Three genera in the Ceratocystidaceae are the respective symbionts of three independent lineages

18 Pages·2015·2.46 MB·English

by  Chase G. Mayers

fungal biology 119 (2015) 1075e1092
journal homepage: www.elsevier.com/locate/funbio
Three genera in the Ceratocystidaceae are the
respective symbionts of three independent
lineages of ambrosia beetles with large, complex
mycangia
Chase G. MAYERSa, Douglas L. MCNEWa, Thomas C. HARRINGTONa,*,
RichardA.ROEPERb,StephenW.FRAEDRICHc,PeterH.W.BIEDERMANNd,
Louela A. CASTRILLOe, Sharon E. REEDf
aDepartmentofPlantPathologyandMicrobiology,IowaStateUniversity,351BesseyHall,Ames,IA50011,USA
bDepartmentofBiology,AlmaCollege,Alma,MI48801,USA
cSouthernResearchStation,USDAForestService,Athens,GA30602,USA
dResearchGroupInsectSymbiosis,MaxPlanckInstituteforChemicalEcology,BeutenbergCampus,Hans-Kn€oll-Str.
8,07745Jena,Germany
eDepartmentofEntomology,CornellUniversity,R.W.HolleyCenterforAgricultureandHealth,Ithaca,NY14853,
USA
fDivisionofPlantSciences,UniversityofMissouri,52AgricultureLab,Columbia,MO65211,USA
a r t i c l e i n f o a b s t r a c t
Articlehistory: ThegenusAmbrosiellaaccommodatesspeciesofCeratocystidaceae(Microascales)thatareob-
Received23May2015 ligate,mutualisticsymbiontsofambrosiabeetles,butthegenusappearstobepolyphyletic
Receivedinrevisedform andmorediversethanpreviouslyrecognized.InadditiontoAmbrosiellaxylebori,Ambrosiella
28July2015 hartigii,Ambrosiellabeaveri,andAmbrosiellaroeperi,threenewspeciesofAmbrosiellaarede-
Accepted3August2015 scribedfromtheambrosiabeetletribeXyleborini:Ambrosiellanakashimaesp.nov.fromXy-
Availableonline19August2015 losandrus amputatus, Ambrosiella batrae sp. nov. from Anisandrus sayi, and Ambrosiella
CorrespondingEditor: grosmanniaesp.nov.fromXylosandrusgermanus.ThegenusMeredithiellagen.nov.iscreated
StephenW.Peterson forsymbiontsofthetribeCorthylini,basedonMeredithiellanorrisiisp.nov.fromCorthylus
punctatissimus.ThegenusPhialophoropsisisresurrectedtoaccommodateassociatesofthe
Keywords: Xyloterini,includingPhialophoropsistrypodendrifromTrypodendronscabricollisandPhialophor-
Ambrosiafungi opsisferrugineacomb.nov.fromTrypodendronlineatum.Eachofthetennamedspecieswas
Scolytinae distinguishedbyITSrDNAbarcodingandmorphology,andtheITSrDNAsequencesoffour
Symbiosis otherputativespecieswereobtainedwithCeratocystidaceae-specificprimersandtemplate
DNA extractedfrom beetlesor galleries. These results support the hypothesis that each
ambrosiabeetlespecieswithlarge,complexmycangiacarriesitsownfungalsymbiont.Co-
nidiophoremorphologyandphylogeneticanalysesusing18S(SSU)rDNAandTEF1aDNA
sequences suggest that these three fungal genera within the Ceratocystidaceae indepen-
dentlyadaptedtosymbiosiswiththethreerespectivebeetletribes.Inturn,thebeetlegen-
era with large, complex mycangia appear to have evolved from other genera in their
* Correspondingauthor.Tel.:þ15152940582.
E-mailaddress:[email protected](T.C. Harrington).
http://dx.doi.org/10.1016/j.funbio.2015.08.002
1878-6146/ª2015TheBritishMycologicalSociety.PublishedbyElsevierLtd.Allrightsreserved.
1076 C.G.Mayersetal.
respectivetribesthathavesmaller,lessselectivemycangiaandareassociatedwithRaffae-
leaspp.(Ophiostomatales).
ª2015TheBritishMycologicalSociety.PublishedbyElsevierLtd.Allrightsreserved.
Introduction Ambrosiella spp. have so far been recovered or reported
from five beetle genera (Harrington et al. 2014), and each of
ThesixrecognizedspeciesofAmbrosiellaBraderexArx&Hen- these genera appear to have relatively large and complex
nebert1965(Sordariomycetes:Microascales:Ceratocystidaceae)are mycangiawithsecretionsdirectlythroughreticulatedmycan-
obligate,mutualisticsymbiontsofambrosiabeetles.Ambrosia gialwalls.WithinthetribeXyleborini,speciesofXylosandrus,
beetlesareanecologicalgroupofmorethan3400speciesofmy- Anisandrus, and Cnestus have large, internal mesonotal
cophagoussapwood-boringbeetlesinthesubfamiliesPlatypo- mycangia in female adults (Francke-Grosmann 1956, 1967;
dinae and Scolytinae (Coleoptera: Curculionidae). Whereas Beaver1989;Kinuura1995;Hulcretal.2007;Stoneetal.2007;
theirbarkbeetlerelativesgenerallyfeedonthenutritiousinner Hulcr&Cognato2010;Cognatoetal.2011a).IntheCorthylini,
bark(secondaryphloem)oftrees,ambrosiabeetlestunnelinthe Corthylusspp.havelong,foldedtubesthatopenintothepro-
nutrient-poor sapwood and depend on mutualistic fungi for coxaeofadultmales(Finnegan1963;Giese1967).IntheXylo-
theirnutrition(Harrington2005).Thebeetlesgenerallydonot terini, female Trypodendron spp. have large, tubular, pleural-
eat wood while boring (Beaver 1989), though the larvae of prothoracic mycangia (Francke-Grosmann 1956, 1967;
someambrosiabeetlesingestfungus-colonizedwood(xylomy- Schneider&Rudinsky1969b).
