compositionanddiversityoffungaldecomposersofsubmerged...

Research ArticleComposition and Diversity of Fungal Decomposers of SubmergedWood in Two Lakes in the Brazilian Amazon State of Para

EveleiseSamiraMartinsCanto 12AnaClaudiaAlvesCortez3 JosianeSantanaMonteiro4

Flavia Rodrigues Barbosa5 Steven Zelski 6 and Joatildeo Vicente Braga de Souza3

1Programa de Pos-Graduaccedilatildeo da Rede de Biodiversidade e Biotecnologia da Amazonia Legal-BionorteManaus Amazonas Brazil2Universidade Federal do Oeste do Para UFOPA Santarem Para Brazil3Instituto Nacional de Pesquisas da Amazonia INPA Laboratorio de Micologia Manaus Amazonas Brazil4Museu Paraense Emilio Goeldi-MPEG Belem Para Brazil5Universidade Federal de Mato Grosso UFMT Sinop Mato Grosso Brazil6Miami University Department of Biological Sciences Middletown OH USA

Correspondence should be addressed to Eveleise Samira Martins Canto eveleisesamirahotmailcom and Steven Zelskizelskisemiamiohedu

Received 25 August 2019 Revised 20 February 2020 Accepted 4 March 2020 Published 9 April 2020

Academic Editor Giuseppe Comi

Copyright copy 2020 Eveleise Samira Martins Canto et al is is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in anymedium provided the original work isproperly cited

Aquatic ecosystems in tropical forests have a high diversity of microorganisms including fungi which are important decomposersof submerged wood Despite the importance of their role in decomposition research concerning the diversity of freshwater fungifrom Brazilian Amazonian environments is scarce e aim of this work was to describe the composition and diversity of fungipresent on submerged wood in two lakes of the Brazilian Amazon (State of Para) Fragments of decaying wood (30 samples perlake) were collected from the Lakes Jua and Maica e wood samples were inspected for 6 months in the presence of fungalreproductive structures Fungi observed in the wood were identified morphologically Twenty-three taxa were identified in theLake Jua (10 sexual and 13 asexual) and 26 taxa in the LakeMaica (17 sexual 9 asexual) ITS sequences were obtained for 14 taxa toaid in identification In the Lakes Jua and Maica the diversity indices were Hrsquo 26514 and Hrsquo 28174 respectively e Soslashrensenindex of the fungal communities in the studied lakes was 03673 is study is the first to describe the fungal biodiversity of twoimportant aquatic environments in Para Brazil

1 Introduction

Freshwater fungi include species that inhabit water for all orpart of their life cycle or any species adapted to colonizepredominantly aquatic or semiaquatic substrates in nature[1] ese organisms serve as key agents in the decompo-sition of submerged dead plant material Fungal biomass is asource of nutrition for other organisms in aquatic ecosys-tems [2 3] Fungal enzymatic activity also modifies plantsubstrates to make them more palatable to invertebrateshredders and scrapers [4]

Freshwater ascomycetes are a group of fungi known toactively participate in the decomposition of submerged

woody debris [5 6] is group is polyphyletic with themajority of species belonging to the subphylum Pezizo-mycotina in the classes Leotiomycetes Sordariomycetes andDothideomycetes [2 7] Members of the Eurotiomycetes [8]and Orbiliomycetes [9] are less common Shearer and Raja[10] reported that freshwater ascomycetes are distributed inrelatively few orders Helotiales Pleosporales SordarialesSavoryellales Microascales Eurotiales and Jahnulales enumber of known taxa has increased in recent years due tobroader sampling and the use of molecular methods foridentification [11 12]

e Amazon basin is arguably the most complex net-work of aquatic habitats on the planet [13] is vast region

HindawiInternational Journal of MicrobiologyVolume 2020 Article ID 6582514 9 pageshttpsdoiorg10115520206582514

contains a variety of aquatic environments that include largerivers lakes swamps and seasonally inundated floodplains[13 14] Aquatic ecosystems can be roughly classified intothree main types clear waters [15] white waters [16] andblack waters [14 17] Water characteristics such as tem-perature dissolved oxygen and amount of organic mattermay influence the diversity of freshwater fungal species [18]e first published studies of freshwater fungi in Amazonianwaters (Peru) were conducted byMatsushima in white waterrivers [19] More recent studies have described new speciesof ascomycetes in the Peruvian Amazon [20ndash24] and in theBrazilian states of Para [25 26] and Amazonas [27 28] Ingeneral the microbial community of the Amazon region isstill underexplored and studies describing the diversity andecology of fungi in the Amazon region are urgently needederefore we sought to investigate new environments suchas the Tapajos River one of the largest clear water tributariesof the Amazon River [17] e present work aimed to de-scribe the composition and diversity of fungi present onsubmerged decomposing wood in two lakes in the TapajosRiver basin near Santarem in the Brazilian state of Para

2 Materials and Methods

21 Study Sites Samples of submerged wood were collectedfrom Lake Jua (2deg25prime57PrimeS 54deg 46prime39PrimeW) and Lake Maica(2deg27prime290PrimeS 54deg 40prime108PrimeW) and essentially the embay-ment of the lower Tapajos and Amazon Rivers respectivelyLake Jua receives water that drains from a stream and theTapajos LakeMaica is amix of surface drainages and the whitewater of the Amazon River [14] (Figures 1 2(a) and 2(b))

22 Water Characterization Physicochemical variableswere measured using a ProfiLine 197iWTWmultiparameterprobe (Wellheim Germany) Temperature (degC) pH elec-trical conductivity (μS) dissolved oxygen concentration(mgL) and turbidity (NTU) were recorded for each samplecollection

23 Sample Collection A single collection was made at eachlocation in Lake Jua in October 2017 and in Lake Maica inNovember 2017 At each site 30 submerged wood fragmentsthat showed signs of active decomposition were collectede lengths of the samples ranged from 6 to 22 cm anddiameters ranged from 6 to 15 cm (Figure 2(c)) esesubstrates were transported in sealable plastic bags(20 cmtimes 10 cm) with a small amount of local water to theLaboratory of Multidisciplinary Teaching in Applied Biol-ogy-Labio Universidade Federal do Oeste do Para(UFOPA) In the laboratory the samples were gently rinsedwith running water placed in moist chambers (plastic boxeswith moist paper towels) and incubated at room temper-ature with 1212 h lightdark conditions

