| The innovation of mammalian teeth is one of the most important reasons for mammalian evolution.The polymorphous teeth provide the ability of efficient mastication,which enable mammals achieve the stable energy sources for survival,and as one of most important factor for mammalian prosperity.Teeth are the most mineralized and hardest organs in the body of mammals,and the variation in shapes,size and number reflects the adaptation to different food resources and feeding strategy.Mammals have evolved different tooth phenotypes that are suggested to be associated with feeding habits,e.g.,hypsodont for herbivorous,secodont for carnivorous,and bunodont for omnivorous.In addition,as the main component of tooth,the microstructure of enamel and dentin also exhibit the adaptive changes according to different feeding habits.For example,the basic unit of enamel,enamel rod,have exhibited specific morphological characteristics among different dietary mammalian lineages.However,some mammalian lineages have lost their teeth(e.g.,baleen whales,pangolins and anteaters,etc)or enamel(e.g.,dwarf sperm whale,pygmy sperm whale,sloths,armadillos and aardvark,etc)during evolution independently,which might be due to their specific feeding habits and strategies.Cetacean is a fascinating group,which live in water environment.Because of special feeding strategy,they possess different tooth characters compared with other mammalian lineages:(1)baleen whales have evolved baleen,instead of teeth;(2)teeth have evolved into multiple unicuspid homodont,instead of functional heterodont in living toothed whales.Although the morphological and physiological adaptation of mammalian teeth to feeding habits and strategies have been demonstrated.Yet,the molecular mechanisms of mammalian tooth adaptation is still unclear.Given these issues,we selected the different dietary mammal lineages,toothless /enamel-less mammal lineages,and different kinds of cetaceans as the research objects,choose the relative tooth-related genes as the candidate genes.Through comparative genomics and evolutionary genomics combined with different analysis methods to discuss the following scientific issues:(1)whether the evolution patterns of teeth related genes have been divergent in different dietary mammals?(2)whether the toothrelated genes become pseudogenized in the toothless / enamel-less lineages? And the selective pressure have been relaxed?(3)the mechanisms of genetic basis for simplified tooth morphology and increasing tooth number in cetaceans after entry into the water environment.To better understand the evolutionary mechanism of tooth-related genes in different mammalian groups in terms of dietary adaptation,tooth loss,as well as number change,The above-mentioned scientific issues are comprehensively explored.Firstly,in order to explore the evolutionary pattern of tooth-related genes among different dietary mammals(herbivores,omnivorous,carnivores),the full CDS of seven enamel-related genes(AMELX,AMBN,ENAM,AMTN,ODAM,KLK4 and MMP20)and six dentine-related genes(DSPP,COL1A1,DMP1,IBSP,MEPE and SPP1)are retrieved from ?60 mammal,which were analyzed by using methods of comparative genomics and evolutionary biology.The results showed:(1)Relatively stronger positive selection was detected in enamel-related genes(ENAM,AMTN,ODAM,KLK4)and dentin-related genes(DSPP,DMP1,COL1A1,MEPE)among herbivorous lineages,in addition,AMELX,AMBN,ENAM,AMTN and MMP20 and COL1A1 have went through accelerated evolution in herbivores.Previous researches have confirmed that these genes are involved in the formation of enamel and dentin,mutations or deletions detected of these genes will lead to severe amelogenesis imperfecta(AI)and dentinogenesis imperfecta(DI).The positive selection of related genes might promote the formation and biomineralization of tooth enamel and dentin to a certain extent,which would make the tooth structure stronger.Thus,it helps the herbivores to form a better enamel layer to enhance the resistance to food fiber and improve the wear resistance of teeth,consequently increasing the working life of teeth.(2)By contrast,only two dentin-related genes(IBSP and SPP1)were detected relatively stronger positive selection,and DSPP,MEPE and SPP1 genes have also underwent accelerated evolution in carnivorous lineages.It may be conducive to dentin development and rapid adaptation in carnivourous lineages.The strong and powerful bite is essential for carnivorous teeth to cut and masticate the food items,thus,solid dentin provides strong support for outer enamel.