cetophagy)(Roeper1995;DeFineLicht&Biedermann2012).The Ambrosiella initially included ambrosia beetle symbionts
major food of both larvae and adults is ambrosial growth of withpercurrentproliferationofconidiogenouscellsvs.sympo-
fungiwithinthesapwoodtunnels.Adiversefungalfloragrows dial proliferation by Raffaelea spp. (Brader 1964; von Arx &
inambrosiabeetlegalleries,butthedominantfungiareobligate Hennebert 1965; Batra 1967). Gebhardt et al. (2005) demon-
symbiontsofambrosiabeetlesandhavenotbeenfoundasfree- stratedthatsomeRaffaeleaspp.havepercurrentandsympodial
livingspecies(Harringtonetal.2010). proliferation,butAmbrosiellaxyleboriBraderexArx&Henne-
Thefungalsymbiontsrelyonthebeetlesfordispersaland bert1965,AmbrosiellahartigiiL.R.Batra1968,andAmbrosiellafer-
are primarily carried by adult beetles in special sacs called ruginea (Math.-Ka€a€rik) L.R. Batra 1968 produced conidia from
mycangia,inwhichthefungigrowinabuddingyeast-likeor phialides. Harrington et al. (2010) limited Ambrosiella spp. to
arthrospore-likephase(Fraedrichetal.2008;Harringtonetal. the phialidic ambrosia fungi within the Ceratocystidaceae and
2014).Inthestudiedcasesofambrosiabeetlemycangia,gland Raffaelea spp. to symbionts within the Ophiostomatales. All
cellssecretematerialintoornearthemycangiumtosupport known Ambrosiella spp. produce a fruity aroma (Harrington
growth of the fungal symbionts (Schneider & Rudinsky 2009),andthesevolatilesmayplayaroleinattractingambrosia
1969a,1969b),andtheoverflowofsporesfromthemycangium beetleswithinthegalleries(Hulcretal.2011).
inoculatesthegalleriesduringconstruction.Ofthetwoweevil As now recognized, most Ambrosiella spp. produce large,
subfamilies,thePlatypodinaeconsistentirelyofambrosiabee- thick-walled,ovoid,terminalaleurioconidiawithinconspicu-
tles and have relatively small and simple mycangia ouscollarettesand/orbasipetalchainsofcylindricaltobarrel-
(Nakashima1975;Cassieretal.1996;Marvaldietal.2002).In shapedphialoconidiaviaring-wallbuilding(Minteretal.1983;
contrast, ambrosia beetles arose at least 10 separate times NagRaj&Kendrick1993;Riggs&Mims2000).Thepresenceof
from bark beetle lineages within the Scolytinae (Farrell et al. deep-seatedphialidesinthesymbiontofTrypodendronscabri-
2001;Jordal&Cognato2012;Kirkendalletal.2015),eachevent collis (tribe Xyloterini), Ambrosiella trypodendri (L.R. Batra)
apparently marked by the development of novel mycangia. T.C.Harr.2010,wasusedtoerectthemonotypicgenusPhialo-
Generally, the Scolytinae mycangia are relatively small and phoropsis L.R. Batra 1968, but Harrington et al. (2010) placed
harbor one or more species of Raffaelea Arx & Hennebert PhialophoropsisinsynonomywithAmbrosiella.However,phy-
1965 (Ophiostomatales) (Cassar & Blackwell 1996; Harrington logeneticanalyseshavegenerallysuggestedthatthetwogen-
& Fraedrich 2010; Harrington et al. 2010, 2014). These small eraaredistinct(Alamoutietal.2009;Harrington2009;Sixetal.
mycangiaincludeoralpouches,pronotalpits,elytralpouches, 2009;Harringtonetal.2010;deBeeretal.2014).
andcoxalenlargements,eachofwhicharesimplemodifica- Atpresent,therearesixnamedspeciesofAmbrosiella:A.
tions of the adult beetle’s exoskeleton, with secreting gland trypodendrifromT.scabricollis(Batra1967);A.ferrugineafrom
cells near the opening of the mycangium (Francke- Trypodendron lineatum (Batra 1967); Ambrosiella xylebori from
Grosmann 1967; Beaver 1989). In contrast, certain genera of Xylosandruscompactus(Brader1964);A.hartigiifromAnisandrus
Scolytinaeexhibitmarkedlylargerandmoreelaboratemycan- dispar (Batra 1967); Ambrosiella beaveri Six, de Beer & W.D.
