Fungal Community Assembly in the Amazonian Dark Earth

12 Pages·2016·0.62 MB·English

MicrobEcol(2016)71:962–973
DOI10.1007/s00248-015-0703-7
SOILMICROBIOLOGY
Fungal Community Assembly in the Amazonian Dark Earth
AdrianoReisLucheta1&FabianadeSouzaCannavan2&
LuizFernandoWurdigRoesch3&SiuMuiTsai2&EikoEuryaKuramae1
Received:15May2015/Accepted:30October2015/Publishedonline:19November2015
#TheAuthor(s)2015.ThisarticleispublishedwithopenaccessatSpringerlink.com
Abstract Here,wecomparethefungalcommunitycomposi- niger, Lithothelium septemseptatum, Heliocephala gracillis,
tion and diversity in Amazonian Dark Earth (ADE) and the and Pestalosphaeria sp. were more abundant in ADJ soils.
respective non-anthropogenic origin adjacent (ADJ) soils Differencesinfungalcommunitycompositionwereassociat-
from four different sites in Brazilian Central Amazon using ed to soil chemical factors in ADE (P, Ca, Zn, Mg, organic
pyrosequencingof18SribosomalRNA(rRNA)gene.Fungal matter, sum ofbases,and basesaturation) and ADJ (Al,po-
community composition in ADE soils were more similar to tential acidity, Al saturation, B, and Fe) soils. These results
each other than their ADJ soils, except for only one site. contributetoadeeperviewofthefungicommunitiesinADE
Phosphorus and aluminum saturation were the main soil and open new perspectives for entomopathogenic fungi
chemical factors contributing to ADE and ADJ fungal com- studies.
munitydissimilarities.Differencesinfungalrichnesswerenot
observed between ADE and ADJ soil pairs regarding to the
Keywords 18SrRNA .Anthrosols .Biochar.Microbial
mostsites.Ingeneral,themostdominantsubphylapresentin
ecology.Pre-Columbiansoil .Pyrosequencing
the soils were Pezizomycotina, Agaricomycotina, and
Mortierellomycotina. The most abundant operational taxo-
nomicunits(OTUs)inADEshowedsimilaritieswiththeen-
tomopathogenic fungus Cordyceps confragosa and the Introduction
saprobes Fomitopsis pinicola, Acremonium vitellinum, and
Mortierellaceae sp., whereas OTUs similar to Aspergillus Amazonian Dark Earth (ADE), also referred to as BTerra
Preta^, was described by Sombroek [1] as a Bwell-drained
soilcharacterizedbythepresenceofathickblackordarkgray
AdrianoReisLuchetaandFabianadeSouzaCannavansharefirst topsoilwhichcontainspiecesofartifacts^.Theanthropogen-
authorship.
ic, pre-Columbian soils occur in 20-ha average spots in the
Electronicsupplementarymaterial Theonlineversionofthisarticle
Amazonian region [2]. ADE is recognized by the elevated
(doi:10.1007/s00248-015-0703-7)containssupplementarymaterial,
amounts of stable carbon (70 times more black carbon) and
whichisavailabletoauthorizedusers.
fertility due to the high concentration of P, Ca, Mg, and Zn,
* EikoEuryaKuramae nutrient holding capacity, and higher pH when compared to
[email protected] adjacent non-anthropogenic origin soils [3, 4]. Despite evi-
dences of human occupation in the Amazon region dating
10,000years BP(before present), Neves and co-workers[5]
1 DepartmentofMicrobialEcology,NetherlandsInstituteofEcology
suggested that ADE formation occurred 2500 to 2000 years
(NIOO/KNAW),Droevendaalsesteeg10,Wageningen6708PB,
TheNetherlands agoasaresultofpopulationincreasingduringthatperiod.Itis
2 CentrodeEnergiaNuclearnaAgricultura(CENA),Universidadede stilluncleariftheADEwasintentionallycreatedorifitwasa
SãoPaulo(USP),Piracicaba,Brazil result of disposals by native settlements. However, the con-
3 CentroInterdisciplinardePesquisasemBiotecnologia(CIP-Biotec), sensus is that the main sources of ADE nutrients originated
UniversidadeFederaldoPampa,SãoGabriel,Brazil from human and animal excrements, plant and animal
FungalCommunitiesinADE 963
residues, mammalian and fish bones, housing material and most studied archaeological ADE sites in Central Amazon
potterydebris,ash,andcharredorganicmaterials[2,5,6]. [18]; (4) Barro Branco (BBO, 03° 18′ 24.76″ S, 60° 32′
Another remarkable characteristic ofADE is the elevated 5.10″ W), located upstream Hatahara in the margin of
microbial diversity and associated bacterial species richness Solimões River close to Manacapuru (AM) under a citrus
[7]. Using culture dependent and independent approaches, orchard(ADE)andcassavaplantation(ADJ).
studiesrevealbacterialandarchaealcommunitiesinADEthat The soil sampling scheme in each site was set by a geo-
aredistinctfromtheadjacentsoilorfromisolatedblackcar- referencedcentralpoint(A)andfourextrapoints1.5mdistant
bon[8–10]. in the cardinal direction (B, C, D, E). Each soil sample was
Significantadvancesinsoilmicrobialecologystudieswere composedbyfivesubsamples(A1,A2,A3,A4,A5,B1,B2,
obtained in the last few years after the adoption of high- B3,B4,B5,etc.)collected0.3maroundthemainpointat0–
throughput 16S ribosomal RNA (rRNA) gene sequencing 10 cm depth using sterile plastic cylinders (5-cm diameter).
technologies[11].Thisapproachwasusedtoinvestigate the Sampling scheme illustration can be viewed in Online
bacterialcommunityassociatedwithbiocharsamplesofADE Resource 1. To minimize the current land use effect, grass
[12]andtheeffectofADEandplantspeciesontheselection layerandlitterwereremoved,andthen,thesoilsampleswere
ofrhizospherebacterialcommunities[4].However,thefungal collected in the space between rows when cultivated. Soil
communitiesassociatedwithADEhavenotyetbeeninvesti- samples werekeptondry ice beforestorage at −20°C. Soil
gatedwithculture-independentmethodsdespitetheecological physicochemical attributes were determined following Raij
importanceoffungiinterrestrialecosystems.Thedegradation et al. [19] in the Soil Fertility Laboratory of the Department
of organic matter, mainly by saprophytic fungi, controls the of Soil Sciences, University of São Paulo (ESALQ-USP).
balance between soil and atmospheric carbon and releases Fieldworkwasconductedunderlegalauthorization(SISBIO
nutrientsforplantuptake[13–15].Thefungalcommunityin 4845833).
