International Journal of Systematic and Evolutionary Microbiology (2004), 54, 2409-2429 DOI 10.1099/ÍJS.0.63246-0

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

Sung-Oui SuhJ Joseph V. McHugh^ and Meredith BlackwelP

Correspondence ^Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA Sung-Oui Suh ^Department of Entomology, University of Georgia, Athens, GA 30602, USA [email protected]

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 were most commonly sampled. Repeated isolation of certain yeasts from the same 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 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-27568'^ = CBS QSSS'^), Candida bol

INTRODUCTION additional fungi and hosts might be discovered. During a study of fungi from the digestive tract of Yeasts and yeast-like endosymbionts have been reported basidiocarp-feeding beetles, about 650 yeasts were isolated from a variety of , including planthoppers, aphids from beetles in 26 families (Suh & Blackwell, 2004). Based and beetles (e.g. Nardon & Grenier, 1989; Noda & Omura, on sequence comparisons of the D1/D2 loop of the large 1992; Suh etal, 2003,2004). Although some endosymbionts subunit (LSU) rRNA gene used for rapid yeast character- from anobiid beetles and planthoppers show an affinity ization, about 30 % of the yeast isolates formed a clade (CT to certain filamentous ascomycetes (Jones & Blackwell, clade) with Candida tanzawaensis, a yeast isolated from 1996; Noda et al, 1995; Noda & Kodama, 1996; Suh et al, mosses in Japan (Nakase et al, 1988). Until the relatively 2001), the majority of the fungal endosymbionts have been recent report of six novel species (Candida ambrosiae, identified as true yeasts (Ascomycetes: Saccharomycetes) Candida canberraensis, Candida caryicola, Candida pruni- (Jones et al, 1999). We were interested in sampling insects cola, Candida pyralidae and Candida xylopsoci), C. tanzawa- more broadly in order to determine if associations between ensis had no known close relatives (Kurtzman, 2001). Currently, only one of the seven previously described Published online ahead of print on 28 June 2004 as DOI 10.1099/ species is known from multiple collections; in fact, C. ijs.0.63246-0. tanzawaensis awaited description for 22 years in the vain Abbreviations: CT clade, Candida tanzawaensis clade; LSU, large hope that another isolate would be discovered (Nakase subunit; SSU, small subunlt. et al, 1988). Here, we describe 16 novel taxa, many with The GenBank/EMBL/DDBJ accession numbers for the sequences multiple isolates, from the gut of fungus-feeding beetles determined In this study are listed In Table 1 and Fig. 1. and compare the characteristics of all 23 members of the

63246 © 2004 lUMS Printed in Great Britain 2409 S.-O. Suh, J. V. McHugh and M. Blackwell large CT clade, thereby recognizing unsuspected diversity in optimized visually. Ambiguous regions were excluded from the ana- what appears to be a largely insect-associated clade. lyses. 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 METHODS designated by the first four letters of the host beetle family and a number for each unique genotype varying by one or more base Yeast isolation and identification. Host beetles were collected pairs from other yeasts from the same beetle family. Maximum- from several different localities in Vermont, the southeastern USA, parsimony analyses were performed using PAUP* 4.0blO (Swofford, and Barro Colorado Island, Panama. Vouchers were deposited at the 2002). Heuristic tree searches were executed using the tree bisection- University of Georgia Collection of , Athens, GA. The reconnection branch-swapping algorithm with random sequence beetles were taken to the laboratory and usually placed in Petri analysis. Bootstrap values of the most parsimonious tree were dishes for 1-3 days without food prior to dissection. Surface disin- obtained from 1000 replications. Base pair differences were counted fection was by submersion in 95 % ethanol for 1-2 min. The alcohol using BLAST2 Sequences (Tatusova 8c Madden, 1999) or from the wash was followed by a 0-7% saline rinse; the rinse liquid was manually aligned sequence database. 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 trans- RESULTS AND DISCUSSION ferred to tubes containing 0-7% saline. The gut segments were crushed in the saline solution with a pipette tip and streaked with a Yeast isolates in the CT clade loop onto the surface of an acidified YM agar plate. Plates were incubated at 25 °C, and single colonies were streaked for purifica- We cultured about 650 yeasts from beetles in 26 families. tion. Cultures were maintained on YM agar or 2 % malt extract agar Most of the isolates were true yeasts (Ascomycota: (Yarrow, 1998). The isolates were initially screened and grouped Saccharomycetes). Phylogenetic analysis of about 600 bp using the D1/D2 loop sequence of the LSU rRNA gene. Selected isolates from each LSU genotype have been deposited at the Agri- of sequence from the D1/D2 region of the LSU rRNA gene cultural Research Service Culture Collection (NRRL) and Centraal- was used for rapid identification and grouping of the bureau voor Schimmelcultures (CBS); holotype specimens were isolates. We discovered a major clade consisting of about deposited at NRRL as lyophilized cultures (Table 1). The morpholo- 30% of all beetle gut yeast isolates obtained using this gical observations and metabolic tests comprising the yeast standard technique. The taxa were closely related to C tanzawa- description were performed on at least one isolate from each LSU ensis and six other previously described Candida species genotype, according to established methods (Yarrow, 1998; Barnett (Kurtzman, 2001). Detailed information about the 164 et al., 2000). Isolates within each clade were crossed in all combina- tions and observed on YM agar, 2 % malt agar and cornmeal agar at yeast isolates in the greatly enlarged CT clade is provided 17 °C for 6 weeks in order to detect ascospore development. Bright- in Table 1. The gut yeasts in the CT clade were isolated field microscopic examination was the usual method of observation, from species in 11 families of beetles: Anthribidae, but some cell preparations were examined more closely with fluores- Carabidae, Ciidae, Endomychidae, Erotylidae, Histeridae, cence microscopy using 0-02% Calcofluor white as an optical Melandryidae, Nitidulidae, Scarabaeidae, Staphylinidae brightener to visualize yeast cell walls more clearly. (including Scaphidiinae) and Tenebrionidae. However, DNA sequencing and sequence analysis. The D1/D2 region of the majority of the yeasts (140 of 164 isolates, Table 1) the LSU rRNA gene was amplified directly without DNA extraction were fi-om species of Erotylidae and Tenebrionidae. The from yeast cells. A cell suspension of one loopful of cells in 50 |.il of CT clade gut yeasts were sorted into 39 groups based on autoclaved water was heated at 95 °C for 5 min, and 2 ]i\ of the their D1/D2 genotype and host beetle ranges. Only three supernatant was used as a template in 50 |il PCRs. For the small isolates (Erot8, Nitil4 and Tene7) had a D1/D2 genotype subunit (SSU) rRNA gene, the nucleic acids were extracted and identical to a previously described species (C. ambrosiae). purified following the procedures of Lee & Taylor (1990). The The other 36 groups were distinct fi-om all other previously primer sets NS1/NS8 and LS1/LR5 were used for PCR amplification of the SSU and LSU rRNA gene, respectively (White et al, 1990; described species (Table 1). Hausner et al., 1993). PCR products were purified using a DNA purification kit (Bio-Rad Laboratories), and purified double-stranded Novel species in the CT clade PCR products were used as templates for sequencing with an ABI PRISM BigDye Terminator cycle sequencing kit. The complete In addition to the D1/D2 region of the LSU rRNA gene sequence of the SSU rRNA gene and the D1/D2 region of the LSU sequences, complete sequences of the SSU rRNA gene rRNA gene were obtained with the primers NSl, NS2, 18H, NS5, (about 1750 bp) were determined for at least one isolate NS8, LSI and LR3 using an ABI PRISM 377 automated DNA from each LSU genotype (see Table 1 for selected isolates), sequencer. GenBank/EMBL/DDBJ accession numbers for DNA sequences from this study are listed in Table 1 and Fig. 1. SSU and these were combined with the LSU rRNA gene rRNA gene sequences of previously described Candida species in sequences in a dataset for better estimation of a phylo- the CT clade were determined to compare with the novel gut yeast geny. A most-parsimonious tree was constructed from the isolates. Previously described species names and GenBank/EMBL/ combined dataset of SSU and LSU rRNA gene sequences DDBJ accession numbers are as follows: C. ambrosiae NRRL YB- by comparing the yeasts with taxa in Ascomycota and 1316^ (AY227712), C. tanzawaensis NRRL Y-17324'^ (AY227713), other yeasts alone (Fig. 1). The 39 gut yeasts we compared C. canherraensis NRRL YB-2417'^ (AY488124), C. caryicola NRRL were representatives of the total 164 isolates in the clade YB-1499'^ (AY488125), C. prunicola NRRL YB-869'^ (AY488126), C. pyralidae NRRL Y-27085"^ (AY488127) and C. xylopsoci NRRL (Fig. 1) with C. tanzawaensis and the other six previously Y-27066^ (AY488128). DNA sequences were ahgned with the described clade members. The clade was well supported multi-alignment program CLUSTAL_X (Thompson et al, 1997) and statistically by 100% bootstrap value (Fig. 1). Of the seven

