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Int J Syst Evol Microbiol 54 (2004), 2409-2429; DOI  10.1099/ijs.0.63246-0
© 2004 International Union of Microbiological Societies

Expansion of the Candida tanzawaensis yeast clade: 16 novel Candida species from basidiocarp-feeding beetles

Sung-Oui Suh1, Joseph V. McHugh2 and Meredith Blackwell1

1 Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA
2 Department of Entomology, University of Georgia, Athens, GA 30602, USA

Correspondence
Sung-Oui Suh
ssuh{at}lsu.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
A major clade of new yeast taxa from the digestive tract of basidiocarp-feeding beetles is recognized based on rRNA gene sequence analyses. Almost 30 % of 650 gut isolates formed a statistically well-supported clade that included Candida tanzawaensis. The yeasts in the clade were isolated from 11 families of beetles, of which Tenebrionidae and Erotylidae were most commonly sampled. Repeated isolation of certain yeasts from the same beetle species at different times and places indicated strong host associations. Sexual reproduction was never observed in the yeasts. Based on comparisons of small- and large-subunit rRNA gene sequences and morphological and physiological traits, the yeasts were placed in Candida ambrosiae and in 16 other undescribed taxa. In this report, the novel species in the genus Candida are described and their relationships with other taxa in the Saccharomycetes are discussed. The novel species and their type strains are as follows: Candida guaymorum (NRRL Y-27568T=CBS 9823T), Candida bokatorum (NRRL Y-27571T=CBS 9824T), Candida kunorum (NRRL Y-27580T=CBS 9825T), Candida terraborum (NRRL Y-27573T=CBS 9826T), Candida emberorum (NRRL Y-27606T=CBS 9827T), Candida wounanorum (NRRL Y-27574T=CBS 9828T), Candida yuchorum (NRRL Y-27569T=CBS 9829T), Candida chickasaworum (NRRL Y-27566T=CBS 9830T), Candida choctaworum (NRRL Y-27584T=CBS 9831T), Candida bolitotheri (NRRL Y-27587T=CBS 9832T), Candida atakaporum (NRRL Y-27570T=CBS 9833T), Candida panamericana (NRRL Y-27567T=CBS 9834T), Candida bribrorum (NRRL Y-27572T=CBS 9835T), Candida maxii (NRRL Y-27588T=CBS 9836T), Candida anneliseae (NRRL Y-27563T=CBS 9837T) and Candida taliae (NRRL Y-27589T=CBS 9838T).


Abbreviations: CT clade, Candida tanzawaensis clade; LSU, large subunit; SSU, small subunit

Published online ahead of print on 28 June 2004 as DOI 10.1099/ijs.0.63246-0.

The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are listed in Table 1Go and Fig. 1Go.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Yeasts and yeast-like endosymbionts have been reported from a variety of insects, including planthoppers, aphids and beetles (e.g. Nardon & Grenier, 1989Go; Noda & Omura, 1992Go; Suh et al., 2003Go, 2004Go). Although some endosymbionts from anobiid beetles and planthoppers show an affinity to certain filamentous ascomycetes (Jones & Blackwell, 1996Go; Noda et al., 1995Go; Noda & Kodama, 1996Go; Suh et al., 2001Go), the majority of the fungal endosymbionts have been identified as true yeasts (Ascomycetes: Saccharomycetes) (Jones et al., 1999Go). We were interested in sampling insects more broadly in order to determine if associations between additional fungi and insect hosts might be discovered. During a study of fungi from the digestive tract of basidiocarp-feeding beetles, about 650 yeasts were isolated from beetles in 26 families (Suh & Blackwell, 2004Go). Based on sequence comparisons of the D1/D2 loop of the large subunit (LSU) rRNA gene used for rapid yeast characterization, about 30 % of the yeast isolates formed a clade (CT clade) with Candida tanzawaensis, a yeast isolated from mosses in Japan (Nakase et al., 1988Go). Until the relatively recent report of six novel species (Candida ambrosiae, Candida canberraensis, Candida caryicola, Candida prunicola, Candida pyralidae and Candida xylopsoci), C. tanzawaensis had no known close relatives (Kurtzman, 2001Go). Currently, only one of the seven previously described species is known from multiple collections; in fact, C. tanzawaensis awaited description for 22 years in the vain hope that another isolate would be discovered (Nakase et al., 1988Go). Here, we describe 16 novel taxa, many with multiple isolates, from the gut of fungus-feeding beetles and compare the characteristics of all 23 members of the large CT clade, thereby recognizing unsuspected diversity in what appears to be a largely insect-associated clade.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Yeast isolation and identification.
Host beetles were collected from several different localities in Vermont, the southeastern USA, and Barro Colorado Island, Panama. Vouchers were deposited at the University of Georgia Collection of Arthropods, Athens, GA. The beetles were taken to the laboratory and usually placed in Petri dishes for 1–3 days without food prior to dissection. Surface disinfection was by submersion in 95 % ethanol for 1–2 min. The alcohol wash was followed by a 0·7 % saline rinse; the rinse liquid was plated on acidified YM agar (Difco YM broth, 2 % plain agar, adjusted to pH 3·5 with HCl) as a negative control. The beetle gut was removed aseptically under a dissecting microscope and transferred to tubes containing 0·7 % saline. The gut segments were crushed in the saline solution with a pipette tip and streaked with a loop onto the surface of an acidified YM agar plate. Plates were incubated at 25 °C, and single colonies were streaked for purification. Cultures were maintained on YM agar or 2 % malt extract agar (Yarrow, 1998Go). The isolates were initially screened and grouped using the D1/D2 loop sequence of the LSU rRNA gene. Selected isolates from each LSU genotype have been deposited at the Agricultural Research Service Culture Collection (NRRL) and Centraalbureau voor Schimmelcultures (CBS); holotype specimens were deposited at NRRL as lyophilized cultures (Table 1Go). The morphological observations and metabolic tests comprising the yeast standard description were performed on at least one isolate from each LSU genotype, according to established methods (Yarrow, 1998Go; Barnett et al., 2000Go). Isolates within each clade were crossed in all combinations and observed on YM agar, 2 % malt agar and cornmeal agar at 17 °C for 6 weeks in order to detect ascospore development. Bright-field microscopic examination was the usual method of observation, but some cell preparations were examined more closely with fluorescence microscopy using 0·02 % Calcofluor white as an optical brightener to visualize yeast cell walls more clearly.


