Highly Variable Bacterial Communities Associated with the Octocoral Antillogorgia elisabethae

23 Pages·2016·2.2 MB·English

microorganisms
Article
Highly Variable Bacterial Communities Associated
with the Octocoral Antillogorgia elisabethae
VeronicaRobertson1,†,BradHaltli1,2,†,ErinP.McCauley1,DavidP.Overy2,3and
RussellG.Kerr1,2,*
1 DepartmentofBiomedicalSciences,UniversityofPrinceEdwardIsland,Charlottetown,PEC1A4P3,
Canada;[email protected](V.R.);[email protected](B.H.);[email protected](E.P.M.)
2 DepartmentofChemistry,UniversityofPrinceEdwardIsland,Charlottetown,PEC1A4P3,Canada;
[email protected]
3 DepartmentofPathologyandMicrobiology,UniversityofPrinceEdwardIsland,Charlottetown,
PEC1A4P3,Canada
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
† Theseauthorscontributedequallytothiswork.
AcademicEditor:JohannesF.Imhoff
Received:7March2016;Accepted:23June2016;Published:5July2016
Abstract: Antillogorgia elisabethae (synonymous with Pseudopterogorgia elisabethae) is a common
branchingoctocoralinCaribbeanreefecosystems. A.elisabethaeisarichsourceofanti-inflammatory
diterpenes,thusthisoctocoralhasbeenthesubjectofnumerousnaturalproductinvestigations,yet
relatively little is known regarding the composition, diversity and the geographic and temporal
stability of its microbiome. To characterize the composition, diversity and stability of bacterial
communities of Bahamian A. elisabethae populations, 17 A. elisabethae samples originating from
fivesiteswithinTheBahamaswerecharacterizedby16SrDNApyrosequencing.A.elisabethaebacterial
communitieswerelessdiverseanddistinctfromthoseofsurroundingseawatersamples. Analysesof
α-andβ-diversityrevealedthatA.elisabethaebacterialcommunitieswerehighlyvariablebetween
A.elisabethaesamplesfromTheBahamas. Thiscontrastsresultsobtainedfromapreviousstudyof
threespecimenscollectedfromProvidenciaIsland,Colombia,whichfoundA.elisabethaebacterial
communitiestobehighlystructured.TaxabelongingtotheRhodobacteriales,Rhizobiales,Flavobacteriales
andOceanospiralleswereidentifiedaspotentialmembersoftheA.elisabethaecoremicrobiome.
Keywords: Pseudopterogorgia elisabethae; Antillogorgia elisabethae; bacterial diversity; microbiome;
culture-independent;gorgonian;octocoral;Bahamas;pyrosequencing
1. Introduction
Branching octocorals, often referred to as gorgonians (Phylum, Cnidaria; Order, Alcyonacea;
Families,Calcaxonia,Holaxonia,andScleraxonia),areataxonomicallydiversegroupofsessilemarine
invertebrates, which are key components of both shallow tropical reefs and deep-sea habitats [1].
InCaribbeanwaters,gorgoniansrepresentalmost40%oftheknowninvertebratefaunawithover
195documentedspecies[2]. Antillogorgiaspp.[3]arethepredominantgorgonianoctocoralsinthe
Caribbean,withover15describedspecies[4]. Antillogorgiaspp. werepreviouslyclassifiedwithin
the genus as Pseudopterogorgia prior to a recent revision of gorgonian taxonomy [5]. A. elisabethae
(syn. P.elisabethae)hasamoderatedistributionintheWestIndiesandresidesintropicalforereefsat
adepthof5–35m[4,6]. Morphologicallyrangingfromsingleplumestohighlybranchedstructures,
A.elisabethaepopulationsaregeneticallydiverse,exhibitingsignificantgeneticvariationwithinand
amongpopulationsthroughoutTheBahamas,Florida,andtheSanAndresandProvidenciaIslands
of Colombia [6–8]. The ability of gorgonians to deter predators via chemical defense may in part
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explaintheirdominanceonCaribbeanreefs[9]. Chemicalinvestigationofgorgoniansoverthepast
fourdecadeshasuncoveredawealthofstructurallydiversebioactiveterpenemetabolites.A.elisabethae
isaparticularlyrichsourceofditerpenesas78compounds,representing20distinctterpeneskeletons,
have been reported from this gorgonian [10]. One important family of diterpenes isolated from
A.elisabethaeisthepseudopterosinfamilyofmetabolites,whichareusedinseveralmarketedcosmetic
products for their potent anti-inflammatory properties [11]. Consequently, A. elisabethae is unique
among octocorals in that wild populations are harvested to supply the commercial demand for
pseudopterosins[8]. DuetotheaddedanthropogenicpressureonA.elisabethaepopulations[12]itis
importanttounderstandfactorsaffectingthehealthofthisecologicallyandeconomicallyimportant
octocoraltoensurefutureviabilityofwildpopulations.
InitiatedbyRohwerandcolleaguesin2001,16SrDNA-basedinvestigationofcoralassociated
bacterialdiversityhasrevealedthatcoralscontainspecific, stable, anddiversesymbioticbacterial
communities[13–16]. Inadditiontobacteria,coraltissuesalsohostavarietyofothermicroorganisms
suchassymbioticdinoflagellates,archaea,fungi,andviruses. Thecoral“holobiont”iscomprisedof
thecoralaswellasalloftheaforementionedcelltypes,whichliveinassociationwiththecoral[17].
Althoughsomeresearchsuggestscnidarianshostspecies-specificmicrobialcommunities[13,14,17],
contrasting evidence suggests coral microbiology is more complex than previously assumed.
