This article isThis article by protected copyright. All rights reserved. 10.1111/jpy.12911doi: le copyediting, paginationbeen throughthe andproofreadingtypesetting, process,may which This article acceptedhas been for publication andundergone fullpeer review but hasnot performinga fluctuation analysis design using th and an we useda process, whichadaptation akey played role in the evolution of group this different evolution experimental importancetheir using Experimental evolutionstudies asmodelcyanobacteria organisms scarce are ABSTRACT Responsibility:Editorial Editor) B.(Associate Palenik Condensed runningtitle: C 2 comparative studybetween Adaptation dynamics andevolutionary rescueunderselection sulfide in cyanobacteria: a type Article :Regular Article DR. ELENA MARTIN
Author for correspondence: e ad to differencesad to between the thisversion and Version Record. of Please citethisarticleas Accepted Article Departamento deBotánicayFisiología Vegetal Oscillatoria Oscillatoria Elena Martín Microcystis
in Málaga, CampusMálaga, de Teatinos s/n,E
the evolution of the organisms.photosynthetic first Forthe time three sp. strain, sp. strain, isolated from spa sulfide). asulfureous (~0.2mM Firstly, total Antonio
- aeruginosa
Clemente - CLEMENTE (Orcid ID CLEMENTE ID (Orcid : 0000 yanobacteria andsulfide yanobacteria M
Flores icrocystis aeruginosa - mail;
ary 2 ,
Ignacio J. J. Ignacio Melero sulfide
- approaches approaches applied have been ontheto shedlight sulfide Moya [email protected] - sensitive strain, unablesensitive togrow strain, above~0.1mM,
and J. María García e spa waterse spa asselective agent, , deFacultad UniversidadCiencias, de
and
- Jiménez - 29071 Málaga, Spain
- ; phone: +34 952134220 +34 ; phone: 0002 O
scillatoria , - 0365 Elena Bañares - Sánchez - 6377)
sp . (c
. With purpose, this
yanobacteria)
- España
we proved despite
1
This article isThis article by protected copyright. All rights reserved. m distributionPoisson ER, e Abbreviations Oscillatoria Key sulfidesevere stress. fast environmental rates,couldincrease deterioration where strains present inhabit. The highdiversity genetic ofa sulfide by possible rare spontaneous adaptationlimits mutations andthe sulfide onthe depend level diversity.genetic sulfideIn conclusion, adaptation sensitive of cyanobacteria determined the survival of increasi Thirdly, using anevolutionary rescue approach, we observedboth that speedofto exposure mM0.4 and sulfide, wasdependentonthetaxa Secondly, applying a protocol,ratchet wetested if thelimitto of sulfide ofadaptation the two that
Accepted rateutation Article M. aeruginosa index index volutionary rescuevolutionary ng sulfide concentrations andpopulations’(deterioration rate) geneticvariation words
sp.;
:
A
: ratchet protocol; 750
evolutionary rescue;
was to sulfideable adapt tothis level byrarespontaneousmutations. , a ;
Oscillatoria q bsorbance at , f ; requency of requency LHW the FM, f ir ir initial sulfide tolerance, finding that M. aeruginosa M. aeruginosa resh massresh sp. ithighest to ~2mM twice tolerance sulfide, level. sulfide λ
= 750nm f luctuation analysis; luctuation
; at lethal at sulfide levels, with a higherdependence on LHW,
- ; resistant allele resistant m La Hediondawaters , a cclimated
its possibilityofsurvivaleven toa
Microcystis aeruginosa ; growth rate s , c M. aeruginosa oefficient ofselection - sensitive strain,even at ; P 0 , f ; irst termirst ofthe CD, c
strains is
adapted to ell density ell ;
; μ, ;
This article isThis article by protected copyright. All rights reserved. selectivepressure increaseswhich the inversely is correlated capacitywith the to proliferate foun (Gonzalez et al.2013) known as‘evolutionary rescue’ (ER), previouseco coulddepend onthe populations to exposed l al.Blount et 2008,Flores contingent, closely toasimilarexposed selection pressure experimental cells culture were foundedfrom having inocula asimilarhistory, and theywere performedwith strai Jiménez etal.2019 ondepending selective used the agent groupsecological ofphotosynthetic capacitadaptation havenotbeenreportedagent until recently. In particular, it was found themaximum that experimental assays evaluating maximumthe cap mutationof organisms andselection allowed given a of tosurpass pressure, level selection 2013, Melero al et survivalcases wasduetotheselection variants of originatedmutation newgenetic by of microorganismsadaptation photosynthetic in toextreme environmentsmost showedthat conferringresistancemutations when thelimit ofacclimationexceeded is A INTRODUCTION
Accepted Article cclimation . d that greater genetic variation greater geneticvariation d that
2007, 2008, Ló
is the mechanism levelsis the toleranceatlow of allowing selectivebut pressure, - Jiménez etal.2019). y forthe sameagent selective
) dependent . It accountmust betaken into that allthe citedwere studiespreviously ns homoghaving a pez . In experimental fact,in populations of and bacteria ithas yeasts, been - ethal ethal conditions natural selection, maythrough aprocess adapt Rodas etal - Moya etal.
on the previous historyon theprevious
or geneticadaptation Although these studiesrevealed that the coupled interplay favors
. microorganisms, this capacity differed andthat
2007, 2008
2008 ( ) Huertas etHuertas al. eneous eco eneous . However, processes it thatevolutionary is known are ,
the possibilitythe ofER eventsand rate that the at survival by is only possible the selection of new , 2012)
varied among different taxonomic and a acity ofanorganismacity a to selective toadapt , . Thus, the possibility thatnatural b - evolutionary history ,
2009
2010, 2011, 2014 Roucoetal.
(Hughes 1999). (Gould 1989, Travisano etal.1995,
, 2011, Fernández
Previous studies
- ( evolutionary history i.e., all i.e., all the - Arjona al. et , Melero
(Costas (Costas on the on the
- This article isThis article by protected copyright. All rights reserved. constrained by phylogeny may orlost relatively rapidly begained response in variation to in However, Clostridium oxidatio Walter etal. 2017 electronas an PSI donorto Bebout and Miller 2004) resistancethe ofPSII overcome toxic effect the of w present are extremely sensitivevery levels to low (<0.1mM) ofthision Klatt 1975 tolerate a varietyof transportelectron and Poulton Canfield 2011) evolution ofthecyanobacteria with eco modelmake organisms themideal evolution, inexperimental especially if aselective agent 2003 Lenski mostofthem performedin action, have been bacteria viruses, andyeasts and (Elena with that,intherecentfieldnoted ofresearchbasedon usemicroorganisms the evolution tostudy lethalconditions under
Accepted thosefrom isolated hereas sulf Article a et 2015) al. , b Sulfi , Padan1979, n to sulf - evolutionary i sulfide
dic conditions playeddic role akey t in , that specifically exports that HS
Garland andRoseGarland However, 2009). th ur . It . It is known strainsisolated that from where environments - ). tolerance cyanobacteriaseemsnottobe in a taxon (Baga (Stal 1995)
Also, it is known Also, isknown it some that cyanobacteria candetoxify sulfide ( de Wit 1987,García andvanGermeden Castenholz 1977,
mplications mplications such sulfideis chosen, as incyanobacteria. (Bell andGonzalez 2011, 2013) Lindseyet al. rinao 1992, Stal 1995,2012)rinao 1992,Stal
or enable PSII or enable (Miller and Bebout
environments throughout evolutionary their history (Cohen etal.1975 , because sulfide , and a channel has, anda channel beendescribed inthe eubacterium (Meyer Kump and 2008, Scott al. et ureous habitats exhibitonemore habitats or ureous differen . maintain by These adaptations oxygenicphotosynthesis sulfide - − independent, anoxygenic photosynthesis withindependent, anoxygenicphotosynthesis Cohen et al.1986
from thecytosol from
inhibits oxygenic photosynthetic and respiratory
a,b 2004) he history and, oflife inparticular, in the , 1986,Hamilton etal.2016,Leister 2017, e unique e unique characteristics ofcyanobacteria . . Cyanobacteria developed. Cyanobacteria theability to
, (Czyzewski Dodds andCastenholz 1990, - Pichel and 1990, Castenholz sulfide - dependent ratheri trait, .