ADE has been poorly characterized and evaluated only by
low-resolutionculture-dependentmethods[16].Theapplica-
tionofhigh-throughputsequencingtechnologieswillexpand DNAExtraction,Amplification,andSequencingof18S
the knowledge of ADE fungal diversity. Comparison to low rRNAGeneFragment
fertilityadjacentsoilswillhelptoelucidatethecarbontrans-
formations by fungi in these soils and to evaluate potential TotalDNAwasextractedfrom250mgofbulksoilintriplicate
land use and climate change effects for future studies. from only three of the five soil samples (A, B, D) using the
Therefore, the aim of this study was to estimate the fungal Power Soil DNA isolation kit (MO BIO Laboratories Inc.,
richnessanddiversityassociatedtoADEandtoadjacentsoils Carlsbad,CA,USA)followingthemanufacturer’sinstructions.
fromfoursitesintheCentralAmazonthroughpyrosequenc- Extracted DNAwas quantified using a NanoDrop ND-1000
ingof18SrRNAgenefragments. spectrophotometer (Thermo Scientific, Wilmington, DE,
USA).The18SrRNAgenefragmentswereamplifiedbypo-
lymerase chain reaction (PCR) using 0.5 μM of the fungal-
MaterialsandMethods specificreverseprimerFR1[20]andthemodifiedversion(to
includeGlomeromycotaarbuscularmycorrhizalfungi)offor-
SiteDescriptionandSoilSampling ward primer FF390w (5′-CGWTAACGAACGAGACCT-3′)
[21].FourPCRreactions(25μL)perextractedsampleDNA
The study area was comprised of four locations in the werecarriedoutusing2.5×reactionbuffercontaining18mM
Brazilian Central Amazonia region near Manaus, the capital ofMgCl ,0.2mMofeachdNTP,0.5μMofeachprimer,25ng
2
of Amazonas state (AM). ADE and the respective adjacent oftemplateDNA,1UTaqpolymeraseFastStartHighFidelity
(ADJ) non-anthropogenic origin soils were collected at (1) (RocheAppliedSciences,Indianapolis,IN,USA),andsterile
Açutuba(ACU,03°05′53.92″S,60°21′19.90″W),located water to 25 μL final volume. The thermocycling conditions
at the margin of Negro River close to the municipality of wereinitialdenaturingat94°Cfor4min,29cyclesof94°C
Iranduba(AM),undercultivationofeggplant(ADE)andpas- for30s,55°Cfor1min(annealingtemperaturewaslowered
ture(ADJ)atthetimeofsampling;(2)Balbina(BAL,01°30′ 2 °C every 2 cycles until 47 °C), and extension at 68 °C for
24.4″S,60°05′34″W),locatedatthePresidenteFigueiredo 2 min. The technical PCR replicate (12 PCR reactions/soil
municipality and characterized by the presence of an undis- replicate) amplicons were pooled and cleaned with the
turbedsecondaryforest. Thissitehas not beendeforestedor QiagenPCR purificationkit(Qiagen,Valencia,CA,USA)to
used for agriculture purposes for at least 20 years [17]; (3) avoidamplificationbias.Atotalof24soilsamples(4sites×2
Hatahara(HAT,03°16′28.45″S,60°12′17.14″W)located soiltypes×3replicates)wereamplifiedusingbarcodedprimers
in a bluff on the margin of Solimões river cultivated with (MIDtags)formultiplexpyrosequencinginaRoche454GS
banana plants (ADE) and pasture (ADJ). This is one of the FLXautomatedsequencer(454LifeSciences,Brandford,CT,
964 A.R.Luchetaetal.
USA)usingtitaniumchemistry.Thecompletelistofbarcoded were transformed (function Log(x+1)) before multivariate
primersislistedinOnlineResource2. analysis. The raw 454 pyrosequencing data of the 18S
rRNA are available at the European Nucleotide Archive
BioinformaticsandStatisticalAnalysis (ENA) (https://www.ebi.ac.uk/ena/) under the study
accessionnumberPRJEB10851.
The18SrRNAgenesequenceswereanalyzedusingQIIME
1.8.0 [22] following the suggested 18S dataanalysistutorial
(http://qiime.org/1.8.0/tutorials/processing_18S_data.html). Results
Multiplex sequence libraries were split into the original
samples based onthe specificbarcodes. The 454 reads were SoilPhysicochemicalProperties
denoised using Denoiser [23] and chimeric sequences
checked with UCHIME [24]. Operational taxonomic units Alltheevaluatedsoilphysicochemicalandfertilityattributes
(OTUs) were clustered considering evolutionary distance of werestatisticallydifferent(p≤0.05)whenADEandADJsoils
0.03 (97 % similarity cutoff) by using UCLUSTalgorithm werecomparedingroups,withtheexceptionoftheK,S,and
[25] and taxonomically affiliated through BLAST search Fe attributes. On average, ADE soils were higher in pH, or-
usingQIIMEBLASTTaxonAssignerdefaultparameters(ap- ganic matter (OM), macronutrients (P, Ca, Mg), and some
plication blastn/megablast, max E value 0.001, min percent- micronutrients(Mn,CuandZn),whileADJsoilshadhigher
age identity 90.0) against SILVA Eukaryotic database (97 levelsofAlandH+Al(Table1).WithintheADEsoilgroup,
SILVA 111 rep set euk) [26, 27]. OTUs not assigned to theHataharasampleshowedthehighestamountsofP,Cu,Fe,
Fungi kingdom, singletons (OTUs containing a unique se- Zn,andMn,whereasintheADJsoilgroup,theAçutubasoil
quenceinthewholeanalysis)aswellasclassifiedasBnohit^ sampleshowedthe highestamountofKandlowestAlcon-
by BLAST search were removed from the dataset. centrationand Alsaturation, comparablewith ADE samples
InconsistencesofSILVAtaxonomicclassificationwereman- (Table1).
uallycorrectedbeforerelativeabundancecalculationbasedon
the OTU BLASTsearch best hit access number and NCBI DiversityofFungalCommunity
taxonomy rank (http://www.ncbi.nlm.nih.gov/taxonomy).