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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

 
DNA sequencing and sequence analysis.
The D1/D2 region of the LSU rRNA gene was amplified directly without DNA extraction from yeast cells. A cell suspension of one loopful of cells in 50 µl of autoclaved water was heated at 95 °C for 5 min, and 2 µl of the supernatant was used as a template in 50 µl PCRs. For the small subunit (SSU) rRNA gene, the nucleic acids were extracted and purified following the procedures of Lee & Taylor (1990)Go. The primer sets NS1/NS8 and LS1/LR5 were used for PCR amplification of the SSU and LSU rRNA gene, respectively (White et al., 1990Go; Hausner et al., 1993Go). PCR products were purified using a DNA purification kit (Bio-Rad Laboratories), and purified double-stranded PCR products were used as templates for sequencing with an ABI PRISM BigDye Terminator cycle sequencing kit. The complete sequence of the SSU rRNA gene and the D1/D2 region of the LSU rRNA gene were obtained with the primers NS1, NS2, 18H, NS5, NS8, LS1 and LR3 using an ABI PRISM 377 automated DNA sequencer. GenBank/EMBL/DDBJ accession numbers for DNA sequences from this study are listed in Table 1Go and Fig. 1Go. SSU rRNA gene sequences of previously described Candida species in the CT clade were determined to compare with the novel gut yeast isolates. Previously described species names and GenBank/EMBL/DDBJ accession numbers are as follows: C. ambrosiae NRRL YB-1316T (AY227712), C. tanzawaensis NRRL Y-17324T (AY227713), C. canberraensis NRRL YB-2417T (AY488124), C. caryicola NRRL YB-1499T (AY488125), C. prunicola NRRL YB-869T (AY488126), C. pyralidae NRRL Y-27085T (AY488127) and C. xylopsoci NRRL Y-27066T (AY488128). DNA sequences were aligned with the multi-alignment program CLUSTAL_X (Thompson et al., 1997Go) and optimized visually. Ambiguous regions were excluded from the analyses. Sequences from newly isolated yeasts were compared with LSU and SSU rRNA gene sequences of other yeasts and fungi obtained from GenBank. Yeast groups based on the D1/D2 sequence were designated by the first four letters of the host beetle family and a number for each unique genotype varying by one or more base pairs from other yeasts from the same beetle family. Maximum-parsimony analyses were performed using PAUP* 4.0b10 (Swofford, 2002Go). Heuristic tree searches were executed using the tree bisection–reconnection branch-swapping algorithm with random sequence analysis. Bootstrap values of the most parsimonious tree were obtained from 1000 replications. Base pair differences were counted using BLAST2 Sequences (Tatusova & Madden, 1999Go) or from the manually aligned sequence database.



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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 consistency index=0·5388. Numbers on tree branches indicate the percentages of bootstrap samplings derived from 1000 samples that supported the internal branches by 50 % or higher.

 

    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Yeast isolates in the CT clade
We cultured about 650 yeasts from beetles in 26 families. Most of the isolates were true yeasts (Ascomycota: Saccharomycetes). Phylogenetic analysis of about 600 bp of sequence from the D1/D2 region of the LSU rRNA gene was used for rapid identification and grouping of the isolates. We discovered a major clade consisting of about 30 % of all beetle gut yeast isolates obtained using this technique. The taxa were closely related to C. tanzawaensis and six other previously described Candida species (Kurtzman, 2001Go). Detailed information about the 164 yeast isolates in the greatly enlarged CT clade is provided in Table 1Go. The gut yeasts in the CT clade were isolated from species in 11 families of beetles: Anthribidae, Carabidae, Ciidae, Endomychidae, Erotylidae, Histeridae, Melandryidae, Nitidulidae, Scarabaeidae, Staphylinidae (including Scaphidiinae) and Tenebrionidae. However, the majority of the yeasts (140 of 164 isolates, Table 1Go) were from species of Erotylidae and Tenebrionidae. The CT clade gut yeasts were sorted into 39 groups based on their D1/D2 genotype and host beetle ranges. Only three isolates (Erot8, Niti14 and Tene7) had a D1/D2 genotype identical to a previously described species (C. ambrosiae). The other 36 groups were distinct from all other previously described species (Table 1Go).

Novel species in the CT clade
In addition to the D1/D2 region of the LSU rRNA gene sequences, complete sequences of the SSU rRNA gene (about 1750 bp) were determined for at least one isolate from each LSU genotype (see Table 1Go for selected isolates), and these were combined with the LSU rRNA gene sequences in a dataset for better estimation of a phylogeny. A most-parsimonious tree was constructed from the combined dataset of SSU and LSU rRNA gene sequences by comparing the yeasts with taxa in Ascomycota and other yeasts alone (Fig. 1Go). The 39 gut yeasts we compared were representatives of the total 164 isolates in the clade (Fig. 1Go) with C. tanzawaensis and the other six previously described clade members. The clade was well supported statistically by 100 % bootstrap value (Fig. 1Go). Of the seven previously described taxa, C. xylopsoci, C. pyralidae, C. prunicola and C. caryicola were basal to the other yeasts in the CT clade. C. ambrosiae, C. tanzawaensis and C. canberraensis, however, were included among the beetle gut yeasts (Fig. 1Go). Within the CT clade, the beetle isolates were resolved into 17 subclades with sufficient divergence to warrant species-level recognition (Kurtzman & Robnett, 1998Go; Kurtzman, 2000Go; Suh & Blackwell, 2004Go) (Fig. 1Go).

As mentioned earlier, only one subclade (including Erot8, Niti14 and Tene7) had D1/D2 sequences identical to the previously described yeast C. ambrosiae, which we consider to be conspecific with these isolates. There were marked differences between C. ambrosiae and the other 16 subclades in the CT clade with 6 bp or more (usually more than 20 bp) difference between the subclades. The genetic variation among multiple isolates within subclades was lower (Table 1Go; Fig. 1Go). For example, yeasts designated Endo2 and Erot24 occurred in a common subclade and differed from each other by 3 bp of D1/D2 sequence. Also, there were minor D1/D2 sequence differences between common subclade members Tene28 and Tene30 or Erot20 and Tene19 (Table 1Go). Within all 17 subclades of the CT clade, however, the sequence variation of D1/D2 among subclade members was always less than 3 bp, within the range of species-level variation recognized in studies of other ascomycete yeast species (Kurtzman & Robnett, 1998Go; Table 1Go).

We observed minor morphological differences among the isolates on YM and cornmeal agars at 5–7 days incubation and in YM broth after 5 days incubation at 25 °C, but morphological variation that consistently distinguished the yeasts within clades was not evident. One morphological feature common to all isolates was the lack of ascospore production. Physiological characters (Table 2Go), however, were more variable and these traits were useful to separate the other species of the CT clade. Below, we characterize and describe 16 novel species of beetle gut yeasts and compare them to the previously described yeasts of the CT clade (Fig. 1Go and Table 2Go).


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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. chickasaworum; 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 (–), raffinose (–), starch (–), D-xylose (–), D-glucose (+); assimilation of D-rhamnose (–), trehalose (+), cellobiose (+), melibiose (–), lactose (–), raffinose (–), inulin (–), D-glucitol (+), D-mannitol (+), galactitol (–), myo-inositol (–), D-glucuronate (–), methanol (–), butane-2,3-diol (–), quinic acid (–), D-glucarate (–), D-galactonate (–), nitrate (–), nitrite (–), ethylamine (+), D-lysine (+), cadaverine (+), creatine (–), creatinine (–), imidazole (–); vitamin requirement, growth without pantothenate (+), niacin (+), p-aminobenzoic acid (+); growth at 25 °C (+), at 30 °C (+), at 40 °C (–); growth on 1 % acetic acid (–); additional tests, starch formation (–), urea hydrolysis (–), Diazonium Blue B reaction (–). Abbreviations: +, positive reaction; –, negative reaction; d, delayed positive reaction; W, weak positive reaction; V, variable reaction; w/o, without.