2410 International Journal of Systematic and Evolutionary Microbiology 54 Table 1. Strains of the novel Candida species and C. ambrosiae isolated in this study, and GenBank/EMBL/DDBJ accession numbers of the sequences

Species Strain designation* LSU rRNA Host beetles and place of collection* Total Nudeotide differences^ gene groups number of CBS NRRL LSU LSU SSU isolatesf rRNA gene rRNA gene

C. guaymorum 9823"^ Y-27568'^ BG 01-7-26-006B-l-l'^ Erot21 Mycotretus interstitialis (Erotylidae) ex imbricate 1 T T basidiocarp, BCI, Panama BG 01-7-26-006A-2-1 Erot21 Iphidus (Hahrodactylus) conspicillatus (Erotylidae) 3 0 0 ex imbricate basidiocarp, BCI, Panama BG 02-7-16-022A-1-1 Erot21 Cydomorphus sp. (Erotylidae) ex polypore, BCI, 3 0 - Panama BG 02-7-20-020A-1-1 Erot21 Iphidus sp. (Erotylidae) ex corticioid fungus, BCI, 1 0 - Panama BG 02-7-20-020B-1-1 Erot21 Cydomorphus sp. (Erotylidae) ex corticioid fungus, 1 0 - BCI, Panama Y-27581 BG 01-7-21-003A-1-1 Scar2 Onthophagus sp. (Scarabaeidae) ex Polyporus 1 0 0 tenuiculus, BCI, Panama C. hokatorum 9824"^ ¥-27571"^ BG 02-7-16-039A-2-l'^ Erot35 Pselaphacus signatus (Erotylidae) ex P. tenuiculus, 10 T T BCI, Panama BG 02-7-14-001E-4-1 Erot35 Mycotretus nitescens (Erotylidae) ex P. tenuiculus, 2 0 - BCI, Panama BG 02-7-16-030B-3-2 Erot35 Iphidus (Megaprotus) delineatus (Erotylidae) ex 3 0 - corticioid fungus, BCI, Panama BG 02-7-18-023B-1-2 Erot35 of Ellipticus gemellatus (Erotylidae) ex 1 0 - Tinctoporellus epimiltinus, BCI, Panama BG 02-7-14-OOlD-l-l Erot35 Pselaphacus sp. (Erotylidae) ex P. tenuiculus, BCI, 1 0 - Panama Y-27558 BG 02-7-14-OOlG-l-l Cara2 Unidentified carabid (Carabidae) ex P. tenuiculus, 1 0 0 BCI, Panama Y-27579 BG 02-7-14-OOlF-l-l Nitil9 Teichostethus testaceous (Nitidulidae) ex P. tenuiculus, 2 0 0 BCI, Panama Y-27576 BG 02-7-14-OOlJ-l-l Melal Unidentified melandryid (Melandryidae) ex P. 1 0 0 tenuiculus, BCI, Panama BG 02-7-14-001K-1-2 Tene27 Unidentified tenebrionid (Tenebrionidae) ex 2 0 0 P. tenuiculus, BCI, Panama C. kunorum 9825"^ ¥-27580"^ BG 02-7-18-017A-l-l'^ Niti25 T. testaceous (Nitidulidae) ex Nodulisporium sp., BCI, 1 T T Panama C. terrahorum 9826"^ ¥-27573"^ BG 02-7-15-019A-2-l'^ Erot41 Iphidus sedecimmaculatus (Erotylidae) ex corticioid 1 T T fungus, BCI, Panama CO p Table 1. cont. CO

Species Strain designation* LSU rRNA Host beetles and place of collection* Total Nucleotide differences! gene groups number of CBS NRRL LSU LSU SSU isolatest rRNA gene rRNA gene

C. emherorum 9827'^ ¥-27606"^ BG 01-7-22-OlOE-l-l"^ Erot24 Triplax alvarengai (Erotylidae) ex Pleurotis sp., BCI, 1 T T Panama BG 01-7-23-002A-1-1 Erot24 Mycotretus scitulus (Erotylidae) ex Pleurotis sp., BCI, 1 0 - Panama BG 01-7-22-012A-1-1-1 Endo2 Unidentified endomychid (Endomychidae) ex 2 3 0 Ripartitella brasiliensis, BCI, Panama C. wounanorum gsis'^ ¥-27574"^ BG 02-7-18-027A-l-l"^ Erot45 Mycotretus dorsonotatus (Erotylidae) ex corticioid 1 T T fijngus, BCI, Panama C. yuchorum gsig'^ ¥-27569"^ BG 01-8-26-OOlA-l-l"^ Erot26 Tritoma atriventris (Erotylidae) ex Lepiota sp., Athens, 3 T T GA, USA C. chickasaworum gsso'^ ¥-27566"^ BG 99-ll-14-10-l-l'^ Erot9 Tritoma sp. (Erotylidae) ex Amanita sp., Athens, GA, 7 T T USA BG 02-2-5-1-1 Erot9 Tritoma sp. (Erotyhdae) ex Pleurotus ostreatus. Baton 7 0 - Rouge, LA, USA BG 02-6-15-003A-1 Erot9 Dacne sp. (Erotylidae), Athens, GA, USA 3 0 - BG 02-6-15-003B-1 Erot9 Tritoma californica (Erotylidae), Athens, GA, USA 1 0 - Y-27561 BG 02-6-15-OlOC-l Ciid7 Unidentified did (Ciidae), Athens, GA, USA 2 0 0 C. choctaworum gssi'^ ¥-27584"^ BG 98-8-18-2"^ Tenel Neomida bicornis (Tenebrionidae) ex Fomitella supina. 12 T T Baton Rouge, LA, USA BG 98-12-9-1-1 Tenel N. bicornis (Tenebrionidae) ex F. supina, St Francisville, 8 0 0 LA, USA BG 02-5-30-OOlB-l Tenel Unidentified tenebrionid (Tenebrionidae), Athens, GA, 7 0 - USA Y-27559 BG 99-2-5-7-1-1 Ciid2 Ceracis curtus (Ciidae) ex F. supina. Baton Rouge, LA, 3 0 0 USA BG 02-5-30-008B-1 Ciid2 Unidentified did (Ciidae), Athens, GA, USA 2 0 - Y-27557 BG 02-3-29-3-1-1 Anth2 Euparius marmoreus (Anthribidae), Sulphur, LA, USA 2 0 0 C. bolitotheri gssi'^ ¥-27587"^ BG 00-8-15-1-1"^ Tenel1 Bolitotherus cornutus (Tenebrionidae) ex sp., 1 T T Athens, GA, USA BG 00-7-30-1-1 Tenel1 B. cornutus (Tenebrionidae) ex Ganoderma sp., 3 0 - Burlington, VT, USA BG 02-3-29-2-2 Tenel1 B. cornutus (Tenebrionidae) ex Ganoderma sp.. 3 0 - Sulphur, LA, USA BG 02-5-27-1-2-3 Tenel1 B. cornutus (Tenebrionidae) ex Ganoderma sp.. 3 0 Baton Rouge, LA, USA Table 1. cont.