Moreover, should it be and Wang 2012) (Cohen al.1986) et
Poulton et et Poulton al. 2010, levels and levels and it is not sulfide t adaptations to sulfide
(Cohen al. et
by
is not sulfide .
t
This article isThis article by protected copyright. All rights reserved. Accepted experiment, inwhich the mechanisms onewasfirst afluctuation analysis with aeruginosa diversity) and rateofincrease the Additionally, oforiginal westudied how thedispersal proxy populations (a for genetic and waters, an to increased theallowing tooriginally survival lethal ofthe history evolutionary 1975a,Castenholzal. 197 sulfide Article beablecould adapt(by to muta the with the increased thelimitwhat is agiventaxon canachieve ofadaptation that under cyanobacteria Arjona et al. 2013 quantum photosyntheticefficiency and yield comparison in to wild growth rateswild than (Fernandez fromadaptation awild proliferates that in sulfide - Therefore, theaimTherefore, ofthisworkwastodet Recently, a tolerant cyanobacterial tolerant cyanobacterial taxa sulfide sulfide
levels presentatLaHedion - . purposes, weperformedFor these of types three Arjona et Arjona et al. 2013) sulfide ( i.e. Oscillatoria
levels observed where is the
, levels? levels? could hypothesizedIt be potentialadaptationwill that the correlate
, acclimation genetic vs. adaptation
sulf sulfide Bañares
levels of a sulfide - - ureous watersmM containing 0.24 type ina cells type, 7) - resistant mutant ofthecyanobacterium
sulfide - sp. . Moreover, italso is toknowrelevant how previous eco the España etal.2016)
sulfide levels increasedlevels ateachratchet wasapplied cycle, in orderto
Microcystis . This strain, isolatedfrom thesulf - sensitive cyanobacteria couldmodulate ERdynamics the tion
would - sulfide sensitive strai sulfide sulfide da spa. Ontheother hand,da spa. a modified ratchet - selection to process) M. aeruginosa, sulfide , such as some of , suchas strains
aeruginosa
- levels modulate thein ERdynamics olate For instance,was found. - resistant strainof free medium, asdecreasedwell maximum as . This
levels. ermine limits the potentialthe of adaptation n isolated fromn isolated non ) , allowing tolerance the species ofthis
finding r
strain, fromisolated non
which let
sulfide ureous La Hedionda spa. ureous spa. LaHedionda
levels oflevels evolutionary experiments. The M. aeruginosa a ise , was obtained bygenetic, wasobtained
us t Microcystis aeruginosa s - Oscillatoria Oscillatoria
type cells the the o disentangle the o disentanglethe - sulfide sulf following ureous waters ureous M. aeruginosa
lower showed lower (Fernandez - sp sulf M.
. question
( than ureous ureous Cohen et - -
:
to This article isThis article by protected copyright. All rights reserved. Accepted ± 36µM( in LHW from ranged 97 tightly sealed without air inthedark 4 bubbles, at °Cbefore use. Total fluctuation analysis storedexperiment. were in plastic bottles Samples were(1.5 L) that collection conducted in triplicate, valuewith a atthetime(CV<3%) of109 µMmat of sample CO, basedmethylene USA)wasused, onthe blue method. concentration ofproportion 65% around35%and respectively.measure To total (LHW)waters tothe according pK and where area, microbial mats coverthewalls. Samples (microorganisms andwater)the usedin experiments from werecollected interior the Article theone external,itself, whichis river walled andoneinternal, for androofed bathing. is La Hediondaspa locatedonthe Manilva River bank(Málaga, and Spain) it and Sample collection MATERIAL AND METHODS theallowing of survival this c role ofthethe diversity andthedeterioration genetic onthenaturalselectionprocess rate, of increase performed ER experiment an b themaximumdetect that Finally, beachieved bybothcyanobacteria. could adaptation we Considering . n
Samples collected ofLHWwere also weekly duringJuneandJuly2016 forthe sulfide = 6). = 6). in situ
levels, as well as levels, aswell to different mechanisms, dispersal understand to inorder the annualthe ±0.06,mean value(7.23 pH , a DR900 Portable Multiparameter Colorimeter (HatchCo., Loveland, sulfide - 207 µMalong sixweekswith the ofcollection a mean of147 value
yanobacterium lethalsulf under concentration determinationconcentration at LaHediondaspa y submitting populations of populations y submitting
a1
of H
2 S (close (close to 7.0),H S
M. aeruginosa Sulfide n idic conditions.idic = 20) found= 20) inLa Hedionda 2 S andHS
sulfide determinations were sulf
(H to different ratesof ide -
species are at a areat species 2
S andHS
has two sectors, concentrations concentrations .
- ) This article isThis article by protected copyright. All rights reserved. Accepted LHW. below) concentrations 21 ranging concentration of200 µM BG11 medium of fluorescent lamps GRO (Sylvania of50density μmol mediumfresh growth axenically maintainedmid in mediumculture in250mL (or mL with cultureflasks, 100 medium). ofgrowth were Cultures mL ventilated cell Articlewas namedO1LH. Chemie,Aldrich Germany), diluted 50%(BG11at Taufkirchen, The mat andgentlyhomogenized samplewasdiluted 900 in mL o dilution process Valmayorthe reservoir Spain). (Madrid, Complutense University ( wasspa. TheMa1Vc bytheVeterinary strain provided School Culture Collection,Algal Microcystis Experiments performed were with the wild Experimental organisms and culture conditions - 50% buffered
Both were maintained as close
aeruginosa M. aeruginosa Microcystis . For this purpose,samples matsmicrobial collected ofwere in June of 2016. -
culture (Greiner;Bio flasks ·
m with 5 mMwith 5 HEPES (pH7.2) . Cultures wereCultures incub .
− and an
2 aeruginosa
, ·
Madrid, -
from aNa s was BG11 24 mM). way, Inthis − 1 - over theover waveband 400 log exponential growth inoculum byweeklytransferring to Oscillatoria
as possible as possible to - Spain) LUX, F36W/GRO,Spain). FeiloTheculture Sylvania,
and 2 S aqueous NaOH master solutionS aqueousNaOH stock (pH13, - 50%. Theculturemedium of Oscillatoria
Oscillatoria and was from isolated non
sp. strain isolated frommats fromLaHedionda ated at20 - type type strain Ma1Vc ofthecyanobacterium -
One, Longwood,NJ, with20mL USA) of the highest . sulfide
,
and
°C underacontinuous photonflux sp –
sp. was following isolated successive a 700 nm,providedby cool sulfide .
concentrations concentrations cultures werecultures axenically grown in50
sulfide
was addedeverydayatafinal - 50%).
f BG11 mediumf BG11 (Sigma concentrations observedin concentrations Oscillatoria Oscillatoria - sulf
in theculture The isolated strain ureous wate ureous - sp. white
was
rs from (see - This article isThis article by protected copyright. All rights reserved. mL of incubation bylinearregression the density fitbetween cell (CD;units in1×10 concentration used. significantnot change e initial concentration was0. andreactions H concentration tested (pH ~13,21 solution above. sulfide concentrations 0.05, (0, were0.1, 0.15,0.2and0.35mM). prepared for Fivereplicates each containing 4mL ofBG11 cells experiment, respectively. Samples of where and Kimura1970) acclimated ( rate growth theIn order totest toxic effect of test:Toxicity ef Acceptedffects persist can Article − 1 · ) and absorbance absorbance atλ =750nm) and (A mL N
Sulfide The numberof cells concentration and weremaintainedfor5 the d at same t
− and 1 were placed were placed in 15mL (Falcon™,BD Biosciences tubes N
was addedmedium tothe dayfrom every aNa 0 fec 2
S volatility are the number the are cell atthe end(after : t of for hours and 2016 Straub (Olson
.
- The sulfide addedThe sulfide th to 24 mM) ly mediumthe pH sulfide m - ) were measured in mid in measured ) were 50% buffered20 with mM (pH7.2),and HEPES increasing 35 mM35 , being thehalf
m on growth rate.
