living in biological soil crust communities of african deserts physiological traits of green algal klebsormidium species streptophyta to cope with desiccation light and temperature gradients

1.Morphology of the four Klebsormidium strains Klebsormidiales, Strepto-phyta BIOTA 14614.7 African Strain A A, BIOTA 14613.5e African Strain B B, SAG 384-1 Arctic Strain C and BIOTA 146

Contents lists available atScienceDirect

Journal of Plant Physiology

j o u r n a l h o m e p a g e :w w w e l s e v i e r c o m / l o c a t e / j p l p h

Living in biological soil crust communities of African

deserts—Physiological traits of green algal Klebsormidium species

(Streptophyta) to cope with desiccation, light and temperature

gradients

a University of Rostock, Institute of Biological Sciences, Applied Ecology & Phycology, Albert-Einstein-Strasse 3, D-18059 Rostock, Germany

b University of Innsbruck, Institute of Botany, Functional Plant Biology, Sternwartestrasse 15, A-6020 Innsbruck, Austria

a r t i c l e i n f o

Article history:

Received 24 July 2015

Received in revised form 31 August 2015

Accepted 2 September 2015

Available online xxx

Keywords:

Aeroterrestrial algae

Biological soil crust

Ecophysiology

Morphology

Photosynthesis

Respiration

a b s t r a c t

GreenalgaeofthegenusKlebsormidium(Klebsormidiales,Streptophyta)aretypicalmembersofbiological soilcrusts(BSCs)worldwide.ThephylogenyandecophysiologyofKlebsormidiumhasbeenintensively studiedinrecentyears,andanewlineagecalledsupercladeG,whichwasisolatedfromBSCsinarid southernAfricaandcomprisingundescribedspecies,wasreported.ThreedifferentAfricanstrains,that havepreviouslybeenisolatedfromhot-desertBSCsandmolecular-taxonomicallycharacterized,were comparativelyinvestigated.Inaddition,Klebsormidiumsubtilissimumfromacold-deserthabitat(Alaska, USA,supercladeE)wasincludedinthestudyaswell.Photosyntheticperformancewasmeasuredunder differentcontrolledabioticconditions,includingdehydrationandrehydration,aswellasunderalight andtemperaturegradient

AllKlebsormidiumstrainsexhibitedoptimumphotosyntheticoxygenproductionatlowphoton flu-encerates,butwithnoindicationofphotoinhibitionunderhighlightconditionspointingtoflexible acclimationmechanismsofthephotosyntheticapparatus.Respirationunderlowertemperatureswas generallymuchlesseffectivethanphotosynthesis,whiletheoppositewastrueforhighertemperatures TheKlebsormidiumstrainstestedshowedadecreaseandinhibitionoftheeffectivequantumyieldduring desiccation,howeverwithdifferentkinetics.Whilethesinglecelledandsmallfilamentousstrains exhib-itedrelativelyfastinhibition,theuniseratefilamentformingisolatesdesiccatedslower.Exceptone,all otherstrainsfullyrecoveredeffectivequantumyieldafterrehydration.Thepresenteddataprovidean explanationfortheregularoccurrenceofKlebsormidiumstrainsorspeciesinhotandcolddeserts,which arecharacterizedbylowwateravailabilityandotherstressfulconditions

©2015Z.PublishedbyElsevierGmbH.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense

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1 Introduction

Onaglobalscale,biologicalsoilcrustcommunities(BSCs)form

themostproductivemicrobialbiomassoftheEarth’s‘CriticalZone’

This zone is also defined as heterogeneous, nearsurface

envi-ronmentinwhichcomplex interactionsinvolvingrocksurfaces,

soil,water,airandlivingorganismsregulatethenaturalhabitat,

andhencedeterminetheavailabilityoflife-sustainingresources

dis-turbedlandscapes,suchasvolcanicareas,glacierforefields,flood

plains andmany drylands,BSCs representthepioneer

commu-∗ Corresponding author.

E-mail address: ulf.karsten@uni-rostock.de (U Karsten).

nities that form the basis for further ecosystem development

as algae, cyanobacteria and lichens, and are characterized as

‘ecosystem-engineers’formingwater-stableaggregatesthathave multi-functionalecologicalrolesinprimaryproduction,nutrient andhydrologicalcycles,mineralization,dusttrapping,weathering andstabilizationofsoils(e.g.Castillo-Monroyetal.,2010).Dueto theextracellularmatrixofcyanobacteriaandalgae,soilparticles areaggregatedformingacarpet-likebiofilm,whichreduceserosion

bywindandwater.Arecentreviewclearlyindicatedthe impor-tantecologicalroleofBSCsforglobalcarbon(C)fixation(about7%

ofterrestrialvegetation)andNfixation(about50%ofterrestrial biologicalNfixation)(Elbertetal.,2012)

Terrestrialfilamentousgreenalgaeofthecosmopolitangenus Klebsormidium (Klebsormidiophyceae, Streptophyta) are often http://dx.doi.org/10.1016/j.jplph.2015.09.002

0176-1617/© 2015 Z Published by Elsevier GmbH This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).

asrivers,lakes,bogs,soil,rocksurfaces,treebark,sanddunes,and

BSCsfromalpineregionsorevendeserts(Rindietal.,2011;Karsten

providedsofarthemostcomprehensivephylogenyofthisgenus

usingITSrRNAandrbcLsequences.Theseauthorsshowedseven

main superclades A–G; which included sixteen well-supported

clades.WhilesupercladeAcontainedspeciesofthecloselyrelated

genusInterfilum(Mikhailyuketal.,2008;Rindietal.,2011),

super-cladesB–FcomprisedallKlebsormidiumspeciessofardescribed

Mostinteresting,anewlineageofKlebsormidiumtermed

super-cladeG,whichwasisolatedfromBSCsinaridsouthernAfricaand

comprisingundescribed species,wasforthefirst timereported

theknownKlebsormidiumspeciesdifferedintheirultrastructure

andtexture ofcellwalls Membersof thesupercladesB, C and

E were characterized as mesophytic and hydrophytic lineages,

becausetheyexhibitedfilamentswithgelatinizedcellwallsthat

easilytransformintoshortfilamentsorevenintoindividualcells

D,FandGweresuggestedasxerophyticlineagesbecauseofdense

andratherstrongfilamentswithoutmucilage(Mikhailyuketal.,

2014).TheseauthorsdescribedsupercladeGmembersalsoas

typ-icalAfricangroup.ThiswasagainsupportedbyRyˇsáneketal.(2015)

whomentionedthatalthoughcollecting200Klebsormidiumstrains

fromEurope,NorthAmericaandAsia,theycouldnotfindasingle

genotypeofsupercladeG

Becauseofthewidebiogeographicdistributionandoccurrence

in so diverse environments, the physiological traits of various

InterfilumandKlebsormidiumspecieshavebeenrecentlystudied

concerningacclimationmechanismsagainstdesiccation,

tempera-ture,visiblelight;UVRandpHgradients(Karstenetal.,2010,2014;

