Penicillium menonorum, a new species related to P. pimiteouiense

Abstract

Penicillium menonorum is described as a new monoverticillate, non-vesiculate species that resembles P. restrictum and P. pimiteouiense. On the basis of phylogenetic analysis of DNA sequences from four loci, P. menonorum occurs in a clade with P. pimiteouiense, P. vinaceum, P. guttulosum, P. rubidurum, and P. parvum. Genealogical concordance analysis was applied to P. pimiteouiense and P. parvum, substantiating the phenotypically defined species. The species P. rubidurum, P. guttulosum, and P. menonorum were on distinct branches statistically excluded from inclusion in other species and have distinct phenotypes.

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ARTICLE
volume 2 · no. 2 121
© 2011 International Mycological Association
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INTRODUCTION
Inthecourseofascreeningprogramtondusefulfungifor
conversion of organic matter into high-value products such
as lipid precursors to biofuels and animal feed formulations,
a Penicillium isolated from garden soil in southern California
was obtained that could not be placed with condence
in any described species. Sequencing of the ITS region
was performed, with sequence analysis showing that this
isolate is phylogenetically related to P. pimiteouiense. DNA
distance from P. pimiteouiense suggested that it might be an
undescribedspecies.
Additional gene loci (β-tubulin, calmodulin, and DNA
replication licensing factor Mcm7) were amplied and
sequenced for this isolate and for phylogenetically and
phenotypicallysimilarspecies.Onthebasisofthephenotypic
and phylogenetic distinctions, this isolate is described as a
newspecies.
MATERIALS AND METHODS
Cultures (Table 1) may be obtained from the Agricultural
Research Service Culture Collection (NRRL), Peoria, IL
(http://nrrl.ncaur.usda.gov). The P. menonorum culture
ex-type is available from theAgricultural Research Service
Patent Culture Collection (http://nrrl.ncaur.usda.gov).
Cultures were maintained on potato-dextrose agar (PDA)
during the course of this study. Colony descriptions were
based on 7 d growth of cultures on Czapek’s yeast autolysate
agar(CYA),maltextractagar(MEA),andglycerolnitrateagar
(G25N)at25 °C,andonCYAat5°Cand37°Casdetailed
by Pitt (1980). Some color names are taken from Ridgway
(1912)andaredesignatedwithanuppercaseRandaplate
number.
Microscope slides were made by teasing apart bits
of mycelium in a drop of lactic acid with cotton blue. A
Zeiss axioscope with DIC optics was used for microscopic
observations. Photomicrographs were taken with a Kodak
14n digital camera attached to the microscope. Micro- and
macro-photographs were sized and placed in a plate using
AdobePhotoshopv.6.0.1.
BiomassforDNAextractionwasgrownin125 mLasks
containing 25 mL malt extract (ME) broth incubated at 25
°C on a rotary platform (200 rpm). Biomass ca. 0.5 g wet
weight was collected by vacuum ltration, placed in micro
centrifuge tubes, and freeze-dried. Freeze-dried mycelium
was ground to a powder with a sterile pipette tip and DNA
was extracted from the powdered biomass using the
CTAB method. Puried DNA was stored in TE buffer (Tris
10 mM, EDTA 1 mM, pH 8.0) at -20 °C until needed. DNA
was amplied using the primers and conditions detailed
in Peterson et al. (2010). Amplied DNA was prepared for
sequencing using ExoSAP-IT (www.usbweb.com). DNA
sequenceswereproducedusing DyeDeoxy v.3.1 reagents
andanABI 3730 DNAsequencer (www.appliedbiosystems.
com). Complementary strand sequences were assembled
and corrected using Sequencher (www.genecodes.com).
Finished sequences were aligned using CLUSTALW
(Chenna et al. 2003), and maximum parsimony trees and
bootstrapproportionswerecalculatedusingPAUPv.4.0b10
(Swofford2003).MrBayesv.3.12(Huelsenbeck&Ronquist
2001,Ronquist&Huelsenbeck2003) wasusedtocalculate
Bayesianposteriorprobabilities.DNAsequencesusedinthis
Penicillium menonorum, a new species related to P. pimiteouiense
StephenW.Peterson1,SamanthaS.Orchard2,andSureshMenon2
1USDA,Agricultural Research Service, National Center for Agricultural Utilization Research, Bacterial Foodborne Pathogens and Mycology
ResearchUnit,1815NorthUniversityStreet,Peoria,Illinois61604USA;correspondingauthore-mail:Stephen.Peterson@ARS.USDA.GOV
2Menon&Associates,Inc.,P.O.Box910033,SanDiego,California92191-0033USA
Abstract: Penicillium menonorum is described as a new monoverticillate, non-vesiculate species that resembles
P. restrictum and P. pimiteouiense. On the basis of phylogenetic analysis of DNAsequences from four loci, P.
menonorum occurs in a clade with P. pimiteouiense, P. vinaceum, P. guttulosum, P. rubidurum, and P. parvum.
GenealogicalconcordanceanalysiswasappliedtoP. pimiteouiense and P. parvum, substantiating the phenotypically
denedspecies.ThespeciesP. rubidurum, P. guttulosum, and P. menonorum were on distinct branches statistically
excluded from inclusion in other species and have distinct phenotypes.
Article info:Submitted:13April2011;Accepted:15June2011;Published:29September2011.
doi:10.5598/imafungus.2011.02.02.02
Key words:
monoverticillate
fungal systematics
congruence analysis
Penicillium
IMA FUNGUS · VOLUME 2 · NO 2: 121–125

