| The Amblyopinae fishes(Perciformes,Gobioidei,Gobiidae).It is widely distributed in the Indo-West Pacific waters,in the Yellow Sea and Bohai Sea of China,East China Sea and South China Sea are also widely distributed.So far,there are 9 effective domestic species reported in Amblyopinae.However,recent studies have found that due to the similarity in external morphology of Amblyopinae fishes,some species have identification errors and have synonymous phenomena.At the same time,many cryptic species have been discovered one after another.Therefore,it is objectively necessary to conduct in-depth research on the classification and phylogeny of the existing Amblyopinae species in China,and to clarify the general evolutionary process and driving mechanism of its species or population.Therefore,this study aims to study the Amblyopinae fishes in the coastal waters of China.First,use DNA barcoding technology,combined with morphological determination,to reorganize and identify the main existing Amblyopinae species in China,and discover possible cryptic species or synonymous phenomena;Then,the mitochondrial genome-wide assay technology was used to analyze the structural characteristics of the mitochondrial genome of this subfamily fish and construct a phylogenetic tree to clarify the phylogenetic relationship and species evolution process of the Amblyopinae,and to explore the driving mechanism of its species radiation evolution;Finally,using the mitochondrial D-Loop gene sequence and nuclear gene S7 molecular markers,the population genetics and molecular evolution of the three species of the Amblyopinae were studied.It is hoped that the evolutionary process and driving mechanism of Amblyopinae species will be explored at the level of the intra-species population,which will lay a foundation for clarifying the phylogenetic relationship and evolutionary mechanism of the main groups of Amblyopinae in China.The main results obtained are as follows:1.DNA barcoding technology was used to amplify the mitochondrial COI and ND2 genes of all the Amblyopinae species,and construct their phylogenetic tree to calculate the K2 P genetic distance between the various species.The results show that the construction of the phylogenetic tree supports the division of all the collected Amblyopinae species into 6genera and 12 species.Three cryptic species have been identified in the Taenioides genus.They are Hainan Taenioides(tentative name Taenioides sp.hainan),Pearl River estuary Taenioides(tentative name Taenioides sp.zhujiang)and Yangtze River estuary Taenioides(tentative name Taenioides sp.changjiang).Genetic distance analysis showed that based on COI gene,the genetic distance of the three cryptic species was 0.059-0.134 with the known model species of Taenioides,which was similar to the average genetic distance of 0.155 among the subfamily of Amblyopinae.Based on the ND2 gene,the genetic distance between the three cryptic species and the known model species was 0.115-0.153,which was similar to the average genetic distance of 0.186 among all the subfamilies of Amblyopinae.Further confirmed that the three cryptic species identified may be effective species.Furthermore,the morphological measurement method was used to determine the countable and measurable traits of the three newly discovered cryptic species.The results showed that the identified T.sp.hainan and T.sp.zhujiang were significantly different from the T.cirratus model species(3 pairs)in the number of whiskers on the ventral surface of the head(7).The number of vertebrae of T.sp.zhujiang(27 pieces)is also significantly different from other Taenioides fishes(29 pieces).At the same time,the three species of Taenioides fishes are significant differences in the six standardized indicators(p<0.05)in head height/body length,head width/body length,body height/body length,kiss to pectoral fin start/body length,kiss to pelvic fin start/body length,and kiss to anus start/body length.Therefore,it is further confirmed that the T.sp.zhujiang,T.sp.hainan,and T.sp.changjiang may be three effective species.Through the Blast comparison of ND2 gene fragments,it was found that the T.sp.zhujiang and T.sp.hainan have very high homology with the reported T.sp.Thailand and T.sp.Gracilis.However,it may be a new recorded species for the first time reported in China,and the T.sp.changjiang has a large difference in gene sequence from the reported Taenioides fishes,so it may be classified as a new species.2.Further analysis of the complete mitochondrial genome sequence of the Amblyopinae fishes showed that: The length of the mitochondrial genome of the Amblyopinae(16552-17245bp)is consistent with the length of the existing teleost mt DNA genome,the base composition and structural order are basically consistent with the typical structure of teleost mt DNA.However,the mitochondrial genome of the Amblyopinae fishes also has its own characteristics.First,the 5'end of the t RNA-Cys non-coding OL region of the Amblyopinae fishes was found to convert RNA primers and DNA synthesis.Compared with the 5'-GCCGG-3' sequence reported in most fishes,5'-CCCGG-3' has base variations.It is speculated that the substitution of bases may form a new pattern and affect light chain replication.The synthesis function of RNA primer may affect the activity of mitochondrial ribonuclease H.Secondly,termination sequence regions(TAS)and conserved sequence blocks(CSB-1,CSB-2 and CSB-3)were