16 novel Candida species from basidiocarp-feeding beetles

21 Pages·2004·0.39 MB·English

International Journal of Systematic and Evolutionary Microbiology (2004), 54, 2409–2429 DOI 10.1099/ijs.0.63246-0
Expansion of the Candida tanzawaensis
yeast clade: 16 novel Candida species
from basidiocarp-feeding beetles
Sung-Oui Suh,1 Joseph V. McHugh2 and Meredith Blackwell1
Correspondence 1Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA
Sung-Oui Suh 2Department of Entomology, University of Georgia, Athens, GA 30602, USA
[email protected]
Amajorcladeofnewyeasttaxafromthedigestivetractofbasidiocarp-feedingbeetles
isrecognizedbasedonrRNAgenesequenceanalyses.Almost30%of650gutisolates
formedastatisticallywell-supportedcladethatincludedCandidatanzawaensis.Theyeastsin
thecladewereisolatedfrom11familiesofbeetles,ofwhichTenebrionidaeandErotylidaewere
mostcommonlysampled.Repeatedisolationofcertainyeastsfromthesamebeetlespeciesat
differenttimesandplacesindicatedstronghostassociations.Sexualreproductionwasnever
observedintheyeasts.Basedoncomparisonsofsmall-andlarge-subunitrRNAgene
sequencesandmorphologicalandphysiologicaltraits,theyeastswereplacedinCandida
ambrosiaeandin16otherundescribedtaxa.Inthisreport,thenovelspeciesinthegenus
CandidaaredescribedandtheirrelationshipswithothertaxaintheSaccharomycetesare
discussed.Thenovelspeciesandtheirtypestrainsareasfollows:Candidaguaymorum(NRRL
Y-27568T=CBS9823T),Candidabokatorum(NRRLY-27571T=CBS9824T),Candida
kunorum(NRRLY-27580T=CBS9825T),Candidaterraborum(NRRLY-27573T=CBS9826T),
Candidaemberorum(NRRLY-27606T=CBS9827T),Candidawounanorum(NRRL
Y-27574T=CBS9828T),Candidayuchorum(NRRLY-27569T=CBS9829T),Candida
chickasaworum(NRRLY-27566T=CBS9830T),Candidachoctaworum(NRRLY-27584T
=CBS9831T),Candidabolitotheri(NRRLY-27587T=CBS9832T),Candidaatakaporum
(NRRLY-27570T=CBS9833T),Candidapanamericana(NRRLY-27567T=CBS9834T),
Candidabribrorum(NRRLY-27572T=CBS9835T),Candidamaxii(NRRLY-27588T
=CBS9836T),Candidaanneliseae(NRRLY-27563T=CBS9837T)andCandidataliae
(NRRLY-27589T=CBS9838T).
INTRODUCTION additional fungi and insect hosts might be discovered.
During a study of fungi from the digestive tract of
Yeasts and yeast-like endosymbionts have been reported
basidiocarp-feeding beetles, about 650 yeasts were isolated
from a variety of insects, including planthoppers, aphids
from beetles in 26 families (Suh & Blackwell, 2004). Based
andbeetles(e.g.Nardon&Grenier,1989;Noda&Omura,
on sequence comparisons of the D1/D2 loop of the large
1992;Suhetal.,2003,2004).Althoughsomeendosymbionts
subunit (LSU) rRNA gene used for rapid yeast character-
from anobiid beetles and planthoppers show an affinity
ization,about30%oftheyeastisolatesformedaclade(CT
to certain filamentous ascomycetes (Jones & Blackwell,
clade) with Candida tanzawaensis, a yeast isolated from
1996; Nodaetal.,1995;Noda&Kodama,1996;Suhetal.,
mosses in Japan (Nakase et al., 1988). Until the relatively
2001),themajorityofthefungalendosymbiontshavebeen
recent report of six novel species (Candida ambrosiae,
identified as true yeasts (Ascomycetes: Saccharomycetes)
Candida canberraensis, Candida caryicola, Candida pruni-
(Jonesetal.,1999).Wewereinterestedinsamplinginsects
cola,CandidapyralidaeandCandidaxylopsoci),C.tanzawa-
morebroadlyinordertodetermineifassociationsbetween
ensis had no known close relatives (Kurtzman, 2001).
Currently, only one of the seven previously described
Published online ahead of print on 28 June 2004 as DOI 10.1099/ species is known from multiple collections; in fact, C.
ijs.0.63246-0. tanzawaensis awaited description for 22 years in the vain
Abbreviations: CT clade, Candida tanzawaensis clade; LSU, large hope that another isolate would be discovered (Nakase
subunit; SSU, small subunit. et al., 1988). Here, we describe 16 novel taxa, many with
multiple isolates, from the gut of fungus-feeding beetles
The GenBank/EMBL/DDBJ accession numbers for the sequences
determined in this study are listed in Table1 and Fig.1. and compare the characteristics of all 23 members of the
63246 G 2004 IUMS Printed in Great Britain 2409
S.-O. Suh, J. V. McHugh and M. Blackwell
largeCTclade,therebyrecognizingunsuspecteddiversityin optimizedvisually.Ambiguousregionswereexcludedfromtheana-
what appears to be alargely insect-associatedclade. lyses.SequencesfromnewlyisolatedyeastswerecomparedwithLSU
and SSU rRNA gene sequences of other yeasts and fungi obtained
from GenBank. Yeast groups based on the D1/D2 sequence were
METHODS designated by the first four letters of the host beetle family and
a number for each unique genotype varying by one or more base
Yeast isolation and identification. Host beetles were collected pairs from other yeasts from the same beetle family. Maximum-
from several different localities in Vermont, the southeastern USA, parsimony analyses were performed using PAUP* 4.0b10 (Swofford,
andBarroColoradoIsland,Panama.Vouchersweredepositedatthe 2002).Heuristictreesearcheswereexecutedusingthetreebisection–
University of Georgia Collection of Arthropods, Athens, GA. The reconnection branch-swapping algorithm with random sequence
beetles were taken to the laboratory and usually placed in Petri analysis. Bootstrap values of the most parsimonious tree were
dishes for 1–3days without food prior to dissection. Surface disin- obtained from 1000 replications. Base pair differences were counted
fectionwasbysubmersionin95%ethanolfor1–2min.Thealcohol using BLAST2 Sequences (Tatusova & Madden, 1999) or from the
wash was followed by a 0?7% saline rinse; the rinse liquid was manuallyalignedsequencedatabase.