24 Isolationand Identificationof FungiPresent onSubmergedWood Every ten days for six months a Stemi DV4 ste-reomicroscope (Zeiss) was used to search for fungal

structures on the collected wood samples e structuresfound were transferred with dissection needles to micro-scope slides containing distilled water e identification ofsexual ascomycetes was performed based on the micro-morphology of ascomata hamathecium asci and asco-spores using an Axioskop 40microscope (Zeiss) as describedpreviously [22 29 30] Aqueous lactophenol cotton bluesolution was used to determine the staining reactions of theapical ascus apparatus Asexual ascomycetes were identifiedby investigating the types of conidiogenesis conidiophoreand conidia using an Axioskop 40 microscope (Zeiss)Isolation of fungal was carried out by transferring the fungalstructures to the surface of Antibiotic Water Agar (20 gLagar and chloramphenicol 250mgL) Potato Dextrose Agar(PDA) (Kasvi) and Malt Extract Agar (MEA) (malt extract30 gL mycological peptone 5gL and 15gL agar pH54plusmn 02) e developed colonies were transferred to PYGagar (125 gL peptone 125 gL yeast extract 5 gL glucose250mgL chloramphenicol and 18 gL agar) [22](Figures 2(d)ndash2(g))

25 DNA Extraction Amplification and SequencingDNA extraction was performed according to the protocol ofFerrer et al [31] Fungi were grown in test tubes containing10mL of potato dextrose broth (120 g potato 10 g dextroseand 1000mL distilled water) for 10 days and transferred toEppendorf tubes (2mL) containing 500 μL of buffer (25mgSDS 70mg NaCl 365mg EDTA 20mL of Tris-HCl and100mL of DI water and 5 μL of szlig-mercaptoethanol) emicrotubes were subjected to freezing (minus20 degC) and heating(65degC for 1 h) in order to rupture the cellular structuresNext 500 μL of a phenolchloroformisoamyl alcohol so-lution (vvv 25241) was added to the microtubes andvortexed until a homogeneous suspension was obtainedesuspension was then centrifuged (14000 rpm 15min) andthe supernatant was transferred to a newmicrotube (15mL)Isopropanol was added to the supernatant in equal volumeand the mixture was homogenized and incubated at minus20degCovernight to precipitate the DNAe precipitated DNAwascentrifuged (14000 rpm for 15min) washed twice with 70ethanol and resuspended in 100 μL of sterile water distilled

Molecular data were gathered by sequencing the ITSregion of rDNA according to the procedure of White et al[32] e reaction yielded a final volume of 50 μL consistingof PCR buffer (10mM Tris-HCl pH 83 50mM KCl)15mM MgCl 2 05 μM of ITS-1 (5rsquo-TCCGTAGGT-GAACCTGCGG-3rsquo) and ITS-4 (5rsquo-TCCTCCGCTTATT-GATATGC-3rsquo) 200 μMdNTPs 15 U TaqDNA polymeraseand 100 ng of fungal DNA ermocycling was performedon an Applied Biosystems ProFlex PCR System with initialdenaturation at 94degC for 5min followed by 35 cycles of DNAdenaturation at 94degC for 1min annealing at 55degC for 1minextension at 72degC for 2min and a final extension at 72degC for10min Two positive controls and one negative control werealso tested Electrophoresis was performed on 15 agarosegel in 1x TBE buffer (100mM Tris base 100mM boric acidand 2mM EDTA pH 80) plus 10 μL100mL of Siber GreenSYBRreg Safe Amplified fragments were visualized under UV

2 International Journal of Microbiology

light e amplification products were purified using a so-lution of polyethylene glycol-PEG (10 g polyethylene glycol800 73 g NaCl and 35mL of water) as previously described[33] Similar volumes of the PEG and amplicon solutionswere transferred to microtubes (15mL) homogenized andincubated (37degC for 15min) e mixture was then centri-fuged (15min at 6000 rpm) the supernatant was discardedand the precipitate was washed twice with 70 ethanol andresuspended in water

e sequencing reaction was performed using theBigDyereg kit (Applied Biosystems) Sequencing was per-formed on the Seq 3130 Genetic Analyzer (Applied Bio-systems) e sequences used in this study were comparedand deposited in the GenBank database (Table 1)

26 Richness Diversity and Equitability of the FungalCommunity e number of individuals was determined foreach piece of wood by the presence or absence of a species onit In this study an individual was quantified on a per-substrate basis One individual was counted if it was foundon a single piece of wood However multiple taxa wereusually found to colonize individual substrates Frequencyrichness (species numbers) alpha diversity (α) and simi-larity of fungi were calculated for each lake To characterizethe diversity of the fungal community the Shannon-Weaverdiversity index (Hprime) was employed [34] is index describesdiversity with a higher value signifying greater heterogeneityand greater diversity e following formula was used toperform these calculations

Hprime minus 1113944(pi)lowast(log 2pi) (1)

where pin niN ni is the individual number of ith taxaand N is the individual number of all taxa

In addition to interpret the Shannon-Weaver index theevenness index (E) was calculated is index is the uni-formity of copy numbers among taxa e evenness tendstoward 0 when one taxon dominates the community andapproaches 1 when the taxa have the same abundance eindex is expressed by the following formula

E Hprimeln S

(2)

where Hprime is the Shannon-Weaver Index based on thenumber of individuals and S is the number of taxa present inthe sample

To evaluate differences in the fungal communities thesimilarity among the samples was calculated using theSoslashrensen index (Sprime) with values ranging from 0 (no simi-larity) to one (absolute similarity) is was calculated usingthe following formula

Sprime 2c

a + b (3)

where a is the total number of taxa collected in Lake Jua b isthe total number of taxa collected in Lake Maica and c is thenumber of common taxa in both lakes A taxa-area curve wasplotted for the two collections to assess the sampling effortStatistical analyses were performed using the PASTprogramversion 32 [35]