investigated sofar weremembers of the supercladesA–F, and

thatdependingonthesuperclademembershipthephysiological

responsepatternssuchasdesiccationtolerancecanbequite

differ-entasexemplarilydescribedforthetwocoexistingKlebsormidium

dissectum (superclade E)and Klebsormidium crenulatum

(super-cladeF) in alpine BSCcommunities of theAlps (Karstenet al.,

desic-cationtoleranceofbothKlebsormidiumspeciescanbeexplained

bydifferencesintheirmorphologicalandstructuralfeaturessuch

asstrongversusweakfilaments(Karstenetal.,2010;Holzinger

dif-ferencesmaybeattributedtodifferentphysiologicaltraits,e.g.K

crenulatum(supercladeF)hasbeenshowntohaveamorenegative

waterpotential(=−2.09MPa)comparedtoK.nitens(superclade

E,=−1.67MPa)(Kaplanetal.,2012)

overviewon thedistributionof BSCs over Africa, pointing to a

concentrationofthesemicrobioticcommunitiesinthesouthern

andsouthwesternpartofthecontinent.Laterandinamore

com-prehensiveway,Büdeletal.(2009)undertookanintensivefield

monitoringand samplingin drylands along a transect

stretch-ingfromtheNamibian–AngolanborderdownsouthtotheCape

Peninsula.TheseauthorsreportedsevenBSCtypeswhichcould

bedifferentiatedonthebasisofmorphologyandtaxonomic

com-position,anddocumented29greenalgalspeciesincludingsome

undescribedKlebsormidiumspecies.Büdeletal.(2009)concluded

thatBSCsareanormaland frequentvegetationelementinarid

andsemi-aridsouthwesternAfrica,andthat rainfrequency and

durationofdryperiodsratherthantotalannualprecipitationare

theecologicalkeyfactorsforthedevelopment,differentiationand

compositionofthemicrobioticcommunities

Asecophysiological studiesongreen algae fromAfrican hot desertBSCsaremissing,andbecauseofthisconspicuously differ-entsupercladeGintheKlebsormidiumphylogeny,ourmajorgoal wastocharacterizeforthefirsttimesomeoftheavailablestrains Photosyntheticactivityundercontrolleddehydrationandrecovery conditionsandalongtemperatureandlightgradientswere com-parativelyinvestigatedinthreeAfricanKlebsormidiumstrains.For comparison,Klebsormidiumsubtilissimumfromacold-desert habi-tat(Alaska,USA,supercladeE)wasincludedinthestudyaswell Themainhypothesiswastoevaluatewhetherphysiologicaltraits are present which influence desiccation, temperature and light toleranceinthesespecificKlebsormidiumgenotypes.Ofparticular interestwastoconsiderwhethertheenvironmentalconditionsof theoriginalhabitatarereflectedintherespectiveresponse pat-terns

2 Materials and methods

2.1 Strainoriginandcultureconditions TheKlebsormidiumstrainsofthesupercladeG-BIOTA14.614.7 (=AfricanStrainA),BIOTA14.613.5e(=AfricanStrainB)andBIOTA 14.614.18.24(=AfricanStrainC)—wereoriginallycollectedand iso-latedbyProf Burkhard Büdelin theframeofthe international BIOTA project(Büdelet al., 2009), and kindlyprovided forthe presentstudy.ThespeciesKlebsormidiumsubtilissimumSAG384-1 (=ArcticStrain)waspurchasedfromTheCultureCollectionofAlgae

at Göttingen University;Germany (international acronym SAG;

habitat and origin, meteorological data, as well as taxonomic assignmentsaresummarizedinTable1

The4Klebsormidiumstockculturesweremaintainedin Erlen-meyer flasks (volume 250–500mL) filled with modified Bold’s BasalMedium(3NBBM;StarrandZeikus,1993),whichisahighly enrichedpurelyinorganicmediumcontainingvarioustrace met-als,fewvitaminsandtriplenitrateconcentration,butnocarbon source.Identicalcultureconditionsand equipmentwasapplied

asdescribedforKlebsormidiumdissectum(KarstenandHolzinger,

2012),i.e.cellswerekeptat20◦Cand35–40␮molphotonsm−2s−1 underalight-darkcycleof16:8hL:D(OsramDaylightLumiluxCool WhitelampsL36W/840,Osram,Munich,Germany).Photonfluence ratemeasurementswereuntertakenwithaSolarLightPMA2132 cosinecorrectedPARsensorconnectedtoaSolarLightPMA2100 radiometer(SolarLightCo.Inc.,Philadelphia,USA).Forall exper-imentsalwaysvitallog-phaseculturesexhibitingcomparablecell densitieswereused

2.2 Lightmicroscopy Algaeof log-phase cultures usedfor physiological measure-ments(AfricanStrainsA–C,ArcticStrain)werealsoinvestigated witha Zeiss Axiovert200Mmicroscope(Carl Zeiss Microscopy GmbH,Jena,Germany),equippedwitha100×1.3NAobjectivelens andanAxiocamMRc5cameracontrolledbyZeissAxiovision soft-ware.Forcontrastenhancement,differentialinterferencecontrast (DIC)wasapplied.Furtherprocessingofimageswasperformed usingAdobePhotoshop(CS5)softwareversion12.1(Adobe Sys-tems,SanJose,CA,USA).Celldimensions(cellwidthandlength) weredeterminedbymeasuringa minimumof 20cells andthe length:width (L:W) ratio was calculated Mean values±SD are shown

2.3 Dehydrationandrecoveryexperiments Forthedesiccationexperimentsanewhighlystandardized

set-upwasappliedtofollow thekineticsofcontrolleddehydration

Table 1

Characterization of the Klebsormidium isolates collected from dry regions Strain number, habitat and origin, taxonomic assignment according to the suggested clades of Rindi

et al (2011) are given The meteorological data include monthly air temperature, monthly rainfall days, monthly precipitation (mm) and annual precipitation (mm) ( www worldweatheronline.com ) The sequence accession numbers include complete and partial sequences, respectively, of the different ribosomal regions SAG: Sammlung für Algenkulturen, Göttingen, Germany.

Assigned species, strain

number and

synonym used in the

text

Habitat and origin Meteorological data Clade assignment

according to Rindi et al.

(2011)

Sequence Accession (18s rRNA, ITS1, 5.8s rRNA, ITS2, 28s rRNA)

Klebsormidium sp.

BIOTA 14614.7

African Strain A

Biological soil crust, BIOTA observatory Grootderm, succulent Karoo, South Africa 28 ◦ 36  44.1  S

16 ◦ 39  45.0  E leg.: Burkhard Büdel

14 March 2001

Air temperature:

13.5–20.6 ◦ C Monthly rainfall days:

0–14 Monthly precipitation:

0 to 34.2 mm Annual rainfall: 56 mm

Klebsormidium sp.

BIOTA 14613.5e

African Strain B

Biological soil crust, BIOTA observatory Koeboes, South Africa,

28 ◦ 45  51.1  S

16 ◦ 98  38.3  E leg.: Burkhard Büdel

12 March 2001

Klebsormidium sp.