plated on acidified YM agar (Difco YM broth, 2% plain agar,
adjusted to pH3?5 with HCl) as a negative control. The beetle gut
was removed aseptically under a dissecting microscope and trans- RESULTS AND DISCUSSION
ferred to tubes containing 0?7% saline. The gut segments were
crushedinthesalinesolutionwithapipettetipandstreakedwitha Yeast isolates in the CT clade
loop onto the surface of an acidified YM agar plate. Plates were
incubated at 25uC, and single colonies were streaked for purifica- We cultured about 650 yeasts from beetles in 26 families.
tion.CulturesweremaintainedonYMagaror2%maltextractagar Most of the isolates were true yeasts (Ascomycota:
(Yarrow, 1998). The isolates were initially screened and grouped Saccharomycetes). Phylogenetic analysis of about 600 bp
using the D1/D2 loop sequence of the LSU rRNA gene. Selected
ofsequencefromtheD1/D2regionoftheLSUrRNAgene
isolates from each LSU genotype have been deposited at the Agri-
was used for rapid identification and grouping of the
cultural Research Service Culture Collection (NRRL) and Centraal-
bureau voor Schimmelcultures (CBS); holotype specimens were isolates. We discovered a major clade consisting of about
depositedatNRRLaslyophilizedcultures(Table1).Themorpholo- 30% of all beetle gut yeast isolates obtained using this
gicalobservationsandmetabolictestscomprisingtheyeaststandard technique. The taxa were closely related to C. tanzawa-
description were performed on at least one isolate from each LSU ensis and six other previously described Candida species
genotype, according to established methods (Yarrow, 1998; Barnett
(Kurtzman, 2001). Detailed information about the 164
etal.,2000).Isolateswithineachcladewerecrossedinallcombina-
yeast isolates in the greatly enlarged CT clade is provided
tionsandobservedonYMagar,2%maltagarandcornmealagarat
in Table 1. The gut yeasts in the CT clade were isolated
17uCfor6weeksinordertodetectascosporedevelopment.Bright-
fieldmicroscopicexaminationwastheusualmethodofobservation, from species in 11 families of beetles: Anthribidae,
butsomecellpreparationswereexaminedmorecloselywithfluores- Carabidae, Ciidae, Endomychidae, Erotylidae, Histeridae,
cence microscopy using 0?02% Calcofluor white as an optical Melandryidae, Nitidulidae, Scarabaeidae, Staphylinidae
brightenertovisualizeyeastcellwallsmoreclearly. (including Scaphidiinae) and Tenebrionidae. However,
the majority of the yeasts (140 of 164 isolates, Table 1)
DNA sequencing and sequence analysis. The D1/D2 region of
were from species of Erotylidae and Tenebrionidae. The
the LSU rRNA gene was amplified directly without DNA extraction
fromyeastcells.Acellsuspensionofoneloopfulofcellsin50mlof CT clade gut yeasts were sorted into 39 groups based on
autoclaved water was heated at 95uC for 5min, and 2ml of the their D1/D2 genotype and host beetle ranges. Only three
supernatant was used as a template in 50ml PCRs. For the small isolates (Erot8, Niti14 and Tene7) had a D1/D2 genotype
subunit (SSU) rRNA gene, the nucleic acids were extracted and identical to a previously described species (C. ambrosiae).
purified following the procedures of Lee & Taylor (1990). The
Theother36groupsweredistinctfromallotherpreviously
primersetsNS1/NS8 andLS1/LR5wereusedforPCRamplification
described species(Table 1).
of the SSU and LSU rRNA gene, respectively (White et al., 1990;
Hausner et al., 1993). PCR products were purified using a DNA
purification kit(Bio-RadLaboratories),andpurifieddouble-stranded Novel species in the CT clade
PCR products were used as templates for sequencing with an ABI
PRISM BigDye Terminator cycle sequencing kit. The complete In addition to the D1/D2 region of the LSU rRNA gene
sequence of the SSU rRNA gene and the D1/D2 region of the LSU sequences, complete sequences of the SSU rRNA gene
rRNA gene were obtained with the primers NS1, NS2, 18H, NS5, (about 1750 bp) were determined for at least one isolate
NS8, LS1 and LR3 using an ABI PRISM 377 automated DNA
fromeachLSUgenotype(seeTable 1forselectedisolates),
sequencer. GenBank/EMBL/DDBJ accession numbers for DNA
