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Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We isolated five yeasts related to Candida mesenterica from the digestive tract, frass, and habitat of beetles in six families inhabiting basidiocarps. Based on rDNA sequence comparisons and phenotypic characters, the yeasts were identified as Kodamaea ohmeri and four undescribed taxa. Phylogenetic analysis of combined small and large subunit ribosomal DNA sequences placed the five taxa in a statistically well supported clade with C. mesenterica, Candida suecica and other yeast species known from basidiocarps, including ‘Endomyces scopularum’ (CBS154.92 and 155.92), Candida fukazawae, Candida fungicola and Candida sagamina. Only one of the new taxa produced ascospores; the other three reproduced only asexually. The yeasts appear to be less closely associated with beetles than with the beetle habitat. The new species and their type strains are Kodamaea laetipori (type strain NRRL Y-27713 T), Candida derodonti (type strain NRRL Y-27711 T), Candida arcana (type strain NRRL Y-27712 T) and Candida plutei (type strain NRRL Y-27715 T).
Key words: diversification, molecular phylogeny, mycophagy, Saccharomycetes, symbiosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The majority of the isolates were ascomycete yeasts that were clustered in several major clades throughout the yeast phylogenetic tree. Some of the yeasts were isolated on numerous occasions from the previously untapped insect gut habitat and apparently were tied to this habitat (Suh and Blackwell 2005; Suh et al 2004a, b). Other yeasts from beetles, however, appeared to be associated primarily with the basidiocarp habitat and only secondarily with the beetle. In this paper we discuss the relationships, phenotypic characters and habitat associations of Kodamaea ohmeri and four new yeasts and describe the new species represented among 16 strains in the Candida mesenterica Diddens & Lodder clade (Saccharomycetes). It is not clear if the habitat of these species is a source for repeated transient infection of the insects or if some of the yeasts have a more specific symbiotic association with the beetles themselves.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Suh et al 2004a, b). Host beetles collected in the southeastern USA and Barro Colorado Island, Panama, (TABLE I) were surface disinfected in 95% ethanol. After the alcohol wash beetles were rinsed in 0.7% saline and 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 and transferred to 0.7% saline. Crushed gut segments were streaked on acidified YM agar, and the plates were incubated at 25 C with single colonies streaked for purification. Selected isolates were deposited at the Agricultural Research Service Culture Collection (NRRL) and Centraalbureau voor Schimmelcultures (CBS); holotype specimens have been deposited at NRRL as lyophilized specimens (TABLE I). Morphological observations and metabolic tests were performed according to established methods (Yarrow 1998, Barnett et al 2000). Isolates within each subclade were crossed in all combinations; in addition all 16 isolates were crossed with tester strains of K. ohmeri of known mating type (NRRL Y-2078, Y-2079 and Y-2080) and observed on YM agar, 2% malt agar, and cornmeal agar, for up to 6 wk to assess ascospore development.
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, b). The D1/D2 region of the large subunit (LSU) rDNA was amplified directly from yeast cells. One loopful of cells was added to 50 µL of autoclaved water and heated at 95 C for 5 min. 2 µL of the supernatant were used as a template in 50 µL polymerase chain reactions (PCRs). Nucleic acid extraction for small subunit (SSU) rDNA followed the procedures of Lee and Taylor (1990). The primer sets NS1-NS8 and LS1-LR5 were used for PCR amplification of SSU and LSU rDNA, respectively (White et al 1990, Hausner et al 1993). 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 PRISMTM BigDye Terminator cycle sequencing kit. Complete sequences of the SSU rDNA and the D1/D2 region of the LSU rDNA were obtained with the primers NS1, NS2, 18H, NS5, NS8, LS1, and LR3 using an ABI PRISM 377 Automated DNA sequencer. A modified primer SS3E (5-GATCCTTCCGCAGGTTCACC-3) sometimes was used instead of NS8.
GenBank accession numbers from this study are listed (TABLE I
). DNA sequences initially were aligned with the multi-alignment program Clustal X (Thompson et al 1997) and optimized visually, and ambiguous regions were excluded from analyses. The sequences from newly isolated yeasts were compared with LSU and SSU rDNA sequences of fungi obtained from GenBank. Yeast isolates were grouped by their identical D1/D2 sequences. Maximum parsimony analyses were performed using PAUP 4.0b10 (Swofford 2002), 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and TABLE II).