BIOTA 14614.18.24

African Strain C

Biological soil crust, BIOTA observatory Grootderm, succulent Karoo, South Africa,

28 ◦ 36  44.5  S

16 ◦ 39  52.4  E leg.: Burkhard Büdel

14 March 2001

Klebsormidium

subtilissimum

SAG 384-1

Arctic Strain

Snow, Port Barrow, Alaska, USA;

cold-desert climate isolated 1952 R.A Lewin

Air temperature:

−29.1–8.3 ◦ C Monthly rainfall days:

4 to 12 Monthly precipitation:

2.3–26.7 mm Annual rainfall:

114 mm

and subsequent rehydration on theeffective quantum yield of

photosystemII(PSII)usingnon-invasivepulseamplitude

modu-lation(PAM)fluorometry(Karstenetal.,2014).Alwayslow-light

acclimatedsamples(35–40␮molphotonsm−2s−1)weremeasured

withaPAM2500(HeinzWalzGmbH,Effeltrich,Germany).Cellsof

eachKlebsormidiumisolatewereconcentratedassmalllightgreen

spotson4replicateWhatmanGF/Fglassfibrefilters(Whatman,

Dassel,Germany).Ontoeachfilterexactly200␮Lofthe

respec-tivehomogeneousalgalsuspension(c.1–2mgchlorophyllaL−1)

wasappliedusinganEppendorfpipette.Usingdefinedstarting

vol-umesoflog-phaseculturesguaranteedreproducibility,sinceitis

difficultorimpossibletoobtainreliablewatercontentsinsmall

quantitiesofalgalfilaments.Themoistfilterswerepositionedon

perforatedmetalgridsontopoffourglasscolumnsinsidea

trans-parent200mLpolystyrolbox.Eachoftheseboxeswasfilledwith

100goffreshlyactivatedsilicagel(SilicaGelOrange,CarlRoth,

Karlsruhe,Germany)andsealedwithatransparenttoplid.The

rel-ativeairhumidity(RH)conditionsinsidetheboxeswererecorded

withaPCE-MSR145S-THminidataloggerforairhumidityand

tem-perature(PCEInstruments,Meschede,Germany).Thedatashowed

thatwithinthefirst30mintheRHdeclinesfromabout30%to10%

andremainsafterthatalmostunchangedatthislowairhumidity

pointingtoareproducibledryatmosphere(Karstenetal.,2014)

Thechambersweremaintainedat22±1◦Cand40␮molphotons

m−2s−1PAR(cultureconditions)(Osramlightsourcesseeabove)

whichreflectsthenaturalaveragetemperaturesinthehabitatsof

thedessertstrainsandpreventslightstressduringmeasurements

Theeffectivequantumyield(F/Fm’)ofphotosystemII(PSII)

wasregularlydeterminedduringthedehydration period(upto

500min dependingon thestrain)using thePAM 2500

accord-ingtheapproachofGentyetal.(1989).F/Fmwascalculatedas

(Fm −F)/Fm withFasthefluorescenceyieldoflight-treatedalgal

cells(40␮molphotonsm−2s−1)andFm  asthemaximum

light-adapted fluorescenceyieldafter employinga 800ms saturation pulseasdescribedbySchreiberandBilger(1993).ThePAMlight probewaspositionedoutsidethecoverlidoftheboxes(always

2mm distance) toguarantee undisturbed RHconditions inside, i.e.allfluorescencemeasurements wereperformedthrough the polystyrollids.ThedistancefromthePAMlightprobetothealgal sample onto theglassfibrefilters wasalwayskeptconstant at

10mm.Sincethewatercontentoftheappliedsmallcellnumbers (200␮Lsuspension)isalmostimpossibletoestimate,we under-tookpreliminaryexperimentsusing25–50timeshigherquantities

ofalgalbiomass,andestimatedthatthefinalremainingwater con-tentaftercontrolleddesiccationusingthedescribedapproachwas between5and10%ofthecontrol(datanotshown).Whileour des-iccationset-upguaranteescomparativeandreproduciblestarting andendpointsofcellularwatercontents,itfailstodescribeany kineticsofwaterloss

Afterthedehydrationperiod,thedriedglassfibrefilterswere transferredtoanewpolystyrolboxwhichwasfilledwith100mL tapwaterinsteadofsilicageltocreateahighhumidityatmosphere (>95%).Thefilterswererehydratedbyadding200␮Lofthe stan-dardgrowthmediumtoeachalgalspotandrecoveryof(Fm −F)/Fm 

wasfollowedwiththesamemethodologyasdescribedabove 2.4 Photosynthesisandrespirationunderlightandtemperature gradients

A Presens Fibox 3 oxygen optode (Presens, Regensburg, Germany)wasappliedtorecordphotosyntheticoxygen produc-tionrates underrisingphoton fluencedensities(PI curves)and respiratoryoxygenconsumptioninthedark.Theoxygensensor wasattachedtoa3mLthermostaticacrylicchamber(typeDW1, HansatechInstruments,Norfolk,UK)combinedwithamagnetic

absorption.Always 2.8mLlog-phaseKlebsormidiumsuspensions

ofknownchlorophyllaconcentrationwereaddedtothechamber

supplementedby0.2mLofaNaHCO3stocksolution(resultingin

2mMNaHCO3finalconcentration)toguaranteesufficientcarbon

supplyduringmeasurements.Thelightpathinsidethemeasuring

chamberwas10mm,andtheappliedlightlevelswerecarefully

determinedwithaHansatechQRT1PARsensor(Hansatech

Instru-ments,Norfolk,UK).Thisquantumsensorwasverticallypositioned

insidethecuvetteallowinglightsource(halogenlamp)calibration

fromtheside(90◦ angle)and therebyovercomingthepotential

difficultiesassociatedwithaccuratelymeasuringPARlightlevels

withinthe small chamber Beforethe onset of increasing

pho-tonfluenceratesrespirationwasmeasuredindarknessfollowed

byexposureof thealgal cells tonine light levelsrangingfrom

0 to 500␮mol photons m−2s−1 PAR (photosynthetically active

radiationfor10min)at22◦Cusingacombinationof4different

HansatechA5neutralfilters(HansatechInstruments,Norfolk,UK)

AllO2rateswerenormalisedtototalchlorophyllaconcentration,

rangingfrom3to6␮g3mL−1,whichweredeterminedaftereachPI

curvemeasurement.Weusedchlorophyllaconcentrationas

ref-erenceparameter,becausecelldensitiesofthe4Klebsormidium

strainsweredifficulttodeterminebecauseofthefilamentous

mor-phologyandthefactofuncontrolleddisintegration intosmaller

fragmentsofsomeoftheisolates.The3mLKlebsormidium

suspen-sionwasfilteredontoaWhatmanGF/Fglassfibrefilterusingaglass

Pasteurpipette,andchlorophyllaandbextractedbyadding3mL

dimethylformamide(DMF)followedbyphotometric

calculatedandfittedwiththemathematicalphotosynthesismodel

characteristicparameters:␣,positiveslopeatlimitingphoton

flu-encerates(␮molO2h−1mg−1Chl.a(␮molphotonsm−2s−1)−1;Ic,

lightcompensationpoint(␮molphotonsm−2s−1);Ik,initialvalue

oflight-saturated photosynthesis(␮molphotons m−2s−1)