5250 75

54deg486primeW 54deg444primeW 54deg402primeW 54deg360primeW


MaicaacuteLake

Juaacute Lake

Tapajoacutes RiverAmazonas

River2deg22

8primeS

2deg27

0primeS

2deg31

2primeS

Scale

Brazil

Paraacute state

N

SamplingsitesSantareacutem city

km

Figure 1 Map of the location of the Lakes Jua and Maica belonging to the basin of the lower Tapajos River Santarem Para Brazil (sourceLima J L)

International Journal of Microbiology 3

(a) (b)

(c) (d)

(e) (f ) (g)

Figure 2 Collection site of Lake Jua (a) Collection site of Lake Maica (b) Wood samples (c) Ascomata on a wood surface (d) Asci in thewater of Dothideomycetes sp (e) Colonies on MEA after 14 days at 25degC (f) Culture in PYG agar (g)

Table 1 Strain code and similarity (ITS Barcode) of 14 taxa isolated from samples of submerged wood collected in the Lakes Jua andMaicaSantarem Para Brazil

CodeGenBank Taxon Accession no Similarity ()MT017512 Pseudallescheria boydii KJ6538231 100MT017513 Curvularia clavata MF0381791 9915MT017519 Curvularia clavata MF0381791 100MT017518 Curvularia petersonii NR_1584481 9715MT017514 Cladosporium holotolerans MN8268231 9784MT017515 Simplicidiella nigra NR_1643831 913MT017516 Jattaea sp KT8237831 9046MT017517 Jattaea sp EU7702231 9032MT017520 Bionectria sp GU8274831 9941MT017523 Gonytrichum macroladum MH8579541 9834MT017524 Fusarium solani JN8822571 9912MT017525 Helicascus thalassioideus KP6371621 9002MT017522 Cladophialophora saturnica NR_1112781 9946MT017521 Calosphaeriales sp KR8172461 9904


In order to determine whether the sample size wassufficient to describe the diversity of the collection sites theaccumulation curve of the taxa found in the samples ofsubmerged wood decomposed in the two lakes under studywas analyzed by the ratio between the number of new taxaand the number of wood samples

3 Results

31 PhysicochemicalCharacteristics In order to characterizethe aquatic environments in this study physicochemicalanalyses of the waters of the Lakes Jua and Maica wereperformed at the time of sampling of wood samples Table 2lists the values of the parameters

32 Sample Size e accumulation curves obtained for thesamples from Lakes Jua and Maica show that the number oftaxa does not asymptote suggesting that we have not ad-equately captured the freshwater fungal species (Figure 3)and additional collections may be necessary

33 Identification of Fungi Morphologically 23 differenttaxa were identified in the Lake Jua with 435 (n 10)being sexual (5 Dothideomycetes and 5 Sordariomycetes)and 565 (n 13) being asexual In the Lake Maica 26 taxawere identified morphologically of which 654 (n 17)were sexual (4 Dothideomycetes and 13 Sordariomycetes)and 346 (n 9) were asexual We carried out DNAanalysis studies with only fourteen taxa (Table 1) It was notpossible to investigate all taxa because most of them wereuncultivated fungi that did not allow DNA extraction ITSsequence data were successfully obtained and used to cor-roborate morphological identification (Table 3)

34 Richness Diversity andEquitability In the Lake Jua thediversity (Hprime) and equitability (E) indices reached 265 and0616255 respectively In this period the five most frequenttaxa were Xylomyces giganteus (18) Pseudoxylomyceselegans (11) Pleosporaceae MT017518 Fluviatispora spand Teratosphaeriaceae MT017515 (9) In the Lake Maicathe diversity and equitability indices reached 282 and0643588 respectively At this site the most frequent taxawere ielavia sp (11) Longicollum biappendiculatum(8) Submersisphaeria sp Aquaticola sp and Moro-sphaeriaceae MT017515 (7) e Soslashrensen index of thefungal communities between the two lakes studied was03673 Eight taxa occurred in both environments Infor-mation on the richness diversity and equitability of the taxaobserved in the two lakes is described in Table 4

4 Discussion

In the present study we described the fungal diversity as-sociated with the decomposition of wood in lakes belongingto the Tapajos River (PA Brazil) is is an important studybecause it is the first to describe the diversity of these or-ganisms in the lakes of the Tapajos River region In the

experimental conditions both lakes presented high diversityand few similarities among the taxa

e Lake Jua had a low pH (49plusmn 02) and low electricalconductivity (93plusmn 02 μS) is was expected because LakeJua is influenced by the Tapajos River [15 36] Its turbiditywas 30plusmn 01 NTU perhaps due to the direct interference ofanthropic actions such as wastewater disposal due to theproximity to the urban area of Santarem e Lake Maicahad neutral pH (67plusmn 02) and high electrical conductivity(42plusmn 02 μS) is may be due to the influence of theAmazon River during the flood season [14] is river has analkaline pH high electrical conductivity and high turbiditydue to the large amount of sediment coming from theAndean regions [15 16] It is important to know thecharacteristics of the water in aquatic fungal diversity studiesbecause previous studies have shown that factors such as pHnutrients substrate type river geomorphology and sea-sonality affect the fungal communities in aquatic environ-ments [37ndash39]

e accumulation curve showed approximate stabilityafter the collection of 20 to 25 samples from each lakestudied (Jua and Maica respectively) Luo et al [40] col-lected 100 samples of grass and bamboo from the LakeDianchi inailand during four sampling events over a one-year period and reached asymptote in 50 and 70 samples Huet al [41] collected 100 samples from a dammed lake and 90samples in a stream obtaining the stabilization of the curvein 60 and 80 samples respectively Cortez [28] collected 60fragments of submerged wood in a lake in the region ofIranduba (Amazonas Brazil) in two periods of the seasonalcycle obtaining a plateau of species in 15 samples collectedin the nonrainy period In this study 30 samples per lakewere collected and appeared sufficient to near a plateau forlocal diversity