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).
Kodamaea laetipori S.-O. Suh et M. Blackwell, sp. nov. FIGS. 2–4
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 C cellulae vegetative subglobosae aut fusiformes (2–6 x 4–9 µm). Plerumque subglobosae, singulae vel binae; pseudohyphae fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 C, candida aut cremea, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 C, pseudohyphae et hyphae verae fiunt. Ascosporae fiunt. Glucosum, maltosum, sucrosum (variabile), trehalosum, cellobiosum (lente) et melezitosum (lente) fermentantur. Galactosum, 
-methyl-D-glucosidum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, L-sorbosum (lente, infirme), D-glucosaminum (lente), D-xylosum, D-arabinosum (infirme), sucrosum, maltosum, trehalosum, -methyl-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum, amylum solubile (lente), glycerolum, ribitolum, D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D-gluconatum, DL-acidum lacticum (lente), acidum succinicum (lente), acidum citricum, ethanolum et propane-1, 2-diolum (lente). Non assimilantur galactosum, D-ribosum, L-arabinosum, L-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, erythritolum, xylitolum, L-arabinitolum, galactitolum, inositolum, D-glucuronatum, methanolum, butano-2, 3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, L-lysinum, cadaverinum et glucosaminum (variabile). Non assimilantur kali nitratum, sodii nitritum, creatinum, creatininum, imidazolum et D-tryptophanum. Amylum non formatur. Bio-tinum externum ad crescentiam necessarium est. Augmentum fiunt in temperatura 35 C. Crescit in medio 100 µg mL–l cycloheximido addito.
HOLOTYPUS.. NRRL Y-27713 T (= CBS 9884), designat stirpem typicum. Isolata a ile coleopterorum (Diaperis sp.: Tenebrionidae) ex Laetiporus sulphureus, Baton Rouge, Louisiana, USA, depositata in Collectione Culturarum ARS (NRRL), Peoria, Illinois, USA. Isotypus NRRL Y-27779 isolata a ile coleopterorum (Diaperis sp.: Tenebrionidae) ex Laetiporus sulphureus, Baton Rouge, Louisiana, USA, depositata in Collectione Culturarum ARS (NRRL), Peoria, Illinois, USA.
After 7 d at 25 C in YM broth, yeast cells are sub-globose to fusiform, (2–6) x (4–9) µm, mostly sub-globose; occurring singly, in pairs, or in short chains (FIG. 3); pseudohyphae present. After 7d at 25 C on YM agar, colonies are white to cream colored, membranous, filamentous, and rough on top. On Dalmau plate culture on cornmeal agar septate hyphae and pseudohyphae with blastoconidia present after 10 d at 25 C (FIG. 4); aerobic growth, white and fuzzy. Only three crosses [(NRRL Y-27713 T X Y-27779), (NRRL Y-27779 X BG 02-5-27-4-3-4), and (BG 02-5-27-4-2-5 X BG 02-5-27-4-5-C2)] produced 1–4 hat-shaped ascospores after 2 wk at 17 C, although the nine strains were crossed in all combinations (FIG. 2). The results of fermentation, assimilation and other physiological tests are listed (TABLE II).
Type strain.. NRRL Y-27713 T (= CBS 9884) is preserved as a lyophilized preparation in the Agricultural Research Service Culture Collection (NRRL), Peoria, Illinois, USA. The strain was isolated from gut of Diaperis sp. (Tenebrionidae) ex Laetiporus sulphureus, Baton Rouge, Louisiana, USA. Isotype NRRL Y-27779 of complementary mating type from gut of Diaperis sp. (Tenebrionidae) ex Laetiporus sulphureus, Baton Rouge, Louisiana, USA.
Etymology.. The species name laetipori (N.L. gen. n.) refers to the genus of the basidiomycete host, Laetiporus sulphureus.
