Morphological Comparison And Temperature Adaptation Of Phoxinus Species In China

Phoxinus,a kind of small cold-water fish,is probably the most widely distributed member in the Cyprinidae family.Its large-span latitudinal distribution creates the species diversity within the genus.However,some of the species still retain high similarities due to the consistency of origin,the sub-species of different geographical distribution,and the existence of sympatric species.In some previous studies,there are confusions about species classification and naming.In addition,systematic studies on the age and growth,phylogenetic relationships,population genetics,and adaptive evolution of Phoxinus species have rarely been reported.Considering the divergent distribution among several temperature zones and differences in phenotypic and physiological traits among species,the Phoxinus fish can be used as good materials for researches on the temperature-based adaptive evolution of species.In this study,we collected biological samples of eight Phoxinus species distributed from the Yangtze River to the main river systems in the north of China.Combined with morphological indicators,molecular markers and bioinformatic analysis were applied to explore morphological kinship and differences among species,phylogenetic relationships,genetic diversity among populations and related environmental factors,as well as molecular mechanisms which had been evolutionarily selected.The main results of this study are as follows:(1)Based on the comparative analysis of three countable characters including lateral scale number(or longitudinal scale number),the number of gill rakes outside the first gill arch and the number of vertebrae,as well as 14 proportional characters including standard length/body height,head length/head height,and caudal peduncle length/caudal peduncle height,it was found that there were significant inter-species differences among the eight Phoxinus species.The principal component analysis of morphological indexes showed that the first two components counted 91.44%of the total variance.In the first principal component,the loading values of standard length/caudal peduncle height,head length/caudal peduncle height,and caudal peduncle length/caudal peduncle height were relatively high.In the second principal component,the loading value of lateral scale number was the highest.Discriminant analysis showed that the comprehensive discriminant accuracy of 14 indexes for 8 Phoxinus species was up to 98.1%after eliminating the three proportional characters of standard length/head length,standard length/caudal peduncle length,and head length/snout length.Morphological cluster analysis showed that the three groups of similar morphologic pairs,namely,P.phoxinus and P.phoxinus ujmonensis,P.brachyurus and P.grumi,P.lagowskii and P.oxycephalus,constituted three basic branches respectively.Because the number of gill rakes outside the first gill arch,standard length/body height,and the depth of caudal fin bifurcation were obviously different from other 7species,P.percnurus formed a single branch and subsequently clustered with other 7species.(2)Samples of P.phoxinus ujmonensis,P.lagowskii,and P.oxycephalus were selected to conduct the comparative analysis of age and growth,owing that resources of these three species were relatively abundant.The dominant standard-length groups of the three Phoxinus species were 41 mm to 50 mm,41 mm to 50 mm,and 51 mm to 60 mm,respectively,while the average standard lengths were 41.16±12.72 mm,55.54±14.51mm,and 62.99±18.28 mm,respectively.And the dominant age groups of the three Phoxinus species were age 2,age 1,and age 1,respectively,indicating that the body length and age of them were in the process of miniaturization.The relationships between standard length and body weight showed that the growth rate of standard length was higher than that of the body weight in P.phoxinus ujmonensis,the growth rate of standard length was lower than that of the body weight in P.lagowskii,and the growth rate of standard length and body weight were equal in P.oxycephalus.The asymptotic lengths(L_?)and growth performance index(?)of these three species presented the tendency of P.phoxinus ujmonensis(L_?=143.70 mm,?=3.47)<P.oxycephalus(L_?=175.95 mm,?=3.63)<P.lagowskii(L_?=218.73 mm,?=3.68).(3)Phylogenetic trees based on concatenated sequences of mitochondrial genes and orthologous genes showed that the three pairs of species(P.phoxinus+P.phoxinus ujmonensis),(P.brachyurus+P.grumi),and(P.lagowskii+P.oxycephalus)could form stable basic branches in each tree,which is consistent with the results of the clusters based on morphological indicators.However,due to the different gene types and numbers for constructing the phylogenetic trees,there were some differences in the relationships of several species among the three trees.For examples,P.percnurus showed a closer genetic relationship with(P.lagowskii+P.oxycephalus)in the tree based on mitochondrial genes,but a closer genetic relationship with(P.brachyurus+P.grumi)in the tree based on orthologous genes.P.eos,distributed in north America,preferentially clustered with(P.tumensis+(P.phoxinus+P.phoxinus ujmonensis))in the tree based on concatenated sequences of 4 mitochondrial genes,but formed a single branch and subsequently clustered with the large branch consisted of the genus distributed in Eurasia in the tree based on concatenated sequences of 13 mitochondrial genes.