and these were combined with the LSU rRNA gene
sequences from this study are listed in Table1 and Fig.1. SSU
sequences in a dataset for better estimation of a phylo-
rRNA gene sequences of previously described Candida species in
the CT clade were determined to compare with the novel gut yeast geny. A most-parsimonious tree was constructed from the
isolates. Previously described species names and GenBank/EMBL/ combined dataset of SSU and LSU rRNA gene sequences
DDBJ accession numbers are as follows: C. ambrosiae NRRL YB- by comparing the yeasts with taxa in Ascomycota and
1316T (AY227712), C. tanzawaensis NRRL Y-17324T (AY227713), other yeasts alone(Fig. 1). The39gutyeastswecompared
C. canberraensis NRRL YB-2417T (AY488124), C. caryicola NRRL
were representatives of the total 164 isolates in the clade
YB-1499T (AY488125), C. prunicola NRRL YB-869T (AY488126),
C. pyralidae NRRL Y-27085T (AY488127) and C. xylopsoci NRRL (Fig. 1) with C. tanzawaensis and the other six previously
Y-27066T (AY488128). DNA sequences were aligned with the described clade members. The clade was well supported
multi-alignment program CLUSTAL_X (Thompson et al., 1997) and statisticallyby100%bootstrapvalue(Fig. 1).Oftheseven
2410 International Journal of Systematic and Evolutionary Microbiology 54
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/ Table 1. Strains of the novel Candida species and C. ambrosiae isolated in this study, and GenBank/EMBL/DDBJ accession numbers of the sequences
ijs
.s
g
m Species Strain designation* LSU rRNA Host beetles and place of collection* Total Nucleotide differencesd
jo
u gene groups number of
rn CBS NRRL LSU LSU SSU
a isolatesD
ls rRNA gene rRNA gene
.o
rg
C. guaymorum 9823T Y-27568T BG 01-7-26-006B-1-1T Erot21 Mycotretus interstitialis (Erotylidae) ex imbricate 1 T T
basidiocarp, BCI, Panama
BG 01-7-26-006A-2-1 Erot21 Iphiclus (Habrodactylus) conspicillatus (Erotylidae) 3 0 0
ex imbricate basidiocarp, BCI, Panama
BG 02-7-16-022A-1-1 Erot21 Cyclomorphus sp. (Erotylidae) ex polypore, BCI, 3 0 –
Panama
BG 02-7-20-020A-1-1 Erot21 Iphiclus sp. (Erotylidae) ex corticioid fungus, BCI, 1 0 –
Panama
BG 02-7-20-020B-1-1 Erot21 Cyclomorphus sp. (Erotylidae) ex corticioid fungus, 1 0 –
BCI, Panama
Y-27581 BG 01-7-21-003A-1-1 Scar2 Onthophagus sp. (Scarabaeidae) ex Polyporus 1 0 0
tenuiculus, BCI, Panama
C. bokatorum 9824T Y-27571T BG 02-7-16-039A-2-1T Erot35 Pselaphacus signatus (Erotylidae) ex P. tenuiculus, 10 T T
BCI, Panama
BG 02-7-14-001E-4-1 Erot35 Mycotretus nitescens (Erotylidae) ex P. tenuiculus, 2 0 –
BCI, Panama
BG 02-7-16-030B-3-2 Erot35 Iphiclus (Megaprotus) delineatus (Erotylidae) ex 3 0 –
corticioid fungus, BCI, Panama
BG 02-7-18-023B-1-2 Erot35 Larva of Ellipticus gemellatus (Erotylidae) ex 1 0 –
Tinctoporellus epimiltinus, BCI, Panama
BG 02-7-14-001D-1-1 Erot35 Pselaphacus sp. (Erotylidae) ex P. tenuiculus, BCI, 1 0 –
Panama
E
Y-27558 BG 02-7-14-001G-1-1 Cara2 Unidentified carabid (Carabidae) ex P. tenuiculus, 1 0 0 x
p
a
BCI, Panama n
s
Y-27579 BG 02-7-14-001F-1-1 Niti19 Teichostethus testaceous (Nitidulidae) ex P. tenuiculus, 2 0 0 io
n
BCI, Panama o
f
Y-27576 BG 02-7-14-001J-1-1 Mela1 Unidentified melandryid (Melandryidae) ex P. 1 0 0 th
e
tenuiculus, BCI, Panama C
a
BG 02-7-14-001K-1-2 Tene27 Unidentified tenebrionid (Tenebrionidae) ex 2 0 0 n
d
P. tenuiculus, BCI, Panama id
a
C. kunorum 9825T Y-27580T BG 02-7-18-017A-1-1T Niti25 T. testaceous (Nitidulidae) ex Nodulisporium sp., BCI, 1 T T ta
n
Panama z
a
C. terraborum 9826T Y-27573T BG 02-7-15-019A-2-1T Erot41 Iphiclus sedecimmaculatus (Erotylidae) ex corticioid 1 T T wa
e
fungus, BCI, Panama n
s
is
2 c
4 la
1 d
1 e
2 S
41 .-O
2 Table 1. cont. .
S
u
h
,
Species Strain designation* LSU rRNA Host beetles and place of collection* Total Nucleotide differencesd J
.
gene groups number of V.
CBS NRRL LSU LSU SSU M
isolatesD c
rRNA gene rRNA gene H
u
g
C. emberorum 9827T Y-27606T BG 01-7-22-010E-1-1T Erot24 Triplax alvarengai (Erotylidae) ex Pleurotis sp., BCI, 1 T T h
a
Panama nd
BG 01-7-23-002A-1-1 Erot24 Mycotretus scitulus (Erotylidae) ex Pleurotis sp., BCI, 1 0 – M
.