Candida derodonti S.-O. Suh et M. Blackwell, sp. nov. FIGS. 5, 6
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 C cellulae vegetative subglobosae aut ellipsoideae (2.5–6.25 x 3.7–7.5 µm), singulae vel binae; pseudohyphae fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 C, candida aut cremea, butyrosa et teres. In agaro farina Zeae maydis confecto post 10 dies ad 25 C, pseudohyphae et hyphae verae fiunt. Ascosporae non fiunt. Glucosum fermentantur. Galactosum, maltosum, -methyl-D-glucosidum, sucrosum, trehalosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, D-glucosaminum, D-ribosum, sucrosum, maltosum, trehalosum, -methyl-D-glucosidum, cellobiosum, salicinum, arbutinum, glycerolum, ribitolum, D-glucitolum, D-mannitolum, gluconolactonum (lente, infirme), 2-keto-D-gluconatum, acidum succinicum, acidum citricum, ethanolum et propane-1, 2-diolum (infirme). Non assimilantur galactosum, L-sorbosum, D-xylosum, L-arabinosum, D-arabinosum, L-rhamnosum, melibiosum, lactosum, raffinosum, melezitosum, inulinum, amylum solubile, erythritolum, xylitolum, L-arabinitolum, galactitolum, inositolum, D-gluconatum, D-glucuronatum, DL-acidum lacticum, methanolum, butano-2, 3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, L-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. Augmentum non fiunt in temperatura 35 C. Non crescit in medio 10 µg mL–l cycloheximido addito.
HOLOTYPUS.. NRRL Y-27711 T (= CBS 9882), de-signat stirpem typicum. Isolata a ile coleopterorum (Derodontus esotericus; Derodontidae) ex Hericium sp., Athens, Georgia, USA, depositata in Collectione Culturarum ARS (NRRL), Peoria, Illinois, USA.
After 7 d at 25 C in YM broth, yeast cells are sub-globose to ellipsoidal, (2.5–6.25) x (3.7–7.5) µm; occurring singly, in pairs or in short chains (FIGS. 5–6); pseudohyphae may be present. After 7d at 25 C on YM agar, colonies are white to cream colored, butyrous, and smooth on surface. On Dalmau plate culture on cornmeal agar septate hyphae and pseudohyphae with blastoconidia present after 10 d at 25 C; aerobic growth, white, shiny, and smooth. Ascospores not produced after 6 wk at 17 C from individual strains or strains crossed in all combinations on half strength cornmeal agar. The results of fermentation, assimilation and other physiological tests are listed (TABLE II).
Type strain.. NRRL Y-27711 T (= CBS 9882) is preserved as a lyophilized preparation in the Agricultural Research Service Culture Collection (NRRL), Peoria, Illinois, USA. The strain was isolated from the gut of Derodontus esotericus (Derodontidae) ex Hericium sp., Athens, Georgia, USA.
Etymology.. The species name derodonti (N.L. gen. n.) refers to the genus of the host beetle, Derodontus esotericus.
Candida arcana S.-O. Suh et M. Blackwell, sp. nov. FIG. 7
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 C cellulae vegetative globosae aut fusiformes (2.5–5 x 3.7–5 µm), singulae vel binae; pseudohyphae non fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 C, candida aut cremea, butyrosa et teres. In agaro farina Zeae maydis confecto post 10 dies ad 25 C, pseudohyphae fiunt. Ascosporae non fiunt. Glucosum (infirme) et trehalosum fermentantur. Galactosum, maltosum, -methyl-D-glucosidum, sucrosum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et D-xylosum non fermentantur. Assimilantur glucosum, D-glucosaminum (lente), sucrosum, maltosum, trehalosum, salicinum (lente), arbutinum (lente), glycerolum (lente), ribitolum, D-glucitolum, D-mannitolum, gluconolactonum (lente), 2-keto-D-gluconatum, D-gluconatum (infirme), acidum succinicum (lente), acidum citricum (lente) et ethanolum (lente). Non assimilantur galactosum, L-sorbosum, D-ribosum, D-xylosum, L-arabinosum, D-arabinosum, L-rhamnosum, -methyl-D-glucosidum, cellobiosum, melibiosum, lactosum, raffinosum, melezitosum, 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 (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 30 C. Crescit in medio 10 µg ml–l cycloheximido addito (lente), non crescit in medio 100 µg mL–l.