The OTU table was rarefied to the lowest number of 18SrRNASequencing
sequences in any sample (1728) before calculation of alpha
diversity indices. Species richness (Chao1 and Abundance Pyrosequencingof18SrRNAgenefromthe24soilsamples
Coverage-based Estimator (ACE)), diversity (Shannon, generated132,764high-qualitysequencesafterdenoisingand
Simpson’sreciprocal)estimators,Good’scoverage,andrare- chimerachecking,withanaveragesizeof351nucleotides.A
factioncurveswerecalculatedinQIIME.Chaoentropyindex totalof105,019sequenceswereusedforfurtheranalysisafter
[28] was calculated on the CHAOEntropy-Online calculator taxonomicclassificationasfungal.Thenumbersofsequences
(https://yuanhan.shinyapps.io/ChaoEntropy/). A bipartite perlibraryrangedfrom1728to6712.Adetaileddescription
OTU network was generated in QIIME and viewed and ofsequencingdepthandnumberofOTUsalongthebioinfor-
edited in Cytoscape 3.2.1 [29]. The fungal OTUs present in matics analyses can be viewed at Online Resource 3. The
allsoilsamples(totalcore)(core_table_100.biomfile),aswell number of picked OTUs ranged between 127 and 172 after
asthecommonOTUsbelongingtoADEorADJsoils(group the removal ofsingletons and library normalization with the
core) were also determined in QIIME (compute_core_ lowest number of sequences (1728) (Table 2). Despite the
microbiome.py). OTUs showing average abundance higher decrease in the number of sequences after quality filtering
than 1 % of the total number of sequences by group (ADE and library normalization (Online Resource 3), the sample
or ADJ) were considered abundant. Differential OTU coverage was approximately 97 % as indicated by Good’s
frequencies between ADE and ADJ soil groups was estimator(Table2).Inaddition,rarefactioncurvesalsopoint-
determined by non-parametric t test followed by Monte edforadequatesequencingeffortsforfungalpopulationcov-
Carlotest(100replicates)afterremovingtheOTUsthatwere erageinthesamples(OnlineResource4).
notrepresentedinatleast25%ofthesamplesusingQIIME Nosignificantdifferencesintheestimatedspeciesrichness
(group_significance.py).Univariateanalyses(ttest,ANOVA, wasobservedbyACEandChao1estimatorswhencomparing
Tukey’stest)wereperformedusingIBMSPSSStatisticsV.22 theADEandADJsoilsamplesinthesamesites,withexcep-
(IBMCorp.,Armonk,NY,USA)software,whereasmultivar- tionofahighernumberofspeciesintheHataharaADEsam-
iateanalyses(canonicalcorrespondenceanalysisandsimilar- pleinrelationtoitsADJsoil(ACEestimator).Regardingthe
itypercentageanalysis)wereperformedusingpaleontological fungal species diversity, Shannon and Chao entropy estima-
statistics (PAST) software package V.3.05 [30]. Count data torsalsopointedtonodifferencesbetweentheADEandADJ
(sequence abundances) and environmental variable values soils. Simpson’s reciprocal indicated lower species diversity
FungalCommunitiesinADE 965
b n
DJ di
A e
s
ersus xpres
v e
E Al
D * * * * * * * *
A ** * ** ns ** ** ns ** * ** * ** ** ns ** ** * ** H+
d
n
b a
b 1b 3a ab Al,
Hatahara 3.73±0.12cd 33.33±1.15a 7.33±1.15bc 0.67±0.06b 6.33±1.15c 1.0±0.0c 3.67±0.58b 8.0±1.13c 103.67±15.7 7.67±0.58bc 0.32±0.11ab 0.03±0.06c 0.3±0.1bc 182.33±27.4 0.4±0.17c 14.0±1.73b 95.67±14.64 63.67±0.58a pH7(CEC), 5)
n 0
BarroBranco 3.47±0.12d 34.67±5.69ab 3.67±2.08c 0.43±0.06b 1.67±0.58c 1.0±0.0c 8.33±2.08a 3.1±0.61c 135.1±30.29ab 2.33±0.58c 0.48±0.09a 0.0±0.0c 0.17±0.15c 193.0±47.13ab 0.53±0.15c 23.67±3.06a 132.00±30.05a 88.67±2.52a xchangecapacityi ’≤Tukeystest(p0.
