| As a class of very important secondary aquatic mammal in the history of species evolution,cetaceans have successfully adapted to the aquatic environment and rapidly radiated into various waters around the world.In order to adapt to aquatic habitat,cetacean external morphology and physiology and ecology have produced a series of adaptive changes.The skeletal internal morphology has undergone tremendous changes from a typical terrestrial form to a semi-aquatic bone sclerosis to a fully aquatic osteoporosis.However,the molecular mechanism of this adaptive change is not yet clear.The finless porpoise(Neophocaena phocaenoides,genus Neophocaena,family Phocoenidae)is a group of small toothed cetaceans which were characterized by spade-shaped teeth,a short,blunt snout and lack of a true dorsal fin,inhabiting coasts of southern and eastern Asia.Currently there are two ways to classify the finless porpoise:the first is to divide it into three subspecies:N.phocaenoides phocaenoides,N.phocaenoides sunameri,and Yangtze subspecies(N.phocaenoides asiaeorientalis);the second is includes the Indo-Pacific finless porpoise(Neophocaena phocaenoides)and the narrow-ridged finless porpoise(N.asiaeorientalis),with two subspecies(Yangtze finless porpoise,N.asiaeorientalis asiaeorientalis,and East Asian finless porpoise,N.asiaeorientalis sunameri)within the latter species.Among them,the Yangtze River finless porpoise is currently the only living in freshwater species,its classification status has been controversial:In addition,freshwater adaptation of the Yangtze finless porpoise is also a hotspot in evolutionary biology research.In view of this,the whole genome de novo sequencing of Yangtze finless porpoise was performed for the first time in this study.Three geographical populations of finless porpoises in Chinese waters were re-sequenced.Population genomics methods were used to study the genetic structure and population history of finless porpoises and establish the taxonomic status of Yangtze finless porpoises,and discussioned their adaptation mechanisms between subspecies.Followed by a comparative phylogenetic method to study the structure of cetaceous bone to reveal the molecular mechanisms of adaptation to evolution of the bone microstructure.Finally,based on the most number of cetacean genome-wide information,we using comparative genomics to explore other aspects of aquatic adaptation.We selected a male Yangtze finless porpoise for whole-genome deep sequencing and de novo assembly to obtain a high-quality genome-wide map.We obtained high-quality data of 265.5 Gb with a sequencing depth of 106.7× and a Contig N50 of 26.7 K.Scaffold N50 is 6.33M,the finless porpoise genome size is about 2.488G.A total of 22,014 protein-coding genes were identified,and the repetitive sequence accounted for 45.18%.Based on the whole genome map of the Yangtze River finless porpoise,48 finless porpoise individuals in Chinese waters(Yellow Sea,South China Sea and the Yangtze River)were completely sequenced.The average sequencing depth was 14.5X and 98.6%coverage,and the largest scale to date was achieved.Cetacean population genomic polymorphism sites were analyzed by population genomics.In order to detect the osmotic adjustment related genes in the Yangtze River and Yellow Sea finless porpoise within the narrow ridge population,XP-EHH method was used to screen the selected regions.Three genes,ADCY1,DYNC2H1,and SLC14A2,were located in the sea finless porpoise and were associated with "kidney water homeostasis," "vasopressin-regulated water reabsorption," and "urea translocation."The Yangtze River finless porpoise has detected the gene ACE2,which encodes a protein that is a monocarboxypeptidase protein and plays an important role in the regulation of renin-angiotensin in the cardiovascular,renal,lung,and central nervous systems.Mice with this gene can cause severe kidney damage and increase urinary protein excretion.Therefore,it is speculated that genes SLC14A2 and ACE2 are key genes for cetacean adaptation to seawater and freshwater environments.The population genetic structure,origin and genetic diversity of the finless porpoise have always been the focus of scientists.Population genetic structure analysis confirmed that there were significant genetic differentiation among the three populations,and there was no gene exchange between the Yangtze River finless porpoise and the two marine populations of the Yellow Sea and the South China Sea.The f3 statistics and Bayes factor analysis showed that there was significant reproductive isolation between the three groups.The "three species division" provides strong statistical support.In addition,based on the dynamic analysis of the population history of PSMC and MSMC models,it was found that the finless porpoises with narrow ridges and wide ridges began to differentiate