| The Pampus fishes (Perciformes, Stromateoidei, Stomateidae) are important commercial species widely distributed in the ndian-Western Pacific Ocean. Only the genus Pampus in family Stomateidae is found in China. So far, six valid species have been confirmed, Pampus argenteus, Pampus echinogaster, Pampus cinereus, Pampus punctatissimus, Pampus minor and Pampus chinensis. It has always been a difficult problem for taxonomists to clarify the Pampus species boundaries with certainty by very similar or variable sibling morphological characteristics, and there are many mistaken descriptions of morphological characteristics. Therefore, it is very important and necessary to take Pampus species as research object. In the present study, samples of six Pampus species were collected from China, Japan, Kuwait and Pakistan, morphological characteristics of Pampus species were described and the correct DNA barcoding were determined. Given the accuracy of data in GenBank, we also corrected some related sequences from GenBank. Mitochondrial genome (mitogenome) sequences of six Pampus species were determined with the genomic organization and structure being analyzed and compared. The genetic divergence level among Pampus species was probed based on mitogenome sequences. Meanwhile, the population genetic structure and phylogeography were investigated for four Pampus species using multiple molecular markers of mitochondrial DNA control region and microsatellite. The main results are presented as follows:1. Morphological characteristics and DNA barcoding of Pampus speciesU Countable characters (including dorsal fin spines and rays, pectoral fin rays, anal fin spines and rays, caudal fin rays, gill rakers on the first gill arch and vertebrae) and morphometric characteristics (including standard length, fork length, head length, postorbital length, snout length, eye diameter, interorbital width, caudal peduncle depth, caudle peduncle length and body depth) were studied. The diagnostic characteristics of Pampus species were described, and a new key of the genus was summarized. Comparison of otolith morphological characteristics of Pampus species showed that there were evident differences among six species.1.2 DNA barcoding of Pampus species were also sequenced to determine their species validity. Combining some congener sequences of COI gene from GenBank, DNA barcoding of each species was analyzed. Phylogenetic relationship also was reconstructed based on Pampus barcoding, which indicated that DNA barcoding was effective for Pampus taxonomy.2. Characterisation of the mitogenomes, phylogeny and evolution of Pampus species2.1 Six complete mitogenomes were sequenced by primer-walking sequencing for six Pampus species (P. argenteus, P. echinogaster, P. cinereus, P. punctatissimus, P minor and P. chinensis), which were a typical circular molecule of 16544 bp,17694 bp,16540 bp、16551 bp,16698 bp and 16534 bp in length, respectively. All the six mitogenomes carried set of 22 tRNAs (except P. echinogaster), two rRNAs,13 protein-coding genes as well as one putative control region and OL. The gene order of which was identical to those of vertebrate mitogenomes. The nucleotide composition of the six mitogenomes was characterized by a strong excess of C relative to G but as light excess of A relative to T on the H-strand. The 13 protein-coding genes of this genus utilized 78 start codons, which were ATG or GTG and identical in the same gene. Five stop codons (TAA, TAG, AGA, TA-and T--) were used in the six mitogenomes. Apart from the conserved blocks TAS and CSB-1, CSB-2 and CSB-3, three putative central conserved sequences blocks (CSB-D, CSB-E and CSB-F) could be recognized.2.2 Combining mitochondrial fragments with different evolution rate, phylogenetic relationship was reconstructed. The results showed that the Pampus species were monophyly group, P. echinogaster and P.minor were early diverged and primitive, P. argenteus and P. cinereus were at the top of phylogenetic tree. The molecular results were inconsistent with that of morphology, possibly due to convergent evolution of the life habits of six species. Results of divergence analysis of Cyt b and COI gene indicated that the genus Pampus had diverged in the Miocene, sine when each species adapted to different living environment under different natural selection pressure, evolved towards different objects, and formed different species with enough variation at last.3. Population genetics of Pampus species3.1 Population genetics of P. argenteus(1) The first hypervariable region (HVR-1) of mitochondrial DNA control region was employed to analyze population genetics of P. argenteus collected from China, Pakistan and Kuwait. A high level of diversity was observed and two significantly distinct lineages were detected. However, the relative frequency of individuals occupying the two major lineages did not differ significantly among sample locations, and meanwhile no differentiated geographical structure was observed in the NJ tree and minimum spanning tree. We inferred that the present distribution of haplotypes was indicator of secondary contact with subsequent high gene flow over the sampled range. The demographic history of P. argenteus examined by mismatch distribution analyses and neutrality tests suggested a sudden population expansion.(2) Genetic differentiation of P. argenteus was studied at seven microsatellite loci. Microsatellite analyses revealed relative high genetic diversity for P. argenteus due to high mutation rate of microsatellite DNA as well as large effective population size of this species. No significant population genetic structure was detected among the six populations, which might be caused by the complicated interaction between marine currents and biological characteristics of P. argenteus.3.2 Population genetics of P. echinogaster(1) The HVR-1 of control region was employed to analyze population genetics of P. echinogaster collected from seven sites along the coast of China. Both the levels of haplotype diversity and nucleotide diversity were moderate. No geographical structure was observed in the NJ tree and minimum spanning tree. The results might be led by the following two aspects:(1) P. echinogaster experienced a large scale habitat expansion after last Glaciations. (2) Both life history characteristics and marine currents shaped the results. The demographic history of P. echinogaster examined by mismatch distribution analyses and neutrality tests suggested a sudden population expansion.(2) Genetic differentiation of P. echinogaster was studied at six microsatellite loci. Microsatellite analyses revealed relative high genetic diversity for P. echinogaster due to high mutation rate of microsatellite DMA as well as large effective population size of this species. No significant genetic structure was detected among the six populations, and there was a panmictic population during its distribution, which might be caused by the complicated interaction between marine currents and biological characteristics of P. echinogaster.3.3 Population genetics of P. chinensis(1) The HVR-1 of control region was employed to analyze population genetics of P. chinensis collected from China and Pakistan. A high level of diversity was observed and two significantly distinct lineages were detected. Chinese and Pakistani populations supported different lineage, respectively. This differentiation resulted likely from isolation of the the edge sea in the Pleistocene caused by the decline of sea level. Another possibility, the study speculated, is the low effective supplement to the new population. Both mismatch distribution analyses and neutrality tests suggested a population expansion of P. chinensis.(2) Genetic differentiation of P. chinensis was studied at seven microsatellite loci. Microsatellite analyses revealed relative high genetic diversity for P. chinensis due to high mutation rate of microsatellite DNA as well as extensive distribution and diverse habitats. Differentiation was also detected between Chinese and Pakistani populations, which might be caused by the complicated interaction between geographic distance and biological characteristics of P. chinensis.3.4 Population genetics of P. minor(1) The HVR-1 of control region was employed to analyze population genetics of P. minor collected from two sites along the coast of China. A high level of diversity was observed. Significant differences were found between the two populations, but no geographical trend was observed, which might be caused by Qiongzhou Strait and P. minor’s life history characteristics. Both mismatch distribution analyses and neutrality tests suggested a population expansion of P. minor.(2) Genetic differentiation of P. minor was studied at three microsatellite loci. Microsatellite analyses revealed relative high genetic diversity of P. minor due to high mutation rate of microsatellite DNA as well as extensive distribution and diverse habitats. Differentiation was also detected between the two populations, which might be caused by the residual currents from east to west all the year round in the Qiongzhou Strait, which restricted the eastward gene flow between the two populations to some extent. |
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