| In this study,Uranoscopidae species and Zeidae species in the coastal waters of Zhejiang Province are taken as the research objects,and their morphological characteristics are described by means of morphological study,and the morphological differences among different species are compared.At the same time,the DNA barcoding was used to identify its species,and the DNA barcoding(12S r RNA and COI gene)sequences were compared and analyzed the sequence differences within and between species,the genetic distances within and between species were calculated,the molecular phylogenetic tree was constructed,and then the evolutionary relationship between Uranoscopidae species and Zeidae species were discussed.In addition,the preliminary genome assembly of Ichthyscopus pollicaris(Vilasri,Ho,Kawai & Gomon,2019)was carried out,which laid the foundation for phylogenetic genomics research of Uranoscopidae species.1.Morphological study and DNA barcoding of Uranoscopidae speciesUranoscopidae species belongs to Osteichthyes,Actinopterygii,Perciformes and Trachinoidei.Fishbase data show that there are 9 genera and 54 species,most of which are warm-water bottom fishes and widely distributed in the Pacific and Atlantic Oceans.Because of the similarity in morphological characteristics,the results of species identification,taxonomic status and biological evolution were affected.In addition,there are some deficiencies and errors in the description of morphological characteristics of the Uranoscopidae species in China.In this study,Uranoscopus japonicus Houttuyn(1782),Uranoscopus tosae(Jordan & Hubbs,1925),Xenocephalus elongatus(Temminck &Schlegel,1843)and I.pollicaris(Vilasri,Ho,Kawai & Gomon,2019)were studied.The main results of this study are as follows:In the morphological studies,the countable and measurable traits of Uranoscopidae species were measured to describe their main morphological characteristics and compare their morphological differences.Among them,there are differences in the fin spines,short spines on the lower edge of preopercle,body color and sides and back of body with spots,which can be identified and classified by comparing the main morphological characteristics.U.japonicus is characterized by 2 dorsal-fins,and the upper dorsal-fin spines was black;irregular white spots on the back of the body;3～4 spines on the lower edge of preopercle.U.tosae is characterized by 6 spines on the lower edge of preopercle;1short spine below the lower edge of preopercle and 2 shoulder spines above the gill foramen.X.elongatus is characterized by 1 dorsal-fins,without dorsal-fin spines;1 small spine above the posterior operculum,no spines on the lower edge of preopercle and no short spines on the opercle;sides and back of body with dark spots.I.pollicaris is characterized by the dorsal-fin is composed of 2 dorsal-fin spines and 18 dorsal-fin soft rays;there were no shoulder spines and brachial spines in the preopercle,inferior operculum and shoulder;the lower part of the anterior branchial cap exposed,and there was a villi-like process above the posterior margin of the branchial cap;There is a feathery periderm above the shoulder;Head is thicker;The upper and lower lips have villi;body color lighter,body dorsal and dorsal-fins with white spots.In the DNA barcoding studies,the DNA barcoding(12S r RNA and COI genes)of four species of Uranoscopidae species were amplified.Combined with the homologous sequences downloaded from Gen Bank,the sequence differences were compared and analyzed,and the phylogenetic tree was constructed.In the study based on the 12 S r RNA gene fragment,the intraspecific genetic distance of U.japonicus was 0.0022,and the intraspecific genetic distance of U.tosae was 0.0024.The genetic distance ranged from0.0622 to 0.2724.The intraspecific genetic distance is less than the interspecific genetic distance.According to the topological structure of the phylogenetic tree,U.japonicus is located in the upper of the NJ tree,which belongs to the early differentiated species,and I.pollicaris is located in the base of the NJ tree,which belongs to the late differentiated species.At the same time,U.japonicus and U.tosae gather together a branch,and then with Uranoscopus oligolepis into a branch,and then together with the Uranoscopus bicinctus,and then together with the X.elongatus into a branch,and finally with the I.pollicaris into a branch,indicating that U.japonicus,U.tosae and U.oligolepis have closer genetic relationship,and they have a slightly distant genetic relationship with the I.pollicaris and X.elongatus.Based on COI gene fragment,the intraspecific genetic distance ranged from 0.0007 to 0.0396;the interspecific genetic distance ranged from 0.1376 to0.2759,in which the genetic distance between U.tosae and Uranoscopus bicinctus was the smallest(0.1376),followed by U.tosae and U.japonicus(0.1923),followed by U.japonicus and I.pollicaris(0.1985),and then X.elongatus and I.pollicaris(0.2285),and U.bicinctus and X.elongatus had the largest genetic distance(0.2759).According