(4)In this study,we developed 12 polymorphic microsatellite loci for P.phoxinus ujmonensis.The analysis of microsatellite and mt DNA markers revealed high(n DNA)and moderate(mt DNA)genetic diversity across seven populations,respectively.Moderate differentiations were also detected among several populations,indicating the impact of habitat fragmentation and divergence.The absence of isolation by distance implied an extensive gene flow,while the presence of isolation by adaptation implied that these populations might be in the process of adapting to divergent habitats.Correlation analysis showed that abiotic factors like dissolved oxygen,p H,total dissolved solids,and conductivity in water,as well as biotic factors like plankton diversity,and fish species diversity had impact on genetic diversity and divergence in P.phoxinus ujmonensis populations.The results of this study will provide an insight into the effect of environmental factors on genetic diversity and contribute to the study of population genetics of sympatric species.(5)In this study,we chose P.phoxinus ujmonensis,P.lagowskii,and P.oxycephalus as object to investigate adaptive evolution among Phoxinus species,as the sampling sites of these three species located in three different temperature zones.Based on transcriptome sequencing,annotating and blasting,a total of 2,163 orthologous genes were identified.In view of the differences in morphology and growth characteristics among the three species,we calculated the relative expression levels(FPKM:Fragments Per Kilobase of transcript per Million fragments mapped)of BMP(bone morphogenetic protein)and GH-IGF(growth hormone,insulin-like growth factor)genes.The average FPKM values of BMP genes and GH-IGF axis genes corresponded to the relationship of growth characteristics of these three species,namely,P.phoxinus ujmonensis<P.oxycephalus<P.lagowskii.Through the selection pressure analysis in the codeml program,57,272,and 206 fast evolving genes,as well as 96,46,and 31 positively selected genes were identified for P.phoxinus ujmonensis,P.lagowskii and P.oxycephalus,respectively.GO enrichment analysis and online annotation in the Uni Prot protein database revealed that the evolutionarily selected genes in P.phoxinus ujmonensis(such as crls1,dhfr,and efs)might play key roles in lipid metabolism,and maintaining mitochondrial function,so as to respond to cold.Meanwhile,they might also positively participate in the transduction of environmental signals,balancing cell proliferation and apoptosis,maintaining homeostasis,and increasing body immunity.In the evolutionarily selected genes shared by warm-adapted P.lagowskii and P.oxycephalus,there were several significant biological processes and physiological functions related to environmental adaptation.For examples,genes like klhl42,cpn1,and cse1l were positively involved in the construction of humoral immunity,which was the first fortress for fish to against external stimuli.At the same time,genes like clcn7 and epas1b positively responded to the stimulation of temperature,dissolved oxygen and other abiotic factors,and further participated in the response to hypoxia.To be noted,genes like klhl22 and related“Cul4-RING E3 ubiquitin ligase complex”might promote the tolerance of P.lagowskii and P.oxycephalus to heat stress.In addition,genes like irx2a,epas1b,tsc22d3,and ugdh were positively involved in the regulation of growth,energy intake,development of bone and other organs,as well as the formation of body axis and other related processes.Moreover,these genes presented selection advantages in the evolution of P.lagowskii,and P.oxycephalus,which could ensure better growth characteristics than P.phoxinus ujmonensis.In conclusion,the small and slender body shape of Phoxinus species was a result of its phenotypic adaptation to the stream environment.During its long term of evolution,different species showed differences in the characteristics related to swimming ability when adapting to divergent environment,such as body axis and tail shape,as well as sensory system development,such as eye size and lateral line.Some highly conserved phenotypic characteristics among species,as well as genetic similarity in evolutionary rate,ensured a relatively stable evolutionary relationship within this genus.In the process of distribution to the lower latitudes and warmer waters,P.lagowskii,and P.oxycephalus were subjected to the selective pressure from the ambient temperature,which showed the adaptive evolution to temperature.Temperature differences in interspecific distributions may also well explain the differences in growth characteristics.