giathataresetentirelywithinthebodyandarecomposedof StonefromCnestusmutilatus(Sixetal.2009);andAmbrosiella
a reticular structure punctuated by gland cells secreting di- roeperi T.C. Harr. & McNew from Xylosandrus crassiusculus
rectly into the mycangium (Francke-Grosmann 1956; (Harringtonetal.2014).InadditiontoX.compactus,A.xylebori
Finnegan 1963; Schneider & Rudinsky 1969b; Stone et al. has been reported from Corthylus columbianus (Batra 1967;
2007).Theselarger,complexmycangiahavespecializedchan- Nord1972)andCorthyluspunctatissimus(Roeper1995).Besides
nelsortubesthatdirecttheoverflowoffungalgrowthtothe An. dispar,A.hartigiihasbeenreportedfromAnisandrus sayi
outsideofthebeetlefortunnelinoculation. and Anisandrus obesus (Hazen & Roeper 1980; Roeper &
ThreeambrosialgeneraintheCeratocystidaceae 1077
French 1981), as well as Xylosandrus germanus (Weber & ofCottonblueonaslideandcoveredwithacoverslipformi-
McPherson1984;Roeper1996).A.ferrugineahasbeenreported croscopicexamination,orthesporemassseparatedandused
from several Trypodendron spp., including T. lineatum, Trypo- forisolationofthefungalsymbiontorforDNAextractionwith
dendrondomesticum,Trypodendronretusum,Trypodendronrufitar- PrepMan(cid:2)Ultra(AppliedBiosystems,FosterCity,CA).
sis,andTrypodendronbetulae(Batra1967;French&Roeper1972;
RoeperandFrench,1981;Roeper1996),andNakashimaetal. DNAextractionandsequencing
(1992) illustrated a fungus from Trypodendron signatum with
similarconidiophoremorphology. IsolatesweregrownatroomtemperatureonMYEA,andDNA
MostoftheaboveidentificationsofAmbrosiellaspp.were wasextractedusingoneoftwomethods:eitherthecultures
based on morphological characters only, and more detailed weregrown2e7dandDNAwasextractedusingPrepMan(cid:2)Ul-
phylogeneticanalysesmayrevealcrypticspeciesandgenera tra, or isolates were grown 4e14 d and extracted using the
among the fungal symbionts. Preliminary DNA sequence ProMega Wizard(cid:2) Genomic DNA Purification Kit (Promega,
analysesandobservationsofculturesfrombeetleswithlarge Madison,WI).
(cid:2)
mycangia suggested that there was more species diversity PrepMan UltrawasusedtoextractDNAfromscrapedfun-
withinAmbrosiellathanpreviouslyrecognized,andeachstud- gal material in beetle galleries or beetle mycangia. In some
(cid:2)
iedambrosiabeetlewithlarge,complexmycangiaappearedto cases, the extracted DNA was concentrated using Amicon
be associated with a single, unique species, either within Ultra-0.5 Centrifugal Filter Devices (EMD Millipore, Billerica,
AmbrosiellaoracloselyrelatedgenusintheCeratocystidaceae. CA). Whole beetles preserved in ethanol were ground with
We studied fungal isolates, beetle galleries, and insect a tissue grinder and Prepman(cid:2) Ultra extraction buffer, and
specimensof14ambrosiabeetlespecieswithlarge,complex the resulting mix was transferred to a microcentrifuge tube
mycangiatodeterminetheidentityoftheirfungalsymbionts forDNAextraction.
andinferanevolutionaryhistoryofthefungi.Ourhypothesis
wasthateachbeetlewouldyieldauniquefungalspecies,and ITSbarcoding
that all species recovered from beetles with large, complex
mycangia would form a monophyletic genus (Ambrosiella) Sequencingoftheinternaltranscribedspacer(ITS)regionof
within the Ceratocystidaceae, stemming from a single evolu- theribosomalDNAforinitialidentificationofunknowncul-
tionaryjumptoambrosiabeetlesymbiosis. tures utilized the general fungal primer ITS1-F (Gardes &
Bruns1993)andITS4(Whiteetal.1990)andthePCRconditions
of Paulin-Mahady et al. (2002) and Harrington et al. (2000).
Materials and methods WhenusingextractedDNAfromgallerymaterial,mycangial
masses,orgroundbeetles,Ceratocystis-specificprimerswere
Beetlecollectionandfungalisolation usedtoamplifytheITSregionintwoparts:primerpairsCera-
to1F (50 GCGGAGGGATCATTACTGAG 30) and ITSCer3.7R (50
MostoftheadultbeetleswerecaughtinflightusingLindgren GTGAAATGACGCTCGGACAG 30) for ITS1 and primer pair
traps with water or polyethylene glycol in collection cups, ITSCer3.1 (50 CAACGGATCTCTTGGCTCTA 30) and ITS4 (50
whilethecupsofothertrapsweredryandhadNoPestinsec- TCCTCCGCTTATTGATATGC 30) for ITS2 (Harrington et al.
ticide strips (Vapona, Spectrum Brands, Middleton, Wiscon- 2014).
sin). The traps were baited with either ethanol lures or All ITS sequences generated from cultures, beetles, and
lineatinflexlure(ContechEnterprises,Victoria,BritishColum- gallerieswerecomparedwiththeITSsequencesofrepresen-
bia)inthecaseofTrypodendronlineatumandTrypodendronscab- tativeCeratocystidaceaeinamanuallyalignedITSrDNAdata-
ricollis. Some adult beetles were caught in-flight with rotary set.Therewereregionsofambiguouslyalignedcharactersin
nettraps.Othermatureadultsweretakendirectlyfromfresh bothITS1andITS2duetonumerousareasofinsertionsand
galleriesbysplittinginfestedwoodsections. deletions (indels), and the indel regions had limited reliable
Mostfungalisolateswereobtainedbygrindingbeetlesand phylogeneticsignal.Nonetheless,thealigneddatasetwasan-
dilutionplating(Harringon&Fraedrich2010;Harringtonetal. alyzedbyUPGMAinPAUP4.0b10(Swofford2002)usinguncor-
2011)orbyplacingwholebeetlesorpartsofbeetlescontaining rected(“p”)distance,andgapsweretreatedasmissingdata.
mycangiaonplatesofSMA(1%maltextract,Difco;1.5%agar,
SigmaeAldrich;and100ppmstreptomycinsulfateaddedafter Phylogeneticanalysis
autoclaving).Isolationswerealsoattempteddirectlyfromam-
brosiagrowthinbeetlegalleriesbyscrapingwithasterilenee- Sequences of the small subunit rDNA (SSU, 18S rDNA) and
dleandtransferringtoSMAorMYEA(2%maltextract,0.2% translation elongation factor 1-alpha (TEF1a) were used for
Difcoyeastextract,1.5%agar). phylogenetic placement of the ambrosia beetle symbionts.