to maintain to Microcystis
= log sulfide . However, e M. aeruginosa
( N 750
after 5d after t on cells of / N sulfide
): - aeruginosa life 0
e cultures )/
it it thatis known t (t
1/2
concentrations close as tothe as possible - of incubation log exponentially growing cultures ) oftotal
Microcystis )
with an initialcelldensitywith an of2.5×10 . t
On the hand, other
= 5d) andattheof start disappeared along dayduetoredox the was estimated the andat start end the
sulfide
2 sulfide culture conditionsculture asindicated S
due to the the due to buffer
- aeruginosa aqueous NaOH master stock aqueous NaOHstock master
,
around around 1 San
downstream biological Jose, CA Jose, Na
, changes in, changes 2
S h
when addit 6 (Eq. 1) , USA) , USA)
cells ions did the the (Crow
sulfide ·
5
This article isThis article by protected copyright. All rights reserved. sulfidedecrease last concentration wasaround 1. (pH ~13,210 solution test. to the mg0.05 FM 0.7, 0.9,1.2and1.5mM). Theinitial biomass tube (four replicates ineach for was treatment) HEPES andwithincreasing (pH7.2) in prepared ( min8 instead of mg) of sample amount the Chl andthe of chlorophyll estimator ofthenumber Instead, ofcells. thebiomass inthe cultures wasestimated present by assingle cellsandproducegrew orcolonies,did not coenobia asoccurs in nature. mustIt highlighted be that, under conditions,culture experimental the Accepted1994) Article days ofincubation at thehighest Sulfide Chl and theweighed. pellet TheChl , using N,N Then, theFM at start (FM filamentousDue tothe of nature N a
50 mL50 (Falcon™) containing tubes 20mL ofBG11
0 a present present in
was addedtothemedium day every from aNa
·
and
(Chl mL toxicity N − - 1 a t dimethylformamide asolvent. as Samples of . This value was chosen because its was becauseits Chl equivalent . Thisvalue chosen . For the estimation . Forthe oftheFM,cultures were cent ) content. Arelationship was established thefresh between mass (FM, - CD =1.1×10 Chl
M. aeruginosa 240 mM)240
due to the decrement the due to hydrogensulfideform onthe a
6 h) = 0.19F . However , as indicated before, asindicated 7
M +1( × A sulfide concentrations sulfide 0
s samples the at initial cell used density inthe toxicity ) and after ulfide a
Oscillatoria 750 concentration wasdetermined accordingto Wellburn a ,
(µg): ( a slight on increase pHcou R
R concentrations concentrations (0, 0.05, 0.1,0.15,0.2,0.35,0.5, 2 2
= 0.982, = 0.980,
t
= 5d (FM
(t sp. absorbance notbeused could 1/2 n n
for
= 5) = 18)
2 tested S
t total ) values were in Eq.1, used the -
aqueous aqueous stock NaOHmaster
- 50% buffered20mM with Oscillatoria
sulfide which couldpartially
rifuged 8000 at rpmfor M. aeruginosa ld be detected inthe detected ld be a
content at 1. at
(Howsley and (Howsley and
sp. were sp. were 5 mM
was similar (Eq. 3) (Eq. 2)
ini
strain as an as an tial
This article isThis article by protected copyright. All rights reserved. LHWw for 3 Set 2consistedof 3×10 40aliquots of weeks, thereby insuring that onemutant could cell enough generate progenyto bedetected. of to added LHWwas the in cells the until Nümbretcht, weregrown Germany).Cells in2mLBG11 inmLprepared centrifug13 and reagent small make thatno enoughto likely pre it of90 1consisted 1). Set independent inoculatedwithcultures Jiménez etal.2019) 2004, 2001, 2007, (Fig.possible results 1),extensively detailed havebeen our in previousstudies insteadcultures ofsolid theinterpretationmedium. oftheexperimentand running The the ( aeruginosa mut aeruginosa Fluctuation ofLHWanalysis 8min,8000 rpmfor pelletwas the frozenforChl and 2016).and Straub 1979)Pearson Accepted Article1943) ations) andations) N
Flores
t was performed.The modificationallowed out carrying experiment the withliquid
In order In order todistinguish betweengenetic = 1×10 .
modifiedstrain, a fluctuationanalysis byLuria initiallydescribed andDelbrück
- López Moya et Moya et al. eeks. eeks. .
6 acclimation Nevertheless, the toxic effect ofthe
cells. Then, cultures were centrifuged, medium the andmL wasdecanted, 2 After 5 d of culture in the presenceAfter the 5dofculturein of -
Rodas al. et . In short, two differentIn short,two setsof experime
2005,
to the selectiveto the agent (LHW) Ma1Vc ofthe - sensitivity sensitivity to LHW
Marvá etal. 2001, 2007,2008a,b,2009,2011,
pellet (selective conditions). (selective pellet Cultures for weregrown 6 6
cells from parental the population, in2mLcultured of e - tubes, sealedwithtubes, a cap (Sar existingmutants present). were were Cultures
2010,
adaptation - resistance transformation in
Fernández - HS form has also been also HS has described form (Olson a
determination. sulfide
-
(i 50% (non 50% .e. ntal cultures were prepared cultures were ntal (Fig - Arjona et Arjona et al. , N
spontaneous preselective , samples centrifuged were at 0
= 1×10
García - selective conditions)
stedt AGandCo, Microcystis 3 -
Villada etal.Villada cells (anumbercells 2013, Microcystis
(Costas al. et
Melero
2002, - .
This article isThis article by protected copyright. All rights reserved. Accepted cells LHWshowing nomutant after andμ exposure wasc termfirst Poisson proportiondistribution) was ofthe the of computedas set1cultures appearance whengenetic of cells resistant adaptation isdetected. The parameter acclimation thesulfide place. exposureto during took neither that onmutations selection spontaneous that occurprior tosulfide norexposure anotherresultObviously, ineach (0resistant cells means cells resistant that byspontaneous mutations arose prior exposure to sulfide. By sulfide). thecontrast, if CV 1issignificantly fromset higher than the CV 2,it inset confirmsand 2,this resistant cells by that appeared acclimation (i.e. consequently, theCVvalueshould inter the random, pre number cells ofresistant must tube per relative be Article thesameto have ofdeveloping resistance. chance theCV Therefore, (SD×100/mean) ofthe variance in the perculture number cells would of (Fig. below 1)because cell every is likely different phenomena toresistance Ifresistant cells sulfide. of arose fluctuation analysis, each ofthem interpreted astheindependent consequence of being two estercellulose filter(Merck Millipore Ltd.,Merck KGaA, Dar Hedionda spa week, each asindicated before,and before filtered usewitha0.22µmmixed durationofthe experiment.fresh the purpose, LHW Forthis samplesfrom collected were La wasreplacedeverytubes tomaintain 3dinorder toxicity asits much aspossib attheculture initial low micromolar In addition, In addition, fluctuationthe the analysis allows estimation ofthemut Two different results can set1experiment befoundinthe whenconductinga to dailyrate of According the - culture (tubeculture - selective mutations occurring before sulfide,selective mutationsoccurring exposureto the highvariation in - to - tube) numbertube) ofresistant cellsshould found 1)and, be (Fig.
be relatively asimilarhigh. If isfound 1 CVin sets value sulfide sulfide
concentration in LHW), LHW the inculture
loss (whichloss one disappeared within dayof ly low. By cellsly low. contrast,if appeared by
culture) couldculture) befound, also indicating
alculated as:alculated mstadt, Germany).
by acclimation, the ,
after theexposure to
ation rate ation of rate (μ) le le throughout P
0
(i.e., the (i.e., the This article isThis article by protected copyright. All rights reserved. Acceptedindependent ofbothisolates LHW measuredcells innon where against thisresistantallele, calculated asfollows: where accordance with Kimura will dueto cells their lower fitness in the ofLHW. numberabsence ofsuchmutants Theaverage Article newrecurrent, mutants generation arise in each bewhich will outcompeted wild bythe type If themutation from Mutation byabsorbance.density presencea hematocytometer, becausethe ofdead interfered cells in estimationthe ofcell (i.e. where , be determined between balance bythe μand rateofselectivethe eliminations ( before additionLHW),the (see was Cellcounting Fig.1). respectively performed using N m q
0 r is thefrequencyof LHW the
–
and and selection equilibrium m N s t
are the the are number ofcells theand at the start at endofthepropagation period are the the are acclimated rates growth of LHW the
a normal wild a normal - selective culture medium (i.e.
and
Maruyama (1966): μ s q
= 1 = = .
- log - / ( - – resistant andtheresistant LHW type LHW
( - e m resistant and allele, resistant P
+ r 0 / / ( s m ) N
s )
t
- -
N sensitive allele toaLHW sensitive
0
)
,
BG11
- s sensitive strainswere usedsensitive to
- is the is the coefficientofselection
50%), respectively. 50%), Four - resistant andtheLHW
- resistant alleleresistant is
(Eq. 4) (Eq. 6) (Eq. 5) s - ), in ), in sensitive sensitive
This article isThis article by protected copyright. All rights reserved. without populations sulfide concentrations ratchet foreach cycle was different for straindueto each their difference in con independenteach populationhasadifferent random to chance experience amutation next the cycle atdifferent times theyreached once density. desiredcell the This means that that highlighted selection levels (i. werecultures inocula occurrenceofmutations thatthe new conferresistance. With objective, this experimental isto design balance a strong and selection pressure size apopulation large enoughto ensure al.2014,Melero et which each adapt,strain wasableto amodifiedprotocolratchet concentrationsincreasing of To studythe adaptation of experimentRatchet with aeruginosa (1 fromisolated onerandomly ofthe culture selected 1afterthe set running fluctuatio strain theoriginal foundedMa1Vc. hand, from the Ontheother LHW compute th
Accepted Article– 1.5 ×10 ferring ferring
Four replicates were prepared were Four replicates foreachoneof the tolerance, asdetailed be The ratchet The ratchet experiment with sulfide 6 sulfide e . ,
resistant cellsresistant culture), per to the wasused prepare LHW m for eachthreeinitial r
(Fig. 2) (Fig. and each each isan replicate population,independent andtheycouldberatcheted to
e. resistance. The choice oftheinitialdose and increase the in - Jiménez , m
three ted ted elevatedwith an andwereexposedto celldensity three s
values, as as indicated inEq.values, of 1.Isolates theLHW . doseswerelow,The initial thisstr because M sulfide Microcystis icrocystis et alet sulfide l ow. . 2019)
sulfide concentrations) ineachratchetcycle (Fig. 2).