e y
n b
o
cd 9ab b b 5bc c 6ab 1a cati nces
Balbina 3.93±0.15b 32.33±10.6 2.67±1.53c 0.70±0.36b 3.33±2.31c 2.00±1.73c 3.67±0.58b 6.03±4.39c 79.37±39.2 9.33±8.5bc 0.37±0.18a 0.0±0.0c 0.1±0.1c 90.67±75.0 0.27±0.12c 13.0±4.36b 73.33±41.3 69.33±17.2 ofbases(SB), nificantdiffere
m g
b bc 4bc 2bc 69b abc b c c c b g,su nosi
Table1PhysicochemicalandfertilityattributesofAmazonianDarkEarth(ADE)andadjacent(ADJ)soils PropertiesADEADJ AçutubaBalbinaBarroBrancoHataharaAçutuba a4.73±0.76abc5.17±0.15a5.20±0.2a4.6±0.4abcpH4.87±0.64ab OM21.67±16.44b49.0±4.58a54.67±9.24a53.0±2.65a36.0±7.81a P204.67±168.36b90.0±49.43bc130.0±34.04bc508.67±88.64a41.0±19.16 K1.0±0.17b0.30±0.0b1.03±0.35ab1.07±0.25ab3.13±2.02a Ca57.67±50.82bc44.67±49.69bc86.33±14.19ab138.33±7.64a35.67±17.0 Mg5.33±2.31bc2.67±2.08c10.67±1.15ab13.67±3.06a6.0±3.61bc S3.33±0.58b3.67±0.58b4.33±0.58b4.33±0.58b5.0±1.0b SB64.0±53.29bc47.63±51.73bc98.03±14.36ab153.07±5.39a44.80±21.0 CEC114.00±34.04b125.63±17.21b140.37±14.2ab200.73±0.6a101.80±10. V%50.67±27.59ab35.0±33.81abc69.67±4.04a76.00±2.65a43.0±17.35 B0.32±0.17ab0.17±0.05ab0.21±0.1ab0.10±0.09b0.23±0.05a Cu1.2±0.35b0.63±0.06bc1.13±0.15b3.27±0.67a0.43±0.23b Zn4.3±1.47bc5.0±1.11bc7.70±5.54b30.77±4.61a1.30±0.62b Fe48.67±22.19c36.33±10.02c66.0±2.0c210.67±36.56a70.0±14.8c Mn3.6±1.57bc4.0±0.69bc4.93±0.75b12.60±3.8a1.37±0.65b Al1.0±1.0d5.0±5.0cd0.0±0.0d0.0±0.0d2.0±2.65d H+Al50.0±19.29b78.0±34.64ab42.33±4.51b48.67±5.77b57.0±13.23 m%3.33±3.51b20.0±20.0b0.0±0.0b0.0±0.0b7.0±10.44b −−−1330.01molL);organicmatter(OM)expressedingdm;PandSexpressedinmgdm;K,Ca,MpH(CaCl2−−33mmolcdm;B,Cu,Zn,Fe,andMnexpressedinmgdm Vsoilbasesaturationindex(%),mAlsaturationindex(%),nsnotsignificantaTheshowedvaluesaretheaverageofthreereplicatesfollowedbystandarddeviation.SamelettersrepresentbIndependentsamplettestcomparingADE×ADJsoilgroups≤≤≤*p0.05;**p0.005;***p0.0005
966 A.R.Luchetaetal.
Table2 EstimatedrichnessanddiversityindicesforthefungalcommunitiesintheAmazonianDarkEarth(ADE)andadjacent(ADJ)soilsfrom
Açutuba(ACU),Balbina(BAL),BarroBranco(BBO),andHatahara(HAT)sites
Speciesrichnessestimators Diversityestimators
Soil NOTUsa ACE Chao-1 1/Dc H′d Chaoentropye Good’sf
type/site
ADE
ACU 136(183,89)b 200.14(239.10,161.17) 197.06(245.57,148.54) 10.30(16.66,3.94) 4.58(5.86,3.31) 3.24(4.13,2.35) 0.97(0.97,0.96)
BAL 132(145,119) 200.28(251.44,149.12) 193.01(262.55,123.48) 6.66(9.27,4.05) 4.19(4.83,3.56) 2.97(3.42,2.52) 0.97(0.98,0.96)
BBO 139(177,102) 210.90(253.77,168.03) 206.80(285.52,128.08) 7.74(11.95,3.53) 4.46(5.33,3.59) 3.16(3.77,2.54) 0.97(0.98,0.96)
HAT 172(182,162) 245.40(254.92,235.87) 248.17(294.53,201.81) 20.21(30.11,10.32) 5.60(5.74,5.46) 3.96(4.06,3.86) 0.96(0.97,0.96)
ADJ
ACU 164(173,155) 236.48(284.49,188.46) 238.93(348.00,129.86) 13.91(15.00,12.82) 5.18(5.29,5.06) 3.66(3.74,3.59) 0.96(0.98,0.95)
BAL 166(186,146) 243.53(269.82,217.23) 246.05(326.08,166.02) 15.54(19.64,11.44) 5.22(5.72,4.73) 3.70(4.03,3.37) 0.96(0.97,0.95)
BBO 127(145,109) 191.69(250.32,133.07) 180.82(221.30,140.33) 14.95(16.42,13.48) 4.94(5.10,4.79) 3.49(3.60,3.37) 0.97(0.98,0.96)
HAT 138(166,110) 188.47(231.82,145.11) 184.10(202.18,166.02) 15.98(26.94,5.01) 5.12(5.93,4.30) 3.61(4.17,3.04) 0.97(0.98,0.97)
aNumberofdeterminedoperationaltaxonomicunits
bTheshowedvaluesrepresenttheaverageofthreereplicatesfollowedbyconfidenceintervals
cSimpson’sreciprocal(1/D)index
dShannonindex
eChaoentropyindex(Chaoetal.2013)
fGood’sestimatedsamplecoverage
inthe ADE fromBALand BBOincomparisonwiththe re- Agaricomycotina fungi (Basidiomycota) (Table 3).
spectiveADJsamples(Table2). Pezizomycotina fungi were statistically significantly (p≤
0.05) more abundant in ADJ soils, and shifts were detected
StatisticalMultivariateAnalysis especially in Balbina and Hatahara sites. Ascomycota
Taphiromycotina (p≤0.005) and Mitosporic Acomycota (p≤
Eventhoughsimilaritieswereobservedinthespeciesrichness 0.05), Chytridiomycota Insertae sedis (p≤0.005), Fungi
anddiversityoftheADEandADJsoilfungalcommunities,the Insertae sedis Mucoromycotina (p≤0.05), and
OTU network and canonical correspondence analysis (CCA) Glomeromycotaphylum(arbuscularmycorrhizalfungi)were
showedtwowell-definedclusterssegregatingthefungalcom- alsomoreabundantinADJsoilsatstatisticalsignificantlevel.
