| In this study,the new record species of fishes in Zhejiang waters(Ambassis gymnocephalus,Branchiostegus albus,Hippichthys penicillus and Arothron firmamentum)were studied using morphological methods to describe their morphological characteristics and compare the morphological differences among species.At the same time,DNA barcoding technology was used to identify the species,and DNA barcode(COI and 12S rRNA gene)sequences were compared and analyzed to calculate intra and interspecific genetic distances,construct phylogenetic trees,and then explore phylogenetic relationships.The main results are as follows:1.Study on morphological characteristics and DNA barcoding of Ambassis gymnocephalusIn this study,Ambassis gymnocephalus samples were analyzed and identified by morphological characteristics and DNA barcoding.The main morphological characters were as follows:the first dorsal-fin rays 7,the second dorsal-fin rays 9,ventral-fin rays 9 and the anal-fin rays 10;the first dorsal fin had 1 barb,followed by 6 stiff spines,the second dorsal fin was small and soft,the dorsal surface of the body was grayish black and the abdomen was silvery white.The intra and interspecific genetic distances of COI gene sequences ranged from 0 to 0.0049 and 0.1271 to 0.2039,respectively,and the average interspecific genetic distance(0.1427)was 142.7 times higher than the average intraspecific genetic distance(0.001).The intra and interspecific genetic distances of 12S rRNA gene sequences ranged from 0 to 0.0059 and 0.0239 to 0.2634,respectively,and the average interspecific genetic distance(0.1311)was 131.1 times higher than the average intraspecific genetic distance(0.001),which were in accordance with the "10 fold rule".The neighbor-joining phylogenetic tree constructed based on COI and 12S rRNA gene sequences showed that all species formed monophyletic populations with high confidence values in the topology of the NJ tree,indicating that DNA barcoding is suitable for the study of phylogenetic relationships in fishes of the Ambassidae.2.Study on morphological characteristics and DNA barcoding of Branchiostegus albusThe morphological characteristics and DNA barcoding studies were conducted on the specimens of Branchiostegus albus.The main morphological characteristics were as follows:dorsal-fin rays 22,pectoral-fin rays 18～19,ventral-fin rays 6,anal-fin rays 12,caudal-fin rays 16～17;no black dorsal midline in front of the dorsal fin,no white spot or white band around the eye,and a yellow horizontal band on the posterior part of the caudal fin.The intra and interspecific genetic distances of COI gene sequences ranged from 0 to 0.0037 and 0.0130 to 0.1884,respectively,and the average interspecific genetic distance(0.0699)was 69.9 time higher than the average intraspecific genetic distance(0.001).The intraspecies and inter species genetic distances of 12S rRNA gene sequences varied from 0 and 0 to 0.1715,respectively,and the average interspecies genetic distance(0.0341)was 34.1 times higher than the average intraspecies genetic distance(0.001),which was consistent with the DNA barcode principle.The topology of the phylogenetic tree showed that the COI homologous sequences were identical,while the results of 12S rRNA homologous sequence analysis showed that the fish named Branchiostegus argentatus and Branchiostegus albus in GenBank were clustered into one group,and it was impossible to distinguish the two fish,indicating that the 12S rRNA gene sequences have limitations.3.Study on morphological characteristics and DNA barcoding of Hippichthys penicillusIn this study,the morphological characteristics and DNA barcoding study were conducted on the specimens of Hippichthys penicillus.The main morphological characteristics were as follows:dorsal-fin rays 27,absence of pectoral,ventral and anal fins;brown tail,white dorsal fin and yellow abdomen.In a study of Syngnathidae fishes based on COI and 12S rRNA gene fragments,the intra and interspecific genetic distances of COI gene sequences ranged from 0 to 0.0058 and 0.1782 to 0.2325,respectively,and the mean interspecific genetic distance(0.1620)was 162.0 times higher than the mean intraspecific genetic distance(0.001).The ranges of intraspecific genetic distance and interspecific genetic distance of 12S rRNA gene sequences were 0～0.0060 and 0.1265～0.2777,respectively,and the average interspecific genetic distance(0.1861)was 186.1 times of the average intraspecific genetic distance(0.001),which met the criterion that the intraspecific genetic distance was smaller than the interspecific genetic distance.In the phylogenetic tree,the same species were all clustered into one team with high confidence,indicating that DNA barcoding can be used as a valid and reliable species identification tool for Syngnathidae fishes.4.Study on morphological characteristics and DNA barcoding of Arothron firmamentumThe morphological characteristics and DNA barcoding study were conducted on the specimens of Arothron firmamentum.The main morphological characteristics were as follows:dorsal-fins rays 14,anal-fins rays 13～14,pectoral-fins rays 15～16,caudal fins rays 11;the body color was grayish black,there was no ventral fin,and the whole body was covered with small spines except around the eyes and the posterior part of the caudal stalk.The intra and interspecific genetic distances of COI gene fragment sequences varied from 0 to 0.0032 and 0.1440 to 0.1887,respectively,and the average interspecific genetic distance(0.1034)was 103.4 times higher than the average intraspecies genetic distance(0.001);the intraspecies genetic distance of 12S rRNA The intraspecies genetic distance and interspecies genetic distance of 12S rRNA gene fragment sequences varied from 0 to 0.0059 and 0.0179 to 0.1916,respectively,and the average interspecies genetic distance(0.0349)was 34.9 times of the average intraspecies genetic distance(0.001),which was consistent with the DNA barcoding principle.The phylogenetic tree was constructed based on the sequences of COI and 12S rRNA gene fragments,and the results showed that all individuals of each formed monophyletic lineages.The magnitude of the interspecific genetic distance of Tetraodontidae was consistent with the proximity of the relatives in the phylogenetic tree,indicating the feasibility of DNA barcoding for the taxonomic identification of Tetraodontidae. |
