Phylogeny and morphology of a Chattonella (Raphidophyceae)

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Phylogeny and morphology of a Chattonella
(Raphidophyceae) species from the Mediterranean
Sea: what is C. subsalsa?
Sascha Klöpper a , Uwe John a , Adriana Zingone b , Olga Mangoni c , Wiebe H.C.F.
Kooistra b & Allan D. Cembella a
a Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570,
Bremerhaven, Germany
b Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy
c Department of Biological Sciences, University Federico II, Via Mezzocannone 8, 80138,
Naples, Italy
Version of record first published: 13 Mar 2013.
To cite this article: Sascha Klöpper , Uwe John , Adriana Zingone , Olga Mangoni , Wiebe H.C.F. Kooistra & Allan D.
Cembella (2013): Phylogeny and morphology of a Chattonella (Raphidophyceae) species from the Mediterranean Sea: what
is C. subsalsa?, European Journal of Phycology, 48:1, 79-92
To link to this article: http://dx.doi.org/10.1080/09670262.2013.771412
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Eur.J.Phycol.(2013),48(1):79–92
Chattonella
Phylogeny and morphology of a (Raphidophyceae)
C. subsalsa
species from the Mediterranean Sea: what is ?
SASCHAKLÖPPER1,UWEJOHN1,ADRIANAZINGONE2,OLGAMANGONI3,
3 WIEBEH.C.F.KOOISTRA2ANDALLAND.CEMBELLA1
1
0
pril 2 12AStlafrzeiodnWeeZgoeonleorgIicnastiAtuntteonfoDroPhorlna,rVainlldaMCaorminuenaRlees,e8a0r1c2h1,ANmapHleasn,dIetalslyhafen12,27570Bremerhaven,Germany
7 A 3DepartmentofBiologicalSciences,UniversityFedericoII,ViaMezzocannone8,80138Naples,Italy
1
3
1 (Received4July2012;revised19October2012;accepted29October2012)
2:
0
at WeanalysedthemolecularandmorphologicalfeaturesofstrainsofChattonellasubsalsaisolatedfromthewesternAdriaticcoast
g ] (MediterraneanSea),withtheaimofconfirmingtheirclassificationandelucidatingtheirphylogeneticpositionswithinthe
n
u Raphidophyceae.Wesequencedpartsoftheribosomaloperon,includingthesmallsubunit(SSU),theinternaltranscribedspacer
h
sc region(ITS)andthelargesubunit(LSU)oftherDNA.Additionally,weanalysedsequencesofthechloroplast-encodedsubunit
r
o psaAofPhotosystemI(PSI)andrbcL,encodingthelargesubunitoftheRubiscogene.Forthreephylogeneticmarkers(LSU,ITS,
f
s
re rbcL),thesequencesofthestrainsfromtheAdriaticSeawereidenticalandfortwomarkers(SSU,psaA)onlyminordifferences
e
e occurred.Allstrainsweresisterto,butwellseparatedfrom,sequencesfromisolatesinculturecollectionsandfromGenBank,thus
M
d farclassifiedasbelongingtoC.subsalsa.Lightandelectronmicroscopyprovidedevidenceformorphologicaldifferences
n
u betweenastrainofC.subsalsa(CCMP217)fromtheGulfofMexicoandtheisolatesfromtheAdriaticSea.Differences
-
ar concernedtheshapeandarrangementofchloroplastsandthepresenceofmucocystsandothersurfacemicrostructures,which
ol wereonlyobservedinisolatesfromtheAdriaticSea.ThisisthefirstevidencefortwodifferenttaxaclassifiedasC.subsalsa,
P
ür whichareclearlyseparatedonthebasisofseveralgeneticmarkersandalsoshowmorphologicaldifferences.Ascomparedwith
f
e strainsassignedtoC.subsalsafromtheNCMA(formerlyCCMP)culturecollection,theAdriaticstrainsmorecloselymatchthe
ut
nstit ournidgeinraalnsepwecsiepsecdieesscnriapmtieo.nN.Tevheisrtwheoleuslds,igmivpelyntthheathsitgrhaimnoCrCphMoPlo2g1i7caalnpdlaostthiceirtygeonfeCtihcaatltloynseilmlailsapresctireasin,sthsehdoeufildnibtieondeosfctrhibeetdrue
I
r C.subsalsamustbedecidedbasedondetailedmorphologicalandmolecularanalysisofmorestrainsfromothergeographical
e
n
e areas.