munities of ADE and ADJ soils from BAL, BBO, and HAT The ADE soils showed significant higher abundance of
(Fig. 1a, b). The same pattern could not be observed for the Basidiomycota Agaricomycotina (p≤0.05) and
fungal communities of ADE and ADJ soils from ACU that Pucciniomycotina (p≤0.05), Fungi Insertae sedis
weremoresimilartoeachotheranddistantfromtheothersite Zoopagomycotina (p≤0.05), and Mortierellomycotina (p≤
assemblages(Fig.1b).CCAalsoindicatedthattheADEfungal 0.05). Shifts in Mortierellomycotina abundance were observed
assemblages were correlated with higher pH, macronutrients, mainlyinACUandHATsites.
sumofbases(SB),percentageofsoilbasesaturation(V%),and Asignificantnumberofsequences,especiallyinADE(p≤
Cu,Zn,andMnconcentrations,whereasADJcommunitywas 0.05),weretaxonomicallyclassifiedonlyatFungidomainand
correlatedwithAl,H+Al,aluminumsaturation(m%),andB environmentalsamplecategorybasedonBLASTaccesstax-
levels(Fig.1b).P and m% contributedwith morethan13 % onomy rank. We cannot affirm whether these results could
eachintheADEversusADJfungalcommunitydissimilarityas represent new fungal species or are resultant of SILVA and
calculatedbysimilaritypercentageanalysis(SIMPER)(Online NCBIdatabaseannotationimprecision.
Resource5).Conversely,theOMandpHcontributed2.5and
0.9%tothedissimilarities,respectively. FungalCoreCommunity
FungalTaxonomy The fungal core community present in all soil samples and
locations computed in QIIME was composed of seven
The phylum Ascomycota, specifically the subphylum OTUs, most of them classified as Ascomycota phylum
Pezizomycotina,wasthemostabundantinallthesoilsamples (Table 4). At species level, they showed similarity to
with exception of BAL ADE that was dominated by Ascomycota Cordyceps confragosa (OTU 822),
FungalCommunitiesinADE 967
Fig.1 Bipartitenetwork (a)
cacct(nB(roABeanoooapnannBdtDlrhlbofneeyOiEenisesdsn)ci)eAiebca,stnnaaiyamtn(n(cliBnCgnAedacdAgCzotçeahoHrluLdAerlentidje)au)paif,sgtabcusawpBeneehanosisanagDtn(r(htr(aArdaaab(lo9e)rAC)O(kn5BHaUDcTnEr%eA)JadUa,)Tnrsct)nhoooidles 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 AAOABBHdçmaaaTulrjtUbaaratociuzhneobaBannarrtaiaa Snnoc Doil ark Earth
(b)
2.4
1.8
1.2
%)
Axis 2 (12.7-3.0 -2.5 -2.0 -1.5 -1.0Aml% HB+Al -0.5 Fe 0.6 OM CEC0.5 MgpCZHunCVM%aPn1.S0B 1.5
-0.6
-1.2
ACU ADE
ACU ADJ
-1.8 BAL ADE
BAL ADJ
BBO ADE
-2.4 BBO ADJ
HAT ADE
HAT ADJ
-3.0
Axis 1 (16.5%)
Lithotheliumseptemseptatum(OTU1344),Aspergillusniger ahigherdiversitywasobserved(Table4).TheADJcorecon-
(OTU 2196), Ophiocordyceps clavata (OTU 2207), and sideringallsiteswascomposedofeightOTUs:twosimilarto
Fomitopsis pinicola (Basidiomycota, OTU 153), and OTUs Mucoromycotina sp. (OTUs 2315 and 2057) and the others
874and1924wereclassifiedasunculturedfungus(Table4). similar to Exophiala dermatitidis (OTU 118), Acremonium
WealsodeterminedthefungalcoresintheADEandADJ vitellinum (OTU 468), Pestalosphaeria sp. (OTU 938),
that were present in all samples of the same group but not Cryptococcusaureus(OTU1002),unculturedBasidiomycota
necessarycompletelyabsentintheotherone.Duetothepar- (OTU1523),andSpathulariaflavida(OTU2120).AfterACU
ticulargroupingpatternsoftheACUsoilsamplesintheOTU ADJ sequence removal, the number of OTUs belonging to
networkandCCAanalyses(Fig.1a,b),wedecidedtocompute ADJfungalcorewasraisedto12OTUs(Table4).
the fungal core in two ways: including and excluding ACU
samples. By considering only the most homogeneous ADE Abundance-BasedAnalyses
andADJsites(BAL,BBO,andHAT),weincreasedthenum-
ber of OTUs in the core. The ADE core considering all sites ThedominantOTUspresentinatleast75%oftheADEand
was composed of six OTUs; of those, three had similarity to ADJ 18S rRNA soil libraries (>1 % of sequences of each
Mortierellaceae sp. (OTUs 991, 1943, and 2141), one to group)wereidentifiedandtestedforstatisticalsignificantdif-
Plectosphaerellasp.(OTU2425),andtheothertwohadsim- ferences in abundance (non-parametric t test followed by
ilaritytounculturedChytridiomycota(OTU1462)anduncul- Monte Carlo test). Of the 30 OTUs that fit this criterion, 12
turedBoletaceae(OTU1134)(Table4).AfterACUADEse- were more abundant in ADE soils, 10 in ADJ soils, and 8
quenceexclusion,theADEcorewasincreasedto15OTUsand showed no significant abundance differences between soil
