the Red Macroalga Porphyra umbilicalis

16 Pages·2013·2.64 MB·English

Diversity and Abundance of the Bacterial Community of
the Red Macroalga Porphyraumbilicalis: Did Bacterial
Farmers Produce Macroalgae?
Lilibeth N. Miranda1*¤, Keith Hutchison2, Arthur R. Grossman3, Susan H. Brawley1
1SchoolofMarineSciences,UniversityofMaine,Orono,Maine,UnitedStatesofAmerica,2DepartmentofMolecularandBiomedicalSciences,UniversityofMaine,Orono,
Maine,UnitedStatesofAmerica,3CarnegieInstitutionforScience,DepartmentofPlantBiology,Stanford,California,UnitedStatesofAmerica
Abstract
Macroalgae harbor microbial communities whose bacterial biodiversity remains largely uncharacterized. The goals of this
study were 1) to examine the composition of the bacterial community associated with Porphyra umbilicalis Ku¨tzing from
SchoodicPoint,ME,2)determinewhetherthereareseasonaltrendsinspeciesdiversitybutacoregroupofbacteriathatare
alwayspresent,and3)todeterminehowthemicrobialcommunityassociatedwithalaboratorystrain(P.um.1)established
inthepresenceofantibioticshaschanged.P.umbilicalisblades(n=5,fall2010;n=5,winter2011;n=2,clonalP.um.1)were
analyzedbypyrosequencingovertwovariableregionsofthe16 SrDNA(V5–V6andV8;147,880totalreads).Thebacterial
taxapresentwereclassifiedatan80%confidencethresholdintoeightphyla(Bacteroidetes,Proteobacteria,Planctomycetes,
Chloroflexi, Actinobacteria, Deinococcus-Thermus, Firmicutes, and the candidate division TM7). The Bacteroidetes
comprisedthemajorityofbacterialsequencesonbothfieldandlabblades,buttheProteobacteria(Alphaproteobacteria,
Gammaproteobacteria)werealsoabundant.Sphingobacteria(Bacteroidetes)andFlavobacteria(Bacteroidetes)hadinverse
abundancesonnaturalversusP.um.1blades.Bacterialcommunitieswerericherandmorediverseonbladessampledinfall
compared to winter. Significant differences were observed between microbial communities among all three groups of
blades examined. Only two OTUs were found on all 12 blades, and only one of these, belonging to the Saprospiraceae
(Bacteroidetes), was abundant. Lewinella (as 66 OTUs) was found on all field blades and was the most abundant genus.
BacteriafromtheBacteroidetes,ProteobacteriaandPlanctomycetesthatareknowntodigestthegalactansulfatesofred
algal cell walls were well-represented. Some of these taxa likely provide essential morphogenetic and beneficial nutritive
factors to P.umbilicalis and may havehad unexpected effects uponevolutionofmacroalgal form aswellasfunction.
Citation:MirandaLN,HutchisonK,GrossmanAR,BrawleySH(2013)DiversityandAbundanceoftheBacterialCommunityoftheRedMacroalgaPorphyra
umbilicalis:DidBacterialFarmersProduceMacroalgae?PLoSONE8(3):e58269.doi:10.1371/journal.pone.0058269
Editor:JoshNeufeld,UniversityofWaterloo,Canada
ReceivedMay17,2012;AcceptedFebruary5,2013;PublishedMarch20,2013
Copyright:(cid:2)2013Mirandaetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:TheworkwassupportedbyNationalScienceFoundationIOS0929558toSHBandIOS0929758toARG,andNationalOceanicandAtmospheric
AdministrationNA060AR4170108toSHB. Thefundershad noroleinstudydesign, datacollectionand analysis,decision topublish, orpreparation ofthe
manuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
¤Currentaddress:TropicalMarineScienceInstitute,NationalUniversityofSingapore,Singapore,Singapore
Introduction pioneersinunderstandingtheobligatorynatureofsomebacterial-
algal associations by discovering that normal algal morphology
Algal-bacterialassociationsareimportanttothebiologyofboth (e.g., of Ulva spp. and related green algae) was lost in axenic
groups of organisms, but the biodiversity of these associations cultures. Normal algal morphology could sometimes be restored
remains largely unexplored andthewaysin whichbacterial-algal byreinfectionwith mixedbacterial culturesorbytheapplication
interactions impact metabolic and developmental features of the of plant hormones [12–14]. Many Bacteroidetes as well as some
partnerorganismsarenotwellunderstood.Arangeofsymbiotic, Proteobacteria and Firmicutes were found to support normal
pathological and opportunistic interactions between macroalgae morphologywhengrowninassociationwithulvaleangreenalgae
and bacteria are being discovered [1]. Depending upon the (e.g., [15–17]), and ‘‘ thallusin’’ was isolated from a Cytophaga sp.
membershipofitsbacterialbiofilm,amacroalgamaysufferserious (Bacteroidetes), crystallized, and demonstrated to be effective in
disease (e.g., [1–4]) or be protected from damaging colonization restoringnormalmorphologyinaxenicculturesofulvaleanalgae
(e.g., [1,5]).Algaldefensive strategies againstbacteria rangefrom [18]. A few Bacteroidetes have just been found to induce
cell wall sloughing to the release of diverse anti-microbial multicellularity in choanoflagellates, close relatives of animals, by
compounds ([1,6] e.g., hydrogen peroxide and other oxidative production of a sulfonolipid morphogen named RIF-1 [19].
burst responses [7,8], furanones that block bacterial communica- Bacterial associations can be expected to be essential to Porphyra
tion[3]).Theclearsymbioticassociationsbetweensomealgaeand umbilicalis,becausethebladephaseofitscloserelative[20],Pyropia
somebacteriaincludebacterialsupplyofvitamins,fixednitrogen, yezoensis,doesnot develop inaxenic culture [21]).
