Comparative Studies Of Epinephelus Moara And Epinephelus Bruneus

Whether Epinephelus moara and E.bruneus,with quite similarity in externalcharacters and distribution,belong to one species or not,has being existed theargument and confusion for a long time.To clarify the questions in classification,study and ratify the genetic status and provide specific molecular markers identifyingthe two grouper species,comparative studies were investigated based on themorphological,cytogenetic and molecular analysis.It provided solid foundation forfurther research,practice and protection of germplasm of E.moara and E.bruneus.Main results revealed as followings:1.Outer shape,color and bands were very similar in E.moara and E.bruneus,but their bars on the body were different.By means of the Fisher Discriminant FoundAnalysis,statistically significant difference would be found to exist between E.moara and E.bruneus (p＜0.001) in meristic and morphometric characters,such as thenumbers of dorsal fin rays,gill rakers and pyloric caeca.Although both species werebasically homologous on the configuration and composition of neurocranium,splanchnocranium,vertebrate,rib and appendicular skeleton,there were manyremarkably differences between them,such as preorbital,post-orbital,rreoperculare,urohyal,predorsal interneural spine and the connection of hypurals and caudal spineand so on.These skeletons could be used as the important characters identifyinggenera or species in fishes.Accordingly,we considered that E.moara and E.bruneuswere different species that could be discriminated by morphological characteristics.2.Cytogenetic characteristic analysis of Epinephelus moara and E.bruneus wasperformed by using karyotype,Ag-NORs,C-banding,FISH with 18S rDNA,5SrDNA and telomere sequences.Although both species shared 2n=48 chromosomes,their karyotypes were different in terms of the number of uni- and bi-armedchromosomes and the localization of nucleolar organizer regions (NORs) revealed byAg-staining and FISH.Karyotypic formulaes were 4SM+44T (NF=52) in E.moaraand 2M+4SM+42T (NF=54) in E.bruneus.Different Ag-NORs sites could beobserved on the short arms or sub-centromere regions of those bi-armed chromosomes in E.moara (chromosome pairs No.9 and 24) and E.bruneus(chromosome pairs No.2,9 and 24).Result of 18S rDNA by FISH was consistent.The signal strength of Ag-NORs was varied within species.Meanwhile,two 5S rDNAsignals were localized on the sub-centromere regions of one pair telocentricchromosomes differed from those chromosomes of 18S rDNA.Most chromosomes exhibited C-banding hetreochromatins and DAPI positivebandings on the centromere regions,some even on the sub-telemere regions in bothspecies.These NORs on chromosome pair No.9 of E.moara and No.2 of E.bruneuswere C-positive heterochromatic sites.Telomere sequence (TTAGGG)n hybridizingsignals were found on the telomere region but not intermediate position of allchromosomes in these two species.E.moara was characterized by uniform signalstrength and size.E.bruneus was different,of which 10 chromosome pairs weresignificantly stronger and larger (about 4 times) than others.All results indicated thatE.moara and E.bruneus,with close relationships,were different species specializedin genus Epinephelus,and E.moara was more primitive than E.bruneus.3.Genetic diversity of E.moara and E.brunues was investigated by AFLP,RAPD and ISSR.997,226 and 148 amplified fragments were recorded,of which thepercentage of polymorphic bands were 36.31%,34.51% and 27.03%,from ten pairsAFLP primers,thirty RAPD primers and twenty-one ISSR primers respectively.Differentiation of inter-species was obvious in both speices.Average genetic distance(D) was 0.2987,0.3178 and 0.1792,Shannon's information index (Ⅰ) was 0.2247,0.2251 and 0.1753,and coefficient of population differentiation (G_st) was 0.7997,0.8298 and 0.6460,respectively.Intra-specific genetic diversity was not at a highlevel in the Fujian south coastal population of E.moara and E.bruneus.Geneticsimilarities (S) were 0.9632 and 0.9630,0.9775 and 0.9691,0.9414 and 0.9769separately.Molecular phylogenetic evolution was estimated based on mitochondrial16S rRNA,Cytb and ND2 gene,as well as nuclear ribosomal ITS1 region.All resultsaffirmed that the genus Epinephelus located at the top of the phylogenetic tree andwas the mostly recently diverged group,and E.moara and E.bruneus were closelyrelated species in genus Epinephelus. Then,four stable SCAR molecular markers,translated from the species-specificISSR and RAPD amplicons,were obtained to discriminate E.moara and E.bruneus.To identify these two species,an improved Nest-Tetra-primer specific PCR assay wasdeveloped and established based on ND2 gene and ITS1 region sequences firstly.Itamplified three and five species-specific molecular markers,respectively.Thismethod provided a highly reliable,precise,and rapid molecular marker technique todiscriminate the two grouper species,as well as a new method of DNA differentiationof closely related species in fishes.Furthermore,the results of verification testsshowed that,in the south sea of Fujian,China,E.moara and E.bruneus had differentniches and no hybridization occurred between them.