HOLOTYPUS.. NRRL Y-27712 T (= CBS 9883), de-signat stirpem typicum. Isolata summus coleopterorum (Derodontus esotericus; Derodontidae), Athens, Georgia, USA, depositata in Collectione Culturarum ARS (NRRL), Peoria, Illinois, USA.
After 7 d at 25 C in YM broth, yeast cells are globose to fusiform, (2.5–5) x (3.7–5) µm; occurring singly, in pairs or in short chains (FIG. 7); pseudohyphae not present. After 7d at 25 C on YM agar, colonies are white to cream colored, butyrous, and smooth on the surface. On Dalmau plate culture on cornmeal agar pseudohyphae present after 10 d at 25 C; aerobic growth, white, shiny and smooth. Ascospores not produced after 6 wk at 17 C from individual strains or strains crossed in all combinations on half-strength cornmeal agar. The results of fermentation, assimilation, and other physiological tests are listed (TABLE II).
Type strain.. NRRL Y-27712 T (= CBS 9883) is preserved as a lyophilized preparation in the Agricultural Research Service Culture Collection (NRRL), Peoria, Illinois, USA. The strain was isolated from surface of Derodontus esotericus (Derodontidae), Athens, Georgia, USA.
Etymology.. The species name arcana (N.L. gen. n.) echoes the species of the host beetle, Derodontus esotericus, and calls attention to the interesting appearance of a second species of closely related yeast on the same host by an undetermined mechanism of diversification.
Candida plutei S.-O. Suh et M. Blackwell, sp. nov. FIGS. 8, 9
In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 C cellulae vegetative ovoidae et cylindratae (2–5 x 4–10 µm), singulae vel binae; pseudohyphae fiunt. Cultura in agaro extramalti et faecis continente post 7 dies ad 25 C, candida aut cremea, margine ciliata. In agaro farina Zeae maydis confecto post 10 dies ad 25 C, pseudohyphae et hyphae verae fiunt. Ascosporae non fiunt. Fermentatio nulla. Assimilantur glucosum, L-sorbosum (infirme), D-xylosum, sucrosum, maltosum, trehalosum, -methyl-D-glucosidum, cellobiosum, salicinum, arbutinum, melezitosum, amylum solubile, glycerolum, ribitolum, D-glucitolum, D-mannitolum, 2-keto-D-gluconatum, acidum succinicum et acidum citricum (infirme). Non assimilantur galactosum, D-glucosaminum, D-ribosum, L-arabinosum, D-arabinosum, L-rhamnosum, melibiosum, lactosum, raffinosum, inulinum, erythritolum, xylitolum, L-arabinitolum, galactitolum, inositolum, gluconolactonum, D-gluconatum, D-glucuronatum, DL-acidum lacticum, methanolum, ethanolum, propane-1, 2-diolum, butano-2, 3-diolum, acidum quinicum, D-glucaratum et D-galactonatum. Assimilantur ethylaminum, L-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. Augmentum non fiunt in temperatura 35 C. Non crescit in medio 10 µg mL–l cycloheximido addito.
HOLOTYPUS.. NRRL Y-27715 T (= CBS 9885), designat stirpem typicum. Isolata a ile coleopterorum (Staphylinidae) ex Pluteus cervinus, Baton Rouge, Louisiana, USA, depositata in Collectione Culturarum ARS (NRRL), Peoria, Illinois, USA.
After 7 d at 25 C in YM broth, yeast cells are ovoid, elongate, and cylindrical, (2–5) x (4–10) µm; occurring singly, in pairs or in short chains (FIG. 8); pseudohyphal growth is also present. After 7d at 25 C on YM agar, colonies are white to cream colored, membranous, filamentous, and rough on top with a fuzzy margin. On Dalmau plate culture on cornmeal agar septate hyphae and pseudohyphae with blastoconidia present after 10 d at 25 C (FIG. 9); aerobic growth, white, fuzzy, and rough rigid top. Ascospores not produced after 6 wk at 17 C from individual strains or strains crossed in all combinations on half strength cornmeal agar. The results of fermentation, assimilation and other physiological tests are listed (TABLE II).