 
Latin diagnosis of Candida guaymorum Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut ellipsoideae (1·25–6·25x1·25–7·5 µm), singulae vel binae; pseudohyphae fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, hebes, teres et margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae fiunt; hyphae verae non fiunt. Ascosporae non fiunt. Glucosum, galactosum (lente), maltosum (infirme, variabilitre), methyl {alpha}-D-glucosidum (infirme, variabilitre), sucrosum (infirme, variabilitre), trehalosum et cellobiosum (lente) fermentantur. Melibiosum, lactosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-glucosaminum, D-xylosum, sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum, glycerolum, ribitolum, D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum (lente, infirme), acidum succinicum, acidum citricum et ethanolum. Non assimilantur L-sorbosum, D-ribosum, L-arabinosum, D-arabinosum, L-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, erythritolum, xylitolum, L-arabinitolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, propane-1,2-diolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, L-lysinum, cadaverinum et glucosaminum. Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, imidazolum et D-tryptophanum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum non fiunt in temperatura 40 °C. Non crescit in medio 10 µg ml–1 cycloheximido addito. Typus: NRRL Y-27568T (=CBS 9823T), designat stirpem typicum. Isolata a ile coleopterorum (Mycotretus interstitialis; Erotylidae), Barro Colorado Island, Panama, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida guaymorum Suh & Blackwell sp. nov.
Candida guaymorum (gu.ay.mo'rum. N.L. m. gen. guaymorum to commemorate the Guaymí, a group of indigenous people of Panama with detailed knowledge of the forest flora).

After 7 days growth in YM broth at 25 °C, cells are globose to ellipsoidal (1·25–6·25x1·25–7·5 µm), and occur singly, in pairs or in short chains (Fig. 2Goa). Pseudohyphae are present. After 7 days on YM agar at 25 °C, colonies are white to cream-coloured with pale-pinkish perimeter on some old colonies, smooth, shiny, flat and filamentous in margin. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae are present; septate hyphae are absent. Aerobic growth is white, shiny and smooth with filamentous margin. No ascospores produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.



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Fig. 2. Budding yeast cells, pseudohyphae and septate hyphae of the novel species. (a) C. guaymorum NRRL Y-27568T; (b) C. bokatorum NRRL Y-27571T; (c) C. kunorum NRRL Y-27580T; (d) C. terraborum NRRL Y-27573T; (e) C. emberorum NRRL Y-27606T; (f) C. wounanorum NRRL Y-27574T; (g) C. yuchorum NRRL Y-27569T; (h) C. chickasaworum NRRL Y-27566T; (i) C. choctaworum NRRL Y-27584T; (j) Candida bolitotheri NRRL Y-27587T; (k) C. atakaporum NRRL Y-27570T; (l) C. panamericana NRRL Y-27567T. (a–f, h–l) Seven days, half-strength cornmeal agar, 25 °C; (g) 5 days, YM broth, 25 °C. Bars, 5 µm.

 
The type strain is NRRL Y-27568T (=CBS 9823T).

Latin diagnosis of Candida bokatorum Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut ellipsoideae (2–6x3–6 µm), singulae vel binae. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, hebes, butyrosa, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae fiunt; hyphae verae fiunt. Ascosporae non fiunt. Glucosum, maltosum (infirme, variabilitre), sucrosum (infirme, variabilitre), trehalosum et cellobiosum fermentantur. Galactosum, methyl {alpha}-D-glucosidum, melibiosum, lactosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-glucosaminum (variabilitre), D-xylosum, D-arabinosum, sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum, glycerolum, ribitolum, D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum (lente, infirme), acidum succinicum, acidum citricum, ethanolum et propane-1,2-diolum (infirme, variabilitre). Non assimilantur L-sorbosum, D-ribosum, L-arabinosum, L-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, erythritolum, xylitolum, L-arabinitolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, L-lysinum, cadaverinum et glucosaminum (infirme, variabilitre). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, imidazolum et D-tryptophanum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum non fiunt in temperatura 40 °C. Variabilitre in medio 10 µg ml–1 cycloheximido addito, non crescit in medio 100 µg ml–1. Typus: NRRL Y-27571T (=CBS 9824T), designat stirpem typicum. Isolata a ile coleopterorum (Pselaphacus signatus; Erotylidae), Barro Colorado Island, Panama, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida bokatorum Suh & Blackwell sp. nov.
Candida bokatorum (bo.ka.to'rum. N.L. m. gen. bokatorum to commemorate the Bókatá, a group of indigenous people of Panama, linguistically related to the Guaymí).

After 7 days growth in YM broth at 25 °C, cells are globose to ellipsoidal (2–6x3–6 µm), mostly subglobose, and occur singly, in pairs or in short chains (Fig. 2bGo). Pseudohyphae may be present. After 7 days on YM agar at 25 °C, colonies are white to cream in colour, butyrous and smooth. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae are present. Septate hyphae may be present. Aerobic growth is white and smooth. No ascospores produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27571T (=CBS 9824T).

Latin diagnosis of Candida kunorum Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae subglobosae aut fusiformes (2–5x3–6 µm), singulae vel binae. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, hebes, butyrosa, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae non fiunt; hyphae verae non fiunt. Ascosporae non fiunt. Glucosum, galactosum (infirme), maltosum (infirme), trehalosum et lactosum (infirme) fermentantur. Sucrosum, methyl {alpha}-D-glucosidum, melibiosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-glucosaminum (lente), D-ribosum (infirme), D-xylosum (lente), D-arabinosum (infirme), sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum (lente), arbutinum, melezitosum, glycerolum, ribitolum, xylitolum (infirme), L-arabinitolum (infirme), D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum, acidum succinicum, acidum citricum (lente), ethanolum et propane-1,2-diolum (infirme). Non assimilantur L-sorbosum, L-arabinosum, L-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, erythritolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, butano-2,3-diolum, acidum quinicum et D-glucaratum. Assimilantur ethylaminum, L-lysinum, cadaverinum et glucosaminum (infirme). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, imidazolum et D-tryptophanum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum non fiunt in temperatura 35 °C. Typus: NRRL Y-27580T (=CBS 9825T), designat stirpem typicum. Isolata a ile coleopterorum (Teichostethus testaceous; Nitidulidae), Barro Colorado Island, Panama, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida kunorum Suh & Blackwell sp. nov.
Candida kunorum (ku.no'rum. N.L. m. gen. kunorum to commemorate the Kuna, a group of indigenous people of Panama with a high degree of literacy and a desire for autonomy).