Fur-thermore,themaximumphotosyntheticrateinthelight-saturated

range(␮molO2h−1mg−1Chl.a)wascalculated

Sincewiththehalogenlampoftheoxygenoptodesystemonly

500␮molphotonsm−2s−1 couldberealized(seeabove),aPAM

2500wasadditionallyusedtoapplyphotonfluenceratesupto

1432␮molphotonsm−2s−1forthedeterminationoflight-induced

rETRandpossiblephotoinhibitoryeffects.Cellsofeach

Klebsormid-iumstrain weretransferredon4replicate WhatmanGF/Fglass

fibrefiltersuntilalightgreenspotwasvisible.Thesemoistfilters

werealsopositionedinthetransparent200mL,tapwaterfilled

polystyrolboxes,andtreatedasalreadydescribed.Algalcellswere

exposedto13photonfluencedensities(PFDs)for30seach(rapid

lightcurves)rangingfrom1upto1432␮molphotonsm−2s−1.The

actiniclightwasprovidedbyaredpowerLED(630nm)ofthePAM

2500.Aftereach lightexposure,asaturatingpulsewasgivento

detectFmandF/Fm .TherelativeelectrontransportrateofPSII

(rETR)wascalculatedaccordingtoKromkampandForster(2003):

rETR= F/Fm PFD

where F/Fm =the effective PSII quantum efficiency and

PFD=photonfluxdensity

All measurements were undertaken under at 22±1◦C

Photosynthesis-irradiance(PI)curves as rETRvs PFDwere

cal-culatedtocheckwhetherthe4strainsofKlebsormidiumexhibit

indicationsofphotoinhibition

Theeffectofrisingtemperaturesonphotosyntheticand

res-piratory response patterns (referenced tochlorophyll a) in the

fourKlebsormidiumstrainswasdeterminedusingaThermoHaake

K20refrigeratedcirculator(ThermoFisherScientificInc.,Waltham,

Massachusetts,USA)connectedtothemeasuringchamber.Allalgal

Fig 1.Morphology of the four Klebsormidium strains (Klebsormidiales, Strepto-phyta) BIOTA 14614.7 (African Strain A) (A), BIOTA 14613.5e (African Strain B) (B), SAG 384-1 (Arctic Strain) (C) and BIOTA 14614.18.24 (African Strain C) (D) taken from 1 month old liquid cultures (A) Cell filament and 2-cell-fragment with clearly visible pyrenoids (arrowheads), cross-wall protuberances (white arrow) and pari-etal chloroplasts with margins protruding towards the cell centre (black arrows) (B) Ovoid and cylindrical individual cells with one pyrenoid per cell (arrowhead) and spherical chloroplast-free districts at the cell poles (arrows) (C) Short cell fila-ments and detaching cells (black arrows) Pyrenoids are marked with arrowheads and cross-wall protuberances with white arrows (D) Cell filament with pyrenoids (arrowheads) and prominent cross-wall protuberances (arrows) Scale bars equal always 10 ␮m.

isolateswereexposedtoninetemperaturestepsrangingfrom5to

45◦Cin5◦CincrementsaccordingtoKarstenandHolzinger(2012) Aftertreatmentwiththehighesttemperature(45◦C) photosyn-thesisandrespirationweremeasuredagainafteratemperature reductionto30◦Ctolookforrecoveryeffects

From the final photosynthetic and respiratory rates (␮mol

O2mg−1 Chl a h−1) the gross photosynthesis:respiration (P:R) ratiosforeachtemperaturewerecalculated

2.5 Statisticalanalysis All oxygen measurements (optode) were carried out with threeindependentreplicates(n=3)whileallmeasurementofthe effectivequantumyield(performedbyPAM)wereperformedin fourindependentreplicates(n=4).Theshowndatarepresentthe respectivemeanvalues±standarddeviation

Statisticalsignificance ofthemeansoftheeffectivequantum yieldofdehydratedandrehydratedsamples,aswellasalloptode dataweretestedwithone-wayANOVAfollowedbyaTukey’s mul-tiplecomparisontesttofindsubgroupsofmeanswithsignificant differences.AnalyseswereperformedwithInStat(GraphPad Soft-wareInc.,LaJolla,CA,USA)

3 Results

3.1 Lightmicroscopy TheAfricanStrainAformeduniseratefilamentsandeachcell containedoneparietalchloroplastwithaprominentpyrenoid sur-rounded bystarch grains (Fig 1a).The chloroplast coveredthe wholelengthofthecellandabout2/3ofthecellcircumference, whilethecentralpartofonelongitudinalmarginoftenprotruded distinctlytowardsthecellcentre(Fig.1a).Mostcrosscellwalls exhibitedprotuberancesofwallmaterialononeoroppositesides

ofthesamecrosscellwallandoccasionallythecrosswallappeared swollen(Fig.1a).Fragmentationintosmallcellfilaments(2–6cells) wasscarcely.Thecelldiameterwashomogenously(Table2 while thecelllengthvariedmore(Table2 resultinginacelllengh:width

Table 2

Cell dimensions (length and width in ␮m) and length:width (L:W) ratio of the four

Klebsormidium strains investigated (n = 20 ± SD) Capital letters (length), small

let-ters (width) and cursive small letters (L:W ratio) indicate significant differences

between the dimensions Data were analysed by one-way ANOVA followed by

Tukey’s post hoc test (p < 0.001) Cell length and width of each strain was compared

by a standard two-sample t test and significantly differences are marked with an

asterisk (p < 0.001).

BIOTA 14614.7 7.8 ± 1.2 A 6.8 ± 0.3 a * 1.2 ± 0.2 a

BIOTA 14613.5e 7.6 ± 1.4 A 4.1 ± 0.4 b * 1.9 ± 0.4 b

SAG 384-1 13.7 ± 4.0 B 5.5 ± 0.5 c * 2.5 ± 0.8 c

BIOTA 14614.18.24 8.0 ± 2.1 A 8.8 ± 1.0 d 0.9 ± 0.3 a

(L:W)ratioof 1.2±0.2 (Table2).Cellsof AfricanStrainBwere

significantly(P<0.001)narrower,butthecelllengthdidnot

dif-fersignificantly(P<0.001)fromAfricanStrainA(Table2).African

StrainBoccurredunicellularlyandtheparietalchloroplastwasless

expanded,covering1/3−2/3ofthecellandusuallydidnotreach

towardsthepolesoftheovoidorcylindricalcells(Fig.1b).The

lon-gitudinalmarginsofthechloroplastweresmoothanditexhibited

onepoorlydevelopedpyrenoid(Fig.1b).Cellsweresurroundedby

athinmucilagelayer(Fig.1b).ThecellshapeoftheArcticStrain

appearedsimilartoAfricanStrainB,however,theywere

signifi-cantlywiderandlonger(Fig.1c,Tab.2).Filamentsshowedastrong tendencytodisintegrationandmostfilamentscontained2–4cells Each cellexhibited one parietalchloroplast embeddinga small pyrenoid(Fig.1c).Thelongitutinalmarginsofthechloroplastwere smooth(Fig.1c).Somecrosscellwallsexhibitedprotuberancesof wallmaterialonoppositesidesofthesamecrosswall(Fig.1c) Cellsweresignificantlylongercomparedtotheotherthreestrains, resultinginaL:Rratioof2.5±0.8(Tab.2).AfricanStrainCformed uniseratefilamentsandthecellwidthandlengthdidnotdiffer significantlyfromeachother(P<0.001;Table2,Fig.1d).Eachcell containedonechloroplasttouchingthecrosswallsandcovering