and growth regulators to the algae, which, in turn, supply the Few studies have attempted to characterize the microbial
bacteriawithfixedcarbon[9].ProvasoliandPintner[10–12]were communities of macroalgae with techniques that surmount the
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BacterialCommunityofPorphyraumbilicalis
well-knownunder-samplingofcommunitiesbyculturetechniques. that were collected in the winter had fewer epiphytes based on
Studiesatappropriategeographicscaleswith16SrDNADGGE, observation with bright-field microscopy before blades were
16S rDNA clone libraries, and more recent metagenomic frozen.
approaches have expanded our understanding of microbial
communities associated with Ulva spp. [22–25], a few brown Laboratory samples
algae (e.g., Fucus vesiculosus, Saccharina latissima [25–26]) and a few Porphyra umbilicalis laboratory blades were derived originally
red algae (e.g., Delisea pulchra, Gracilaria vermiculophylla [22,25]). fromasinglebladecollectedatSchoodicPointonApril3,2008;
Thesestudieshavebeguntoaddressthescalesofvariationofthe this is the same site where the field samples for this study were
microbial community in space and time and have described collected. Laboratory cultures in WES-PES seawater [48] were
differences related to the macroalgal host’s taxonomy. Some initially and repeatedly treated (106over 2 years, 3 d each) with
investigators have also questioned whether there is a taxonomi- 100mgL21penicillinGand25mgL21dihydrostreptomycin(16
cally-specificcoremicrobiomeonanalgalhostoronlyaconstant PenStrep[48])toreduce/eliminatebacteria.Bladesforthisstudy
coreofmicrobialgeneactivitiesthatcanbeprovidedbydifferent were part of the 7th generation of the original isolate. Filtered
sets of microbial taxa on the algal host [27]. These questions aerationwasprovidedbyairstones,andtheP.umbilicalisbladesfor
require additional studies, at greater sampling depth, on more thisstudyweregrowntomaturitybyattachingthemtothesideof
algae,suchascanbeprovidedbypyrosequencing.Pyrosequencing transparent Nalgene containers (3 L) using aquarium seaweed
of the variable regions of the 16S rDNA [28] has transformed clips. The cultures were incubated in Percival 136LL VL
microbialecologyandisprovidingnovelinformationaboutmany incubators (Percival Scientific, Inc., Perry, IA) at 10uC, a
naturalandanthropogenichabitats[29–31].Itsrecentapplication 14:10 L:D photoperiod and irradiance of 60 mmol photon m22
tomicrobialcommunitiesofseveraltypesofcoralreefalgaefound s21. Two clonal blades (hereafter ‘‘Lab’’) were frozen and stored
different microbial communities on different algal genera and forDNAextraction.Thesebladesexperiencedidenticallaboratory
suggested that coral disease is being elevated by the increase in growth conditions and reproduction in the laboratory was
fleshy algal-associated bacteria inreef environments[32–33]. exclusivelybythegenerationofneutralspores[37],whichmakes
Here we describe the bacterial community associated with P. the blades clonal. This is the isolate (‘‘P.um.1’’, UTEX LB2951)
umbilicalis by high-throughputpyrosequencing ofthe16SrDNA. thatisthesubjectofaJGIwhole-genomesequencingprojectonP.
Thisalgaiscommerciallyimportantashumanfood(‘‘laver’’),and umbilicalis [49].
it is widely distributed on rocky intertidal shores of the north
Atlantic[34–37].ExistingdataonmicrobesofPorphyrasensulato Genomic DNA isolation and amplification
(i.e., including Pyropia yezoensis, ‘‘nori’’) are limited to culture and Each sample consisted of an entire blade, allowing recovery of
cloning studies or intensive analysis of a few pathogenic bacteria bacterialivingonorinsideanypartoftheblade.Eachbladewas
belonging to Flavobacteria and Gammaproteobacteria ([38–46]). groundinliquidnitrogenusingasterilemortarandpestle.Abouta
It is clear, however, that there is potential for discovery of much 200ml volume of the powdered tissue was transferred into an
new microbial biodiversity associated with this group of algae, as 1.5 ml microcentrifuge tube containing lysis buffer and RNase A
demonstratedbytherecentdescriptionoftheclassPhycisphaerae solution. Tubes were placed on a rotator for 12min at room
(Planctomycetes) from a Porphyra sp. [47]. The purpose of our temperature, and then genomic DNA was extracted using the
studies was to describe the natural community on blades DNeasyHPlant Mini Kit (Qiagen, Valencia, CA).
(gametophyes) in the intertidal zone at two different times, to ToexplorethediversityofbacteriaassociatedwithP.umbilicalis,
compare the natural community to that remaining on clonal, we obtained sequences of the V5–V7 and V8–V9 regions of the
antibiotic-treatedbladesthatgrownormallyinlaboratoryculture, 16S rRNA gene. Whole blade material was used for DNA
and to discover whether a core microbiome exists over the three extraction to minimize the potential loss of bacteria living inside
conditions. blades, but this also leads to the extraction of chloroplast 16S
rDNA, which would be amplified by most primers that amplify
Methods bacterial 16S rDNA sequences. To overcome this problem, we
used a modified primer set 799f [59-AMCVGGATTAGA-
Ethics Statement
TACCCBG] [50] and 1492r [59-GYTACCTTGTTAC-
Thescientificresearchandcollectingpermitswereobtainedfor GACTT] [51] that amplifies most bacterial sequences while
the described field studies from the United States Department of excluding amplification of chloroplast 16S rDNA [50]. PCR
theInterior,NationalParkService,AcadiaNationalPark(permit reactions were performed using the GoTaqH Green Master Mix
#s:ACAD-2008,2009, 2010,2011-SCI-0004). (Promega, Madison, WI) in a 25ml total volume (about 80ng
genomic DNA as template, 1 ml each of 10mM forward and
Field samples reverse primers, 9.5ml sterile water and 12.5 ml GoTaq premix).