Species Strain designation* LSU rRNA Host beetles and place of collection* Total Nucleotide differences^ gene groups number of CBS NRRL LSU LSU SSU isolatest rRNA gene rRNA gene

Y-27562 BG 99-8-11-1-1 Erot3 Megalodacne fasciata (Erotylidae) ex Ganoderma 4 0 0 applanatum, Athens, GA, USA C. atakaporum gsss'^ Y-iysyo'^ BG 02-7-21-Nhu-l-l-2"^ Erot28 Triplax festiva (Erotylidae) ex Inonotus cuticularis. Baton 1 T T Rouge, LA, USA C. panamericana gssé'^ Y-iysó?'^ BG 01-7-26-006B-2-l'^ Erot20 M. interstitialis (Erotylidae) ex imbricate mushroom, BCI, 1 T T Panama Y-27582 BG 01-7-26-006C-1-1 Stapl Unidentified staphylinid (Staphylinidae) ex imbricate 1 0 0 basidiocarp, BCI, Panama Y-27590 BG 02-5-27-1-2-4 Tenel9 B. cornutus (Tenebrionidae) ex Ganoderma sp.. Baton 1 1 0 Rouge, LA, USA C. bribrorum gsss'^ Y-27572'^ BG 02-7-14-0010-3-1^^ Erot38 Larvae of Pselaphacus sp. (Erotylidae) ex P. tenuiculus, 1 T T BCI, Panama BG 02-7-14-002A-2-1 Erot38 Megalodacne audouini (Erotylidae) ex basidiocarp, BCI, 3 0 - Panama BG 02-7-14-002B-2-1 Erot38 Pupa of Megalodacne sp. (Erotylidae) ex basidiocarp, 1 0 - BCI, Panama BG 02-7-20-004A-1-1 Erot38 Megalodacne sp. (Erotylidae) ex Ganoderma sp., BCI, 1 0 - Panama Y-27591 BG 02-7-14-002E-2-1 Tene28 Unidentified tenebrionid (Tenebrionidae) ex basidiocarp, 2 0 0 BCI, Panama Y-27592 BG 02-7-14-0021-1-1 Tene30 Unidentified tenebrionid (Tenebrionidae) ex basidiocarp, 2 1 0 BCI, Panama Y-27564 BG 99-8-11-1-4-2 Erote M. fasciata (Erotylidae) ex G. applanatum, Athens, GA, 1 2 1 USA C. maxii gssó'^ Y-27588'^ BG 01-7-21-006A-l-l'^ Tenel7 Unidentified tenebrionid (Tenebrionidae) ex polypore, 2 T T BCI, Panama C. anneliseae gssy'^ Y-27563'^ BG 99-8-11-1-2-2"^ Erot4 M. fasciata (Erotylidae) ex G. applanatum, Athens, GA, 2 T T USA BG 02-7-21-Nhu-l-l-C Erot4 T. festiva (Erotylidae) ex I. cuticularis. Baton Rouge, LA, 1 0 - USA Y-27583 BG 02-5-23-003E-5 Stap5 Unidentified Scaphidiinae (Staphylinidae), Athens, GA, 1 0 0 USA Y-27585 BG 98-9-2-2-4 Tene4 Platydema ruficorne (Tenebrionidae) ex decayed polypore. 11 0 0 Baton Rouge, LA, USA CO p CO

Table 1. cont.

Species Strain designation* LSU rRNA Host beetles and place of collection* Total Nucleotide differences! gene groups number of CBS NRRL LSU LSU SSU isolatest rRNA gene rRNA gene

BG 99-8-18-1-1 Tene4 Diaperis nigronotata (Tenebrionidae) ex Inonotus 4 0 0 ludovicianus. Baton Rouge, LA, USA S BG 00-6-14-1-1 Tene4 Neomida ferruginea (Tenebrionidae) ex basidiocarp. Baton 6 0 - o Rouge, LA, USA BG 01-7-21-OlOA-l-l Tene4 Unidentified small diaperine (Tenebrionidae) ex polypore, 0 - BCI, Panama BG 01-7-21-OlOB-l-l Tene4 Unidentified tenebrionid (Tenebrionidae) ex polypore, BCI, 0 - Panama BG 02-6-15-01 lB-2 Tene4 Alobates sp. (Tenebrionidae), Athens, GA, USA 0 - BG 02-5-30-001B-6 Tene4 Unidentified tenebrionid (Tenebrionidae), Athens, GA, USA 0 - Y-27577 BG 02-7-18-032A-1-1 Mela2 Unidentified melandryid (Melandryidae) ex polypore, BCI, 0 0 Panama BG 02-7-18-032C-1-1 Mela2 Larva of unidentified melandryid (Melandryidae) ex polypore, 0 - BCI, Panama Y-27575 BG 02-7-18-032B-2-1 HistS Unidentified histerid (Histeridae) ex polypore, BCI, Panama 0 1 Y-27560 BG 02-5-23-003C-4 CiidS Unidentified ciid (Ciidae), Athens, GA, USA 0 0 C. taliae 9838"^ ¥-27589"^ BG 01-7-23-018C-l-l'^ Tenel8 Unidentified tenebrionid (Tenebrionidae) ex polypore, BCI, T T Panama C. ambrosiae Y-27565 BG 99-8-11-1-C2 Erot8 M. fasciata (Erotylidae) ex G. applanatum, Athens, GA, USA 0 0 Y-27586 BG 99-8-18-1-4-1 Tene7 D. nigronotata (Tenebrionidae) ex I. ludovicianus. Baton 0 0 Rouge, LA, USA Y-27578 BG 01-7-26-005A-1-1 Nitil4 T. testaceous (Nitiduhdae) ex polypore, BCI, Panama 0 0

*CBS, Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; NRRL, Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, IL, USA; LSU, Mycology Laboratory, Department of Biological Sciences, Louisiana State University, Baton Rouge, LA, USA; BCI, Barro Colorado Island. tEach isolate came from one beetle. Total numbers include the yeasts isolated on different collection dates. tThe D1/D2 sequences for the LSU rRNA genes were determined for all yeast isolates and deposited in GenBank/EMBL/DDBJ. Accession numbers of LSU rRNA gene sequences are AY242241, AY242244, AY242246, AY242249, AY242253, AY242254, AY242257, AY242258, AY242260, AY242262, AY242263, AY242273, AY242274, AY242277, AY242278, AY242284, AY242288, AY242298, AY242312, AY242345, AY242350-AY242352, AY309784-AY309919 and AY426946-AY426949. The sequence of the SSU rRNA gene was determined for at least one isolate from each LSU group. See Fig. 1 for GenBank/EMBL/DDBJ accession numbers for SSU rRNA gene sequences. The base pair differences are from sequence comparisons between type strain (T) and other isolates in each species in the D1/D2 region of the LSU rRNA gene (about 600 bp) and the SSU rRNA gene (about 1750 bp). C ambrosiae isolates from this study were compared with its type strain, NRRL YB-1316'^. Expansion of the Candida tanzawaensis clade