g
e
W
d Key words: Chattonella subsalsa; compensatory base changes; harmful algae; Mediterranean Sea; molecular phylogeny;
e
r morphology;psaA;Raphidophyceae;rbcL;rDNA
f
Al
g
n
u
ft
Sti Introduction heterodynamicflagellaemergingfromagrooveatthe
y [ anterior end of the cell. A flagellum covered with
b TheRaphidophyceae(describedbyChadefaud,1950,
ed andemendedbySilva,1980)areclassifiedwithinthe tripartite hairs is directed forward and pulls the cell,
d whileaflagellumwithnohairstrailsbackwards.Cells
oa stramenopiles,togetherwithotherprominentprotists,
wnl such as diatoms and chrysophytes. In a recent evolu- ofseveralspeciescontainavariablenumberofejecto-
somes, which take the form of easily discharged
Do tionary tree of eukaryotes (Keeling et al., 2005), the
mucocysts. Numerous chloroplasts are densely
stramenopiles are placed with the alveolates (includ-
packed in the periphery of the cell, just below the
ing dinoflagellates and ciliates) within the chromal-
surface, and vary in colour from green to yellow-
veolates,asoneofthefive‘supergroups’identifiedin
greentogoldenbrown.Marineraphidophytescontain
thissystem.Alternatively,sixgroupsareproposedin
chlorophylla,c and/orc ,anddiadinoxanthin,fucox-
the classification by Adl et al. (2005), who also con- 1 2
anthin and violaxanthin as the major carotenoids.
siderwithcautionthegroupingofstramenopileswith
There are also pigment variants, which can be
alveolates.
restricted to particular genera; for example,
Members of the class Raphidophyceae are unicel-
FibrocapsaandHaramonasspeciesdifferinthepre-
lular,nakedflagellates,whichswimbymeansoftwo
senceoffucoxanthinolinFibrocapsabut19-butanoy-
loxyfucoxanthininHaramonas(Bjørnland&Liaaen-
Correspondenceto:UweJohn.E-mail:[email protected] Jensen,1989;Mostaertetal.,1998).
ISSN0967-0262(print)/ISSN1469-4433(online)/13/010079-92©2013BritishPhycologicalSociety
http://dx.doi.org/10.1080/09670262.2013.771412
S.Klöpperetal. 80
Eleven marine raphidophyte species have been particularlyproblematic.Chattonellamarina,C.anti-
described withinsix genera (Chattonella, Fibrocapsa, qua and C. ovata were first described based upon
Haramonas, Heterosigma, Chlorinimonas and morphologicalcharactersvisiblebylightmicroscopy,
Viridilobus),thelatestadditionbeingViridilobusmar- but they were subsequently shown to exhibit high
inus (Demir-Hilton et al., 2012). Among these raphi- sequence similarities for different genetic markers
dophytes,sevenspecieshavebeenassociatedwithfish (Hirashita et al., 2000; Bowers et al., 2006; Hosoi-
kills(Hallegraeffetal.,1998).Thereisnoevidenceof Tanabe et al., 2006; Demura et al., 2009). Based on
harmfuleffectscausedbythethreeknownHaramonas the most recent molecular and morphological find-
speciesorbyChlorinimonassublosa,perhapsbecause ings, it has been suggested that differences among
their sand-dwelling habit limits contact with fish these three taxa warrant them to be accorded at most
shoals. Several harmful events have been caused by varietal status (Demura et al., 2009), the valid name
3 speciesinthegenusChattonella,includingthesubject for the species being C. marina (Subrahmanyan) Y.
1
0 of thepresent paper, C. subsalsa(Imai & Yamaguchi, Hara&Chihara,whichcomprisesC.var.marina,var.
2
ril 2012). The monotypic genus Heterosigma and the antiqua (Hada) Demura & Kawachi, and var. ovata
Ap genus Fibrocapsa, containing F. japonica and a (Y. Hara & Chihara) Demura & Kawachi. In the
7 recently described new genotype from the phylogenetictreespresentedinthepublicationsmen-
1
3 Mediterranean(Klöpperetal.,2008),arealsoichthyo- tioned above, C. subsalsa diverged early from the
1
2: toxic.Harmfulraphidophytebloomsarebestdocumen- lineage with other Chattonella species, whereas C.