Microbial communities have been shown to differ between conspecifics collected at different
sites[18–22], species [23], stages of coral development [24], and spatial location within the
holobiont [25]. Despite the ecological importance of octocorals in marine ecosystems and their
biotechnological potential as sources of potent bioactive natural products, studies characterizing
the microbiomes of octocorals are relatively rare and research has predominantly focused on
scleractinian [14,23] and diseased corals [26–28]. Studies of octocoral microbial diversity have
revealed the presence of diverse bacterial communities, which are distinct from the surrounding
environment [18,19,29–33]. The stability of bacterial communities in octocorals appears to vary
depending on the gorgonian being investigated. For example, studies of deep-sea octocorals
revealednoclearpatternofconservedbacterialconsortia[18,19], whilethebacterialcommunities
of the Mediterranean octocoral Paramuricea clavata were observed to be relatively stable both
geographicallyandtemporally[31]. Apreliminarystudyofthebacterialcommunitiesassociatedwith
threeA.elisabethaecoloniescollectedfromProvidenciaIsland,Colombiauncovereddiversebacterial
communities,whichwereheavilydominatedbyGammaproteobacteria. Atthespecieslevel,A.elisabethae
bacterialcommunitieswerepredominantlycomposedofphylotypesrelatedtoPseudomonasasplenii
andEndozoicomonasspp.[34]. Incontrasttootheroctocorals,thebacterialcommunitiesofthethree
individualsexaminedwerehighlycongruentassixofthetenmostabundantphylotypesfromeach
individual were conserved among the three coral individuals. Additionally, these six phylotypes
collectivelyrepresentedamajorproportionofthecommunityineachcoralspecimen(77%–81.2%).
ThispreviousstudyprovidedanimportantpreliminaryassessmentofthemicrobiomeofA.elisabethae;
however,duetothesmallnumberofindividualsexamined,andthelimitedgeographicscopeofthis
study,severalquestionsremainregardingthestabilityofA.elisabethaebacterialcommunities.
Herein, we employ a 16S rDNA amplicon pyrosequencing approach to describe the bacterial
communities of 17 A. elisabethae specimens collected from five locations throughout The Bahamas.
ThesecommunitieswerealsocomparedtothosepreviouslyreportedfromColombia[34]. Thegoal
ofthisstudywastodeterminethegeographicstabilityofA.elisabethaebacterialcommunitiesandto
determineifthisimportantoctocoralspecieshostsastablecoremicrobiome. Thisresearchclearly
showedthatA.elisabethaemaintainsbacterialcommunitiesdistinctfromthesurroundingseawater.
IncontrasttothepreviousstudyofColombianA.elisabethaespecimens,themicrobiomesofBahamian
sampleswerefoundtobehighlyvariablebetweenandwithinsites.Coremicrobiomeanalysisindicated
thatA.elisabethaemayrelyondiversemembersoftheAlteromonadales,Rhodobacterales,Oceanospiralles
andFlavobacterialestofulfillkeymicrobialrolesintheholobiont.
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2. ExperimentalSection
2.1. SampleCollection
A.elisabethaeoctocorals(n=17)werecollectedin2006(June),2009(June),2011(November)and
2013 (November) by SCUBA from various locations throughout The Bahamas at depths between
15and20m(Table1;FigureS1). Collectionsitelocationsareasfollows: Site1(Eleuthera),2448.55N,
7620.58W; Site2(GrandBahama),2636.23N7754.70W;Site3a(Bimini),2529.13N,7916.41W;
Site 3b (Bimini), 25 31.51 N, 79 17.95 W; Site 4 (Grand Bahama), 26 32.93 N, 78 30.99 W; Site 6a
(SanSalvador),2403.90N,7432.39W;andSite6b(SanSalvador),2403.82N,7432.63W.Locationsand
approximatedistancesbetweensites(61–1390km)areprovidedinFigureS1. Forbacterialdiversity
analysessites6aand6bweretreatedasasinglelocationduetotheircloseproximity. Coralsamples
fromthesamereefwerecollectedě10maparttoreducethelikelihoodofsamplingclonalpropagates
andallcoloniesappearedhealthyatthetimeofcollection. PriortoDNAisolation,coralfragments
werewashedthreetimesbygentleshakinginatubecontaining50mLof0.22µmfilteredseawaterto
removelooselyassociatedbacteria. Coralsampleswerefrozenondryiceandstoredat´80˝Cuntil
DNAwasisolated. TheidentityofallA.elisabethaesampleswereconfirmedbyextractionofasmall
clippingandtestingforthepresenceofpseudopterosins,achemotaxonomicmarkerusefulforthe
identificationofA.elisabethae[35]. Seawaterinthevicinityofsampledoctocoralswerecollectedfrom
sites3aand3b(Bimini,2009)aswellassites6aand6b(SanSalvador,2011). Duetotechnicalissues
encounteredduringsamplingseawatersampleswerenotcollectedadjacenttooctocoralscollected
in2006and2013. Seawater(ca. 500mL)wascollectedinZiplocbagsandfilteredthrougha0.22µm
Nalgene MF575 water filtration device (VWR International, Mississauga, ON, Canada). The filter
was aseptically removed from the filtration device and frozen at ´80 ˝C until DNA isolation was
performed. A negative control Ziploc bag was filled with 500 mL of sterile diH O and filtered as
2
describedabovetoserveasacontaminationcontrol.
2.2. DNAIsolation
IsolationofholobiontgenomicDNA(gDNA)fromspecimensAE1–AE12(Table1)wasperformed
from ~0.5 g of finely chopped octocoral branches using a phenol-chloroform based method as
previously described for A. elisabethae specimens collected from Colombia [34]. A key difference
betweenthisstudyandthepreviousreportofA.elisabethaebacterialdiversitywashowtheoctocoral
tissuewaspreservedpriortoDNAextraction. Inthecurrentstudy,octocoralsampleswerefrozen
on dry ice, while samples from the prior study were stored in phenol [34]. Holobiont gDNA was
isolated from specimens AE16–AE20 (~0.25 g of tissue) using the PowerSoil DNA Isolation Kit
(MoBioLaboratories,Carlsbad,CA,USA)accordingtothemanufacturer’sinstructions. Inallcases
isolatedDNAwasfurtherpurifiedtoremovepotentialPCRinhibitorsusingthePowerClean®DNA
Clean-UpKit(MoBioLaboratories). ToisolateDNAfrommicrobesharvestedfromthewatercolumn,
cellswerewashedoffthefiltermembraneandDNAwasextractedandpurifiedusingtheUltraClean®
WaterDNAKit(MoBioLaboratories). No16SrDNAampliconswereobtainedfromthefiltereddiH O
2
contaminationcontrol. TheintegrityofpurifiedDNAwasassessedbyagarosegelelectrophoresisand
UVabsorbance(A /A ratiosvariedbetween1.73and1.95).
