the R.kanagurta mitochondrial genomes,especially inthe codon usage.All protein-coding genes were encoded on the H-strand,except ND6gene which was encoded on L-strand,. GTG as an initiation codon was found only inCOXI gene, all genes started with an standard codon ATG.2. Phylogenetic analysis in20Scombridae fishesWe got the other19Scombridae fishes mitochondrial genome seuqences fromGenBank. The Transition/Transversion (R values) ranged from2.07to4.59. TheKa/Ks values for13protein coding genes of mitochondrial genomes of20Scombridae fishes were analyzed with the PAML4.3software is far less than1.0,which appears to13protein coding genes without the positive selection.From thesemitochondrial sequence datas, three kinds of data sets were analyzed:(1) the totalcomplete mitochondrial nucleotide sequences;(2) total protein-coding nucleotidesequence;(3) each single protein-coding nucleotide sequence. All the data sets wasaligned using clustal X software and reconstructed phylogenetic relationship withneighbor-joining (N-J) in MEGA4.0and bootstrap analysis was performed with1000replications respectively. According to the test results, mitochondrial protein-codinggenes can be roughly divided into three groups, good (ND5，ND4，COXI and Cytb)、medium(ND2, COXI, ND1,COXIII and ND6) and poor(ND3,ND4L,ATP8,and ATP6).The reconstructed phylogenetic relationship showed Scombridae fishes wereprimitive.3. Genetic diversity and molecular phylogeography of K.pelamisThe PCR products of Cytb and D-loop sequences of K.pelamis were directlysequenced. In the Cytb gene,we defined132distinct haplotypes among149K.pelamisCytb sequences.The haplotype diversity (Hd)in our study ranged from0.964to1.000,and nucleotide diversity (π) ranged from0.00835to0.01167. Geneticdistances between populations ranged from0.008to0.011and from0.008to0.012within populations. Pairwise Fst statistics ranged from-0.05373to0.09712suggestedthat lower level of differentiation and no significance was observed in8 K.pelamispopulations. The hierarchical analyses such as molecular variance(AMOVA), Neighbor-joining (NJ) tree and median-joining (MJ) network analysisexhibited that:8K.pelamis populations formed a panmictic population. Neutral testand Mismatch analysis indicated that all the K.pelamis populations have notexperienced a population expansion.In the D-loop sequence analyses,146distinct haplotypes were detected in149K.pelamis D-loop sequences. Haplotype diversity (Hd) in this study ranged from0.964to1.000, and nucleotide diversity (π) ranged from0.03765to0.04484. Geneticdistances between populations ranged from0.040to0.045similar to the geneticdistances within populations. Pairwise Fst statistics ranged from-0.02283to0.05706suggested that lower level of differentiation and no significance was observed in8K.pelamis populations. The hierarchical analysis such as molecular variance(AMOVA), Neighbor-joining (NJ) tree and median-joining (MJ) network analysisexhibited that:8K.pelamis populations formed a panmictic population. Neutral testand Mismatch analysis suggested that all the K.pelamis populations have notexperienced a population expansion.