Genetic Analysis Of Ulva Prolifera—the Causative Species Of The Yellow Sea Green Tides

Green tides have become a global serious problem as a result of increasedeutrophication in coastal waters. North America, Europe, Asia and Australia wereseverely affected by this ecological issue in recent years. The great amount ofmacroalgae biomass accumulated in the costal areas brought serious influences bothto the ecology and to the economy. The Yellow Sea green tides have occurred insummer successively for five years since2007. They are characterized by their largescale, long distance drifting, great loss caused, and periodical occurrence. The YellowSea green tides have become an ecological disaster stably appear. The dominatingspecies of the Yellow Sea green tides was identified as Ulva prolifera (=Enteromorpha prolifera) both morphologically and molecularly. It was the same onespecies caused the green tides in the Yellow Sea annually. However, U. prolifera waswidely distributed along the coast of China, studies at the species level were notadequate either to figure out the origin of the biomass or to explain the mechanism ofthe green tide formation. It is essential to study the genetic variances and geneticdistances among the free-floating samples at different sites in different years and theattached population samples along the Chinese coasts at the intra-species level.There were few studies about the intra-species genetic variance of the Ulvaspecies. Specific molecular markers for the study of genetic diversity of U. proliferawere not available. Considering the extremely high plasticity of U. proliferamorphological characters and the uncertainty of the original location of the samplesbecause of the green tides, U. pertusa was chosen as the material to establish themethods for the genetic diversity study at the intra-species level for Ulva species.Twelve U. pertusa populations from three coastal areas of China: Qingdao, Yantai andDalian, were applied to ISSR analysis. The selected four ISSR (inter-simple sequencerepeat) primers amplified120polymorphic bands totally. Genetic diversity wasgreater within Qingdao populations. Analysis of molecular variance (AMOVA) showed the greatest variance within populations, much less variance amongpopulations and among areas. Unweighted pair-group mean analysis (UPGMA)indicated that clustering of U. pertusa individuals mainly relates to their populationsand geographic distances separating those populations. Genetic differentiation andlimited gene flow among U. pertusa populations were indicated by ISSR analysis.Analysis of the genetic variation within and among U. pertusa populations indicatedthat ISSR method is appropriate to study the genetic diversity at the intra-species levelfor Ulva species.Six attached and seven green-tide-forming (free-floating) U. prolifera sampleswere collected at different sites distributed from the north of the Yellow Sea to thesouth of the East China Sea, from2007to2009.52individuals (4individuals fromeach sample) were taken in ISSR analysis. Based on the results of the90polymorphicbands from four ISSR primers, the genetic diversity level of the floating samples wasfound to be lower than that of the attached samples. UPGMA and principal coordinateanalysis (PCoA) suggested that free-floating U. prolifera samples in the Yellow Seafrom2007to2009had a close genetic relationship, and the free-floating samples wereseparated from the attached samples. Genetic differentiation and limited gene flowamong attached U. prolifera populations were indicated by AMOVA analysis.Based on the genetic similarity of the Yellow Sea free-floating U. prolifera,specific molecular markers were developed to identify the bloom-forming populationprecisely and quickly. ISSR fingerprints were used to identify the specific bands forthe free-floating population, and then primers were designed from the sequences ofthe specific bands. In this procedure, ISSR markers were transformed into SCAR(sequence characterized amplified region) markers. Five pairs of SCAR primerswere designed. However, after specificity tested by seven floating samples collectedfrom the blooming biomass from2007to2010and five attached samples collectedfrom different coastal areas, only one SCAR primer pair (Flo₅5L) was recognized asthe specific marker for the bloom-forming U. prolifera population in the Yellow Sea.It is a useful tool for detecting the green tides causative biomass at an early stage.In recently two years, new opinions about the species composition of the Yellow Sea green tides and the genetic variance of the bloom-forming species appeared.Species diversity of the bloom-forming algae was considered to be lower estimatited,and large genetic variance was claimed to be existed among free-floating algaesamples by some studies.In this situation, inter-and intra-species genetic diversity of the bloom-formingalgae in the Yellow Sea was studied. At the species level, free-floating green algaewere collected at different sites in the Yellow Sea in2010and2011. Referring to priorstudies of the Yellow Sea green tides from2007to2009, information about182free-floating samples and155attached Ulva samples were also taken intoconsideration. Results of morphology and molecular phylogenetic analysis comfirmedthat the Yellow Sea green tides from2007to2011were dominated by one species–U.prolifera, though species diversity of the Ulva genus existed along the north coast ofChina. At the intra-species level, genetic variance and genetic relationships wereanalyzed among the free-floating samples and attached population samples along theChinese coast collected in the last five years. Results of molecular phylogeneticanalysis and the SCAR marker amplification showed that the Yellow Sea free-floatingsamples in the last five years formed a single genetic entity, which was different fromthe attached populations. On the other hand, the free-floating U. prolifera exhibitedsome different phenotypic characters, such as denser branches, higher growth rate andadditional flexibility to the adversity, which were essential for them to adapt thefree-floating life on the sea surface. These evidences implied that it is a new ecotypeof U. prolifera–the free-floating type that caused the world's largest green tides. Thisconclusion is meaningful to figure out the origin of the bloom-forming biomass, toclarify the mechanism of the green tide formation and to explain the reoccurrence ofthe ecological phenomenon.