identified in the control region,but central conserved sequences(CSB-D,CSB-E and CSB-F)were not identified,which may be related to mitochondrial metabolism and heavy chain replication.The phylogeny and evolution of the Chinese Amblyopinae fishes were analyzed using the mitochondrial whole genome sequence.The results showed that: Amblyopinae and Oxudercinae are the closest to their relatives in the evolutionary tree,and they nest in each other in the phylogenetic tree.Then it formed a sister group with Sicydiinae,and finally formed the Gobiidae with the Gobionellinae.This result indicates that the Amblyopinae cannot constitute a monophyletic group independently,and the classification relationship between the Amblyopinae and the Oxudercinae may need to be redefined.In order to further reveal the evolutionary mechanism of the Amblyopinae,we further analyzed the adaptive evolution of the subfamily of Amblyopinae at the mitochondrial genome level.Based on the clustering results of the constructed ML phylogenetic tree,combined with the goby ecotype,the Amblyopinae and Oxudercinae branches are calibrated as the foreground branch,and the other remaining branches the branch-site model test is performed for the background branch,and the positive selection test is performed for each gene.The results show that,compared with other groups,the foreground branch has screened positive selection sites in 4 genes(ATP6,ATP8,ND2 and ND4),and a total of 16 extremely significant positive selection sites have been detected.These mutations may cause amino acid changes.Polarity changes may lead to changes in protein structure,which may have caused mitochondrial ATP synthesis and function.This evolutionary process may be related to the changes in energy requirements of Amblyopinae and Oxudercinae fishes adapting to lowoxygen tolerance in tidal flats and intertidal caves.This result indicate that adaptive evolution may play an important role in the radiation evolution and speciation of the Amblyopinae.3.The mitochondrial D-Loop gene and nuclear gene S7 were used to study the population genetic structure and molecular evolution of three important species in the Amblyopinae.The population genetics and molecular evolution studies of 90 samples of Odontamblyopus lacepedii from 4 populations along the coast of China(Dandong,Qingdao,Lianyungang,and Shanghai Chongming Island)have shown that: Mitochondrial D-Loop genes define a total of 47 haplotypes,with a diversity index of 0.950 and a total nucleotide diversity index of 0.0054,showing high genetic diversity.ML cluster analysis showed that in the study area,only some individuals in Qingdao population showed obvious genetic differentiation among O.lacepedii populations,but there was no significant differentiation among other populations,and the FST value between populations was also small,at 0.0175-0.1798,and the Nm value between populations is relatively large,between 2.2802 to 28.0877,only the Qingdao population and other populations have a significant genetic differentiation.The AMOVA test showed that the differences between groups mainly existed within the group(90.36%),further confirming that the differentiation mainly came from within the group.Based on the calculation of the molecular clock theory,the expansion time of the O.lacepedii population was about 55,000 to 110,000 years ago,which was in the late Pleistocene.It is speculated that in the late Pleistocene,the shallow sea shelf areas of the Yellow and Bohai Sea were exposed to land,and the population replanting and population expansion events of O.lacepedii in the Yellow and Bohai Seas after the glacial period may be due to the lack of significant population genetic structure of O.lacepedii important reason.The analysis of the nuclear gene S7 gene showed that 74 haplotypes were defined based on83 individuals,the diversity index reached 0.997,and the total nucleotide diversity index reached 0.0086,showing high genetic diversity.The ML cluster analysis showed that there was no significant differentiation among O.lacepedii populations in the study area,and the FST value between the populations was also small,ranging from-0.0053 to 0.0037,and they were not significant,and the Nm value between the populations was larger,between-240.885 to 161.312.The AMOVA test further showed that 100.08% of the differences between the four geographic populations existed within the populations,while only-0.08% existed between the populations.The genetic distances between the four geographic populations were all less than 0.01,indicating that there was almost no genetic differentiation between the populations.This result is slightly different from that of the D-Loop gene.The reason may be related to the slower evolution rate of nuclear genes compared to mitochondrial genes,so the resolution is relatively low.However,the research results of mitochondrial D-Loop genes indicate that the Qingdao population has a statistically significant differentiation from other groups,so it may be treated differently in future resource management and protection.A total of 89 samples of Odontamblyopus rebecca from 4 coastal populations of China(Wenling?Ruian?Jiulong River and Pearl River Estuary)were studied on population genetics and molecular evolution.The results showed that: mitochondrial D-Loop gene defines 54 haplotypes,the diversity index reaches 0.954,and the total nucleotide diversity index reaches0.0087,showing high