After 7 days growth in YM broth at 25 °C, cells are subglobose to fusiform (2–5x3–6 µm), mostly subglobose, and occur singly, in pairs or in short chains (Fig. 2cGo). After 7 days on YM agar at 25 °C, colonies are white, membranous and butyrous with smooth margin. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae and septate hyphae are absent. Aerobic growth is white and smooth. No ascospores produced after 6 weeks at 17 °C from the single strain on YM agar or half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27580T (=CBS 9825T).

Latin diagnosis of Candida terraborum Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut ellipsoideae (4–6x5–7 µm), singulae vel binae. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, hebes, butyrosa, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae fiunt; hyphae verae non fiunt. Ascosporae non fiunt. Glucosum (lente), galactosum (infirme), maltosum (lente), sucrosum (lente) et trehalosum (lente) fermentantur. Methyl {alpha}-D-glucosidum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-glucosaminum (infirme), D-xylosum (infirme), sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum, glycerolum, ribitolum, D-glucitolum, D-mannitolum, gluconolactonum (infirme), 2-keto-D-gluconatum, D-gluconatum (infirme), acidum succinicum, acidum citricum, ethanolum (lente) et propane-1,2-diolum (infirme). Non assimilantur D-sorbosum, D-ribosum, L-arabinosum, D-arabinosum, D-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, erythritolum, xylitolum, D-arabinitolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, D-lysinum, cadaverinum et glucosaminum (infirme). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, imidazolum et D-tryptophanum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. 30 °C crescit neque 35 °C. Non crescit in medio 10 µg ml–1 cycloheximido addito. Typus: NRRL Y-27573T (=CBS 9826T), designat stirpem typicum. Isolata a ile coleopterorum (Iphiclus sedecimmaculatus; Erotylidae), Barro Colorado Island, Panama, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida terraborum Suh & Blackwell sp. nov.
Candida terraborum (ter.ra.bo'rum. N.L. m. gen. terraborum to commemorate the Térraba, a group of indigenous people of Panama, survivors of epidemics that once decimated their numbers).

After 7 days growth in YM broth at 25 °C, cells are globose to ellipsoidal (4–6x5–7 µm), mostly subglobose, and occur singly, in pairs or in short chains (Fig. 2dGo). Big and elongated cells are also observed. After 7 days on YM agar at 25 °C, colonies are off-white, butyrous and smooth. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae are present; septate hyphae are absent. Aerobic growth is white with fuzzy margin. No ascospores produced after 6 weeks at 17 °C from the single strain on YM agar or half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27573T (=CBS 9826T).

Latin diagnosis of Candida emberorum Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut ellipsoideae (1·25–5x2·5–6·25 µm), singulae vel binae. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, hebes, butyrosa, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae fiunt; hyphae verae non fiunt. Ascosporae non fiunt. Glucosum, galactosum, trehalosum et cellobiosum (lente, infirme) fermentantur. Maltosum, methyl {alpha}-D-glucosidum, sucrosum, melibiosum, lactosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-glucosaminum (lente, infirme), D-xylosum, sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum (lente, infirme), glycerolum, ribitolum, D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum (lente, infirme), acidum succinicum, acidum citricum et ethanolum. Non assimilantur D-sorbosum, D-ribosum, L-arabinosum, D-arabinosum, D-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, erythritolum, xylitolum, D-arabinitolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, propane-1,2-diolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, D-lysinum, cadaverinum, glucosaminum et D-tryptophanum (infirme). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum et imidazolum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum non fiunt in temperatura 40 °C. Non crescit in medio 10 µg ml–1 cycloheximido addito. Typus: NRRL Y-27606T (=CBS 9827T), designat stirpem typicum. Isolata a ile coleopterorum (Triplax alvarengai; Erotylidae), Barro Colorado Island, Panama, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida emberorum Suh & Blackwell sp. nov.
Candida emberorum (em.be.ro'rum. N.L. m. gen. emberozrum to commemorate the Emberá, a group of indigenous people of Panama, knowledgeable in botanical lore).

After 7 days growth in YM broth at 25 °C, cells are globose to ellipsoidal (1·25–5x2·5–6·25 µm), mostly globose or subglobose, and occur singly, in pairs or in short chains (Fig. 2eGo). Pseudohyphae may be present. After 7 days on YM agar at 25 °C, colonies are white to cream-coloured with pale-pinkish perimeter, smooth and butyrous. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae are present; septate hyphae are absent. Aerobic growth is white, dull and smooth with filamentous margin. No ascospores produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27606T (=CBS 9827T).

Latin diagnosis of Candida wounanorum Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut ovoidae (2–4x3–6 µm), plerumque subglobosae, singulae vel binae. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, butyrosa. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae non fiunt; hyphae verae non fiunt. Ascosporae non fiunt. Glucosum (lente), galactosum (infirme) et trehalosum (lente) fermentantur. Maltosum, methyl {alpha}-D-glucosidum, sucrosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-glucosaminum (lente), D-xylosum, sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum (infirme), glycerolum, ribitolum, D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, acidum succinicum (lente), acidum citricum et ethanolum. Non assimilantur D-sorbosum, D-ribosum, L-arabinosum, D-arabinosum, D-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, erythritolum, xylitolum, D-arabinitolum, galactitolum, inositolum, D-gluconatum, D-glucuronatum, DL-acidum lacticum, methanolum, propane-1,2-diolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, D-lysinum et cadaverinum. Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, glucosaminum, imidazolum et D-tryptophanum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. In temperatura 30 °C crescit neque 35 °C. Non crescit in medio 10 µg ml–1 cycloheximido addito. Typus: NRRL Y-27574T (=CBS 9828T), designat stirpem typicum. Isolata a ile coleopterorum (Mycotretus dorsonotatus; Erotylidae), Barro Colorado Island, Panama, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida wounanorum Suh & Blackwell sp. nov.
Candida wounanorum (wou.na.no'rum. N.L. m. gen. wounanorum to commemorate the Wounan, a group of indigenous people of Panama known for their artistry in using plant materials; they inhabited the same region as the Emberá).

After 7 days growth in YM broth at 25 °C, cells are globose to oval (2–4x3–6 µm), mostly subglobose, and occur singly, in pairs or in short chains (Fig. 2fGo). After 7 days on YM agar at 25 °C, colonies are off-white with fuzzy white spots, butyrous and smooth. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae and septate hyphae are absent. Aerobic growth is white, shiny, smooth and filamentous in margin. No ascospores produced after 6 weeks at 17 °C from the single strain on YM agar or half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27574T (=CBS 9828T).