(3)Among omnivorous primates,relatively stronger positive selection of AMBN and ENAM gene mainly focused on catarrhini clade.Previous studies have confirmed that AMBN and ENAM genes are the main genes for enamel formation,which not only participate in the construction of enamel,but also regulate the thickness of enamel,mutation or deletion of these two genes would lead to severe enamel defects.Genes exhibit the significantly positive selection in primates,suggesting that tooth enamel formation may be enhanced to adapt to a wider range of food resources.Interestingly,a significantly positive association between the evolutionary rate of enamel-related genes(ENAM,ODAM,KLK4,MMP20)and the average enamel thickness was found in primates.It is suggested that the evolution of enamel-related genes is conducive to the development of enamel in primates.(4)ENAM gene was under the divergent selection among different dietary bats,besides,the evolutionary rate in fruit-eating bats was significantly higher than in insect-eating bats.This is consistent with the finding that the tooth complexity of fruit-eating bats is higher than that of insectivorous bats.To explore the convergent evolution of tooth-related genes among similar feeding lineages,we reconstructed the ancestral sequences of related genes and screened the convergent amino acid substitution sites.The results showed seven parallel substitution sites were identified across herbivorous lineages,whilst 14 parallel substitution sites were found in carnivorous lineages.Our results revealed that mammalian tooth-related genes have experienced variable evolutionary patterns according to the different feeding habits,which provide some new insights into the molecular basis of dietary adaptation in mammals.Secondly,to further explore the molecular mechanisms of mammalian tooth loss and enamel loss,base on the previous studies,we identified two one-copy genes,ACPT and C4orf26,which were pseudogenized in toothless and enamel-less mammals.Subsequently,we scanned and identified ACPT and C4orf26 genes in other tetrapod lineages(anura,chelonia,squamata,crocodilia,aves).The results showed:(1)ACPT gene is absent in teethless non-mammalian tetrapods(anura,chelonia,aves)and intact in teethed lineages(squamata,crocodilia).In addition,two shared deletion sites were found in exons 4 and 5 of ACPT in all living baleen whales,which supported the hypothesis that mineralized dental structure have been degenerated in the common ancestor of baleen whales.Further selection pressure analysis showed that the selection pressure of ACPT gene was completely relaxed in both toothless and enamel-less mammals(? value was nearly 1).The inactivation time suggested that inactivating mutation of ACPT gene resulted from enamel degeneration in armadillos;on the contrary,inactivating mutation of ACPT resulted in enamel loss in aardvarks.In addition,we cannot estimate the causal relationship for ACPT inactivating and enamel loss in Kogia breviceps,due to lack of fossil evidence.(2)C4orf26 gene was absent in non-mammalian lineages,whether toothed or toothless.C4orf26 gene was pseudogene or absent among toothless mammals(e.g.,baleen whales,pangolins),but intact in enamel-less mammals(e.g.,armadillos,sloths and aardvarks)and the rest of the toothed mammal lineages.Selective pressure analysis found that the selective constraints of C4orf26 have been relaxed in toothless mammals,contrarily,this gene was under strong purifying selection among enamel-less mammals.Based on our results,we proposed that C4orf26 gene was the specific gene in mammals,which was responsible for tooth loss.It has been found that odontoclasts play a key role in loss of deciduous tooth and pathological tooth loss in mammals,some related genes(ACP5,BCAR1,CTSK,ITGAV,MMP9,PLCG2,TNFRSF11 A and TNFSF11)are involved in the process of differentiation and activation of odontoclasts,as well as hydrolyzation of dentin.In order to understand the evolutionary pattern of odontoclast-related genes in toothless mammals,we performed sequence alignment and selective pressure analysis.The results showed: All odontoclast-related genes were intact.Compared with the toothed mammalian lineages,three odontoclast-related genes(BCAR1,MMP9 and PLCG2)have underwent accelerated evolution,which might increase the activity of odontoclasts and further promote the loss of teeth.This research further improved the mechanisms of evolutionary basis of tooth loss / enamel loss.Finally,in order to understand the mechanisms about tooth simplification of extant whales,we collected the OPC(Orientation patch count,which can quantify the tooth shape)from previous studies and carried out some statistical analyses.Besides,we screened the “whale-single-copy dataset” that were established by ourselves to try to find some key genes,which may be related to tooth simplification for whales.The results showed:(1)the OPC of archaeoceti was second to extant herbivores,and significantly