Panama B
la
BG 01-7-22-012A-1-1-1 Endo2 Unidentified endomychid (Endomychidae) ex 2 3 0 c
k
w
Ripartitella brasiliensis, BCI, Panama e
C. wounanorum 9828T Y-27574T BG 02-7-18-027A-1-1T Erot45 Mycotretus dorsonotatus (Erotylidae) ex corticioid 1 T T ll
fungus, BCI, Panama
C. yuchorum 9829T Y-27569T BG 01-8-26-001A-1-1T Erot26 Tritoma atriventris (Erotylidae) ex Lepiota sp., Athens, 3 T T
GA, USA
C. chickasaworum 9830T Y-27566T BG 99-11-14-10-1-1T Erot9 Tritoma sp. (Erotylidae) ex Amanita sp., Athens, GA, 7 T T
USA
BG 02-2-5-1-1 Erot9 Tritoma sp. (Erotylidae) ex Pleurotus ostreatus, Baton 7 0 –
Rouge, LA, USA
BG 02-6-15-003A-1 Erot9 Dacne sp. (Erotylidae), Athens, GA, USA 3 0 –
In BG 02-6-15-003B-1 Erot9 Tritoma californica (Erotylidae), Athens, GA, USA 1 0 –
tern Y-27561 BG 02-6-15-010C-1 Ciid7 Unidentified ciid (Ciidae), Athens, GA, USA 2 0 0
atio C. choctaworum 9831T Y-27584T BG 98-8-18-2T Tene1 Neomida bicornis (Tenebrionidae) ex Fomitella supina, 12 T T
na Baton Rouge, LA, USA
l
Jo BG 98-12-9-1-1 Tene1 N. bicornis (Tenebrionidae) ex F. supina, St Francisville, 8 0 0
u
rn LA, USA
a
l BG 02-5-30-001B-1 Tene1 Unidentified tenebrionid (Tenebrionidae), Athens, GA, 7 0 –
o
f USA
S
ys Y-27559 BG 99-2-5-7-1-1 Ciid2 Ceracis curtus (Ciidae) ex F. supina, Baton Rouge, LA, 3 0 0
te
m USA
a
tic BG 02-5-30-008B-1 Ciid2 Unidentified ciid (Ciidae), Athens, GA, USA 2 0 –
a Y-27557 BG 02-3-29-3-1-1 Anth2 Euparius marmoreus (Anthribidae), Sulphur, LA, USA 2 0 0
n
d C. bolitotheri 9832T Y-27587T BG 00-8-15-1-1T Tene11 Bolitotherus cornutus (Tenebrionidae) ex Ganoderma sp., 1 T T
E
vo Athens, GA, USA
lu
tio BG 00-7-30-1-1 Tene11 B. cornutus (Tenebrionidae) ex Ganoderma sp., 3 0 –
n
a Burlington, VT, USA
ry
BG 02-3-29-2-2 Tene11 B. cornutus (Tenebrionidae) ex Ganoderma sp., 3 0 –
M
ic Sulphur, LA, USA
ro
b BG 02-5-27-1-2-3 Tene11 B. cornutus (Tenebrionidae) ex Ganoderma sp., 3 0 –
io
lo Baton Rouge, LA, USA
g
y
5
4
h
ttp
:// Table 1. cont.
ijs
.s
gm Species Strain designation* LSU rRNA Host beetles and place of collection* Total Nucleotide differencesd
jo gene groups number of
u
rn CBS NRRL LSU isolatesD LSU SSU
a
ls rRNA gene rRNA gene
.o
rg Y-27562 BG 99-8-11-1-1 Erot3 Megalodacne fasciata (Erotylidae) ex Ganoderma 4 0 0
applanatum, Athens, GA, USA
C. atakaporum 9833T Y-27570T BG 02-7-21-Nhu-1-1-2T Erot28 Triplax festiva (Erotylidae) ex Inonotus cuticularis, Baton 1 T T
Rouge, LA, USA
C. panamericana 9834T Y-27567T BG 01-7-26-006B-2-1T Erot20 M. interstitialis (Erotylidae) ex imbricate mushroom, BCI, 1 T T
Panama
Y-27582 BG 01-7-26-006C-1-1 Stap1 Unidentified staphylinid (Staphylinidae) ex imbricate 1 0 0
basidiocarp, BCI, Panama
Y-27590 BG 02-5-27-1-2-4 Tene19 B. cornutus (Tenebrionidae) ex Ganoderma sp., Baton 1 1 0
Rouge, LA, USA
C. bribrorum 9835T Y-27572T BG 02-7-14-001D-3-1T Erot38 Larvae of Pselaphacus sp. (Erotylidae) ex P. tenuiculus, 1 T T
BCI, Panama
BG 02-7-14-002A-2-1 Erot38 Megalodacne audouini (Erotylidae) ex basidiocarp, BCI, 3 0 –
Panama
BG 02-7-14-002B-2-1 Erot38 Pupa of Megalodacne sp. (Erotylidae) ex basidiocarp, 1 0 –
BCI, Panama
BG 02-7-20-004A-1-1 Erot38 Megalodacne sp. (Erotylidae) ex Ganoderma sp., BCI, 1 0 –
Panama
Y-27591 BG 02-7-14-002E-2-1 Tene28 Unidentified tenebrionid (Tenebrionidae) ex basidiocarp, 2 0 0
BCI, Panama
Y-27592 BG 02-7-14-002I-1-1 Tene30 Unidentified tenebrionid (Tenebrionidae) ex basidiocarp, 2 1 0
BCI, Panama
Y-27564 BG 99-8-11-1-4-2 Erot6 M. fasciata (Erotylidae) ex G. applanatum, Athens, GA, 1 2 1 Ex
p
USA a
n
C. maxii 9836T Y-27588T BG 01-7-21-006A-1-1T Tene17 Unidentified tenebrionid (Tenebrionidae) ex polypore, 2 T T sio
n
BCI, Panama o
C. anneliseae 9837T Y-27563T BG 99-8-11-1-2-2T Erot4 M. fasciata (Erotylidae) ex G. applanatum, Athens, GA, 2 T T fth
e
USA
C
BG 02-7-21-Nhu-1-1-C Erot4 T. festiva (Erotylidae) ex I. cuticularis, Baton Rouge, LA, 1 0 – a
n
d
USA id
a
Y-27583 BG 02-5-23-003E-5 Stap5 Unidentified Scaphidiinae (Staphylinidae), Athens, GA, 1 0 0
ta
USA nz
a
Y-27585 BG 98-9-2-2-4 Tene4 Platydema ruficorne (Tenebrionidae) ex decayed polypore, 11 0 0 w
a
Baton Rouge, LA, USA en
s
is
2 c
4 la
1 d
3 e
2 S
41 .-O
4 .
S
u
h
Table 1. cont. ,J
.
V
.