∼3/4ofthecellcircumference(Fig.1d).Thelongitudinalmargins

ofthechloroplastweresmoothanditcontainedoneclearly vis-iblepyrenoidsurroundedbystarchgrains(Fig.1d).Sometimes, prominentcrosswallprotuberancesoccurred(Fig.1d)

3.2 Lightrequirementsofphotosynthesis Thephotosyntheticoxygenproductionwasstimulatedbyrising photonfluencedensitiesinall4Klebsormidiumisolatesasreflected

inPIcurvesfromwhichcharacteristicparametersforthe descrip-tionofthelightrequirementswerederived(Fig.2;Table.3).African StrainsBandCandtheArcticStrainexhibitedlight-saturatedPI curves withmaximum oxygenproduction rates between 137.5

Fig 2. Dark respiration and photosynthetic oxygen evolution as function of increasing photon fluence densities up to 500 ␮mol photons m −2 s −1 in the four Klebsormidium isolates BIOTA 14614.7 (African Strain A), BIOTA 14613.5e (African Strain B), BIOTA 14614.18.24 (African Strain C) and SAG 384-1 (Arctic Strain) (n = 3, mean value ± SD) The dotted line represents in the 3 African Strains a fitted curve of the data measured according the photosynthesis model of Webb et al (1974)

Table 3

Photosynthesis-irradiance curve parameters of the Klebsormidium strains studied Data were recorded as oxygen evolution using an oxygen optode at 25 ◦ C and for the 3 African Strains fitted with the photosynthesis model of Webb et al (1974) without photoinhibition ␣: positive slope at limiting photon fluence rates (␮mol O 2 mg−1Chl.

a (␮mol photons m−2s−1)−1); I c : light compensation point (␮mol photons m−2s−1); I k : initial value of light-saturated photosynthesis (␮mol photons m−2s−1) In addition, the chlorophyll a: chlorophyll b ratios are given Data represent mean values ± SD of 3 replicates The significance of differences among the strains, as indicated by different letters (capital letters for ␣; small letters for I c ; cursive capital letters for I k ) was calculated by one-way ANOVA (p < 0.001) and Tukey’s post hoc test.

Klebsormidium sp BIOTA 14614.7 (African Strain A) 5.73 ± 1.39 A 14.65 ± 2.27 a 18.83 ± 2.69 A 2.43 ± 0.26 a Klebsormidium sp BIOTA 14613.5e (African Strain B) 10.93 ± 2.56 B 4.44 ± 0.78 b 19.99 ± 3.16 A 2.10 ± 0.31 a Klebsormidium sp.BIOTA 14614.18.24(African Strain C) 12.40 ± 1.96 B 5.86 ± 1.19 b 14.11 ± 2.71 B 2.07 ± 0.17 a Klebsormidium subtilissimum SAG 384-1 (Arctic Strain) 8.29 ± 2.63 B 10.55 ± 2.09 a 23.56 ± 3.34 A 1.86 ± 0.15 b

Fig 3. The effect of increasing photon fluence densities up to 1432 ␮mol photons

m −2 s −1 on the relative electron transport rate (rETR, ␮mol electrons m −2 s −1 ) in 3 of

the studied Klebsormidium strains (n = 4, mean value ± SD) BIOTA 14614.7 (African

Strain A), BIOTA 14613.5e (African Strain B), and SAG 384-1 (Arctic Strain) BIOTA

14614.18.24 (African Strain C) showed the same curve shape as BIOTA 14613.5e

(African Strain B) and hence was not shown All measurements were done at

22 ± 1 ◦ C.

and182.9␮molO2h−1mg−1 Chl.a.Incontrast,AfricanStrainA

didnot reachlight-saturationuptotheapplied500␮mol

pho-tonsm−2s−1,resultinginamaximumoxygenproductionofonly

79␮molO2h−1mg−1Chl.a(Fig.2).Noneoftheinvestigated

Kleb-sormidiumsamplesexhibitedanyindicationofphotoinhibition,at

leastuptothehighestphotonfluencerateof500␮molphotons

m−2s−1tested.Todoublecheckthatindeednophotoinhibitionis

visibleinthe4Klebsormidiumstrains,relativeelectrontransport

ratesasfunctionofincreasingphotonfluencerateupto1432␮mol

photonsm−2s−1weremeasured(Fig.3 andnophotoinhibitory

effectcouldbedetermined.WhileAfricanStrainAshoweda

rel-ativelylow␣value(photosyntheticefficiencyinthelightlimited range)of5.73␮molO2h−1mg−1Chl.a(␮molphotonsm−2s−1)−1, this valuewas significantlyhigher in theAfrican StrainsBand

Cand theArcticStrain(p<0.05)(Table3).The4Klebsormidium strainsexhibitedrathersimilarIcvalues(lightcompensationpoint) between4.4and 14.7␮molphotonsm−2s−1,and Ik values (ini-tiallightsaturationpoint)rangingfrom14.1to23.6␮molphotons

require-mentsforphotosynthesis(Fig.2).Inaddition,theChl.atoChl.b ratiosinallinvestigatedstrainsweresimilarbetween1.86and2.43

3.3 Temperaturerequirementsofphotosynthesisandrespiration Theeffectofincreasingtemperaturesongrossphotosynthetic oxygenproductionandrespiratoryoxygenconsumptioninthe4 Klebsormidiumstrainsclearlypointedtoisolate-specificdifferences (Fig.4).TheAfricanStrainsAandCshowedasimilarresponse pat-ternwithrisingphotosyntheticactivityfromlowvaluesat5–10◦C

uptomaximumvaluesbetween30and35◦C,followedbyasharp decline and complete inhibition between 35 and 45◦C (Fig.4) AlthoughAfricanStrainBexhibitedalsolowoxygenevolutionrates between5and10◦C,andoptimumphotosynthesisat30–35◦C,this straincouldmuchbettercopewith40◦Cwherestill50%ofthe maximumcouldberecorded,whileat45◦Cphotosynthesiswas notdetectable(Fig.4).Incontrast,theArcticStrainshowedalready

at5and10◦Crelativelyhighoxygenevolutionrates,andabroad optimumphotosyntheticactivitybetween15and35◦C,followed

byadrasticlossat40and45◦C(Fig.4)