. C. rhagii NRRL Y-2594T (AB018172/U45729) C. guaymorum NRRLY-27568T(AY242238/AY242350) C. guaymorum NRRL Y-27581 (AY242178/AY242288) bokatorum NRRL Y-27571T(AY426950/AY309798) bokatorum NRRL Y-27558 (AY426951/AY309803) bokatorum NRRL Y-27576 {AY426953/AY309806) bokatorum NRRL Y-27579 (AY426952/AY309804) bokatorum NRRL Y-27748 (AY426954/AY309807) • C. kunorum NRRL Y-27580T(AY426955/AY309809) 93jC. chickasaworum NRRL Y-27561 (AY426958/AY309833) C. chickasaworum NRRLY-27566T(AY242154/AY242263) 9_8_r C. emberorum NRRL Y-27606T (AY242168/AY242277) C. emberorum NRRL y-TllAl (AY242175/AY242284) 1821 C. wounanorum NRRL Y-27574T (AY426957/AY309813) C. yuchorum NRRL Y-27569T(AY242169/AY242278) C. terraborum NRRL Y-27573T{AY426956/AY309810) C. choctaworum NRRL Y-27584T(AY242136/AY242241) C. choctaworum NRRL Y-27557 (AY426959/AY309858) C. choctaworum NRRL Y-27559 (AY242188/AY242298) 100 iC. bolitotheri NRRL Y-27587T(AY242142/AY242249) Ic bolitotheri NRRL Y-27562 (AY242148/AY242257) canberraensis NRRL YB-2417T(AY488124/AY013718) C. panamericana NRRL Y-27582 (AY242233/AY242345) C. panamericana NRRL Y-27567T (AY242164/AY242273) C. panamericana NRRL Y-27590 (AY426961/AY309874) C. atakaporum NRRL Y-27570T (AY426960/AY309872) C. ambrosias NRRL YB-1316T (AY227712/AY013716) C. ambrosiae NRRL Y-27586 (AY242140/AY242246) C. ambrosiae NRRL Y-27565 (AY242153/AY242262) C. ambrosiae NRRLY-27578 {AY242201/AY242312) C. tanzawaensis NRRL Y-17324T(AY227713/U44811) C. bribrorum NRRL Y-27572T(AY426962/AY309875) C. bribrorum NRRL Y-27591 (AY426963/AY309881) C. bribrorum NRRL Y-27592 (AY426964/AY3Û9883) • C. bribrorum NRRL Y-27564 (AY242151/AY242260) C. anneliseae NRRL Y-27563T(AY242149/AY242258) C. anneliseae NRRL Y-27585 (AY242240/AY242352) C. anneliseae NRRL Y-27577 (AY426966/AY309916) C. anneliseae NRRL Y-27583 (AY426965/AY309888) C. anneliseae NRRL Y-27575 (AY426967/AY309918) C. anneliseae NRRL Y-27560 (AY426968/AY309919) C. fa/Zae NRRL Y-27589T (AY242145/AY242254) 100 C. max/7 NRRL Y-27588T(AY242144/AY242253) C. xylopsoci NRRL Y-27066T (AY488128/AY013719) C. pyralidae NRRL Y-27085T (AY488127/AY013715) 90' C. prunicola NRRLYB-869T(AY488126/AY013714) C. caryicola NRRL YB-1499T(AY488125/AY013717) • 5 changes

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.

previously described taxa, C. xylopsoci, C. pyralidae, C. As mentioned earlier, only one subclade (including ErotS, prunicola and C. caryicola were basal to the other yeasts in Nitil4 and Tene7) had D1/D2 sequences identical to the the CT clade. C. ambrosiae, C. tanzawaensis and C. previously described yeast C. ambrosiae, which we consider canberraensis, however, were included among the beetle to be conspecific with these isolates. There were marked gut yeasts (Fig. 1). Within the CT clade, the beetle iso- differences between C. ambrosiae and the other 16 sub- lates were resolved into 17 subclades with sufficient diver- clades in the CT clade with 6 bp or more (usually more than gence to warrant species-level recognition (Kurtzman & 20 bp) difference between the subclades. The genetic varia- Robnett, 1998; Kurtzman, 2000; Suh & Blackwell, 2004) tion among multiple isolates within subclades was lower (Fig. 1). (Table 1; Fig. 1). For example, yeasts designated Endo2 and http://ijs.sgmjournals.org 2415 S.-O. Suh, J. V. McHugh and M. Blackwell

Erot24 occurred in a common subclade and differed from Erotylidae), Barro Colorado Island, Panama, depositata in each other by 3 bp of D1/D2 sequence. Also, there were Collectione Culturarum (NRRL), Peoria, IL, USA. minor D1/D2 sequence differences between common sub- clade members Tene28 and Tene30 or Erot20 and Tenel9 Description of Candida guaymorum Suh & (Table 1). Within all 17 subclades of the CT clade, however, Blacltwell sp. nov. the sequence variation of D1/D2 among subclade members was always less than 3 bp, within the range of species-level Candida guaymorum (gu.ay.mo'rum. N.L. m. gen. guay- variation recognized in studies of other ascomycete yeast morum to commemorate the Guaymi, a group of indi- species (Kurtzman & Robnett, 1998; Table 1). genous people of Panama with detailed knowledge of the forest flora). We observed minor morphological differences among the isolates on YM and cornmeal agars at 5-7 days incubation After 7 days growth in YM broth at 25 °C, cells are globose and in YM broth after 5 days incubation at 25 °C, but to ellipsoidal (1-25-6-25 x 1-25-7-5 |im), and occur singly, morphological variation that consistently distinguished the in pairs or in short chains (Fig. 2a). Pseudohyphae are yeasts within clades was not evident. One morphological present. After 7 days on YM agar at 25 °C, colonies are white feature common to all isolates was the lack of ascospore to cream-coloured with pale-pinkish perimeter on some production. Physiological characters (Table 2), however, old colonies, smooth, shiny, flat and filamentous in margin. were more variable and these traits were useful to separate After 10 days Dalmau plate culture on corn meal agar the other species of the CT clade. Below, we characterize at 25 °C, pseudohyphae are present; septate hyphae are and describe 16 novel species of beetle gut yeasts and absent. Aerobic growth is white, shiny and smooth with compare them to the previously described yeasts of the CT filamentous margin. No ascospores produced after 6 weeks clade (Fig. 1 and Table 2). at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength cornmeal Latin diagnosis of Candida guaymorum Suh et agar. See Table 2 for a summary of physiological and other Blaclcwell sp. nov. characteristics. In medio liquido dextrosum et peptonum et extractum The type strain is NRRL Y-27568'^ ( = CBS 9823"^). levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut ellipsoideae (1 •25-6-25 x 1 •25-7-5 ^m), singu- Latin diagnosis of Candida boliatorum Suh et lae vel binae; pseudohyphae fiunt. Cultura in agaro extramalti Blackwell sp. nov. et faecis continente post 7 dies ad 25 °C, albida, hebes, teres et margine ciliata. In agaro farina Zeae maydis confecto In medio liquido dextrosum et peptonum et extractum post 10 dies ad 25 °C, pseudohyphae fiunt, hyphae verae non levidinis continente post 7 dies ad 25 °C cellulae vegetativae fiunt. Ascosporae non fiunt. Glucosum, galactosum {lente), globosae aut ellipsoideae (2-6 x 3-6 ^m), singulae vel binae. maltosum {infirme, variabilitre), methyl a-D-glucosidum Cultura in agaro extramalti et faecis continente post 7 dies {infirme, variabilitre), sucrosum {infirme, variabilitre), ad 25 °C, albida, hebes, butyrosa, margine ciliata. In agaro trehalosum et cellobiosum {lente) fermentantur. Melibio- farina Zeae maydis confecto post 10 dies ad 25 °C, pseudo- sum, lactosum, melezitosum, raffinosum, inulinum, amylum hyphae fiunt, hyphae verae fiunt. Ascosporae non fiunt. solubile et D-xylosum non fermentantur. Assimilantur Glucosum, maltosum {infirme, variabilitre), sucrosum glucosum, galactosum, D-glucosaminum, D-xylosum, sucro- {infirme, variabilitre), trehalosum et cellobiosum ferment- sum, maltosum, trehalosum, methyl a-V)-glucosidum, cello- antur. Galactosum, methyl a-D-glucosidum, melibiosum, biosum, salicinum, arbutinum, melezitosum, glycerolum, lactosum, melezitosum, raffinosum, inulinum, amylum ribitolum, D-glucitolum, D-mannitolum, gluconolactonum, solubile et D-xylosum non fermentantur. Assimilantur 2-keto-D-gluconatum, D-gluconatum {lente, infirme), acidum glucosum, galactosum, D-glucosaminum {variabilitre), succinicum, acidum citricum et ethanolum. Non assimilantur D-xylosum, D-arabinosum, sucrosum, maltosum, trehalosum, L-sorbosum, D-ribosum, L-arabinosum, D-arabinosum, L- methyl a-D-glucosidum, cellobiosum, salicinum, arbutinum, rhamnosum, melibiosum, lactosum, raffinosum, inulinum, melezitosum, glycerolum, ribitolum, D-glucitolum, D- amylum solubile, erythritolum, xylitolum, L-arabinitolum, mannitolum, gluconolactonum, 2-keto-D-gluconatum, D- galactitolum, inositolum, D-glucuronatum, DL-acidum lacti- gluconatum {lente, infirme), acidum succinicum, acidum cum, methanolum, propane-l,2-diolum, butano-2,5-diolum, citricum, ethanolum et propane-\,2-diolum {infirme, acidum quinicum, D-glucaratum et D-galactonatum. Assimi- variabilitre). Non assimilantur L-sorbosum, D-ribosum, lantur ethylaminum, L-lysinum, cadaverinum et glucos- L-arabinosum, L-rhamnosum, melibiosum, lactosum, raffino- aminum. Non assimilantur kali nitratum, sodii nitritum, sum, inulinum, amylum solubile, erythritolum, xylitolum, creatinum, creatininum, imidazolum et D-tryptophanum. L-arabinitolum, galactitolum, inositolum, D-glucuronatum, Amylum non formatur. Biotinum externum ad crescentiam DL-acidum lacticum, methanolum, butano-2,3-diolum, necessarium est. Augmentum non fiunt in temperatura 40 °C. acidum quinicum, D-glucaratum et D-galactonatum. Non crescit in medio 10 )ig ml~^ cycloheximido addito. Assimilantur ethylaminum, L-lysinum, cadaverinum Typus: NRRL Y-27568'^ ( = CBS 9823'^), désignât stirpem et glucosaminum {infirme, variabilitre). Non assimilantur typicum. Isolata a He coleopterorum {Mycotretus interstitialis; kali nitratum, sodii nitritum, creatinum, creatininum.