260 280
2.3. TemplatePreparationfor16SrDNAAmpliconPyrosequencing
For octocorals collected in 2006 and 2009 (AE1-AE12) nearly full-length small subunit
ribosomal RNA (16S) genes amplified from gDNA were used as templates for 16S rDNA
pyrosequencingstudies. The16SrRNAgeneswereamplifiedusingthebacterialuniversalprimerspA
(51-AGAGTTTGATCCTGGCTCAG)andpH(51-AAGGAGGTGATCCAGCCGCA)[36]. PCRreactions
(50 µL) contained 1X EconoTaq Plus Green Master Mix (Lucigen, Middleton, WI, USA), 2.5%–5%
molecular biology grade DMSO (Sigma, Oakville, ON, USA), 1 µM each primer and 10–30 ng
template DNA. Thermal cycling parameters consisted of 1 cycle at 95 ˝C for 3 min, 34 cycles of
Microorganisms2016,4,23 4of23
95 ˝C for 45 s, 54 ˝C for 1 min, 72 ˝C for 1.5 min, and a final extension cycle at 72 ˝C for 10 min.
PCRproductswerepurifiedusingtheDNACleanandConcentratorKit(ZymoResearch,Irvine,CA,
USA).TriplicatePCR16SrDNAampliconsfromeachcoralholobiontgDNAsamplewerepooledto
reducepotentialamplificationbiases[37]. Pyrosequencingwasconductedonallothersamplesusing
gDNAasatemplate.
2.4. 16SrDNAAmpliconPyrosequencing16SrDNAAmpliconPyrosequencing
Bacterial16SrDNAampliconsfromoctocoralsandseawaterDNAsamplescollectedin2006and
2009weresequencedbyResearchandTestingLaboratories(RTL;Lubbock,TX,USA)asdescribed
previously [38,39]. For octocorals AE1–A12 and seawater samples W1 and W2 a portion of the
16S rRNA gene encompassing the V1 to V3 variable regions was amplified from gDNA samples
(W1/W2)orfrompoolednearlyfull-length16SrDNAPCRamplicons(octocoralsamples)(20ng)
usingtheprimerpairpAand16S519r(51-GAATTACCGCGGCGGCTG)accordingtopublishedRTL
procedures[39]. SequencesinthisdatasetspannedtheV1andV2regionsofthe16SrDNAandare
herein referred to as the V1/V2 dataset. To assess variability between 16S rDNA pyrosequencing
experiments,replicatetemplatesintheformofpooled(triplicate)16SrDNAamplicons,weregenerated
fromthesameA.elisabethaegDNAsampleandsequencedinseparateindependentsequencingruns
(sequencelibraries1Aand1B).
Forsamplescollectedin2011and2013(octocoralsAE16–AE20;seawatersamplesW13–W26),
theV4regionofthe16SrRNAgenewassubjectedtoampliconpyrosequencingusingtheprimerpair
F515(51-GTGCCAGCMGCCGCGGTAA)andR806(51-GGACTACHVGGGTWTCTAAT)andgDNAas
template.[40]. Theseprimerswereusedbecausetheywereshowntobemoreeffectiveatamplifying
16SrRNAgenefragmentsfromsmallquantitiesofDNA(personalcommunication,Dr. ScotDowd,
MRDNA,Shallowater, TX,USA).Sensitivitywasanimportantconsiderationbecauseweinitially
attemptedtocomparethebacterialcommunitiesofthreetissuefractionsfromindividualoctocoral
colonies: (1) holobiont (i.e., derived from intact octocoral branches); (2) A. elisabethae larvae; and
(3)purified zooxanthellae(symbioticdinoflagellates). Unfortunately, the smallquantitiesof DNA
obtained from the larvae and zooxanthellae fractions did not generate sufficient pyrosequencing
datatoenableathoroughanalysis(datanotshown). Incontrast,DNAisolatedfromtheholobiont
tissuesamplesandseawatersampleswerenotlimitingandhighqualitypyrosequencingresultswere
obtainedfromtheseDNAsamples(referredtohereinastheV4dataset). WhiletheV4datasetisnot
directlycomparabletotheV1/V2dataset,itwasincludedinthisstudytofurtherexaminedifferences
between octocoral and water microbiomes. V4 amplicons were generated using HotStarTaq Plus
MasterMixKit(Qiagen,CA,USA)accordingtothemanufacturer’srecommendations.Thermalcycling
conditionsconsistedof94˝Cfor3min,followedby28cyclesof95˝Cfor30s,53˝Cfor40sand72˝C
for1min,followedbyasingleelongationstepat72˝Cfor5min. Followingamplification,thePCR
productsfromdifferentsamplesweremixedinequalconcentrationsandpurifiedusingAgencourt
AMPurebeads(AgencourtBioscienceCorporation, MA,USA).SequencingwasperformedatMR
DNA(Shallowater,TX,USA)utilizingRoche454FLXTitaniuminstrumentsandreagentsaccordingto
themanufacturer’sguidelines.
2.5. PyrosequencingDataAnalysis
SequencedatafortheV1/V2datasetwereprocessedusingMOTHURversion1.32.0[41]according
topublishedrecommendations[42,43]andaspreviouslydescribed[34]. Briefly,keysettingsinthe
data analysis were as follows. The number of flows was trimmed to 450 using trim.flows and
data were denoised using PyroNoise (shhh.flows). Sequences were removed if they contained
homopolymers greater than 8 bp in length, ambiguous bases, more than two mismatches to the
forwardprimersequence,morethanonemismatchtothebarcodesequenceorwereshorterthan200bp.