Porphyra umbilicalis was collected from the intertidal zone at PCRwasperformedusingthefollowingprotocol:94uCfor2min
Schoodic Point, Acadia National Park, Maine (44u 209 1.68" N, followedby30cyclesof20sat94uC(denaturation),40sat53uC
68u3929.14"W)onOctober28,2010(fall)andJanuary3,2011 (annealing),and40sat72uC(extension),withafinalextensionat
(winter). Five P. umbilicalis blades were collected individually in 72uC for 7 min. Two products of different size were obtained: a
plastic bags using random numbers’ sampling along a 30m mitochondrial product (ca. 1100bp in length) and a bacterial
transect placed through the middle of the intertidal zone in each product(ca.750bp).Thebacterialproductwasexcisedfromthe
season. Positions for fall blades along the transect were 0 m (F1), agarosegelandrecoveredusingtheillustraTMDNAandGelBand
3 m (F2), 5 m (F3), 7m (F4), 13m (F5); winter blades were PurificationKit(GEHealthcare,Piscataway,NJ).Becausespecies
collected at 3 m (W1), 10m (W2), 16m (W3), 26m (W4) and diversityinamicrobialassemblagefromthesamesamplecanvary
28m(W5).Bladesweretransportedbacktothelaboratoryonice, due to different variable regions used in analysis [52–54], we
rinsedbrieflyin15uCautoclavedseawaterwithin2 hofcollection compared the bacterial composition during fall 2010 and winter
to remove any superficially-attached microbes, frozen in liquid 2011usingtwoprimersetsthatrecovereddifferenthypervariable
nitrogen, and stored at 280uC. The P. umbilicalis blade samples regionsofthe16SrDNA.The750bpPCRproductwasusedas
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BacterialCommunityofPorphyraumbilicalis
templateforasecondroundofPCRfollowingthesameprogram samplewiththelowestnumberofsequences);however,weconfine
as above, with the primer pairs 799f and 1192r (59- ouranalysesofV5V6totaxondiscoveryinsupportoftheV8data
CRTCCMCACCTTCCTC), for the V5–V7 region, and 1192f (see Results and Discussion). A large proportion of sequences in
(59-GAGGAAGGTGKGGAYG) -1492 r for the V8-V9 region. the V5V6 library remained unclassified below the domain
The PCR products were purified using the DNA Clean and Bacteria (Table 1), and these were asymmetrically distributed
ConcentratorTM -5kit (Zymo Research, Irvine,CA) prior to 454 among samples.
sequencing. Rarefaction analysis in mothur was used to examine the
adequacy of the sampling depth for OTUs formed at 0.03
Bacterial Tag-Encoded FLX 454-Pyrosequencing distance. V8 samples were rarefied by group over number of
Bacterialtag-encodedFLX16SrDNAampliconpyrosequenc- samplesobtainedforfall(n=5),winter(n=5)andlab(n=2).V8
ing (bTEFAP) [55] was conducted by the Research and Testing samples were normalized to the number of sequences in the
Laboratory (RTL; Lubbock, TX) using the Genome Sequencer smallestsample(L2,n=1943sequences,Table1).Alpha-diversity
FLX instrument with Titanium protocols and reagents (Roche wasstudiedinthecuratedsamplesbeforeandafternormalization.
Applied Science, Indianapolis, IN). All PCR products used as Theobservedrichnessofsamplesat0.03,0.05,and0.10distance
sequencing templates were adjusted to 20ngml21. Two pyrotag (i.e., 97%, 95%, 90% sequence similarity) was compared to the
sequence libraries were generated by RTL from each of the 12 minimumpredictedrichnesscalculatedbytheChao1estimatorin
Porphyra blades using the primers given above, with barcoded mothur. Diversity was examined by the inverse Simpson index.
sequences in each library recovered by sequencing from V5 The microbiome was further analyzed by identifying OTUs
through V7 or from V8 through V9. Raw sff files were provided formedat0.03distancethatheld$1%ofsequencesintheV8and
byRTLforanalysis,andtheseareavailableintheSequenceRead V5V6 samples, and OTUs (rare or abundant) that were shared
Archive (SRA, www.ncbi.nlm.nih.gov) under accession over at least one sample of each of the three groups (fall, winter,
SRA065667/SRP018227.Weusedthemothur(v.1.27.0)software lab) were recovered with the Venn command in mothur. The
package [56] for curation and analysis of sequences. Mothur’s UPGMA algorithm using distance as calculated with the Jaccard
implementationofPyroNoisewasusedwithadefaultof450flows coefficient was used to examine the dissimilarity of samples by
indenoising,andsequencesweretrimmedtoaminimumlengthof membershipwithadendrogramofthenormalizedV8subsample
200bp with default parameters in mothur. Sequences were while a distance matrix calculated using the Yue-Clayton theta
aligned against the silva database in mothur, filtered, trimmed, similarity coefficient was used to produce a dendrogram that
andpre-clusterediftheywerewithin2basesofamoreabundant describes the samples based on abundance and membership.
sequence.Chimerasweredetectedwithmothur’simplementation Dendrogramstructurewastestedforstatisticalsignificancewitha
of uchime, using each library as the reference set. The Gold
reference database as an option in chimera.uchime (mothur.org)
Table1.Statisticsforthesequencesretrievedfromthe16S
often works best for microbial communities that are well-studied,
rDNAover theV5–V6 andV8 variableregions.
and it identified only about a third of the chimeric sequences
foundbyusingtheP.umbilicalispyrosequencingdataasaninternal
reference to detect chimeras with the chimera.uchime command
[57].Sequences identified as being chimericinthe12samplesin A.Totalsequences
each dataset (V5V7: 39.6%+/28.4% [mean, SD]; V8V9: V5–V6 V8
33.2%+/25.5% [mean, SD]) were removed using the mothur Totalsequences 106,366 41,514
pipeline.