and to estimatetheand to maximum Microcys
aeruginosa
aeruginosa aeruginosa
was carriedin out.Arequisite this experimental
doses (10,doses 30and90µM tis
aeruginosa and and Oscillatoria
Oscillatoria Oscillatoria sulfide
started started with four
(Huertas 2010,Rouco etal. doses It tested. must be
sp. sulfide sulfide ain wasain from isolated non sp. to
- - resistant resistant cultures of resistant inoculum,resistant -
) andfour controls sensitive cells were sensitive concentration to the exposureto the independent independent sulfide
different n analysis
M. - This article isThis article by protected copyright. All rights reserved. Accepted(pH 7.2)and corr the were inoculated initially with5mg Microcystis Oscillatoria 200 µM populations for eachthree initial dosesof ratchet tubesand treated inoculatedwith were thesamedensityused beginning cell at ofthe the 405, andfrom405to607µM ratcheteach werecycle asfol concentration beginning the at of the a1.5experiment andlater samethe concentration(Fig. 2) sulfide showed cultures cellgrowth if treated alower thancon Articlesimilar tothatobserved incontrols,theywere transferred to the ratchet next cycle to were compared those ones.in control treated If showed cultures a theexperiment.during cell Afterculture, 7dof concentrations in (pH~13, 21 added every day tothe culture medium fromNa a maintain to order 50 μmol addition contained BG11 inmLincubated 15 tubes sulfu reous reous waters.
In the experimentIn the with experiment (i.e., 6×10 . Culture
sulfide ·
m -
aeruginosa 24 mM)24 medium andculture was changedweekly tomaintain a constant pH − sp. and the sp. andthe increase ineachratchet cycle waslower those usedin than the 2
·
. Control s - − 50% buffered mM with 20 corrand the HEPES,pH7.2,
tubes tubes were closed maintained capand with a continuous irradiance under of sulfide 1 , at 20 , at Each (replicate) population was foundedwith6×10 esponding
ratchet cycles (100µMeachtime).
°C. Culturesfor weregrown least each at 7d sulfide concentrations close as to as possible (Falcon™) lows: fromlows: 10to30,fromto 270,from270 30to90,from 90 5 sulfide Oscillatoria cells).
sulfide concentrat
FM in 20mL in FM BG11 . startingAt the point ofeachratchet bothcycle, control containingmL 4 medium. culture All culture media
. Each ratchet cycle entailed . Eachratchet cycleentailed a 3
addition sulfide
ion was oneusedfor cultivation the of the sp. thefirst ratchet cycle was started with four , grownand were 7d. Changes foratleast in
(300, 400 and 500 µM) plus the the at (300,control 400and500µM)plus 2 S - aqueous aqueous stock NaOHmaster solution trols, week theyremained another at - 50%, bufferedmM with20 HEPES
T ubes (50 mLubes (50 volume,
the desiredthe level, - fold increase. Changes in sulfide cell cell 5 - fold in increase cells, which were - sulfide concentration esponding esponding e nriched cultures
dose. sulfide ; however, Falcon™ sulfide In sulfide
was
)
This article isThis article by protected copyright. All rights reserved. Accepted Fig.rates, 3a) andthree modesdispersal (control environmental deteriorati performedwith by stress effects deterioration oftheenvironmental dispersal and mode onthe consequently, isableto orgrow aswell as before, occursEvolutionary rescue whenapopulation exposedtoselective survives conditions and, rate. deterioration and in rescue Evolutionary gro samethe Consequently, biomass control. asthe the number wasculture ofdays expressed in byeachreplicate termsselective taken under ofdays Article cell further growth waso population tohavewas considered reachedthe maximum of whenno capacity adaptation andwerenot transferred, analyz to nextwere transferred the ratchet c effecttoxic of and 0. sulfide as forthe concentration. ratcheteach werecycle performed by increasing 100 µMthepreexisting wth was a function of evolutionarywth wasafunction their potential toto adapt selective the conditions. 8 units (which was8 units(which with medium restored the that couldslightlymask renewal) the , We different imposed nine conditions,consistingof ofthree combinations experiments, culturesreachedIn both treated ifthe abiomass similartocontrols,they at at the endofthe7d sulfide M. aeruginosa sulfide Daily Daily Microcystis , anexperiment basedonthedesign fromand González Bell sulfide , as indicated before, asindicated M
bserved after 90dofto bserved exposure e ratchet on speeds (control (control on speeds icrocystis
aeruginosa additi of incubation incubation ed again after the ratchetend ofthenext cycleperiod. A xperiment. However ons and
aeruginosa ycle. Culturesycle. witha lower biomass controlswere than
(Fig. 3).(Fig. . the mediumthe showed anincrease pH weekly
- no deterioration
under under
- no dispersal renewal ofrenewal medium culture were performed at evenat , sulfide
at concentrations above 1.5mM
higher rates. In orderto study the sulfide -
, slow or fastdeterioration, slowor selection: effect of d of effect selection: - , localor globaldispersal; Fig.
additions sulfide dynamics under ofER sulfide
conditions toreach conditions . Results were. Results (2011)
between 0.5 -
dependent, as ispersal ispersal
was
This article isThis article by protected copyright. All rights reserved. aliquots weretransferred correspondin tothe from same the newthree tubesnewmedium. with dispersal simulation,For global alltwelve populations Eppendorfin an mixed. tubeand Frommixture, three50 this µL same wasdispersal achieved bymixing thethree cell cultures (three populations) subjected to the bytransferringapopulationwas achieved aliquot medium(50 µL)to new (Fig each transferred medium weekto new different inthree ways (Fig for 4weeks,level which after number the wascomputed ofevents ER(Fig fo level treatments, respect next gradient transf addition in 4mL ofBG11 of1.5×10 acelldensity established at atstarting the lowest sulfide necessary which is toobserveERevents. Three independent werecultures foreach used 210, 240 ( fourincluded 3b). Cultures each under subjected treatment were tofour 0
Accepted Article, 30,60,90µM),G2( erred to the next erred tothenext sulfide
Tomim To simulate three different the en concentration in gradient;each in total, the experiment comprised 108populations r 12weeks transfer beforethe G4level. to treatments, keptatG4 Inall were cultures . µM;
concentration 3b). purpose, (Fig. Forthis 75µL was ofeachcell culture placed
Fig. 3a). Fig. 3a). In thelastgradient (G4) every four every or theandfast environmentaltwo weeksin slow deterioration increasing sulfide ic the three dispersal modes threecultures dispersal the ic (from all were modes) deterioration all - 50% bufferedmM with20 corr the HEPES,pH7.2,with ively ively (Fig sulfide
level weremixed 3b)level (Fig. indicated as before and 50 twelve µL sulfide 30 sulfide , 60,90,
. level (G1 gradient). Each p
3a). Inthe3a). nodeterioration treatment, c gradient
concentrations 120 µM),G3 5
at differentat Populations we rates. cells vironmental deterioration modes, cultureswere
· g newmedia.
mL
all t per gradient, increasing 30
( - 1 60
he concentrations and was grown in15mLand wasgrown (Falcon tubes , 90,120,150µM)andG4 ( opulation of
sulfide The same was followed The procedure aliquots wereto transferred
stress gradients (G), stress whi .
3b). Control (no dispersal) M. aeruginosa ultures remained at G1 ultures remained at tested re transferred to the µ esponding .
were lethal, 3a) M each time:M each G1 .