0
at tedforEastAsianwaters,wheretheyheavilyaffectthe subsalsastrainsfromgeographicallydistinctlocations
g ] extensivefisheryandmaricultureactivities(Toriumi& showednodifferences.
n
u Takano,1973;Yoshimatsu,1987).Nevertheless,raphi- Herewepresentananalysistoclarifythesystema-
h
c dophytebloomshavealsoafflictedother coastal areas ticsofC.subsalsaandisolatesofChattonellafromthe
s
r
fo bothrecentlyandoverthepastfewdecades,including western Adriatic coast. We used five phylogenetic
s
re intheMediterraneanSea(Hollande&Enjumet,1957; markers:thesmallsubunit(SSU),internaltranscribed
e
Me Tregouboff,1962;Margalef,1968;Mikhail,2007). spacer(ITS)regionandlargesubunit(LSU)ofnuclear
d Tracking raphidophyte distributions in recent rDNA,andthepsaAandrbcLgenes,whicharechlor-
n
u investigations and historically, such as from time- oplast encoded.We also examined strains usinglight
-
ar series data, has proven difficult because samples and electron microscopy to (1) seek morphological
Pol usually do not preserve well enough with most fixa- differencesthatmightcorrelatewiththephylogenetic
r
ü tives to display the representative morphological resultsand(2)showwhichstrainsbestagreewiththe
f
ute characteristicsofthisalgalgroup.Thecellsaremor- originalspeciesdescriptionofBiecheler(1936).
stit phologicallyplasticandextremelydelicate,andtend
In to disintegrate with nearly all common fixatives,
r
ne although Katano et al. (2009) recently reported that Materialsandmethods
e
g HEPES-buffered paraformaldehyde and glutaralde-
We hydedeliverpromisingresultsforshort-termfixation Cultures
d
e ofChattonellaspecies.Asaconsequenceofthelack Surface water samples were collected by bucket along the
r
Alf ofarigidcellwall,raphidophytesarehighlyvariable shallow sandy coast in front of Rimini, Italy (Adriatic Sea,
g incellshape,dependingonphysiologicalconditions 44○04′23″N,12○34′50″E)inAugust2004duringabloomof
n
Stiftu (eItmaali.,, 22000060);. BInansdp-eScciehsmoidfttheet gael.n,u2s0C04h;atBtoonwelelars, cCuhlatuttroensewllearecfe.stsaubblsisahlseadiannd10F0ibmrloccualptsuaresgplpa.ssMfloanskosclionnKal
[ growthmedium(Kelleretal.,1987)andmaintainedat21°C
y cellscanbeoval,globularoranteriorlyroundedand
b with a 14:10 h light:dark photoperiod at a photon flux
ed posteriorlypointed. density of 80 μmol m−2 s−1. All cultures were harvested in
oad There is increasing interest in investigating mole- the middle of exponential growth by centrifugation
nl cular aspects of raphidophytes, to classify them phy- (4000× g, 15°C,15 min).Chattonella subsalsaCCMP217
w
o logenetically and to explore relationships on the from the Provasoli–Guillard National Center for Marine
D specific- or even intra-specific level. Molecular stu- Algae and Microbiota (NCMA) (formerly CCMP), East
dies on raphidophytes initially focused on classifica- Boothbay,ME,USA,andallotherstrainsfromculturecol-
tion of the entire class within a phylogenetic system lectionswereculturedformolecular,morphologicalandpig-
(Cavalier-Smith & Chao, 1996). In several recent mentanalysisunderidenticalconditions(strainsandorigins
investigations gene sequences have been analysed arelistedinTable1).
from different species to trace phylogenetic relation-
ships within the class (Hirashita et al., 2000; Bowers
DNAextraction,amplification,sequencingand
et al., 2006; Hosoi-Tanabe et al., 2006; Yamaguchi
analyses
etal.,2008,2010;Demuraetal.,2009;Band-Schmidt
et al., 2012) and provide molecular tools for species DNA was extracted from 50 ml of culture in exponential
identification(e.g.Tyrrelletal.,2002).Theclassifica- growth phase, harvested as above, followed by cell lysis
tion of three species within the genus Chattonella is usingTissueLyser(Qiagen,Hilden,Germany)forcelllysis.