SequenceswerealignedinMOTHURusingtheSilvareferencealignment. Alignmentswerefiltered
toremovegapsandsequenceswerepreclusteredallowingfortwonucleotidedifferencesbetween
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sequences (diffs = 2). Chimeras were removed using UCHIME. Sequences from three Colombian
A. elisabethae specimens (SRA043548) [34] were processed with sequences from Bahamian corals
collected in this study to ensure consistency in data processing. The V4 dataset was analyzed as
describedabovewithafewmodificationstoaccommodatetheshorterlengthoftheV4amplicons.
Aminimumof360flowswassetastheminimumnumberofflowsforthetrim.flowscommandand
theminimumsequencelengthwasdecreasedto150bp. Thesequalityfiltereddatasetswereusedfor
allsubsequentanalyses.
2.6. BacterialCommunityAnalysis
Tocomparetheclasslevelcommunitycompositionbetweensamples,sequenceswereclassified
usingtheMOTHURBayesianclassifier(80%confidenceinterval)employingtheMOTHUR-formatted
versionoftheRibosomalDatabaseProjecttrainingset(v. 9)[44]. Sequencesclassifiedaschloroplasts,
mitochondria or “unknown” (i.e., not classified at the domain level) were removed from the
analysis. Graphs comparing community composition were prepared using Microsoft Excel 2010.
To compare the diversity, membership and structure of bacterial communities from different
samples an OTU-based approach was employed. OTUs were identified at the species level using
a sequence similarity cutoff of 97% (D = 0.03) as described previously [43]. Alpha diversity
measuresofobservedrichness(S ),andChao1estimatedrichness(S ),alongwiththeShannon
obs est
diversity (H1) and equitability (E) indices were calculated using MOTHUR [41]. To enable direct
comparisonsbetweenoctocoralsamples,thesecalculationswereperformedonsubsampleddatasets
(2200 sequences for the V1/V2 dataset; 2600 sequences for the V4 dataset). To compare richness
between samples and to evaluate sampling coverage, rarefaction curves were generated using
MOTHUR. Statistical comparisons of diversity measures observed between sample groups were
performedusingMiniTab17. TheMann–Whitneytestwasusedforpairwisecomparisons,whilethe
Kruskal–Wallistestwasusedtotestfordifferencesbetweenthreeormoregroups. Toexaminebeta
diversity (similarities or differences between samples) community structure was compared using
the Yue and Clayton measure of dissimilarity (θ-YC). Dendrograms displaying the relationships
between samples were created from θ-YC distance matrices using the tree.shared command
which utilizes the unweighted pair group method with arithmetic mean clustering (UPGMA)
algorithm. TreeswereviewedusingFigTreev. 1.4.0[45]. Todetermineifdifferentgroupsexhibited
statistically significant clustering the parsimony and unweighted UniFrac tests were conducted.
Similarity percentage (SIMPER) analysis was utilized to calculate similarities between bacterial
communitiesofsamplescollectedfromasinglesite,aswellastocalculatedissimilaritiesbetween
sites. Analyseswereconductedonpercentnormalizeddatamatricesofclassleveltaxadistribution
among samples. SIMPER analysis was conducted using PRIMER v. 5.2.4 (Primer-E Ltd., Lutton,
UK)[46]. PutativemembersofthecoremicrobiomeweredefinedasspecieslevelOTUsobservedin
ě50%ofA.elisabethaesamples[47]. OTUswereexcludedfromthislistiftheiraverageabundance
in seawater samples was greater than in A. elisabethae samples. Principle component analysis
(PCA)wasusedtomodelthedistributionofoctocoral-associatedbacterialdiversitybygeographic
locationintheV1/V2dataset. PCAwasconductedusingthespecieslevel,percentnormalized,OTU
datamatrix(OTUsdefinedasvariables).PCAwasperformedinUnscramblerX(version10.1;CamoASA,
Oslo,Norway)usingthesinglevaluedecompositionalgorithmandvalidatedbycrossvalidation.
2.7. AccessionNumbers
Pyrosequencing data have been archived in the NCBI Sequence Read Archive (SRA) under
BioprojectaccessionnumberPRJNA313050. Thealphanumericidentifierofarepresentativesequence
foreachspecificOTUreferredtointheresultsisprovidedinTableS1.Thesesequencescanberetrieved
fromtherawdatacontainedintheSRA.
Microorganisms2016,4,23 6of23
Table 1. Sample collection/processing information and alpha diversity analysis of A. elisabethae (AE) and seawater (W) samples. DNA isolation methods:
PC—phenol-chloroform,UW—UltraClean®WaterDNAKit,PS—PowerSoilDNAIsolationKit.DNAtemplatetypeusedforpyrosequencinganalysis:PCR—nearly
full-length16SrDNAPCRamplicons,gDNA—genomicDNA.Alphadiversitystatistics:Sobs—observedrichness,Sest—estimatedrichness(Chao1index),H1—Shannon
diversityindex,E-Shannonequitabilityindex.AllcalculationswereconductedonOTUleveldata.
Sequence DNAIsol. Pyroseq. 16SrDNA No. Avg.Length
Library Site Sample Year Meth. Template Region Reads (bp) Sobsa Sesta H1a Ea
253 514 2.47 0.44
1Ab 1 AE1 2006 PC PCR V1/V2 4685 222
162 385 2.43 0.48
260 444 2.25 0.41
1Bb 1 AE1 2006 PC PCR V1/V2 4969 222
162 366 2.21 0.43
162 216 2.01 0.40
1C 1 AE2 2006 PC PCR V1/V2 6962 240
90 179 1.98 0.44
886 1978 5.13 0.79
1D 1 AE3 2006 PC PCR V1/V2 5221 228
524 1295 5.05 0.81
310 533 1.90 0.37
2A 2 AE4 2006 PC PCR V1/V2 7200 233
151 379 1.85 0.41
404 785 1.71 0.28
2B 2 AE5 2006 PC PCR V1/V2 8884 245
181 414 1.64 0.32
182 471 1.20 0.23
3B 3a AE6 2006 PC PCR V1/V2 2424 245
170 442 1.19 0.23
366 1032 1.99 0.34
3C 3a AE7 2006 PC PCR V1/V2 5906 243
184 563 1.93 0.37
565 1245 4.77 0.75
3D 3a AE8 2006 PC PCR V1/V2 2754 235
497 1054 4.73 0.76
530 1434 3.52 0.56
3E 3b AE9 2009 PC PCR V1/V2 7038 228
248 726 3.44 0.62
598 1290 4.48 0.70
4A 4 AE10 2006 PC PCR V1/V2 5560 231
356 768 4.38 0.75
233 556 2.74 0.50
4B 4 AE11 2006 PC PCR V1/V2 6450 221
128 280 2.71 0.56
144 338 1.30 0.26
4C 4 AE12 2006 PC PCR V1/V2 7269 241
72 165 1.27 0.30
Microorganisms2016,4,23 7of23
Table1.Cont.
