A PPLIED AND E NVIRONMENTAL M ICROBIOLOGY,July2004,p.4064–4072Vol.70,No.7 0099-2240/04/$08.00ϩ0DOI:10.1128/AEM.70.7.4064–4072.2004
Copyright©2004,American Society for Microbiology.All Rights Reserved.
A Single Species,Micromonas pusilla(Prasinophyceae),Dominates the
Eukaryotic Picoplankton in the Western English Channel
Fabrice Not,1Mikel Latasa,2Dominique Marie,1Thierry Cariou,1
西方音乐史论文Daniel Vaulot,1*and Nathalie Simon1
Station Biologique,UMR7127CNRS,INSU and Universite´Pierre et Marie Curie,29680Roscoff,France,1
and Institut de Cie`ncies del Mar(C.S.I.C.),08003Barcelona,Spain2
Received17September2003/Accepted11March2004
The class Prasinophyceae(Chlorophyta)contains several photosynthetic picoeukaryotic species described
from cultured isolates.The ecology of these organisms and their contributions to the picoeukaryotic community
in aquatic ecosystems have received little consideration.We have designed and tested eight new18S ribosomal
DNA oligonucleotide probes specific for different Prasinophyceae clades,genera,and species.Usingfluorescent
in situ hybridization associated with tyramide signal amplification,these probes,along with more general
probes,have been applied to samples from a marine coastal site off Roscoff(France)collected every2weeks
between July2000and September2001.The abundance of eukaryotic picoplankton remained high(>103cells
ml؊1)during the sampling period,with maxima in summer(up to2؋104cells ml؊1),and a single green algal
species,Micromonas pusilla(Prasinophyceae),dominated the community all year round.Members of the order
Prasinococcales and the species Bathycoccus prasinos(Mamiellales)displayed sporadic occurrences,while the
abundances of all other Prasinophyceae groups targeted remained negligible.
Several studies have demonstrated the importance of eukaryotic picoplankton(cell size,0.2-to3-m)in terms of biomass and productivity in the euphotic zone of oceanic oligo-trophic waters(15),as well as in coastal waters(10).To date,only ϳ40species belonging to nine algal classes(Chlorophyceae,Pra-sinophyceae,Trebouxiophyceae,Prymnesiophyceae,Bolido-phyceae,Eustigmatophyceae,Pinguiophyceae,Bacillariophyceae, and Pelagophyceae)of photosynthetic picoplanktonic eukaryotes have been formerly described(41).However,phylogenetic anal-yses of sequences retrieved from natural samples in different oceanic regions have demonstrated much higher diversity,since many of these sequences do not correspond to any described taxa (19).The contributions of the different taxonomic groups to the picoplanktonic biomass,diversity,and ecology are poorly known because simple and reliable methods to detect and
quantify such organisms in natural samples are lacking.Pigment signatures, scanning electron microscopy,and serial dilution cultures suggest that the classes Prasinophyceae(division Chlorophyta),Pelago-phyceae(division Heterokontophyta),and Prymnesiophyceae are major components of the picoplankton biomass in different ma-rine systems(20,35).
Among these,the class Prasinophyceae contains several photosynthetic picoeukaryote species.This class is considered to be the most primitive in the green lineage and to have given rise to all other green algal classes,as well as to the land plants (34).Members are known to be common in temperate and cold regions and can occur as prominent constituents of ma-rine picoplankton(38).Within these organisms,genera such as Ostreococcus,Bathycoccus,and Micromonas have been de-scribed in coastal waters(4b,6).Micromonas pusilla(the only described species in the genus Micromonas)has been identified as a major component of the picoplanktonic community in several oceanic and coastal regions,such as the Mediterranean Sea(39),the Norwegian Sea(37),and central California wa-ters(35).However,the techniques used to establish these facts (microscopic identification of cells presenting few morpholog-ical characteristics or serial dilution cultures)are time-consum-ing and incompatible with extensive ecological studies.In con-sequence,the precise distributions and the seasonal dynamics of an apparently very common picoplankter,such as M.pusilla, are poorly known.
