Molecular Evolution Of Three Antimicrobial Peptide Loci Of Pelophylax Nigromaculatus And A Study On Genetic Strueture Of Chinese Populations

Antimicrobial peptides (AMPs) system is one of the most important innate immune defense systems of amphibians. High genetic diversities have been maintained in AMPs genes by balancing selection and positive selection after gene duplications. After gene duplications, there should be some AMP genes that are subjected to strong purifying selection to support the basic survival needs of animal populations if many other molecules can freely occur genetic variations to co-evolve with specific pathogens. To test our hypothesis, we selected the Pelophylax nigromaculatus that extensively distributed throughout Palaearctic and Oriental Regions in China as the research object, and investigated the gene copy number, allele number and mutation patterns of three broad-spectrum AMP genes (Temporin, Esculentin-1N and Esculentin-2Na).7unique Esculentin-1N haplotypes and8unique Temporin haplotypes were observed at least three times from a single individual according to the data from four different geographical populations (HRB, DY, WH and GD). So, there were at least four Esculentin-1N or Temporin loci. All the8Temporin haplotypes encoded one mature peptide, and population analyses (n=20) also showed no genetic variations across mature peptide coding regions. Compared with the Exon2, the intron and3’UTR region each presented a sudden increased genetic diversity and significantly higher than that of intron region of neutral gene FIBI7. This result ruled out selective sweep and recent gene duplication events, but supported that the strong purifying selection and gene conversions had forced Temporin gene with a low genetic variation. Esculentin-1N haplotypes encoded four types of mature peptides. Although only four mutations were found among7haplotypes,3of them were non-synonymous sites. Neutrality test, differences of the proportion of radical (pr) and conservative (pc) non-synonymous, and nature selection tests all showed signatures of positive selection on Esculentin-1N genes, but no significant P value was found lower than0.05. This Evidence supported that low level positive selection shaped a relative high diversity of Esculentin-1N gene. Esculentin-2Na was defined as a single-locus gene. Analyses of Esculentin-2Na gene of127individuals from6different geological populations showed that125individuals encoded an identical mature peptide;73%of mutations were synonymous substitutions; parameters of neutrality tests were significantly lower than0(P<0.05); dN/dS values were significantly lower than1(P=0.0176); no codon sites were found under positive selection (P=1), and92%codon sites had a post mean ω value that lower than0.2with a posterior probability higher than95%; haplotypes network was star-like and separated by short branches. All the signatures indicated that the mature peptide coding region of Esculentin-2Na gene was under strong purifying selection. These results indicated that Temporin and Esculentin-2Na gene are subjecting to strong negative selection, which suggested the two genes had extremely important functions of basic immune defense. Esculentin-1N genes are under low level positive selection, which represented the adaptive evolution of AMP genes.To provide more valuable information for studying the evolution of AMPs genes, we studied the genetic structures and genetic variations of the same127individuals by using mitochondrial control region and nuclear FIBI7gene and TYR gene. Results supported the existence of mainland lineage and northeast lineage, and mainland lineage were clustered into sub-lineage A and sub-lineage B. Sub-lineage B only contained shared haplotype of TS and WH populations, and sub-lineage A, however, contained shared haplotype of TS\WH\DY\GD, which indicating the origin of sub-lineage B is in inner area of middle-lower yangtze area as Wuhan area, but refugium for sub-lineage A was more close to coastal area.