ClassifiedBacteria 79,532 37,671
Curation of the V5V7 sequences to reduce pyrosequencing
UnclassifiedBacteria 26,834 3,843
errorsresultedinadatasetinwhichsequenceshadameanlength
of 266 bases (range: 249–281). Sequences began near E. coli
position800[58]andendednearposition1066;thisdatasetthus B.Totalsequencesbysample
(plastidsequencesremoved
beginsintheconservedregionprecedingV5andextendsthrough
variableregionV6toendintheconservedregionforwardofthe V5V6 V8
V7;thisdatasetisreferredtointheResultsastheV5V6dataset. Fallblade1 8154 2613
Curation of the V8V9 sequences produced a dataset in which Fallblade2 3516 3864
sequence mean length was 222 bases (range 197–232 bases)
Fallblade3 10,296 2438
beginning near E. coli position 1190 and ending near position
Fallblade4 8405 2936
1407. This means that these sequences include most of the
conserved region before V8 and end just prior to the V9 region; Fallblade5 7393 2863
thisdataset isreferred toas V8intherestof thisstudy. Winterblade1 7875 4362
Winterblade2 2658 3771
Taxonomic classification, estimation of community
Winterblade3 2581 4279
richness and statistical analysis
Winterblade4 18129 4874
Sequences were classified using the Ribosomal Database
Winterblade5 22790 5397
Project’s rdp9 reference set in the mothur pipeline at an 80%
Labblade1 8210 2174
confidence level, whichis regarded as 89% accurate in classifica-
tionatthegenuslevel[59].Operationaltaxonomicunits(OTUs) Labblade2 6359 1943
wereformedat0.03,0.05and0.10distances,whicharegenerally Total 106,366 41,514
considered to correspond to species-, genus- and family-level
Totalsequencesarethoserecoveredafterqualitycuration(denoising,
classifications, using the average neighbor algorithm in mothur.
trimming,chimeraremoval,andremovalofP.umbilicalischloroplast
WepresentalphaandbetadiversitymetricsforV8samplesbefore
sequences).
and after normalization (i.e., normalized in mothur to the V8 doi:10.1371/journal.pone.0058269.t001
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BacterialCommunityofPorphyraumbilicalis
weighted unifrac test in mothur. Thetayc (based on membership in overall sequence abundance and in the number of OTUs
and abundance of OTUs) and Jaccard (based on membership) formedat0.03distance(Figs1,2;TablesS1,S2,S3inFileS1).The
distance matrices were used in Analysis of Molecular Variance V5V6libraryidentified6ofthe7samephylainfieldblades,but
(AMOVA)totestwhetherthevariationamongfall,winterandlab TM7 was found, whereas Deinococcus-Thermus (Fig.1) was not.
normalized V8 samples was greater than variation between Both of these phyla were identified at trace levels from the
samples within each group. The level of significance for each respective libraries. Proportional assignment of sequences to
experiment’s AMOVA tests was set at 0.0125 (4 tests in each different phyla in the V5V6 library was similar to V8 except for
experiment). Biplots of OTUs significantly (p-value #0.05) apotentiallyequalabundance(buthighvariabilityamongblades)
associated with the axes of the non-metric dimensional scaling ofunclassifiedChloroflexicomparedtoBacteroidetes(Fig.1).The
(nmds)ordinationusedtoplotrelationshipsofthe12sampleswere penicilin-streptomycintreated,clonallaboratorybladesalsohada
plotted using the statistical computing package R (v.2.5.1[60]), high proportion of Bacteroidetes but the proportions of Sphingo-
including the vegan package (v. 2.0–5, [61]). Axis 1-axis 3 plots bacteria(1%)comparedtoFlavobacteria(75%)onlabbladeswere
from a 3-dimensional nmds plot (R=0.99, stress=0.0399) are closetoinverseproportionsoftheseclassesonfieldblades(Figs.1,
shown;calibrationswith3abundantOTUsthatwerefoundonly 2; Tables S1, S2 in File S1). Alphaproteobacteria and Gamma-
inwinterorfalloron labbladesshowedthatOTUssignificantly proteobacteria were also common on lab blades whereas
associated with axis 3 show affinity for the winter group; OTU Actinobacteria, Deinococcus-Thermus, Firmicutes and TM7
vectors that point away from the axis 1 label have lab group appeared absent, no Betaproteobacteria or Deltaproteobacteria
affinities; OTU vectors that point toward the axis 1 label in the werefound,andChloroflexiwererarewithinbothV8andV5V6
biplot graph have fallaffinities. libraries on laboratory blades (Fig. 1, Tables S1, S2 in File S1).
We obtained relevant environmental data for the Maine coast The laboratory blades completed their normal [37] northwestern
close to our study site from a coastal GoMOOS environmental Atlanticlifehistory(i.e.,reproductionsolelybyneutralspores)with
station (http://www.neracoos.org/gomoos; Accessed 2012 April agenerationtimeasshortas64daysatthetimeofsamplingoflab
16) from October 1–31, 2010 (fall) and December 1–31, 2010 bladesfor thisstudy.
(winter).PhotoperiodwasestimatedfromtheEldridgeTideandPilot Sixty-one genera were found within the V8 library among the
Book2012[62].Althoughouranalysisofthefallandwinterblades classifiedbacterialsequences,and76generawerefoundwithinthe
likelyshowsseasonaltrends,weemphasizethatanadditionalyear V5V6 library. Forty-eight of the genera recovered from the V8
ofsamplingineachseasonwouldberequiredtoconfirmthemas and V5V6 sequences represent known (named) taxa (Table 2).
seasonal differences, as opposed to two different time points, Most of these were members of the Saprospiraceae (Sphingobac-
because for logistical reasons, sampling was performed only once teria),Flavobacteria,AlphaproteobacteriaandGammaproteobac-
per season. teria.Labbladeslacked26ofthese(Fig.1,Table2).Asignificant
proportionofsequencesremainedunclassifiedatlowertaxonomic
Results levels, including within the Saprospiraceae and Cytophagaceae
(Sphingobacteria) and Flavobacteriales (Flavobacteria) of the
Classification of the reads Bacteroidetes, the Chloroflexi, the Sphingomonadales (Alphapro-
A total of 106,366 (V5V6) and 41,514 (V8) sequences teobacteria), and within the unclassified Bacteria (Fig. 1, Tables
(Table 1) were obtained for analysis from raw reads following S2–S3 in File S1). Lewinella (Saprospiraceae) was particularly
curation that included denoising, trimming, identification and abundantonP.umbilicalisblades,representingatotalof3.35%of
removal of chimeras (see Methods), and removal of a small all sequences recovered in theV8 library (all from field samples),
number of chloroplast sequences. Sequences assigned to the which clustered into 66 OTUs, by far the most OTUs within a
Cyanobacteria_Chloroplast category by the rdp9 database in described genus (Tables S2–S3inFile S1).