Type strain.. NRRL Y-27715 T (= CBS 9885) is preserved as a lyophilized preparation in the Agricultural Research Service Culture Collection (NRRL), Peoria, Illinois, USA. The strain was isolated from gut of unidentified staphylinids beetle (Staphylinidae) ex Pluteus cervinus, Baton Rouge, Louisiana, USA.
Etymology.. The species name plutei (N.L. gen. n.) refers to the genus of the basidiomycete host, Pluteus; coincidentally, it also recalls the fuzzy colony margin appearing as a breastwork.
Phylogenetic relationships of new species.— –
A single most parsimonious tree resulted from analysis of approximately 2400 bp of combined sequence data of SSU and LSU rDNA from the new species and selected ascomycetes, including both Pezizomycetes and Saccharomycetes (FIG. 10); the analyses included all close relatives of the new strains. Members of the basal ascomycetes (Taphrinomycotina) were out-group taxa. The new species were distinguished from each other and from previously described species in a well supported C. mesenterica clade of 13 species, consisting of three subclades. The sister clade of the C. mesenterica clade included species of Clavispora, Metschnikowia and their asexual relatives (FIG. 10) (Kurtzman and Robnett 1997).
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). Although the individual branches were not well supported by bootstrapping, C. plutei showed a close relationship to C. fungicola; K. laetipori was a sister taxon of C. sagamina and C. fukazawae. Kodamaea laetipori was isolated not only from the gut of tenebrionid and scarabaeid beetles but also from the surface of the beetle and frass in the L. sulphureus basidiocarp. Candida plutei, however, was found only in the gut of a staphylinid beetle from the basidiocarp of P. cervinus. Candida plutei, K. laetipori, and other previously described Candida species in the clade are not clearly distinguishable morphologically, although physiological characters are sufficient to identify these taxa (TABLE II).
Subclade B was basal to subclade A, and consisted of K. ohmeri, including the two new isolates from this study. In a separate analysis of the D1/D2 loop sequences only (data not shown) the four known species of Kodamaea and Candida restingae Rosa, Lachance, Starmer, Barker, Bowles & Schlag-Edler (Lachance et al 1999, Rosa et al 1999) and the two new K. ohmeri isolates from this study formed a well supported monophyletic group as expected.
Members of subclade C received strong support as a monophyletic grouping that was a sister of subclades A and B. Both of the new taxa, C. derodonti and C. arcana, were associated with a derodontid beetle. Candida derodonti was isolated from the beetle gut, and C. arcana was present on its outer surface (TABLE I). The two new species are closely related but are clearly distinguished at the level of species by both molecular and physiological characters (FIG. 10 and TABLE II). Candida mesenterica and Candida suecica Rodrigues de Miranda & Norkrans are basal members of the subclade.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In the case of the yeasts in the C. mesenterica clade, however, the ties between the yeasts and insects do not seem to be strong, based on these criteria. The trend within the clade, including both the new isolates and most previously described species, appears to be for closer associations with the habitat than with the insects, especially the basidiocarp in the case of subclade A members.
In subclade A previously described members (C. fungicola, C. sagamina, C. fukazawae, and ‘E. scopularum’ ) were isolated from mushrooms (de Hoog 1998, Nakase et al 1999). Kodamaea laetipori was isolated not only from the gut of tenebrionid and scarabaeid beetles in basidiocarps, but also from the surface of the beetle body and frass in L. sulphureus. Candida plutei, on the contrary, was found only in the gut of a staphylinid beetle collected in the agaric, P. cervinus (TABLE I). ‘Endomyces scopularum,’ a member of this basidiocarp-associated subclade, has been a source of taxonomic confusion. Three strains parasitic on agaric basidiocarps deposited as E. scopularum in the CBS culture collection were studied using DNA sequences (Kurtzman and Robnett 1995, Suh et al 2001). CBS 131.86 was a Sporothrix (Ophiostomatales); CBS 154.92 and 155.92 from basidiocarps of Tricholoma from Germany, however, were identified as true yeasts with identical SSU and LSU rDNA sequences. CBS 154.92 and CBS 155.92 are considered currently to belong to an undescribed species of Candida (Suh et al 2001).