At the Lakes Jua and Maica we observed 23 and 26 taxarespectivelye number of unique taxa was 15 and 18 in theLakes Jua and Maica respectively (Table 3) ese results aresimilar to those observed by Cortez [28] who identified 25taxa in a black water lake in Amazonas State When com-pared to other works [41ndash43] the total number of taxa in thisstudy was lower is may be related to differences in thenumber of samples per period the quantity of collectionsmade and the types of environments studied In additionprevious studies have stated that physicochemical factors ofwater play an important role in the richness of ascomycetespresent in freshwater and may be a factor in these lakes [44]

e Lake Jua presented greater richness of asexual fungi(565) and Maica of sexual fungi (654) In the Lake Jua

Table 2 Characterization of the physicochemical parameters of thewater of the Lakes Jua andMaica Santarem Para Brazil during thecollections

Parameters Lake Jua Lake MaicapH 49plusmn 02 67plusmn 02Electrical conductivity (microS) 93plusmn 02 42plusmn 02Water temperature (degC) 30plusmn 01 30plusmn 01Total O2 (mgL) 78plusmn 01 65plusmn 01Turbidity (NTU) 30plusmn 01 269plusmn 01


Lake JuaacuteLake Maicaacute

5 10 15 20 25 30 350Number of samples

0

5

10

15

20

25

30

Num

ber o

f tax

a

Figure 3 Accumulation curve of the number of fungi taxa found in relation to the sampling effort of collecting submerged fragments ofdecomposed wood in the Lakes Jua and Maica Santarem Para Brazil

Table 3 Frequency of taxa observed in samples of submerged wood collected in the Lakes Jua and Maica Santarem Para Brazil

TaxaLake Jua Lake Maica

FAB FRE () FAB FRE ()Xylomyces giganteus Goh W H Ho K D Hyde amp K M Tsui 18 15 0 0Pseudoxylomyces elegans (Goh W H Ho K D Hyde amp K M Tsui) Kaz Tanaka amp Hiray 13 11 0 0Teratosphaeriaceae MT017515lowastlowast 11 9 0 0Pleosporaceae MT017518lowastlowast 11 9 0 0Fluviatispora sp 11 9 0 0Cancellidium applanatum Tubaki 9 7 1 1Cumulospora splowast 7 6 0 0Cordana terrestris (Timonin) M Hern-Rest Gene amp Guarro 7 6 0 0Chloridium sp 5 4 5 6Lasiosphaeria sp 4 3 0 0Jobellisia splowast 4 3 0 0Aquaticola sp 3 2 6 7Taeniolella splowast 3 2 2 2Morosphaeriaceae MT017525lowastlowast 2 2 6 7Microascaceae MT017512lowastlowast 2 2 0 0Diaporthales sp1 MT017516 MT017517lowastlowast 2 2 0 0Gonytrichum sp MT017523lowastlowastlowast 2 2 0 0Monodictys sp 2 2 0 0Unidentified Pleosporales 3 1 1 5 6Acrogenospora sphaerocephala (Berk amp Broome) M B Ellis 1 1 1 1Cladophialophora sp MT017522lowastlowast 1 1 0 0Curvularia sp MT017513 MT017519lowastlowast B L Jain 1 1 1 1Stilbella splowast 1 1 0 0Dothideomycetes sp 0 0 3 4Dothideomycetes sp1 0 0 3 4ielavia splowast 0 0 9 11Longicollum biappendiculatum Zelski F R Barbosa Raja A N Mill amp Shearer 0 0 7 8Submersisphaeria sp 0 0 6 7Annulatascus-like sp 0 0 5 6Savoryellales sp 0 0 3 4Aniptodera sp 0 0 2 2Pseudohalonectria-like splowast 0 0 2 2Annulatascaceae-like sp 0 0 2 2Ophioceras-like splowast 0 0 2 2Sordariomycetes sp MT017521lowastlowast 0 0 2 2Nectriaceae sp MT017520lowastlowast 0 0 2 2Cladosporiaceae MT017514lowastlowast 0 0 2 2Nectriaceae MT017524lowastlowast 0 0 1 1Potamomyces armatisporus K D Hyde 0 0 2 2Helicosporium sp 0 0 1 1Sporoschisma sp 0 0 3 4TOTAL 121 100 84 100lowastFirst records for the Brazilian Amazon FAB absolute frequency FRE relative frequency lowastlowastITS Sequence obtained


this may be related to the influence of a stream that drainsinto this environment supporting the increase of this taxonin the lake [45 46] In the Lake Maica the sexual speciesrichness was greater ese results resemble those reportedby Hu et al [41] who found greater sexual richness in alentic environment Cortez [28] also found similar resultsand attributed these results to pluviometric action becauserainwater can carry fungi from soil or vegetation to the lake

e five most frequent taxa in the Lake Jua werePseudoxylomyces elegans Xylomyces giganteus Pleospor-aceae MT017518 Fluviatispora sp and TeratosphaeriaceaeMT017515 In the Lake Maica the five most frequent taxawere ielavia sp Longicollum biappendiculatum Sub-mersisphaeria sp Aquaticola sp and MorosphaeriaceaeMT017525 Aquaticola sp and Longicollum biappendicula-tum were the most frequent taxa described by Cortez [28]Abdel-Aziz [43] in a study on submerged stems of Phrag-mites australis (Cav) Trin ex Steud listed seven species thatwere common in this study (Aniptodera sp Annulatascus-like Aquaticola sp Ophioceras sp Pseudohalonectria spielavia sp and Monodictys-like) Raja et al [42] infreshwater environments of the Florida Peninsula sharedfive taxa (Aniptodera sp Ophioceras sp Submersisphaeriaaquatica Fluviatispora reticulata and Longicollum biap-pendiculatum) It was observed that these taxa occurred inboth lentic and lotic environments According to Sheareret al [24] the composition of freshwater ascomycetes speciesis similar between Florida tropical South America (Peru)and tropical Southeast Asia

