Molecular Identification, Species Composition And Population Development Of Green Tide Algae In The Yellow Sea

A serious outbreak of green tide continuously struck the Yellow Sea of China from2007to2011. More seriously, the largest outbreak of green tide in the sea area ofQingdao in2008hampered the regatta sailing during the Beijing Olympic Games. Thusgovernment and researchers had paid much attention to green tide. Ulva is the maincomponents of green tide algae. The research hotpots include taxonomic identificationand genetic origin of the green tide algae, and outbreak mechanism, forecast and earlywarming, prevention and control management and comprehensive utilization of greentide. This paper dealt with the molecular identification and population succession ofgreen tide algae in the Yellow Sea. In the second chapter, the morphological featuresand molecular identification of the main pure culture green tide algae were preliminarilyinvestigated. The molecular identification and species composition of shore-based greentide algae were studied in the next chapter. The fourth chapter focused on the speciescomposition and their succession of early free-floating green tide algae in the southernYellow Sea from2009to2011, and on the4-year (from2008to2011) variation of thedominant outbreak population U. prolifera in this Sea. In the final chapter, thefeasibility of ITS sequences and rbcL sequences as gene barcode of Ulva green tidealgae was explored.The morphological characteristics of4molecularly identified and subsequentlypurely cultured species of green tide algae originally collected from the Yellow Seawere investigated. The external morphological and anatomical features of U. prolifera,U.compressa and U.linza were in accordance with those described in CommercialMarine Algae of China (Zeng,1962), the two former with tubular structure and U.linza with distromatic blades. Ulva sp. didn t differ with U.linza in the externalmorphological and anatomical features. In conclusion, in the culture condition inlaboratory, the three species, U. prolifera, U.compressa and U.linza, could be identifiedfrom each other in term of taxonomic features, but couldn t be done for U.linza andUlva sp., so the molecular method is necessary. The15samples with similarmorphological features sorted from green tide algae collected in different habitats. Allthe15samples possessing distromatic blades were classified into two groups, with12samples for LPP group and3samples for Ulva sp. group by the phylogenetic tree of ITSDNA sequences.12samples of LPP group were further classified into two groups, i.e.,U.prolifera and U.linza.Shore-based investigation along the coastline of Jiangsu Province in2009and in2010showed that filamentous Ulvaceae algae were prevalent in various environmentsof this Province including floating rafts for Porphyra cultivation, landbased culturingponds, estuaries, embankments, intertidal stones, wharfs, and hard muddy coasts. Thenuclear encoded internal transcribed spacer (ITS) and the associated5.8S rDNA regionsas well as5S rDNA spacer sequences were sequenced and analyzed for the green algaecollected from the coastline of the Jiangsu Province in2009（22samples）and in2010(36samples). The phylogenetic analysis of ITS sequence and5S rDNA spacersequences revealed that the samples of2009fell into five distinct clades: the U.prolifera clade, the U. compressa clade, the Ulva sp. clade, the U.linza clade and theBlidingia sp. Clade. The similar pattern for the samples collected in2010was revealedtoo. The sequences of Ulva sp., U. compressa, and Blidingia sp. were completely inagree with those floating in the sea area, respectively. Regionally, the all early floatinggreen tide algae of sea area almost occurred in the seashore of Rudong, Dafeng, andSheyang. All the species of early floating green tide algae were distributed in laver raftand coastal levee, however, U. prolifera was predominant in the culture pond and tidalriver with low salinity. Further analysis with combination of ITS DNA sequence and5SrDNA spacer sequence found that the ITS sequence of U.prolifera sampled from culture pond and tidal river, identical with HM031181, differed in2bp with those of thedominant green tide algae of Yellow Sea in2008(numbered as RD802, the same in thenext text), the5S rDNA spacer sequence had3hypotypes, of which hypotypeⅡpredominated. The same3hypotypes of5S rDNA spacer sequence occurred forfree-floating