Molecular Basis Of Local Adaptation And Speciation Of The Euchiloglanis Fish Complex(Teleostei: Siluriformes)

How to survive and adapt to the complex natural environment has always been a core issue for evolutionary success.Local adaptation is an important aspect of biological adaptation to the natural environment,and it is prevalent in nature.It plays an important role in the shaping of biodiversity,the maintenance of species genetic variation,the expansion of species distribution,and ecological speciation.Although the evidence of local adaptation is widespread,the genetic foundation for it has so far been poorly understood.Fishes have a strong ability to adapt to the environment and eventually lead to speciation,making fish the most abundant group in vertebrates.In addition,their distribution is strictly linked to water system.Moreover,their genetic diversity and population structure are easily affected by geological events.Compared with other vertebrates,there is a close relationship between fish distribution and the evolution of water basins.Thus,fish,especially freshwater fish,are ideal organisms for studying local adaptation and speciation.Fish from the genus Euchiloglanis Regan,1907 are a group of demersal and non migratory freshwater catfishes(Teleostei: Siluriformes).Three species(E.dorsoarcus,E.longus and E.phongthoensis)distributed in the Red River,and other three species(E.davidi,E.kishinouyei and E.longibarbatus)distributed in the upper Yangtze River in the Hengduan Mountain Region of China have been described so far.The three species distributed in the upper reaches of the Yangtze River in the Hengduan Mountains are very similar in their morphology,and their genetic diversity is low at the molecular level.The separation of these three species supposed to be recent and may actually be in the process of speciation.There may be no complete reproductive isolation and perhaps there is gene flow between each other(here we treat the three Euchiloglanis fishes distributed in the Hengduan Mountain Region as “Euchiloglanis fish complex”).Thus,the Euchiloglanis fish complex is a particularly ideal model for studying local adaptation and speciation.Based on the above considerations,this research takes the Euchiloglanis fish complex as our research goal.The molecular basis of local adaptation and speciation was discussed from the perspective of multivariate morphometrics,geometric morphometrics,phylogeography and population genomics.The scientific questions to be answered are as follows:(1)Which phenotypic characteristics may play an important role in the process of local adaptation and speciation?(2)How about the genetic differentiation and phylogeography of the complex species?(3)Which genes and ecological factors may play a key role in the process of local adaptation and speciation?The main results of the present study are as follows:1.Multivariate morphometrics and geometric morphometrics were used to study the morphological differentiation of the Euchiloglanis fish complex.A total of 233 samples from 8 populations were selected,and 11 anatomical coordinates and 52 morphological characteristics were selected for multivariate morphological measurements;besides,a total of 226 samples from 8 populations were selected,and 20 and 17 landmarks of the dorsal view and ventral view were selected for the geometric morphometric analysis,respectively.The results are as follows:(1)The results from the Principal Component Analysis(PCA)and Linear Discrimination Analysis(LDA)based on the standardized multivariate morphological data show that samples from Jinsha River and Qingyi River have great morphological differentiation,and can be distinguished from the individuals of Dadu River and the Yalong River by a few morphological characteristics.The morphological differences among the different populations of the Euchiloglanis fish complex are mainly manifested in the head relative size,mouth size,the origin of the pectoral fins,pelvic fins,dorsal fin,anal fin and adipose fin,eye diameter,jaw extension,gill width,anus position,the length of the dorsal and adipose fin,and caudal peduncle.(2)The results from PCA and Canonical Variable Analysis(CVA)based on the landmarks of truss-based morphometric data were consistent with the results from the multivariate morphometric analysis:(1)For the samples from Qingyi River,the head is extraordinary flat,the origin of the dorsal fin falls a relatively posterior position,and the total dorsal fin display a longer length,the origin of the adipose fin also posteriorly locates with shortest length,and they have the longest anal fin;(2)For the populations of the Jinsha River,the degree of head flat is the minimum,the starting point on the dorsal fin falls a relatively anterior position,and the dorsal fin length is the shortest,while the starting point on the dorsal fin falls a relatively front position,and the adipose fin length is the longest,the mouth is relatively the smallest,and the starting point of anal fin and anus falls a relatively front position;(3)For the samples from Yalong River,the mouth is the largest,and the starting point of the anal fin and anus falls a relatively posterior position;(4)The anal fin lengths are not very different and the caudal peduncle for the populations from the Dadu River and the Yalong River is the highest,while for the populations from the Jinsha River is the shortest.2.Genetic differentiation and phylogeography of the Euchiloglanis fish complex were analyzed based on mitochondrial DNA(mtDNA)cyt b gene sequences and 10 microsatellite markers.The results are as follows:(1)In total,360 mtDNA cyt b sequences were obtained and 125 haplotypes were recovered.The phylogenetic trees and networks constructed based on haplotypes indicated that the Euchiloglanis fish complex was monophyletic,and the populations were split into four independently evolving lineages,with the lineages appearing to reflect geographical associations with rivers;(2)Ten polymorphic microsatellite loci were used to genotype 192 individuals of 7 populations.The results