Cancellidium applanatum found in Lake Jua and Maicawas also reported by Zelski et al [22] in a survey of asco-mycetes in wood submerged in freshwater habitats in Peruis species is considered a generalist and can occur inherbaceous and woody substrates of lotic and lentic habitatsis species as well as Monodictys sp can be consideredresistant to dry periods [47] and its presence is not sur-prising considering that the Lake Jua also undergoes con-siderable periods of drought In addition Acrogenosporasphaerocephala has recently been reported on leaf material in

rivers and streams in the Caatinga biome as well as insubmerged wood in a lake in Manaus Amazonas [5 28]Nine taxa were identified as new records for the BrazilianAmazon as shown in Table 1

e Lakes Jua and Maica presented a diversity of Hprimeindex 265 and 282 respectively Hu et al [41] inailandcompared an artificial lake and a stream obtaining a diversityindex of 234 and 368 respectively indicating that factorssuch as the composition of riparian forest damming ofrivers and anthropogenic actions may affect the communityand the diversity of fungi in freshwater habitats Cortez [28]studied the diversity in a lake in the state of Amazonasmaking seasonal comparisons and obtained indices of 260in the rainy season and 213 in the nonrainy season isstudy inferred that seasonality may affect fungal diversity Inthe Nile Abdel-Aziz [43] also performed seasonal com-parisons with indices of 308 and 244 for winter andsummer respectively Hyde et al [48] emphasized that thenumber of taxa per wood sample is higher in the tropics thanin temperate habitats According to Graccedila et al [49] thevariation in low diversity rates in tropical and subtropicalareas may be related to ecological andmethodological issuese diversity indexes of the present work are similar to thosefound in other lentic environments (meansim 28) and cor-roborate the assertion that diversity in lotic environments isgreater (meansim 460) [41 50ndash52]

Soslashrensenrsquos similarity index (03673) revealed low simi-larity among the studied lakes is may be due to thestructural and physicochemical differences found during thestudy that are responsible for the composition of the mycotain aquatic ecosystems [37 38 53] According to Leff [54] themicrobial composition is affected in streams and rivers bythe unidirectional flow of water (which is reflected in or-ganismsrsquo life strategies) mixing and aeration of water andthe importance of allochthonous materials

Studies on fungi that decompose submerged wood inaquatic environments are lacking in the Amazon regionus the present work was motivated to contribute to theknowledge of the diversity of aquatic fungi in this region

Table 4 Comparison of diversity (indicesH E and S of the fungi community present in the samples of the Lakes Jua andMaica SantaremPara Brazil

Sampling Lake Jua October 2017 Lake Maica November 2017Sample size 30 30Number of ascomycetes (sexual) 51 67Number of ascomycetes (asexual) 70 14Average number of taxa per sample 40 28Singleton taxa 15 18Overlap in both lakes 8

Five most common taxa Xylomyces giganteus 18 (18) ielavia sp 9 (11)Pseudoxylomyces elegans 13 (11)

Longicollum biappendiculatum 7 (8)Pleosporaceae MT017518 11 (9) Submersisphaeria sp 6 (7)

Fluviatispora sp 11 (9) Aquaticola sp 6 (7)Teratosphaeriaceae MT017515 11 (9) Morosphaeriaceae MT017525 6 (7)

Species richness (R) 23 26Shannon-Weaver (Hprime) 265 282Evenness (E) 0616255 0643588Soslashrensen (Sprime) 03673


e results show that there is a rich and balanced diversityis study provides more insight into the dynamics offreshwater fungi in clear water habitats and indicates howthe community is distributed in the Amazonian lakes It isnoteworthy that further studies are needed to strengthen thisline of research and increase available information onbiogeography ecology and taxonomy of freshwater asco-mycetes species in the Brazilian Amazon

Data Availability

e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

e authors declare that they have no conflicts of interest

Acknowledgments

e authors would like to thank the Universidade Federal doOeste do Para-UFOPA and the Laboratorio de MicologiaInstituto Nacional de Pesquisas da Amazonia-INPA forprovision of laboratory facilities Laboratorio de Tecnologiasde DNA-UFAM responsible for DNA sequencing samplesWe thank Prof Dr Carlos Ivan Vildoso and Dr Huzefa Rajafor their collaboration is study was funded in part by theCoordenaccedilatildeo de Aperfeiccediloamento de Pessoal de Nıvel Su-perior-Brasil (CAPES) (finance code 001rdquod)

References

[1] M K M Wong T-K Goh I J Hodgkiss et al ldquoRole of fungiin freshwater ecosystemsrdquo Biodiversity and Conservationvol 7 no 9 pp 1187ndash1206 1998

[2] E B G Jones and K-L Pang ldquoTropical aquatic fungirdquoBiodiversity and Conservation vol 21 no 9 pp 2403ndash24232012

[3] H-P Grossart and K Rojas-jimenez ldquoAquatic fungi tar-geting the forgotten in microbial ecologyrdquo Current Opinion inMicrobiology vol 31 pp 140ndash145 2016

[4] V Gulis K Kuehn and K Suberkropp ldquoe role of fungi incarbon and nitrogen cycles in freshwater ecosystemsrdquo inFungi in Biogeochemical Cycles G M Gadd Ed pp 404ndash435Cambridge University Press Cambridge UK 2006

[5] F R Barbosa H A Raja C A Shearer and L F P GusmatildeoldquoSome freshwater fungi from the Brazilian semi-arid regionincluding two new species of hyphomycetesrdquo CryptogamieMycologie vol 34 no 3 pp 243ndash258 2013

[6] P O Fiuza L A Costa A O Medeiros V GulisL F P Gusmatildeo and P Gusmatildeo ldquoDiversity of freshwaterhyphomycetes associated with leaf litter of Calophyllumbrasiliense in streams of the semiarid region of Brazilrdquo My-cological Progress vol 18 no 7 pp 907ndash920 2019

[7] M Blackwell ldquoe Fungi 1 2 3 51 million speciesrdquoAmerican Journal of Botany vol 98 no 3 pp 426ndash438 2011

[8] X-Y Liu D Udayanga Z-L Luo et al ldquoBackbone tree forChaetothyriales with four new species of Minimelanolocusfrom aquatic habitatsrdquo Fungal Biology vol 119 no 11pp 1046ndash1062 2015

[9] A Swe R Jeewon S B Pointing and K D Hyde ldquoDiversityand abundance of nematode-trapping fungi from decaying

litter in terrestrial freshwater and mangrove habitatsrdquo Bio-diversity and Conservation vol 18 no 6 pp 1695ndash1714 2009