Kodamaea ohmeri, a sexually reproducing member of the C. mesenterica clade, is a member of subclade B (FIG. 10). It was reported previously from the widest variety of substrates of any member of the C. mesenterica clade, including leaves and fruits, films on brine and fermented foods and clinical isolations. This species also is known from a basidiocarp (e.g., ATCC 64326, ex Suillus variegatus (Sw.: Fr.) O. Kuntze fruiting body, Czechoslovakia). Our discovery of two new isolates collected in association with mushroom-feeding beetles in widely separated localities, suggests that K. ohmeri might be more common in basidiocarps than has been recognized previously. Other members of the clade, Kodamaea kakaduensis Lachance, Bowles, Starmer & Barker, Kodamaea nitidulidarum Rosa, Lachance, Starmer, Barker, Bowles & Schlag-Edler, Kodamaea anthophila Rosa, Lachance, Starmer, Barker, Bowles & Schlag-Edler and C. restingae, are well known from nitidulid beetles and Drosophila in associations with decaying cactus flowers in Brazil and with Hibiscus and morning glory flowers in Australia and Hawaii (Lachance et al 1999, 2001; Rosa et al 1999). A direct connection between the Kodamaea strains from flowers and basidiocarps through the nitidulid associates seems implausible because the beetles are very different in their habitat and geographical distribution.
New subclade C isolates were from the gut and body surface of Derodontus esotericus, a generalist feeder on basidiocarps (Leschen 1994). We seldom encountered this beetle in our collecting and do not know if our two isolates are an indication that there is a group of closely related yeasts associated with these beetles or if the discovery of these new taxa is a coincidence. Previously known clade members, C. mesenterica and C. suecica, were isolated from beer and seawater, respectively.
Geographical distribution.— – Most species of the C. mesenterica clade have restricted distributions and have not been collected often from North American or the Western Hemisphere. Our isolates came from Georgia and Louisiana, USA, except for one Panamanian strain of K. ohmeri, the only known broadly distributed species. Except for K. ohmeri, the other new isolates were the first members of the clade to be reported from North American, and only K. nitidulidarum of the clade members had been reported from the Western Hemisphere. Many clade members are known only from the original studies and have not isolated since that time.
Sexual reproduction.— – The generic designation of K. laetipori is somewhat problematic and points to the need for a revision of yeast taxonomy at the level of genus. Although the species of Kodamaea discussed here have morphological features in common with the type species of Pichia, P. membranifaciens (E.C. Hansen) E.C. Hansen, they are in distantly related clades. The placement of K. laetipori in Kodamaea in this study is based primarily on its placement near Kodamaea species as the closest sexual relatives, albeit in a different subclade.
We seldom have encountered sexual reproduction among the yeasts isolated from beetles in basidiocarp habitats, even when closely related strains are crossed. Kodamaea laetipori, the new taxon discovered in this study with sexual reproduction, has been isolated not only from the gut contents of beetles but also from the surface of the beetles and frass in the mushroom habitats of the host beetles, suggesting that K. laetipori might be acquired from the environment by insects. In contrast none of the other isolates described in this paper produced ascospores, even in crosses of strains with identical D1/D2 sequences or tester strains of known mating types of K. ohmeri, although they do not have identical D1/D2 sequences and might not be expected to cross. It should be noted that the majority of the crosses among the nine strains of K. laetipori did not result in ascospore formation. Of special interest in this connection is K. ohmeri, K. kakaduensis, K. nitidulidarum and K. anthophila, all of which possess heterothallic mating systems (Kurtzman 1998, Lachance et al 1999, Rosa et al 1999). Although the D1/D2 loop sequences of our two new isolates were identical to those of K. ohmeri, we did not observe ascospore formation when they were crossed. Because we had only two isolates the probability that we have both mating types is only 25%; ascospores, however, were not formed with the tester strains of known mating types. This is an important aspect of the biology of beetle-associated isolates, because information on recombination or lack thereof is important to hypotheses of diversification and isolation with insect hosts (Suh et al 2003).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: ssuh{at}lsu.edu
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LITERATURE CITED |
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de Hoog GS. 1998. Endomyces Reese. In: Kurtzman CP, Fell JW, eds. The yeasts, a taxonomic study. 4th ed. Amsterdam: Elsevier. p 194–196.