AnewChattonellagenotype 81
ntriesinbold rbcL DQ273995 DQ273989 DQ273994 AF015581JX067595 JX067596 JX067594JX067600
e
k
n
a
B 5 69 584 3
n 0 00 100 0
e A 6 66 666 6
s.G psa X067 X067X067 X067X067X067 X067
er J JJ JJJ J
b
m
u
n
n
o
April 2013 Bankaccessi ITS AF136761 JX067592 AY704166AF153196 JX067586 AY858865 AB217657
17 Gen
3
g ] at 02:1 markerand 28SrDNA JX067556 JX067557 AB217640 JX067559JX067562 JX067568 JX067558 JX067555
n c
hu eti
c n
s e
r g
d Meeresfo gin,applied 18SrDNA AB217626 AY788925 AY788923 JX026934JX026936 JX026940JX026938JX026933 JX026931
un ori
ar- on
Wegener Institute für Pol ees,includinginformation Origin SetoInlandSea,JapanAichi,JapanHarima-Nada,Japan Kagoshima,JapanJapan Kagoshima,JapanHarima-Nada,JapanJapan HongKongGulfofMexico,USARimini,AdriaticSea,Italy Rimini,AdriaticSea,ItalyRimini,AdriaticSea,ItalyHarima,JapanRimini,AdriaticSea,Italy California,USA–
d ctr
Alfre eneti 1
ung hylog olate 02 9 ’Japan 4
d by [Stift presentedp Strain/is CCMP205CCMP205NIES-1 CCMP204‘C.Tomas’1Japan CCMP216NIES-603‘C.Tomas CCMP217CRIM-B CRIM-CCRIM-DCCMP218FRIM-A CCMP227‘’1893
e
d e
oa th
Downl Table1.Listofselectedstrainsandisolatesforindicatesequencesobtainedforthisstudy. Species Chattonellamarinavar.antiqua(Hada)Demura&Kawachi2009 Chattonellamarina(Subrahmanyan)Hara&Chihara1982var.marina Chattonellamarinavar.ovata(Y.Hara&Chihara)Demura&Kawachi2009 ChattonellasubsalsaBiecheler1936ChattonellasubsalsaAdriatic Chattonellasp.Fibrocapsacf.japonica Heterosigmaakashiwo(Hada)HadaexHara&Chihara1987 1TranscribedfromGenBank.
S.Klöpperetal. 82
DNA isolation was performed with a DNeasy Mini Kit were calculated with PhyML (Guindon & Gascuel, 2003)
according to the manufacturer’s protocol (Qiagen, Hilden, using a BIO-NJ (Neighbour-Joining) tree as a starting tree
Germany). The PCR reaction mix for all primer pairs con- andtheGTRevolutionarymodel,withagammadistribution
sistedof5µlof10×HotMasterTaqBuffer,1µldNTPMix parameter estimated from the data. The best model for the
10 mM and 0.5 µl Taq DNA polymerase (all Eppendorf, analyses was chosen using Modeltest (Posada & Crandall,
Hamburg, Germany) in 40.5 µl water. Different primers 1998, 2001). Bootstrap values were calculated with 100
(0.5µl,10pmolµl−1)wereaddedpair-wise. replicates.
Theprimers1F(5′-AACCTGGTTGATCCTGCCAGT-3′) Analyses of the secondary structure were based on the
and 1528R (5′-TGATCCTTCTGCAGGTTCACCTAC-3′) comparison of the ITS2 sequences of the CCMP217 and
(Medlin et al., 1988) were used for amplification of SSU CRIM-C strains with a secondary structure model for
rDNA. The SSU PCR was performed with a Mastercycler ChattonellaintheITS2Ribosomaldatabase(http://its2-old.
Gradientsystem(Eppendorf,Hamburg,Germany)andcom- bioapps.biozentrum.uni-wuerzburg.de).TheITS2secondary
3 prisedafirstdenaturingstepat95°Cfor7min,followedby structures of CCMP217 and the CRIM-C strains were
1
0 35 cycles of 95°C for 2 min, 54°C for 4 min and 72°C for obtained with mfold (Zucker, 2003). These results were
2
pril T10TTmAinA.GTCheATLASU-3′p)raimnderDsiwr-e2rCe(D5I′-RC-CFT(5T′G-AGCTCCCCGGCTTGGTATAT tbhreanckuetsendottaotiomnaonfuathlleyraibdojusostmtahledasetacboansdea.rTyhsetrnuucmtubreesr oinf
A
7 CAAGA-3′), according to Scholin et al. (1994). The PCR compensatorybasechanges(CBCs)betweenthetwoaligned
1
3 stepswereaholdat94°Cfor9min,followedby20cyclesof sequences was calculated with CBCAnalyser (Wolf et al.,
2:1 94°Cfor30s,55°Cfor30sand72°Cfor9m.ForITS,we 2005)and4SALE(Seibeletal.,2008).
0 used ITS A (5′-CCAAGCTTCTAGATCGTAACAAGGHT
g ] at TCGCCGTTATAGAGRTT-3T′)CAanGdCIRTGSGB-3′)(5(′A-CdCacThGiCetAaGl.T,C1G99A6C),AwKitAh Morphologicalobservations
n
hu PCRstepscomprisingaholdat94°Cfor5min,followedby Cells were observed during the exponential growth phase.