3b). L 150
was , 180, sulfide ocal ocal ch TM
) This article isThis article by protected copyright. All rights reserved. comparison theoverall of 1andin sets 2of analysis,wasdetermined fluctuation aone the using hypothesisofsimilarThe null CV forthenumber cells ofresistant values tube, per computed analysis. Statistical statistical were analyses outusingcarried RCore Team and apost performed time three without exceedingrecovery) initialpre their m just thehigher rateobserved theG4than growth before (i.e., treatment an absolute increase in nm w was renewed weekly we all tubes G2 toG3andfrom G4. G3to µM andfrom90to120 concentration from G2level was from G1to from from 0to30µM; 30to60µM; to90 60 from tubes of all same the with mixture among ofthesame tubes ondepending dispersal mode, the explainedbefore: as without anymixture(nodispersal), sulfide when there was achangeof
Acceptedw Article as detected). as detected). This definitionis agross because populations may undergo ER and (decline as measured every week ineachas population density. toestimate cell
An ER event was scored if the growth rate wasscoredifthegrowthAn ERfour rate atG4wasafter event weeks level similaror Tomaintain Then, aftertwo(fastdeterioration) or four(slow weeksat deterioration) the same level, thepopulations transferred were one.to next the - hoc Student re tightlyclosed caps, with
sulfide
. Tomonitor growth rates along experiment, the absorbance at 750 the s. wereThe ERdata event analyz - Newman m sulfide
concentrations concentrations
sulfide sulfide mean oftheLHW values
-
Keuls (SNK) test computedKeuls forapercentage of 95%. The level (g
. A similarprocedurewas followedwhen changing from level, as level, as below.indicated sulfide sulfide lobal dispersal).lobal The increase in and pH - stress density. The complete experiment was
concentration (localdispersal)mixture orwith additions weremade daily as constan - ed using Model a I, two sensitive andLHW sensitive (2013)
t during as possible incubations
The transfersweredifferent .
- tailed
-
and sulfide re sistant cells of Z the mediumthe - test. test. The w ay ANOVA
, This article isThis article by protected copyright. All rights reserved. lowdispersal showed avery in set2, with CV 1 significantlyset higher( for proliferating cells in set 1 occurredin culturesfromproliferation all 2(Table set 1). Whereas numberthe of resistant LHW. time, this growthwas in17ofAfter detected ofset1,whereas cell 90cultures cell the oftheCultures wild aeruginosa Fluctuation oftheanalysis LHW almost at 900 undetectable µM rangethe meanoverall maximum of0.30 value sulfide 70% by strain wasca.0.7doublings In Toxic effect of RESULTS RCoreusing Team (2013). withtheBartletttest. checked All werethe tests performed accordance (1999), in with Zar homogeneity boththe ofvariances in Student a post Student tailed M. aeruginosa 100 µM (Fig. 4). 100 µM(Fig. The
Accepted Articlesulfide - hoc Student - free to0.31 conditions the the additiondaily - from 100to350 free medium, culture the
.
t sulfide - computed forthemutation test. The ERdata event - Newman - type
on wild m
value of value Microcystis
LHW of 50
M · -
Keuls (SNK) test computedKeuls (SNK) test forape - d type strainsof sulfide - sulfide 1 -
, and wasnegatively affected it by resistant cultures ranged overfiveorders resistant ofmagnitude, it - 0.01 doublings sensitivity toLHW M Oscillatoria acclimated growth( rate sulfide
. Higher concentrations. Higher inhibited aeruginosa
(Fig. 4). (Fig.
were analyz 0.02 doublings - selection equilibrium, wasperformedaone using
and completelybeing inhibited atconcentrations M - t
- icrocystis sp. increased linearly fromsp. increasedlinearly 0.22
test and the ANOVA, previously were ·
d strain were strain were incubated for 6weekswith - 1 - ed using I, a Model two
resistance in transformation at at
100 ·
d
aeruginosa - 1 , m when M )
of sulfide rcentage of95%. The Microcystis sulfide sulfide
and
m additions , growthbeing Oscillatoria
, decreasing ca. additions additions were in - P way ANOVA and
<0.0001) <0.0001) than aeruginosa , reachingan
0.01 in Microcystis
sp.
-
This article isThis article by protected copyright. All rights reserved. respectively 6). In (Fig. t sulfide Oscillatoria (from 405µM 100to sulfide ofbeginning ratchet the experiment wereable to up adapt were different, asexpected. Inthe case of adaptationto The limits ofgenetic genetic of Limits adaptation to absencethe of s the of frequency ( the mean both of ( significantly decreased fortheLHW value cellssensitive of absenceIn the oftheselectiveagent (i.e. Mutation algalculturethe collection withdaily additions of cellsresistant 2.1×10 wasestimated as LHWduring exposure. The spontaneous mutation rate(µ) of LHW t ofset2(Tablethat 1). Consequently, could inferred it be that LHW Accepted Article exposureo the toLHW spontaneous mutations by rare rather thanbyspecificadaptation
A LHW randomlychosen additions (Fig. 5). Tha - selection equilibrium sp m ,
maximum wereobserved adaptationvalues atmM 1.5mM, and2 mM 1.8 values, acoefficientvalues, ofselection ( when the initial Microcystis q - ) ofLHW elective agent, waselective agent, 9.4×10 resistant cellswas 0.4±0.03 resistant t is t is to say, resistance the to su lfide P his case, his case, <0.01, Student <0.01, Student ; see Fig. 4). In the Fig.4).Inthe ratchet; see experiment performedwith -
aeruginosa resistant alleles inwild alleles typepopulationsresistant of . sulfide
- sulfide resistant fromculture set1wasisolated and resistant maintained in sulfide sulfide
of - 7
, mutations cellper per generation (Table 1
concentrations were 0.3, 0.4 and 0.5 mM, 0.3,concentrations were 0.4 and0.5 M
t was 0.5±0.2doublings resistance increasedmore 2 resistance than
BG11 - M. in test) bytest) resistant 20%in cells. overallUsing the icrocystis
Microcystis aeruginosa - 7 sulfide . -
50% medium) the doublings s 0.2 mM0.2 ) of0.222wascomputed. The estimation
aeruginosa
in
, 11 foundedof 12cultures the at aeruginosa M. aeruginosa sulfide
·
the dailyaddition of the d - 1
(
and n
. ·
= 4). = 4). That is, - m - d sensitive cells cells sensitive to LHW resistant cells arose prior cellsarose prior resistant
-
and 1 Oscillatoria value oftheLHW value ( n
increased 4 = 4), whereasthe = 4), M. aeruginosa Oscillatoria Oscillatoria - fold (from0.9 to2 m
405 sp.
). -
fold
sp. µM - , in -
This article isThis article by protected copyright. All rights reserved. Acceptedenvironmental deterioration rate and modedispersal significantly affect of enhancement of occurredEvolutionary rescue under combinationsall ofpopulation dispersal modes rate and rate deterioration and in rescue Evolutionary to exposed the initial higher sp., 90(10, 30or µM ofadaptationhighest capacity in (Fig.was detected foreachstrain 4). Ontheotherhand 6). (Fig. That inboth is, atconcentrationscases, above the the thenextreach higher concentration concentration wasuntil the µM >90 Articleaeruginosa sulfide (d periods differences canbeanalyz test did not concentrations 2 above mM (Camacho 2005) al. et above 0 sulphur(S detected mM
Oscillatoria because thehighestcapabecause sulfide
Because eachr concentration, especially at case thehighestconcentrations. the In of . 4 mM. well as This phenomenon occurs highin natural ) togrowthe same cell concentration ascontrol atthecultures higher next , werethere no differences (exceptin one ofthereplicates) in time to needed ; see Fig. 4). Fig.4). However,; see ratchet inthe experiment with
sp. ratchet experiment,this wasobservedat concentrations 1 around mM sulfide sulfide 0 ) precipitation in tubeswhentotal cultures . However, because eplicate evolved anindependent as population, within . M
; Fig. 5). However,; Fig.5). there differences were inthe of case ed. fourThus, the culturereplicatesfoundedrequi icrocystis concentration, ranging from (Fig. 2to12events 7). Whereas both sulfide city of(up cultures adaptation to2mM)wasreached bythose M. aeruginosa
concentrations (0.5mM aeruginosa sulfide
the the change inturbidity could affect growth rate, we
in this ratchetin this experiment. , regardless oftheinitialconcentrations tested under under sulfide , there werenodifferences inthe
sulfide sulfide
selection: effect of dispersal dispersal of effect selection: sulfide - sulfide Oscillatoria
levels atlevels whichnogrowth ; Fig6).
