Studies On The Identification Of The Excitation-emission Matrixes Of Phytoplankton In The East China Sea Based On Wavelet Analysis

Phytoplankton is the main source of primary production in both marine and freshwater habitats.Some of the species occasionally grow very fast or "bloom" and accumulate into dense,visible patches near the surface of the water,then the harmful algal blooms(HABs) break out.The HABs is very severe in the costal waters of the China Sea.They destroy the zoological balance of the water.Worse still,some species produce potent neurotoxins that can be transferred through the food web,where they affect and even kill the higher forms of life such as zooplankton,shellfish,fish,birds,marine mammals and even humans.This phenomenon becomes very serious.It becomes an urgent need for determining the algae causing HABs in situ,rapidly and reliably.The excitation-emission matrixes(EEMs) has present 'fingerprint feature' to phytoplankton,which makes it potential to be utilized in the detection of the phytoplankton communities and the identification of HAB causative species when HAB breaks out.However,the utilization of fluormetric methods in discriminating phytoplankton just focus on some divisions that present obvious fluorescent characteristics. There is neither applied fluormetric method that directly aims at the discrimination of the phytoplankton communities in given sea area of the China Sea,nor fluormetric method for the identification of HAB causative species in situ when HABs break out.This paper just focuses on the above problems.Based on the analysis of the characteristics of the phytoplankton communities in coastal water of the China Sea, thirty-eight phytoplankton species,that is Alexandrium tamarense,Amphidinium carterae, Scrippsiella trochoidea,Gymnodinium spp.,Gymnodinium simplex,Prorocentrum donghaiense,Prorocentrum minimum,Prococentrum marinum,Karenia mikimotoi; Pseudo-nitzschia pungens,Skeletonema costatuma,Thalassiosira rotula,Nitzschia closterium, Leptocylindrus danicus,Ditylum brightwellii,Odontella cf_sinensis,Coscinodiscophyceae Coscinodiscus cf_<sub>radiatus,Coscinodiscophyceae Coscinodiscus sp.,Chaetoceros curvisetus, Chaetoceros debilis,Chaetoceros didymus;Isochrysis galbana,Prymneium parvum,Gephyrocapsa oceanic,phaeocystis globosa,Distephanus speculum;Platymonas helgolanidica,platymonas subcordiforus,Micromonas pusilla,Dunaliella salina,Chlorella pynenoidosa;Heterosigrna akashiwo,Chattonella marine;Rhodomonas sp.,Rhodomonas salina;Synechococcus sp.,Spirulina sp.and Anabaena sp.,most of which are HAB causative species and dominant species in the East China Sea,were selected and cultured in the laboratory.They belong to seven divisions and thirty genuses.The EEMs were measured and the scanning precision,culturing recurrence,growth stability(at different growth stages, under different culturing lights and temperatures),as well as the difference between EEMs of different species are analyzed.Based on this,the feasibility of the utilization of EEMs in discriminating phytoplankton species is expounded.With the utilization of wavelet analysis, the identifying methods of the HAB causative algae and dominant species in the East China Sea are established based on both the continuous and the discrete EEMs,aiming at the commercial fluorescence speetrophotometer and the ones that would be developed by us, respectively.In last section,based on the chlorophyllous eoncentration,the primary semi-quantitative fluormetric method for the in vivo phytoplankton species was established in the lab.All these work can provide important groundwork for the monitoring of the HAB causative phytoplankton species that in the East China Sea.The main conclusions of this paper are as follows:1.On the sum-up of the HAB causative species as well as the domain species in the coastal waters of the China Sea,and the analysis of the composing characteristics of the phytoplankton communities,the following conclusions are gotten:a.during the non-HAB period,there are always more than one phytoplankton dominant species.They usually belong to one or two divisions.The common predominance of one dominant species is less than 75%,while the predominance of one division are usually more than 90%,or even close to 100%.b.The HABs were mostly caused by only one species,the predominance of which are usually more than 90%,or even close to 100%.The HABs caused by two species become more and more.When such HABs take place,the general predominance of the two species are more than 90%,or even close to 100%.2.Two criteria are utilized in the analysis of the feasibility to the discrimination of phytoplankton species by EEMs.The criteria are Relative Standard Deviation(RSD) and Included Angle Cosine(IAC).The results present that it is possible to identify phytoplankton species based on EEMs.The part with Ex= 400～600 nm,Em= 650～670 nm are selected as the optimal EEMs.Under the culturing conditions in this paper,most phytoplankton species present good scanning precision,culturing recurrence,and growth stability('at different growth stages,under different culturing irradiances,as well as different culturing temperatures).Also,there are obvious differences between most phytoplankton species that belonging to different genuses under our experimental conditions.3.Two wavelets(db7 and coif2) are utilized to extract the characteristics of the EEMs. Different scale vectors and time-series vectors are obtained.The discriminating characteristic spectra of both scale and time-series are selected by Fisher discriminant method.Classifying results under different conditions are analized by Bayes discriminant method.Clustering analysis is utilized to extract the reference spectra from both the scale and time-series discriminating characteristic spectra obtained by both wavelets.A two-rank "database of reference spectra" is established for each wavelet.Based on the databases,a fluormetric method for the identification of phytoplankton species is established by nonnegative least squares(NNLS).This method can identify phytoplankton species at both the genus level as well as the division level.The correct discriminating rates are more than 86%and 96%,respectively.Some HAB causative species (Prorocentrum donghaiense,Alexandrium tamarense,Thalassiosira nordenskioldii and Chaetoceros socialis) can be identified by this fluormetric method when the cellular density is about 10⁷ cell L^-1 in the actual sea waters.The dominant species in the phytoplankton communities can be identified at the division level when the cellular density of the community is 10⁵～10⁶ cell L^-1. 4.Twelve excitation wavelengths,they are,400 nm,430 nm,450 nm,460 nm,470 nm, 490 nm,500 nm,510 nm,525 nm,550 nm,570 nm及590 nm,are selected to compose a discrete EEM.The emission wavelengths are 650～700 nm to the EEM.5.Two wavelets(db3 and coif2) are utilized to extract the characteristics of the discrete EEMs.Classifying results under different conditions are analized by Bayes discriminant method.Different scale vectors and time-series vectors are obtained.The discriminating characteristic spectra of both scale and time-series are selected by Fisher discriminant method.The reference spectra of both the scale and time-series discriminating characteristic spectra obtained by both wavelets were obtained by clustering analysis.A two-rank "data base of reference spectra" is established for both wavelets,respectively. Based on this,a fluormetric method for the identification ofphytoplankton species is established by nonnegative least squares(NNLS).This method can identify phytoplankton species at both the genus level as well as the division level.The correct discriminating rates are more than 85%and 96%,respectively.Some HAB causative species (Prorocentrum donghaiense,Thalassiosira nordenskioldii and Chaetoceros socialis) can be identified by this fluormetric method when the cellular density is about 10⁷ cell L^-1 in the actual sea waters.The dominant species in the phytoplankton communities can be identified at the division level when the cellular density of the community is 10⁵～10⁶ cell L^-1.6.Based on the chlorophyllous concentration,a primary semi-quantitative fluormetric method for determining the in vivo phytoplankton species was established in the lab.The detecting limit is about 8μg L^-1,and the optimal linear range is 20～200μg L^-1.The innovation in this paper is the establishment of the reference spectra for the main phytoplankton species in the East China Sea.Based on this,28 dominant species in the 979 laboratory mixtures and the HAB causative species during the single type HAB periods can be identified at the genus level.The correct discriminating rates are more than 85%at the genus level,and that are more than 96%at the division level.