The aim of this work was to identify and study the seasonal variations of the dominant taxa in the picoeukaryote commu-nity at a coastal site of the western English Channel in the vicinity of a long-term oceanographic observation site(31). Oligonucleotide probes targeting18S rRNA coupled tofluo-rescent in situ hybridization associated with tyramide signal amplification were used to detect the picoplanktonic taxa(22). Eight new oligonucleotide probes specific for Prasinophyceae were designed and validated on pure cultures.These probes,as well as more general probes targeting the Chlorobionta and the eukaryotes,allowed the study in detail of the dynamics of the dominant taxa along a seasonal time series between July 2000and September2001.
MATERIALS AND METHODS
Cultures.Nineteen unialgal strains of picoeukaryotes belonging to different phylogenetic clades of the Prasinophyceae were selected(Table1).They were grown in Nalgene(Rochester,N.Y.)flasks at20°C in K medium(11).In order to test the new probes designed in this study,cells were harvested during the mid-exponential growth phase.For each culture,4.5ml was harvested andfixed with paraformaldehyde(1%final concentration)for1h.These cultures were filtered on0.2-m-pore-size Anodiscfilters(Whatman International Ltd.,Maid-stone,England).Thefilters were dehydrated in an ethanol series(50,80,and 100%;3min each)and stored atϪ80°C until further hybridization tests were
performed.
*Corresponding author.Mailing address:Station Biologique,UMR 7127,CNRS et Universite´Pierre et Marie Curie,Place George Teis-sier,BP74,29682Roscoff Cedex,France.Phone:332-98-29-23-70. Fax:332-98-29-23-24.E-mail:vaulot@sb-roscoff.fr.
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Natural samples.Natural samples were collected twice a month between July 2000and September 2001at 0.5-m depth with 5-liter Niskin bottles off Roscoff,France,at the ASTAN station (48°46ЈN,3°57ЈW).The water was pre filtered through a 200-m-pore-size mesh and further processed in the laboratory.Tem-perature,salinity,and concentrations of phosphate,nitrate,and ammonium were measured by standard oceanographic methods.
For fluorescent in situ hybridization (FISH),90ml of seawater was pre filtered through 3-m-pore-size Nuclepore filters (Whatman International Ltd.)and fixed with 10ml of 10%paraformaldehyde for 1h.Samples were then filtered onto 0.2-m-pore-size Anodisc filters under a maximum pressure of 200mm Hg and dehydrated in an ethanol series (50,80,and 100%;3min each).The filters were stored at Ϫ80°C.
For flow cytometry analyses,1.5ml of pre filtered (3-m pore size)samples was fixed with a mixture of 1%paraformaldehyde and 0.1%glutaraldehyde (final concentrations)and then deep frozen in liquid nitrogen and stored at Ϫ80°C.For high-performance liquid chromatography (HPLC)pigment measure-ments,the Ͻ200-m seawater fraction (1liter)was collected on a GF/F filter (Whatman International Ltd.).The 3-to 200-m fraction was collected on a 3-m-pore-size Nuclepore filter.Finally,the Ͻ3-m fraction was collected on a GF/F filter.The filtrations were conducted under a pressure of 200mm Hg,and all of the filters were immediately deep frozen in liquid nitrogen and stored at Ϫ80°C.
Flow cytometry.Total photosynthetic-cell counts were obtained from fixed seawater samples using a FACSsort flow cytometer (Becton Dickinson,San Jose ´,Calif.),as described previously (18).Photosynthetic picoeukaryotes were dis-criminated from cyanobacteria using Cytowin software (available from www.sb-roscoff.fr/Phyto/cyto.html).
HPLC.Pigment analyses were performed using the method of Zapata et al.(43),with minor modi fications as described by Latasa et al.(14).The contribu-tions of different algal groups to the total chlorophyll a (Chl a )was estimated using CHEMTAX (17).While the contributions of the Mamiellales,Prasinococ-cales,and Pseudoscour fieldiales,which possess prasinoxanthin,could be com-puted,those of the other clades (prasinoxanthinless Prasinophyceae)cannot be distinguished fr
om those of other Chlorophyta.
FISH associated with tyramide signal ampli fication.In situ hybridization with horseradish peroxidase-labeled probes,signal ampli fication,and target cell de-tection were performed as described previously by Not et al.(22).The only difference was the use of a more viscous antifading reagent,AF1(Citi fluor Ltd.,London,United Kingdom),instead of AF3in order to preserve the hybridized slides longer (up to 2weeks in the dark at 4°C)without signi ficant loss of fluorescence.