968 A.R.Luchetaetal.
ACU), ADEversuscADJ * ns ** * ns * ns * ns ns ** ns ns ns ns * ** * * ** * ns
(
a c c
b b b b c b
u a b a b b b a a b b a b a a b b b b a a b a
Açut DJ 7.99 0.10 0.09 1.34 0.07 1.32 2.28 0.15 0.00 0.19 1.83 0.29 0.00 0.03 0.57 2.60 0.78 0.07 0.72 0.25 3.05 0.21
sfrom HATA 56.73± 0.06± 0.19± 2.33± 0.08± 3.80± 6.71± 0.64± 0.00± 0.27± 4.78± 3.59± 0.00± 0.04± 0.50± 4.32± 2.37± 0.04± 3.63± 0.17± 9.51± 0.23±
soil c c b b bc
DJ) E 05b 03b 03b 00b 03b 24b 29a 58a 07b 19b 21c 84b 50a 09a 12a 61a 37c 15a 41a 00a 26a 00c
djacent(A HATAD 34.22±8. 0.02±0. 0.02±0. 0.00±0. 0.02±0. 7.77±1. 3.92±5. 2.28±0. 0.04±0. 0.14±0. 1.62±0. 2.58±1. 0.41±0. 0.08±0. 1.16±0. 18.52±2. 0.71±0. 0.50±0. 2.28±1. 0.00±0. 23.73±5. 0.00±0. 5)
a 0
arth(ADE)and BBOADJ 51.54±6.40ab 0.04±0.03b 0.39±0.29ab 0.58±0.31b 0.08±0.09b 2.57±0.59b 0.19±0.07b 0.06±0.06b 0.21±0.09a 0.12±0.12b 2.66±0.49bc 9.38±0.29a 0.02±0.03a 0.00±0.00a 0.06±0.06b 4.48±0.54b 4.88±1.76a 0.02±0.03b 1.58±0.03abc 0.15±0.07a 21.01±7.33ab 0.00±0.00c ’≤keystest(p0.
E u
anDark ADE ±2.21ab ±0.15a ±0.00c ±0.00b ±0.00b ±0.94b ±0.19b ±1.31ab ±0.00b ±0.03b ±0.32c ±3.44a ±0.00a ±0.00a ±0.13b ±1.77b ±0.59bc ±0.12ab ±0.12c ±0.00a ±2.69ab ±0.00c cesbyT
micalclassification)ofAmazoni ADEBALADJBBO ±4.70c47.28±6.41ab49.92 ±0.03b0.17±0.15b0.58 ±0.00c0.54±0.22a0.00 ±0.00b10.47±2.28a0.00 ±0.03b0.12±0.06b0.00 ±4.93a8.85±1.00b6.35 ±0.03b0.79±0.58b0.14 ±1.10ab0.02±0.03b1.22 ±0.00b0.02±0.03b0.00 ±0.07b0.12±0.15b0.02 ±0.45c1.81±1.49c0.87 ±0.55b1.23±0.34b12.38 ±0.15a0.10±0.09a0.00 ±0.00a0.00±0.00a0.00 ±0.03b0.25±0.15b0.42 ±2.61b10.15±2.57b7.39 ±0.09c3.05±0.79ab1.77 ±0.46a0.17±0.17b0.35 ±0.26bc4.75±2.61a0.46 ±0.00a0.15±0.03a0.00 ±0.35a9.93±0.49b18.13 ±0.03ab0.02±0.03ab0.00 LVA111database representnosignificantdifferen
a(basedonBLASTbesthittaxono bADEACUADJBAL ±6.41abc39.68±8.18abc27.37 ±0.03b0.04±0.03b0.04 ±0.00c0.00±0.00c0.00 ±0.00b0.00±0.00b0.00 ±0.10a0.15±0.12b0.02 ±3.45b3.70±1.71b33.29 ±0.22b1.10±0.50ab0.10 ±0.00b0.04±0.03b1.10 ±0.00b0.00±0.00b0.00 ±0.83ab8.06±7.35a0.08 ±0.50c7.02±1.02a0.75 ±0.86b4.92±1.43b0.81 ±0.27a0.23±0.27a0.41 ±0.00a0.58±0.67a0.00 ±0.09b0.10±0.17b0.14 ±1.33a9.12±2.38b7.68 ±0.24bc0.79±0.32c0.44 ±0.34b0.35±0.15ab0.98 ±0.44ab4.53±1.24a0.83 ±0.00a0.10±0.07a0.00 ±6.25ab19.46±3.13ab25.95 ±0.00c0.02±0.03ab0.02 afterBLASTsearchagainst97SI ystandarddeviation.Sameletters
Table3Relativeabundance(%)ofthefungi18SrRNAgenetaxBalbina(BAL),BarroBranco(BBO),andHatahara(HAT)sites aaSubphylumACUPhylum AscomycotaPezizomycotina44.27 Saccharomycotina0.08 Taphrinomycotina0.00 MitosporicAscomycota0.00 UnclassifiedAscomycota0.46 BasidiomycotaAgaricomycotina(norank)7.66 Basidiomycotaenvironmental0.15samplesPucciniomycotina0.00 Ustilaginomycotina0.00 BlastocladiomycotaBlastocladiomycotaInsertaesedis3.43 ChytridiomycotaChytridiomycotaInsertaesedis1.04 Chytridiomycotaenvironmental1.68samplesEntomophthoromycotaEntomophthoromycotainsertae0.23sedisEntorrhizomycotaEntorrhizomycotaInsertaesedis0.00 FungiincertaesedisKickxellomycotina0.15 Mortierellomycotina16.78 Mucoromycotina0.85 Zoopagomycotina0.25 GlomeromycotaGlomeromycotaInsertaesedis4.07 UnclassifiedGlomeromycota0.00 UnculturedfungusEnvironmentalsamples18.89 Unclassifiedsequence0.00 nsnotsignificantaTaxonomicclassificationbasedontheNCBIrankofthebesthitbTheshowedvaluesaretheaverageofthreereplicatesfollowedbcIndependentsamplettestcomparingADE×ADJsoilgroups≤≤*p0.05;**p0.005
FungalCommunitiesinADE 969
Table4 AmazonianDarkEarth(ADE)andadjacent(ADJ)soilsgeneralfungalOTUcoreandspecificsoiltypecores(ADEorADJ)followedbythe
bestBLASThitandtheNCBItaxonomicalclassification
NCBItaxonomicclassificationa
OTUnumber Soilgroup Accessnumber Phylum Subphylum Specie
OTU153 ADE/ADJ AY705967 Basidiomycota Agaricomycotina(norank) Fomitopsispinicola
OTU822 ADE/ADJ AB111495 Ascomycota Pezizomycotina Cordycepsconfragosa
OTU874 ADE/ADJ JN054669 nd nd Unculturedfungus
OTU1344 ADE/ADJ AY584662 Ascomycota Pezizomycotina Lithotheliumseptemseptatum
OTU2196 ADE/ADJ GQ903337 Ascomycota Pezizomycotina Aspergillusniger
OTU2207 ADE/ADJ JN941726 Ascomycota Pezizomycotina Ophiocordycepsclavata
OTU1924 ADE/ADJ JN054669 nd nd Unculturedfungus
OTU991 ADE EU688964 Fungiincertaesedis Mortierellomycotina Mortierellaceaesp.