Latin diagnosis of Candida yuchorum Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut ellipsoideae (2·5–7x2·5–7 µm), plerumque globosae, singulae vel binae. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, teres, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae et hyphae verae fiunt. Ascosporae non fiunt. Glucosum, galactosum (lente), trehalosum et cellobiosum (lente) fermentantur. Maltosum, methyl {alpha}-D-glucosidum, sucrosum, melibiosum, lactosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-xylosum, sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, glycerolum, ribitolum, D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum (lente), acidum succinicum et acidum citricum. Non assimilantur D-sorbosum, D-glucosaminum, D-ribosum, L-arabinosum, D-arabinosum, D-rhamnosum, melibiosum, lactosum, raffinosum, melezitosum, inulinum, amylum solubile, erythritolum, xylitolum, D-arabinitolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, ethanolum, propane-1,2-diolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, D-lysinum, cadaverinum, glucosaminum et D-tryptophanum (lente). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum et imidazolum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum non fiunt in temperatura 40 °C. Non crescit in medio 10 µg ml–1 cycloheximido addito. Typus: NRRL Y-27569T (=CBS 9829T), designat stirpem typicum. Isolata a ile coleopterorum (Tritoma atriventris; Erotylidae), Athens, GA, USA, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida yuchorum Suh & Blackwell sp. nov.
Candida yuchorum (yu.cho'rum. N.L. m. gen. yuchorum to commemorate the Yuchi, native Americans of the southeastern USA; their public worship was tied to the corn harvest when food was shared).

After 7 days growth in YM broth at 25 °C, cells are globose to ellipsoidal (2·5–7x2·5–7 µm), mostly globose or subglobose, and occur singly, in pairs or in short chains (Fig. 2gGo). Pseudohyphae and septate hyphae may be present. After 7 days on YM agar at 25 °C, colonies are cream-coloured, smooth and shiny with filamentous margin. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae and septate hyphae may be present. Aerobic growth is white and dull. No ascospores produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27569T (=CBS 9829T).

Latin diagnosis of Candida chickasaworum Suh & Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut fusiformes (2–6x2–8 µm), singulae vel binae. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, butyrosa, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae et hyphae verae fiunt. Ascosporae non fiunt. Glucosum, galactosum et trehalosum fermentantur. Maltosum, methyl {alpha}-D-glucosidum, sucrosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-glucosaminum (lente, infirme), D-xylosum, sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum (variabilitre), glycerolum, ribitolum, xylitolum (infirme, variabilitre), D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum (variabilitre), acidum succinicum, acidum citricum, ethanolum et propane-1,2-diolum (infirme, variabilitre). Non assimilantur D-sorbosum, D-ribosum, L-arabinosum, D-arabinosum, D-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, erythritolum, D-arabinitolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, D-lysinum, cadaverinum et glucosaminum (variabilitre). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, imidazolum et D-tryptophanum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est (variabilitre). In temperatura 30 °C crescit neque 35 °C. Non crescit in medio 10 µg ml–1 cycloheximido addito. Typus: NRRL Y-27566T (=CBS 9830T), designat stirpem typicum. Isolata a ile coleopterorum (Tritoma sp.; Erotylidae), Athens, GA, USA, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida chickasaworum Suh & Blackwell sp. nov.
Candida chickasaworum (chic.ka.sa.wo'rum. N.L. m. gen. chickasaworum to commemorate the Chickasaw, native Americans who in earlier times made use of many endemic and introduced southeastern USA plants).

After 7 days growth in YM broth at 25 °C, cells are globose, subglobose, to fusiform (2–6x2–8 µm), and occur singly, in pairs or in short chains (Fig. 2hGo). Pseudohyphae and septate hyphae may be present. After 7 days on YM agar at 25 °C, colonies are white to cream-coloured, butyrous and smooth with mycelial edge. After 10 days Dalmau plate culture on corn meal agar at 25 °C, elongated pseudohyphae and septate hyphae may be present. Aerobic growth is white to cream-coloured with mycelial margin. No ascospores produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27566T (=CBS 9830T).

Latin diagnosis of Candida choctaworum Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut ovoidae (2–6x2–8 µm), plerumque globosae et subglobosae, singulae vel binae. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, butyrosa, teres. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae et hyphae verae may fiunt. Ascosporae non fiunt. Glucosum, galactosum (variabilitre) et trehalosum fermentantur. Maltosum, methyl {alpha}-D-glucosidum, sucrosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-sorbosum, D-glucosaminum, D-ribosum (variabilitre), D-xylosum, L-arabinosum (infirme, variabilitre), D-arabinosum, trehalosum, cellobiosum, salicinum, arbutinum, glycerolum, erythritolum, ribitolum, xylitolum (variabilitre), D-arabinitolum (variabilitre), D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum (variabilitre), acidum succinicum, acidum citricum, ethanolum et propane-1,2-diolum (infirme, variabilitre). Non assimilantur D-rhamnosum, sucrosum, maltosum, methyl {alpha}-D-glucosidum, melibiosum, lactosum, raffinosum, melezitosum, inulinum, amylum solubile, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, D-lysinum, cadaverinum et glucosaminum (variabilitre). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, imidazolum et D-tryptophanum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum non fiunt in temperatura 40 °C. Crescit in medio 100 µg ml–1 cycloheximido addito. Typus: NRRL Y-27584T (=CBS 9831T), designat stirpem typicum. Isolata a ile coleopterorum (Neomida bicornis; Tenebrionidae), Baton Rouge, LA, USA, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida choctaworum Suh & Blackwell sp. nov.
Candida choctaworum (choc.ta.wo'rum. N.L. m. gen. choctaworum to commemorate the Choctaw, native Americans warriors and code-talkers, many of whom were removed from their native lands in the southeastern USA).

After 7 days growth in YM broth at 25 °C, cells are globose to oval (2–6x2–8 µm), and occur singly, in pairs or in short chains (Fig. 2iGo). Pseudohyphae may be present. After 7 days on YM agar at 25 °C, colonies are white to cream-coloured, smooth and butyrous with slightly wrinkled edge. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae and septate hyphae may be present. Aerobic growth is white, shiny, smooth and slightly fuzzy. No ascospores produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27584T (=CBS 9831T).

Latin diagnosis of Candida bolitotheri Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut subglobosae (2–6x2–6 µm), singulae vel binae. Pseudohyphae fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, teres, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae fiunt; hyphae verae fiunt. Ascosporae non fiunt. Glucosum, galactosum et trehalosum fermentantur. Maltosum, methyl {alpha}-D-glucosidum, sucrosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-sorbosum (variabilitre), D-glucosaminum, D-ribosum (variabilitre), D-xylosum, trehalosum, cellobiosum, salicinum, arbutinum, glycerolum, erythritolum, ribitolum, xylitolum, D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum (lente, infirme), acidum succinicum, acidum citricum et ethanolum. Non assimilantur L-arabinosum, D-arabinosum, D-rhamnosum, sucrosum, maltosum, methyl {alpha}-D-glucosidum, melibiosum, lactosum, raffinosum, melezitosum, inulinum, amylum solubile, D-arabinitolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, propane-1,2-diolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, D-lysinum, cadaverinum et glucosaminum. Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, imidazolum et D-tryptophanum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum non fiunt in temperatura 45 °C. Variabilitre in medio 10 µg ml–1 cycloheximido addito, non crescit in medio 100 µg ml–1. Typus: NRRL Y-27587T (=CBS 9832T), designat stirpem typicum. Isolata a ile coleopterorum (Bolitotherus cornutus; Tenebrionidae), Athens, GA, USA, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida bolitotheri Suh & Blackwell sp. nov.
Candida bolitotheri (bo.li.to.the'ri. N.L. n. gen. bolitotheri is named for the coleopteran host, Bolitotherus cornutus, occurring in Ganoderma basidiocarps from Vermont to Louisiana, USA).