Epi fluorescence microscopy and image acquisition.The hybridized cells were observed with an Olympus (Tokyo,Japan)BX 51epi fluorescence microscope equipped with a mercury light source and an 100ϫUVFL (Olympus,Tokyo,Japan)objective.Excitation-emission filters were 360/420for DAPI (4Ј,6Ј-dia-midino-2-phenylindole)and 490/515for fluorescein isothiocyanate.For each sample,10randomly chosen microscopic fields were counted by eye.For probes with broad taxonomic speci ficity (e.g.,CHLO02),Ͼ500cells were counted.Because of the large number of hybridizations and the time required for each analysis,it was not possible to count replicates for each sample.However,for 33samples,three replicates (i.e.,three hybridizations with the same probe on three different filters)were analyzed,and the average error was 15%(range,2to 38%).Design of 18S rRNA oligonucleotide probes.The oligonucleotide probes (Ta-ble 2)were designed with ARB software 陶瓦
(16)using a small-subunit rRNA database containing Ͼ30,000complete and partial sequences.In addition to published sequences,our database also contained unpublished partial eukaryote sequences retrieved from three coastal sites.Although the probes could have been designed based only on the public sequences,the additional sequences
TABLE 1.Origins and culture conditions of picoplankton strains
RCC a no.Genus and species Strain Light (E m Ϫ2s Ϫ1)Cell diameter (m)Origin
116Ostreococcus tauri OTTH 05951000.8Thau Lagoon
143Ostreococcus sp.EUM 13BBL 1000.8Tropical Atlantic Ocean 141Ostreococcus sp.EUM 16BBL 1000.8Tropical Atlantic Ocean 114M.pusilla CCMP b 4901002North Atlantic 299M.pusilla NOUM 171002Equatorial Paci fic 372M.pusilla Naples 1002Gulf of Naples 373M.pusilla
Skagerrak 1002Skagerrak 417Mantoniella squamata CCMP 4801003–5North Sea
113 B.prasinos CCMP 18981002Mediterranean Sea 369Coccoid CCMP 12051002–6Sargasso Sea
287Coccoid NOUM 15100 2.5Equatorial Paci fic Ocean
261P.marina TAK 9801404Takapoto Atoll (Paci fic Ocean)370P.provasolii CCMP 12031002–4North Atlantic 135P.provasolii CCMP 11991002–4Gulf of Mexico 251Pycnococcus sp.ROS 94011002English Channel 253Pycnococcus sp.ROS 94041002English Channel 136P.capsulatus CCMP 14071003–6Sargasso Sea 134Prasinococcus sp.CCMP 11941003–5Gulf of Mexico 137
Prasinoderma sp.CCMP 12201003–8Gulf of Mexico
a Roscoff Culture Collection (www.sb-roscoff.fr/Phyto/RCC/).
b
CCMP (Provasoli-Guillard National Center for Culture of Marine Phytoplankton,West Boothbay Harbor,Maine [/]).
TABLE 2.Novel oligonucleotide probes targeting prasinophycean taxa
Probe name
Sequence
Target group
Position of 16S rRNA (Escherichia coli )
Closest sequence not targeted
溶液聚合Taxonomy No.of mismatches
PRAS015Ј-ACG GTC CCG AAG GGT TGG -3ЈPseudoscour fieldiales clade V 193Tilletia caries
2PRAS035Ј-GCC ACC AGT GCA CAC CGG -3ЈPrasinococcales
620Friedmannia israeliensis 2PRAS045Ј-CGT AAG CCC GCT TTG AAC -3ЈMamiellales (except the genus Dolichomatix )
651Choricystis minor 1PRAS055Ј-GCC AGA ACC ACG TCC TCG -3ЈClade VIIA,RCC 287,CCMP 1205
651Pyramimonas olivacea 3PRAS065Ј-AAT CAA GAC GGA GCG CGT -3ЈEnvironmental clade VIIB 651Scutopus ventrolineatus 3MICRO015Ј-AAT GGA ACA CCG CCG GCG -3ЈM.pusilla 211Ostreococcus tauri 1BATHY015Ј-ACT CCA TGT CTC AGC GTT -3Ј B.prasinos 651Uncultivated bacteria
3OSTREO01
5Ј-CCT
CCT
CAC
CAG
GAA
GCU -3ЈOstreococcus
647
Corynebacterium genitalium
3
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FIG.1.Phylogenetic tree of the Prasinophyceae obtained by the neighbor-joining method and based on analyses of complete 18S rRNA gene sequences.The speci ficities of the different probes designed in this study are presented.The clades were named according to the system of Guillou et al.(8).The numbers on the branches correspond to bootstrap values (done on 1,000replicates).