Hausner G, Reid J, Klassen GR. 1993. On the subdivision of Ceratocystis s. l., based on partial ribosomal DNA sequences. Can J Bot 71:52–63.
Kurtzman CP. 1998. Pichia E. C. Hansen emend. Kurtzman. In: Kurtzman CP, Fell JW, eds. The yeasts, a taxonomic study, 4th ed. Amsterdam: Elsevier. p 273–352.
——— Robnett CJ.1995. Molecular relationships among hyphal ascomycetous yeasts and yeastlike taxa. Can J Bot 73:S824–S830.[CrossRef]
———, ———. 1997. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large subunit (26S) ribosomal DNA gene. J Clin Microbiol 35:1216–1223.[Abstract]
Lachance M-A, Bowles JM, Starmer WT, Barker JSF. 1999. Kodamaea kakaduensis and Candida tolerans, two new ascomycetous yeast species from Australian Hibiscus flowers. Can J Microbiol 45:172–177.[CrossRef][Medline]
———, Starmer WT, Rosa CA, Bowles JM, Barker JSF, Janzen DH. 2001. Biogeography of the yeasts of ephemeral flowers and their insects. FEMS Yeast Res 1:1–8.[Medline]
Lee SB, Taylor JW. 1990. Isolation of DNA from fungal mycelia and single spores. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols—a guide to methods and applications. San Diego, California: Academic press. p 282–287.
Leschen RAB. 1994. Fungal host use in two species of Derodontus Leconte (Coleoptera: Derodontidae). Coleopterists Bulletin 48:126–130.
Nakase T, Suzuki M, Sugita T, Suh S-O, Komagata K. 1999. Three new species of anamorphic yeasts phenotypically and phylogenetically related to Candida mesenterica: the description of Candida fungicola sp. nov., Candida sagamina sp. nov., and Candida fukazawae sp. nov. isolated from fruit bodies of mushrooms. Mycoscience 40: 465–476.[CrossRef]
Rosa CA, Lachance MA, Starmer WT, Barker JSF, Bowles J, Schlag-Edler B. 1999. Kodamaea nitidulidarum, Candida restingae, and Kodamaea anthophila, three new related yeast species from ephemeral flowers. Int J Syst Biol 49:309–318.
Suh S-O, Blackwell M. 2005. The beetle gut as a habitat for new species of yeasts. In: Vega FE, Blackwell M, eds. Insect fungal associations: Ecology and Evolution. New York: Oxford University Press. p 244–256.
———, Gibson CM, Blackwell M. 2004a. Metschnikowia chrysoperlae sp. nov., Candida picachoensis sp. nov. and Candida pimensis sp. nov., isolated from the green lacewings Chrysoperla comanche and Chrysoperla carnea (Neuroptera: Chrysopidae). Int J Syst Evol Microbiol 54:1883–1890.
———, Kurtzman CP, Blackwell M. 2001. The status of Endomyces scopularum-a filamentous fungus and two yeasts. Mycologia 93:317–322.[CrossRef]
———, McHugh JV, Blackwell M. 2004b. Expansion of the Candida tanzawaensis yeast clade: 16 new Candida species from basidiocarp-feeding beetles. Int J Syst Evol Microbiol 54:2409–2429.
———, Marshall CJ, McHugh JV, Blackwell M. 2003. Wood ingestion by passalid beetles in the presence of xylose-fermenting gut yeasts. Mol Ecol 12:3137–3145.[CrossRef][Medline]
Swofford DL. 2002. PAUP. Phylogenetic Analysis Using Parsimony (*and other methods), version 4.0b10. Sunderland, Massachusetts: Sinauer Associates.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tool. Nucleic Acids Res 25:4876–4882.
White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols—a guide to methods and applications. San Diego, California: Academic Press. p 315–322.
Yarrow D. 1998. Methods for the isolation, maintenance and identification of yeasts. In: Kurtzman CP, Fell JW, eds. The yeasts, a taxonomic study, 4th ed. Amsterdam: Elsevier. p 77–100.
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