Genomic Insights Into The Phylogeny Of Zokors And Speciation Of Eospalax

Zokor is a small,herbivorous mammal that lives underground and plays an important role in the grassland ecosystem.Myospalacinae subfamily contains two existing genus,Eospalax and Myospalax.The Eospalax consists of six species,including E.fontanierii,E.cansus,E.baileyi,E.smithi,E.rothschildi and E.rufescens,all of which are distributed in China.Previous studies on the phylogeny of zokors were performed by using a few different molecular markers or phenotypic data,which hampered further development of research on zokors.This study utilized whole genome DNA sequences to examine the phylogeny of Myospalacinae in Spalacidae and the phylogenetic relationships among six Eospalax species within Myospalacinae.Moreover,we explored the factors that contributed to inconsistencies between gene trees and species tree.Demographic history and the effect of tectonic events,vegetation and climate on speciation of the six species were investigated.Specific research results can be summarized as follows:(1)Based on the Hi Fi long-read and Hi-C sequencing,a high-quality chromosome-level reference genome was assembled for E.fontanierii.The genome size was 3.18 Gb,Contig N50 was 52.54 Mb,total repeat sequence accounted for49.32%,and the predicted number of genes was 21,656.This zokor genome is the most consistent,accurate,and complete compared with others.(2)Comparative genomic analysis proved that Myospalacinae were more closely related to Rhizomyinae than to Spalacinae.The divergence of Myospalacinae and Rhizomyinae occurred 26.9 million years ago(Mya),while the Spalacinae diverged 2My earlier.Discordance of gene trees and species tree for these three subfamilies resulted from interspecies introgression.(3)There exist discordances between the NJ tree constructed by whole genome SNPs and the ML tree constructed by 13 protein-coding genes of mitochondria.Discordances between gene trees and species tree were also detected by coalescent analysis.The discordances on ancient nodes are mainly caused by ancient introgression,while on recently radiative nodes are mainly caused by incomplete lineage sorting(ILS).(4)The speciation of Eospalax was influenced by climate,geological events,and vegetation.At 3.60 Mya,the uplifting of the Tibetan Plateau resulted in the bifurcation of the genus Eospalax into two clades at 3.22 Mya,one at high altitude and the other at low altitude.With the expansion of C4 plants on the Loess Plateau in1.60 Mya,1.45 Mya E.cansus settled in the Loess Plateau and speciation.Ice age approaching drove some individuals to migrate southward,hence the speciation of E.rufescens on Qinling mountain and E.rothschildi in south China.As for the high-altitude clade,the formation of the Yellow River in 1.2 Mya isolated some high-altitude individuals to the north of the Yellow River,which contributed to the speciation of E.baileyi-I.Ice age drove some individuals to migrate southward to seek refuge,so the divergence of E.baileyi-II and E.smithi occurred in 0.75 Mya.(5)The highly differentiated islands between two species are due to ancestral polymorphism sorting.Ontology enrichment analysis of positively selected genes for each species revealed that functional pathways related to habitat adaptation,such as immunity,cardiac development,DNA repair and nervous system development,help each species adapt to its specific living environment.In conclusion,this paper systematically and comprehensively revealed the phylogeny and speciation of Eospalax,the effects of incomplete lineage sorting and introgression on the inconsistencies of gene trees and species tree,the causes of highly differentiated regions among species,the driving forces of speciation,and the mechanisms of species adaptation.