Entomopathogens of Amazonian stick insects and locusts are members of the Beauveria species

17 Pages·2014·1.79 MB·English

by  Spatafora, Joseph W.

Entomopathogens of Amazonian stick insects and locusts are
members of the Beauveria species complex (Cordyceps sensu stricto)
Sanjuan, T., Tabima, J., Restrepo, S., Læssøe, T., Spatafora, J. W., &
Franco-Molano, A. E. (2014). Entomopathogens of Amazonian stick insects and
locusts are members of the Beauveria species complex (Cordyceps sensu stricto).
Mycologia, 106(2), 260-275. doi:10.3852/106.2.260
10.3852/106.2.260
Allen Press Inc.
Version of Record
http://cdss.library.oregonstate.edu/sa-termsofuse
Mycologia,106(2),2014,pp.260–275.DOI:10.3852/106.2.260
#2014byTheMycologicalSocietyofAmerica,Lawrence,KS66044-8897
Entomopathogens of Amazonian stick insects and locusts are members
of the Beauveria species complex (Cordyceps sensu stricto)
Tatiana Sanjuan1 clade, suggest that the holomorphs of these species
Laboratorio de Taxonom´ıa y Ecolog´ıa de Hongos, may include Beauveria or Beauveria-like anamorphs.
Universidad de Antioquia, calle 67 No. 53 – 108, The varying host specificity of the beauverioid
A.A. 1226, Medellin, Colombia
Cordyceps species suggest the potential importance
Javier Tabima of identifying the natural host taxon before future
Silvia Restrepo consideration of strains for use in biological control
Laboratorio de Micologia y Fitopatologia, Universidad of pest locusts.
de Los Andes, Cra 1 No. 18A- 12, Bogota´, Colombia Key words: Anamorph-teleomorph connection,
entomopathogenicfungi,hostspecificity,Neotropics,
Thomas Læssøe
Orthoptera, phylogeny
DepartmentofBiology,Universitetsparken15DK-2100,
Copenhagen Ø, Denmark
Joseph W. Spatafora INTRODUCTION
Department of Botany and Plant Pathology, Oregon
Entomopathogenic fungi of the genus Cordyceps
State University, Corvallis, Oregon 97331
sensuKobayasietMains(Mains1958)haveacomplex
Ana Esperanza Franco-Molano taxonomy in spite of being extensively studied since
Laboratorio de Taxonom´ıa y Ecolog´ıa de Hongos, the 19th century. A recent phylogenetic classification
Universidad de Antioquia, calle 67 No. 53 – 108, based on molecular data separated the species into
A.A. 1226, Medellin, Colombia
four genera (Cordyceps s. str., Elaphocordyceps, Meta-
cordyceps and Ophiocordyceps) across three families,
Cordycipitaceae, Clavicipitaceae and Ophiocordycipi-
Abstract: In the Amazon the only described species
taceae, with numerous species of undetermined
of Cordyceps sensu stricto (Hypocreales, Cordycipita-
phylogenetic affinity retained in a residual Cordyceps
ceae) that parasitize insects of Orthopterida (orders
sensulato(Sungetal.2007b).InthestudyofSunget
Orthoptera and Phasmida) are Cordyceps locustiphila
al. (2007b) the species of cordycipitoid fungi record-
and C. uleana. However, the type specimens for both
ed on Orthoptera were classified in Cordycipitaceae
taxa have been lost and the concepts of these species
for C. locustiphila Henn. and Ophiocordycipitaceae
are uncertain. To achieve a more comprehensive
forOphiocordycepsamazonica(Henn.)G.H.Sung,J.M.
understanding of the systematics of these species,
Sung, Hywel-Jones & Spatafora. Species such as C.
collections of Cordyceps from the Amazon regions of
uleana Henn. from Peru, C. lilacina Moreau from
Colombia, Ecuador and Guyana were subjected to
Congo and C. neogrillotalpae Kobayashi, C. stiphrodes
morphological,ecologicalandmolecularphylogenet-
Syd. and C. ctenocephala Syd. from New Guinea were
ic studies. Phylogenetic analyses were conducted on
not assigned to any family and retained in Cordyceps
partial sequences of SSU, LSU, TEF, RPB1 and RPB2
s.l. due to the lack of conclusive information.
nuclearloci.Twonewspeciesareproposedincluding
Cordyceps locustiphilaandC. uleanaweredescribed
C. diapheromeriphila, a parasite of Phasmida, and C.
by Hennings (1904) from collections sent by Ernesto
acridophila, a parasite of the superfamily Acridomor-
Ule from Peru´. He described C. locustiphila as a
pha(Orthoptera),whichisbroadlydistributedacross
species with gregarious or solitary, claviform and
theAmazon.ForC.locustiphilaalectotypificationand
yellow stromata, but surprisingly no information
an epitypification are made. Cordyceps locustiphila is
regarding partspores was given, although this charac-
host specific with Colpolopha (Acridomorpha: Roma-
leidae), and its distribution coincides with that of its ter is commonly used in Cordyceps taxonomy. The
host. The phylogenetic placement of these three original illustration shows the fungus emerging from
species was resolved with strong support in the the abdomen and the coxa (the basal segment of the
Beauveria clade of Cordyceps s. str. (Cordycipitaceae). insect leg attached to the thorax) of an adult locust
This relationship and the morphological similarity of (Orthoptera: Acrididae). While C. locustiphila is
their yellow stromata with known teleomorphs of the frequently collected in the Amazon, there have been
few taxonomic studies of this species (Evans 1982).