Species Strain designation* LSU rRNA Host beetles and place of collection* Total Nucleotide differencesd M
c
gene groups number of H
CBS NRRL LSU isolatesD LSU SSU ugh
rRNA gene rRNA gene
a
n
d
BG 99-8-18-1-1 Tene4 Diaperis nigronotata (Tenebrionidae) ex Inonotus 4 0 0 M
ludovicianus, Baton Rouge, LA, USA .B
BG 00-6-14-1-1 Tene4 Neomida ferruginea (Tenebrionidae) ex basidiocarp, Baton 6 0 – lac
k
Rouge, LA, USA w
e
BG 01-7-21-010A-1-1 Tene4 Unidentified small diaperine (Tenebrionidae) ex polypore, 3 0 – ll
BCI, Panama
BG 01-7-21-010B-1-1 Tene4 Unidentified tenebrionid (Tenebrionidae) ex polypore, BCI, 2 0 –
Panama
BG 02-6-15-011B-2 Tene4 Alobates sp. (Tenebrionidae), Athens, GA, USA 1 0 –
BG 02-5-30-001B-6 Tene4 Unidentified tenebrionid (Tenebrionidae), Athens, GA, USA 1 0 –
Y-27577 BG 02-7-18-032A-1-1 Mela2 Unidentified melandryid (Melandryidae) ex polypore, BCI, 1 0 0
Panama
BG 02-7-18-032C-1-1 Mela2 Larva of unidentified melandryid (Melandryidae) ex polypore, 1 0 –
In BCI, Panama
te Y-27575 BG 02-7-18-032B-2-1 Hist5 Unidentified histerid (Histeridae) ex polypore, BCI, Panama 1 0 1
rn
a Y-27560 BG 02-5-23-003C-4 Ciid5 Unidentified ciid (Ciidae), Athens, GA, USA 1 0 0
tion C. taliae 9838T Y-27589T BG 01-7-23-018C-1-1T Tene18 Unidentified tenebrionid (Tenebrionidae) ex polypore, BCI, 1 T T
a
lJ Panama
o
u C. ambrosiae Y-27565 BG 99-8-11-1-C2 Erot8 M. fasciata (Erotylidae) ex G. applanatum, Athens, GA, USA 1 0 0
rn
al Y-27586 BG 99-8-18-1-4-1 Tene7 D. nigronotata (Tenebrionidae) ex I. ludovicianus, Baton 2 0 0
o
f Rouge, LA, USA
S
y Y-27578 BG 01-7-26-005A-1-1 Niti14 T. testaceous (Nitidulidae) ex polypore, BCI, Panama 1 0 0
s
te
m
atic *CBS,CentraalbureauvoorSchimmelcultures,Utrecht,theNetherlands;NRRL,AgriculturalResearchServiceCultureCollection,NationalCenterforAgriculturalUtilizationResearch,Peoria,
a IL, USA; LSU, Mycology Laboratory, Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, USA; BCI, Barro Colorado Island.
n
d DEach isolate came from one beetle. Total numbers include the yeasts isolated on different collection dates.
E
v dTheD1/D2sequencesfortheLSUrRNAgenesweredeterminedforallyeastisolatesanddepositedinGenBank/EMBL/DDBJ.AccessionnumbersofLSUrRNAgenesequencesareAY242241,
o
lutio AY242244,AY242246,AY242249,AY242253,AY242254,AY242257,AY242258,AY242260,AY242262,AY242263,AY242273,AY242274,AY242277,AY242278,AY242284,AY242288,AY242298,
n AY242312,AY242345,AY242350–AY242352,AY309784–AY309919andAY426946–AY426949.ThesequenceoftheSSUrRNAgenewasdeterminedforatleastoneisolatefromeachLSUgroup.
a
ry SeeFig.1forGenBank/EMBL/DDBJ accessionnumbers forSSUrRNAgenesequences. Thebasepairdifferencesarefromsequencecomparisonsbetweentypestrain(T)andotherisolatesin
M
ic eachspeciesintheD1/D2regionoftheLSUrRNAgene(about600bp)andtheSSUrRNAgene(about1750bp).C.ambrosiaeisolatesfromthisstudywerecomparedwithitstypestrain,NRRL
rob YB-1316T.
io
lo
g
y
5
4
Expansion of the Candida tanzawaensis clade
Fig. 1. Consensus of two most-parsimonious trees obtained from combined SSU and LSU rRNA gene sequence data.
Candida rhagii was chosen as the outgroup taxon based on analyses including a broader range of taxa (not shown).
GenBank/EMBL/DDBJ accession numbers after the names of yeast species are for SSU and LSU rRNA gene sequence,
respectively. Tree length=514; consistency index=0?6381; homoplasy index=0?3619; retention index=0?8444; rescaled
consistencyindex=0?5388.Numbersontreebranchesindicatethepercentages ofbootstrapsamplingsderivedfrom1000
samples that supported the internal branches by 50% or higher.
previously described taxa, C. xylopsoci, C. pyralidae, C. As mentioned earlier, only one subclade (including Erot8,
prunicola and C. caryicola were basal to the other yeasts in Niti14 and Tene7) had D1/D2 sequences identical to the
the CT clade. C. ambrosiae, C. tanzawaensis and C. previouslydescribedyeastC.ambrosiae,whichweconsider
canberraensis, however, were included among the beetle to be conspecific with these isolates. There were marked
gut yeasts (Fig. 1). Within the CT clade, the beetle iso- differences between C. ambrosiae and the other 16 sub-
lates were resolved into 17 subclades with sufficient diver- cladesintheCTcladewith6 bpormore(usuallymorethan
gence to warrant species-level recognition (Kurtzman & 20 bp)differencebetweenthesubclades.Thegeneticvaria-
Robnett, 1998; Kurtzman, 2000; Suh & Blackwell, 2004) tion among multiple isolates within subclades was lower
(Fig. 1). (Table 1;Fig. 1).Forexample,yeastsdesignatedEndo2and
http://ijs.sgmjournals.org 2415
S.-O. Suh, J. V. McHugh and M. Blackwell
Erot24 occurred in a common subclade and differed from Erotylidae), Barro Colorado Island, Panama, depositata in
each other by 3 bp of D1/D2 sequence. Also, there were Collectione Culturarum(NRRL), Peoria, IL, USA.
minor D1/D2 sequence differences between common sub-
clade members Tene28 and Tene30 or Erot20 and Tene19
Description of Candida guaymorum Suh &
(Table 1).Withinall17subcladesoftheCTclade,however,
Blackwell sp. nov.
thesequencevariationofD1/D2amongsubclademembers
was always lessthan 3 bp, within therange ofspecies-level Candida guaymorum (gu.ay.mo9rum. N.L. m. gen. guay-
variation recognized in studies of other ascomycete yeast morum to commemorate the Guaymı´, a group of indi-
species(Kurtzman & Robnett, 1998; Table 1). genous people of Panama with detailed knowledge of the
forest flora).