genetic diversity.ML cluster analysis showed that in the study area,the 4 populations of O.rebecca differentiated into 2 groups.Group A is composed of Jiulong River population,Pearl River Estuary population,part of Wenling population and Ruian population;Group B is other Wenling population and Ruian population.In addition,the haplotype network diagram also supports the differentiation of these two groups.The differentiation index of the two groups was 0.115,and it was extremely significant(P<0.01),indicating that there is a moderate degree of genetic differentiation in the O.rebecca population.The AMOVA test showed that the differences between groups mainly existed within the group(88.5%),further confirming that the differentiation mainly came from within the group.Based on the calculation of molecular clock theory,the differentiation time of the two taxa was about 55 000-110 000 years ago,in the late Pleistocene.It is speculated that the geographical separation caused by the sea level rise and fall in the late Pleistocene led to the occurrence of different geographical groups of O.rebecca.The differentiation,and the subsequent mixing of the differentiated taxa,the secondary connection has resulted in the existing pedigree and geographic pattern of O.rebecca.The analysis of the nuclear gene S7 gene showed that a total of 45 haplotypes were defined based on 90 individuals,the diversity index reached 0.899,and the total nucleotide diversity index reached 0.0057,showing high genetic diversity.ML cluster analysis showed that there was no significant differentiation among O.rebecca populations in the study area,and the FST value between the populations was also small,ranging from-0.0095 to 0.0015,and none of them were significant,while the Nm value between populations was relatively large,ranging from-82.3331-344.3276.The AMOVA test showed that 100.71% of the differences between the 4 geographic populations exist within the population,while only-0.71% exist between the populations(FST:-0.00075).The genetic distances between the 4 geographic populations are all less than 0.01,further indicating that there is almost no genetic differentiation among population differences.This result is slightly different from the results of the D-Loop gene.The reason may be related to the slower evolution rate of nuclear genes compared with mitochondrial genes,so the resolution is relatively low.However,the research results of mitochondrial D-Loop genes indicate that compared with other groups,the Wenling and the Ruian group have a statistically significant differentiation,so they may need to be treated differently in the future resource management and protection.A total of 104 samples of Taenioides.sp.changjiang from 6 populations(Weishan Lake?Luoma Lake?Hongze Lake?Gaoyou Lake?Chaohu Lake and Taihu Lake)in the Yangtze River Estuary and its adjacent waters were carried out population genetics and molecular evolution studies,the results show that: The mitochondrial D-Loop gene defines 12 haplotypes,the diversity index reaches 0.748,and the total nucleotide diversity index reaches0.0055,showing low genetic diversity.ML cluster analysis showed that within the scope of the study,there has been significant genetic differentiation among the T.sp.changjiang populations,among which the genetic differentiation between Taihu Lake and other populations,Luoma Lake and Weishan Lake and other populations is the most significant.The FST value between groups is between-0.0340-0.6355,and the gene flow between groups is between 0.4117-9.6051.The AMOVA test showed that 69.4% of the differences among the six geographic populations existed within the populations,and 30.6% existed between the populations,further indicating that there is a certain genetic differentiation among the T.sp.changjiang populations.It is inferred that the population genetic structure of the T.sp.changjiang may have invaded the major freshwater lakes with the help of water transfer engineering,which suggests that the T.sp.changjiang may adapt to the new invasion environment through the change of genetic material.The analysis of the nuclear gene S7 gene showed that a total of 17 haplotypes were defined based on 111 individuals,the diversity index reached 0.501,and the total nucleotide diversity index reached 0.0013,showing low genetic diversity.The ML cluster analysis once again showed that there was no significant genetic differentiation among the T.sp.changjiang populations in the study area,and the FST value between the populations was also small,ranging from-0.0287 to 0.2222,while the gene flow Nm between the populations was relatively large,from-345.3276 to 209.5840.The AMOVA test further showed that 95.89% of the differences between the 6 geographical groups existed within the groups,while only 4.11% existed between the groups,and the genetic distances between the 6 geographical groups were all less than 0.01.The reason may also be related to the slower evolution rate of nuclear gene S7 compared to mitochondrial genes,so the resolution is relatively low.However,the results of the mitochondrial D-Loop gene showed that there has been significant genetic differentiation among different populations during the invasion of the T.sp.changjiang.The correlation between this genetic differentiation and the success of the invasion still needs to be further studied in the future. |
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