Submitted14Jan2013;acceptedforpublication5Mar2013. The type specimen of C. locustiphila was studied only
1Correspondingauthor.E-mail:[email protected] by Petch, who made a revision of Cordyceps species
260
SANJUANETAL.: AMAZONIAN CORDYCEPS 261
parasitizing Orthoptera (Petch 1934). He considered trips were made to a lowland tropical forest of the inter-
O. amazonica, collected in Brazil, and O. striphodes, Andes valley at the municipal forest, Mariquita, Tolima
collected in New Guinea, as synonyms of C. Department,inJan2011,andthesecondattheElAmargal
BiologicalStationNuqui,Choco´ Department,inMar2012.
locustiphila because all have clavate stromata with a
All areas are 20–200 m, average temperatures is 28 C, and
shortstalk.ConcerningtheC.uleanatype,Petchsaid
relative humidity is 90%. Specimen collection at each site
‘‘C. uleana has clavae with a short stout stalk and a
occurred over a 2 h interval and avoided human trails.
globose head with strongly projecting perithecia.’’
Collecting involved careful examination of leaf litter,
Petch did not report any information about part-
downed wood and elevated plant structures (e.g. leaves,
spores or the host. In addition, Petch (1933) twigs) to detect the emergence of stromata from insect
compared in detail the type of C. uleana, preserved cadavers.Additionaldataonforestcharacteristics,including
in alcohol, with two specimens collected from stick habitat and live, unparasitized host specimens also were
insects (Phasmida) in Madagascar. He identified collected.Driedspecimenswereplacedinplasticbagswith
them as C. uleana due to their similarity in the silicagelandstoredattheAntioquiaUniversityHerbarium
globosestromataheadandprojectingperithecia.The (HUA).
partsporesoftheMadagascar materialis3–9mmlong Morphological observations.—Collected material as well as
(TABLE I). Finally Moureau (1949) identified two material provided by the National Herbarium of Colombia
specimens from Congo on Mantodea ootheca as C. (COL), the National Herbarium of Ecuador (QCNE) and
uleanabecausetheyhadyellow,globosestromatawith Dr M. Catherine Aime’s personal collection (MCA) were
anechinulatesurface.Hedescribedthepartsporesas rehydratedinsterilizedwaterandstainedwithCongoredor
5–10 mm long and filiform (TABLE I). lactophenol cotton blue. Perithecia, asci, ascospores and
partspores were examined in a Leica Dm 1000 compound
As we have summarized above, there is no
microscope and fluorescent Olympus B60 microscope.
consensus on the taxonomy, phylogenetic position
Methuen Handbook of Color (Kornerup and Wanscher
orecologicalrelationship ofCordycepsonOrthoptera
1984) wasused forcolordescriptions ofstromata.
and Phasmida, particularly for C. locustiphila and C.
uleana sensu Hennings. Unfortunately, during the DNA extraction, PCR and sequencing.—In the field small
Second World War (WWII), Hennings’s types depos- pieces of fresh tissue from stromata were placed in 50 mL
ited in Berlin were destroyed. The only original CTAB extraction buffer (1.4 M NaCl; 100 mM Tris-HCl
pH8.0;20mMEDTApH8.0;2%CTABw/v),andDNAwas
material supporting these species is a copy of an
extractedfollowingthemethodinKepleretal.(2011).Five
original illustration deposited in Kew Botanical
nuclear loci were amplified: small subunit ribosomal RNA
Garden and the original paper (Hennings 1904).