higher than extant omnivores and carnivores,which suggested that archaeoceti were completely carnivorous,but their teeth may retain some characteristics of their herbivorous ancestors.In addition,our results also exhibited a stable decreasing trend along with the dietary change from herbivorous to omnivorous,until to carnivorous.The OPC in living toothed whales are lowest.The results suggested that tooth morphology of cetacean ancestors was simplified probably due to dietary switch.In addition,compared with cetacean ancestors and extant carnivores whose teeth possessed the ability to chew,the teeth of living whales are greatly simplified.The results suggested that the loss of chewing ability may be a key factor for the tooth simplification in living toothed whales.(2)Through screening whale-single-copy sets of cetaceans,we were lucky to find that ODAM gene became psuedogene in all cetacean species(except for Physeter catodon),which was consistent with tooth loss in baleen whales and simple enamel microstructure in toothed whales.Base on sequence alignment,we found odontoceti and mysticeti have their own shared inactive mutation,respectively.In addition,selective pressure have been relaxed among some relative pseudogenized branches,such as crown odontoceti,crown mysticeti and termimal branch of Dasypus novemcinctus,etc.However,the ancestral branch of odontoceti was detected that have been under purifying selection.Considered that ODAM is functional gene in extant P.catodon,we speculated that ODAM is functional gene in odontoceti's LCA.This hypothesis is consistent with fossil evidence that the ancestors of toothed whales still have heteromorphic dentition and the enamel layer is relatively complex.In order to explore the molecular evolutionary mechanisms of increased tooth number in extant odontoceti,10 related genes(APC,AXIN2,EDA,EDAR,LTBP3,MSX1,OSR2,PAX9,SPRY2 and SPRY4)and 35 species were selected to carry out analysis.Regression analysis between the evolutionary rate of genes,which involved in tooth number regulation,and tooth number in mammals,five genes(APC,AXIN2,EDA,LTBP3 and SPRY2)showed a significantly positive relevance,and SPRY4 exhibited a significantly negative relevance.Further evolutionary analyses were carried out in cetacean group,odontoceti group and mysticeti group,respectively.The results showed that: the evolutionary rate of APC,EDA and EDAR genes in cetaceans was significantly higher than that in other groups,showing accelerated evolution.This might be associated with the fact that the number of teeth of cetaceans may have changed to adapt to their unique feeding patterns after they entered the water.Further research showed that the accelerated evolution was also found in APC,EDA and EDAR genes.Meanwhile,the evolutionary rate of EDAR gene was significantly different between Mysticeti and Odontoceti,which was related to their special evolutionary process.In addition,the evolutionary rate of SPRY2 gene showed the significance between the ancestral branch and terminated branch of Odontoceti.The evolutionary rate of Odontoceti ancestral branch was much higher(?11 times)than other clades,which indicated that SPRY2 gene have went through accelerated evolution.Moreover,we detected one positively seleted signal that distributed in the ancestral branch of Odontoceti in EDA gene.Bese on the results mentioned above,we speculated that the increase in tooth numbers may have occurred in the LCA of living cetaceans,and in the LCA of Odontoceti,the multi-tooth trait was further strengthened,thus increasing their ability to hunt in the water enviroment.In all,the tooth simplification of cetaceans was related to loss of mastication ability and inactivation of ODAM gene,and the tooth simplification may have occurred in the LCA of living toothed whales and baleen whales,respectively.However,the increase of tooth number might have occurred in in the LCA of living cetaceans,and then the multi-tooth trait was further strengthened in the LCA of living toothed whales.Totally,we mainly discussed three aspects deeply and systematically to explore the evolutionary model of tooth-related genes: mammalian dietary adaptation,tooth /enamel loss of mammals,and tooth changes in cetaceans.We first revealed the characteristics of mammalian dietary adaptation from the perspective of tooth-related genes evolution,and further improved the genetic mechanism of tooth loss / enamel loss in mammals.Importantly,we investigated the genetic basis of tooth simplification and increasing tooth number in cetaceans for the first time,which increased the knowledge of mechanism about aquatic adaptation in cetaceans based on the perspective of tooth evolution and adaptation. |
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