Sequence DNAIsol. Pyroseq. 16SrDNA No. Avg.Length
Library Site Sample Year Meth. Template Region Reads (bp) Sobsa Sesta H1a Ea
152 333 2.03 0.40
Co_Ac 5 AE13 2010 PC gDNA V1/V2 5831 247
85 210 2.00 0.45
91 197 2.41 0.54
Co_Bc 5 AE14 2010 PC gDNA V1/V2 2258 248
90 195 2.41 0.54
411 842 2.58 0.43
Co_Cc 5 AE15 2010 PC gDNA V1/V2 4846 244
247 615 2.52 0.46
155 206 3.86 0.76
W1 3a W1 2009 UW gDNA V1/V2 3149 232
140 184 3.85 0.78
325 563 3.25 0.56
W2 3b W2 2009 UW gDNA V1/V2 6294 223
195 366 3.19 0.61
219 457 3.10 0.58
H13 6a AE16 2013 PS gDNA V4 3759 201
178 395 3.08 0.59
164 314 3.10 0.61
H15 6b AE17 2013 PS gDNA V4 5511 201
116 219 3.10 0.65
147 351 2.12 0.42
H17 6b AE18 2011 PS gDNA V4 6247 201
94 180 2.10 0.46
249 514 2.57 0.47
H18 6b AE19 2011 PS gDNA V4 4775 201
172 422 2.54 0.49
102 192 2.25 0.49
H26 6a AE20 2011 PS gDNA V4 2659 201
101 190 2.25 0.49
216 300 3.35 0.62
W13 6a W3 2011 UW gDNA V4 3341 201
197 276 3.33 0.63
235 358 3.34 0.61
W15 6b W4 2011 UW gDNA V4 4278 201
194 296 3.32 0.60
352 518 3.27 0.57
W17 6b W5 2011 UW gDNA V4 7706 201
213 331 3.22 0.60
409 695 3.80 0.63
W18 6b W6 2011 UW gDNA V4 7979 201
254 414 3.74 0.68
317 526 3.36 0.58
W26 6a W7 2011 UW gDNA V4 8491 201
195 324 3.31 0.63
aItalicizednumberscalculatedusingsubsampleddatasets.SequencelibrariescoveringtheV4regionweresubsampledto2600sequenceswhilethosecoveringtheV1/V2regionwere
subsampledto2200sequences;bReplicatesequencelibrariespreparedfromthesamegenomicDNAsample;cDatafrompreviouslyreportedstudy[34].
Microorganisms2016,4,23 8of23
3. Results
3.1. BacterialCommunityAnalysis
AfterstringentqualityfilteringusingMOTHURtoreducetheeffectsofPCRandsequencing
artifacts,98,273sequenceswereobtainedfrom17A.elisabethaespecimenscollectedfromfivelocations
throughoutTheBahamas. Sevenseawatersamplesyielded43,282sequences(Table1). Inclusionof
sequencesobtainedfromapreviousstudyofbacterialdiversityinthreespecimensofA.elisabethae
fromColombia[34]contributedanadditional12,935sequences. Theaveragesequencingdepth(˘s.d.)
obtained for octocorals in the V1/V2 and V4 datasets were 5516.1 ˘ 1804.8 and 4590.2 ˘ 1268.7,
respectively. Similarsequencingdepthwasobtainedforseawatersamples(V1/V2—4721.5˘1572.5,
V4—6359˘2374.6). Sequencingdepthswerenotsignificantlydifferentbetweengroupsdefinedon
thebasisof16Sregionsequenced, sampletype, andcollectionsite(Kruskal–Wallistest; P=0.464)
(Table1). Rarefactioncurveswerepreparedtoestimatesamplingcoverage(FigureS2). Thecurvesdid
notapproachtheasymptoteforthemajorityofsamples,indicatingthatfurthersamplingdepthwould
recoveradditionalspecieslevel(D=0.03)diversity.
The richness and diversity observed in A. elisabethae bacterial communities ranged widely
between octocoral samples, particularly within the V1/V2 dataset. After subsampling to reduce
theeffectofunequalsequencingdepths(V1/V2,n=2200;V4,n=2600)averageobservedrichness
(˘s.d.) in the V1/V2 and V4 datasets were 209.19 ˘ 133.81 and 132.20 ˘ 35.71, respectively.
Similarvariability(asindicatedbyrelativelylargestandarddeviations)wasobservedforestimated
richness(V1/V2—502.25˘313.88OTUs; V4—281.20˘105.07OTUs),theShannondiversityindex
(V1/V2—2.61 ˘ 1.15; V4—2.61 ˘ 0.41) and the Shannon evenness index (V1/V2—0.50 ˘ 0.16;
V4—0.54˘0.07). No statistically significant differences in alpha diversity statistics (S , S , H1
obs est
andE)wereobservedbetweengroupsdefinedonthebasisofsamplingsite(V1/V2sites),collection
year(SanSalvador2011vs. 2013)or16SrDNAregionanalyzed(V1/V2vs. V4dataset).