(SSU) andlargesubunit ribosomal RNA(LSU),elongation
Thus, it is necessary to define the position of these
factor-1a(TEF),andthelargestandsecondlargestsubunits
species based on morphological and molecular
ofRNApolymeraseII(RPB1andRPB2).Nuclearribosomal
phylogenetic analyses of freshly collected specimens. internaltranscribedspacerregion(ITS)alsowasamplified
We clarify species and type concepts of Amazonian and sequenced for all samples. PCR amplification was
Cordyceps s. str. on the superorder Orthopterida performed in 25 mL MasterAmp 23 PCR premix E
(Orthoptera and Phasmida; Grimaldi and Engel (Epicenter, Madison, Wisconsin), 0.2 mM of each dNTP’s,
2005) through the examination of morphological 0.5 mM amplification primers, 0.1–0.2 mg template DNA
and 1.25 U Taq DNA polymerase (Fermentas, Glen Burnie,
characters, host association, geographic distribution
Maryland). Amplification of SSU and LSU were performed
andphylogeneticanalysesofmoleculardataobtained
respectivelywithNS1/NS4 (Whiteet al.1990)andLROR/
from specimens of C. locustiphila-like and C. uleana-
LR5 primers (Vilgalys and Sun 1994). ITS was amplified
like material from Colombia, Ecuador and Guyana,
with primers ITS1f and ITS4 (White et al. 1990). Amplifi-
cation of TEF was performed with the primers 983F and
MATERIALSANDMETHODS 2218R (Rehner and Buckley 2005). Amplification of RPB1
was performed with primers cRPB1-1aF and cRPB1-CaR
Field collections.—Most specimens in this study were (Castlebury et al. 2004), and amplification of RPB2 was
collected in Colombia, Department of Amazonas, Munici- performed with primers fRPB2-5f2 and fRPB2-7cR (Liu et
palityofLaChorrera,whichisaUitotoindigenousterritory al. 1999). The PCR reactions were performed in a thermo-
and Municipality of Leticia Town in El Zafire reserve cycler 1000 (BIORAD, Hercules, California) programmed
(4u09210S,69u539550W).Twokindsofforestsweresampled: as follows: 94 C for 3 min; 10 cycles of 94 C for 30 s, 55 C
(i) ‘‘chagra’’ which is characterized by low canopy and for1min,and72Cfor2min;35cyclesof94Cfor30s,50C
mixed forest surrounded by the typical indigenous agricul- for1min,and72Cfor2min;onecycleof72Cfor3min
turecrops,and(ii)high,intactcanopyforestwithrelatively and 4 C indefinitely (Kepler et al. 2011). Sequencing was
lowhumandisturbancethatislimitedtousebyindigenous performed with the amplification primers at Macrogen
peoples for hunting and timber. Initial collections were (Seoul, South Korea) sequencing service. New DNA
made in Mar and then May–Jul 2010 and 2011, which sequences of genes generated in this study were submitted
typically is the rainy season in this region. Additional field to GenBank(TABLE II).
262 MYCOLOGIA
1
–
1 8
Partspores Size(m)m * 3–5(8)1 33)4–7(10) 3–30.6–0.8 3–101 36–)8–120. 3–41 301 3–81
3 ( 2 5 ( 3 1 6
5 m 5 4 4 5
m) 3–3. 4m 3–4 4.5– 2.4– 3.5– 3 4–4.
ci (m 3 3 3 3 3 3 3 3
As e 0 0 0 0 0 0 0 0 4 a.
Siz 0–20 0–30 0–45 0–30 0–35 0–40 0–18 5–15 30 pter
5 7 6 5 0 0 5 3 0 mi
1 2 2 2 2 2 1 1 4
e
H
0 0 0 0 ) 0 0
30 44 25 35 00 25 38 e:
– – – – 4 – – H
(mm) 3250 3200 3200 3250 (–700)–350(– 00–110 3200 3320 ptera,
cium Size 50ca. 00–600 50–730 00–350 40–650 20–6003280 3001 75–350 20–680 Coleo
Perithe nt 1 ed5 4rsed d3 ed4 ed3 ed3 ed,2 d6 ea,Co:
Arrangme mmersed,aggregates emi-immers mmersedtosemi-immewidewallubimmerseaggregates emi-immers emi-immersaggregates emi-immers emi-immerswidewall ubimmerse Ma:Mantod
I S I S S S S S S
a,
4 d
m) –7 –4 2– mi
ata Size(m 10–15 37–205 37–273 6–8 3302 (3–)7–16 35515 330–40 3252.5 Ph:Phas