[10] C A Shearer and H A Raja Freshwater AscomycetesSpringer Berlin Germany 2010 httpfungilifeillinoisedu

[11] S Hongsanan S S N Maharachchikumbura and K D HydeldquoAn updated phylogeny of Sordariomycetes based on phy-logenetic and molecular clock evidencerdquo Fungal Diversityvol 84 no 3 pp 25ndash41 2017

[12] H Zhang W Dong K D Hyde et al ldquoTowards a naturalclassification of Annulatascaceae-like taxa introducingAtractosporales ord nov and six new familiesrdquo Fungal Di-versity vol 85 no 1 pp 75ndash110 2017

[13] F Aprile A J Darwich G W Siqueira and F R R SantosldquoApplication of hydrological and limnological studies onbuilding model for water circulation of meromictic blackwater lakes at the centralrdquo International Research Journal ofEnvironment Sciences vol 2 no 7 pp 58ndash63 2013

[14] H Sioli ldquoeAmazon limnology and landscape ecology of amigthtropical river and itrsquos basinrdquo Monographie Biologicae SpringerScience amp Business Media vol 56Berlin Germany 1983

[15] K O Konhauser W S Fyfe and B I Kronberg ldquoMulti-element chemistry of some Amazonian waters and soilsrdquoChemical Geology vol 111 no 1-4 pp 155ndash175 1994

[16] A-M Aucour F-X Tao P Moreira-turcq P SeylerS Sheppard and M F Benedetti ldquoe Amazon River be-haviour of metals (Fe Al Mn) and dissolved organic matter inthe initial mixing at the Rio NegroSolimotildees confluencerdquoChemical Geology vol 197 no 1-4 pp 271ndash285 2003

[17] E M Latrubesse E Y Arima T Dunne et al ldquoDamming therivers of the Amazon basinrdquo Nature vol 546 no 7658pp 363ndash369 2017

[18] E Chauvet J Cornut K R Sridhar M-A Selosse andF Barlocher ldquoBeyond the water column aquatic hyphomy-cetes outside their preferred habitatrdquo Fungal Ecology vol 19pp 112ndash127 2016

[19] T Matsushima Matsushima Mycological Memoirs (N 7Kobe Kobe Hyogo Japan 1993

[20] S E Zelski H A Raja A N Miller F R BarbosaL F P Gusmatildeo and C A Shearer ldquoLongicollum biappen-diculatum gen et sp nov a new freshwater ascomycete fromthe Neotropicsrdquo Mycosphere vol 5 no 2 pp 539ndash545 2011

[21] S Zelski H A Raja A N Miller and C A ShearerldquoChaetorostrum quincemilensis gen et sp nov a newfreshwater ascomycete and its Taeniolella-like anamorph fromPerurdquo Mycosphere vol 2 no 5 pp 593ndash600 2011

[22] S E Zelski J A Balto C Do H A Raja A N Miller andC A Shearer ldquoPhylogeny and morphology of dematiaceousfreshwater microfungi from Perurdquo IMA Fungus vol 5 no 2pp 425ndash438 2014

[23] S E Zelski H A Raja A N Miller and C A ShearerldquoConioscypha peruviana sp nov its phylogenetic placementbased on 28S rRNA gene and a report of Conioscypha graciliscomb nov from Perurdquo Mycoscience vol 56 no 3pp 319ndash325 2015

[24] C A Shearer S E Zelski H A Raja J P Schmit A NMillerand J P Janovec ldquoDistributional patterns of freshwater as-comycetes communities along an Andes to Amazon eleva-tional gradient in Perurdquo Biodiversity and Conservationvol 24 no 8 pp 1877ndash1897 2015

[25] R F d Santos H M P Sotatildeo J S Monteiro L F P Gusmatildeoand A H Gutierrez ldquoConidial fungi associated with leaf litterof red cedar (Cedrela odorata) in Belem Para (eastern BrazilianAmazon)rdquo Acta Amazonica vol 48 no 3 pp 230ndash238 2018


[26] J S Monteiro and L F P Gusmatildeo ldquoAn emendation ofFusticeps and two new species from the Brazilian AmazonForestrdquo Mycotaxon vol 123 no 1 pp 431ndash437 2013

[27] P O Fiuza B M de Paiva Ottoni-Boldrini J S MonteiroN R Catena N Hamada and L F P Gusmatildeo ldquoFirst recordsof ingoldian fungi from the Brazilian Amazonrdquo BrazilianJournal of Botany vol 38 no 3 pp 615ndash621 2015

[28] A C A Cortez Influencia da sazonalidade e domodo de coletana diversidade de fungos decompositores de madeira submersade ambientes aquaticos da regiatildeo amazonica (Tese de dou-torado) Universidade Federal do Amazonas ManausAmazonas Brasil 2016 httpstedeufamedubrbitstreamtede56325Tese20-20Ana20Claudia20A20Cortezpdf

[29] L Cai K D Hyde and C K M Tsui ldquoGenera of freshwaterfungirdquo in e University of Hong Kong K D Hyde EdFungal diversity Press San Diego CA USA 1st edition 2006

[30] K Seifert and W Gams ldquoe genera of hyphomycetesrdquoPersoonia vol 27 pp 119ndash129 2011

[31] C Ferrer F Colom S Frases E Mulet J L Abad andJ L Alio ldquoDetection and identification of fungal pathogens byPCR and by ITS2 and 58S ribosomal DNA typing in ocularinfectionsrdquo Journal of Clinical Microbiology vol 39pp 2873ndash2879 2001

[32] T J White T Bruns S Lee and J Taylor ldquoAmplification anddirect sequencing of fungal ribosomal RNA genes for phy-logeneticsrdquo in PCR Protocols A Guide to Methods and Ap-plications N Innis D Gelfand J Sninsky and TWhite Edspp 315ndash322 Academic Press Cambridge MA USA 1990

[33] I S Dun and F R Blattner ldquoCharons 36 to 40 multi enzyonehigh capacity recombination deficient replacement vectorswith polylinkers and ploystuffersrdquo Nucleic Acids Researchvol 15 no 6 pp 2677ndash2698 1987