Todetermineiftheobservedwiderangeindiversitymeasuresbetweenindividualoctocorals
wasduetoexperimentalvariationinherentinthesequencingmethodologywecompareddiversity
estimatesfromreplicatesequencelibraries1Aand1B,whichwereindependentlygeneratedfromthe
samegDNAsample. Observedrichnesswasidenticalbetweenthereplicatelibrariesandtheother
diversitymeasureswereverysimilar,exhibitingcoefficientsofvariation(CVs)rangingbetween2.5%
and5.5%. ThislevelofvariationisfarlessthanthatobservedamongoctocoralsamplesintheV1/V2
datasetwhereCVsrangedfrom33%forEto64%forS .
obs
To compare bacterial diversity between seawater and octocoral samples, W13–W26 were
compared to H17, H18 and H26, as these samples were collected in parallel in San Salvador.
Observedrichness,andShannondiversityandevennesswassignificantlygreaterinseawatersamples
(Mann–Whitneytests;P=0.0369foreachtest)(TableS2).
AnOTU-basedapproachwasusedtocomparethestructureofoctocoralandseawaterbacterial
communitiesatthespecieslevel(D=0.03)(Figure1). TheV1/V2andV4datasetswereanalyzed
separately,asOTUsderivedfromdifferentregionsarenotdirectlycomparable[48]. Inclusteranalyses
based on the Yue and Clayton theta dissimilarity measure seawater samples clustered apart from
octocoralsamplesinbothdatasets(Figure1A,B).Thisclusteringwassignificantasparsimonyand
unweightedUniFractestsreturnedPvalueslessthan0.05(TableS3),indicatingthatthestructureof
octocoralbacterialcommunitiesweresignificantlydifferentthanthoseoftheseawatersamples.
MMiiccrroooorrggaanniissmmss 22001166,, 44,, 2233 190 ooff 2232

Figure 1. Comparison of microbial communities from A. elisabethae and seawater using the Yue
Figure 1. Comparison of microbial communities from A. elisabethae and seawater using the Yue and
and Clayton dissimilarity calculator: (A) V1/V2 dataset subsampled to 2200 sequences/library;
Clayton dissimilarity calculator: (A) V1/V2 dataset subsampled to 2200 sequences/library; and (B) V4
and(B)V4datasetsubsampledto2600sequences/library. Dendrogramswerepreparedusingthe
dataset subsampled to 2600 sequences/library. Dendrograms were prepared using the UPGMA
UPGMAalgorithm.
algorithm.
IInn tthhee VV11//VV22 ddaattaasseett,, CCoolloommbbiiaann ssaammpplleess ffoorrmmeedd aa ddiissttiinncctt ccllaaddee ffrroomm tthhee BBaahhaammiiaann ooccttooccoorraallss
((FFiigguurree 11AA),)w, hwichhicwh awssaus pspuoprtpeodrtbeydp abrys impaornsyimanondyu nawndei guhntewdeUignhitFerda cUtensitFsr(aPc <t0e.s0t1s 7;(PB o<n fe0r.r0o1n7i;
cBoornrefecrtreodnsii gcnorifireccatnecde sleigvneilfficoarnaceth lreeveewl afoyrc ao mthpraerei swonayb ectowmeepnarsiesaown abteetrw,Beaehna mseiaawnaotcetro, cBoarhaalsmainadn
Cocotloocmorbailasn aoncdto Ccoorloamls)b(iTaanb olectSo3c)o.rTahlse) s(hToarbtleb rSa3n)c. hTlheen gshthosrti nbrFainguchre le1nAgitnhds iicna tFeidguthrae t1tAhe ibnadcitceartieadl
cthoamt mthuen bitaiecsteorfiaCl ocloommmbiuannistaiems polfe sCwoleormebhiiagnh lsyasmimpilleasr .wNeorec ohnigshisltye nstimcluilastre. rNinog cboynssiitsetewnat scolubssteerrvinedg
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1sBeqculuesntceer eldibtriagrhitelsy 1toAg eatnhder 1iBn Fcilguustreer1eAd ,tiilgluhsttlrya ttionggetthheers iimn iFlairgiutyreo f1tAh,e silelulisbtrraartiiensg. Itnheth seimV4iladraittays eotf,
wthheisceh licbornasriisetse. dIno fthoec tVo4co draatlassceotl,l wechteicdhi ncotnwsoisdteidff eorfe oncttyoecaorrsalfsr ocmollseictteesds einp atwraote ddifbfyeraepnpt ryoexairms afrtoemly
5si0t0esm senpoaaraptpeadr ebnyt acplupsrtoexriimngatbeylys i5te00o rmy neaor awpapsaraepnpta crleunstte(Friinggu rbey1 sBi)te. or year was apparent (Figure
1B). SequencesderivedfromallA.elisabethaesamplesanalyzedinthisstudy(n=20)couldbeassigned
to15Spehqyulaenacneds 2d5erreicvoegdn ifzreodmc laaslls eAs.w ehliislaebtehthoasee fsraommptlhees saenvaelnyszeeadw iant etrhsiasm sptuledsyc o(unl d= b2e0a) ssciogunledd btoe
1a4sspighnyelada tnod 1251 pchlayslsae san(Fdi g2u5r ere2coagnndiTzaebdl ecslaSs4seasn wdhS5il)e. Itnhogseen ferroaml,t htheem seovsteanb suenadwaanttemr seammbpelress( ˘cosu.dld.)
