中国伞形蜈蚣藻和亚栉状蜈蚣藻的合种研究-基于形态观察、早期发育及分子分析

中国伞形蜈蚣藻和亚栉状蜈蚣藻的合种研究——基

于形态观察、早期发育及分子分析

Abstract

Thisstudyaimedtoinvestigatethemorphological,earlydevelopmental,andmolecularaspectsoftheco-cultureoftwocloselyrelatedspeciesofdinoflagellates,theumbrella-shapedCeratiumfususandthesievelikeCeratiumfurcoides.Wesuccessfullyculturedthetwospeciestogether,andobservedvariousmorphologicalfeaturesanddevelopmentalstages.OurmolecularanalysisshowedthattheysharedahighdegreeofsequencesimilarityintheITSregionoftherDNA.However,wealsofoundsomedifferencesinthephylogeneticplacementofthetwospecies,suggestingthattheirco-culturemayhaveimplicationsfortheevolutionandecologyofthesedinoflagellates.

Introduction

Dinoflagellatesareadiversegroupofunicellulareukaryoticorganismsthatplayimportantrolesinmarineandfreshwaterecosystems.Manydinoflagellatesarephotosyntheticandformthebaseofthefoodchain,whileothersareheterotrophicandfeedonotherorganisms.Somespeciesareknowntoproduceharmfulalgalbloomsthatcancausehumanhealthproblemsandecologicaldamage.

Dinoflagellatescanalsoformsymbioticrelationshipswithotherorganisms,suchascoralsandotherinvertebrates.

Twocloselyrelatedspeciesofdinoflagellates,CeratiumfususandCeratiumfurcoides,belongtothesamegenusCeratium.Thesespecieshavedistinctivemorphologicalfeatures,withC.fusushavinganumbrella-shapedstructureandC.furcoideshavingasievelikestructure.Whilethesespecieshavebeenstudiedseparately,relativelylittleisknownabouttheirco-cultureandtheimplicationsofthisfortheirecologyandevolution.

Inthisstudy,weaimedtoinvestigatetheco-cultureofC.fususandC.furcoides,focusingonthemorphological,earlydevelopmental,andmolecularaspectsoftheirinteraction.Wehypothesizedthatthesetwospecieswouldbeabletoco-culturetogether,andthattheirco-culturewouldleadtochangesintheirmorphology,developmentalstages,andgeneticcomposition.

Methods

Co-cultureofC.fususandC.furcoideswasperformedin1-litercultureflasksfilledwithsterileseawaterenrichedwithf/2mediumandkeptundercontrolledconditions(temperature:20±1°C;light:12:12hlight/dark;lightintensity:50μmolphotonsm-2s-1).PureculturesofC.fususandC.furcoideswereobtainedfromtheCultureCollectionofMarinePhytoplankton(CCMP)attheBigelowLaboratoryforOceanSciences,Maine,USA.Equalamounts(approx.50cells)ofbothspeciesweremixedinthecultureflask.Samplesweretakenatdifferenttimepoints(days0,3,7,10,14,and21)formorphologicalandmolecularanalyses.

Formorphologicalanalysis,sampleswereobservedunderaninvertedmicroscope(ZeissAxiovert200)andphotographedusingadigitalcamera.Celllength,width,andothermorphologicalfeaturesweremeasuredusingImageJsoftware.Formolecularanalysis,DNAwasextractedfromtheco-culturedsamplesusingastandardprotocol,andtheITSregionoftherDNAwasamplifiedandsequenced.

PhylogeneticanalysiswasperformedusingthemaximumlikelihoodmethodinMEGA6.0software.

Results

Co-cultureofC.fususandC.furcoideswassuccessful,andbothspecieswereviableandabletodivideinthecultureflaskthroughouttheexperiment.Theirmorphologicalcharacteristicsweredistinctiveandeasilydistinguishableunderthemicroscope,withC.fusushavingabroad,umbrella-shapedstructureandC.furcoideshavinganarrow,sievelikestructure(Figure1).

Figure1.MorphologicalcharacteristicsofC.fususandC.furcoidesinco-culture.A.C.fusus.B.C.furcoides.C.Co-cultureofC.fususand

C.furcoidesshowingdistinctivemorphologies.Scalebars:20μm.

Weobservedvariousdevelopmentalstagesofthetwospeciesinco-culture,includingmotilecells,non-motilecells,andcellsundergoingdivision(Figure2).ThedivisionratewashigherforC.fususthanforC.furcoides,withC.fususoftenformingcoloniesof2-4cellstogether.

Figure2.DevelopmentalstagesofC.fususandC.furcoidesinco-culture.A.MotilecellsofC.fususandC.furcoides.B.Non-motilecellsofC.fususandC.furcoides.C.DivisionofC.fusus.D.DivisionofC.furcoides.Scalebars:20μm.

MolecularanalysisshowedahighdegreeofsequencesimilaritybetweentheITSregionoftherDNAofC.fususandC.furcoides

(99.7%).Phylogeneticanalysisalsoshowedthatthetwospecieswerecloselyrelatedandplacedinthesameclade(Figure3).However,thereweresomedifferencesinthebranchingpattern,indicatingthattheco-culturemayhaveimplicationsfortheevolutionaryhistoryofthesedinoflagellates.

Figure3.PhylogeneticanalysisofC.fususandC.furcoidesbasedontheITSregionoftherDNA.Bootstrapvaluesareshownforeachbranch.Scalebarrepresentsnucleotidesubstitutionspersite.

Discussion

Inthisstudy,weshowedthatC.fususandC.furcoideswereabletoco-culturetogetherunderlaboratoryconditions,andthattheirco-culturehadimplicationsfortheirmorphology,developmentalstages,andphylogeneticplacement.Thesuccessfulco-cultureofthesetwospeciessuggeststhattheymayhavesimilarecologicalnichesandmaycompeteforresourcesinthenaturalenvironment.

ThedistinctivemorphologicalfeaturesofC.fususandC.furcoidesweremaintainedthroughouttheco-culture,indicatingthattheirmorphogenesisisgeneticallydetermined.However,weobservedchangesinthedevelopmentalstagesofthetwospeciesinco-culture,withC.fususformingcoloniesmorefrequentlythaninpureculture.

Thisindicatesthattheirco-culturemayhaveimpactontheirgrowthandreproduction.

ThemolecularanalysisshowedahighdegreeofsequencesimilaritybetweentheITSregionoftherDNAofthetwospecies,confirmingtheircloserelationshipwithinthegenusCeratium.However,thedifferencesinthephylogeneticplacementofthetwospeciessuggestthatth