We observed minor morphological differences among the
isolates onYM andcornmealagars at5–7 days incubation After7 daysgrowthinYMbrothat25uC,cellsareglobose
and in YM broth after 5 days incubation at 25uC, but toellipsoidal(1?25–6?2561?25–7?5 mm),andoccursingly,
morphologicalvariationthatconsistentlydistinguishedthe in pairs or in short chains (Fig. 2a). Pseudohyphae are
yeasts within clades was not evident. One morphological present.After7 daysonYMagarat25uC,coloniesarewhite
feature common to all isolates was the lack of ascospore to cream-coloured with pale-pinkish perimeter on some
production. Physiological characters (Table 2), however, oldcolonies,smooth,shiny,flatandfilamentousinmargin.
were more variable and these traits were useful to separate After 10 days Dalmau plate culture on corn meal agar
the other species of the CT clade. Below, we characterize at 25uC, pseudohyphae are present; septate hyphae are
and describe 16 novel species of beetle gut yeasts and absent. Aerobic growth is white, shiny and smooth with
comparethemtothepreviouslydescribedyeastsoftheCT filamentousmargin.Noascosporesproducedafter6 weeks
clade (Fig. 1and Table 2). at 17uC from individual strains on YM agar or strains
crossed in all combinations on half-strength cornmeal
Latin diagnosis of Candida guaymorum Suh et agar.SeeTable 2forasummaryofphysiologicalandother
Blackwell sp. nov. characteristics.
In medio liquido dextrosum et peptonum et extractum The type strain is NRRL Y-27568T(=CBS9823T).
levidinis continente post 7 dies ad 25uC cellulae vegetativae
globosae aut ellipsoideae (1?25–6?2561?25–7?5 mm), singu-
Latin diagnosis of Candida bokatorum Suh et
laevelbinae;pseudohyphaefiunt.Culturainagaroextramalti
Blackwell sp. nov.
et faecis continente post 7 dies ad 25uC, albida, hebes, teres
et margine ciliata. In agaro farina Zeae maydis confecto In medio liquido dextrosum et peptonum et extractum
post 10 dies ad 25uC, pseudohyphae fiunt; hyphae verae non levidinis continente post 7 dies ad 25uC cellulae vegetativae
fiunt. Ascosporae non fiunt. Glucosum, galactosum (lente), globosaeautellipsoideae (2–663–6 mm),singulaevelbinae.
maltosum (infirme, variabilitre), methyl a-D-glucosidum Cultura in agaro extramalti et faecis continente post 7 dies
(infirme, variabilitre), sucrosum (infirme, variabilitre), ad 25uC, albida, hebes, butyrosa, margine ciliata. In agaro
trehalosum et cellobiosum (lente) fermentantur. Melibio- farina Zeae maydis confecto post 10 dies ad 25uC, pseudo-
sum, lactosum, melezitosum, raffinosum, inulinum, amylum hyphae fiunt; hyphae verae fiunt. Ascosporae non fiunt.
solubile et D-xylosum non fermentantur. Assimilantur Glucosum, maltosum (infirme, variabilitre), sucrosum
glucosum, galactosum, D-glucosaminum, D-xylosum, sucro- (infirme, variabilitre), trehalosum et cellobiosum ferment-
sum, maltosum, trehalosum, methyl a-D-glucosidum, cello- antur. Galactosum, methyl a-D-glucosidum, melibiosum,
biosum, salicinum, arbutinum, melezitosum, glycerolum, lactosum, melezitosum, raffinosum, inulinum, amylum
ribitolum, D-glucitolum, D-mannitolum, gluconolactonum, solubile et D-xylosum non fermentantur. Assimilantur
2-keto-D-gluconatum,D-gluconatum(lente,infirme),acidum glucosum, galactosum, D-glucosaminum (variabilitre),
succinicum,acidumcitricumetethanolum.Nonassimilantur D-xylosum,D-arabinosum,sucrosum,maltosum,trehalosum,
L-sorbosum, D-ribosum, L-arabinosum, D-arabinosum, L- methyl a-D-glucosidum, cellobiosum, salicinum, arbutinum,
rhamnosum, melibiosum, lactosum, raffinosum, inulinum, melezitosum, glycerolum, ribitolum, D-glucitolum, D-
amylum solubile, erythritolum, xylitolum, L-arabinitolum, mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-
galactitolum, inositolum, D-glucuronatum, DL-acidum lacti- gluconatum (lente, infirme), acidum succinicum, acidum
cum, methanolum, propane-1,2-diolum, butano-2,3-diolum, citricum, ethanolum et propane-1,2-diolum (infirme,
acidumquinicum,D-glucaratumetD-galactonatum.Assimi- variabilitre). Non assimilantur L-sorbosum, D-ribosum,
lantur ethylaminum, L-lysinum, cadaverinum et glucos- L-arabinosum,L-rhamnosum,melibiosum,lactosum,raffino-
aminum. Non assimilantur kali nitratum, sodii nitritum, sum, inulinum, amylum solubile, erythritolum, xylitolum,
creatinum, creatininum, imidazolum et D-tryptophanum. L-arabinitolum, galactitolum, inositolum, D-glucuronatum,
Amylum non formatur. Biotinum externum ad crescentiam DL-acidum lacticum, methanolum, butano-2,3-diolum,
necessariumest.Augmentumnonfiuntintemperatura40uC. acidum quinicum, D-glucaratum et D-galactonatum.