2416 International Journal of Systematic and Evolutionary Microbiology 54 Expansion of the Candida tanzawaensis clade

Table 2. Physiological characteristics of novel Candida species and C. ambrosia from this study

Taxa: 1, C. guaymorum; 2, C. bokatorum; 3, C. l^unorum; 4, C. terrahorum; 5, C emherorum; 6, C. wounanorum; 7, C. yuchorum; 8, C. c/îicfc- flsaworum; 9, C. c/ioctaworum; 10, C. bolitotheri; 11, C. atakaporum; 12, C. panamericana; 13. C hribrorum; 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 forma- tion ( •), 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.

Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Fermentation of carbon compounds D-Glucose -1- + /d -1- d -1- d -1- -1- + /d -1- -1- -1- -1- d -l-/d/w -1- -1- D-Galactose d - w w + /d w d + /d V + /d - d V - - d + /d Maltose w/- w/- w d ------w/- - - - d/w Methyl a-D-glucoside w/------V Sucrose w/- w/- - d ------d/w iz,a-Trehalose -1- +/d -1- d + d -1- + /d -l-/d/w d d -1- -1- w d/w d -1- Lactose - - w ------Cellobiose d +/d - - d/w - d - - - - d/w - w - d V Melezitose ------w/- Inulin ------w/- Assimilation of carbon compounds D-Galactose -1- + -1- -1- -1- -1- -1- -1- -1- -1- -1- -1- -1- -1- V -1- -1- D-Sorbose ------+ /d V - - V - V - w/- D-Glucosamine -I-/W V d w d/w d - d/w + /d -1- d + /d -I-/W d -l-/d/w -1- -I-/W D-Ribose - - w - - - - - V V - - V -1- V - +/d D-Xylose -1- -1- d w -1- -1- -1- + /d + /d -l-/d/w -1- + -1- - -l-/d/w d -1- L-Arabinose ------w/- - - - V - - - -l-/d/w D-Arabinose - + /d/v/ w ------l-/d/w - w - V w -l-/d/w -1- - Sucrose -1- + -1- -1- -1- -1- -1- -1- - - -1- -1- -1- -1- -1- -1- -1- Maltose -1- + -1- -1- -1- -1- -1- -1- - - -1- -1- -1- -1- -1- -1- -1- Methyl a-D-glucoside -1- + -1- -1- -1- -1- -1- -1- - - -1- -1- -1- -1- -1- -1- -1- Salicin -1- + d -1- -1- -1- -1- -1- -1- -1- -1- -1- -1- -1- -1- -1- -1- Arbutin -1- -1- -1- -1- -1- -1- -1- -1- -1- -1- -1- + /d -1- -1- -1- -1- -1- Melezitose -1- -1- -1- -1- d/w w - V - - -1- + -1- -1- -1- -1- -1- Soluble starch ------w ------Glycerol -1- -l-/d/w -1- -1- -1- -1- -1- -1- + /d -1- -1- -1- -1- d -l-/d/w -1- -1- Erythritol ------1- -1- - - -1- -1- -1- -1- -1- Ribitol -1- -1- -1- -1- -1- -1- -1- + /d -1- -1- -1- -1- -1- -1- -1- -1- -1- Xylitol - - w - - - - w/- V -I-/W - - +/d w + /d d + /d D-Arabinitol - - w - - - - - V - - - - - V - -1- D-Glucono-l,5-lactone + + /d/v/ -1- w + /d -1- -1- -l-/d/w +/d + /d -1- -1- -1- d + /d -1- -1- 2-Keto-D-gluconate + -1- -1- -1- + -1- -1- -1- -1- -1- -1- -1- -1- -1- -1- -1- -1- D-Gluconate d/w d/w -1- w d/w - d V V d/w w -1- -1- -1- -1- -1- -l-/d/w DL-Lactate ------d ------Succinate -1- + /d -1- -1- -1- d -1- -1- -I-/W -l-/d/w d -1- +/d -1- + /d -1- -1- Citrate -1- + d -1- + /d -1- -1- -l-/d/w +/d -1- d -1- + -1- + /d -1- -1- Ethanol -I-/W + -1- d + -1- - -l-/d/w +/d + /d -1- - + -1- -l-/d/w -1- -1- Propane-l,2-diol - w/- w w - - - w/- w/- - w - - - w/- - - Assimilation of nitrogen compound s D-Glucosamine -I-/W w/- w w + /V/ - -1- V V + lv/ w V V w V w V D-Tryptophan - - - - w - d - - • d ------

http://ijs.sgmjournals.org 2417 S.-O. Suh, J. V. McHugh and M. Blackwell

Table 2. cont.

Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Vitamin requirements w/o vitamins ------V - - - - - w/o myo-Inositol + + + + + + + + + + + + + d + + + w/o Biotin ------V w/- w/- - + /W w/- - - - - w/o Thiamin + + + + + + + + + + + + /w + d V d + w/o Biotin and Thiamin ------V w/- w/- - V w/- - - - - w/o Pyridoxine + + /d + + + + + + + + + + + + + + + w/o Pyridoxine and Thiamin + + + + + w + + + + + + /w + /W d + + + Growth tests Growth at 35 °C V V - - + /W - + - V V + +/d V - V w + 0-01% Cycloheximide - w/------+ w/- - - w - V - V 0-1% Cycloheximide ------+ - - - w/- - - - - 50% D-Glucose + + + + +/w w + +/d + /d +/d w + +/d + + /d/w + + 60% D-Glucose - - - w - - - w/- + /d/w V - + w/- - V - + /d/w 10% NaCl + + /w - w V + + + /W + /w + /d/w + + + /d/w + + /d/w + + 16% NaCl • • • • • • • • V • w w/- w/- • w/- • w/- imidazolum et D-tryptophanuni. Amylum non formatur. binae. Cultura in agaro extramalti et faecis continente post Biotinum externum ad crescentiam necessarium est. 7 dies ad 25 °C, albida, hebes, butyrosa, margine ciliata. In Augmentum non fiunt in temperatura 40 °C. Variabilitre agaro farina Zeae maydis confecto post 10 dies ad 25 °C, in medio 10 \ig ml~' cycloheximido addito, non crescit in pseudohyphae non fiunt, hyphae verae non fiunt. Ascosporae medio 100 \ig mr\ Typus: NRRL Y-2757l'^ ( = CBS 9824'^), non fiunt. Glucosum, galactosum {infirme), maltosum désignât stirpem typicum. Isolata a He coleopterorum {infirme), trehalosum et lactosum (infirme) fermentantur. {Pselaphacus signatus; Erotylidae), Barro Colorado Island, Sucrosum, methyl a-D-glucosidum, melibiosum, cellobiosum, Panama, depositata in Collectione Culturarum (NRRL), melezitosum, raffinosum, inulinum, amylum solubile et Peoría, IL, USA. D-xylosum non fermentantur. Assimilantur glucosum, galac- tosum, D-glucosaminum (lente), D-ribosum (infirmé), Description of Candida bokatorum Suh & D-xylosum (lente), D-arabinosum (infirme), sucrosum, Blaclcwell sp. nov. maltosum, trehalosum, methyl a-D-glucosidum, cellobiosum, salicinum (lente), arbutinum, melezitosum, glycerolum, Candida bokatorum (bo.ka.to'rum. N.L. m. gen. bokatorum ribitolum, xylitolum (infirme), L-arabinitolum (infirme), to commemorate the Bókatá, a group of indigenous people D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D- of Panama, linguistically related to the Guaymi). gluconatum, D-gluconatum, acidum succinicum, acidum citricum (lente), ethanolum etpropane-l,2-diolum (infirme). After 7 days growth in YM broth at 25 °C, cells are globose Non assimilantur L-sorbosum, L-arabinosum, h-rhamnosum, to ellipsoidal (2-6 x 3-6 |im), mostly subglobose, and occur melibiosum, lactosum, raffinosum, inulinum, amylum solu- singly, in pairs or in short chains (Fig. 2b). Pseudohyphae bile, erythritolum, galactitolum, inositolum, D-glucuronatum, may be present. After 7 days on YM agar at 25 °C, colonies DL-acidum lacticum, methanolum, butano-2,3-diolum, are white to cream in colour, butyrous and smooth. After acidum quinicum et D-glucaratum. Assimilantur ethylami- 10 days Dalmau plate culture on corn meal agar at 25 °C, num, L-lysinum, cadaverinum et glucosaminum (infirme). pseudohyphae are present. Septate hyphae may be present. Non assimilantur kali nitratum, sodii nitritum, creatinum, Aerobic growth is white and smooth. No ascospores creatininum, imidazolum et D-tryptophanum. Amylum non produced after 6 weeks at 17 °C from individual strains formatur. Biotinum externum ad crescentiam necessarium on YM agar or strains crossed in all combinations on half- est Augmentum non fiunt in temperatura 35 °C. Typus: strength cornmeal agar. See Table 2 for a summary of NRRL Y-27580'^ ( = CBS 9825'^), désignât stirpem typicum. physiological and other characteristics. Isolata a He coleopterorum (Teichostethus testaceous; The type strain is NRRL Y-2757l'^ ( = CBS 9824"^). Nitidulidae), Barro Colorado Island, Panama, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA. Latin diagnosis of Candida i(unorum Suh et Blaclcwell sp. nov. Description of Candida ¡(unorum Suh & Blackwell sp. nov. In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae Candida kunorum (ku.no'rum. N.L. m. gen. kunorum to subglobosae aut fusiformes (2-5 x 3-6 |im), singulae vel commemorate the Kuna, a group of indigenous people of

2418 International Journal of Systematic and Evolutionary Microbiology 54 Expansion of the Candida tanzawaensis clade

L¿p"7 CO

n «I

(i)

(I)

Ñ \^

Fig. 2. Budding yeast cells, pseudohyphae and septate hyphae of the novel species, (a) C. guaymorum NRRL Y-27568^; (b) C. bokatorum NRRL Y-2757l"^; (c) C. kunorum NRRL Y-27580"^; (d) C. terraborum NRRL Y-27573"^; (e) C. emberorum NRRL Y-27606'^; (f) C. wounanorum NRRL Y-27574"^; (g) C. yuchorum NRRL Y-27569"^; (h) C. chickasaworum NRRL Y- 27566"^; (i) C. choctawomm NRRL Y-27584"^; (j) Candida bolitotheri NRRL Y-27587"^; (k) C. atakaporum NRRL Y-27570"^; (I) C. panamericana NRRL Y-27567^. (a-f, h-l) Seven days, half-strength cornmeal agar, 25 °C; (g) 5 days, YM broth, 25 °C. Bars, 5 \im.

Panama with a high degree of literacy and a desire for plate culture on corn meal agar at 25 °C, pseudohyphae autonomy). and septate hyphae are absent. Aerobic growth is white and smooth. No ascospores produced after 6 weeks at 17°C After 7 days growth in YM broth at 25 °C, cells are sub- from the single strain on YM agar or half-strength cornmeal globose to fusiform (2-5 x 3-6 |im), mostly subglobose, agar. See Table 2 for a summary of physiological and other and occur singly, in pairs or in short chains (Fig. 2c). After characteristics. 7 days on YM agar at 25 °C, colonies are white, membranous and butyrous with smooth margin. After 10 days Dalmau The type strain is NRRL Y-27580'^ ( = CBS 9825'^). http://ijs.sgmjournals.org 2419 S.-O. Suh, J. V. McHugh and M. Blackwell