mothur were all identified as Porphyra umbilicalis chloroplast
sequences (best BLAST hit, followed by hits to four other Alpha-diversity metrics of microbial blade communities
bangialeanplastids)andremoved(1.1%[V5V6]and0.3%[V8– (V8 tags)
V9] of sequences before this final curation) from trimmed and
A total of 2,526 OTUs were formed at 0.03 distance from
chimera-checked sequences before tabulation of curated se-
sequences in the V8 library (Table S3 in File S1). Most of these
quences (Table 1). Most of the curated sequences (V5V6:
OTUswereformedfromonly1sequence(73.8%)andonly3.6%
74.8%; V8: 90.7%) were classified to a particular bacterial
ofOTUswerecomprisedof$20sequences.Rarefactionanalysis
phylum and class (or lower taxon). The proportion (25.2%,
ofindividualsamples(Fig.3)beforeandafternormalizationfound
V5V6;9.3%,V8) of sequences that wereunclassified below the
that the sampling depth was best at describing the microbial
levelofBacteria(Table1)probablyrepresentsamixtureoftrue
community in the laboratory blades and some winter blades;
biodiversity that was not classified as well as complex chimeras
rarefactioncurvesapproachedaplateauforL1,L2,W3andW4.
thatwerenotidentifiedbychimera.uchime(seeMethods).Some
Todeterminehowwellthesamplingdepthdescribedrichnessby
V5V6 samples were dominated by these sequences, however,
group(fallvswintervslab),weconsideredrarefactionoverblades
andwelimitouruseofV5V6datatoobtaininformationonthe
withineachgroupinmothur(Fig.4).Thisanalysisshowedthata
core microbiome and taxon memberships of the classified
sample size of 5 blades was too low to describe the microbial
Bacteria (Table 1).
community of P. umbilicalis at the fall sampling date; this sample
size was more suitable for the winter time point, although
Taxonomic overview of bacterial communities on additional sampling would clearly find additional OTUs (0.03
Porphyra umbilicalis distance).Thegrouprarefactionanalysisshowedthatanalysisof2
Seven phyla (Bacteroidetes, Proteobacteria, Actinobacteria, bladesfromtheclonallaboratorycultureswassatisfactory(Fig.4).
Chloroflexi, Planctomycetes, Firmicutes, Deinococcus-Thermus) The number of OTUs observed at 0.03 (‘‘species’’), 0.05
were identified in the V8 library (Figs 1, 2; Table S1 in File S1). (‘‘genus’’)and0.10(‘‘family’’)distancewascomparedtoestimates
The classes Sphingobacteria (Bacteroidetes) and Alphaproteobac- oftheminimumtruepredictedrichnesswiththeChao1estimator
teria(Proteobacteria)dominatedfieldbladesintheV8libraryboth (Fig.5,TableS4inFileS1).ThenumberofobservedOTUsata
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BacterialCommunityofPorphyraumbilicalis
greyforabsenceinallblades;whiteforabsencewhenthetaxonwas
recoveredinatleastoneblade(n=12),lightpink=.0to1%;orange
=.1% to 10%; red =.10% to 50%; and dark red =.50%. These
categories make it possible to compare blades across libraries, but
strictly the colors apply only vertically within each sample. The
taxonomiclevelreferstophylum(2),class(3),order(4),family(5)and
genus(6)hereandinthesupplementaltables.
doi:10.1371/journal.pone.0058269.g001
0.03distance ranged betweensamples from336–448(F4,F1)for
fallblades,183–420(W3,W5)forwinterblades,andwaslowestat
74–79(L2,L1)forthelabblades.TheChao1estimatorpredicted
manyadditionalOTUs:arangeof1006–1868(F4,F5)OTUsfor
fall blades, 588–1672 (W3, W5) for winter blades, and 196–222
(L2, L1) for lab blades (Fig. 5, Table S4 in File S1). The relative
order of observed OTU richness in individual samples was
virtually identical with (L2 #L1#W3 #W4 #W2 W1=F4#F2
#W5#F3 #F1#F5) and without (L2 #L1#W3 #W4
#W2#W1#F4#W5#F2#F3#F5#F1)consideringtheOTUs
belonging to unclassified Bacteria in the V8 library (1,087 of the
2,526 total OTUs). Normalization of sampling depth (see
Methods, Fig. S1) reduced the total number of OTUs to 1,705
OTUs (0.03 distance) with a range of 219–369 (F2, F3) OTUs
observed over individual fall samples and a Chao1 prediction of
618–1604 (F2, F1); individual winter samples ranged from 108–
185 observed OTUs (W3, W5) and 440–834 predicted OTUs
(W3,W5)for.Normalizedsamplesforlabbladeshad74observed
OTUs and 196 Chao1 predicted OTUs. The inverse Simpson
diversity index was nearly identical for each sample before and
after normalization, and it was lowest at about 1.5 for W4 and
highestat27forF1(Fig.5,TableS4;W4<W3,W5,L1<W1
< L2 < F2,W2 < F4,F3,F5,F1). Similar trends were
observedforthe‘‘genus’’-level(95%similarity)and‘‘family’’-level
(90%similarity) OTUs (Table S4inFile S1).
TheBacteroidetes(866OTUs)andProteobacteria(510OTUs;
337 in Alphaproteobacteria and 96 in Gammaproteobacteria)
were OTU-rich over all samples (Fig. 2B, Table S3). The
proportionofOTUsinBacteroideteswashighinSphingobacteria
in field blades compared to Flavobacteria, and this relationship
was reversed for lab blades (Fig. 2, Table S3 in File S1).
Unclassified Bacteria (Table 1) formed a mean of 37% of all fall
OTUs,41%ofwinterOTUs,and18%oflabOTUs(TableS1in
FileS1,Fig.2B);mostoftheseOTUswererepresentedbysingle
sequences.