U. prolifera, with the same ITS DNA sequence as RD802, but hypotypeⅠwas only found in RD802.The results from samples of2009showed that U. linza was the main component ofgreen tide algae in base shore of Lianyungang, and its ITS DNA sequence and5S rDNAspacer sequence differed largely with those collected from base shore of Rudong,Dafeng and Sheyang, and those of free-floating U. linza. It was suggested that thefree-floating green tide algae would not originated from base shore of Lianyungangcoastline in terms of those above differences. More samples from culture pond and tidalriver were collected in the investigation journey of2010. The U. prolifera group withITS sequence identical to RD802and hypotypesⅡof5S rDNA spacer sequence. This U.prolifera group might be the hybrid progeny of U. prolifera proup (RD802), with ITSsequence identical to RD802and hypotype of5S rDNA spacer sequence, and U.prolifera group prevailed in culture pond (with ITS sequence identical to HM031181and hypotypeⅡof5S rDNA spacer sequence). It need more evidence to support theabove inference.From2009to2011, we investigated the green tide algae species composition andcommunity succession in the early stage of green tide in the southern Yellow Sea bymolecular phylogenic analysis. The results showed that the green tide algae species inthe early stage were Ulva sp., U.compressa, U.linza, U.prolifera and Blidingia sp., andthe three-year composition of green tide algae were identical. As the monitoring resultof three years, large-scale green tide algae first occurred in Rudong sea in mid-to-lateApril of every year, the dominant bloom forming alga were Ulva sp. and U.compressaother than U. prolifera, the dominant free-floating green tide algae U. prolifera emergedin mid May every year, and subsequently its biomass mushroomed accounting for90% of the whole biomass of green tide algae until June, and then the floating algae groupcontinued to drift to the north. However, the sea area of Dafeng and Sheyang hardlywitnessed the community succession pattern mentioned above. Only one hypotype of5S rDNA spacer sequence (hypotypeⅠ) was found from the samples of early floatinggreen tide algae collected in2009with ITS sequence identical to RD802, however,4hypotypes were found from the samples in2010with the same ITS sequence as RD802,which were hypotypeⅠidentical to RD802, hypotypeⅡ to the dominant species foundin culture pond, hypotype Ⅲ and Ⅳ. Hypotype Ⅲ and Ⅳ were hardly found in theearly floating samples collected from Dafeng sea area, which were mixed with Alligatoralternanthera, indicating samples of hypotype Ⅲ and Ⅳ were brought to there throughtidal river. There appeared two hypotypeⅠand Ⅱ of5S rDNA spacer sequence amongthe samples of early floating green tide algae collected in2011with ITS sequenceidentical to RD802, and the occurrence frequency of the latter hypotype increasedcompared with in2010. The analysis on ITS sequence and5S rDNA spacer sequence ofalgae collected from2008to2011, leading to green tide in theYellow Sea, showed thatthe proportion of hypotypeⅡ to hypotypeⅠincreased largely year by year, with zero ofthe proportion in2008,0.098in2009,0.429in2010, and4.625in2011. So, thehypotypeⅡsuperseded hypotyeⅠto become the dominant hypotype in2011.Using the rbcL sequences and ITS sequences of10species (9species from Ulva andthe other one was Blidingia minima), part of which were download from GenBank andthe other obtained from the common green algae collected during pre investigation ofour laboratory along coastline of China，I explored the DNA barcoding of green tidealgae. Although rbcL and ITS seperately was not fit to be used as DNA barcoding interm of the DNA barcoding judgement standard of the maximum intra-specific geneticdistance being less than the minimum inter-specific genetic distance, they coulddiscriminate7and8species of the10selected species. The variation of minimuminter-specific genetic distance of rbcL sequence was lower than that of ITS, indicatingthat the former was more conservative that the latter and thus more useful for idenfication at the higher taxonomic level. ITS sequence can very good distinguish theother species of green tide algae except LPP complex. Because the arcticle researchresults confirmed the5S rDNA spacer sequence could effectively distinguish LPPcomplex. So author put forward ITS sequence combined with5S rDNA spacer sequencecould be as green tide algae DNA barcode.