to the topological structure of the phylogenetic tree,all the reference sequences were clustered with the self-measured U.japonicus,U.tosae,X.elongatus and I.pollicaris,and all species formed a single-line population with high confidence values on the topological structure of the NJ tree.In the base composition of 12 S r RNA and COI gene sequences,the average A+T content was slightly higher than the average G+C content,which was consistent with the existing research results on marine fish.The genetic distance between species was consistent with the phylogenetic tree,which indicated the effectiveness of DNA barcoding for the Uranoscopidae species.In the genome survey of Ichthyscopus pollicaris studies:Genome survey sequencing was used for sequencing.After filtering,the clean data amount was 72.27 Gb,the sequencing quality was normal and the sequencing error rate was normal.In addition,the total number of K-mer was 56,507,961,416,the depth of K-mer was 91,the genome size of the sample was about 598 Mb,the revised was 590 Mb,the heterozygosity was 0.45 %,and the proportion of repeat sequences was 36.02 %.A genome of 532.06 Mb in size was obtained by assembling a genome of I.pollicaris from scratch.The length of Contig N50 was 1.48 kb,and the length of Scaffold N50 was 11.24 kb.In the assembly results,the number of Scaffolds were longer than 100 bp was 480,035,and the number of Scaffolds were longer than 2 kb was 42,345.The genomic sketch of I.pollicaris assembled in this study laid the foundation for further phylogeny genomics research of the Uranoscopidae species.2.Morphological study and DNA barcoding of Zeidae speciesZeidae species belongs to Osteichthys,Actinopterygii,and Zeiformes,which is a warm fish widely distributed in the Pacific Ocean,Atlantic Ocean and Indian Ocean.There are 13 species belonging to 7 genera in the world and 9 species belonging to 7 genera in China.In this study,Zenopsis nebulosa(Temminck & Schlegel,1845)and Zeus faber Linnaeus(1758)were studied.The main results of this study are as follows:In the morphological studies,the characteristics of countable and measurable traits of Z.faber and Z.nebulosa were measured,the main morphological characteristics were described,and their morphological differences were compared.It was found that the dorsal-fin spines of Z.faber and Z.nebulosa were slender and hard,and there were fin spines in the anal fin.The dorsal-fin spines and dorsal-fin soft rays had fin membrane and extended into linear shape,and some samples could reach the tail.Body height,close to half the full length;big mouth;big eyes;the gill pore is wide.In addition,there is a large black spot in the middle of both sides of the body of Z.faber,which is the main identification feature,and no obvious spots were found on the body side of Z.nebulosa,which could be identified from other traits.In the DNA barcoding studies,24 sequences were detected in the 12 S r RNA gene sequence analysis,including 16 haplotypes,which were 3 haplotypes of Z.faber and 13 haplotypes of Z.nebulosa.The intraspecific and interspecific genetic distances were calculated based on 12 S r RNA gene.The intraspecific genetic distance of Z.faber was0.0039,and the intraspecific genetic distance of Z.nebulosa was 0.0089.The genetic distance between species ranged from 0.0535 to 0.3648.The genetic distance between Z.nebulosa and Zenopsis stabilispinosa was the smallest(0.0535),and the genetic distance between Z.stabilispinosa and Parazen pacificus was the largest(0.3648).A phylogenetic tree was constructed based on the 12 S r RNA gene sequence.The results showed that Z.nebulosa firstly clustered with Z.stabilispinosa,then with Z.faber,then with Cyttopsis rosea,then with Parazen pacificus,and finally with Zenion japonicum.The analysis of the COI gene,32 COI sequences were detected,including 10 haplotypes,7 haplotypes in Z.faber and 3 haplotypes in Z.nebulosa.The intraspecific genetic distance of Z.faber was0.0017,and that of Z.nebulosa was 0.0009.The genetic distance between species ranged from 0.0400 to 0.2011,of which the genetic distance between Z.nebulosa and Zenopsis conchifer was the smallest(0.0400),followed by Z.nebulosa and Z.stabilispinosa(0.0479),followed by Z.stabilispinosa and Z.conchifer(0.0521),followed by Z.nebulosa and Z.faber(0.1596),and Z.faber and P.pacificus had the largest genetic distance(0.2011),which met the standard that intraspecific genetic distance was less than interspecific genetic distance.The phylogenetic tree was constructed based on COI gene fragment sequence.The results showed that all the reference sequences were clustered with the self-tested Z.faber firstly clustered with Z.nebulosa,Z.conchifer clustered with Z.nebulosa,then clustered with Z.stabilispinosa,and then clustered with Z.faber,and then clustered with P.pacificus,C.rosea and Z.japonicum respectively.All species had high confidence values in the topological structure of NJ tree.In summary,the results based on12 S r RNA and COI gene fragments are basically consistent. |