4066NOT ET AL.A PPL .E NVIRON .M ICROBIOL .
allowed us to confirm that the targeted regions were conserved on the corre-sponding sites of coastal representatives of taxa belonging to the clades targeted. When the probes were designed with the ARB Probe Design function,care was taken to maximize the number of mismatches to nontarget sequences and to position these mismatches near the center of the probe.The theoretical speci-ficities of the new probes were checked using the Probe Match function of the ARB software.Oligonucleotide probes with a5Јamino link(C6)were purchased from MWG(Courtaboeuf,France).The probes were then labeled with horse-radish peroxidase(Roche Diagnostic Boehringer,Meylan,France)as described previously(40).The optimal formamide concentration was determined empiri-cally to be40%in the hybridization buffer,and specificity tests of the new probes were performed at this concentration.These probes are available in the rRNA probe database for protists and cyanobacteria(www.sb-roscoff.fr/Phyto /Databases/RNA_probes_intr
oduction.php).Because probes with broad taxo-nomic specificity usually do not work equally well for all targeted organisms,the three probes EUK1209R,CHLO01,and NCHLO01were used in combination to estimate all eukaryotes(22).Finally,probe CHLO02,specific for the subregnum Chlorobionta(Chlorophyta and Streptophyta),was applied(30).To our knowl-edge,the division Streptophyta has no representative in the pelagic marine systems.Consequently,in this study,cells labeled by the probe CHLO02were considered to belong to the division Chlorophyta.
Tree construction.PAUP*version4.0beta10(33)was used to perform phylogenetic analyses of complete18S rRNA sequences using neighbor-joining methods.Cyanophora paradoxa,Pavlova gyrans,Mesostigma viride,and Chara foetida were included in the analyses as an outgroup,and the tree was rooted with C.paradoxa.Bootstrapping(1,000replicates)allowed the evaluation of tree significance.Trees were drawn using TreeView(Roderic Page,University of Glasgow,Glasgow,United Kingdom).
RESULTS
启动子Design and specificity tests of18S ribosomal DNA(rDNA) probes targeting Prasinophyceae.No unique characteristic ex-ists that unites all Prasinophyceae taxa to the exclusion of other Viridiplanta
e or members of other algal phyla(34).This is also supported by molecular phylogenies that show the paraphyletic structure of the class Prasinophyceae(21).Therefore,it is not possible to identify a single probe targeting all Prasinophyceae species.Based on comparative18S rRNA sequence analyses, we designed eight oligonucleotide probes(Fig.1).The probes PRAS01,PRAS03,PRAS04,PRAS05,and PRAS06are spe-cific,respectively,for the orders Pseudoscourfieldiales(clade V only);Prasinococcales(clade VI);Mamiellales(clade II, with the exception of Dolichomastix);clade VIIA,containing the coccoid strain CCMP1205;and clade VIIB,composed of sequences retrieved from the Pacific Ocean.The probes OSTREO01,BATHY01,and MICRO01are specific for the genus Ostreococcus and the species Bathycoccus prasinos and M.pusilla,respectively(Table2).
The specificities of the newly designed probes were evalu-ated by whole-cell hybridization of well-characterized refer-ence strains(Table3).Among the probes targeting Prasino-phyceae orders and clades(PRAS01to-06),only PRAS04 hybridized all,and only,target strains.Since no isolate of clade VIIB has yet been established in culture,positive controls could not be performed for PRAS06,but the probe did not show any nonspecific labeling with the strains tested.PRAS01, PRAS03,and PRAS05hybridized only target strains,but not all of them(Table3).We encountered two different problems. First,PRAS01conferred a goodfluorescence signal when hy-bridized with the two strains R
CC261(Pseudoscourfieldia ma-rina)and RCC253(Pycnococcus sp.),but no signal was ob-served with the strain RCC135(Pycnococcus provasolii). Second,RCC369(CCMP1205)and RCC287,targeted by PRAS05,and Prasinoderma strain RCC137,targeted by PRAS03,presented a heterogeneous signal after hybridization: onlyϳ50%of the cells showed a positive signal.The problem encountered with these probes was also observed with the general Chlorophyta probe CHLO02with the same strains. The three genus-and species-specific probes(OSTREO01, BATHY01,and MICRO01)labeled all,and only,the taxa for which they were designed(Table4).