c
rs 35cyclesof94°Cfor20s,57°Cfor10sand70°Cfor5min. Due to the radical changes in the morphology of raphido-
sfo TheprimersforrbcLwere130F(5′-AACWACWACTTGG phyte cells exposed to different fixatives, all light micro-
re ATTTGGAA-3′) and 1600R (5′-GCATGAATATGMTG scopical (LM) observations were conducted with living
e
Me WACCAT-3′)(Yoonetal.,2002),withPCRstepscomprising cells. Staining with Neutral Red vital stain (Merck,
d a hold at 94°C for 2 min, followed by 39 cycles of 94°C Darmstadt, Germany) was performed by adding a drop to
n
u for 20 s, 46°C for 30 s and 70°C for 5 min. Finally, for living cells under the cover slip and waiting until the red
ar- amplifying psaA, the primers were F3 (5′-GCTTACCGTG liquid permeated the cells. Examination was carried out
Pol TAGATCCAGTTCC-3′) and R3 (5′-CCTTCTAATTTA with Zeiss Axiophot and Axiovert 200 microscopes
ür CCAACAACTG-3′)(Beszteri,2005);PCRconditionswere equipped with an AxioCam photocamera and evaluated
e f aholdat94°Cfor3min,followedby39cyclesof94°Cfor with AxioVision (Rel. 3.1) (all Carl Zeiss, Oberkochen,
nstitut 30Ts,h4e4.P5C°CRfoprro3d0ucs,tsanwde7re0°Cpufroifire5dmwinit.h a Qiagen PCR GeArmsarnayp)h.idophytesaredelicate, severalpreparation proto-
r I PurificationKitandsequencedwithanABI3130XLsequen- colswereperformedfortheelectronmicroscopicalinvestiga-
e
en cer,usingtheBigDyeTerminator3.1CycleSequencingKit tions. For scanning electron microscopy (SEM), cells were
eg (Applied Biosystems, Warrington, UK). The 10-µl reaction fixedwithosmiumtetroxide(atafinalconcentrationof1.5%
W
mix contained 1.5 µl of 5× sequencing buffer, premix Big for 5 min at 4°C), dehydrated in a graded ethanol series,
d
e Dyepolymerasesand4.5µlwater.Finally,1µlprimerwas critical-pointdried,andsputter-coatedwithgold.Themate-
r
Alf added.Thesequencingreactionconditionswerethesameas rial was examined with a JSM-6500F scanning electron
g forPCR.Additionalprimerswereusedforlongsequences; microscope(JEOL,Peabody,MA,USA).
Stiftun AfoAr-S3S′)U, t1h0es5e5Fwer(e5′5-G28GFTG(5G′-TGGCCGAGTTGAGACTCTCGCTATCGTCTT-C3′C), culFtuorre trsaanmsmpliesssiownereelecfitrxoend mwicitrhoscgoluptyara(TldEeMhy)d,esoamnde
y [ 536R (5′-AATTACCGCGGCKGCTGGCA-3′) and 1055R osmium tetroxide as described by Zingone et al. (1995).
d b (5′-ACGGCCATGCACCACCACCCAT-3′) (Scholin et al., The best results, however, were obtained by the following
de 1994); and for psaA, 870F (5′-ggnggwytatggttaagtga-3′) procedure, modified from Eikrem & Moestrup (1998):
oa (Yoonetal.,2002). 20 ml of plankton culture were fixed for 30 min with
wnl All publicly available sequences of Chattonella and glutaraldehyde in filtered seawater (final concentration
Do selected other raphidophytes (as of 1 May 2012) and 4%). After three washes with 0.1 M cacodylate buffer
sequences generated in this study were aligned with (pH 7.5), a solution containing 0.5% ferricyanide and
ClustalW and finally manually corrected in Bioedit 0.5% osmium in 0.1 M cacodylate buffer was added to
(Version 7). Alignments are available in the supplementary the cell pellet and kept overnight at 4°C. After washing
information(TableS2). steps with buffer and deionized water, specimens were
Reduced (haplotype) Neighbour Joining (NJ) trees, con- stained with 4% aqueous uranyl acetate at room tempera-
taining only selected Chattonella strains and the raphido- turefor90min.Aftersequentialdehydrationatsixethanol
phyte Heterosigma akashiwo as outgroup, were obtained concentrations (final 100%), and two treatments with pro-
with Mega 5 software (version 5.05) (Tamura et al., 2011). pylene oxide, pellets were kept in a 1:1 mixture of pro-
The analysis included polymorphic representatives of C. pylene oxide and Epon embedding resin (Sigma–Aldrich
subsalsa,thenewAdriaticisolates,andtheC.marinacom- Fluka, Buchs SG, Switzerland) for a minimum of 8h. For
plex. Bootstrap values were based on 1000 replicates. final polymerization, the pellet was placed in Epon at
Maximum likelihood (ML) phylogenetic trees for all genes 50°C for 12 h. Thin sections were prepared on a MT X
AnewChattonellagenotype 83
ultramicrotome (RMC products, Boeckeler, Tucson, AZ, Secondarystructureandcompensatorybasechanges
USA),placedongrids,andsubsequentlysoakedfor5min
The secondary structure models of ITS2 for
in lead citrate. Thin sections were viewed with a LEO
Chattonella subsalsa CCMP217 and the Adriatic
912AB transmission electron microscope (LEO,
ChattonellaCRIM-Cdifferedonlyinhelix3,whereas
Oberkochen, Germany).