Inallsamplesrespirationat5and10◦Cwasnotdetectableor verylow, while temperaturesbetween>10◦C and 30–35◦C led

toa continuous,andalmostlinearincreaseinrespiratory activ-ityuptothemaximumvaluesbetween−41.4and−84.9␮molO2

h−1mg−1Chl.a(p<0.001)(Fig.4).Furtherincreasesin

tempera-Fig 4. Photosynthetic oxygen development and respiratory oxygen consumption in ␮mol O 2 h−1mg−1Chl a measured at 200 ␮mol photons m−2s−1as function of increasing temperatures in the four studied strains of Klebsormidium (n = 3, mean value ± SD), BIOTA 14614.7 (African Strain A), BIOTA 14,613.5e (African Strain B), BIOTA 14,614.18.24 (African Strain C) and SAG 384-1 (Arctic Strain) Significances among the treatments were calculated by one-way ANOVA (p < 0.001) Different capital (net photosynthesis) and small letters (respiration) represent significant differences among the temperatures as revealed by Tukey’s post hoc test.

Fig 5. Photosynthesis and respiration in ␮mol O 2 h −1 mg −1 Chl a measured at

200 ␮mol photons m −2 s −1 at 45 ◦ C (see Fig 3 ) and after transfer back to 30 ◦ C in

the four studied strains of Klebsormidium (n = 3, mean value ± SD), BIOTA 14614.7

(African Strain A), BIOTA 14,613.5e (African Strain B), BIOTA 14614.18.24 (African

Strain C) and SAG 384-1 (Arctic Strain) Significances among the treatments were

calculated by one-way ANOVA (p < 0.001) Different capital (photosynthesis) and

small letters (respiration) represent significant differences among the temperatures

as revealed by Tukey’s post hoc test.

tureupto45◦CledtoamoderatedecreaseinrespirationofAfrican

StrainC, whilethe remainingKlebsormidiumstrainsshowedan

unaffectedrespiratoryrateatthishightemperature(Fig.4).The

photosynthesisandrespirationdataindicateconspicuously

differ-enttemperaturerequirementsforbothphysiologicalprocesses

After treating all samples with 45◦C, temperature was

decreasedto30◦Candtheshort-termrecoverypotentialof

pho-tosynthesisandrespirationevaluated(Fig.5).ThethreeAfrican

Strainsshowedverysimilarresponsepatterns,i.e.withdeclining

temperaturesthenegativephotosynthethicratespartially

recov-eredtozeroor slightlypositive values.Butalsotherespiratory

activityreturnedtolowrates.Incontrast,theArcticStrain

exhib-itedlesshigh,butstillstrongnegativephotosyntheticactivityand

arespirationvaluesimilartothatbetween20and25◦C(Fig.5)

The photosynthesis:respiration quotient (P:R) in all 4

Kleb-sormidium isolates decreased with increasing temperatures

(p<0.001)(Fig.6).Whileat5◦CthehighestP:Rvaluewasonly

observedinAfricanStrainA,allothersamplesshowedanincrease

from 5 to 10◦C (maximum value) followed by a continuous

decrease(Fig.6).TheP:Rratiosdroppedinallinvestigated

Kleb-sormidiumisolatestonegativevalues,i.e.inAfricanStrainsBandC

onlyat45◦C,inAfricanStrainAat40and45◦C,andintheArctic

Strainalreadyat30◦Candallhighertemperatures(Fig.6)

3.4 Dehydrationandrehydration

Thestandardizedmethodologicalapproachwiththesilicagel

filledpolystyrolboxesandPAMmeasurementsfromtheoutside

allowedcomparativeeffective quantumyield determinationsin

allKlebsormidiumsamplesduringthedehydrationand

rehydra-tionintervals,indicatingdifferentresponsepatterns(Figs.7,8)

WhileAfricanStrainBshowedacontinuousdecreaseofF/Fm

frombeginningonoveraperiodof350mindowntozerosignal,all

otherisolatesexhibitedanunchangedmaximumeffective

quan-tumyieldforsometimebeforeathresholdwasreachedafterwhich

thefluorescencesignalsstronglydroppedtovaluesbetween0and

20%(Fig.7).TheintervalofunaffectedF/Fmwas230–250min

intheAfricanStrainsAandCand,andonly100minintheArctic Strain.Aftertheseperiodstheeffectivequantumyielddecreased within100to130mintozeroinbothAfricanStrains,whileittook

>200mintoreach20%ofthecontrolintheArcticStrain,whichwas theminimumsignal(Fig.7)

After500mindehydrationintervalallKlebsormidiumsamples wererehydrated.WhilebothAfricanStrainsBandCtogetherwith theArcticStrainfullyrecoveredwithin1000–1500min,inAfrican Strain Aonly 20%of thecontrol F/Fm values wererecorded, evenaftera2000minrehydrationinterval(Fig.8).AfricanStrain

Cexhibitedthefastedrecoverykinetics

4 Discussion

4.1 Habitatandphylogeny

Inthepresentstudy,weexaminedforthefirsttimethe mor-phology and ecophysiologicaltraits ofthree differentstrainsof Klebsormidium isolated fromBSCcommunities of the succulent Karoo,SouthAfricawhichisknownassemi-aridhotdesert(Büdel

etal.,2009).Thisregionischaracterizedbyhightemperaturesand extremelylowprecipitationrangingfrom0to34.2mmpermonth resultinginanannualrainfallofonly56mm(Table1).The investi-gatedBIOTAstrainsbelongphylogeneticallytotheKlebsormidium supercladeG(Rindietal.,2011),whichis describedonlyas so-called‘Africanclade’withoutanyfurtherinformationontaxonomy

orphysiology.ForcomparisonweselectedKlebsormidium subtilissi-mumfromacold-deserthabitat(Alaska,USA,supercladeE),which

ischaracterizedbymuchlowertemperatures,butalsorelatively lowprecipitationrangingfrom2.3to26.7mmpermonthsumming

uptoanannualrain-/snowfallof114mm(Table1).Incontrastto thethreeAfricanstrains,K.subtilissimum(ArcticStrain)isawell describedspecies (Fig.1C)(Silvaetal.,1972).ThethreeAfrican StrainsA,BandCexhibitedsignificantlydifferentmorphologies particularlyreflectedinthecellwidthwhichrangedfrom4.1to

timeunderidenticalcontrolledconditionsandhenceanypotential environmentaleffectonthemorphologycanbeexcluded Conse-quently,weassumethatthethreeAfricanKlebsormidiumstrains representnewspecies,whichhavestilltobedescribed