Latin diagnosis of Candida terraborum Suli et The type strain is NRRL Y-27573 ( = CBS 9826 ). Blaclcwell sp. nov. In medio liquido dextrosum et peptonum et extractum Latin diagnosis of Candida emberorum Suh ef levidinis continente post 7 dies ad 25 °C cellulae vegetativae Blackwell sp. nov. globosae aut ellipsoideae (4-6 x 5-7 |im), singulae vel binae. In medio liquido dextrosum et peptonum et extractum Cultura in agaro extramalti et faecis continente post 7 dies levidinis continente post 7 dies ad 25 °C cellulae vegetativae ad 25 °C, albida, hebes, butyrosa, margine ciliata. In agaro globosae aut ellipsoideae (1-25-5 x 2-5-6-25 iim), singulae farina Zeae maydis confecto post 10 dies ad 25 °C, vel binae. Cultura in agaro extramalti et faecis continente pseudohyphae fiunt, hyphae verae non fiunt. Ascosporae non post 7 dies ad 25 °C, albida, hebes, butyrosa, margine ciliata. fiunt. Glucosum (lente), galactosum (infirme), maltosum In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, (lente), sucrosum (lente) et trehalosum (lente) fermentantur. pseudohyphae fiunt, hyphae verae non fiunt. Ascosporae non Methyl a-D-glucosidum, melibiosum, lactosum, cellobiosum, fiunt. Glucosum, galactosum, trehalosum et cellobiosum melezitosum, raffinosum, inulinum, amylum solubile et (lente, infirme) fermentantur. Maltosum, methyl a-D- D-xylosum non fermentantur. Assimilantur glucosum, galac- glucosidum, sucrosum, melibiosum, lactosum, melezitosum, tosum, D-glucosaminum (infirme), D-xylosum (infirme), raffinosum, inulinum, amylum solubile et D-xylosum non sucrosum, maltosum, trehalosum, methyl a-D-glucosidum, fermentantur. Assimilantur glucosum, galactosum, D- cellobiosum, salicinum, arbutinum, melezitosum, glycerolum, glucosaminum (lente, infirme), D-xylosum, sucrosum, mal- ribitolum, D-glucitolum, D-mannitolum, gluconolactonum tosum, trehalosum, methyl a-D-glucosidum, cellobiosum, (infirme), 2-keto-D-gluconatum, D-gluconatum (infirme), salicinum, arbutinum, melezitosum (lente, infirme), glycero- acidum succinicum, acidum citricum, ethanolum (lente) et lum, ribitolum, D-glucitolum, D-mannitolum, gluconolacto- propane-\,2-diolum (infirme). Non assimilanturD-sorbosum, num, 2-keto-D-gluconatum, D-gluconatum (lente, infirme), D-ribosum, L-arabinosum, D-arabinosum, D-rhamnosum, acidum succinicum, acidum citricum et ethanolum. Non melibiosum, lactosum, raffinosum, inulinum, amylum solu- assimilantur D-sorbosum, D-ribosum, L-arabinosum, D- bile, erythritolum, xylitolum, D-arabinitolum, galactitolum, arabinosum, D-rhamnosum, melibiosum, lactosum, raffino- inositolum, D-glucuronatum, DL-acidum lacticum, methano- sum, inulinum, amylum solubile, erythritolum, xylitolum, lum, butano-2,3-diolum, acidum quinicum, D-glucaratum et D-arabinitolum, galactitolum, inositolum, D-glucuronatum, D-galactonatum. Assimilantur ethylaminum, D-lysinum, DL-acidum lacticum, methanolum, propane-\,2-diolum, cadaverinum et glucosaminum (infirme). Non assimilantur butano-2,3-diolum, acidum quinicum, D-glucaratum et kali nitratum, sodii nitritum, creatinum, creatininum, D-galactonatum. Assimilantur ethylaminum, D-lysinum, imidazolum et D-tryptophanum. Amylum non formatur. cadaverinum, glucosaminum et D-tryptophanum (infirme). Biotinum externum ad crescentiam necessarium est. 30 °C Non assimilantur kali nitratum, sodii nitritum, creatinum, crescit neque 35 °C. Non crescit in medio 10 |xg ml~^ creatininum et imidazolum. Amylum non formatur. Biotinum cycloheximido addito. Typus: NRRL ¥-27573"^ (=CBS externum ad crescentiam necessarium est. Augmentum non 9826^), désignât stirpem typicum. Isolata a He coleopterorum fiunt in temperatura 40 °C. Non crescit in medio 10 ng ml~^ (Iphiclus sedecimmaculatus; Erotylidae), Barro Colorado cycloheximido addito. Typus: NRRL Y-27606'^ ( = CBS Island, Panama, depositata in Collectione Culturarum 9827^), désignât stirpem typicum. Isolata a He coleopterorum (NRRL), Peoría, IL, USA. (Triplax alvarengai; Erotylidae), Barro Colorado Island, Panama, depositata in Collectione Culturarum (NRRL), Peoria, IL, USA. Description of Candida terraborum Sufi & Blaclcwell sp. nov. Description of Candida emberorum Suh & Candida terraborum (ter.ra.bo'rum. N.L. m. gen. terrabo- Blackwell sp. nov. rum to commemorate the Térraba, a group of indigenous Candida emberorum (em.be.ro'rum. N.L. m. gen. embero- people of Panama, survivors of epidemics that once deci- zrum to commemorate the Emberá, a group of indigenous mated their numbers). people of Panama, knowledgeable in botanical lore). After 7 days growth in YM broth at 25 °C, cells are globose After 7 days growth in YM broth at 25 °C, cells are globose to ellipsoidal (4-6 x 5-7 |im), mostly subglobose, and occur to ellipsoidal (1-25-5 x 2-5-6-25 |im), mostly globose or singly, in pairs or in short chains (Fig. 2d). Big and subglobose, and occur singly, in pairs or in short chains elongated cells are also observed. After 7 days on YM agar (Fig. 2e). Pseudohyphae may be present. After 7 days on at 25 °C, colonies are off-white, butyrous and smooth. After YM agar at 25 °C, colonies are white to cream-coloured 10 days Dalmau plate culture on corn meal agar at 25 °C, with pale-pinkish perimeter, smooth and butyrous. After pseudohyphae are present; septate hyphae are absent. 10 days Dalmau plate culture on corn meal agar at 25 °C, Aerobic growth is white with fuzzy margin. No ascospores pseudohyphae are present; septate hyphae are absent. produced after 6 weeks at 17 °C from the single strain on Aerobic growth is white, dull and smooth with filamentous YM agar or half-strength cornmeal agar. See Table 2 for a margin. No ascospores produced after 6 weeks at 17 °C summary of physiological and other characteristics. from individual strains on YM agar or strains crossed in all

2420 International Journal of Systematic and Evolutionary Microbiology 54 Expansion of the Candida tanzawaensis clade combinations on half-strength cornmeal agar. See Table 2 smooth and filamentous in margin. No ascospores pro- for a summary of physiological and other characteristics. duced after 6 weeks at 17 °C from the single strain on YM agar or half-strength cornmeal agar. See Table 2 for a The type strain is NRRL Y-27606'^ ( = CBS 9827'^). summary of physiological and other characteristics.