the other three helices were identical. The sequences
were 92% identical (201/218) (Fig. S6). Comparison
Pigmentanalysis
ofthesecondarystructuremodelsrevealedoneCBC,
Five 10-ml samples of cultures grown as described above which was confirmed with both CBCdetect and
werefilteredonto25mmWhatmanGF/Ffiltersandstoredat 4SALEprograms.
–80°Cforhigh-performanceliquidchromatography(HPLC)
analyses.Filteredsampleswereextractedin100%methanol
3 andthepigmentswereseparatedonan1100HewlettPackard Morphology
1
0 liquidchromatograph(HewlettPackardCompany,PaloAlto,
2
pril CViAd,usUsiSAet)aflo.ll(o1w99ed6).byThdeioddieodaerraayrradyetedcettieocntoarccwoarsdisnegt taot AAddrriiaattiiccwCehreat3to7n.6el±la1..7Cµhmatt(omneelalna±cSel.lDs.)flroonmg atnhde
A
7 440 nm to determine chlorophylls and carotenoids. The 24 (± 1.7) µm wide (Table 2). Under LM, cell shape
1
3 detector was calibrated with pigment standards obtained appeared rather variable, being oval, pear- or lemon-
2:1 from the International Agency for 14C Determination, VKI shaped(Figs6,7).Somecellsshowedashortcolour-
at 0 WaterQualityInstitute,Hørsholm,Denmark. less tail-like protrusion at the posterior end. Both
] flagella exceeded the cell length by one and a half
g
un Results times. Theanteriorly directedflagellum pulls the cell
h
c forward showing a regular helicoidal motion, while
ors Phylogeny thisflagellumisseentoassumeasinusoidalshapein
f
es Representative NJ haplotrees of Chattonella strains, veryslowlymovingcells(Fig.6).Thetrailingflagel-
r
e
e including newly obtained isolates from the Adriatic lumappearstostabilizeandsteerthecell.Theswim-
M
d (CRIM), based on five selected markers from rDNA ming behaviour can be described as a steady
n
u andnuclear-encodedplastidDNA,areshowninFigs movementalongaconvolutedpath.Swimmingvelo-
ar- 1–5. All markers yielded similar tree topologies for city increases with rising temperature. Numerous
Pol theChattonellastrains.TheC.marinastrains,includ- green to golden, roundish to barrel-shaped chloro-
ür ingallavailablevariants,togetherwithanundescribed plasts are densely packed below the cell surface
f
e Chattonella from the CCMP culture collection, (Figs 6, 7). In addition to the chloroplasts, two kinds
ut
stit formed a sister clade to C. subsalsa and the new of structures are visible at the cell surface: circular
n strains from the Adriatic. Within this C. subsalsa bodies of c. 1.5 µm diameter with a sub-central
I
er clade,therewasaclearsubdivisionintothepreviously depression, and more numerous and smaller (c.
n
ge knownC.subsalsaandthenewlyisolatedChattonella 0.3 µm) refringent bodies (Fig. 7), which are inter-
e
W genotypefromtheAdriatic. spersedamongthelargerones.Thesmallerbodiesare
d ThenucleotidedifferencesbetweenC.subsalsaand staineddarkbrownbyosmiumtetroxide.Oneormore
e
r
Alf theAdriaticgenotypewerenoticeablyhigherthanthe round, reddish or brownish inclusions up to 5 µm
g differences within the C. marina complex (Figs 1–5 diameter are seen at times towards the posterior end
n
u and see Demura et al., 2009). Whereas the rather ofthecell.
ft
Sti slowly evolving SSU showed only three base substi- Staining of living cells with Neutral Red killed
y [ tutions (out of 1773 aligned nucleotides) between themandcausedimmediateejectionoflargenumbers
d b previously described C. subsalsa and the new of mucocysts with a maximum length of c. 30 μm
e
d Adriatic strains, the highly variable D1/D2 region of (Figs 8, 9). The mucocysts were pointed proximally
a
nlo theLSU(671bp)yielded25substitutions(Figs1,2). and gradually widened distally, ending in a slightly
w TheITS-region(ITS1,5.8SrDNAandITS2;820bp) enlarged terminal opening. Such mucocysts were
o
D also exhibited 25 substitutions, but, due to the high defined as ‘oboe-shaped’ by Biecheler (1936), who
degree of sequence difference, the C. subsalsa clade firstdescribedC.subsalsa.