After 7 days growth in YM broth at 25 °C, cells are globose to subglobose (2–6x2–6 µm), mostly globose, and occur singly or in short chains (Fig. 2jGo). Pseudohyphae may be present. After 7 days on YM agar at 25 °C, colonies are white to cream-coloured with very pale-pinkish from centre to edge, and smooth to slightly wrinkled centre with mycelial edge. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae are present. Septate hyphae may be present. Aerobic growth is white, shiny and smooth with filamentous margin. No ascospores produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27587T (=CBS 9832T).

Latin diagnosis of Candida atakaporum Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae subglobosae aut cylindratae (3–5x5–9 µm), singulae vel binae. Pseudohyphae fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, butyrosa, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae et hyphae verae fiunt. Ascosporae non fiunt. Glucosum et trehalosum (lente) fermentantur. Galactosum, maltosum, methyl {alpha}-D-glucosidum, sucrosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-glucosaminum (lente), D-xylosum, D-arabinosum (infirme), sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum, amylum solubile (infirme), glycerolum, ribitolum, D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum (infirme), DL-acidum lacticum (lente), acidum succinicum (lente), acidum citricum (lente), ethanolum et propane-1,2-diolum (infirme). Non assimilantur D-sorbosum, D-ribosum, L-arabinosum, D-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, erythritolum, xylitolum, D-arabinitolum, galactitolum, inositolum, D-glucuronatum, methanolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, D-lysinum, cadaverinum, glucosaminum (infirme) et D-tryptophanum (lente). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum et imidazolum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum non fiunt in temperatura 40 °C. Non crescit in medio 10 µg ml–1 cycloheximido addito. Typus: NRRL Y-27570T (=CBS 9833T), designat stirpem typicum. Isolata a ile coleopterorum (Triplax festiva; Erotylidae), Baton Rouge, LA, USA, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida atakaporum Suh & Blackwell sp. nov.
Candida atakaporum (a.ta.ka.po'rum. N.L. m. gen. atakaporum commemorates the Atakapa, native Americans of the northwestern coast of the Gulf of Mexico and their unique language).

After 7 days growth in YM broth at 25 °C, cells are subglobose, ellipsoidal or cylindrical (3–5x5–9 µm), and occur singly, in pairs or in short chains (Fig. 2kGo). Pseudohyphae and septate hyphae may be present. After 7 days on YM agar at 25 °C, colonies are off-white with membranous margin and butyrous with small filamentous spots. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae and septate hyphae are present. Aerobic growth is off-white and fuzzy. No ascospores produced after 6 weeks at 17 °C from the single strain on YM agar or half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27570T (=CBS 9833T).

Latin diagnosis of Candida panamericana Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut ovoidae (3–6·25x3·75–7 µm), plerumque subglobosae, singulae vel binae. Pseudohyphae fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, teres, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae fiunt; hyphae verae fiunt. Ascosporae non fiunt. Glucosum, galactosum (lente), trehalosum et cellobiosum fermentantur. Maltosum, methyl {alpha}-D-glucosidum, sucrosum, melibiosum, lactosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-glucosaminum, D-xylosum, sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum, glycerolum, ribitolum, D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum, acidum succinicum et acidum citricum. Non assimilantur D-sorbosum, D-ribosum, L-arabinosum, D-arabinosum, D-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, erythritolum, xylitolum, D-arabinitolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, ethanolum, propane-1,2-diolum, butano-2,3-diolum, acidum quinicum et D-glucaratum. Assimilantur ethylaminum, D-lysinum, cadaverinum et glucosaminum (variabilitre). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, imidazolum et D-tryptophanum. Amylum non formatur. Vitaminae externae ad crescentiam necessaria non sunt. Augmentum non fiunt in temperatura 40 °C. Non crescit in medio 10 µg ml–1 cycloheximido addito. Typus: NRRL Y-27567T (=CBS 9834T), designat stirpem typicum. Isolata a ile coleopterorum (Mycotretus interstitialis; Erotylidae), Barro Colorado Island, Panama, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida panamericana Suh & Blackwell sp. nov.
Candida panamericana (pan.a.mer.i.can'a. N.L. f. adj. panamericana to call attention to its broad distribution spanning the regions from Louisiana to Panama).

After 7 days growth in YM broth at 25 °C, cells are globose to oval (3–6·25x3·75–7 µm), mostly subglobose, and occur singly, in pairs or in short chains (Fig. 2lGo). Some cells form clusters. Pseudohyphae are present. After 7 days on YM agar at 25 °C, colonies are white to off-white in colour, smooth, glistening and flat with filamentous margin. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae are present. Septate hyphae may be present. Aerobic growth is white, shiny and smooth with filamentous margin. No ascospores produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27567T (=CBS 9834T).

Latin diagnosis of Candida bribrorum Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut ellipsoideae (3–8x3–8 µm), plerumque subglobosae, singulae vel binae. Pseudohyphae fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, butyrosa, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae et hyphae verae fiunt. Ascosporae non fiunt. Glucosum, galactosum (variabilitre), maltosum (infirme, variabilitre) et trehalosum fermentantur. Methyl {alpha}-D-glucosidum, sucrosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-sorbosum (variabilitre), D-glucosaminum, D-ribosum (variabilitre), D-xylosum, L-arabinosum (variabilitre), D-arabinosum (variabilitre), sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum, glycerolum, erythritolum, ribitolum, xylitolum, D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum, acidum succinicum, acidum citricum, ethanolum et propane-1,2-diolum (variabilitre). Non assimilantur D-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, D-arabinitolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, D-lysinum, cadaverinum et glucosaminum (variabilitre). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, imidazolum et D-tryptophanum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum non fiunt in temperatura 40 °C. Infirme crescit in medio 10 µg ml–1 cycloheximido addito, variabilitre in 100 µg ml–1. Typus: NRRL Y-27572T (=CBS 9835T), designat stirpem typicum. Isolata a ile coleopterorum (Pselaphacus sp.; Erotylidae), Barro Colorado Island, Panama, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida bribrorum Suh & Blackwell sp. nov.
Candida bribrorum (bri.bro'rum. N.L. m. gen. bribrorum to commemorate the Bribri, a small group of indigenous people of northern Panama, who apply ecological principles and recognize symbiotic relationships).

After 7 days growth in YM broth at 25 °C, cells are globose to ellipsoidal (3–8x3–8 µm), mostly subglobose, and occur singly, in pairs or in short chains (Fig. 3Goa). Pseudohyphae are present. After 7 days on YM agar at 25 °C, colonies are white to cream in colour, butyrous and smooth surface with slightly fuzzy spots. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae and septate hyphae are present. Aerobic growth is white to cream-coloured with fuzzy margin. No ascospores produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.