Peterson, Orchard & Menon
ARTICLE
122 ima funGuS
study are deposited in GenBank (www.ncbi.nlm.nih.gov)
withaccessionnumbersHQ646566–HQ646603,AF033460–
AF033462, AF033464, AF037431, and AF037434. Data
setsandtree diagramsaredepositedatTREEBASE(www.
treebase.org).
Theinitialsearchtondphylogeneticallyrelatedspecies
was performed by BLAST searches of GenBank using the
ITSsequencefromthenewspecies.
RESULTS
Penicillium menonorumS.W.Petersonsp. nov.
MycoBank MB519297
(Fig.1A–D)
Etymology:NamedforMenon&Associateswhosescientists
isolatedthefungus.
A speciebus aliis conidiophoris brevibus, conidiis scaberulis,
colorein substratonutritorioCYApallidecaesio atque augmentoin
temperatura37°Cdistinguendum.
Typus: USA: California:isolatedfromgardensoil,2009(BPI
881018–holotypus;cultureex-holotypeNRRL50410).
ColoniesonCYA(Fig.1A)attaining17–20mmdiamafter7d
growthat25°C, velutinous-silky, radially sulcate peripherally,
centrally raised ca. 2–3 mm, sporulation moderate, central
regionpalebluishgray(courtgrayR-47),peripheralareawhite;
no exudate or soluble pigments; no sclerotia or ascomata;
reverse yellowish brown centrally (buckthorn brown R-15)
to pale brownish-yellow (warm buff, R-15) peripherally. On
MEA(Fig.1B) attaining17–19mmdiamafter 7dgrowthat
25 °C, mycelium loosely woven, wooly,umbonate 3–4 mm
deep centrally, sporulation moderate, white peripherally,
courtgray (R-47)centrally; noexudateorsolublepigments;
nosclerotiaor ascomata; reverse yellowish browncentrally
tobrownishyellowperipherally.OnG25Nattaining8–10mm
diam after 7 d growth at 25 C, umbonate, wooly 1–2 mm
deep,whitetocourtgray;noexudateorsolublepigment;no
sclerotia or ascomata; reverse white to buff. Incubation for
7 d on CYA at 5 °C produced no growth or germination of
conidia.Incubationfor7donCYAat37°C produced colonies
of29–32mmdiam,resemblinggrowthonCYAat25°C,but
clear exudate moderately abundant, the reverse color is a
darker, more uniform shade of brown. Conidiophores (Fig.
1C) smooth-walled, hyaline, 5–15(–20) × 1.5–2.0 µm, non-
vesiculate,withanapicalwhorlof(1–)2–5phialides5–7(–9)
×2.5–3.5µm, conidiasphericaltosubspherical,(2–)2.5–3.5
µm(Fig.1D),withroughenedtorugosesurface.
Table 1.Provenanceofisolatesusedinthisstudy.
Species NRRL Accession No. Origin
Penicillium erubescens MB335726a(syn.Eupenicillium
erubescens)
6223 SouthAfrica:Pretoria:isolatedfromnurserysoil,
1967,cultureex-type
Penicillium guttulosumMB266689 907 USA:Utah:isolatedfromsoil,1927,cultureex-type
Penicillium menonorumMB519297 50410 USA:California:isolatedfromgardensoil,2009,
culture ex-type
Penicillium parvum MB289101(syn.Eupenicillium parvum) 2095 Nicaragua:isolatedfromsoil,July1945,A.G.
Kevorkian, culture ex-type
6032 Papua-NewGuinea:isolatedfromsoil,ca.1973,S.
Udagawa, culture ex-type of P. papuanum MB319290
35488 Ghana:Tafo:isolatedfromsoil,ca.1949
35492 Venezuela:isolatedfromsoil,ca.1976,D.T. Wicklow
Penicillium pimiteouienseMB460126 2063 NewGuinea:isolatedfromtentcloth,ca.1944,G.W.
Martin
25542 USA:Illinois:Peoria:isolatedfromhumankidneycell
cultureplate,April1996,J.T. Hjelle, culture ex-type
26932 USA:Illinois:Peoria:isolatedfromhumankidneycell
cultureplate,November1997,M.A. Miller-Hjelle
26933 USA:Illinois:Peoria:isolatedfromhumankidneycell
cultureplate,November1997,M.A. Miller-Hjelle
28602 USA:Illinois:Peoria:isolatedfromhumankidneycell
cultureplate,July1998,J.T. Hjelle
Penicillium rubidurumMB319295(syn. Eupenicillium
rubidurum)
6033 Papua-NewGuinea:isolatedfromsoil,1975,culture
ex-type
Penicillium vinaceumMB281754 739 USA:Utah:isolatedfromsoil,1927,cultureex-type
740 Unknown:obtainedfromM.B. Morrow,1936
aMB=MycoBank(http://www.mycobank.org/).