from the Bayesian genetic clustering analyses based on the microsatellite loci were consistent with the results of phylogenetic trees that based on mtDNA cyt b haplotypes;(3)The Euchiloglanis fish complex appeared to have a moderate level of genetic diversity,and the genetic differentiation between two rivers reached a high degree of separation;(4)The results from the mantel tests based on mtDNA cyt b and microsatellite loci suggested that the genetic distance was significantly correlated with the geographical distance;(5)Population expansion signals were detected from mismatch distribution analyses,neutrality tests,and Bayesian skyline plots.The expansion times were concurrent with Quaternary interglacial fluctuations;(6)The results from the divergence time estimation showed that the Euchiloglanis fish complex diverged from its sister clade Pareuchiloglanis sinensis at around 1.3 Ma;within the Euchiloglanis fish complex,the split of the Dadu and Yalong Rivers was at about 0.7 Ma(95% HPD = 0.5-1.0);the divergence time between the Dadu-Yalong and Jinsha-Qingyi River populations occurred at about 1.0 Ma;this divergence time is in concordance with recent geological events,including the Kun-Huang Movement(1.2-0.6 Ma)and the lag time(<2.0 Ma)of river incision in the Hengduan Mountain Region.3.The population genomic analyses were performed based on RAD-seq(Restriction-site Associated DNA sequencing)data for the Euchiloglanis fish complex.Eight populations of the Euchiloglanis fish complex containing 213 samples,and three P.sinensis as outgroup were sequenced by Illumina HiSeq 4000.A total of 339.42 Gb raw data were yielded,and 226.52 Gb clean data was obtained after quality filtered.A total of 6606 SNPs were identified across the populations,and the results based on these SNPs or RAD-tags containing SNPs are as follows:(1)The phylogenetic relationships were reconstructed by Bayesian Inference(BI),Maximum Parsimony(MP),and Maximum Likelihood(ML)methods.The topologies of the BI,MP,and ML trees were similar,and indicated that the Euchiloglanis fish complex was divided into four monophyletic lineages according to the rivers.The results from PCA and Discriminant Analysis of Principal Components(DAPC)support the results of phylogenetic analysis,indicating a close relationship between populations of the Dadu River and the Yalong River;(2)The SOM(Self-organizing feature map)model was used to identify the nucleotide diagnostic sites between different rivers of Euchiloglanis fish complex.A total of 1891 nucleotide diagnostic sites were obtained;(3)The results from the gene flow analysis indicated that the gene flow from Dadu River to Yalong River reached 3.735,which explained why the samples from Dadu River and Yalong River have close genetic relationships and relatively small morphological differentiation;(4)The results from the Bayesian coalescent species delimitation showed that the Euchiloglanis fish complex can be divided into three species,one is from Jinsha River,one is from Qingyi River,and another one is from Dadu River and Yalong River;(5)LOSITAN and ARLEQUIN were used to perform outlier loci analysis.The selected sites shared by the two methods were ranged from 38-163 within each pairwise river systems.Furthermore,the selective loci shared by LOSITAN and ARLEQUIN analyses within each pairwise rivers were used to do functional annotation based on the database of Swissprot and GO to screen candidate genes associated with local adaptation of the Euchiloglanis fish complex.Candidate genes associated with local adaptation and speciation for the Euchiloglanis fish complex are as follows:(1)genes related to transmembrane transport were the MYH family,MYO1 H,Myosin IC,EXOC3,DNM1,STOM,and SLC2A10;(2)genes associated to synaptic transmission were CA family,ACTL6 A,PPFIA1,TAAR8;(3)genes involved to transcriptional regulation were ZSCAN29,ZNF257,KANSL3,HCFC1;(4)genes regulated a variety of cellular processes,including cellular growth,senescence and differentiation were ID1,MYOCD,MKL2,PRKCD,MYH10,DHX32,and ANK2;(5)TUBD1,TUBB2 A are key participants in processes such as mitosis and intracellular transport;(6)LRP1B was a potential cell surface proteins that bind and internalize ligands in the process of receptor-mediated endocytosis;(7)genes of SLC family were involved in phosphate transporter;(8)genes of PRKCE,RAB32 and RAB38 were involved in immune response or played a role in the maturation of phagosomes that engulf pathogen;(9)TRPV4 was a gene related to the regulation of systemic osmotic pressure.(6)The results from the GO enrichment based on the GO annotation of difference SNPs and selective loci between pairwise river showed that the GO terms were mainly related to: cell differentiation,regulation of cellular component organization;regulation of cell morphogenesis;tissue development;growth;transcription;synthesis process;metabolism;transmembrane transport and ion regulation;synaptic transmission;cellular response to hypoxia;cellular response to biotic stimulus,and immune response;(7)The cluster analysis based on the climatic factors showed that the difference of precipitation,temperature,and altitude was the key climatic factors that caused local adaptation and speciation for the Euchiloglanis fish complex.In summary,this study uses a step-by-step approach to analyze the genetic basis of the Euchiloglanis fish complex in the process of local adaptation and speciation.Firstly,the morphological differentiation of this fish complex was analyzed by using the methods of multivariate morphometrics and geometric morphometrics.Secondly,the genetic differentiation of this fish complex was studied based on the mitochondrial cyt b gene sequences and 10 polymorphic microsatellite markers.Finally,based on the whole genome scanning of RAD sequencing,the key genetic changes related to phenotypic variation during the local adaptation and species formation were analyzed.This provides information and examples for the study of the causes and adaptability of species diversity in the Hengduan Mountains of Southwest China.