Study On The Evolution Of Duplicate Genes In Tree Sparrow Genome And Their Role In Rapid Adaptive Evolution Of Sperm

Environmental heavy metal pollution can adversely affect sperm either directly or indirectly,if sperm fails to rapidly adapt to the heavy metal stress,a decrease in population fitness and subsequent population collapse caused by poor sperm quality and male fertility will occur.Therefore,when faced with the direct and strong selective pressure,only the sperm can buffer the detrimental effects caused by heavy metal pollution through rapid adaptive evolution,there would be a hope for the maintenance and development of the stable population.Preliminary studies of our group have found adaptive and compensatory sperm morphology changes and motility improvements,which indicated sperm underwent rapid adaptive evolution,in tree sparrow（Passer montanus）inhabiting polluted areas over the past 65 years,yet the genetic mechanisms that enable rapid sperm adaptation to environmental degradation are still not clear.In addition,as the important sources of genetic variations,duplicate genes are not only one of the primary driving forces of genome evolution,but also the booster of adaptation to environmental change and stress.However,little is known about the role of duplicate genes in the context of rapid adaptation and evolution of sperm.Based on the previous research foundation,this research still took tree sparrow as the study subject.Meanwhile,Baiyin（BY）area which is heavily polluted with heavy metals（mainly Cu,Zn,Pb and Cd）for more than 65 years and the relatively unpolluted Liujiaxia（LJX）area were selected as polluted and control sites,respectively.On the basis of a high-quality chromosome-level reference genome of tree sparrow which was assembled through the combination of the third sequencing technology（Hi Fi reads）and Hi-C sequencing,the identification and subsequent characterization of duplicate genes were conducted in the tree sparrow genome.Meanwhile,the candidate genes associated with tree sparrow sperm evolution in BY were identified through the combination of comparative population genetics and transcriptomics.All the findings were used to explore the molecular basis of rapid adaptive sperm evolution happened in the backdrop of environmental heavy metal stress and the role played by duplicated genes in this process.The main results are as follows:1.Completing the characterization analysis of the assembled reference genome of tree sparrow.The total size of the assembled tree sparrow reference genome was 1.28 Gbp,and 1.16 Gbp of which were anchored into 36 chromosomes.The 8largest assembled chromosomes were defined as macrochromosomes while the remaining smaller chromosomes were defined as microchromosomes.The differences of GC content,gene density and repeat density existed between macro-and microchromosomes.Compared with the previously published Illumina-based draft genome assembly,the newly chromosome-level assembly of tree sparrow showed great improvements of continuity and completeness.The high-quality reference genome was not only a critical foundation for exploring the genomic characteristics and evolutionary dynamics of duplicate genes in tree sparrow,but also a prerequisite for the follow-up population genetic and transcriptome analyses.2.Duplicate genes and their adjacent transposable elements co-evolved in tree sparrow genome.At least 16.84%of tree sparrow genome assembly was composed of transposable elements.TIR elements of DNA transposons（7.80%）were the most abundant transposable elements,and followed by LTR retrotransposons（5.43%）.Most of the intact LTR retrotransposons in tree sparrow genome were endogenous retroviruses.Relative to TIR elements and LTR retrotransposons,LINE（3.02%）and SINE（0.05%）retrotransposons showed lower proportion and activity.In addition,8gene families including C2H2ZNF,OR,PIM,PAK,MROH,HYDIN,HSF and ITPRIPL were significantly and simultaneously expanded in tree sparrow genome through segmental duplications,and these families had similar distribution trend with LTR retrotransposons.The similar distribution of significantly expanded gene families and LTR retrotransposons suggested that the duplicate genes may co-evolve with the adjacent transposable elements in tree sparrow genome.3.Duplicate genes showed testis-specific expression pattern.A large proportion of members in the 8 significantly gene families,especially in OR（94%）and C2H2ZNF（88%）,were transcriptionally inactive.In addition,the highly transcribed genes from the other 6 gene families generally exhibited testis-specific expression pattern.Above results indicated the duplicate genes may obtain new gene functions relevant to male reproduction through specifically expressed in tree sparrow testis.Meanwhile,the duplicate genes which were unable to play an active role in testis may rapidly lose their transcriptional activity,and thus became pseudogenes.4.Identifying PIM1 as a candidate gene associated with rapid adaptive evolution of sperm in tree sparrow.29 protein-coding genes showed signs of positive selection were preliminarily screened out based on genome-wideπand F_ST,and 23 of which were belong to the significantly expanded gene families in tree sparrow.This phenomenon indicated that the duplicated genes might be important targets of selection under environmental heavy metal stress.Furthermore,to increase the validity of the result,the CLR values and differential expression analysis were combined to detect the candidate genes.As the overlap between the positively selected genes and differentially expressed genes,the gene encoding the serine/threonine protein kinase PIM1 was identified to be a candidate gene for sperm evolution in BY.5.The role of duplicate genes PIM1 in rapid adaptive evolution of sperm.In contrast to other amniotic genomes（including chicken）that harbor only a single copy of PIM1,a total of 449 copies of PIM1 were detected in tree sparrow genome and 142of them were found to be complete for the conserved protein kinase domain.The expression pattern of PIM1 was shifted from extensive expression in multiple tissues to testis-specific expression.Significantly higher expression level of PIM1 was detected in the testis sampled from BY tree sparrows than LJX（0.01<P<0.05）,which was consistent with our earlier finding of longer and faster swimming sperm in BY.In addition,there was no difference in PIM1 copy numbers between BY and LJX populations（P>0.05）.The results showed that the upregulated PIM1 expression may be an important mechanism which enable the rapid adaptive evolution of sperm,however,the higher expression level was not driven by increased copy numbers caused by further expansion of PIM1 in BY population.In conclusion,a large number of duplicate genes added into tree sparrow genome through segmental duplications were detected in our research,and these duplicate genes may obtain new gene functions that related to male reproduction.Among the duplicate genes,PIM1 may be critical in the rapid adaptive evolutionary process of sperm under environmental heavy metal stress.These results suggested that the large number of duplicate genes,which generated during the long evolution history of tree sparrow genome,may facilitate the speed of sperm adaptation and create new opportunities for the maintenance of stable population reproduction and development when facing the serious threat of environmental degradation.Therefore,this study could provide a reference for further exploration of evolutionary significances of duplicate genes on the long-term avian species evolution and rapid adaptation in a short time.