Penicillium menonorum sp. nov.
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volume 2 · no. 2
DNA sequences from the β-tubulin locus included all
or part of 4 exon and 4 intron regions.After alignment the
data set included 703 base positions. The calmodulin data
includedallorpartof4exonand3intronregionsandaligned
with726basepositions.The ID regions included the ITS1,
ITS2,5.8S rDNA, andca.650 bases fromthe28S rDNAin
analignmentof1141bases.DNAreplicationlicensingprotein
(Mcm7)wascomposedofanaminoacidcodingregionof616
bp length. Penicillium erubescens was chosen as the out-
group on the basis of phylogenetic trees previously published
(Petersonet al.1999,Peterson2000).
The most parsimonious trees, bootstrap proportion and
Bayesian posterior probabilities for individual data sets
were determined and the trees were compared for strongly
supported contradictory branch points. Strongly supported
nodesarethosewith > 90 % of the bootstrap sampleanda
Bayesianposteriorprobability of > 0.90.Theindividuallocus
trees contained no strongly supported contradictions that
wouldprecludecombiningthedata.Thedatafromthefourloci
werecombinedtocalculateasinglephylogenetictree(Fig.2).
TheveisolatesofP. pimiteouiense occur on a single
strongly supported branch; three isolates of P. parvum
and the single isolate of P. papuanum occur on a different
strongly supported branch, and the two P. vinaceum isolates
occur on another strongly supported branch. Penicillium
rubidurum and P. guttulosum are most closely related to
each other and form a sibling group to P. pimiteouiense,
while P. menonorum is positioned basal in the tree to this
threespeciesbranch.
DISCUSSION
Penicillium menonorum is similar phenotypically to P.
pimiteouiense, P. restrictum, P. striatisporum, P. vinaceum,
P. rubidurum, P. erubescens, and P. parvum. Penicillium
restrictum, P. malacaense, P. kurssanovii, P. griseolum, and P.
striatisporum, which phenotypically resemble P. menonorum,
arephylogenetically positionedindifferentclades(Peterson
& Horn 2009). Other species bearing some resemblance
to P. menonorum are either not represented by extant ex-
type cultures or the type cultures are not readily available.
Penicillium menonorum differs from P. pimiteouiense by
producing conidiophores in a basal layer rather than from
aerial hyphae and a bluish gray (Court gray R-47) color
on CYA versus white in P. pimiteouiense. Additionally, P.
pimiteouiense produces yellow exudate and a brown soluble
pigment, neither of which appear in P. menonorum after 7 d
incubation.On differentmedia (e.g.,yeast extractmaltagar
incubatedat25 °C) orafterextendedincubation, a clearto
rosy exudate often appears in P. menonorum. Penicillium
restrictum produces somewhat longer conidiophores (up to
60µm)andhassmallercolonies(<10mmdiam)at37°Cthan
P. menonorum (29–32 mm diam). Penicillium striatisporum
produces rosy colored colonies on Czapek’s agar and has
striate conidia. Penicillium vinaceum produces copious
exudate in yellow to vinaceous colors, yellow to brown
solublepigments,andadarkbrowncolonyreverseonCYA,
and colonies grown at 37 °C are somewhat smaller (8–20
mmdiam) than thoseofP. menonorum.Penicillium parvum
typically has mycelium that varies from white to yellow to red
in color, while the P. menonorummyceliumisuniformlywhite.
Penicillium parvum usually makes brown or purple-brown
exudate, a brown soluble pigment, and has a colony reverse
that is deep reddish-brown versus P. menonorum, which
has no exudate or soluble pigments and a yellow brown
colony reverse after 7 d incubation. Penicillium rubidurum
produces white to orange or rosy-buff mycelium, red-brown
exudate, a dark brown colony reverse, and produces conidia
on M40Y medium but not on CYA. Penicillium menonorum
produces no exudate or soluble pigment and has a yellow
brown reverse and has abundant conidiogenesis on CYA.
Penicillium erubescens produces white, pink or esh color
mycelium, reddish-brown exudate, and gray-red to magenta
to vinaceous purple soluble pigments, with colony reverse
either similarly colored or brown. Each of these species is
Fig. 1. Penicillium menonorumNRRL50410.A.Coloniesgrown7dat25°ConCYAshowingtheradialsulcationandfaintblue-graycentralcolor
characteristicofthespecies.Bar=1cm.B.Coloniesgrown7dat25°ConMEAhavingwoolyconsistencyanddarkenedcentralareawherethe
fungusissporulating.Bar=1cm.C.Conidiophores,phialidesandconidia.Bar=10µm.D.Roughenedconidia.Bar=10µm.
AB C D