rdycepsspecies Strom Features Claviform Claviform Claviform,echinulatedryGlobular,scattered.caespitoseGlobosus,gregariousGlobose,gregarious,equinulateClaviform,solitary Claviform, Cylindrical,simple Or:Orthoptera,
o
sonofbeauverioidC Host Colpolophasp.(Or) Colpolophasp.(Or) nymphandadult:Acridomorpha(Or)Locustidaeadult(Or) Oothec(Ma) Diapheromeridaeadult(Ph) Staphylinidaelarvae(Co),(He)Scarabeidaeadult(Co) Gryllotalpa(Or) originaldescription;
ri n a a a e
gicalcompa Distributio Peru Colombia,Ecuador Colombia,Ecuador,GuyanaPeru Congo EcuadorGuyana JapanKore NewGuineNepal NewGuine ovivedinth
o r
TI.AmorpholABLE Specieswithyellowstromata C.locustiphilaHenning(1904)C.locustiphilasensuSanjuanetal.C.acridophilasp.nov.Sanjuan&Franco C.uleanaHenning(1904) C.uleanasensuMoureau(1949)C.diapheromeriphilasp.nov.Sanjuan&RestrepoC.staphylinidicolaKobayasi&Shimizu(1982)C.scarabeicolaKobayasiandShimizu(1976)C.neogryllotalpaeKobayasiandShimizu(1976) *Informationnotp
SANJUANETAL.: AMAZONIAN CORDYCEPS 263
1 04853 790366 57 26 6
31853026 464546111 2 13 4026 275 2
B2 910224890809809809809809 809809809809809809900224224 384 384384 384891224224 224891384 573
P 65688888 888888655 0 00 0655 560 4
R 4Q4QQQQQ QQQQQQ4QQ 0 00 04QQ Q40 5
EFDEFHHHHH HHHHHHEFDD JX JXJX JXEFDD DEFJX AY
3 92631 578 44 1 07 7
3617635967961795386297364557080103677
B1 963223885808808808808808908808808808088808808913420964890385384385385385385385885223224384385385965886384384223
P 8568888868888886886000000065500086005
R 4Q4QQQQQ4QQQQQQ4Q4400000004QQ0004400Q
AYDEFHHHHHEFHHHHHHEFDAYEFJXJXJXJXJXJXJXEFDDJXJXJXAYEFJXJXD
ankno. TEF KC519366AF543782DQ522317EF468743AY531907HQ880996AY531890HQ880990AY531912EF469057HQ881005DQ376246HQ880999AY531899HQ881004AY531920DQ862013AY489611AF543778EF468801JQ958614JQ958616JQ958613JQ958615 JQ958617JQ958618EF468747DQ522325DQ522359JQ958610JQ958611JQ958612AY489616EF468754JQ958619 DQ522332
B
Gen nLSU C519370F543792Q518752 F339520 Q862029Y489716F543789F468850Q895536Q895538Q895541Q895537Q895542Q895539Q895540F468807Y184962F339555Q895534Q895533 F339522F468813Q895535Q958597Y184966
KAD A DAAEJJJJJJJEAAJJ AEJJA
nSSU C519368F543769Q522537F468949 F339570 Q862027Y489684F543765F468995Q895527Q895528 Q895526 Q895531Q895532F468953Y184973F339604Q895530Q895529 F339572F468960Q895525Q958598Y184977
KADE A DAAEJJ J JJEAAJJ AEJJA
sstudy ITS KC519371 AY531981 DQ862044 JQ958600JQ958602 JQ958601JQ958607JQ958604JQ958605 JQ958599JQ958603JQ958608 JQ958606JQ958609
hi
t
n
nformationforspecimensusedi Host/substratum Lepidoptera:SphingidaeCowdungHemiptera:CoccidaePoaceaeColeoptera:CurculionidaeOrthoptera:AcridiidaeHemiptera:PentatomidaeColeoptera:ScarabaeidaeColeopteraSoilHomoptera:DelphacidaeColeoptera:CerambycidaeColeoptera:ScotlitydaeColeoptera:ScarabaeidaeColeoptera:CurculionidaeSoil BarkPoaceae Acrididae:Ommatolampissp.Proscopidae:ApiosceliscolumbicaAcrididae:Ommatolampissp.Acrididae:Ommatolampissp.Romaleidae:TropidacriscristataAcrididae:Ommatolampissp.Romaleidae:Prionacrissp.Lepidoptera,pupaLepidoptera,larvaeCoccusviridis(Hemiptera)Phasmida:DiapheromeridaePhasmida:DiapheromeridaePhasmida:DiapheromeridaeLepidoptera,pupaLepidopteraRomaleidae:ColpolophasinuataRomaleidae:Colpolophasp.Lepidoptera,pupa
i
e
c
n
e
pecimenvoucherandsequ Taxon AkanthomycesaculeatusAphysiostromastercorariumAschersoniabadiaBalansiaepichloe¨BeauveriaamorphaBeauveriaaustralisBeauveriabassiannaBeauveriabrongniartiiBeauveriacaledonicaBeauveriacaledonicaBeauveriakipukaeBeauveriamalawesiBeauveriapseudobassianaBeauveriasungiiBeauveriavarroaeBeauveriavermiconiaBionectriacf.aureofulvaBionectriaochroleucaClavicepspurpureaConoideocrellaluteorostrataCordycepsacridophilaCordycepsacridophilaCordycepsacridophilaCordycepsacridophilaCordycepsacridophilaCordycepsacridophilaCordycepsacridophilaCordycepsbifusisporaCordycepscardinalisCordycepsconfragosaCordycepsdiapheromeriphilaCordycepsdiapheromeriphilaCordycepsdiapheromeriphilaCordycepsgunniCordycepskyusyue¨nsisCordycepslocustiphilaCordycepslocustiphilaCordycepsmilitaris