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Fig. 3. Budding yeast cells, pseudohyphae and septate hyphae of the novel species. (a) C. bribrorum NRRL Y-27572T; (b, c) C. maxii NRRL Y-27588T; (d) yeast cells of C. anneliseae NRRL Y-27563T; (e) C. taliae NRRL Y-27589T. (a–c) Seven days, YM broth, 25 °C; (d, e) 7 days, half-strength cornmeal agar, 25 °C. Bars, 5 µm.

 
The type strain is NRRL Y-27572T (=CBS 9835T).

Latin diagnosis of Candida maxii Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut subglobosae (2–6·25x3·75–6·25 µm), singulae vel binae. Pseudohyphae et hyphae verae fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae et hyphae verae fiunt. Ascosporae non fiunt. Glucosum (lente), trehalosum (infirme) et cellobiosum (infirme) fermentantur. Galactosum, maltosum, methyl {alpha}-D-glucosidum, sucrosum, melibiosum, lactosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-glucosaminum (lente), D-ribosum, D-arabinosum (infirme), sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum, glycerolum (lente), erythritolum, ribitolum, xylitolum (infirme), D-glucitolum, D-mannitolum, gluconolactonum (lente), 2-keto-D-gluconatum, D-gluconatum, acidum succinicum, acidum citricum et ethanolum. Non assimilantur D-sorbosum, D-xylosum, L-arabinosum, D-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, D-arabinitolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, propane-1,2-diolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, D-lysinum, cadaverinum et glucosaminum (infirme). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, imidazolum et D-tryptophanum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. 30 °C crescit neque 35 °C. Non crescit in medio 10 µg ml–1 cycloheximido addito. Typus: NRRL Y-27588T (=CBS 9836T), designat stirpem typicum. Isolata a ile coleopterorum (Tenebrionidae), Barro Colorado Island, Panama, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida maxii Suh & Blackwell sp. nov.
Candida maxii (max.i'i. N.L. m. gen. maxii for Max Vallone, whose grandparents generously support the Mycological Society of America).

After 7 days growth in YM broth at 25 °C, cells are globose to subglobose (2·5–6·25x3·75–6·25 µm). Pseudohyphae and septate hyphae are present (Fig. 3b, cGo). After 7 days on YM agar at 25 °C, colonies are white in colour, ridged, glistening and flat with filamentous margin. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae and septate hyphae are present. Aerobic growth is white, shiny and smooth with filamentous margin. No ascospores produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27588T (=CBS 9836T).

Latin diagnosis of Candida anneliseae Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut ellipsoideae (1·25–7x1·25–7 µm), singulae vel binae. Pseudohyphae fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, teres, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae fiunt; hyphae verae fiunt. Ascosporae non fiunt. Glucosum et trehalosum (lente, infirme) fermentantur. Galactosum, maltosum, methyl {alpha}-D-glucosidum, sucrosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum (variabilitre), D-sorbosum (variabilitre), D-glucosaminum, D-ribosum (variabilitre), D-xylosum, D-arabinosum, sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum, glycerolum, erythritolum, ribitolum, xylitolum, D-arabinitolum (variabilitre), D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum, acidum succinicum, acidum citricum, ethanolum et propane-1,2-diolum (infirme, variabilitre). Non assimilantur L-arabinosum, D-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, D-lysinum, cadaverinum et glucosaminum (variabilitre). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, imidazolum et D-tryptophanum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum non fiunt in temperatura 40 °C. Variabilitre in medio 10 µg ml–1 cycloheximido addito, non crescit in medio 100 µg ml–1. Typus: NRRL Y-27563T (=CBS 9837T), designat stirpem typicum. Isolata a ile coleopterorum (Megalodacne fasciata; Erotylidae), Athens, GA, USA, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida anneliseae Suh & Blackwell sp. nov.
Candida anneliseae (an.ne.lis.e'ae N.L. n. gen. anneliseae for Annelise Berler, whose grandparents generously support the Mycological Society of America).

After 7 days growth in YM broth at 25 °C, cells are globose to ellipsoid (1·25–7x1·25–7 µm), and occur singly, in pairs or in chains (Fig. 3dGo). Pseudohyphae may be present. After 7 days on YM agar at 25 °C, colonies are white to cream-coloured, pale-pinkish from centre to edge and smooth with slightly winkled mycelial edge. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae are present. Septate hyphae may be present. Aerobic growth is white, shiny, smooth, and the growth consistent throughout mycelium with filamentous-like periphery. No ascospores produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27563T (=CBS 9837T).

Latin diagnosis of Candida taliae Suh et Blackwell sp. nov.
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut ovoidae (2·5–5x3·75–6·25 µm), plerumque ellipsoideae, singulae vel binae. Pseudohyphae et hyphae verae fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 °C, albida, butyrosa, teres, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae fiunt; hyphae verae non fiunt. Ascosporae non fiunt. Glucosum, galactosum (lente) trehalosum (lente) et cellobiosum (lente) fermentantur. Maltosum, methyl {alpha}-D-glucosidum, sucrosum, melibiosum, lactosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, galactosum, D-glucosaminum, D-xylosum (lente), D-arabinosum, sucrosum, maltosum, trehalosum, methyl {alpha}-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum, glycerolum, erythritolum, ribitolum, xylitolum (lente), D-glucitolum, D-mannitolum, gluconolactonum (lente), 2-keto-D-gluconatum, D-gluconatum, acidum succinicum, acidum citricum et ethanolum. Non assimilantur D-sorbosum, D-ribosum, L-arabinosum, D-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, D-arabinitolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, propane-1,2-diolum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, D-lysinum, cadaverinum et glucosaminum (infirme). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, imidazolum et D-tryptophanum. Amylum non formatur. Biotinum externum ad crescentiam necessarium est. Augmentum non fiunt in temperatura 40 °C. Non crescit in medio 10 µg ml–1 cycloheximido addito. Typus: NRRL Y-27589T (=CBS 9838T), designat stirpem typicum. Isolata a ile coleopterorum (Tenebrionidae), Barro Colorado Island, Panama, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA.

Description of Candida taliae Suh & Blackwell sp. nov.
Candida taliae (tal'i.ae. N.L. n. gen. taliae for Talia Berler, whose grandparents generously support the Mycological Society of America).

After 7 days growth in YM broth at 25 °C, cells are globose to oval (2·5–5x3·75–6·25 µm), mostly ellipsoidal, and occur singly, in pairs or in short chains (Fig. 3eGo). Pseudohyphae and septate hyphae are present. After 7 days on YM agar at 25 °C, colonies are off-white, butyrous and mostly smooth with bumpy areas in centre. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae are present; septate hyphae are absent. Aerobic growth is white, shiny and smooth with filamentous margin. No ascospores produced after 6 weeks at 17 °C from the single strain on YM agar or half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

The type strain is NRRL Y-27589T (=CBS 9838T).