Non crescit in medio 10 mg ml21 cycloheximido addito. Assimilantur ethylaminum, L-lysinum, cadaverinum
Typus: NRRL Y-27568T (=CBS 9823T), designat stirpem et glucosaminum (infirme, variabilitre). Non assimilantur
typicum.Isolataailecoleopterorum(Mycotretusinterstitialis; kali nitratum, sodii nitritum, creatinum, creatininum,
2416 International Journal of Systematic and Evolutionary Microbiology 54
Expansion of the Candida tanzawaensis clade
Table 2. Physiological characteristics of novel Candida species and C. ambrosia from this study
Taxa: 1, C. guaymorum; 2, C. bokatorum; 3, C. kunorum; 4, C. terraborum; 5, C. emberorum; 6, C. wounanorum; 7, C. yuchorum; 8, C. chick-
asaworum; 9, C. choctaworum; 10, C. bolitotheri; 11, C. atakaporum; 12, C. panamericana; 13. C. bribrorum; 14, C. maxii; 15, C. anneliseae;
16, C. taliae; 17, C. ambrosiae from this study. The following characteristics are invariable in all species compared. Fermentation of
melibiose (2), raffinose (2), starch (2), D-xylose (2), D-glucose (+); assimilation of D-rhamnose (2), trehalose (+), cellobiose (+),
melibiose (2), lactose (2), raffinose (2), inulin (2), D-glucitol (+), D-mannitol (+), galactitol (2), myo-inositol (2), D-glucuronate (2),
methanol (2), butane-2,3-diol (2), quinic acid (2), D-glucarate (2), D-galactonate (2), nitrate (2), nitrite (2), ethylamine (+), D-lysine
(+), cadaverine (+), creatine (2), creatinine (2), imidazole (2); vitamin requirement, growth without pantothenate (+), niacin (+),
p-aminobenzoic acid (+); growth at 25uC (+), at 30uC (+), at 40uC (2); growth on 1% acetic acid (2); additional tests, starch forma-
tion (2), urea hydrolysis (2), Diazonium Blue B reaction (2). Abbreviations: +, positive reaction; 2, negative reaction; d, delayed positive
reaction; W, weak positive reaction; V, variable reaction; w/o, without.
Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Fermentation of carbon compounds
D-Glucose + +/d + d + d + + +/d + + + + d +/d/W + +
D-Galactose d 2 W W +/d W d +/d V +/d 2 d V 2 2 d +/d
Maltose W/2 W/2 W d 2 2 2 2 2 2 2 2 W/2 2 2 2 d/W
Methyl a-D-glucoside W/2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 V
Sucrose W/2 W/2 2 d 2 2 2 2 2 2 2 2 2 2 2 2 d/W
a,a-Trehalose + +/d + d + d + +/d +/d/W d d + + W d/W d +
Lactose 2 2 W 2 2 2 2 2 2 2 2 2 2 2 2 2 2
Cellobiose d +/d 2 2 d/W 2 d 2 2 2 2 d/W 2 W 2 d V
Melezitose 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 W/2
Inulin 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 W/2
Assimilation of carbon compounds
D-Galactose + + + + + + + + + + + + + + V + +
D-Sorbose 2 2 2 2 2 2 2 2 +/d V 2 2 V 2 V 2 W/2
D-Glucosamine +/W V d W d/W d 2 d/W +/d + d +/d +/W d +/d/W + +/W
D-Ribose 2 2 W 2 2 2 2 2 V V 2 2 V + V 2 +/d
D-Xylose + + d W + + + +/d +/d +/d/W + + + 2 +/d/W d +
L-Arabinose 2 2 2 2 2 2 2 2 W/2 2 2 2 V 2 2 2 +/d/W
D-Arabinose 2 +/d/W W 2 2 2 2 2 +/d/W 2 W 2 V W +/d/W + 2
Sucrose + + + + + + + + 2 2 + + + + + + +
Maltose + + + + + + + + 2 2 + + + + + + +
Methyl a-D-glucoside + + + + + + + + 2 2 + + + + + + +
Salicin + + d + + + + + + + + + + + + + +
Arbutin + + + + + + + + + + + +/d + + + + +
Melezitose + + + + d/W W 2 V 2 2 + + + + + + +
Soluble starch 2 2 2 2 2 2 2 2 2 2 W 2 2 2 2 2 2
Glycerol + +/d/W + + + + + + +/d + + + + d +/d/W + +
Erythritol 2 2 2 2 2 2 2 2 + + 2 2 + + + + +
Ribitol + + + + + + + +/d + + + + + + + + +
Xylitol 2 2 W 2 2 2 2 W/2 V +/W 2 2 +/d W +/d d +/d
D-Arabinitol 2 2 W 2 2 2 2 2 V 2 2 2 2 2 V 2 +
D-Glucono-1,5-lactone + +/d/W + W +/d + + +/d/W +/d +/d + + + d +/d + +
2-Keto-D-gluconate + + + + + + + + + + + + + + + + +
D-Gluconate d/W d/W + W d/W 2 d V V d/W W + + + + + +/d/W
DL-Lactate 2 2 2 2 2 2 2 2 2 2 d 2 2 2 2 2 2
Succinate + +/d + + + d + + +/W +/d/W d + +/d + +/d + +
Citrate + + d + +/d + + +/d/W +/d + d + + + +/d + +
Ethanol +/W + + d + + 2 +/d/W +/d +/d + 2 + + +/d/W + +
Propane-1,2-diol 2 W/2 W W 2 2 2 W/2 W/2 2 W 2 2 2 W/2 2 2
Assimilation of nitrogen compounds
D-Glucosamine +/W W/2 W W +/W 2 + V V +/W W V V W V W V
D-Tryptophan 2 2 2 2 W 2 d 2 2 2 d 2 2 2 2 2 2
http://ijs.sgmjournals.org 2417
S.-O. Suh, J. V. McHugh and M. Blackwell
Table 2. cont.
Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Vitamin requirements
w/o vitamins 2 2 2 2 2 2 2 2 2 2 2 V 2 2 2 2 2
w/o myo-Inositol + + + + + + + + + + + + + d + + +
w/o Biotin 2 2 2 2 2 2 2 V W/2 W/2 2 +/W W/2 2 2 2 2
w/o Thiamin + + + + + + + + + + + +/W + d V d +
w/o Biotin and Thiamin 2 2 2 2 2 2 2 V W/2 W/2 2 V W/2 2 2 2 2
w/o Pyridoxine + +/d + + + + + + + + + + + + + + +
w/o Pyridoxine and Thiamin + + + + + W + + + + + +/W +/W d + + +
Growth tests
Growth at 35uC V V 2 2 +/W 2 + 2 V V + +/d V 2 V W +
0?01% Cycloheximide 2 W/2 2 2 2 2 2 2 + W/2 2 2 W 2 V 2 V
0?1% Cycloheximide 2 2 2 2 2 2 2 2 + 2 2 2 W/2 2 2 2 2
50% D-Glucose + + + + +/W W + +/d +/d +/d W + +/d + +/d/W + +
60% D-Glucose 2 2 2 W 2 2 2 W/2 +/d/W V 2 + W/2 2 V 2 +/d/W
10% NaCl + +/W 2 W V + + +/W +/W +/d/W + + +/d/W + +/d/W + +
16% NaCl 2 2 2 2 2 2 2 2 V 2 W W/2 W/2 2 W/2 2 W/2
imidazolum et D-tryptophanum. Amylum non formatur. binae. Cultura in agaro extramalti et faecis continente post
Biotinum externum ad crescentiam necessarium est. 7 dies ad 25uC, albida, hebes, butyrosa, margine ciliata. In
Augmentum non fiunt in temperatura 40uC. Variabilitre agaro farina Zeae maydis confecto post 10 dies ad 25uC,
in medio 10 mg ml21 cycloheximido addito, non crescit in pseudohyphae non fiunt; hyphae verae non fiunt. Ascosporae
medio100 mg ml21.Typus:NRRLY-27571T(=CBS9824T), non fiunt. Glucosum, galactosum (infirme), maltosum
designat stirpem typicum. Isolata a ile coleopterorum (infirme), trehalosum et lactosum (infirme) fermentantur.
(Pselaphacus signatus; Erotylidae), Barro Colorado Island, Sucrosum, methyl a-D-glucosidum, melibiosum, cellobiosum,
Panama, depositata in Collectione Culturarum (NRRL), melezitosum, raffinosum, inulinum, amylum solubile et
Peoria, IL, USA. D-xylosum non fermentantur. Assimilantur glucosum, galac-
tosum, D-glucosaminum (lente), D-ribosum (infirme),
Description of Candida bokatorum Suh & D-xylosum (lente), D-arabinosum (infirme), sucrosum,
Blackwell sp. nov. maltosum, trehalosum, methyl a-D-glucosidum, cellobiosum,
salicinum (lente), arbutinum, melezitosum, glycerolum,
Candidabokatorum(bo.ka.to9rum.N.L.m.gen.bokatorum
ribitolum, xylitolum (infirme), L-arabinitolum (infirme),
tocommemoratetheBo´kata´,agroupofindigenouspeople
D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-
of Panama, linguistically related to theGuaymı´).
gluconatum, D-gluconatum, acidum succinicum, acidum
citricum(lente),ethanolumetpropane-1,2-diolum(infirme).
After7 daysgrowthinYMbrothat25uC,cellsareglobose
Non assimilanturL-sorbosum,L-arabinosum,L-rhamnosum,
toellipsoidal(2–663–6 mm),mostlysubglobose,andoccur
melibiosum, lactosum, raffinosum, inulinum, amylum solu-
singly, in pairs or in short chains (Fig. 2b). Pseudohyphae
bile,erythritolum,galactitolum,inositolum,D-glucuronatum,
maybepresent.After7 daysonYMagarat25uC,colonies
DL-acidum lacticum, methanolum, butano-2,3-diolum,
are white to cream in colour, butyrous and smooth. After
acidum quinicum et D-glucaratum. Assimilantur ethylami-
10 days Dalmau plate culture on corn meal agar at 25uC,
num, L-lysinum, cadaverinum et glucosaminum (infirme).
pseudohyphaearepresent. Septatehyphaemay bepresent.
Non assimilantur kali nitratum, sodii nitritum, creatinum,
Aerobic growth is white and smooth. No ascospores
creatininum, imidazolum et D-tryptophanum. Amylum non
produced after 6 weeks at 17uC from individual strains
formatur. Biotinum externum ad crescentiam necessarium
onYMagarorstrainscrossedinallcombinationsonhalf-
est. Augmentum non fiunt in temperatura 35uC. Typus:
strength cornmeal agar. See Table 2 for a summary of
NRRL Y-27580T (=CBS 9825T), designat stirpem typicum.
physiological and other characteristics.
Isolata a ile coleopterorum (Teichostethus testaceous;
The type strain is NRRL Y-27571T(=CBS9824T). Nitidulidae), Barro Colorado Island, Panama, depositata
in Collectione Culturarum(NRRL),Peoria, IL, USA.
Latin diagnosis of Candida kunorum Suh et
Blackwell sp. nov. Description of Candida kunorum Suh &
Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum
levidinis continente post 7 dies ad 25uC cellulae vegetativae Candida kunorum (ku.no9rum. N.L. m. gen. kunorum to
subglobosae aut fusiformes (2–563–6 mm), singulae vel commemorate the Kuna, a group of indigenous people of
2418 International Journal of Systematic and Evolutionary Microbiology 54