For mycangial examination, adult female Xyleborini were Taxaselected(Table1)includedarepresentativeofeachsym-
dissectedin20%lacticacidonadeepwellslideusingfinefor- biontandotherrepresentativesofthenewlyrecognizedgen-
cepsandascalpel.Anincisionwasmadejustposteriortothe eraintheCeratocystidaceae,whichwerepreviouslytreatedas
scutellumtoexposetheinteriorofthebeetlewithoutdamag- Ceratocystisspp.orThielaviopsisspp.(deBeeretal.2014).The
ingthemycangium,whichsitsdirectlybeneaththemesono- representativetaxaarewellcharacterized,exceptforthefor-
tum and is attached to the scutellum. Fine forceps were mer members of the Ceratocystis moniliformis complex, now
thenusedtogentlyteaseoutthemycangiumandscutellum. treated as Huntiella. Our Huntiella moniliformis isolate C792
The mycangium/scutellum was either transferred to a drop from a Populus sp. in Minnesota is probably an undescribed
1078 C.G.Mayersetal.
Table1eCultures,specimens,andGenBankaccessionsforrepresentativespecies.
Cultureorspecimen Associated GenBankaccessionno
identificationnumbers ambrosiabeetle
ITS SSU TEF1a
Ambrosiella
A.batrae C3130(CBS139735) Anisandrussayi KR611322 KR673881 KT290320
A.beaveri C2749(CBS121750) Cnestusmutilatus KF669875 KR673882 KT318380
A.grosmanniae C3151(CBS137359) Xylosandrusgermanus KR611324 KR673884 KT318382
A.hartigii C1573(CBS404.82) Anisandrusdispar KF669873 KR673885 KT318383
A.nakashimae C3445(CBS139739) Xylosandrusamputatus KR611323 KR673883 KT318381
A.roeperi C2448(CBS135864) Xylosandruscrassiusculus KF669871 KR673886 KT318384
A.xylebori C3051(CBS110.61) Xylosandruscompactus KF669874 KR673887 KT318385
Ambrosiellasp. M257 Eccoptopterusspinosus KR611325
Meredithiella
M.norrisii C3152(CBS139737) Corthyluspunctatissimus KR611326 KR673888 KT318386
Meredithiellasp. M260 Corthylusconsimilis KR611327
Phialophoropsis
P.ferruginea M243(BPI893129) Trypodendronlineatum KR611328 KR673889 KT318387
Phialophoropsissp. C2230(CBS460.82) Trypodendrondomesticum KC305146 KR673890 KT318388
Phialophoropsissp. CBS408.68 Trypodendronretusem KC305145
P.trypodendri SUTT Trypodendronscabricollis KR611329
CeratocystisandotherCeratocystidaceae
C.adiposa C999(CBS183.86) ¼JN604448 KR673891 HM569644
C.fagacearum C927(CBS129242) ¼KC305152 KR673892 KT318389
C.fimbriata C1099(ICMP8579) AY157957 KR673893 HM569615
C.norvegica C3124(UAMH9778) DQ318194 KR673894 KT318390
Endoconidiophoracoerulescens C301(CBS100.198) KC305116 KR673895 HM569653
Huntiellamoniliformis C1007(CBS204.90) ¼DQ074739 KR673896 KT318391
Huntiellasp. C792 KR611330 KR673897 KT318392
H.moniliformopsis C1934(DAR74609) ¼NR119507 KR673898 HM569638
Thielaviopsisethacetica C1107 ¼KJ881375 KR673899 HM569632
species,whileH.moniliformisisolateC1007(CBS204.90,CMW geneshowedtheGTRþIþGmodeltobemostappropriate.
11046)fromIndiahastheITSsequenceofHuntiellaomanensis MrBayes 3.2.1 (Ronquist & Huelsenbeck 2003) was used for
(DQ074739).OthertaxaintheMicroascalesincludedPseudalle- Bayesian analysis with this GTR þ I þ G model. A single
scheriaspp.(mixedspecies;Pseudallescheriaellipsoideafor18S MCMC runwith four chains(one cold,three heated)ranfor
rDNA (U43911) and Pseudallescheria angusta for TEF1a) and 600000generations,whichwassufficienttobringtheconver-
Gondwanamycescapensis(18SrDNA,FJ176834).Outgrouptaxa gencediagnosticsbelow0.01;aburn-inof25%wasappliedbe-
were Plectosphaerella cucumerina (18S rDNA, AF176951) and forecreatingamajorityruleconsensustreewiththefunction
Neurosporacrassa(18SrDNA,X04971). “sumt”.ThetreewasvisualizedwithFigTree.
The SSU sequences were amplified and sequenced using A full heuristic, maximum parsimony (MP), 10000-repli-
avarietyofprimers(Whiteetal.1990;Vilgalys2005),typically cate bootstrap analysis and 50 % majority rule consensus
usingtheoverlappingsequencesfromNS-1/NS-6andSR-9R/ treewascreatedwithPAUPtoaddbootstrapsupportvalues
NS-8, but sometimes overlapping sequences were obtained to the Bayesian inference tree. All characters had equal
with NS-1/NS-4, NS-3/NS-6, and NS-5/NS-8. These overlap- weights,andtheheuristicsearchwasperformedwithsimple
pingsequencesyieldedanalignedsequenceofapproximately stepwiseaddition.TheMPanalysisusedthesamecombined
1700bp. datasetbuttreatedgapsasanewstate(5thbase).