lackedstrongbootstrap support(Fig. 3).Geneticdis- Under SEM (Figs 10–13), cells of the Adriatic C.
tancesbetweenthetwosetsofstrainswerestrikingly subsalsa appeared pear- or lemon-shaped (Fig. 12)
highintheplastidencodedpsaA(1371bp)andrbcL with a groove in the anterior part of the cell, slightly
(627 bp), the steps separating them being 91 (psaA: displaced from the cell apex, from which the two
Fig. 4) and 24 (rbcL: Fig. 5). More detailed raphido- approximately equal flagella originated. Within the
phyte phylogenies, based on ML and including all groove,ontheproximalpartofoneflagellum,mastigo-
investigated strains, are shown in the supplementary nemeswerepresent,formingamesh-likestructurecon-
information(FigsS1–S5).Theytoosupportthesister nectingtheflagellumtothecellsurface(notshown).The
relationshipsbetweentheChattonellastrainsfromthe whole cell surface bore globular bodies, 0.3 µm in
AdriaticandC.subsalsa. diameter, some of which were arranged in irregular
S.Klöpperetal. 84
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Figs1–5. Neighbourjoining(NJ)phylogenetictreesofselectedsequences(allhaplotypes)offivegeneticmarkerswithaccompany-
ingbasepairdifferencesandbootstrapvalues(inparentheses).Branchlengthsindicategeneticdistances(scalebarsareshownin
individual trees). Seealso ML treesinFigs S1–S5.Raphidophytesother thanChattonellawere usedas outgroups(Heterosigma
akashiwoorFibrocapsacf.japonica).1.NJtreeofeightselectednuclearSSUsequencesofChattonellaspp.2.NJtreeofsixselected
nuclearLSUsequencesofChattonellaspp.3.NJtreeofsixselectedITSsequencesofChattonellaspp.4.NJtreeoffiveselected
psaAsequencesofChattonellaspp.5.NJtreeofsixselectedrbcLsequencesofChattonellaspp.
AnewChattonellagenotype 85
s e
nt nc
e a
m Fr
onofC.subsalsa.Measure ChattonellasubsalsaMignot1976 AreaofSète,Mediterranean, roundedanteriorly,pointedorroundedposteriorly 50 –1520 roundish/oval regular n/a green yes n/a n/a
pti
3 scri
pril 201 quentde France
orschung ] at 02:13 17 A andtheoriginalandasubse ChattonellasubsalsaBiecheler1936 AreaofSète,Mediterranean, roundedanteriorly,pointedposteriorly,attimesglobular–3050 –1525 ovoid regular n/a ‘’grassgreen yes oboe-shaped n/a
eresf 217),
e P
M M 0) 0)
d C 2 2
n C a = =
Institute für Polar- u mtheGulfofMexico( ChattonellasubsalsCCMP217 GulfofMexico lemon/pear-shaped,oval –35.440.0(37.6±1.4,n –18.326.6(23.4±2.1,n peanut-shape irregular ~60 greenish/golden no – ~1.5×celllength
r o
ne fr
e a
g s
e al
aded by [Stiftung Alfred W mtheAdriaticSeaandC.subsparentheseswhereavailable. ChattonellasubsalsaAdriaticCRIM-A&CRIM-B AdriaticSea lemon/pear-shaped,oval –34.840.2(37.6±1.7,n=20) –18.326.6(24.0±1.7,n=20) roundish regular >60 greenish/golden yes oboe-shaped ~1.5×celllength
nlo froin
Dow forChattonellahemean±S.D. Length(µm) Width(µm) Shape Arrangement Number Colour Presence Shape Relativelength
at
datwith
al
phologicherange, feature s
Table2.Moraregivenast Strainorigin Morphological Cellshape Celldimension Chloroplast Mucocyst Flagella
S.Klöpperetal. 86
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re Figs6–13. ChattonellasubsalsafromtheAdriaticSea,LM(Figs6–9)andSEM(Figs10–13).6.LivingcellofstrainCRIM-Bwith
f
Al theundulatedflagellumdirectedanteriorly(af),theposteriorflagellumdirectedbackwards(pf)andthechloroplasts(ch).7.Slightly
g swollenlivingcellofstrainCRIM-Bshowingchloroplasts(ch),andlargerbutton-like(arrows)andsmallerrounded(arrowheads)
n
u surfacebodies.8.CellsofstrainCRIM-AstainedwithNeutralRedshowingamassivedischargeofoboe-shapedmucocysts(mu).9.