concentration was
ed ER the dynamics environments sulfid
red differentred time
- Microcystis strain
e sp., we
(Fig. 5).In Oscillatoria
This article isThis article by protected copyright. All rights reserved. experiment performing tosimulate tried evolutionthe of twocyanobacteriataxaunder selection by demonstrations very are difficult and very hence scant hasbeenThis assumption widelyacce appearance better of newgenotypes adapted onesthan newnatural previous to conditions. Neodarwinism postulates selection and that variability pressure genetic are involved in the DISCUSSION (SNK test). weredispersal), there no differences environmentalbetween deterioration spe wereOnlywhen (SNKdeterioration observed whole pool test). the mixedgenetic was (global fast determineddeterioration number the ofER significantly higherinslowthan events,being in submitted to same the modes (SNK test hinders fast deterioration rate the achievement ofER local eventsunder and nodispersal dispersalwithout independently of deterioration rate) the 7). Theoccurrence (Fig. of were ERwhenpopulations dispersed(and globally events, werepresentedtheand, forthis reason, mode number ER afunction ofdispersal of eventsas variance in than ER contribution fromthe washigher (Table 2). of
Accepted Article M. aeruginosa deterioration (SNK however, test); nosignificant differences betweenslowandconstant According to ANOVAAccording the
s
for the first the timefor . Several studies . Several have addressed the different mechanisms allowingthe
). When geneticmade the interchange was among only populations ,
under under with the exception Onthe ofno(SNKhand, deterioration other test). the sulfide sulfide , to our knowledge, , toour
concentration (local of dispersal), speed the environmentalthe
selective conditions, the interaction ofselective conditions, interaction both did the factors not
P - values, thecontributionvalues, ofthedispersal modethe to pted bythepted community, scientific butexperimental , wassignificantly under higherthan localdispersal, or
three differen environmental deterioration (Table2) environmental deterioration ( Sniegowski
t ‘evolution t ‘evolution in action’
2005). In this study, we sulfide ed treatments
This article isThis article by protected copyright. All rights reserved. Acceptedsulfide aeruginosa thanbyacclimation.rather result This qualitative issimilar to foundinthat Ma1G the prior arose to exposure toLHW the 150µM (ca. inthefluctuationanalysis The results observed experiment indicated thatLHW demonstrated thatthisorganism to couldgenetically adapt the lethal anoxygenic photosynthesisdependent suggesting that it could exhibit strainthis m showed fromisolated La spa,where Hedionda Cohen growthand then low rate, at aeruginosa Article environmentthe which from grow ≥0.1 mM different tolerances tothe presence of tolerance. limitand the ion ofadaptationtothis bedifferentcould depending ontheinitial a selective agentfor most photosyntheticmicroorganisms, begenetic could orph (Miller and Bebout 2004,Fernández ha studies ofproliferation cy
even when submitted even whensubmitted to , et Despite Despite the of high sensitivity As expected Fernández
al sulfide ve analyz
. strain exposed to the strain exposedthe to sulfu was isolated from
1986
exposure , anobacteria foundtodayanobacteria sulf in - Garcí Arjona al. et 2013 ed of the process , aximum growth rates when rates submittedaximum to growth Microcystis a - , whereas , whereas the O1LH Pichel sulfide each each
0.9 mM sulfide sulfide
aeruginosa and strain was isolated strain wasisolated
concentrations (<0.1 mM)concentrations (<0.1 isexpected ( -
sulfide - Arjona et 2013) al. ). More Castenholz, 1990) free Microcystis reous waters from waters Bañosde reous Los Vilo 240 µM (ca. - sulfide (Cohen et (Cohen et 1986 al. sulfide sulfide resistant photosynthesisoxygenic oreven
waters, soinhibition ofphotosynthesis, oxygenic
adaptation froman evolutionary pointof view over, spontaneous the mutation rates inboth and . Growthof
additions concentration from ranged 0.1 Oscillatoria sulfi Oscillatoria idic environments, howev
aeruginosa (Miller de , in agreement with . Onthecontrary, ) by rare spontaneous) byrare mutations . Inway, this the tolerance ,
M. aeruginosa
Camacho 2000,2005) al. et
strain main
and Bebout
0.1 sp. wild strains type showed to sulfide - 0.35 mM sulfide tained the abilitytained the to , w
was not detected at was notdetected
2004). Oscillatoria e unequivocally e unequivocally er, few very sulfide sulfide Castenholz, 1977
levels ofLHW. - 0.2 mM. Infact, 0.2 - resistant cells resistant sulfide Microcystis ysiological sulfide
levels in levels in additions to sulfide
. M. sp. was
- , , ,
This article isThis article by protected copyright. All rights reserved. Acceptedsulfide fact that speciesseveral of genus the mM sulfide hand, other the of O1LH strain rolethat the of previous levelof the times maximal thanthe higher typestrain. ofthewild tolerance Nodifferences found were in experimental couldsurvive under culture 0.4mM aeruginosa in a proliferate in selective situations without agent, wouldsince it bedisplaced wild bythe type LHW in the Articleexplained the large decrease in and photosynthetic efficiencyrates photosynthetic an increase in anda size observed, morphologywere disrupted as aswell a 50%decrease in bydecreased the 70%in was lowcomparedwithobtained intheMa1G that mentioned above,growth strain the as rate physiolothe significantly by20%compared that ofMa1Vc to strain, wild which could beanindicationof strains.in both The equilibriumselection ofca. de Vilo; units per inmutantscell per generation). Moreover, a similar valuemutation of of were ofmagnitudecases thesame(2.1×10 order sulfide sulfide . Nevertheless, onlyafew. Nevertheless, populations derived experimental were able togrow ca.2 at
App levels can -
lying theratchetprotocol,lying adaptation to - sulfide increased up the to twice LHW free environment. exposure gical ofgical cost mutation the conferring resistant cells.resistant However, cases, inboth the achieve al ≥1.5mM (Camacho et concentrations
adaptation regardless ofthe initial m , the conce highest
value ofLHWvalue sulfide sulfide
1
sulfide
m Oscillatoria
exposure not did ultiinfluence resistant cells . These changes phy on cell - - Oscillatoria resistant cellsresistant in resistant mutant cellresistant per mutant 10 ntration ntration tested. This result isin agreement withthe , asexpected, developed agreater tolerance to sulfide (Fernández
sulfide proliferate proliferate in environments natural where sulfide
( concentration. concentration. Thus, almost every - Bañares 7 sulfide in this study,in this and7.1×10 sulfide sulfide sulfide
tolerance. However, this reduction
additions -
Arjona etal.2013) in the in the Ma1Vc of strain - España et al. España et
siology could siology could relevant be notso - concentration used,suggesting free medium decreased - mate resistant genotypewould not 6
wild . , foura concentration
1996, 2000) sulfide - type was cells found
2016)
resistance. Onthe - . Inthese cells, 7 in LosBaños , which
because they Microcystis -
This article isThis article by protected copyright. All rights reserved. later. Inparticular, highest the ER events scores were recordedunderglobal dispersa variationby bothgenetic anddeterioration rate, effectbut the oftheformer isgreater thanthe 2019 (Bell is variation one ofthepremises selection, ofnatural demonstrations experimental are scant survivalpopulation a at lethal level of and variation) speed the of environmental ( stre caseofthe waters, LaHediondaspa microorganismsmay inducefluctuating hypolimnia ofstratifiedmarine or lakes andfreshwatersediments, growth the of where concentration, sulf like analyzfurther morphological characteristics ofthe (Cohen cyanobacteria concentrations changes induces in the photosynthetic characteristics of in moderate themodulate end aeruginosa in capacity 2000, 2005) or maintain photosynthesisoxygenic relatively insensitiveto exhibit
Accepted ArticleOscillatoria am flow. toanalyz So, we wanted , and Liu Although some sulfide
- González, 2009,2011, high of levels et et al.
Oscillatoria is very sensitive to is verysensitive to . Interestingly, culture previous conditions highest modulate the adaptation ed to shed more sheded to process light onthe to ofadaptation -
d sp. factThis couldberelatedtothe pres that the 2019 riven anoxygenicphotosynthesis - point of maximum its Onthecontrary, adaptation. the ofpresence ) . Our indicated results that occurrenceofthe ERwasmodulated events et et al. ur in concentrations springs, others change with sulfide
but not in but not sulfide 1986, - rich environmentsrich maintain relatively a constant
sulfide (0.5 mM) theadaptationtohighest allowed ofthis levels ion
Lindsey
Camacho M. aeruginosa sulfide sulfide e how the population dispersal(aproxye howthepopulation of genetic
sulfide and, as aconsequence,and, as sub
vertical gradientsvertical of et al.et sulfide -
content changes resistant strains obt et al. et
(evolutionary rescue). (evolutionary rescue). Although genetic 2013, . This could be explained fact that bythe 2005) (Belkin (Belkin
concentration) changeconcentration) affectcould to
Orr . and The photosynthetic
and and
along the year the dependingalong onthe sulfide
sulfide
Uncless Padan 1983, ence ofhighence ained ained in t - lethal lethal sulfide
(Camacho (Camacho
2014,
sulfide time, the like sulfide his will study be .