4.2 Lightrequirementsofphotosynthesis ThePI-curveparameters␣,IcandIkonlyrevealedsmall differ-encesamongtheinvestigatedfourKlebsormidiumisolates(Fig.2,

thoseofIc andIkasverylow.Theinvestigatedsamplesthusall exhibitveryminorlightrequirementsforsaturated photosynthe-sis,whichhasbeenreportedbeforeforotherKlebsormidiumand closelyrelatedInterfilumspecies(Karstenetal.,2010,2014;Karsten

algae(indicated byhigh␣andlow Ic/Ik values) isusually cou-pledtomoreorlessstrongphotoinhibitionunderenhancedphoton fluence rates (Bischof et al., 1998 and references therein), and althoughouroxygenoptodemeasurementswererecordedonly

upto500␮molphotonsm−2s−1,theadditionallymeasuredrapid light curves(rETR) withaPAM2500up to1432␮mol photons

m−2s−1 didalsonotindicateanyphotoinhibition.The conspicu-ouslylowlight requirementissurprisingfortheAfricanstrains

asSouthAfricaisknownforhighinsolation.Averynice explana-tionforlowlightadaptationunderhighsolarradiationconditions

isprovidedbyGrayetal.(2007).TheseauthorsinvestigatedBSCs

ofNorthAmericandesertswheretheabundantgreenmicroalgae verticallyoccupiedmicroenvironmentswithinthesoilcrust,which contributestomassiveself-shadingandhenceprotectionfromthe

Fig 6.Photosynthesis:respiration (P:R) quotient as function of increasing temperatures in the four Klebsormidium isolates BIOTA 14,614.7 (African Strain A), BIOTA 14613.5e (African Strain B), BIOTA 14614.18.24 (African Strain C) and SAG 384-1 (Arctic Strain) (n = 3, mean value ± SD) Significances among the treatments were calculated by one-way ANOVA (p < 0.001) Different capital letters represent significant differences among the temperatures as revealed by Tukey’s post hoc test.

Fig 7. The effect of controlled desiccation on the effective quantum yield (F/Fm  ) of photosystem 2 as regularly measured with a PAM 2500 during the experiment (400–500 min) in the four Klebsormidium isolates BIOTA 14614.7 (African Strain A), BIOTA 14,613.5e (African Strain B), BIOTA 14614.18.24 (African Strain C) and SAG 384-1 (Arctic Strain) (n = 4, mean value±SD) Effective quantum yield values of control algae under 40 ␮mol photons m −2 s −1 PAR was determined as 0.54–0.59 and standardized

to 100% for better comparison All measurements were done at 22 ± 1 ◦ C.

damagingin-situlightfield.Theresultingdifferential

susceptibil-itytoenhancedphotonfluenceratesexpressedbyindividualtaxa

indicatesacomplexspatialarrangementofthealgalspeciesinsuch

amicrohabitat,whichseemstobestructuredinresponsetothe

verticalattenuationofirradiance(Grayetal.,2007).Under

nat-uralconditions, Klebsormidiumspeciesoftenformmulti-layered

structuresinterwoven withtheuppermillimetres of soil

parti-clesandothermicroorganisms,whichmostprobablycontribute

toahighdegreeofself-shadingandhencephotoprotectionof indi-vidualfilamentsinsidesuchanassemblage(Karstenetal.,2010)

IncontrasttothethreeAfricanisolatesfromBSCs,precise habi-tatinformation ontheArcticStrainismissing.Thisspecies was isolatedfromsnowinNorthernAlaska,whichischaracterizedas cold-desertregion.Butwhetheritoccurredascrust-like assem-blageorbiofilminorontopofsnowisnotreported.PortBarrow (Alaska) is located at 71◦N and hence the annual surface

inci-Fig 8.The effect of controlled rehydration on the effective quantum yield (F/Fm  ) of photosystem 2 in the desiccated algal samples (see Fig 6 ) as measured with a PAM 2500 during the experiment (1000–2000 min depending on the response) in the four Klebsormidium isolates BIOTA 14614.7 (African Strain A), BIOTA 14613.5e (African Strain B), BIOTA 14614.18.24 (African Strain C) and SAG 384-1 (Arctic Strain) (n = 4, mean value ± SD) Effective quantum yield values of control algae under 40 ␮mol photons m −2 s −1

PAR was determined as 0.54–0.59 and standardized to 100% for better comparison All measurements were done at 22 ± 1 ◦ C.

dentsolarradiation is about40%less compared totheequator

incombinationwithdiurnalweatheraswellasseasonalchanges

betweenpolardayandnight(Thomasetal.,2008).Consequently,

theArcticStrainlikelyreceivesmuchlowerinsolationcompared

totheAfricanstrains.ThefourinvestigatedKlebsormidiumisolates

exhibitedallahighphotophysiologicalplasticitywhichisin

agree-mentwithotherinvestigatedKlebsormidiumspecies(Karstenetal.,

traits but also correspondingto findings in streptophyte green

algaeobtainedfromhydro-terrestrialhabitats(Kaplanetal.,2013;

Streptophytaisconsideredascloselyrelatedtolandplants,asetof

potentialprotectivemechanismscanbeconsideredthathavebeen

developedbyalgaeandembryophytestocounteract

photoinhibi-tion.Thesemechanismslimittheextentofphotodamage(Raven,

2011),andinclude,forexample,partialavoidancebyrestricting

thenumberofphotonsincidentonthephotosyntheticapparatus

bychloroplastmovement.Otheravoidingprocessestypicallyaim

todissipateexcitationofphotosyntheticpigments,forexample,by

non-photochemicalquenching(e.g.xanthophyllcycle)or

photo-chemicalquenching(e.g.alternativeelectrontransportpathways)

E)genomeclearlyindicatesthepresenceofabasicsysteminvolved

inhigh-lightprotection,whichisconsideredasfundamental

mech-anismforalgaladaptationtoterrestrialhabitats(Horietal.,2014)

Thissystemincludescyclicelectronflowactivityatphotosystem

I,whichisactivatedunderradiationstressanddesiccation(Hori

etal.,2014).Thecyclicelectronflowisassumedtoincreasethe

protongradientacrossthethylakoid membrane,which induces

non-photochemicalquenchingandATPbiosynthesis,followedby

thedissipationofexcessradiationenergy(Horietal.,2014)