OTU1943 ADE EU688964 Fungiincertaesedis Mortierellomycotina Mortierellaceaesp.
OTU2141 ADE EU688964 Fungiincertaesedis Mortierellomycotina Mortierellaceaesp.
OTU2425 ADE HQ871881 Ascomycota Pezizomycotina Plectosphaerellasp.
OTU1462 ADE GQ995336 Chytridiomycota nd unculturedChytridiomycota
OTU1134 ADE EF024156 Basidiomycota Agaricomycotina(norank) unculturedBoletaceae
OTU310b ADE GU369995 nd nd Unculturedmarineeukaryote
OTU339b ADE DQ198797 Basidiomycota Pucciniomycotina Atractiellasolani
OTU362b ADE GU568155 nd nd Unculturedsoilfungus
OTU548b ADE JN941713 Ascomycota Pezizomycotina Ophiocordycepsnutans
OTU1526b ADE AF026592 Basidiomycota Agaricomycotina(norank) Bjerkanderaadusta
OTU1878b ADE ABIS01004081 Ascomycota Pezizomycotina Coccidioidesposadasii
OTU2075b ADE EU417636 Glomeromycota Incertaesedis UnculturedGlomus
OTU2282b ADE AB196322 Fungiincertaesedis Kickxellomycotina Ramicandelaberlongisporus
OTU2475b ADE AB901634 nd nd Unculturedeukaryote
OTU118 ADJ DQ823107 Ascomycota Pezizomycotina Exophialadermatitidis
OTU468 ADJ HQ232212 Ascomycota Pezizomycotina Acremoniumvitellinum
OTU938 ADJ AF104356 Ascomycota Pezizomycotina Pestalosphaeriasp.
OTU1002 ADJ DQ437076 Basidiomycota Agaricomycotina(norank) Cryptococcusaureus
OTU1523 ADJ EF441962 Basidiomycota nd UnculturedBasidiomycota
OTU2120 ADJ Z30239 Ascomycota Pezizomycotina Spathulariaflavida
OTU2315 ADJ JF414214 Fungiincertaesedis Mucoromycotina Mucoromycotinasp.
OTU2057 ADJ JF414228 Fungiincertaesedis Mucoromycotina Mucoromycotinasp.
OTU1853b ADJ HQ333479 Ascomycota Mitosporic(norank) Heliocephalagracilis
OTU1086b ADJ AB032629 Basidiomycota Agaricomycotina(norank) Cryptococcusflavus
OTU2235b ADJ JF836023 Ascomycota Taphrinomycotina Archaeorhizomycesborealis
OTU2242b ADJ GQ995264 Chytridiomycota nd UnculturedChytridiomycota
ndnotdetermined(nd)
aTaxonomicclassificationbasedonbestBLASThitaccessaftersearchagainstSILVAdatabase(97SILVA111taxamapeuks)(Quastetal.2013)
bPresentonlyinBalbina,BarroBranco,andHataharasites
origins(Fig.2).OTU822,similartoC.confragosa,showed Discussion
thehighestnumberof18SrRNAsequences(17.8%)andwas
moreabundantintheADEsoils(Fig.2a)thaninADJsoils. Up to date, the fungal community in ADE has been charac-
ThesecondmostabundantwasOTU2196(6.4%),similarto terized only by culture-dependent methods [16] and poorly
A. niger, and significantly more abundant in ADJ soils described when compared to Bacteria and Archaea commu-
(Fig. 2b) than ADE soils. Chytridiomycota-like OTUs also nities [7–10, 12, 31, 32]. To our knowledge, this is the first
showedhighabundancelevelsbutwithoutdifferencesbased study assessing the soil fungal composition and diversity of
onthesoilorigin(Fig.2c). ADE sites in the Brazilian Central Amazonia compared to
970 A.R.Luchetaetal.
Fig.2 Differential frequencies ofmost abundant OTUs (>1 % ofthe significantmostabundantinAmazonianDarkEarth(ADE)(a),inadja-
groupsequences)determinedbynon-parametricttestusingMonteCarlo cent(ADJ)soils(b),andshowingnosignificantdifferencesbetweenthe
simulation (100 replicates). Plots representing the OTUs statistically soiltypes(c)
their respective adjacent non-anthropogenic origin soils by Despite the lower fungal species richness and diversity ob-
using high-throughput 18S rRNA gene sequencing. No dif- served in our study, elevated ratios of amino sugar and
ferenceinthefungusspeciesrichnesswasobservedbetween muramic acid in soil microbial biomass indicated a general
ADE and ADJ soils, with the exception of the HATsite, in predominance of fungi over bacteria in the ADE samples
which higher species richness in ADE was found with the [6].