Gut yeasts close to Candida ambrosiae Kurtzman (Table 1Go)
After 7 days growth in YM broth at 25 °C, cells are globose to ellipsoidal (2·5–5x2·5–6·25 µm), and occur singly, in pairs or in chains. Pseudohyphae are present. After 7 days on YM agar at 25 °C, colonies are white to cream-coloured, rough and partly powdery with wrinkled mycelial edge. After 10 days Dalmau plate culture on corn meal agar at 25 °C, pseudohyphae are present. Aerobic growth is white, dull, dry, powdery; consistent growth throughout mycelium with branch-like periphery. No ascospores produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength cornmeal agar. See Table 2Go for a summary of physiological and other characteristics.

CT clade: a major clade of gut yeasts from basidiocarp-feeding beetles
The repeated isolation of C. tanzawaensis-like yeasts from beetles first attracted our attention to these fungi. As mentioned above, about 30 % of all 650 yeast isolates from our ongoing study are members of the clade. Except for a few isolates we placed in C. ambrosiae, none had been described previously (Fig. 1Go). Most of the 16 novel CT clade members appear to be common associates of basidiocarp-feeding beetles, and some of the yeasts have broad geographical distributions. The 22-year interval between the isolation of C. tanzawaensis and its description, and the absence of any additional reports in the intervening 21 years since the description (Nakase et al., 1988Go), contrasts with the common occurrence of the novel CT yeasts and C. ambrosiae in the specialized gut habitat in which we have found them thus far. The discovery of so many novel species of undescribed yeasts suggests that the microbial flora of the beetle gut is distinctive and has not been well examined previously. We do not know the basis of the apparent widespread relationship between these organisms, but it must be significant to beetles or yeasts or both because the associations are so common. In one case an unsuspected genetic resource that might be tied to such a function has been suggested for a yeast associate of certain wood-ingesting beetles (Suh et al., 2003Go).

Host specificity of yeasts and dispersal in the CT clade
Our data reveal several examples of highly specific associations between certain beetles and yeasts. The same yeast was associated repeatedly with Neomida bicornis (Tenebrionidae); this yeast (Tene1 of C. choctaworum) was isolated from the beetle gut at least eight times from five different localities in southern Louisiana over a 5-year period (Table 1Go). Another specialized association occurred between Bolitotherus cornutus (Tenebrionidae) and Tene11 of C. bolitotheri in all samples examined from Vermont to southwestern Louisiana (Table 1Go). We also observed that several CT clade yeasts were present in the gut of insects at different stages in the insect life histories. For example, Erot38 (C. bribrorum) has been found in both a pupa and an adult of Megalodacne audouini, and Mela2 (C. anneliseae) was isolated from larvae and adults of the same melandreid beetle species. Our indirect evidence, therefore, supports a view that certain yeasts are present during the entire life cycle of their beetle hosts and that parental transmission occurs early in successive generations (Suh & Blackwell, 2004Go). Previous studies have shown maternal transmission of yeasts to insects, including examples such as the yeast-like endosymbionts of anobiid beetles that are transmitted to their offspring by contamination of the egg shell (Jurzitza, 1979Go).

Several beetle species in the Tenebrionidae are associated with closely related yeasts. An example of a close association is Tene4 (C. anneliseae), isolated from species of Diaperis, Neomida, Platydema, Alobates and several unidentified taxa. These yeasts have identical D1/D2 genotypes and small, but consistent, differences in physiological traits to distinguish among them. Other CT clade yeasts also show evidence of past host-switching among distantly related beetles. For example, isolates of C. anneliseae (Mela2, Hist5 and Ciid5) are identical to Tene4 in D1/D2 genotype, although the beetle hosts are not closely related and belong to four different families spread throughout a broad geographical range in our sampling. We will need to investigate the possibility of more variable DNA markers to explain transmission among phylogenetically distinct hosts. We believe that these beetles most likely acquired their yeast associates comparatively recently, perhaps by relatively rare host-switching within a common habitat.

Diversification and resolution of taxa within the CT clade
Identification of yeast taxa based on morphological and physiological characteristics has been problematic and often leads to misidentification (Kurtzman & Phaff, 1987Go; Price et al., 1978Go). For this reason molecular methods increasingly are being used to identify yeasts; additionally, molecular markers, especially DNA sequences, have become the standard for the description of yeast species. The variable D1/D2 sequence of the LSU rRNA gene (about 600 bp) has been determined for currently recognized ascomycete yeasts (Kurtzman & Robnett, 1995Go, 1997Go, 1998Go; Suh & Blackwell, 2004Go). In addition to helping to delimit species, comparisons of the D1/D2 sequences also are a useful tool for rapid identification of yeasts and detection of novel species (e.g. Kurtzman, 2000Go) with a few exceptions among closely related species, such as Saccharomyces bayanus and Saccharomyces pastorianus (Kurtzman & Robnett, 1998Go).

In our study, SSU rRNA gene sequences, as expected, did not provide enough variation to distinguish all species in the CT clade. Identical sequences were determined for about 1750 bp of the SSU rRNA gene for three pairs of taxa: C. tanzawaensis and C. ambrosiae, C. guaymorum and C. bokatorum, and C. maxii and C. anneliseae. The more variable D1/D2 loop sequences of the LSU rRNA gene, which sometimes may be too variable to align among phylogenetically distant groups when comparing a broad range of yeasts, were useful in distinguishing CT clade yeasts (Suh et al., 2004Go). The differences in D1/D2 sequences, varying from 6 to more than 60 bp between species and less than 3 bp for multiple isolates of a species, revealed some close relationships. For example, the strains Endo2 and Erot24 in C. emberorum are only 3 bp different in D1/D2 region, and there are only minor differences within the species of C. panamericana and C. bribrorum in that region (Table 1Go). The closely related CT clade yeasts additionally were supported by other taxonomic characters, and more than 80 physiological tests have been useful in distinguishing the taxa (Table 2Go). In addition to identification of CT clade species using BLAST searches and phylogenetic analyses based on DNA, identification was possible using physiological traits.


    ACKNOWLEDGEMENTS
 
We thank Louisiana State University undergraduate students Christine Ackerman, Katie Brillhart, Cennet Erbil, Nhu Nguyen, Ebony Spikes and Amy Whittington for their skilled assistance in all phases of this study. Dr Donald Windsor, Dr Annette Aiello, Ms Oris Acevedo and Ms Maria Leone graciously provided information and helped with logistics for collecting at the Smithsonian Tropical Research Institute, Barro Colorado Island, Panama. Drs Jean Euzéby and Richard Warga gave expert advice on Latin names and descriptions. We acknowledge the curators and culture collections that preserve the germ plasm derived from and used in our studies: NRRL, CBS and ATCC. Use of the GenBank public database also is acknowledged. The work was supported by the National Science Foundation, Biodiversity Surveys and Inventories Program (DEB-0072741) and by the Louisiana State University Boyd Professor Fund.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
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