s
Taxon, no. 77262321810596-15a19694622147862152251256770327760185516812694292271-3281140561288511343179220179222179219179221118118672018672611098893609101247186272186714155776404588617921817921793623
I. r
TIABLE Vouche HUAATCCBCCAEGARSEFARSEFARSEFARSEFARSEFARSEFARSEFARSEFARSEFARSEFARSEFARSEFG.J.S.CBSGAMNHJHUAHUAHUAHUAMCAQCNEQCNEOSCOSCCBSQCNEQCNEMCAOSCEFCCHUAHUAOSC
264 MYCOLOGIA
1 581589 9053 68 9 0 14434
1 3 2 332222834541144 4 545 3452355
B2 892 224 893 224224224224224224910224224584224911573224224 224 224893893 911911224224224224224
P 6 5 6 555555655856455 5 566 6655555
R 4 Q 4 QQQQQQ4QQQQ45QQ Q Q44 44QQQQQ
F F F FY FF FF
E D E DDDDDDEDDDDEADD D DEE EEDDDDD
0 4 35 7 49 0 3 4 5 6 72390
0791814 8797720835829293949 97895497890
B1 890886886887223888888 223966964223223965909223965584223909223966223966223909223 223888888888909966223223223223224
P 6666566 5885586588565858565 56666855555
R 4444Q44 Q44QQ44Q4QQ4Q4Q4Q4Q Q44444QQQQQ
FFFF FF YY YF Y F Y Y F FFFFY
EEEEDEE DAADDAEDADDEDADADED DEEEEADDDDD
kno. TEF F468795F468759F468762F468761Q522335F468776F468778 Q522338Y489629Y489615Q522328Q522330Y489618F469061Q522341F543777F543772Q522343F469063Q522344F543781Q522347F543784Q522348F469065Q522349C519365Q522350F468781F468782F468783F469066Y489627Q522351Q522327F543775F543774Q522353
n EEEEDEE DAADDAEDAADEDADADEDKDEEEEADDAAD
a
B
Gen nLSU F468846F468819F468821F468823F339524F468836F468838B044637Q518767Y489734Y489721Q518759Q518761Y489723F469077F3395261739648428F339545F469078Y545726F54379147832F543793Q518772F469080Q518773C519369F339536F339537F339565F339549F469081Y489732Y184968Q518758F543787F339530F339531
EEEEAEEADAADDAEAUUAEAAUADEDKAAAAEAADAAA
nSSU F468991F468964F468966F468968F339574F468981F468984B044631Q522553Y489702Y489689Q522545Q522547Y489691F469124F3395763240548427F339594F469125Y545722F54376832409F543771Q522558F469127Q522559C519367F339585F339586F339614F339598F469128Y489700Y184979Q522544F543763F339579F339580
EEEEAEEADAADDAEAUUAEAAUADEDKAAAAEAADAAA
S
T
I
r
olo nis
Host/substratum LepidopteraLepidoptera,limacodidpupaLepidoptera,limacodidpupaColeoptera:ScarabeidaeColeoptera,StaphylinidpupaLepidopteraLepidopteraLepidoptera:NoctudidaeHymenomycetes:InonotusEuascomycetes:Elaphomycessp.Euascomycetes:Elaphomycessp.Euascomycetes:Elaphomycessp.Euascomycetes:Elaphomycessp.Coleoptera:ScarabaeidaeArachnidaPoaceae:FestucarubraRosaceae:Fragariasp.NematodeHemipteraadultLaxmanniaceae:woodHemiptera:CoccidaeHymenomycetes:TrametesversicLepidopterapupaLepidopterapupaLepidoptera:NoctudidaeLepidoptera:NoctudidaeHymenomycete:AgaricArachnidaHemiptera:CoccidaeHymenomycetes:ColtriciaperensoilsoilLepidoptera:IragoidesfasciataDiplopodaLepidopteraColeopteraHemiptera
s
de a
ued Taxon CordycepsninchukisporaCordycepsochraceostromataCordycepspruinosaCordycepspruinosaCordycepsscarabeicolaCordycepsstaphylinidicolaCordycepstakeomontanaCordycepstakeomontanaCordycepstuberculataCosmosporacoccineaElaphocordycepscapitataElaphocordycepsfractaElaphocordycepsjaponicaElaphocordycepsophioglossoiElaphocordycepssubsessilisEngyodontiumaranearumEpichloe¨typhinaGlomerellacingulataHaptocilliumsinenseHirsutellasp.HydropisphaeraerubescensHypocrealuteaHypocrellanectrioidesHypomycespolyporinusIsariafarinosaIsariafarinosaIsariatenuipesIsariatenuipesLecanicilliumantillanumLecanicilliumaranearumLecanicilliumattenuatumLecanicilliumfusisporumLecanicilliumpsallioteLeuconectriaclusiaeMariannaeapruinosaMetacordycepschlamydosporiMetacordycepstaiiMetarhiziumanisoplaeMetarhiziumflavoridae