ft
Sti DetailedmicrographofmucocystsdischargedfollowingNeutralRedstainingofacellofstrainCRIM-A.10.Cellsurfaceofacellof
y [ strainCRIM-Ashowingroundedbodiesarrangedincirclesaroundacentraldepression(arrows).11.Detailofthecellsurfaceofstrain
b CRIM-Awitharingofsmallroundedbodiessurroundinganaperture(arrow).12.WholecellofstrainCRIM-Awithstretchedflagella
ed disappearinginfilterholes.13.CellofstrainCRIM-Asurroundedbynumerousmucocysts(mu).Scalebars=10µm(Figs6–9,12,
d
a 13),1µm(Fig.10)and0.5µm(Fig.11).
o
nl
w
o
D ringsof7–10(Fig.10)surroundingasmoothdepression; which were of comparable thickness (Fig. 14). The
depressions at times contained an aperture of variable ectoplasm consisted of a loose matrix of large
size and position (Fig. 11). The areas separating the vacuoleswithembeddedchloroplasts.Inpreparations
individualringswerefilledwiththesameroundbodies made without ferricyanide, numerous round osmio-
asthoseintherings(Figs10,12).Somecellsseenunder philicglobuleswereseenbelowthecellsurface(Fig.
SEM were surrounded by large numbers of ejected 16). In the chloroplasts, about 15 lamellae were
mucocysts, the majority sticking flattened to the filter observed running parallel to the long axis, each con-
(Fig.13).Theoriginofthemucocystsonthecellsurface sistingoftwothylakoids(Figs 15,18). Thepyrenoid
wasnotclearlydetectable. was located in the proximal (inner) part of the chlor-
UnderTEM(Figs14–18),therewasacleardiffer- oplast,embeddedintheendoplasm(Figs14,15,18).
entiation between the peripheral part of the cell (the Theproximalendsofthelamellaepenetratedintothe
ectoplasm) and the central part (the endoplasm), matrixofthepyrenoidasasinglemoreorlessdilated
AnewChattonellagenotype 87
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e Figs14–18. ChattonellasubsalsafromtheAdriaticSea,TEM.14.WholecellofstrainCRIM-Aintransversesectionwithclearly
g
We separatedendoplasm(en)andectoplasm(ec)andcellorganelles.15.SectionintheflagellarareaofacellofstrainCRIM-B.Notethe
d mastigonemes(ma)attachedtotheanteriorflagellum(af)andtherhizoplast(rh).16.DetailofcellofstrainCRIM-Cwithosmiophilic
re granules(arrowheads)atthesurface.17.Transversesectionacrosstheanteriorflagellum(af)ofacellofstrainCRIM-B,showing
f
Al severalrowsofmastigonemesattachedtotheflagellarmembrane.18.SectionofacellofstrainCRIM-AshowingtheGolgibody(g)
g adjacenttothenucleus(nu)intheflagellarareaandothercellularorganelles,suchaspyrenoids(py)withinthechloroplasts(ch),and
n
u mitochondria(m).Scalebars=5µm(Figs14,18),1µm(Fig.15),2µm(Fig.16)and0.2µm(Fig.17).
ft
Sti
[
y
d b thylakoid (Figs 15, 18). The endoplasm was com- connected the bases of the flagella with the anterior
e
d posed of a dense, granular matrix and included, in surfaceofthenucleus(Fig.15).
a
nlo additiontotheproximalpartsofthe chloroplasts,the
w nucleusandalltheothercellorganelles.Thenucleus Chattonella subsalsa CCMP217. Under LM, the
o
D occupiedmostoftheendoplasmandwaspointedatits shape of C. subsalsa CCMP217 cells was variable,
anterior end, protruding towards the flagellar bases whichistypicalformostraphidophytespeciesinculture
(Figs15,16).Severalelongated,andincross-section andinnaturalsamples(e.g.Band-Schmidtetal.,2012).
roundish,mitochondriawerelocatedintheendoplasm Healthycellstendedtobepyriformwithacolourlesstail
aroundthenucleus.Theanteriorflagellumboremas- attheposteriorend,whichwasmissingincellsassum-
tigonemes with a conical basal part (Figs 15, 17). inganovalshape(Figs19–21).Thechloroplastswere
StacksofmanyflattenedandelongatedGolgivesicles green to golden in colour, had oval or peanut-like
werepresentbetweenthenucleusandthechloroplasts shapes,andwereirregularlypackedatthecellperiphery
in the area close to the flagellar pit (Figs 14, 18). (Figs19–21).Nootherstructurewasvisibleonthecell
Vesiclesofunknownoriginwereobservedcontaining surface,apartfromverysmallandirregularlydistributed
preformed flagellar hairs (not shown). A rhizoplast refringentgranules(Fig.19).