| Sand lance, Ammodytes personatus, extremely similar with its congener A.hexapterus on morphological characteristics, is a quintessentially forage fish and ofgreat significance being a commercially prominent species in the northwestern Pacific.Two distinct lineages were found throughout the distribution of A. personatus inprevious studies. Dasyatis akajei is a typical ovoviviparous member of the familyDasyatidae in the northwestern Pacific, with habitats commonly in demersal sandyareas, besides, it can also survive in the freshwater only in Xijiang River and adjacentrivers of China. In the present study, mitochondrial genome (mitogenome) sequencesof these two Ammodytes species were determined with the genomic organization andstructure being analyzed and compared. The genetic divergence level between twolineages of A. personatus was probed based on mitochondrial genome sequences.Meanwhile, the population genetic structure and phylogeography were investigatedfor D. akajei using multiple molecular markers of mitochondrial DNA control region,microsatellite and AFLP. Moreover, the phylogenetic relationship of five Dasyatisspecies was assessed. The main results are presented as follows:1. Characterisation of the mitogenomes of A. personatus and A. hexapterusThe complete mitogenomes of the first member of the suborder Trachinoideiwere determined for A. hexapterus, A. personatus S and A. personatus N, which is atypical circular molecule of16,527bp,16,537bp and16,527bp in length, respectively.The length of all genes and control region in A. personatus N mitogenome is identicalto those of A. hexapterus, while is different from those of A. personatus S on the16SrRNA gene (2bp indels) and control reigion (8bp indels). All the three mitogenomescarry set of22tRNAs,2rRNAs,13protein-coding genes as well as a putative controlregion and OL, the gene order of which is identical to those of canonical vertebrate mitogenomes. The nucleotide composition of the three mitogenomes is characterizedby a strong excess of C relative to G but a slight excess of A relative to T on theH-strand. Anti-G bias and anti-C bias were found at the third codon positions of H-and L-strand, respectively, ultimately reflected in the codon usage on each strand. Theprotein-coding genes in all three mitogenomes employed the same start codon ATGexcept for COI gene being started with GTG. Five stop codons (TAA, TAG, AGG,TA-, and T--) were used in the three mitogenomes. The protein-coding genes of A.hexapterus utilized the same stop codon with those of A. personatus S and A.personatus N, but with ND1and ND5genes as the exception. Except for ATP8gene,a significant pattern of Ka/Ks (P<0.01) was observed in all protein-coding genes,suggesting these genes were under a strong purifying selection. All tRNA genes couldputatively form typical cloverleaf secondary structures with the exception oftRNASer(AGY). Apart from the conserved blocks TAS and CSB-1,-2, and-3, all of thesix putative central conserved sequences blocks (CSB-A to CSB-F) could berecognized.2. Probe into the genetic divergence level between two lineages of A. personatusbased on mitogenomic sequencesFourteen and nine mitogenome sequences were determined for south and northlineages of A. personatus, respectively, between which the2bp indels in16S rRNAgenes were identified. The high sequence divergence could be detected between themitogenomic sequences of the two lineages. Apart from the control region, the similarlevel of sequence divergence was observed between two lineages as well as betweenthe south lineage and A. hexapterus, which was higher than that between the northlineage and A. hexapterus (except for ATP8and ATP6genes). While, the lowest levelof sequence divergence was observed within each lineage. A molecular phylogeneticanalysis of two lineages of A. personatus and A. hexapterus was conducted usingBayesian inferences multiple models based on the complete mitogenomes excludingthe ND6genes and control region. Well-resolved phylogenies constructed in thepresent study strongly supported the close relationship of the north lineage and A.hexapterus, which was obviously divergent from the south lineage. The result confirmed that the south and north lineage were genetically distinct as having beendifferent species.3. Population genetic structure and molecular phylogeography of D. akajei(1) The first hypervariable region of mitochondrial DNA control region of474bpwas analyzed for107individuals from7localities within the distribution range of D.akajei. A high level of haplotype diversity was observed in D. akajei. Nogenetically-differentiated geographical clade was observed in the NJ and BI trees aswell as the minimum spanning tree. Significant level of genetic structure was detectedbetween four marine populations (TZ, WZ, ND and ZZ) with two exceptions, whichwere unusual for elasmobranchs detected previously over such short geographicaldistance. However, limited sampling suggested that the freshwater population was notparticularly distinct (P>0.05), but additional samples would be needed to confirm it.Demersal and slow-moving characters likely have contributed to the geneticallyheterogeneous population structure. The demographic history of D. akajei examinedby mismatch distribution analyses, neutrality tests and Bayesian skyline analysessuggested a sudden population expansion dating to upper Pleistocene.(2) A total of207loci were identified by4primer combinations from87individuals of7D. akajei populations. A high level of Nei’s gene diversity wasobserved in D. akajei. No significant genealogical clusters associated with samplingsites were revealed on the UPGMA tree. A small but significant geneticdifferentiation among four marine samples (TZ, WZ, ND and ZZ) was detected.However, no genetic differentiation was detected between marine and freshwaterpopulations. Except for the TZ vs. WZ, the gene flow estimates demonstrated lowlevel of effective immigrants, suggesting limited dispersal between pairwise marinepopulations. Mantel test showed no significant correlation of genetic divergence withgeographical distance.(3) Ten dinucleotide microsatellite DNA markers were developed based on theenriched microsatellite library constructed, and was used for genetic analysis of4D.akajei populations. A high expected heterozygosity and polymorphism informationcontent (PIC) were detected, indicating a high level of genetic diversity. A weak but significant genetic heterogeneity was found between pairwise marine populations,which was consistent with the result of AFLP analysis. While, bayesian clusteringshowed that all individuals shared on gene pool. Mantel test revealed the geneticdivergence was not significantly correlated with geographical distance. Demersal andslow-moving characters are probably responsible for the genetic structure pattern.4. Phylogeny and evolution of five Dasyatis speciesThe control region structure of five Dasyatis species was firstly analyzed as wellas Pteroplatytrygon violacea and Neotrygon kuhlii. The length of control regions from7species was larger than1860bp, of which the nucleotide substitutions account foronly1.3%. In the termination associated sequence domain, two types of repeatsequences were found for7species with the exception of D. laevigata, and the TASmotifs were located in the first type one. The conserved sequence boxes were absentin the central conserved domain (CD), but were all identified in the conservedsequence blocks domain (CSB-1~-3). Apart from P. violacea, repeat sequences couldbe detected at the3’ end of control region for these species, and were distinct fromone another on the motifs, length and repeat numbers. A molecular phylogeny of fiveDasyatis species was constructed based on mitochondrial sequence data of COI, Cyt band control region in multiple perspective. The well-resolved topologies indicated thatD. zugei was the earliest diverging taxon with Dasyatis. The clade formed by D.akajei and D. bennetti was placed as a sister taxon to the clade formed by D. izuensisand D. laevigata. The monophyletic clade formed by these4Dasyatis speciessubsequently clustered with P. violacea, and then with D. zugei. These resultsstrongly supported the non-monophyly of Dasyatis. Besides, the obviously highdivergence between Dasyatis species and N. kuhlii also supported they aretaxonomically different genera. |
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