n
I.Conti no. 38.1655691569310684 571812623 1110021140507123311099011099110640571235309.8556429513567.95125253609320883889-10476479125256279111007902350.85726.73a402.78164.70101270478.755413101244571431452037
I r
e
TABLE Vouch EGSEFCCEFCCNHJ ARSEFNHJ OSCCBSOSCOSCOSCOSCOSCCBSATCCFAUCBSNHJATCCATCCGJSATCCOSCEFCCOSCHUACBSCBSCBSCBSCBSCBSARSEFCBSARSEFARSEFARSEF
SANJUANETAL.: AMAZONIAN CORDYCEPS 265
RPB2 Q522418Q522420468911468913468914 468920 468924468925Q522432 Q522436468933Q522457 468940468941 468943469116469118Q522462Q522463Q522465Q522467469119469053Q522470 ollections,ds;EFCC,JulianH.collectionUniversity
GenBankno. nSSUnLSUTEFRPB1 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 haca,NewYork,USA;ATCC,AmericanTypeCultureCureauvoorSchimmelcultures,Utrecht,theNetherlanction;EGS,E,G.Simmonspersonalcollection;GAM,mAntioquiaUniversity,Medellin,COL;KEW,mycologygelHywel-Jonespersonalcollection;OSCOregonState
TII.ContinuedABLE ITSVoucherno.TaxonHost/substratum CBS744.73NomuraeaatypicolaArachnidaOSC110987OphiocordycepsacicularisColeopteraARSEF5692OphiocordycepsagriotidisColeopteraOSC128576OphiocordycepsbrunneipunctataColeopteraKEW53484OphiocordycepsentomorrhizaColeopteralarvaeEFCC3101OphiocordycepsgracilisLepidopteralarvaeEFCC10125OphiocordycepsheteropodaHemiptera:CicadaenymphOSC110993OphiocordycepsmelolonthaeColeoptera:ScarabeidaeEFCC9247OphiocordycepsnigrellaLepidopteralarvaeOSC110994OphiocordycepsnutansHemiptera:PentatomidaeEFCC7287OphiocordycepssinensisLepidopterapupaeKEW78842OphiocordycepssoboliferaHemiptera:CicadaenymphOSC110998OphiocordycepssphecocephalaHymenoptera:VespidaeOphiocordycepstricentriHemiptera:CicadaeadultOSC128574OphiocordycepsunilateralisHymenoptera:FormicidaeOSC111003OphiocordycepsvariabilisDipteralarvaeCBS109876OphionectriatrichosporaonlianaARSEF2181PurpureocilliumlilacinumsoilCBS431.87PurpureocilliumlilacinumnematodeCBS284.36PurpureocilliumlilacinumsoilEGS38.166PhytocordycepsninchukisporaBeilschmiediaerythrophloiaCBS145.70PochoniabulbillosarootofPiceaabiesGJS91-164RoumegueriellarufulaGloboderarostochiensis(Nematoda)EFCC6564ShimizuomycesparadoxusSmilacaceae:SmilaxsieboldiiCBS116.25SimplicilliumlamellicolaHymenomycete:AgaricusbisporusCBS101267SimplicilliumlanosoniveumUredinales:HemileiavastatrixCBS102308SphaerostilbellaberkeleyanaHymenomycete:PolyporeARSEF1915TorrubiellaratticaudataArachnidaCBS101237TorrubiellawallaceiLepidopteraCBS384.81VerticilliumepiphytumUredinalesCBS460.88VerticilliumincurvumHymenomycete:Ganodermalipsiense AEG,AnthonyE.Glennpersonalcollection;ARSEF,USDA-ARSCollectionofEntomopathogenic,ItManassas,Virginia,USA;BCC,BIOTECCultureCollection,KlongLuang,Thailand;CBS,CentraalbEntomopathogenicFungalCultureCollection,Chuncheon,Korea;FAU.,F.A.UeckerpersonalcolleMillerMycologicalHerbariumAthens,Georgia;GJS,G.J.Samuelspersonalcollection;HUA,HerbariuofRoyalBotanicalGarden,KEW,Surrey,UK;MCA,MarieCatherineAimepersonalcollection;NHJ,NiHerbarium,Corvallis,Oregon,USA;QCNE,NationalHerbariumofEcuador,Quito,ECU.
266 MYCOLOGIA
Sequence alignment and phylogenetic analyses.—Sequences- analyses, respectively (FIG. 1, SUPPLEMENTARY FIG. 1).
were edited with Geneious Pro 4.8.5 (Drummond et al. TheOrthopteraandPhasmidapathogens—hereafter,
2009)andcombinedwithsequencesfromCordycipitaceae, namedtheOrthopteridaclade—arenestedwithinthe
Clavicipitaceae and Ophiocordycipitaceae used by Sung et Beauveria clade (MLBS 5 67, PP 5 0.95) and
al. (2007b) and sequences of Beauveria from Rehner et al.
comprise three clades with strong support (MLBS 5
(2011). (All sequences in this study are in TABLE II.)