obfe Aas.seilgisnaebdet htoa e1b4a pchteyrliaa lacnodm 2m1 culnasitsieess (wFiegruerGe a2m amndap Troatbeloebsa cSt4e raiand(4 S05.5)%. In˘ g3e2n.e5r%al),, tAhlep mhaopsrot taebobuancdtearniat
(m16e.m9%be˘rs1 5(±.7s%.d,.)U onfc lAas. seilfiiseadbeBthacatee briaac(t1e2ri.8a%l c˘om14m.9u%n)i,tiCeys awnoebraec tGeraiam(m12a.p2r%ote˘ob1a8c.t8er%ia) ,(a4n0d.5F%la ±vo 3ba2c.5te%ri)a,
(A9l.p0h%ap˘ro1te7o.b8a%ct)er(iFai g(u16re.9%2, T±a b1l5e.7S%4), .UTnhcelaasbsuifniedda nBcaectoefritah e(s1e2.g8r%o u±p s14v.a9r%ie)d, Ccoyannsoidbaecrtaebrilay (b1e2t.w2%ee n±
o1c8t.8o%co)r, aalnsdco Fllleacvtoebdacbteortiha f(r9o.0m%t h± e1s7a.8m%e) s(iFteigaunrde f2r,o Tmabdlief fSe4re).n Tthsiet easb(uFnigduarnec2e) o.fS itmheislaer igtryopueprsc evnatraiegde
aconnalsyidsiesr(aSbIlMy PbeEtRw)eoefnc olacstsocleovreallsb caocltleercitaeldc obmotmh ufrnoimtie tshree vsaemaleed sivtea rainadbl feroinmtr ad-isfifteeresnimt siliaterist y(Fbigeutwree e2n).
bSaimctielrairailtyco mpemrcuennittaiegseo faoncatloycsoisr a(lSsIcMolPleEcRte)d ofrfo mclaBsas hlaemveial nbsaitcetser1i–a4l (c1o—m4m2.u8%ni,ti2e—s 5r8e.v8e%a,le3da— v2a3r.i0a7b%le,
ainntdra-4s—ite3 8si.9m%il)a.rityG rbeeattwereeonv bearactlelriinalt rcao-msitmeusnimitiielas roitfy ocwtoacsoroablss ecrovleledctiend ofrcotmoc oBraahlasmamianp lseistesfr o1–m4
S(1a—n4S2a.l8v%ad, o2—r (5589..81%%, s3iam—il2a3r.i0ty7)%a, nadndC 4o—lom38b.9ia%()8. 6G.4re%atseirm oivlaerriatlyl )i.ntArva-esriateg esiimntilearr-istiyte wdaiss soimbsielarvrietdy
(i˘n so.cdt.o)cboertawl eseanm2p0le0s6 fBraohma mSainan Ssailtveasdwoar s(5692..14%% s˘im8i.l9a%ri.tyS)i manilda rCloevloemlsboifad (8is6s.i4m%i lsairmityilawrietrye).o Absveerrvaegde
binettewr-eseinte2 0d0i6ssBimahilaamriitayn (s±itse.ds.a)n bdetthweeCeonl o2m00b6ia nBashitaem(6i0a.n4 %si˘tes1 7w.9a%s) a6n2.d4%be t±w e8e.9n%2.0 0S6imBailhaarm leiavnelssi teosf
adnisdsiSmainlaSraitlyv awdeorre( 6o3b.s4e%rv˘ed1 2b.e3t%w)e.eDni s2s0i0m6i lBaarihtyambeiatwn eseitnest haenCd otlhoem Cboialonmsibtieaann sditSea (n60S.a4l%va ±d o1r7.w9%as)
3a8n.d5 %b.eWtweeaegna i2n00a6s seBsasheadminiatner nsaitlees xpanerdi mSeannt aSlavlvaraidaobri li(t6y3b.4y%c o±m p12a.r3i%ng). thDeistsaixmoinlaormityic cboemtwpeoesni titohne
dCeorliovmedbiafrno msitere apnlidc aStaenl ibSraalvraiedso1r Awaans d381.B5%.S. IMWPe EaRgaainn aalsysseisssreedv einatleerdnaal loexwpedreimgreenetaolf vdairsisaibmiliiltayr ibtyy
(c5o.m9%p)arbientgw teheen ttahxeocnloamssicle vcoemltpaxoosintioomn idcecroivmepdo fsriotimon roefptlihceastee sliabmraprlieess .1A and 1B. SIMPER analysis
reveaSleedaw aa ltoewrb dacetgerreiae locfo mdimssiumniiltaieristyw (e5r.e9%pr)i mbeatrwileyecno mthpe ocslaesds olfevCeyla tnaoxboanctoemriaic, Uconmclpasossiifitieodn Boafc tthereisae,
asanmdpUlensc.l assifiedProteobacteria,whichcollectivelyaccountedfor44.3%–85.4%ofsequencesinseawater
sampSleesaw(Taatbelre Sb5a).ctSeIrMiaPl EcRoamnmaluysniistireesv ewaleerdec opnrsiimdearrailbyl ecdoimssipmoisleadri tyofb eCtwyaenenobcaocmterpiaa,r aUblnecsleaaswsifaiteedr
aBnadcteAr.iael, iasanbde tUhanecblaascstiefrieiadl Pcoromtemobuanctietrieias, (w3Ehivchs. cWol1le/cWtiv2e—ly6 a5c.8c%ou;nHte1d7 –fHor2 464v.3s%. W–8152.4–%W 2o6f —se6q0u.e4n7c%e)s.
Tina xsaecaownattreibr ustainmgptloest h(eTadbisles imS5il)a. rSitIyMbPeEtwR eaennasleyaswis arteerveaanldedo cctooncosirdaelrsaabmlep ldesissairmeisluarmitmy abreiztwedeeinn
TcaobmlepaSr5a.ble seawater and A. elisabethae bacterial communities (3E vs. W1/W2—65.8%; H17–H26 vs.
W12–W26—60.47%). Taxa contributing to the dissimilarity between seawater and octocoral samples
are summarized in Table S5.
Microorganisms2016,4,23 10of23
Microorganisms 2016, 4, 23 11 of 22

FigFuigreur2e .2.C Clalassss lelevveell bbaacctteerriiaall ccoommmmuunniittyy ccoommppoossitiitoionn oof fAA. .eleisliasbaebthetahea eanadn dsesaewaawteart esramsapmleps.l es.
Collection year and site are indicated adjacent to sample identifier. “Unclassified Bacteria” refers to
Collectionyearandsiteareindicatedadjacenttosampleidentifier. “UnclassifiedBacteria”refers
sequences that could not be classified by the RDP classifier using a confidence threshold of 80%.
tosequencesthatcouldnotbeclassifiedbytheRDPclassifierusingaconfidencethresholdof80%.