Arieli Arieli
sulfide Carja andPlotkin Carja - resistant
et al.et et al. et le sulfide et al.et vels donot
l, 2005) 1996,
1991) M. . In , This article isThis article by protected copyright. All rights reserved. conse cyanobacteriumin this variation ahighpercentage ofphenotypic several in trai sulfide 1980, extrapolated toexpl Ma1Vc strain of trajectory.evolutionary mustEven so,this assertion made be carefully andrest achievement of variabilitygenetic ofthe population speedand the ofdeteriorationfor aredecisive the concentration the aresubjected towhich cultures showedintheratchet(as th results), oflikelihood adaptation tolethal levels of coli de Meaux to deteriorationis adaptation onmore slower based mutations ofsmaller beneficial mutations,withrapi hypothesis with the that therateof environmental canmodulatedeterioration the effect of deterioration studied rate. The results found underlocalorwithout dispersal areinagreement Moreover,events. at geneticvariation, the lowest the orlocal without dispersal, respectively) fastdeterioration a doesnotER rate favourthe However, variationor whenthegenetic is lower evenminimal wassimulated under (as the overcome in ofregardless theenvironmental rate. deterioration variationThis suggests thatahigh genetic
Accepted Article M. aeruginosa .
quence ofvariation, ma genetic Arnold
From an ecological pointFrom of anecological view, could hypothesized it be that the to adaptation Takin in naturalpopulations of , sulfide
2009). fact, In Lindsey g into g into alltheseaccount results, that wecanhypothesize even whenthe et al. et sulfide Microcystis
populations iscrucial forgenetic anditcould adaptation be enoughto stress stress in range the any we examinedunder of speed 2001). ain macro
resistance, ergo, dynamics thepopulation willinfluence the
aeruginosa - d change favouring afewmutations oflarge effect, whereas evolutionary Microcystis et et al. king the non king the (2013) obtained (2013) similar experimental in results , since , since micro sulfide
processes ( processes
aeruginosa
- by Ma1Vc is not constrained by not by Ma1Vcis the initial genetic contribution less significant
- ER event under reduction occurs any evolutionary be routescannot Stanley 1979,
may occur.easily The reason is that
Eldredge environmental change. effect (Collins ricted tothe and ts isa
Cracraft e E. and
This article isThis article by protected copyright. All rights reserved. Accepted r kindly 2015 87314 projectsThis workwasfinanciallysupported by the CGL2014 ArticleACKNOWLEDGEMENTS CONFLICT OF INTEREST: declare The authors that havethey noconflict ofinterest. how the ofc taxa adaptation mechanisms to sulfide in variation populationsnatural of (Bañares - 072984 grant (Ministerio deEconomía072984 grant yCompetitividad, Spain). Dr. C.Henry Eric -
P (Ministerio deEconomía Spain). yCompetitividad, EM The experimental evolution approaches addressedstudy ustoexplore inthis allowed levels abovenormally conditions. lethal evised English the style and usage. sulfide yanobacteria. Further studies, involving couldmore lineagesofcyanobacteria clarify - España
tolerance evolve could these in microorganisms. et al.et 2006, 2007, sulfide
and constraints the to this process in tworepresentative M. aeruginosa
López - Rodas
et al. et could evenovercom could 2006a,
b) - 53862 . Consequently, ahigh genetic -
C was foundedbyBES - e a sudden change e asudden in P and CGL2017
-
- This article isThis article by protected copyright. All rights reserved. AcceptedG. & Bell, G. Bell, S.&Belkin, Padan,E.1983.Low potential promotes redox sulphide Bañares Bañares ArticleBañares Bagarinao, T. an as 1992.Sulphide environmentalfactor and toxicant: tolerance and Arnold, S.J.,Pfender, M.E.&Jones, A. G. 2001.The B.,Arieli, Padan,E.&Shahak, Y. 1991. experiencing environmental deterioration. environmental change. evolution hydrogen in 85:159 pH compensationpoint, in cyanobacterium the Inter Microcystis aeruginosa photochemical andnonphotochemical of quenching, instrains cyanobacterium the variability associatedGenetic with photosynthetic pigment concentrati strain. photosynthetic cyanobacterium Reul, M.H. A., Mariné, & Flores adaptations in organisms. aquatic betweenmicromacroevolution.bridge and thylakoids of
- - - & España, E.,López España, E.,Fernández España, E.,López
- Gonzalez, Gonzalez, A. 2011. Adaptation andevolutionary rescue in metapopulations A.Gonzalez, rescueprevent 2009.Evolutionary can extinction following strain invariability photosyntheticuse the exemplifiedof carbon, bythe inorganic Microb. Ecol. - 62.
Oscillatoria limnetica performance sulphide between the Microcystis aeruginosa
- - 71:860 Rodas, V.,Salgado, C., Costas, E. Rodas, V.,Costas, E.,Salgado,C. Oscillatoria limnetica Ecol. Lett. . FEMS - Arjona, García M.M., - 72. Microb. Ecol.
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Aquat. Toxicol.Aquat. Moya, A. 2016. 12:942 Sulfide . J. Biol. Chem.J. Biol. : acomparative study ofmorphologyand - - 8. Science induced sulfide Genetica.
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Microbiol 60:449 Microcystis aeruginosa Microcystis
- adaptive landscape adaptive landscape conceptualas a Sánchez, J.,Hernández M. 24:21 -
Sulphide resistanceSulphide in the resistant mutant and resistant mutant the wild 332:
266:104 - 112 55. – 1327 .
62. & & 129 –
- 113: quinone quinone reductase activity in Flores Flores
- - : 11. 30. 3091 - 9
and l
– - - 32. Moya, A.2006. Moya, A.2007. - 8.
ight on, and . Aquat. Bot. -
dependent - López, M., - type type
This article isThis article by protected copyright. All rights reserved. Accepted S. Collins, & DeMeaux, J.2009. Adaptation to different of rates environmental change in Cohen, Cohen, Y., Jørgensen, B.B., Revsbech,N.P. R.1986. &Poplawski, hydrogenAdaptation to Cohen, Y., Jørgensen, B.B., dependentanoxygenic Paden,E.&Shilo, M. 1975a.Sulfide Castenholz, R. W. 1977. The effect ofsulfideontheblue O. Carja, ArticleCamacho, A.,Vicente, E. &Miracle, M. R.2000.Ecologyofadeep Camacho, A., Rochera, C.,Silvestre, J.J., Vicente, E.&Martin, W. H. 2005.Spatial Camacho, A., Garcia Blount, Z.D.,Borland,C. Z. cyanobacterium Environ. Microbiol. ofoxygenic andanoxygenicphotosynthesissulfide among cyanobacteria. in cyanobaphotosynthesis the Yellowstone Park. National variability. Hydrobiol. (= strategies.ecological andphysical chemicalofacold gradient ofdifferent sulphurousspring:the role dominance of inorganic c (Spain). underwater the lightfieldin and to three cyanobacterial isolates from Lake Arcas Sci ofin an akey innovation Y., E.&ShiloM.1975b. Padan Planktothrix . USA
& Plotkin, J. B.& J. Plotkin, 2019. FEMS Microbi. Ecol.
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-
This article isThis article by protected copyright. All rights reserved. AcceptedGarcía García García Flores ArticleFlores Flores Fernández S.F.Elena, R. &Lenski, E.2003.Evolution experimentsmicroorganisms: with dynamics the 4, 6 Dictyos experimentalenvironments: an model analyzing rapidthe adaptation of Martín in7strainsofathermophiliccapacity cyanobacterium. temperature and acidification. of toxic dinoflagellate the E. &López Phytolog. habitatnatural environment (sulphureous m waters)throughpreselective López 95:697 evolutionin the ofdinoflagellate fromsulphureous water Los Bañosde Vilo (SSpain). involved in theadaptation microorganisms ofphotosynthetic ex to the López and genetic ofadaptation.bases ------Moya, A., Rouco, M.,García Moya, A., Costas, E.,Bañares Moya, A., Costas, E.&López Villada, L.,Rico,M., Altamirano, M.,Sánchez Villada, L.,López Pichel, F. R. &Castenholz, ‐ - Trinitrotoluene by rare preselective mutations Arjona, M. Arjona, M. M - - - - Rodas, V. 2005. Rodas, V., Flores Costas,E.& phaerium chlorelloides Otero, L. &Costas,E.2002. 703.
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This article isThis article by protected copyright. All rights reserved. AcceptedHughes, A. L. 1999. I. Huertas, I. Huertas, Howsley, R.&Pearson, H. Hamilton, T. L.,Bryant, D. A. &Macalady, J. L. 2016. ofbiologyinplanet The role ArticleGould, J.L. Gonzalez, A., Ronce,O.,Ferriere,R., &Hochberg, rescue: M.E.2013.Evolutionary an Garland, T., Jr. Rose, &M. R.2009. M. Oxfor B phytoplankton differently: approach. evidencethroughamechanistic phytoplankton differently. species different phytoplankton toadapt tocontamination:groups herbicideswill affect cyanobacteria. in photosynthesis Environ. Microbio cyanobacterialevolution: primary production in low USA. B emerging inters at the focus 730 pp Applications ofSelection Experiments. algaecide. aeruginosa E. 2004 iol. Sci. iol. .