100, PP 5 1.00). These three clades also are
Glomerella cingulata and the various species of the
supported in the separate analysis of each of the
Bionectriaceae, Nectriaceae and Hypocreaceae were used
as the outgroup taxa based on phylogenetic analyses of individual five genes (SUPPLEMENTARY TABLE 1). The
Castlebury et al. (2004), Sung et al. (2007a) and Kepler et concordance of the five independent gene genealo-
al. (2011). A preliminary alignment of the sequences was gies (Taylor et al. 1999) provides additional support
performedwithClustalW(Thompsonetal.1994)aspartof for recognition of two different phylogenetic species
BioEdit 7.0. (Hall 1999). A re-alignment of SSU and LSU that parasitize locusts and one that is a pathogen of
was performed with Muscle (Edgar 2004) with the default stick insects (FIG. 1).
settings from GeneiousPro 4.8.5 (Drummond et al.2009);
To analyze the relationships and host affiliation of
re-alignmentofproteincodinggenesTEF,RPB1andRPB2
theOrthopteridacladefurther,phylogeneticanalyses
was performed with MAFFT (Katoh and Toh 2010) as part
were conducted on the combined dataset of ITS (13
of the CIPRES gateway (Miller et al. 2010). Finally, each
sequences of 586 bp) and TEF (11 sequences of
alignment wasrefined manually inBioEdit.
998 bp) with C. staphylinidicola as the outgroup,
Maximumlikelihood(ML)analyseswereperformedwith
RAxML-VI-HPC 2.0 using a GTR-GAMMA model of evolution availablefromTreeBASE(S13829).C. locustiphila,C.
(Stamatakis 2006) with 1000 bootstrap replicates. All five diapheromeriphila sp. nov. and C. acridophila sp. nov.
genes were concatenated into a single dataset and 11 data each received support that was consistent with the
partitions were defined: one each for SSU and LSU, plus previous five-gene phylogenetic tree (MLBS 5 100,
nine for each of the three codon positions for the protein PP 5 1.00) (FIG. 4). C. locustiphila was restricted to
coding genes TEF, RPB1 and RPB2 (Kepler et al. 2012). the genus Colpolopha (Romaleidae: Orthoptera) and
Bayesian Inference was performed with MrBayes 3.1.2
C. diapheromeriphila was restricted to the Diapher-
(Huelsenbeck and Ronquist 2001) and partitions were
omeridaefamily(Phasmida).C.acridophilahadthree
specified as in RAxML analyses. 10000000 MCMCMC
subclades weakly to moderately supported (MLBS 5
generations were performed, using a sample frequency of
45, 71, 80; PP 5 0.59, 0.67, 0.92 respectively) that do
500generationsandaburn-inof25%ofthetotalrun.Two
not correspond with a particular family of Acrido-
runs using four chains each (one cold and three heated
chains) were performed and each run was examined with morpha (FIG. 2). However, these three groups corre-
Tracer1.5(DrummondandRambaut2007)toverifyburn- spond to three geographic zones: (i) Napo area
inparametersandconvergenceofindividualchains.Forthe (Ecuador) close to the Andes foothills; (ii) western
analysis of combined ITS and TEF we performed RAxML AmazonbasininColombia,Peru´ andBrazil;and(iii)
and Bayesian analyses with the same parameter mentioned Amazon region of Guyana. Within each locality the
beforebutwithpartitionspergene.ForITStheGTR+G+I hosts of the specimens collected belong to different
model was used, while the HKY + G model was applied to families of Acridomorpha that normally are distribut-
TEF. Each of these models were obtained by using the
ed throughout the Amazon region (FIG. 2).
findings of Rehner et al. (2011) and Kepler et al. (2012)
andcorroboratedbymodelreconstructionwithJModelTest
2.1.3 (Posada 2008). Nodes were considered supported by TAXONOMY
bootstrapvaluesgreaterthan70%andposteriorprobability
Cordyceps locustiphilaHenn.,Fungi amazoniciII.acl.
equalto orgreater than 0.95.
Ernesto Ule collecti. Hedwigia 43:246 (1904).
Lectotype (selected here): Hennings, P. Fungi ama-
RESULTS
zonici II. a cl. Ernesto Ule collecti. Tab IV f. 3.
Fifty-eight sequences were obtained from the 12 (1904) FIG. 3a–d
specimens analyzed (TABLE II). The concatenated Stromata gregarious, claviform, simple, bright yel-
alignment was 4770 bases long, with 1094 bases from low (4B4), 12–20 mm long. Fertile head clavate,
the SSU, 926 bases from LSU, 1007 bases from TEF, slightly echinulate from protruding perithecial osti-
1049 bases from RPB2 and 690 bases from RPB1. oles,brightyellow(4A4),5–732–5mm.Stipefleshy,
The alignment is available from TreeBASE (S13829). terete,sometimescaespitose,grayishyellow(4B6),3–5
Phylogenetic analyses resolved three families of 3 1–2 mm. Perithecia semi-immersed, perpendicular
entomopathogenicfungi,Cordycipitaceae,Ophiocor- orientation,ovoid,550–600(–650)3250–320mm(n5
dycipitaceae and Clavicipitaceae, with strong statisti- 20),walllessthan50mmwide.Ascicylindrical,270–300
cal support (bootstrap, MLBS 5 100, posterior 3 4 mm (n 5 40), apical apparatus 3 3 4 mm.