Hydrological conditions at ASTAN station.Due to the strong tidal mixing,the coastal waters off Roscoff are perma-
TABLE3.Specificity tests of the oligonucleotide probes for Prasinophyceae order level clades
Species RCC no.
Specificity a
PRAS01PRAS03PRAS04PRAS05PRAS06CHLO02
M.pusilla114ϪϪ؉ϪϪ؉Ostreococcus tauri116ϪϪ؉ϪϪ؉Mantoniella squamata417ϪϪ؉ϪϪ؉B.prasinos113
ϪϪ؉ϪϪ؉Coccoid strain369ϪϪϪ؎Ϫ؎Coccoid strain287ϪϪϪ؎Ϫ؎P.marina261؉ϪϪϪϪ؉P.provasolii135؊ϪϪϪϪ؊Pycnococcus sp.253؉ϪϪϪϪ؉Prasinoderma sp.137Ϫ؎ϪϪϪ؎Prasinococcus sp.134Ϫ؉ϪϪϪ؉P.capsulatus136Ϫ؉ϪϪϪ؉
a Boldface indicates the theoretical specificity,whileϩandϪindicate the results of in situ hybridization tests(ϩ,brightfluorescent signal;Ϫ,no detectable fluorescent signal;Ϯ,weakfluorescent signal).
TABLE4.Specificities of oligonucleotide probes for
Mamiellales genera and species
Species RCC no.
Specificity a
BATHY01OSTREO01MICRO01
Ostreococcus tauri RCC116Ϫ؉Ϫ
Ostreococcus sp.RCC143Ϫ؉Ϫ
Ostreococcus sp.RCC141Ϫ؉Ϫ
B.prasinos RCC113؉ϪϪ
M.pusilla RCC114ϪϪ؉
M.pusilla RCC299ϪϪ؉
M.pusilla RCC373ϪϪ؉
M.pusilla RCC372ϪϪ؉
a See Table3,footnote a,for explanation of symbols.令牌网
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nently mixed all year round (32).During the sampling period,the temperature varied between 10.2(March 2001)and 16.6°C (August 2001).The nitrate and phosphate concentrations fol-lowed a similar pattern of variation,with minima in summer (0.4and 0.10M,respectively)and maxima in late fall and winter (15.3and 0.66M,respectively)(Fig.2).
Photosynthetic pigments.During the period studied,Chl a concentrations in the 0.2-to 200-m fraction (total phyto-plankton)varied according to the classical pattern observed in this area,with minima in winter (0.2g liter Ϫ1)and maxima in summer (2.5g liter Ϫ1),corresponding to the diatom bloom (32).The 2001bloom occurred in late June (Fig.3A).Maxi-mum concentrations of picoplanktonic Chl a occurred later,at the end of July (753ng liter Ϫ1).Large phytoplankton (Ͼ3-m diameter)and picoplankton (Ͻ3-m diameter)contributed approximately equally to the Chl a biomass under nonbloom conditions.During bloom periods (July 2000and July 2001),large phytoplankton dominated the total Chl a biomass.Over-all,picophytoplankton made up 34%of the total Chl a throughout the study period.
Within the picoplanktonic Chl a fraction,the contribution of the division Chlorophyta was almost always Ͼ25%(45%,on average)(Fig.3B),except at the end of May,when it plum-meted below 10%,although picoplanktonic Chl a decreased only slightly (Fig.3A).Within the Chlorophyta,the contri-butions of the Mamiellales,Prasinococcales,and some of the Pseudoscour fieldiales (containing prasinoxanthin)to pico-planktonic Chl a ,as estimated by CHEMTAX,were signi ficant all year round and largely dominant (Ͼ80and up to 100%)during spring and summer 2001.
Overall composition of the picoeukaryotic community.The abundances of the photosynthetic picoeuk
aryotes determined by flow cytometry varied between 1ϫ103and 2ϫ104cells ml Ϫ1.Maximum abundances were recorded in May and July 2001(Fig.4A).The abundance of picoeukaryotic cells de-tected by epi fluorescence microscopy after in situ hybridization with a combination of the probes EUK1209R,CHLO01,and NCHLO01varied between 1.3ϫ103and 1.35ϫ104cells ml Ϫ1(Fig.4A).Cells belonging to the division Chlorophyta (tar-geted by CHLO02)dominated the picoeukaryotic community all year long (1ϫ103to 1.5ϫ104cells ml Ϫ1;85%,on average,of the total number of picoeukaryotes)(Fig.4A).