ACE index. This finding diverges from the bacterial com- Previous studies revealed that ADE samples from different
munity richness that was described being 25 % greater in originsharborsimilarbacterialandarchaealcommunitiesaswell
ADE soils than in ADJ [7]. Culture-dependent [8] and asbacterialfunctionalgenes(e.g.,bph,encodingforabiphenyl
culture-independent analysis [12] also showed a higher bac- dioxygenase) that are distinct from adjacent soils of non-
terial diversity in ADE in comparison with ADJ soils. anthropogenicorigin[9,33].Inthisstudy,weobservedthesame
However, for fungi, we have detected no differences in fun- patternforfungalcommunities.TheADEfungalcommunitiesin
gal diversity in ACU and HATsoils. Only the reciprocal of three of the four evaluated sites (BAL, BBO, and HAT) were
Simpson’s index estimated a lower diversity in BAL and more similar to each other than their respective ADJ soils at
BBO ADE, indicating possible fungal species dominance. OTU level analysis, thus suggesting an effect of past land use
FungalCommunitiesinADE 971
on the fungal community selection. Nevertheless, the same fresh organic matter instead of direct oxidation of recalcitrant
groupingpatternwasnotobservedfortheACUsitewherethe BC.However,thepotentialforlowerBCoxidationratesbythe
fungal communities could not be segregated by soil type and Agaricomycotinafungicannotbediscardedandshouldbein-
were more dissimilar from the other ADE and ADJ sites. vestigatedinthefuture.Wewereunabletoaffirminthisstudy
Currently, the ACU ADE have intensively been used for agri- if the decomposition offresh organic matter priming affected
cultureunderannualcroprotationsystem(e.g.,eggplant,cow- recalcitrant BC decomposition. Controversial results are ob-
pea, cabbage, zucchini, cucumber, passion fruit, papaya) [34] served in the literature showing positive effect of glucose on
and also showed the lowest amount of organic matter among BCoxidationinBC/sandymixture[46]andnoprimingeffect
the surveyed ADE soils. Shifts in fungal communities in onBCmineralizationbytheincorporationof13C-labeledplant
Amazonian soils due to land use changes, e.g., conversion of residues to ADE in long-term experiments [39]. In the other
native forest to pasture and agriculture, have already been de- direction, Glaser and Knorr [38] determined significant
scribed[35],buttheextensionofthealterationsinADElanduse amounts of biological BC production under humid tropical
onthemicrobialcommunitiesarestillscarce[4].Weobservedan conditionsandattributedittotheblackpigmentaspergilinpro-
increase in the common ADE OTUs (ADE fungal core) after ducedbyAspergillusniger.DespitethepresenceofA.nigerin
ACUsampleremoval,butwecannotaffirmthatthiseffectwasa thegeneralfungalcore,theOTU2196similartothisspecies
result of the ACU ADE transformations in response to more wassignificantlyabundantintheADJsoils.A.nigerisaver-
intensivelanduseorduetonaturaldifferencesinADEagesor satile ubiquitous fungus, commonly found in soil and litter
formationprocesses.ThelowconcentrationofAlandaluminum [47],andabletoproduceandsecreteenzymesandsiderophore
saturationinADJsoilfromACUpointstopriorlimeapplication molecules[48]andsolubilizeinorganicP[49].
beforesampling,whichcouldexplaintheout-groupingofADJ In this study, we also observed a high abundance of 18S
samples.Lehmann[36]suggeststhatthespecificmicrobialcom- rRNAsequencessimilartothefungalspeciesC.confragosa,a
positioninADEisaresultofitsuniqueconditionsratherthanthe pathogen of arthropods and other fungal species [50].
cause.Indeed,thehigheramountsofnutrients,mainlyP,Ca,Zn, Entomopathogenic fungi, like Cordyceps and
and Mg, and higher SB and V% were associated with ADE Ophiocordyceps,arecommonlyfoundinundisturbedtropical
fungalcommunities,whereasAlandaluminumsaturationswere humidforestssoilandlitterandcancontrolinsectoutbreaks
moreassociatedtothefungalcommunitiesinADJsoils.High [51]. C. confragosa, also known as Lecanicillium lecanii
levelsofAland Mn indirectly caused by soilpHacidity have (Zimm.)duringitsanamorphicstage,isaparasiteofthegreen
been described as a limiting factor for crop production in coffeescale(Coccusviridis,Hemiptera)[52]andcoffeeleaf
Amazoniansoils[37].SignificantcorrelationsofAlcontentsin rustfungus(Hemileiavastatrix)[53].Inagriculturalenviron-
ADE and ADJ soils with bacterial rizhospheric and bph gene ments, soil can act as the fungus propagule reservoir during
communitystructureswerealsoobserved[4,33];however,fur- the dry seasons and absence of the target insects [54]. We
ther studies are still necessary to confirm this assumptions for observed a dominance of C. confragosa-like OTUs in the
fungalcommunitiesintheseenvironments. BBOADEsoilthatwascultivatedwithacitrusorchardand
The microbialfunctions in ADEare stillunclear[36],and speculatethatthisfunguscouldbeactingintheinsectbiolog-
mosthypothesisreliesonblackcarbon(BC)oxidation,mainly ical control. Further studies are necessary to explore these
byfungi[6]orBCbiologicalproduction[38].Duetoitspoly- predictions. Our findings indicated that beyond the impor-
cyclicaromaticstructure,BCcannotbeconsideredanavailable tance in C transformations, ADE soils could be a source of
sourceofCformicrobialgrowth[3,39];however,itmaybe newentomopathogenicfungi.
mineralizedbymicrobialco-metabolism[9].Inoursurvey,we
observed a significantly higher abundance in ADE of OTUs
showing similarity to the brown rot fungi F. pinicola [40] as Conclusions
well as the saprophytic fungi A. vitellinum [41] and
Mortierellaceaesp. LN07-7-4. Aremarkablecharacteristicof OurstudyrevealedthatfungicommunitiesinADEweremore
theBasidiomycotabrownrotfungiistheselectivedegradation similar to each other than to the adjacent soils, even when
ofwoodpolysaccharides,whichavoidsligninmolecules[42]. considering the different origins and ages of formation. The
Inthesameway,Mortierellaspp.andAcremoniumspp.were concentrationsofsoilPandAlwerethemainchemicalprop-
found in thermophilic compost and vermicompost [43–45]. ertiesassociatedtothefungalassemblagesinADEandADJ
Mortierellales fungi were more associated to manure silage soils, respectively. However, other potential factors driving
and hay compost than hardwood composts [45]. ADE fungi communities beyond the soil chemical attributes
Mortierellaceawasalsodescribedasdominantinsoilsamples might be further investigated. Recently, it was demonstrated
fromprimaryfloretsandagriculturalareasinAmazonia[35]. thatplantspeciescaninfluencerhizosphericbacterialcommu-
Basedontheseresults,wehypothesizethatthemostabundant nitiesinADE[4].ThemostabundantOTUsintheADEsoils
fungal species in ADE are involved in the decomposition of showedsimilaritytosaprophyticfungispeciesrelatedtofresh