Sample and group structure
Although subsampling reduced the number of OTUs (Table
S4), the normalized community remained representative of the
parent sample (cf. Fig 2A and 2C, Table S1 in File S1) and was
used to test whether microbial communities on blades were
dissimilartoeachotherandwhethertherewassignificantstructure
among the fall vs winter vs lab microbial communities. The
dendrogramproducedbyclusteringsamplesbasedupontheYue-
Clayton similarity coefficient (thetayc) (Fig. 6) had significant
structure(branch-weightedunifrac:F-L,p,0.006;F-W,p,0.001;
L-W, p,0.001). Microbial communities of lab blades segregated
fromothersamples,andthepositionofmicrobialcommunitieson
fallandwinterbladesinthedendrogramdidnotrelatecloselyto
blade positions along the linear transect in the intertidal zone. A
dendrogram based upon distances from the Jaccard calculator
(Fig. S2) also showed significant structure (branch-weighted
Figure1.Taxonomicclassificationofsequencesonindividual unifrac: F-L, p=0.006; F-W, p,0.001; L-W, p,0.001). Lab
bladesofPorphyraumbilicalis(%abundancenormalizedtothe communities and fall communities formed two discrete clusters,
number of sequences in each sample) using sequences andtherewasmoretrendinthedendrogramtowardrelevanceof
recovered from both V8 and V5V6 libraries. Color codes are
theblade’stransectposition.Themicrobialcommunitiesfromthe
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BacterialCommunityofPorphyraumbilicalis
Figure2.BacterialcommunityrecoveredintheV8libraryfromPorphyraumbilicalisinfall(n=5blades),winter(n=5blades)andlab
P.um.1(n=2blades).Pie-chartsforthemeanproportionofA)sequences,B)OTUs(0.03distance),andC)sequencesinnormalizedsamplesthat
belongtoparticularphylaandclasses.
doi:10.1371/journal.pone.0058269.g002
threegroups(fall,winter,lab)weresignificantlydifferent(p,0.007 2494 of the Hyphomonadaceae (Alphaproteobacteria) was also
forgroupandallpair-wisetestsineachanalysis,TableS5inFile present on all 12 blades, but it was uncommon on most field
S1) from each other in AMOVA based on two different analyses blades. The sampling depth (Table 1) across fall (n=14,714
(Jaccard, thetayccalculators, see Methods). sequences)andwinter(n=22,683sequences)wasabout26higher
forwinterinthecuratedsequences,butamongthesecoreF-W-L
The core microbiome and most abundant OTUs OTUs,sixwere$36moreabundantinwinterthanfall,fourwere
WeidentifiedsharedOTUs(0.03distance)ineachlibrary(V8, evenlydistributed,andtwowere$36moreabundantinfallthan
V5V6) without sample normalization in order to use the greatest winter(Table3).Ineachseason,therewastaxonomicdiversityin
sequencingdepthacrosssamplestoidentifythenumberofOTUs the core microbiome (Table 3, Fig. 8, Table S9 in File S1), and
shared among groups (i.e., sequences merged across samples although most of these OTUs were very abundant, several were
withinfall,winter,andlabgroups).OTUs(0.03distance)formed not.
from V5V6 sequences for this analysis represented 4,599 OTUs, Fall and winter microbial communities on blades shared 269
and2,309ofthesewerefoundinBacteriathat classifiedtophyla OTUs(Fig.7).ThesharedOTUs(TableS6inFileS1)belonged
and lowertaxonomic levels. toIamia(Actinobacteria);Aureispira,Haliscomenobacter,Lewinella,and
The core microbiome (Fig. 7, Table 3) of P. umbilicalis based unclassified Sphingobacteriales including Saprospiraceae and
upontheV8analysisfound13OTUsincommon,and11OTUs Chitinophagaceae (Sphingobacteria); Tenacibaculum (Flavobac-
hadanabundanceofatleast1%ofallseqeuencesonatleastone teria); Rhodopirellula (Planctomycetacia); Hellea, Loktanella, and a
ofthe12blades,includingGranulosicoccus(Gammaproteobacteria). large proportion of unclassified Rhodobacteraceae (Alphaproteo-
EightofthecoretaxaweremembersoftheSphingobacteria;four bacteria); andGranulosicoccus (Gammaproteobacteria).
ofthesebelongtotheSaprospiraceae,includingonefoundacross Twenty-threeofthemostsequence-richOTUsintheV8library
all12bladesathighabundance,beingrepresentedbymorethan (N.B. 37 OTUs contained $1% of all sequences on at least one
1%ofallsequencesfromeachblade(OTU2521,seeTable3.A). field blade) were at least 36more abundant in fall compared to
Aswiththefullcommunityanalyzedabove,themajority(12of13) winter (Table S7 in File S1). Of the eight OTUs that had $1%
of the core OTUs were Bacteroidetes and Proteobacteria, abundance on at least one lab blade, six OTUs were more than
including four OTUs belonging to Alphaproteobacteria. OTU 36asabundantinwinter,and3OTUswerenotfoundamongfall
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BacterialCommunityofPorphyraumbilicalis
Table2.Taxonomicsummaryofnamedgenerafoundinthe Bacilli(Firmicutes) Aeribacillus*
V5V6orV8libraries (I)and taxafound inonly onelibrary Anoxybacillus*
(V5V6,II; V8,III).