4.3 Temperaturerequirementsofphotosynthesis

Increasingtemperatureshadastrongeffectonrespiratory

oxy-genconsumption and photosyntheticoxygenproduction inthe

fourKlebsormidiumstrainsandrevealedisolate-specificdifferences (Fig.4).Whilethecold-desertArcticStrainshowedoptimal pho-tosynthesisalreadyat15◦C(followedbyabroadtolerancerange), thethreehot-desertAfricanStrainsexhibitedmaximum photosyn-theticratesbetween30and35◦C.At40◦ConlyAfricanStrainBhad positiveoxygenproductionrates,whileinallotherisolates pho-tosynthesiswascompletelyinhibited.Thesedataindicateforthe firsttime,thatthetemperatureconditionsofthenaturalhabitatare reflectedintheecophysiologicalresponsepatternsofthestudied Klebsormidiumstrains,pointingtospecificadaptations(genotypes)

asdiscussedforacidophilicpopulationsofKlebsormidium(ˇSkaloud

etal.,2014).Anotheraspectistheconspicuouslydifferent tem-perature requirement of photosynthesis and respiration While respiratoryrateswerenotdetectableorverylowat5and10◦C

inallstudiedstrains,highestratesoccurredbetween30and45◦C, i.e.respirationunderhighertemperaturesismoreefficiently func-tioningthanphotosynthesis,whiletheoppositeistrueforlower temperatures,wherephotosynthesistypicallyexhibitsenhanced activity rates compared to respiration This is also reflected in theP:Rratios(Fig.5 whichconfirmapositivenetcarbongain andhencebiomassformationoverabroadrangebetween tem-perate(5◦C)andhotconditions(35–40◦C)intheAfricanStrains, whilethecold-desertArcticStrainexhibitedpositiveP:Rvalues onlyundertemperateconditionsbetween5and25◦C.These dif-ferent tolerance widthsreflect thenatural hot and cold desert habitats,respectively.Thedisproportionateeffectoftemperature

onphotosynthesisand respirationinterrestrialgreen algaehas alsobeen documentedin theclosely related K.crenulatum and

K.dissectum,bothfromalpineBSCs(Karstenetal.,2010;Karsten

habitats(Karstenetal.,2014),aswellasinPrasiolacrispafrom Antarctica(Davey,1989).Prasiolacrispaisamacroalgalmember

oftheTrebouxiophyceae(Chlorophyta),andhencefroman evolu-tionaryviewfardistantfromKlebsormidium.Nevertheless,similar physiologicalresponsepatternswithrisingtemperaturesstrongly supporttheassumptionthatterrestrialgreenalgaeingeneral,and

photosynthesisisprimarilycontrolledbylight-relatedprocesses

suchas,forexample,lightabsorptionandenergytransfer,

respi-rationistypicallyinfluencedbytemperature(AtkinandTjoelker,

2003).Respirationofplantsandalgaeisacomplexprocess

con-sistingofvarious enzymes,which exhibitdifferenttemperature

optimaandthatarelocalizedindifferentcellularcompartments

Themostsensitiverespiratoryenzymewouldalwaysactasa

bot-tleneckaffectingthewholeprocess(AtkinandTjoelker,2003).The

latterauthorsdiscussedtheunderlyingacclimationmechanisms

whichinclude, for example,temperature-dependentchanges in

substrateavailability, themaintenance of homeostaticlevels of

ATPsynthesisacrosstemperaturegradientsand/orreduction in

theproductionofreactiveoxygenspecieswhichareharmfulfor

allmetabolicfunctions

4.4 Dehydrationandrehydration

Thedesiccationexperimentswereundertakenbyusingafully

standardizedapproach(Karstenetal.,2014)toguarantee direct

comparisonbetweenthe4strainsstudied.While AfricanStrain

Bexhibitedacontinuousdecreaseintheeffectivequantumyield

fromthebeginningonoveraperiodof350mindowntozerosignal,

theother3samplesshowedanunchangedmaximumF/Fmfor

sometimebeforeathresholdwasreachedafterwhichthe

fluores-cencesignalsstronglydecreased(Fig.7).Theintervalofunaffected

F/FmwasmuchlongerinAfricanStrainsAandC,comparedto

theArcticStrain.ThesedifferencesintheF/Fmkineticsduring

desiccationcanbeexplainedbytheisolate-specificmorphologies

AfricanStrainsAandCarecharacterizedbylonguniserate

fila-mentsandmuchthicker,mechanicallymorestablecellscompared

tothesinglesmallcellsorshortcellfilamentsofAfricanStrainB

andtheArcticStrain,respectively(Fig.1).Similarresponsepatterns

hadbeenrecentlyreportedforvariousisolatesofthecloselyrelated

Interfilum(Karstenetal.,2014),whichexhibitedalsostriking

dif-ferencesinmorphology.Single-cellstrainsofInterfilumweremuch

moresensitivetodesiccationthanalgalcellsinhabitingan

aggre-gate,colonyorbiofilm,allrepresentingmorphologicalstructures

whichhamperoratleastretardwaterloss

Recovery kinetics of F/Fm after rehydration of the dried

Klebsormidiumsamplesalsoexhibitedconspicuousstrain-specific

differences.ExceptAfricanStrainA,allotherisolatesfully

recov-ereduponrehydrationwithfluidwater.AfricanStrainAshowed

only20%ofthecontroleffectivequantumyield,whichpointsto

somedamageinthephotosyntheticapparatus.Therecovery

kinet-icsoftheAfricanStrainsBandCandtheArcticStrainweresimilarto

thoseofInterfilum(Karstenetal.,2014)andanaeroterrestrialgreen

algalbiofilmoccurringontopofabuildingfac¸ade(Häubneretal.,

2006).ThisbiofilmalsoshowedaquickrecoveryofF/Fmafter

artificialmoistening(Häubneretal.,2006).Suchhighdehydration

toleranceinconjunctionwithhighrecoveryratesseemstobea

typicalfeatureofmanyterrestrialgreenalgaefromalpine,dune

anddrylandBSCs(DeWinderetal.,1990;Grayetal.,2007;Karsten

2013).The underlyingmechanisms havebeenrecentlytouched

forthefirsttimeinKlebsormidiumcrenulatumusinga

transcrip-tomicapproach(Holzingeretal.,2014).Theseauthorsreported

alsoastronginhibitionofF/Fmduringdesiccation,but,for

exam-ple,inparalleltheup-regulationoftranscriptsforphotosynthesis,

energyproduction,reactiveoxygenspeciesmetabolism,aswellas

ofplantproteinsinvolvedinearlyresponsetodesiccation(ERD)or

enzymesforoligosaccharidebiosynthesis.Themostimportant

con-clusionwasthefirstexperimentalproofthatstreptophytegreen

algaeexhibitsimilarmoleculareventsduringdesiccationstress

thanland plantspointing toancestralmechanismsfor the

suc-cessfulcolonizationofterrestrialhabitats(Holzingeretal.,2014)

Futurestudiesshouldtakethestrainandcladespecificdifferences shownhereintoaccount,andourbiologicalunderstandingofthe underlyingmechanismswilllargelybenefitwhennotonly investi-gatingoneselectedmodelorganism

Acknowledgements

The present study was undertaken during thefirst author’s sabbatical attheUniversity of Innsbruck,and itwas supported

by a grant from the Deutsche Forschungsgemeinschaft (DFG) (KA899/16-1/2/3/4)(U.K.),aswellasaFWFgrantP24242-B16and FWFgrant I 1951-B16(A.H.) Our sincere thanks are extended

toProf.BurkhardBüdel(UniversityofKaiserslautern)for provid-ingtheBIOTAstrains,aswellastoDr.TatianaMikhailyuk(M.H Kholodny Instituteof Botany, National Academy of Sciences of Ukraine)forinformationconcerningthephylogeneticpositionsof theisolatesused

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