Comparative Genomics On The Adaptive Evolution Of Cavefish

Underground caves are found on all continents,except Antarctica.The most striking of the cave environment characteristic is the complete lack of sunlight,which means that there is no photosynthesis,and therefore no primary producers.Although some caves house large bats and maybe rich in food,most cave areas rely on food accidentally brought in through seasonal floods.Therefore,nutritional factors may be an important selection pressure for metabolism,biological behavior,and sensory adaptation.As the most successful taxa of vertebrates living in underground caves,cavefish are limited by the dark underground cave environment and face severe survival challenges,including the lack of photosynthesis in the cave,due to which the organic matter in the caves is poor and accidental.The vision of cavefish is lost and they must rely on other senses for their activities.Therefore,although cavefish have multiple origins,all typical cavefish face similar environmental selection pressures and have similar evolutionary characteristics such as eye degradation,loss of pigment.The evolution of cavefish has always received great attention and many degenerative mechanisms have been revealed,but there are still problems that have not been resolved.For example,what role does natural selection play in the adaptive evolution of cavefish? What is the genomics basis for the convergent phenotype of cavefish?Although two cavefish genomes have been published,the difference in the ploidy of the two genomes makes comparative analysis difficult.Therefore,it is necessary to sequence the genomes of other representative cavefish.Triplophysa rosa belongs to a large cavefish group in China.Owing to the multiple cave species and the existence of closely related surface species of this genus,it has become the best choice for genome research.Therefore,this research takes the cavefish T.rosa as the main research object.Through its transcriptome,whole-genome sequencing and combined with the existing cavefish genome data to answer the above questions from the perspectives of comparative transcriptomics and comparative genomics.The main research contents are as follows:1.First,comparative transcriptome analysis of T.rosa and T.bleekeri was conducted.Sequencing of the transcriptomes of both species,to identify differentially expressed genes(DEGs)and genes under positive selection,revealed 4,802 DEGs and 50 genes under positive selection(dN/dS >1,FDR < 0.1).For T.rosa,we identified one Gene Ontology category related to vision that was significantly enriched in the downregulated genes.Specifically,we found that many of the downregulated genes,including pitx3,lim2,crx,gnat2,rx1,rho,prph2,and β|γ-crystallin,are associated with lens/retinal development and maintenance.However,compared with those in the surface loach,the low dS rates and high dN rates of the protein-coding sequences in T.rosa indicate that changes in amino acid sequences might be involved in the adaptation and visual degeneration of cave loaches.We also found that genes associated with light perception and light-stimulated vision evolved at higher rates(some genes with dN/dS > 1,but FDR > 0.1).The findings of this study indicate that the degradation of cavefish vision is probably associated with both gene expression and amino acid changes,and provide new insights into the mechanisms underlying the degeneration of cavefish eyes.In addition,this study showed that the cavefish genome may have a special evolutionary pattern.However,the transcriptome data do not contain all genes from the genome,and a comparative transcriptome study based on T.rosa may not be representative.Therefore,it is necessary to further sequence the whole genome of T.rosa cavefish.2.Genome sequencing and assembly of T.rosa were performed.In this study,Illumina,PacBio sequencing,and Hi-C technology were used to assemble the T.rosa genome.A total of 141 Gb of Illumina clean reads,11.6 Gb of PacBio clean reads,and 111.29 Gb of Hi-C sequencing data were obtained.We applied these sequencing data to assemble the T.rosa genome,and constructed a chromosome-level genome: 25 chromosomes with a total length of 638.47 Mb;the total length of the whole genome is 675.5 Mb.A total of 26,107 protein-coding genes were annotated in the genome,and approximately 95% of the genes were functionally annotated through a search in public databases.The integrity evaluation of the gene set showed that the integrity of the T.rosa gene set was as high as 95.3%,indicating high-quality genome assembly and gene set prediction.Then,a preliminary comparative genomic analysis was carried out by combining Astyanax mexicanus cavefish andSinocyclocheilus anshuiensis cavefish.The analysis results showed that doubling the genome resulted in more genes with redundant copies and greatly reduced the number of single-copy orthologous genes.Therefore,it is difficult to obtain enough genes of S.anshuiensis for analysis to reveal the evolutionary mechanism of the cavefish genome.In general,the high-quality genome assembled in this work not only provides valuable genome resources for future research on the population and protection of T.rosa but also lays foundation for further research on the dark adaptation mechanism of the cavefish.3.The whole genomes of the three cavefishes were directly aligned,and four-fold degenerate sites(4D)were obtained to evaluate the evolution rate of the genome.Then,using the existing carp genome data,the gene set of S.anshuiensis genome was split into subgenomes,and the whole genomic gene sets from three unique cavefishes and their closely related surface fish pairs were compared.The comparative analyses revealed several shared features of cavefish genome evolution:(1)Cavefishes have lower mutation rates than their surface fish relatives;(2)Nonsynonymous to synonymous substitution rate ratios(dN/dS)were higher in cavefishes than in surface fishes,consistent with the genome-wide relaxation of purifying selection.Consequently,cavefish genomes suffer from an increased burden of mutational load,including mutations that change their protein hydrophobicity profiles,and are thus likely to be harmful.(3)Nevertheless,genes belonging to the lipid metabolism ontology category are under relaxed purifying selection in all cavefish genomes,which may be associated with the nutrient-poor habitat of cavefish.Despite these similar genome-wide evolutionary patterns,there is no overlap in positively selected genes in different cavefish lineages,indicating that the phenotypic convergence in cavefish is not caused by convergent evolution of the same sets of genes.The analyses of previously identified candidate genes of cave phenotypes support this conclusion.Our work reveals previously uncharacterized patterns of cavefish genome evolution and provides new comparative insights into the evolution of vision-related genes in cavefish and cave mammals.In summary,this study used comparative transcriptomes to reveal the molecular characteristics of eye degeneration in T.rosa and the possible mechanism of cavefish genome evolution.Then,using the next and third generation sequencing technologies,the genome of T.rosa cavefish was assembled to the chromosome level,and a high-quality genome and gene set were obtained.Combining existing cavefish genome data,the comparative genomics analysis was conducted and revealed the special evolution pattern of cavefish genome.This study provides new insights into the evolutionary model of cave fish genomes,and provides new enlightenment for the in-depth study of the convergent evolution mechanism of cave organisms.