Peterson, Orchard & Menon
ARTICLE
124 ima funGuS
easily distinguished from P. menonorumonthesebases.
Raper & Thom (1949) regarded P. guttulosum to be a
synonym of P. janthinellum, differing primarily by the production
of copious amounts of exudate. Penicillium guttulosum as
represented by Gilman & Abbott’sex-type strain is distinct
from P. janthinellum as well as the species studied here.
Penicillium guttulosumculturesonCYAresemblethecultures
of P. vinaceum, differing most noticeably in the production of
dark purple exudate in large quantities, while P. vinaceum
exudateismoreredincolor.Penicillium rubidurum colonies
also resemble P. vinaceum and P. guttulosum but produce
pale yellow exudate. Pitt (1980) treated P. papuanum as a
P. pimitiouiense
NRRL2063
NRRL28602
NRRL26933
NRRL26932
NRRL25542T
100/1.00
81/1.00
98/1.00 P.rubidurumNRRL6033
P.gu+ulosumNRRL907
P.menonorumNRRL50410T
90/1.00
NRRL35488
NRRL35492
NRRL2095T
P.papuanumNRRL6032T
P.parvum
100/1.00
100/1.00
P.vinaceumNRRL739T
P.vinaceumNRRL740
P.erubescensNRRL6223T
10
64/0.94
CombineddatafromBT2,CF,IDandMcm7loci,
3186totalcharacters,2798areconstant,204are
variablenotparsimonyinformaKve,200areparsimony
InformaKve;1mpt,CI=0.8348,RC=0.7190
Fig. 2.Phylogenetictreecalculatedusingmaximumparsimonycriterionfortheconcatenateddatasetcomposedofbeta-tubulin,calmodulin,ITS
and28SrDNA,andDNAreplicationlicensingprotein(Mcm7).Bootstrapproportions/Bayesianposteriorprobabilitiesareplacedoninternodes.

Penicillium menonorum sp. nov.
ARTICLE
125
volume 2 · no. 2
synonym of P. parvum and they are in the same strongly
supportedclade(Fig.2).Phenotypically,theyareverysimilar
toeachother.Additionalisolatesofeachspeciesareneeded
to further assess the phylogenetic and phenotypic distinctions
ofthesespecies.
Phylogenetic systematics (Hennig 1966) is based on the
principlethatspeciesmustbemonophyletic.Tayloret al.(2000)
presented the genealogical concordance phylogenetic species
recognition(GCPSR)conceptasameansofdeterminingthe
boundariesofspeciesinfungi.Dettmanet al.(2006)showed
experimentallythatGCPSRiseffectiveinrecognizingspecies
boundaries in the genus Neurospora.GCPSRcanbeapplied
to P. pimiteouiense and P. parvum in this study and the species
aresupportedbytheGCPSRprinciples.Penicillium vinaceum,
P. guttulosum, P. rubidurum, and P. menonorum are each on
distinct branches, but the boundaries of the species cannot be
determinedfromthesingleisolatesavailablehere.Phenotypic
distinctions make each of these species recognizable and the
phylogenetic placement of the species is consistent with the
phenotypicdescriptionsofthespecies.
ACKNOWLEDGEMENTS
Amy McGovern provided highly skilled technical support that is
greatly appreciated. Patricia Eckel kindly translated the diagnosis
into Latin. Mention of trade names or commercial products in this
publicationissolelyforthepurposeofprovidingspecicinformation
and does not imply recommendation or endorsement by the U.S.
Department of Agriculture. USDA is an equal opportunity provider
andemployer.
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