Within the Chlorophyta,organisms belonging to the order Mamiellales (Prasinophyceae),targeted by the probe PRAS04,dominated year round (Fig.4B).Within the Mamiellales,M.pu-silla was the dominant species during the sampling period (75%of the cells detected by PRAS04,on average)(Fig.4C)and accounted for 45%of picoplanktonic eukaryotes.The sec-ond most abundant species detected was B.prasinos ,which accounted for 12%(range,1to 67%)of the Mamiellales and 8%of the picoplanktonic eukaryotes (Fig.4C).Cells belonging to the genus Ostreococcus were less abundant,with an average of 3%(range,0to 19%)of the Mamiellales and 1.4%of the eukaryotes (Fig.4C).
The second most abundant clade within the division Chlo-rophyta was the order Prasinococcales,tar
geted by the probe PRAS03and accounting for an average of 3.4%(range,0to 34%)of the Chlorophyta cells (Fig.4B).Cells belonging to the other clades were detected at very low abundances all year long.Probes PRAS01,PRAS05,and PRAS06targeted an av-erage of 0.6,0.4,and 0.6%,respectively,of cells belonging to the division Chlorophyta (Fig.4B).Only 16%,on average,of the cells detected by the probe speci fic for the division Chlo-rophyta were not targeted by the Prasinophyceae probes used in this study.
Seasonal variation of picoeukaryotic community.The pico-eukaryotic community exhibited a marked seasonal cycle,with minima of abundance in early winter and maxima (ϳ10times the winter abundances)in midsummer.The patterns of abun-dance variation during the sampling period were similar for total picoplanktonic eukaryotes,photosynthetic picoeukary-otes,Chlorophyta,and Mamiellales and for the species
M.
甲酸钾
FIG.2.Variations in temperature and phosphate and nitrate concentrations at the ASTAN station between July 2000and September 2001.
4068NOT ET AL.A PPL .E NVIRON .M ICROBIOL .
pusilla .On a smaller temporal scale,three major peaks of abundance were observed in 2001,in late spring and summer (mid-May,mid-June,and the end of July)(Fig.4A).For the Mamiellales and for M.pusilla ,an additional peak was ob-served in late August (Fig.4C).The other clades and genera detected in this study showed more sporadic proliferations throughout the year.The species B.prasinos was detected all year long but became more abundant during spring (1.8ϫ103cells ml Ϫ1in March 2001)(Fig.4C).The cells targeted by the probe PRAS03(order Prasinococcales)reached signi ficant abundance in early fall (1.2ϫ103cells ml Ϫ1)and late spring (600cells ml Ϫ1).In fall 2000,they accounted for up to 34%of the cells targeted by the CHLO02probe (Fig.4B).Spectacular decreases in the cell abundances of all the taxa detected by our probes and in flow cytometry counts were observed on May 30,2001.
DISCUSSION
New probes targeting Prasinophyceae.Phylogenetic analy-ses of 18S rDNAs recovered directly from samples collected in different oceanic regions have revealed an unsuspected diver-sity within eukaryotic picoplankton (20).In most gene libraries analyzed,including libraries constructed from sam
ples col-lected in the western English Channel (26),prasinophycean sequences were well represented (42).The set of probes pre-sented in this study covers most known clades (Fig.1)and,compared to the few previously published probes for prasino-phycean taxa,have improved speci ficities.Indeed,PRAS04has no mismatch with any Mamiellales sequence known to date,in contrast to PRAS02(2),which has one mismatch with B.pra-sinos .The probes Micro/Manto1and Micro/Manto2,designed in 1996(12)for Micromonas and Mantoniella ,present
matches
FIG.3.(A)Variations in Chl a biomass as measured by HPLC (total Ͻ200-m and fraction Ͻ3-m)at the ASTAN station.(B)Contributions of the division Chlorophyta and of the orders Mamiellales,Prasinococcales,and Pseudoscour fieldiales to the picoplankton fraction (Ͻ3-m diameter)according to the CHEMTAX algorithm applied to HPLC data.
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