Amplification of TEF1a used the forward amplification
primer EFCF1a (50 AGTGCGGTGGTATCGACAAGCG 30) or Speciesdescriptions
EFCF1.5 (50 GCYGAGCTCGGTAAGGGYTC 30) and the reverse
primer EFCF6 (50 CATGTCACGGACGGCGAAAC 30) following Forgrowthratestudies,selectedisolatesweregrownonMYEA
theprotocolofOliveiraetal.(2015).Sequencingwasgenerally plates.Plugsfromtheleadingedgeofgrowthtakenwithaster-
performed with the PCR primers as well as the internal ile#1corkborerwereplacedupsidedownonthreenewMYEA
primers EFCF2 (50 TGCTCAACGGGTCTGGCCAT 30) and EFCF3 platesperisolate,andtheplateswereincubatedat25(cid:2)Cfor
(50 ATGGCCAGACCCGTGAGCA 30). The aligned sequences 4e6d.ColordescriptionsofculturesfollowedRayner(1970.).
wereapproximately1200bp.
AcombinedSSUandTEF1adataset(TreeBaseURL:http:// Results
purl.org/phylo/treebase/phylows/study/TB2:S17680) of 2781
characterswasusedforphylogeneticanalysis.Modeltesting
Using ITS rDNA sequences as a barcode to delineate puta-
usingModelTest2.1.7v20141120(Darribaetal.2012)onboth
tive species, we were able to associate an Ambrosiella sp.
the combined dataset and the separate datasets for each
with each of 14 studied species of ambrosia beetles with
ThreeambrosialgeneraintheCeratocystidaceae 1079
Fig1eConidiophoresandconidiaofAmbrosiellaspp.(A,B).A.beaveriisolateC2749(CBS121750,expara-type).(A).phia-
loconidiophore.(B).aleurioconidiophore.(CeF).A.nakashimae.(C,D).ingallery,specimenBPI893134(holotype).(E,F).Isolate
C3445(CBS139739,ex-type).(GeI).AhartigiiisolateC3450(CBS139746).(G).phialoconidiophore.(H,I).aleurioconidiophore
anddetachedaleurioconidia.(JeL).A.batraeisolateC3130(CBS139735)and(M,N).isolateC3045(CBS139736).(OeR).A.
grosmanniae.(OeQ)ingallery,specimenBPI893133.(R).IsolateC3125(CBS137357).(S,T).A.roeperiisolateC2448(CBS
135864,ex-type).(UeW).A.xyleboriisolateC2455.AllphotosbyNomarskiinterferencemicroscopyofmaterialstainedwith
cottonblue.Bar[10mm.
large,complexmycangia:eightspeciesfromtribeXyleborini beetles,orfromsporulationinbeetlegalleries.Galleryspor-
(Cnestusmutilatus,Xylosandrusamputatus,Xylosandrusgerma- ulation and isolates on MYEA showed fungi with macro-
nus, Xylosandrus crassiusculus, Xylosandrus compactus, Ani- and microscopic characteristics expected of Ambrosiella
sandrus dispar, Anisandrus sayi, and Eccoptopterus spinosus), spp. (Figs 1 and 2), including a fruity aroma when grown
twofromtribeCorthylini(CorthyluspunctatissimusandCorthy- on MYEA. Each of the beetle species yielded a different
lusconsimilis),andfourfromtribeXyloterini(Trypodendronlin- Ambrosiella sp. based on unique ITS sequences (Fig 3) and
eatum, T. domesticum, T. scabricollis, and T. retusum.) The ITS morphology (conidiophores and/or culture characteristics).
sequences were obtained from pure cultures or from DNA No species of ambrosia fungus was found associated with
extracted from dissected mycangial spore masses, whole more than one beetle species.
1080 C.G.Mayersetal.
Fig2eGrowthandsporulationofambrosiafungiingalleriesofambrosiabeetlesfromthreebeetletribes.(AeD).Ambrosiella
nakashimaeingalleries(BPI893134)ofXylosandrusamputatus.(A).growthintunnels.(B,C).protoperitheciaandaleurioco-
nidia.(D).chainedaleurioconidiaonaleurioconidiophore.(E,F).MeredithiellanorrisiiingalleriesofCorthyluspunctatissimus.
(E).Growthingallery(BPI893135).(F).Branchedaleurioconidiophore(BPI893137).(GeJ).M.norrisiiinculture(C3152,CBS
139737,BPI893136,ex-type).(G).detachedsinglealeurioconidium.(H).detachedaleurioconidiumwithconidiophorecell
attached.(I,J).Branchedaleurioconidiophores.(KeO).PhialophoropsisferrugineaingalleriesofTrypodendronlineatum.(KeN).
SpecimenBPI893130.(K).Gallerygrowth.(LeN).Deep-seatedphialides.(O).Deep-seatedphialide(specimenBPI407710).All
photosbyNomarskiinterferencemicroscopyofmaterialstainedwithCottonblue.Bar[10mm.
Beetleassociations mycelium (Fig 2AeD). Isolations from the fungal growth in
five beetlegalleriesyieldedan Ambrosiella sp. whose ITS se-
Xyleborini quencedifferedfromthatofA.beaveribyhavinganextraT
Two Cnestus mutilatus females caught in a trap in Barrow inastringofTsattheendofITS2(Fig3).