in the Pleistocene(100KYA),which was characterized by the obvious alternation of climate cooling,glacial and interglacial periods,and two groups over time.The degree of differentiation becomes higher and higher,and then gradually differentiates into two independent populations as they enter the last Glacial Maximum(LGM)(30 KYA).LGM not only promoted the differentiation of the wide-ridged and narrow-necked finless porpoise,but also contributed to the further differentiation of the Yangtze finless porpoise from narrow-ridged populations at 1-10KYA due to the changes in sea level caused by it.Sea level changes during LGM dispersed the ancestral narrow-ridges finless porpoises into the Yangtze River region,and subsequent sea level declines between 4000-1000 years ago caused the Yangtze River estuary to dry up,prompting the ancestral groups of the Yangtze and Yellow Seas to rapidly and violently isolate themselves geographically.Based on the above evidence,we propose that the Yangtze finless porpoise should be significantly different from the wide-ridges finless porpoise and marine narrow-ridges finless porpoise and fully adapt to the freshwater environment of the species Neophocoena asiaeorientalis.This finding helps the Yangtze River finless porpoise to receive better attention and protection as the flagship species of the Yangtze River ecosystem and biodiversity conservation after the functional extinction of the Baiji.In order to adapt to aquatic life,the extant whale bone microstructure presents a non-pathological osteoporosis.We investigated the possible relationship between bone microstructure and the molecular evolution of genes using bone histological parameters and an index of 'root-to-tip,dN/ds under a phylogenetic framework across mammals with different habitats.Further,we detected positive selection along ancestral branches of cetaceans in genes that are functionally associated with bone modeling and remodeling.Some interesting findings included the following:1)Genes involved in osteoclast function were identified to be under accelerated evolution in cetaceans,suggesting their important roles in bone remodeling during the adaptation to a water environment;2)Genes in the Wnt pathway related to osteoblasts have experienced divergent evolution between cetaceans and other terrestrial mammals;3)Several genes encoding bone collagens were identified to be under selective pressure and to have a significant relationship with bone microstructure in cetaceans.Among them,the COL1A2 gene,which is the main protein that constitutes the bone mineral,was screened for positive selection in whales,ancestry of bats and manatees.The branch site model of PAML software detected 9 positive selection sites of cetaceans,whose amino acid substitutions are both proline and hydroxyproline in the Gly-X-Y repeat structure,which may affect the thermo-stability of the triple helical structure of collagen that further changed the bone micro-structure Our results suggested that variable evolutionary pressures have shaped the genetic adaptation of osteoporosis in cetaceans,which is eventually driven by habitats and ecological behaviors.To further explore the adaptation of cetaceans to aquatic habitats,we performed comparative genomics analysis on 7 cetaceans and 10 mammals,reconstructed phylogenetic relationships,estimated differentiation time,identified gene family contraction and expansion,and selected pressure analysis.The cetacean oxidative stress-related gene families,such as the peroxiredoxin(PRDX)family,glutathione peroxidase(GPx),and the OGT gene family,have been found to have expanded but most copies of the sequence are incomplete.This expansion produces multiple copies and is accompanied by the evolution of pseudogenes.It is an adaptive model for cetacean tolerance to hypoxia stress.In addition,there were positive selections related to cerebellar development-related genes such as AHI1,CC2D2A,FANCD2,STT3A,and TTBK2,and neuronal differentiation-related gene families showed contraction,suggesting that they may be associated with smaller brains of Phocoenidaes.Based on de novo sequencing and population resequencing analysis of the Yangtze finless porpoise,the population evolutionary history and taxonomic status of the Yangtze finless porpoise were revealed.For the first time,the independent status of the Yangtze finless porpoise was proposed,providing an important basis for its protection and management.In addition,through comparative genomics analysis,it provides a molecular mechanism for adaptive changes in bone microarchitecture,oxidative stress,and immunity resulting from the adaptation of cetacean aquatic habitats,laying a foundation for a comprehensive understanding of the genetic mechanisms of secondary aquatic adaptation in cetaceans. |
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