Phylogenetic And Adaptive Evolution Analysis Of The Pacific White Shrimp, Litopenaeus Vannamei

Except Insects, crustacean is one of the groups with most species in Athropoda.As a species in Crustacea, Pacific white shrimp, Litopenaeus vannamei, is animportant economic breeding species in China, which produce millions of tons everyyear. In present, most of the shrimp researches are focused on aquaculture, diseasecontrol and genetic breeding, while few of them are focused on phylogeny andevolutionary biology.As the development of next-generation sequencing technology, a growingnumber of shrimp genomes and transcriptomes are sequenced and published gradually,which may provide good resources for the exhaustive research on shrimpphylogenetic and adaptive evolution analysis. In this study, we performedphylogenetic analysis of arthropods and estimated the divergence time of shrimpbased on the amount of transcriptome data. We also developed a new method toanalyze the phylogeny of shrimp under Crustacea. Besides, novel bioinformaticsmethods were implemented to analyze the adaptive evolution of shrimp. There arethree major sections included in this thesis as follows:1. Phylogenetic analysis of L. vannameiThrough using TreeFam methodology, we extracted19,982gene families fromfull genes of nine arthropods. Then,85single-copy genes were selected from them forthe phylogenetic analysis of Athropoda. The phylogenetic tree constructed bysingle-copy genes was similar with the trees constructed by custom molecularmarkers, and also has high confidence on each branch, which indicated that these85single-copy genes could be used as good molecular marker for the Arthropodaphylogenetic analysis. After estimating the divergence time of each species, we foundthat Crustacea was divergent at about395million years ago. And there were a great many of gene gain and gene lose when the ancestor evolved to shrimp and Daphnia,indicating that there is significant bias between the genomes of Daphnia and shrimps.L. vannamei and Penaeus monodon were also divergent early and a lot of gained orlosed gene families were found between them, which suggest these two Penaeidea arequite different each other. These results rightly supported the opinion of upgrading thesix subgenus of Penaeus to the stage of genus. Furthermore, we found that there aremany GO term with one shrimp gene gain significantly while the other shrimp losesignificantly. Among these GO terms, cellular component related gene families weresignificantly gained in P. monodon, while nervous system related gene gamilies weresignificantly enriched in L. vannamei. The bias of gene gain and gene lose betweentwo shrimps may help them adapting to their new environments, respectively.We developed a new method for the metazoans phylogenetic analysis based onthe short sequences segments, namely K-strings. We collected23,223key K-stringswhich make significant contribution for the phylogenetic tree construction. Throughusing this new method, we constructed a phylogenetic tree of Crustacea, and foundthat this tree is topological similar with the custom molecular phylogentic tree. L.vannamei was phylogentic close with Farfantepenaeus californiensis, while far fromP. monodon. The divergence time of Penaeidea was estimated at about95millionyears ago. Besides, we collected427key K-strings of Crustacea and map them on theoriginal protein sequences. The results showed that these phylogentic important keyK-strings are totally tend to accumulated at the conserved regions of the proteinsequences, which indicating that these key K-strings can be thought as evolutionaryelements that suffering selective pressure and play important role in crustaceansphylogeny.2. Horizontal gene transfer analysis of L. vannameiGenerally, horizontal gene transfer (HGT) events could help receipients gainsome new phylotypes or functions to support for the receipients adapting to newenvironments. In recent years, a growing number of genes have been reported asbeing horizontally transferred from prokaryotes to eukaryotes, most of them involvingarthropods. As a member of the phylum Arthropoda, L. vannamei has to adapt to the complex water environments with various symbiotic or parasitic microorganisms,which provide a platform for horizontal gene transfer.In this section, we analyzed the genome-wide HGT events in L. vannamei.Through homology search and phylogenetic analysis, followed by experimental PCRconfirmation,14genes with HGT event were identified:12of them were transferredfrom bacteria and two from fungi. Structure analysis of these genes showed that theintrons of the two fungi-originated genes were substituted by shrimp DNA fragment,two genes transferred from bacteria had shrimp specific introns inserted in them.Furthermore, around other three bacteria-originated genes, there were three largeDNA segments inserted into the shrimp genome. One segment was a transposon thatfully transferred, and the other two segments contained only coding regions ofbacteria. These structure alterations provide evidence that mature RNA may be thesubstrate for HGT events. Among14HGT genes, most of them were detected to beexposed to negative selection pressure and they appeared to be functional. Functionalprediction of these14genes showed that6of them might be related to energymetabolism, and4others related to defense of the organism. And most of themexpressed differently at five development stage of shrimp laveral, which indicatingthese genes are important for the shrimp development. Considering the functions ofthese14HGT genes, we thought they may helpful for the improvements of shrimpenergy metabolism and defense mechanism.3. Adaptive evolution analysis of L. vannameiDuring the long evolutionary history, because of the natural selection, shrimpswill evolve some adaptive mechanisms for the environment adaptation. Generally,these adaptive mechanisms can be directly displyed on the newly generatedphylotypes or biological characters, but it was actually displayed on the geneticmaterials mutations.We collected many transcriptomes of six shrimps: L. vannamei, Fenneropenaeuschinensis, P. monodon, Pandalus latirostris, Macrobrachium rosenbergii, Astacusleptodactylus. After removing redundant sequences, we finally obtained average12,000protein-coding genes, and select1,669groups of orthologs from them. Based on these orthologs, we constructed a conserved phylogenetic tree of these six shrimps.The topology of this tree is similar with normal trees constructed by molecularmarkers, and also correspondent with the custom morphological classification. Then,we set the branches of three Penaeidea and two Caridea as foreground branchesrespectively to analyze the bias of adaptive mechanisms between Penaeidea andCaridea. We also set the branches of four seawater shrimps and two freshwatershrimps as foreground branches respectively to analyze the adaptive mechanisms ofdifferent water environments.We obtained231and110positive selected genes from Penaeidea and Caridea,respectively. It was found that most of the positive selected genes in Penaeidea areenriched in the functional related GO terms of ribosome biogenesis, membrane andresponse to stimulus. The genes enriched in the ribosome biogenesis and proteintranslation suffer positive selection pressure may be benefit for the protein synthesisin Penaeidea, while the positive selected genes enriched in membrane compositionmay be co-evolved with the genes in protein synthesis. It was interesting to find thatmany positive selected genes are enriched in stimulus response related GO terms inPenaeidea. As Penaeidea have excellent giant nerve fibers to conduct impulses at thevelocity highest among animals, it seems the nervous systems of Penaeidea wereadaptive evolved during the long evolution history. When compared with Penaeidea,the positive selected genes in Caridea are primarily enriched in methylation relatedfunctions, which suggest that Caridea may improve its adaptation through methylationmodification.When analyze the adaptive evolution of the shrimps under seawater or freshwater,we obtained174and360positive selected genes among them, respectively. Becauseosmoregulation is one of the most important biological processes in water adaptationof shrimps, we surveyed genes related to osmoregulation among these positiveselected genes. As expected, we found two genes, NKA and NKCC, positive regulatethe osmoregulation in seawater shrimps. Both genes are responsible for establishingelectrochemical gradients across biological membrane, which were considered benefitfor the seawater shrimps adapt to high salinity environments. In contrast, there is a positive selected gene RHOGTP8in freshwater shrimps can negative regulateosmoregulation procedures. RHOGTP8can maintain the intracellular ionconcentration, which may helpful for the freshwater adaptation. Therefore, all theseosmoregulation related positive selected genes are considered to play important rolein the improvements of water environment adaptation mechanisms.