Staphylococcus*
TM7 (unclassified)*
II.TaxafoundbyV5V6andnotV8
I.GenerarecoveredbyV5V6orV8tags
Actinobacteria Propionibacterium
Actinobacteria(Actinobacteria): Ilumatobacter*
Lacinutrix
Iamia*
Pibocella
Propionibacterium*
Flavobacteria(Bacteroidetes) Tamlana
Sphingobacteria(Bacteroidetes) Aureispira*
Winogradskyella
Haliscomenobacter*
Zobellia
Lewinella*
Chloroflexi Caldilinea
Saprospira*
Planctomycetes Planctomyces
Flavobacteria(Bacteroidetes) Aureispira*
Alphaproteobacteria(Proteobacteria) Jannaschia
Haliscomenobacter*
Ketogulonicigenium
Lewinella*
Oceanibulbus
Saprospira*
Pelagicola
Maribacter
Tropicimonas
Pibocella
Deltaproteobacteria(Proteobacteria) (nonerecoveredinV8)
Tamlana
Peredibacter
Tenacibaculum
Bdellovibrio
Winogradskyella
Desulforhopalus
Zobellia
Gammaproteobacteria(Proteobacteria) Alteromonadaceae
Caldilinea(Chloroflexi) Caldilinea*
Aestuariibacter
Deinococci(Deinococcus-Thermus) (allunclassified)*
Congregibacter
Planctomycetacia(Planctomycetes) Planctomyces*
TM7 (nonerecoveredinV8)
Rhodopirellula
III.TaxafoundbyV8butnotV5V6
Phycisphaerae(Planctomycetes) Phycisphaera
Sphingobacteria(Bacteroidetes) Auresipira
Alphaproteobacteria(Proteobacteria) Hellea
Saprospira
Hyphomonas
Krokinobacter
Litorimonas
Deinococcus-Thermus (nonerecoveredinV5V6)
Robiginitomaculum*
Alphaproteobacteria(Proteobacteria) Roseovarius
Jannaschia*
(Alphaproteobacteria)
Ketogulonicigenium*
Betaproteobacteria(Proteobacteria) (nonerecoveredinV5V6)
Loktanella*
Deltaproteobacteria(Proteobacteria) Bacteriovorax
Oceanibulbus*
Gammaproteobacteria(Proteobacteria) Pseudoalteromonas
Pelagicola
Neptunomonas
Sulfitobacter
Acinetobacter
Tropicimonas
Sphingopyxis *Notpresentinlaboratoryblades
doi:10.1371/journal.pone.0058269.t002
Deltaproteobacteria(Proteobacteria) Bacteriovorax*
Peredibacter*
sequences(TableS7inFileS1).ThemostabundantOTUsinfield
Bdellovibrio* samples (Table 3) included ones belonging to the Saprospiraceae
Desulforhopalus* (Sphingobacteria) including two of the 66 OTUs classified to
Gammaproteobacteria(Proteobacteria) Aestuariibacter Lewinella(Table3,TableS3inFileS1),aswellastoFlavobacteria,
and Alphaproteobacteria. The most abundant OTUs on both
Pseudoalteromonas*
laboratory blades ($1%) belonged to the Bacteroidetes, Plancto-
Psychromonas*
mycetes, and Proteobacteria including Sphingopyxis in Alphapro-
Granulosicoccus
teobacteria(Table3).Biplots(Fig.9)ofthenmdsordinationofthe
Arenicella samples and OTUs that were significantly (p,0.05) associated
Cocleimonas* withoneormoreofthenmdsaxesdemonstratedthat:1)different
Congregibacter OTUs belonging to the Alphaproteobacteria (Fig. 9A) are
associated strongly with all axes (fall, winter, lab), 2) different
Neptunomonas*
OTUs belonging to Flavobacteria (Fig. 9B) were significantly
Acinetobacter*
associatedwitheitherfallorlabblades,butnotwinterblades,and
3) most Saprospiraceae OTUs (Fig. 9C) significantly associated
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BacterialCommunityofPorphyraumbilicalis
Figure3.RarefactionanalysisofV8samplesfromeachfall,winterandlabbladeshowingthemean(±95%C.I.)numberofOTUs
(0.03distance)asafunctionofsequencingdepth.NotetheoverlapofcurvesforF1,F3,F5(top);W1,W3,W5(middle);L1,L2(bottom).PanelA
containstheanalysisforsamplesbeforenormalization;PanelBshowsrarefactionanalysisforthenormalizedsamples.
doi:10.1371/journal.pone.0058269.g003
with an axis were found on fall blades but a few OTUs were OTUs). Many other OTUs in these taxonomic groups occurred
significantlyassociatedwithwinterblades(Fig.S3,seeTableS9in on the blades, but not in significant association with one of the
File S1 for data on the entire set of 67 significantly associated nmds axes(see Fig.1, Tables S2,S3in FileS1).
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BacterialCommunityofPorphyraumbilicalis
most bacteria remaining on blades were Bacteroidetes belonging
totheFlavobacteria.Thetwodayswhenwesampledbladesfrom
theMaineshorewereinmid-fallandwinter,andseveralstatistical
comparisonsfoundthatthemicrobialcommunitiesweredifferent
fromeachotheronthesetwodaysandalsothateachwasdifferent
fromthemicrobialcommunityonlabblades.Fallbladeshadthe
highest observed and predicted (Chao1) richness and the highest
diversity(inverseSimpsonIndex).OnlytwoOTUswerefoundon
all blades in this study, and only an OTU belonging to the
Saprospiraceae (Bacteroidetes) was found abundantly on all
blades.AlthoughthegenusLewinellawasfoundonallfieldblades,
noneofits66OTUswasuniversallypresentontheseblades.This
pyrosequencingstudyincreasesourunderstandingofthemicrobial
communitiesoftheredalgaeandmakesitpossibletocomparethe
bacteria associated with marine red and green algae, which have
different cell wall compositions but are two of the three known
groups of algae formedin primaryendosymbiosis.