[34] C J Krebs ldquoEcological methodologyrdquo in University of BritishColumbia Harper amp Row Manhattan NY USA 2rd edi-tion 1989

[35] O Hammer D A T Harper and P D Ryan ldquoPAST pa-leontological statistics software package for education anddata analysisrdquo Paleontologia Electronica vol 4 no 1 pp 1ndash92001

[36] W P Duncan and M N Fernandes ldquoPhysiochemicalcharacterization of the white black and clearwater rivers ofthe Amazon Basin and its implications on the distribution offreshwater stingrays (Chondrichthyes Potamotrygonidae)rdquoPan-American Journal of Aquatic Sciences vol 5 no 3pp 454ndash464 2010

[37] S Duarte F Barlocher J Trabulo F Cassio and C PascoalldquoStream-dwelling fungal decomposer communities along agradient of eutrophication unraveled by 454 pyrosequencingrdquoFungal Diversity vol 70 no 1 pp 127ndash148 2014

[38] J Heino M Tolkkinen A M Pirttila H Aisala andH Mykra ldquoMicrobial diversity and community-environmentrelationships in boreal streamsrdquo Journal of Biogeographyvol 41 no 12 pp 2234ndash2244 2014

[39] P Song S Tanabe R Yi M Kagami X Liu and S BanldquoFungal community structure at pelagic and littoral sites inLake Biwa determined with high-throughput sequencingrdquoLimnology vol 19 no 2 pp 241ndash251 2018

[40] J Luo J F Yin L Cai K Q Zhang and K D HydeldquoFreshwater fungi in Lake Dianchi a heavily polluted lake inYunnan Chinardquo Fungal Diversity vol 16 pp 93ndash112 2004

[41] D Hu L Cai H Chen A H Bahkali and K D HydeldquoFungal diversity on submerged wood in a tropical stream and

an artificial lakerdquo Biodiversity and Conservation vol 19no 13 pp 3799ndash3808 2010

[42] H A Raja J P Schmit and C A Shearer ldquoLatitudinalhabitat and substrate distribution patterns of freshwater as-comycetes in the Florida Peninsulardquo Biodiversity and Con-servation vol 18 no 2 pp 419ndash455 2009

[43] F A Abdel-Aziz ldquoFreshwater fungi from the river nileEgyptrdquo Mycosphere vol 7 no 5 pp 741ndash756 2016

[44] M A S Graccedila V Ferreira C Canhoto et al ldquoA conceptualmodel of litter breakdown in low order streamsrdquo Interna-tional Review of Hydrobiology vol 100 no 1 pp 1ndash12 2015

[45] F Barloche and B Kendrick ldquoDynamics of the fungal pop-ulation on leaves in a streamrdquo Journal ofEcology vol 62 no 3pp 761ndash779 1974

[46] J Webster and E Descals ldquoMorphology distribution andecology of conidial fungi in freshwater habitatsrdquo in Biology ofConidial Fungi M Dekker Ed vol 1 pp 459ndash468 AcademicPress Cambridge MA USA 1981

[47] K D Hyde S Fryar Q Tian A H Bahkali and J XuldquoLignicolous freshwater fungi along a north-south latitudinalgradient in the AsianAustralian region can we predict theimpact of global warming on biodiversity and functionrdquoFungal Ecology vol 19 pp 190ndash200 2016

[48] K D Hyde S Hongsanan R Jeewon et al ldquoFungal diversitynotes 367ndash490 taxonomic and phylogenetic contributions tofungal taxardquo Fungal Diversity vol 80 no 1 pp 1ndash270 2016

[49] M A S Graccedila K Hyde and E Chauvet ldquoAquatic hypho-mycetes and litter decomposition in tropical - subtropical loworder streamsrdquo Fungal Ecology vol 19 pp 182ndash189 2016

[50] K D Hyde and T K Goh ldquoFungi on submerged wood In lakebarrine north queensland Australiardquo Mycological Researchvol 102 no 6 pp 739ndash749 1998

[51] T K Goh and K D Hyde ldquoFungi on submerged wood andbamboo in the Plover Coverdquo Fungal Diversity vol 3pp 57ndash85 1999

[52] W H Ho K D Hyde and I J Hodgkiss ldquoFungal com-munities on submerged wood from streams in Brunei HongKong and Malaysiardquo Mycological Research vol 105 no 12pp 1492ndash1501 2001

[53] C Wurzbacher S Rosel A Rychła and H P GrossartldquoImportance of saprotrophic freshwater fungi for pollendegradationrdquo Plos One vol 9 no 4 pp 1ndash12 2014

[54] L G Leff ldquoFreshwater habitatsrdquo Encyclopedia of Microbiol-ogy Elsevier vol 4e pp 1ndash4 Amsterdam Netherlands 2019


contains a variety of aquatic environments that include largerivers lakes swamps and seasonally inundated floodplains[13 14] Aquatic ecosystems can be roughly classified intothree main types clear waters [15] white waters [16] andblack waters [14 17] Water characteristics such as tem-perature dissolved oxygen and amount of organic mattermay influence the diversity of freshwater fungal species [18]e first published studies of freshwater fungi in Amazonianwaters (Peru) were conducted byMatsushima in white waterrivers [19] More recent studies have described new speciesof ascomycetes in the Peruvian Amazon [20ndash24] and in theBrazilian states of Para [25 26] and Amazonas [27 28] Ingeneral the microbial community of the Amazon region isstill underexplored and studies describing the diversity andecology of fungi in the Amazon region are urgently needederefore we sought to investigate new environments suchas the Tapajos River one of the largest clear water tributariesof the Amazon River [17] e present work aimed to de-scribe the composition and diversity of fungi present onsubmerged decomposing wood in two lakes in the TapajosRiver basin near Santarem in the Brazilian state of Para

2 Materials and Methods

21 Study Sites Samples of submerged wood were collectedfrom Lake Jua (2deg25prime57PrimeS 54deg 46prime39PrimeW) and Lake Maica(2deg27prime290PrimeS 54deg 40prime108PrimeW) and essentially the embay-ment of the lower Tapajos and Amazon Rivers respectivelyLake Jua receives water that drains from a stream and theTapajos LakeMaica is amix of surface drainages and the whitewater of the Amazon River [14] (Figures 1 2(a) and 2(b))