County, Georgia in September 2013 were ground and plated Xylosandrus germanus adults collected in eight different
on SMA, and the recovered isolates had both aleurioconidia USA states (Georgia, Iowa, Michigan, Missouri, New York,
and phialoconidia typical of Ambrosiella beaveri (Six et al. Ohio, Tennessee, and Virginia) and Europe (Germany, the
2009.)TheITSsequenceoftheseisolateswasidenticaltoacul- Netherlands, and Switzerland) yielded a unique Ambrosiella
ture(CBS121750)fromtheholotypeofA.beaverifromC.muti- sp.AfemalebeetletrappedinflightinMissouriwasdissected
latusinMississippi(Sixetal.2009). toremoveandobservethemycangiumanditssporecontents,
GalleriesofXylosandrusamputatusintwostemsofCinnamo- andacultureoftheAmbrosiellasp.wasrecoveredfromthere-
mum camphora in Lowndes County, Georgia in August 2014 movedsporemass.Themycangiumsatjustbelowthemeso-
werelinedwithathick,grey-whitemyceliumwithaleurioco- notumandwasattachedtothescutellum(Fig4A).Whenthe
nidiophores, and protoperithecia were scattered in the mycangial contents were freed, the spore mass maintained
ThreeambrosialgeneraintheCeratocystidaceae 1081
Fig3eUnrootedUPGMAdistancetreeofITSrDNAsequencesofAmbrosiellaspp.fromCnestus,Xylosandrus,andAnisandrus
spp.;Meredithiellaspp.fromCorthylusspp.;Phialophoropsisspp.fromTrypodendronspp.;andseveralrepresentativesofthe
Ceratocystidaceae.SingleasterisksindicatesequencesobtainedfromDNAextractedfromwholebeetlesormycangialspore
masses;doubleasterisksindicatesequencesfromDNAextractedfromgallerygrowth.Sequenceswithoutasterisksarefrom
DNAextractedfromcultures.CountryorUSAstate(twoletterabbreviation)oforiginofthebeetleisindicated.
theshapeofthemycangium(Fig4B,C),andthesporemass ThreeXylosandruscompactusbeetlestrappedinGeorgiain
didnotdisperseinwater,lacticacid,oroil(IsoparM).When 2007yieldedisolatesofAmbrosiellaxylebori,andeachhadan
mountedandstained(Fig4DeF),contentsofthemycangium ITS sequence identical to a culture (CBS 110.61 ¼ C1650)
wereobservedtobeahomogenousmassofarthrospore-like fromtheholotype,whichwasfromX.compactusintheIvory
cells, similar to those reported by Harrington et al. (2014) Coast(Fig3).
frommycangiaofXylosandruscrassiusculus.TheITSrDNAse- FreshAmbrosiellahartigiiisolateswereobtainedfromAni-
quencesofallisolatesfromX.germanuswereidenticalexcept sandrus dispar collected in Austria and the Czech Republic.
atonebasepositionneartheendofITS2,andtheseITSse- Theseisolates(includingCBS 139746¼ C3450, fromAustria)
quences closely matched two GenBank accessions sporulatedheavily,unliketheisolatefromtheholotypespec-
(HQ538467 and HQ670423) from Korean X. germanus speci- imen(CBS404.82¼C1573,fromGermany).TheITSsequences
mens(Fig3). ofallA.hartigiiisolateswereidentical,exceptfortheAustrian
Xylosandrus crassiusculus adult beetles from Georgia, Mis- isolate,whichhadonebasesubstitutionaswellasanaddi-
souri,Ohio,andSouthCarolinayieldedisolateswithITSse- tionalTneartheendoftheITS2region(Fig3).
quences matching that of Ambrosiella roeperi (Harrington AnAnisandrussayiadulttrappedinBooneCounty,Missouri
et al. 2014). An additional adult X. crassiusculus was trapped in May 2013 yielded a novel Ambrosiella sp. with branching
in Taiwan in July 2014 and yielded an isolate of A. roeperi aleurioconidiophores and disarticulating aleurioconidia
withanITSsequencesimilartootherrecoveredA.roeperise- (Fig 1JeN). Additional An. sayi adults trapped with a rotary
quences, but the sequence most closely matched that of nettrapinMontcalmCounty,MichiganinMay2014yielded
aSouthCarolinaisolatefromX.crassiusculus(Fig3). the same fungal species. More specimens were trapped in
1082 C.G.Mayersetal.
Fig4eMycangiumandexcisedsporemassofAmbrosiellagrossmaniaefromafemaleXylosandrusgermanus.(A).excised
mycangium(myc)andattachedscutellum(sc).(BeF).mycangialsporemass.(B).ventralaspect.(C).dorsalaspect.(D,E).
pressedwithcoverslip.(D).fullsporemass.(E).detailbetweenlobes.(F).edgeofsporemass.(B,C)bystereomicroscope,
unstainedmaterial.AllotherphotosbyNomarskiinterferencemicroscopyofmaterialstainedwithcottonblue.For(A)and
(D),bar[100mm;for(F),bar[10mm.