probability, PP 5 1.00) in the ML and Bayesian Ascospores filiform, hyaline, 225 3 0.8 mm (n 5 10);
SANJUANETAL.: AMAZONIAN CORDYCEPS 267
FIG.1. Bayesian50%majorityruleconsensusphylogenetictreebasedoncombineddatasetofSSUrRNA,LSUrRNA,TEF,
RPB1andRPB2ofCordycepsspeciesthatparasitizeOrthopterida.BayesianposteriorprobabilitiesandMLbootstrapsupport
aregivenrespectivelyatfirstandsecondposition,aboveorbelowthebranches.Bionectriaceae,Nectriaceae,Hypocreaceae,
Clavicipitaceae, Ophiocordycipitaceae are collapsed for emphasis of Cordyipitaceae, but a fully expanded tree is available
(SUPPLEMENTARY FIG.1).
268 MYCOLOGIA
FIG. 2. Host associations of C. acridophila complex species mapped on phylogeny inferred from Bayesian inference and
maximumlikelihoodanalysesofcombinednuclearrDNAITSandelongationfactorTEFwithC.staphylinidicolaasoutgroup.
Hostidentitycodesareindicatedatbranchtips.ThemapoftheAmazonregionindicatessamplinglocalitiesandtheirrelation
tothecentersofdistributionforthefollowingacrididtaxa:1.Napo,2.AmazonNianwest,3.Guyana.Hostidentificationsymbols
include Diapheromeridae (Phasmida) Apioscelis columbica (Proscopidae) Ommatolampisspp.(Acrididae).Romaleidae:
&Tropidacris cristataXPrionacris compressa cColpolopha spp.
breakingintotruncatepartspores,3–5(–8)31mm(n specimen (QCNE 186267, FIG. 3C) was identified as
550). Co. latipennis Sta˚l, which is distributed in Ecuador
Host: On imago of Colpolopha cf. sinuata Sta˚l and Peru (Eades 2012). Specimen QCNE 186267 was
(1873), Orthoptera: Romaleidae. collected in the Amazon foothills of Ecuador, the
Known distribution: Colombia and Ecuador. sameregioninwhichspecimenC.Padilla(1423)also
Specimens examined: COLOMBIA. TOLIMA: Mariquita was collected.
MunicipalForest,5u119290N,74u549400W,560m.Jan2011,
T.Sanjuan881(EPITYPE,HUA179218).CHOCO:Nuqu´ı,
Cordyceps acridophila T. Sanjuan et A.E. Molano-
BiologicalStationElAmargal,5u349390N,77u309490S,40m.
26 Jan 2000, T. Sanjuan 208 (PARATYPE, HUA 179217). Franco, sp. nov. FIG. 3e–g
ECUADOR. ORELLANA: Tiputini Research Station, MycoBank MB801975
00u409S, 76u099040W, 235 m. 16 Jun 2004, T. Læssøe 11514 Etymology: Referring to the host from superfamily group
(PARATYPE, QCNE 186267). NAPO: Jatunsacha Reserve, Acridomorpha.
1u049S,77u369W,450m.18Jul2004,C.Padilla1423. Stromata capitate-stipitate, gregarious, or solitary,
Notes:Thehostofthelectotypeislikelyaspeciesof 7–27 mm long. Fertile part clavate, papillate when
Colpolopha because it has a thin and conspicuous fresh and echinulate when dry, pale yellow (4A2/3),
prothoracic crest typical of this genus (FIG. 3a). bright yellow, 6–12 3 (2.5–) 4–5 mm. Stipe fleshy,
Colpolopha has wide distribution in the Amazon, with terete, cylindrical, grayish yellow (4B5), 5–14 3 2–
the center in the Amazonian foothills of Peru, 3 mm. Perithecia immersed when young and semi-
Colombia and Ecuador but is also known from Costa immersedwhenmature,perpendicularinorientation.
Rica (Carbonell 2004). The host of the epitype was Ostioles pale yellow (4A/B5), ovoid to ellipsoid, 450–
identified as Co. sinuata Sta˚l (determination by C. 7303200–440mm(n520),wall50–100mmwide.Asci
Carbonell), as well as another collection examined hyaline, cylindrical 260–450 3 3–4 mm, apical appara-
from Colombia (HUA 179217). The host species of tus 2.4–3 3 3–4 mm (n 5 40). Ascospores parallel,
the epitype has been reported only from lowlands in smooth, filiform, hyaline, breaking regularly into
Colombia (Eades 2012). The host of the Ecuadorian truncatepartspores,(3–)4–7(–10)31mm(n550).