22 Water Characterization Physicochemical variableswere measured using a ProfiLine 197iWTWmultiparameterprobe (Wellheim Germany) Temperature (degC) pH elec-trical conductivity (μS) dissolved oxygen concentration(mgL) and turbidity (NTU) were recorded for each samplecollection

23 Sample Collection A single collection was made at eachlocation in Lake Jua in October 2017 and in Lake Maica inNovember 2017 At each site 30 submerged wood fragmentsthat showed signs of active decomposition were collectede lengths of the samples ranged from 6 to 22 cm anddiameters ranged from 6 to 15 cm (Figure 2(c)) esesubstrates were transported in sealable plastic bags(20 cmtimes 10 cm) with a small amount of local water to theLaboratory of Multidisciplinary Teaching in Applied Biol-ogy-Labio Universidade Federal do Oeste do Para(UFOPA) In the laboratory the samples were gently rinsedwith running water placed in moist chambers (plastic boxeswith moist paper towels) and incubated at room temper-ature with 1212 h lightdark conditions

24 Isolationand Identificationof FungiPresent onSubmergedWood Every ten days for six months a Stemi DV4 ste-reomicroscope (Zeiss) was used to search for fungal

structures on the collected wood samples e structuresfound were transferred with dissection needles to micro-scope slides containing distilled water e identification ofsexual ascomycetes was performed based on the micro-morphology of ascomata hamathecium asci and asco-spores using an Axioskop 40microscope (Zeiss) as describedpreviously [22 29 30] Aqueous lactophenol cotton bluesolution was used to determine the staining reactions of theapical ascus apparatus Asexual ascomycetes were identifiedby investigating the types of conidiogenesis conidiophoreand conidia using an Axioskop 40 microscope (Zeiss)Isolation of fungal was carried out by transferring the fungalstructures to the surface of Antibiotic Water Agar (20 gLagar and chloramphenicol 250mgL) Potato Dextrose Agar(PDA) (Kasvi) and Malt Extract Agar (MEA) (malt extract30 gL mycological peptone 5gL and 15gL agar pH54plusmn 02) e developed colonies were transferred to PYGagar (125 gL peptone 125 gL yeast extract 5 gL glucose250mgL chloramphenicol and 18 gL agar) [22](Figures 2(d)ndash2(g))

25 DNA Extraction Amplification and SequencingDNA extraction was performed according to the protocol ofFerrer et al [31] Fungi were grown in test tubes containing10mL of potato dextrose broth (120 g potato 10 g dextroseand 1000mL distilled water) for 10 days and transferred toEppendorf tubes (2mL) containing 500 μL of buffer (25mgSDS 70mg NaCl 365mg EDTA 20mL of Tris-HCl and100mL of DI water and 5 μL of szlig-mercaptoethanol) emicrotubes were subjected to freezing (minus20 degC) and heating(65degC for 1 h) in order to rupture the cellular structuresNext 500 μL of a phenolchloroformisoamyl alcohol so-lution (vvv 25241) was added to the microtubes andvortexed until a homogeneous suspension was obtainedesuspension was then centrifuged (14000 rpm 15min) andthe supernatant was transferred to a newmicrotube (15mL)Isopropanol was added to the supernatant in equal volumeand the mixture was homogenized and incubated at minus20degCovernight to precipitate the DNAe precipitated DNAwascentrifuged (14000 rpm for 15min) washed twice with 70ethanol and resuspended in 100 μL of sterile water distilled

Molecular data were gathered by sequencing the ITSregion of rDNA according to the procedure of White et al[32] e reaction yielded a final volume of 50 μL consistingof PCR buffer (10mM Tris-HCl pH 83 50mM KCl)15mM MgCl 2 05 μM of ITS-1 (5rsquo-TCCGTAGGT-GAACCTGCGG-3rsquo) and ITS-4 (5rsquo-TCCTCCGCTTATT-GATATGC-3rsquo) 200 μMdNTPs 15 U TaqDNA polymeraseand 100 ng of fungal DNA ermocycling was performedon an Applied Biosystems ProFlex PCR System with initialdenaturation at 94degC for 5min followed by 35 cycles of DNAdenaturation at 94degC for 1min annealing at 55degC for 1minextension at 72degC for 2min and a final extension at 72degC for10min Two positive controls and one negative control werealso tested Electrophoresis was performed on 15 agarosegel in 1x TBE buffer (100mM Tris base 100mM boric acidand 2mM EDTA pH 80) plus 10 μL100mL of Siber GreenSYBRreg Safe Amplified fragments were visualized under UV








E Hprimeln S

(2)



Sprime 2c

a + b (3)


5250 75



MaicaacuteLake

Juaacute Lake


River2deg22

8primeS

2deg27

0primeS

2deg31

2primeS

Scale

Brazil

Paraacute state

N


km



(a) (b)

(c) (d)

(e) (f ) (g)






3 Results





4 Discussion












0

5

10

15

20

25

30

Num

ber o

f tax

a





















Data Availability




Acknowledgments


References

































































E Hprimeln S

(2)



Sprime 2c

a + b (3)


5250 75



MaicaacuteLake

Juaacute Lake


River2deg22

8primeS

2deg27

0primeS

2deg31

2primeS

Scale

Brazil

Paraacute state

N


km



(a) (b)

(c) (d)

(e) (f ) (g)






3 Results





4 Discussion












0

5

10

15

20

25

30

Num

ber o

f tax

a





















Data Availability




Acknowledgments


References



























































(a) (b)

(c) (d)

(e) (f ) (g)






3 Results





4 Discussion












0

5

10

15

20

25

30

Num

ber o

f tax

a





















Data Availability




Acknowledgments


References




























































3 Results





4 Discussion












0

5

10

15

20

25

30

Num

ber o

f tax

a





















Data Availability




Acknowledgments


References





























































0

5

10

15

20

25

30

Num

ber o

f tax

a





















Data Availability




Acknowledgments


References










































































Data Availability




Acknowledgments


References



























































compositionanddiversityoffungaldecomposersofsubmerged...

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