ChattahoocheeNationalForest,GeorgiainJuneandJuly2014, Ceratocystissp.(“CspXapi1”)fromanambrosiabeetleinKorea,
andfourbeetlesweregroundanddilutionplated,yieldingthe perhapsAnisandrusapicalis(formerlyXyleborusapicalis).
same Ambrosiella sp. The ITS sequences of isolates from all
threelocationswereidenticalandmostsimilartothatofA. Corthylini
hartigii(Fig3). Isolation from the galleries of Corthylus punctatissimus in
An Eccoptopterus spinosus adult was trapped in Taiwan in youngblackmaple(Acernigrum)saplingsinIowainAugust
July 2014 and stored in ethanol. Although fungal isolation 2013yieldedafunguswithanITSsequenceclosetobutdis-
was not possible, the mesonotal mycangium was dissected tinct fromall known Ambrosiella. The sporulation in galler-
and yielded a dual-lobed spore mass similar in morphology ies and cultures formed terminal aleurioconidia on many
to that recovered from the mycangia of X. germanus (Fig 4). short side branches (Fig 2EeJ). Surface-sterilized males
DNAextractedfromthesporemassfromthemycangiumof taken from these Iowa galleries, as well as galleries and
E. spinosus yielded a unique ITS sequence (GenBank beetles collected in Michigan from Acer saccharum in Octo-
KR611325) somewhat close to that of A. xylebori but most ber 2013, yielded isolates with the identical ITS sequence
closely matching a sequence (HQ670422) of an unidentified (Fig 3).
ThreeambrosialgeneraintheCeratocystidaceae 1083
Fig5ePhylogenetictreefromBayesiananalysisofthecombinedTEF1aandSSU(18S)rDNAdatasetofambrosiabeetle
symbiontsandrepresentativesoftheMicroascales.PosteriorprobabilitiesfromBayesiananalysisandbootstrapsupport
values(>50%)frommaximumparsimonyanalysisareindicatedonbranchlabels.Thickenedbrancheswithasterisksin-
dicateposteriorprobabilityof1.0and100%bootstrapsupport.Tribesandthetypeofmycangiumexhibitedbythehost
beetlesinthegeneraassociatedwithCeratocystidaceaeareindicatedinthethreeshadedboxes.Diagramaticillustrationsof
conidiophoresoftheambrosiabeetlesymbiontsareincluded;solidarrowsindicateconsistentobservationinaspecies,and
thedashedarrowsindicaterareorambiguousobservations.ThetreeisrootedtoNeurosporacrassaandPlectosphaerella
cucumerina.
MaleCorthylusconsimilisbeetlesfrom La Esperanza,Mex- from female beetles from each location yielded an identical
ico, collected in 2007, were stored in ethanol. DNA was ITS sequence (KR611329) using the Ceratocystidaceae-specific
extracted from the prothorax of one of the specimens and primers,andthesequencewassimilartothatofthesymbi-
yielded an ITS sequence (KR611327) similar to the symbiont ontsofotherTrypodendronspp.(Fig3).
from C. punctatissimus but differing at 10 base positions TrypodendonlineatumbeetlesweretrappedinAlaskausing
(Fig 3). A dissection of the prothorax revealed a long, coiled Lineatinlureandcollectioncupsthatweredryandcontained
tube connected to the procoxal cavity and containing a ho- theinsecticideVapona(Reichetal.2014).Severalfemalespec-
mogenoussporemassofcellssimilartothoseseeninmeso- imensweredriedandshippedtoIowaforisolations,butno
notalmycangiaofXyleborinifemales(Harringtonetal.2014). Ambrosiellasp.wasisolated.GalleriesinaninfestedlogofPicea
sp.collectedinColoradoin2014werealsoexamined.Thegal-
Xyloterini leries (BPI 893129, 893130) were packed with conidiophores
Trypodendronscabricollisbeetlestrappedin2013from10loca- withdeep-seatedphialidesbutnoaleurioconidia(Fig2KeN).
tionsinMissouriwerestoredinethanol.TheDNAextractions Attempts to isolate the fungus from the galleries were not
1084 C.G.Mayersetal.
successful, but DNA was extracted from the fungal growth. Trypodendronspp.,andassignmentoftheCorthyluspunctatissi-
TheITSsequencesfromtheextractedDNAfromfemalebee- mussymbionttoanewgenus.
tlestrappedinAlaskaandfromthegallerygrowthinColorado AMBROSIELLAArx&Hennebertemend.T.C.Harr.,Myco-
wereidentical (KR611328) and similar to those ofthe fungal taxon111:354.2010.
symbiontsfromotherTrypodendronspp.(Fig3). Typespecies:AmbrosiellaxyleboriBraderexArx&Hennebert.
A culture deposited as Ambrosiella ferruginea (CBS 460.82) AmbrosiellabeaveriSix,DeBeerandW.D.Stone,Antonie
was isolated in 1971 from Trypodendron domesticum in Ger- vanLeeuwenhoek96:23.2009.
many. No conidiophores were seen in this culture, but its MycoBankMB504757.
ITSsequencedifferedonlyslightlyfromthatoftheT.lineatum Comments e This species was probably introduced to the
andT.scabricollissymbionts(Fig3). Southeastern USA with its beetle host, Cnestus mutilatus,
TheITSsequence(KC305145)ofanotherA.ferrugineaisolate from Asia (Six et al. 2009). The culture from the holotype of
fromTrypodendronretusuminWisconsin(CBS408.68¼MUCL Ambrosiellabeaveri(CBS 121750)and two isolates (C3180and
14520) was similar to, but distinct from, the ITS sequence C3181) from Cnestus mutilatus in Georgia produce chains of
fromthethreeotherTrypodendronassociates(Fig3). phialoconidiafromdeep-seatedphialides(Fig1A)andtermi-
nal aleurioconidia onbranchedconidiophores(Fig1B),asil-
Phylogeneticanalysis lustratedbySixetal.(2009).
Ambrosiella nakashimae McNew, C. Mayers, and T. C.