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This article isThis article by protected copyright. All rights reserved. a µ (mutants cell One CV theno.of LHW of cells with No. of cultures inthe rank: No. of Ma1Vc T P ABLE
Accepted<0.0001 (significant) Article - tailed Z tailed
replicate cultures: replicate 1
Microcystis aeruginosa . Fluctuation analysis ofLHW analysis Fluctuation - test fo
s ·
generation r compar
- resistant cellsresistant culture: per
- ison of CVs:ison of 1
)
strain
- sensitivity toLHW sensitivity
> 10 10 1 0
- 1 10 5 4 0 - - 10 10 6 4
6 5
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resistance transformationresistance inthe 9 6 1 1 73 90 Set 1 2.1× 2.351
10
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10.891 7
a 40 0 0 0 0 40 Set 2 0.214
This article isThis article by protected copyright. All rights reserved. Error Dispersal × deterioration Deterioration Dispersal ofvariation Source areindicateddeterioration inFig sulphide conditions.Modesoflethal ofenvironmental population andrates dispersal T Accepted Article ABLE
2
.
Two
-
way ANOVAfor thecomparison of ERof the events
18 4 2 2 df
ure
3 46.66 14.59 50.07 86.51 SS
6 2 4 8
2.59 3.64 25.03 43.25 MS
2 8
7 9
1.40 9.65 16.69 F
Microcystis aeruginosa
0.27 0.001 7.65×10 P - value
- 5
at at This article isThis article by protected copyright. All rights reserved. that foundsimilar biomassto incontrol cultureswere transferred not (asterisk) andwere nextto the sulfide level cycleor ratchet 90 (30, and Culturesnot reaching 270µMsulfide) a showing the concentrationsamefinal cell (indicated bydots) as control ones transfer were densitylow cell (whitecultures) and were maintained7 d ineachtreatment. Cultures concesulfide pH 7.2),and4replicates of aeruginosa F be similar. anexperimentalused as the Inthiscase, control. number cells ofresistant cultures inall must were foundedfromso culture, varianceof samepopulationthe the parental the is parental earlycelldensity)appear (high or (lowcelldensity)late d occurredevents inmost and cultures (clearcultures) ofthe that raremutationalevents could cellsresistant cultures inall wouldbe different. The diagram showsthatnomutational also propagationbeforeculture exposure waters to sulfide (i.e., genetic adaptation),thenumb cultureswouldin all similar. be arose Ifresistant byrare cells occurring duringmutations cellsresistant arose wasreacheddots) andthen transferredselective tothe agent (La water, Hedionda LHW). If non of cultures F FIGURE LEGENDS AcceptedIG ArticleIG . 1 . -
selective conditions (BG11 2. Ratchet p 2. Ratchet . Fluctuation analysis. Fluctuation schematic diagram possible results. In Set and 1,different
Microcystis aeruginosa strain ntrations ntrations (10, 30prepared. and90µM)were All startedcultures withthe same rotocol scheme. the initial 4replicates phase, of During cultures during exposure toLHW the (acclimation), thenumber ofresistant cells
containing growthmedium control (BG11 three - 50% medium) higha very density cell until (indicated by
treatments
(started froma small inoculum) propagated were under
containing the same the containing medium increasingwith
uring propagation. Set2replicates - 50%, 20mMHEPES, Microcystis er of er red red This article isThis article by protected copyright. All rights reserved. with 3replicateseach)were founded with this concentrations, were 12tubes in total, mixed, 12new andthe concentrations populations (4 mixedfounded the dispersal, with Onglobal culture. all tubes from 4different the sulfide same concentration(onlyone mixed, sulfide shown)were andth were made directly. Onthe local dispersal condition, the 3 populations orreplicatesat the ondepending dispersal condition the condition,applied. In thenodispersal thetransferences deteriorationthree conditions transferred everyweektofresh were medium inadifferen respectively. Populations remained G4inalltreatments. 4weeksin (b) All fromcultures the gradient wa and fastdeterioration treatments G2 wentthrough transfernext andG3,butthe to the G4gradient to the (where all sulfide concentrations are supposedtobelethal). assayed Slow treatment, remained populations o was assayed inthreedifferent populations, butonly isshown. one Inthenodeterioration 90, 120and150µM)Each G4(150,180,210and240µM)as indicated. concentration fromnew sulfidegradient 60and90 G1 (0,30, (represented bydifferent of intensities were these concentrations gray)and increased in each Each gradient(a) sulfide (fromG1 toG4)comprised sulfide fourincreasing concentrations F to sameexposure the concentration(not shown). sulfide maximum ofcellgrowthwas whennofurther after90 capacity adaptation observed d of wereas they different). A population(rep for adetailed descriptionfor oftheconcentrationstested nexttransference tothe sulfide level(ratchetcycle) isnot shown Material andMethods (see maintained the at concentrationsame untilsulfide they reached th Accepted ArticleIG . 3.
Schematic diagram rescue oftheevolutionary in experiment s 2weeks,onslow performedorfastdeterioration 4or treatments, after n the initialsulfiden the gradient the for12weeks,until transfer licate) was tohave considered reachedthe mixed culture. scheme In the only
µM) to G2(30,µM) to 60,90and120µM),G3(60,
M. aeruginosa
e new 3 populations e new3populations were e control biomass. The Microcystis aeruginosa
and Oscillatoria t way
sp . . , This article isThis article by protected copyright. All rights reserved. inthehorizontal axisforindicated eachinitial concentration. Eachofthefourreplicates 0.4mM(a), 0.5 and (b) mM (c). in concentration increase at sulfide ratchetThe each cycle is ofmMgrown inthe presence 0.2 to sulfide, three different of initial levels 0.3 sulfide: mM Oscillatoria culturescontrol (with0.2mM during the experiment sulfide) ratchet cycles performedwith F concentrations strain. forthis area ratchetindicates the atwhichcycle cellswere the subjected tolet founded thecycle are at beginningofeachratchet shown withdifferent colors. The shaded i 30µM(b)and90(c). (a), The increase insulfide concentrationateach ratchet cycle is grownpreviously ofsulfide, inthe absence three to different initiallevels of 10 sulfide: µM Microcystis aeruginosa culturescontrol (withoutsulfide)during ratchet the experiment cyclesperformed with F growthin the medium. ( increment ofthenumber ( ofcells Mic F regardless the concentrations sulfide tested. transferences fromG2areshown asanexample, G1to process wasthesame butthe ndicated in horizontalaxisforthe eachinitial concentration. Eachofthefourreplicates Oscillatoria AcceptedIG ArticleIG IG . 4. . 6. . 5 rocystis . Time for required under cultures increasing concentrations sulfide as as togrow dense
Time for required under cultures increasing concentrations sulfide as as togrow dense Effect ofsulfide
aeruginosa sp. O1LH str
sp., black circles, sp., black
exposure Ma1Vc strain. The experimentexposure ofcells, withthe started and ain. ain. The experiment with started ofcells,previously the exposure Oscillatoria
n
= 5dofat4) after culture increasing on the acclimated( rate growth M. aeruginosa
sp .
strains. Growth wasestimated rate bythe , gra y circles,
n = 5) orChl= 5) m ) ofthewild hal hal sulfide sulfide concentrations a concentration - type
This article isThis article by protected copyright. All rights reserved. 0.05) significantindicate differences detectedbyStudent gra light dispersal each Treatments mode at represented are bywhite (contbars lethal rescue evolutionary (ER) observedin events F 0.7→mMcycle 0.8 7d,sotheynot took are represented inorderto simplify thefigure. concentrations strain. forthis For the three initial concentrations, everysulfide ratchet the till area ratchetindicates the atwhichcycle cellswere the subjected tole founded thecycle are at beginningofeachratchet shown withdifferent colors. The shaded Accepted ArticleIG . 7 .
. sulfide
Effect ofenvironmental mode change deterioration anddispersal number onthe of y bars (
concentrations (maximumconcentrations Verticallines on show 12ERbars events). slow deterioration) and gra dark Microcystis aeruginosa y deterioration). (fast bars Different letters - Newma
n - Keuls (SNK) post
populations submittedto thal thal sulfide rol - no deterioration), - hoc test ( SD ( n
=3). =3). P < This article is protected by copyright. All rights reserved. This article is protected rights by All copyright. reserved. Accepted Article
This article is protected by copyright. All rights reserved. This article is protected rights by All copyright. reserved. Accepted Article
This article is protected by copyright. All rights reserved. This article is protected rights by All copyright. reserved. Accepted Article
This article is protected by copyright. All rights reserved. This article is protected rights by All copyright. reserved. Accepted Article
This article is protected by copyright. All rights reserved. This article is protected rights by All copyright. reserved. Accepted Article
This article is protected by copyright. All rights reserved. This article is protected rights by All copyright. reserved. Accepted Article
This article is protected by copyright. All rights reserved. This article is protected rights by All copyright. reserved. Accepted Article