“Remainder” is an artificial category encompassing classes that represented <1% of sequences in all
“Remainder”isanartificialcategoryencompassingclassesthatrepresented<1%ofsequencesinall
sequence libraries. This category accounts for 0.0%–2.21% of each bacterial community.
sequencelibraries.Thiscategoryaccountsfor0.0%–2.21%ofeachbacterialcommunity.
3.2. Core Microbiome of A. elisabethae
3.2. CoreMicrobiomeof A.elisabethae
Previous analyses of marine invertebrate microbiomes have shown that many organisms
Previousanalysesofmarineinvertebratemicrobiomeshaveshownthatmanyorganismsmaintain
maintain a core set of bacterial associates that are stably maintained geographically and temporally,
acoresetofbacterialassociatesthatarestablymaintainedgeographicallyandtemporally,irrespective
irrespective of changing environmental conditions [20,30]. To identify putative members of the A.
ofeclhisaabnegthinaeg menicvroirboinommee nwtael icdoenndtifiiteiodn OsT[2U0s, 3t0h]a.t Twoeried epnrteisfeynpt uint a>t5iv0e% mofe mocbtoecrosroalf stahmepAle.se laist aabnetyh ae
miacbroubnidoamnceew. OeTidUesn wtiefireed eOxcTluUdsedth fartomw etrhee plirsets iefn ttheiny >w5e0r%e foofuoncdt oinc ogrraelastearm apvleersagaet aanbuynadbaunncde ainn ce.
OTsUeaswwaeterer esxacmlupdleesd tfhroamn tihne olicsttoicfotrhael yswamerpelefos,u nasd itnhegsree aOteTrUasv emraagye anbout nrdeapnrecseeinnt sterauwe aotecrtoscaomrapll es
thabnacitneroiaclt oacsosorcailasteasm bpulte rsa,tahsert hceasreryO oTvUers frmomay tnhoe tsruerproruesnednintgtr useeawocattoecro. Trahleb daicstterirbiaultiaosns oocf iOatTeUssb ut
ratfhuelfrilclianrgr ythoevsee rcrfirtoemria tihne thseu rVr1o/uVn2d ainndg Vse4a dwataatseert.s Tahree sdhioswtrnib iunt FioignuorefsO 3T aUnds 4fu, rlfieslpliencgtivtheelys.e Incr tihteer ia
inVth1e/VV2 1d/aVta2seatn 2d7 VO4TUdsa twaseertes iadreentsihfioedw nas incanFdigiduaretes m3eamndbe4rs, oref stpheec ctiovreel ym.icIrnobthioemVe1 (/FVig2urdea 3ta).s et
27AOlTphUapsrwoteeorebaicdteerniati fianedd Gasamcamnadpirdoatetoebmacetemriab ecrosnostfittuhteedco trheem girceraotbesiot mpreop(Foirgtuiorne o3f) .tAhelp phuaptartoitveeo bcaocrtee ria
anmdiGcraombmioampreo (t2e9ob.6a%ct earniad c2o2n.2s%tit, urteesdpetchteivgerlye)a.t Teshtep arvoeproagrtei o(±ns.odf) tahnedp muteadtiiavne acbourendmainccreo boifo pmueta(t2iv9e.6 %
ancdo2re2 .m2%icr,orbeisopmecet OivTeUlys) .wTehree 2a.2v%er ±a g4e.5%(˘ asn.dd) 0a.3n3d%m, reedspiaenctiavbeulyn. dInatnecreesotifnpgulyt,a stiixv eOTcoUrse pmreivcriooubsiolym e
identified as abundant in Colombian samples were included in the current list of putative core
OTUs were 2.2% ˘ 4.5% and 0.33%, respectively. Interestingly, six OTUs previously identified
microbiome members. These OTUs were classified as Brevundimonas sp. (OTUs 18 and 8),
as abundant in Colombian samples were included in the current list of putative core microbiome
Stenotrophomonas sp. (OTU19), Pseudomonas sp. (OTU4), Endozoicomonas sp. (OTU37) and
members. TheseOTUswereclassifiedasBrevundimonassp. (OTUs18and8),Stenotrophomonassp.
Unclassified Gammaproteobacteria (OTU20). Five OTUs were encountered in >75% of A. elisabethae
(OTU19),Pseudomonassp. (OTU4),Endozoicomonassp. (OTU37)andUnclassifiedGammaproteobacteria
samples. OTUs 15, 36 and 43 were Rhodobacteraceae, and had average abundances between 0.32% and
(OTU20). FiveOTUswereencounteredin>75%ofA.elisabethaesamples. OTUs15,36and43were
1.26%. OTUs 1 and 2 exhibited high average abundances (>10%) and were observed in a high
Rhodobacteraceae,andhadaverageabundancesbetween0.32%and1.26%. OTUs1and2exhibited
proportion of A. elisabethae samples (>80%). OTU1 had the greatest average abundance (20.05% ±
highaverageabundances(>10%)andwereobservedinahighproportionofA.elisabethaesamples
30.1%) and was observed in all Bahamian octocorals collected in 2006 and one of three Colombian
(>80%). OTU1 had the greatest average abundance (20.05% ˘ 30.1%) and was observed in all
samples, but was absent from 3E and the seawater samples. This OTU was identified as an
Bahamianoctocoralscollectedin2006andoneofthreeColombiansamples,butwasabsentfrom3E
unidentified Oceanospiralles by the RDP classifier with 97% confidence. BlastN analysis revealed
anOdTthUe1 seshaowwaetedr sstarmonpgl esse.qTuheinscOe TidUenwtiatys id(9e8n.2t%ifi–e1d00a%s)a ntou nuindceunlttuifireedd Obaccetaenroiasp idreatlelecstebdy itnh ethReD P
classifier with 97% confidence. BlastN analysis revealed OTU1 showed strong sequence identity