A Bayesian consensus tree of the combined SSU and TEF1a Harr.,sp.nov.(Fig1CeF).
datasetplacedthefungalsymbiontswithintheCeratocystida- MycoBankMB812571.
ceae(Fig5).Therewasstrongsupportforgroupingtheambro- Etymology.NamedafterToshioNakashima,whocharacter-
siabeetlesymbiontswithCeratocystisadiposa(Butler)Moreau izedfungisporulatinginthegalleriesofnumerousambrosia
1952, Ceratocystis fagacearum(Bretz) J. Hunt 1956, Ceratocystis beetlesinJapan.
norvegica J. Reid & Hausner 2010, and Huntiella spp. There Typus: USA: Georgia: Lowndes Co., 30(cid:2) 490 06.600 N, 83(cid:2) 280
appeared to be three lineages of ambrosia symbionts, and 48.300 W, Xylosandrus amputatus gallery in Cinnamomum cam-
these three lineages correlated with the three respective phora, Aug 2014, S. Cameron, holotype (BPI 893134); ex-type
hostbeetletribes:Xyleborini,Corthylini,andXyloterini(Fig5). C3445(CBS139739).
Therewasstrongsupport(1.0priorprobability,100%boot- Coloniesonmaltextractyeastagar7e32mmafter4dand
strap) for the Xyleborini associates as a monophyletic clade, 25e50mmafter6dat25(cid:2)C,surfacemyceliumwhite,flat,be-
andallofthesespeciesformedaleurioconidiaonbranching comingsmokegraytoolivegreenwithcreampatches,reverse
conidiophores (Fig 5). The Xylosandrus amputatus associate cream white, becoming dark green to black, odor weak,
hadsequencessimilartothoseofAmbrosiellabeaveri,theAni- slightlysweetat6e10d.Sporodochiawhitetograyonsurface,
sandrus sayi associate had sequences similar to those of superficial.Aleurioconidiophores(Fig1E)hyalinetobrownwith
Ambrosiella hartigii, and the Xylosandrus germanus associate age,simpletobranched,12e115(155)mmlong,composedof
had sequences most similar to those of Ambrosiella roeperi one or more cells, producing a single terminal aleurioconi-
andAmbrosiellaxylebori(Fig5).ThesinglespeciesfromtheCor- dium or a chain of aleurioconidia, conidiogenous cell with
thyliniappearedtogroupseparatelyfromtheAmbrosiellaspp. acollarette.Aleurioconidia(Fig1F)globosetosubglobose,hya-
fromtheXyleboriniandappearedtobemorecloselyrelatedto line to light brown with age, thick-walled, aseptate, 6e10
C.adiposaandC.norvegica,aswellastothegenusHuntiella(for- (14) (cid:4) 6e10 mm. Phialoconidiophores uncommon, with moder-
merlytheCeratocystismoniliformiscomplex).Thesequencesof atelyseatedphialides,producingbasipetalchainsofconidia
theTrypodendronsymbiontsweremostsimilartothoseofC. byring-wallbuilding.Phialoconidiaellipsoidaltoglobose,hya-
fagacearum(Fig5). line to light olive brown with age, becoming thick-walled,
Comparisonofmarginallikelihoodsfromtopologicaltest- aseptate6e10(12)(cid:4)5e9mm.Protoperithecia(Fig2B,C)superfi-
inginMrBayesshowedthatitwasmorelikelythatthethree cial, spherical, tan to brown, 55e70 mm diam. Gallery growth
lineages of ambrosia beetle symbionts were polyphyletic dense,flat,whitetogrey,becomingbrownwithage.Aleurioco-
((cid:3)10,793.39) rather than a single, monophyletic group nidiophores(Fig1C)similartothoseinculture,9e70mmlong,
((cid:3)10,803.71). Phialophoropsis (Xyloterini associates) was more endinginaphialidethatmayhaveadistinctcollarette.Aleur-
likelytobeaseparatecladedistinctfromtheXyleboriniassoci- ioconidiaingalleries(Fig1D)largerthaninculture,terminalor
ates(i.e.,Ambrosiella)((cid:3)10,793.91),ratherthanformingasingle in chains, globose to suboblate, thick walled, aseptate, hya-
monophyleticgroupwithit((cid:3)10,795.35).TheCorthyluspuncta- line,maybecomelightbrownwithage,(8)9e12.5(15)(cid:4)(7.5)
tissimus associate was clearly outside of Ambrosiella sensu 8.5e11.5(16)mm.
stricto(Fig5),buttheC.punctatissimusassociatewasfoundto Otherculturesexamined:USA:Georgia:LowndesCo.,30(cid:2) 490
bemorelikelygroupedwiththeXyleboriniassociatesasasister 06.600 N, 83(cid:2) 280 48.300 W, Xylosandrus amputatus gallery in C.
group((cid:3)10,789.59)thannot((cid:3)10,794.99)inthemarginallikeli- camphora,Aug2014,S.Cameron,CBS139740(C3443).
hoodtesting. Notes.ThesymbiontofX.amputatusissimilartoA.beaveri
bothinDNAsequencesandmorphology,especiallythealeur-
Taxonomy ioconidiophores, but differs in growth rate (7e32 mm vs.
28e36 mm diam after 4 d at 25C), rarity of phialoconidio-
Phylogenetic analysis and morphological characters sup- phores,andtheproductionofprotoperithecia.Thisisthefirst
portedrecognitionofthreenewspeciesofAmbrosiella,resur- reportofprotoperitheciainambrosiabeetlesymbionts,which
rection of the genus Phialophoropsis for the symbionts of arethoughttobeasexual(Harringtonetal.2010).Noperithecia