Figure4.Rarefactionofsequencesbynumber(inanyorder)of
bladessampledineachgroupwithintheV8library(i.e.,‘‘1’’on Algal microbial communities and the core microbiome
thex-axiswouldnotrefertoeitherF1orW1).Means(695%C.I. The microbial communities of two red macrophytes from
asbrokenlines)areplotted. temperatelatitudes,Deliseapulchra[22]andGracilariavermiculophylla
doi:10.1371/journal.pone.0058269.g004
[25]werestudiedwith16SrDNAclonelibraries,andover5,000
nearly full length 16S rDNA seqeuences recently described [24]
Lewinellawasalwayspresentonfieldblades(n=10),butnotthe the microbial community of Ulva australis (Chlorophyta). These
sameOTU.SeveralofthemostabundantOTUsinLewinellawere offer among the best comparisons available to our results on P.
sequence-rich, and these OTUs affect distances between samples umbilicalis. The microbial community of G. vermiculophylla is of
withingroupsinnmdsplots(Fig.9C,Table3;OTUs2302,2475); specialinterestbecauseithasagar-richcellwalls,sufatedgalactans
however the majority of the OTUs in Lewinella were represented that are structurally similar to porphyran in the Porphyra cell wall
by singlesequences. [63]. Lachnit et al. [25] found that Gracilaria was richest in
ThecoremicrobiomeasdeterminedfromtheV5V6sequences BacteroidetesfollowedbyProteobacteria,asfoundforP.umbilicalis
(Table 1) found 19 OTUs shared between fall, winter and lab inthepresentstudy.Theyfoundtemporallyconsistentdifferences
samples,and13ofthesebelongedtoclassifiedBacteriawithinthe in the composition of the microbial community at the level of
Sphingobacteria,Flavobacteria(7OTUsincludingoneassignedto phylumbetweenGracilaria,thegreenalgaUlvaandthebrownalga
Lacinutrix), Chloroflexi, Planctomycetes (both classses including Fucus vesiculosus from individuals collected within 20m of each
Phycisphaera in Phycisphaerae), and an OTU in the Alphaproteo- other.Longfordetal.’ssimilarstudy[22]nearSydney,Australia,
bacteria (Table 4). Two classified OTUs from Flavobacteria and foundrepresentativesof7phylaonDeliseapulchara (Rhodophyta);
Alphaproteobacteria were more than 1% abundant on both BacteroidetesandProteobacteriawerealsoprominentonthered
blades. OTUs present at $1% of total sequences of at least one alga Delisea in that study. More recently, Burke et al. [24]
field blade in the V5V6 library (Table S8 in File S1) included examinedthemicrobialcommunityofU.australiswithover5,000
(Table 4) 2 Sphingobacteria, 1 Flavobacteria and 1 Chloroflexi nearly full-length 16S rDNA sequences, finding that 74% of all
that were identified at $1% abundance on at least 3 blades sequences classified to Alphaproteobacteria (Proteobacteria) and
(n=10).OnlyanOTUclassifiedtoChloroflexiwasfoundonmost 13% to Bacteroidetes. Given that only 6 OTUs (a Lewinella, 4
(n=8) bladesat $1%abundance. unclassified Rhodobacteriaceae and Erythrobacter) were found in
common across the 6 samples of Ulva blades in their study, they
Environmental conditions proposed [24] that Ulva’s obligately symbiotic microbes must
Environmental conditions were different preceding the two operate as a lottery that in functionally-equivalent, but OTU-
different collection times on the Maine shore. October air (7– distinctive, subsets can supply the host with its microbial
16uC) and water (11–13uC) temperatures were higher, and the requirements (e.g., vitamins, and plant growth regulators such as
photoperiod was longer (12 L:12D) in contrast to December the morphogen thallusin). This concept was supported by their
whenair(25–10uC)andwater(7–9.5uC)temperatureswerelower recent metagenomic study [27], which showed a highly shared
and thephotoperiodwas shorter (9L:15D) functional genome across different microbial taxa occupying the
surfacesofblades,despitethelowsimilarityinthecompositionof
Discussion species-level OTUs onindividual blades[24].Our study suggests
something similar for Porphyra blades, because even using the
Pyrosequencing of microbial communities associated with highersequencingdepthofpyrosequencing,only1OTUfromthe
Porphyra umbilicalis identified 8 phyla containing 76 genera, Saprospiraceae was found in common over all 12 blades in our
including 48 genera that have been isolated and formally study. However, the natural microbial community of the
described. Bacteria belonging to the Bacteroidetes (especially temperate Northern Hemisphere P. umbilicalis has some striking
Sphingobacteria) were most abundant and also comprised the similarity to that reported from the temperate Southern Hemi-
most species-level OTUs; however, the Proteobacteria, especially sphereU.australis[24](cf.ourFig1andTablesS1–S3inFileS1
Alphaproteobacteria, were also abundant and OTU-rich. Clonal [24]). At the level of genus, this includes the Saprospiraceae
laboratorybladesof‘‘P.um.1’’,theP.umbilicalisisolatethatispart Lewinella and Haliscomenobacter, unclassified Flavobacteriaceae,
of a whole genome sequencing project, had much reduced unclassified Planctomycetaceae (as well as Rhodopirellula for P.
microbial richness and diversity compared to field blades, and umbilicalis), and the Proteobacteria Krokinobacter, Loktanella, Mar-
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BacterialCommunityofPorphyraumbilicalis
Figure 5. OTUs richness and diversity. Richness (A) of V8 OTUs (0.03 distance) observed and those predicted (Chao1) for samples before
normalizationanddiversity(B)calculatedforsamplesbeforeandafternormalizationwiththeinverseSimpsonindex.
doi:10.1371/journal.pone.0058269.g005
ibacter, Roseovarius, unclassified Roseobacteriaceae, and Winograds- nearly 26 higher coverage of the V5V6 library. However,
kyella.Thewidespreadlevelofhorizontalgenetransferamongtaxa Chloroflexi were abundant in the V5V6 library compared to
withinandamongmicrobialtaxa[64–65]supportstheconceptof being a small component of the microbial community in the V8
a lottery of species, but there still appear to be some particular library.BothDeinococcus-ThermusandTM7wererecognizedat
macrophyte-associated taxa that deserve special attention in trace levels in sequences within the V8 and V5V6 libraries,
understanding microbial-macroalgal interactions. respectively;additionalsamplingdepthineithermightrevealboth
phyla. It is well-known that the 16S rDNA evolves at different
Variability in recovery of microbial taxa in the V5V6 ratesalongitslength[66],whichcontributestosomevariabilityin
library compared to V8 library description of the microbial community when different variable
regionsofthe16Sgeneareused.AsreportedintheResults,the
There was little variation in the taxa found to be present
primers we used to assay the V5V6 regions recognized more
betweenthetwolibraries,especiallywhentakingintoaccountthe
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