Latin diagnosis of Candida wounanorum Suh et The type strain is NRRL Y-27574'^ ( = CBS 9828"^). Blackwell sp. nov. Latin diagnosis of Candida yuchorum Suh et In medio liquido dextrosum et peptonum et extractum Blackwell sp. nov. levidinis continente post 1 dies ad 25 °C cellulae vegetativae globosae aut ovoidae (2-4 x 3-6 |xm), plerumque subglo- In medio liquido dextrosum et peptonum et extractum bosae, singulae vel binae. Cultura in agaro extramalti et levidinis continente post 7 dies ad 25 °C cellulae vegetativae faecis continente post 7 dies ad 25 °C, albida, butyrosa. In globosae aut ellipsoideae (2-5-7 x 2-5-7 iim), plerumque agaro farina Zeae maydis confecto post 10 dies ad 25 °C, globosae, singulae vel binae. Cultura in agaro extramalti et pseudohyphae non fiunt, hyphae verae non fiunt. Ascosporae faecis continente post 7 dies ad 25 °C, albida, teres, margine non fiunt. Glucosum {lente), galactosum (infirmé) et cïliata. In agaro farina Zeae maydis confecto post 10 dies ad trehalosum {lente) fermentantur. Maltosum, methyl a-D- 25 °C, pseudohyphae et hyphae verae fiunt. Ascosporae non glucosidum, sucrosum, melibiosum, lactosum, cellobiosum, fiunt. Glucosum, galactosum (lente), trehalosum et cellobio- melezitosum, raffinosum, inulinum, amylum solubile et sum (lente) fermentantur. Maltosum, methyla-D-glucosidum, D-xylosum non fermentantur. Assimilantur glucosum, galac- sucrosum, melibiosum, lactosum, melezitosum, raffinosum, tosum, D-glucosaminum {lente), D-xylosum, sucrosum, inulinum, amylum solubile et D-xylosum non fermentantur. maltosum, trehalosum, methyl a-n-glucosidum, cellobiosum, Assimilantur glucosum, galactosum, D-xylosum, sucrosum, salicinum, arbutinum, melezitosum {infirme), glycerolum, maltosum, trehalosum, methyl a-D-glucosidum, cellobiosum, ribitolum, D-glucitolum, D-mannitolum, gluconolactonum, salicinum, arbutinum, glycerolum, ribitolum, D-glucitolum, 2-keto-D-gluconatum, acidum succinicum (lente), acidum D-mannitolum, gluconolactonum, 2-keto-D-gluconatum, D- citricum et ethanolum. Non assimilantur D-sorbosum, gluconatum (lente), acidum succinicum et acidum citricum. D-ribosum, L-arabinosum, D-arabinosum, D-rhamnosum, Non assimilantur D-sorbosum, D-glucosaminum, D-ribosum, melibiosum, lactosum, raffinosum, inulinum, amylum solu- L-arabinosum, D-arabinosum, D-rhamnosum, melibiosum, bile, erythritolum, xylitolum, D-arabinitolum, galactitolum, lactosum, raffinosum, melezitosum, inulinum, amylum inositolum, D-gluconatum, D-glucuronatum, DL-acidum solubile, erythritolum, xylitolum, D-arabinitolum, galactito- lacticum, methanolum, propane-l,2-diolum, butano-2,3- lum, inositolum, D-glucuronatum, DL-acidum lacticum, diolum, acidum quinicum, D-glucaratum et D-galactonatum. methanolum, ethanolum, propane-\,2-diolum, butano-2,3- Assimilantur ethylaminum, D-lysinum et cadaverinum. Non diolum, acidum quinicum, D-glucaratum et D-galactonatum. assimilantur kali nitratum, sodii nitritum, creatinum, Assimilantur ethylaminum, D-lysinum, cadaverinum, gluco- creatininum, glucosaminum, imidazolum etD-tryptophanum. saminum et D-tryptophanum (lente). Non assimilantur kali Amylum non formatur. Biotinum externum ad crescentiam nitratum, sodii nitritum, creatinum, creatininum et imida- necessarium est. In temperatura 30 °C crescit neque 35 °C. zolum. Amylum non formatur. Biotinum externum ad Non crescit in medio 10 ^g ml~^ cycloheximido addito. crescentiam necessarium est. Augmentum non fiunt in Typus: NRRL ¥-27574"^ ( = CBS 9828'^), désignât stirpem temperatura 40 °C. Non crescit in medio 10 |ig ml~^ typicum. Isolata a He coleopterorum (Mycotretus dorsonotatus; cycloheximido addito. Typus: NRRL Y-27569'^ ( = CBS Erotylidae), Barro Colorado Island, Panama, depositata 9829^), désignât stirpem typicum. Isolata a He coleopterorum in Collectione Culturarum (NRRL), Peoria, IL, USA. (Tritoma atriventris; Erotylidae), Athens, GA, USA, depos- itata in Collectione Culturarum (NRRL), Peoria, IL, USA. Description of Candida wounanorum Suh & Blackwell sp. nov. Description of Candida yucliorum Suh & Blackwell sp. nov. Candida wounanorum (wou.na.no'rum. N.L. m. gen. wounanorum to commemorate the Wounan, a group of Candida yuchorum (yu.cho'rum. N.L. m. gen. yuchorum to indigenous people of Panama known for their artistry in commemorate the Yuchi, native Americans of the south- using plant materials; they inhabited the same region as eastern USA; their public worship was tied to the corn the Emberá). harvest when food was shared). After 7 days growth in YM broth at 25 °C, cells are globose After 7 days growth in YM broth at 25 °C, cells are globose to oval (2-4 X 3-6 ^m), mostly subglobose, and occur to ellipsoidal (2-5-7 x 2-5-7 um), mostly globose or sub- singly, in pairs or in short chains (Fig. 2f). After 7 days on globose, and occur singly, in pairs or in short chains YM agar at 25 °C, colonies are off-white with fuzzy white (Fig. 2g). Pseudohyphae and septate hyphae maybe present. spots, butyrous and smooth. After 10 days Dalmau plate After 7 days on YM agar at 25 °C, colonies are cream- culture on corn meal agar at 25 °C, pseudohyphae and coloured, smooth and shiny with filamentous margin. septate hyphae are absent. Aerobic growth is white, shiny. After 10 days Dalmau plate culture on corn meal agar at http://ijs.sgmjournals.org 2421 S.-O. Suh, J. V. McHugh and M. Blackwell

25 °C, pseudohyphae and septate hyphae may be present. 25 °C, colonies are white to cream-coloured, butyrous and Aerobic growth is white and dull. No ascospores produced smooth with mycelial edge. After 10 days Dalmau plate after 6 weeks at 17 °C from individual strains on YM agar culture on corn meal agar at 25 °C, elongated pseudohyphae or strains crossed in all combinations on half-strength and septate hyphae may be present. Aerobic growth is white cornmeal agar. See Table 2 for a summary of physiological to cream-coloured with mycelial margin. No ascospores and other characteristics. produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half- The type strain is NRRL Y-27569'^ ( = CBS 9829"^). strength cornmeal agar. See Table 2 for a summary of physiological and other characteristics. Latin diagnosis of Candida chickasaworum Suh & Blacl(well sp. nov. The type strain is NRRL Y-27566'^ ( = CBS 9830"^). In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae Latin diagnosis of Candida choctaworum Suh globosae aut fusiformes (2-6 x 2-8 |im), singulae vel binae. ef Blaclcwell sp. nov. Cultura in agaro extramalti et faecis continente post 7 dies In medio liquido dextrosum et peptonum et extractum ad 25 °C, albida, butyrosa, margine ciliata. In agaro farina levidinis continente post 7 dies ad 25 °C cellulae vegetativae Zeae maydis confecto post 10 dies ad 25 °C, pseudohyphae et globosae aut ovoidae (2-6 x 2-8 \xm), plerumque globosae hyphae verae fiunt. Ascosporae non fiunt. Glucosum, et subglobosae, singulae vel binae. Cultura in agaro extramalti galactosum et trehalosum fermentantur. Maltosum, methyl et faecis continente post 7 dies ad 25 °C, albida, butyrosa, oi-D-glucosidum, sucrosum, melibiosum, lactosum, cellobio- teres. In agaro farina Zeae maydis confecto post 10 dies ad sum, melezitosum, raffinosum, inulinum, amylum solubile 25 °C, pseudohyphae et hyphae verae may fiunt. Ascosporae et D-xylosum non fermentantur. Assimilantur glucosum, non fiunt. Glucosum, galactosum {variabilitre) et trehalosum galactosum, D-glucosaminum {lente, infirme), D-xylosum, fermentantur. Maltosum, methyl a-D-glucosidum, sucrosum, sucrosum, maltosum, trehalosum, methyl a-D-glucosidum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, cellobiosum, salicinum, arbutinum, melezitosum [variabili- inulinum, amylum solubile et D-xylosum non ferment- tre), glycerolum, ribitolum, xylitolum {infirme, variabilitre), antur. Assimilantur glucosum, galactosum, D-sorbosum, D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D- D-glucosaminum, D-ribosum {variabilitre), D-xylosum, gluconatum, D-gluconatum {variabilitre), acidum succini- L-arabinosum {infirme, variabilitre), D-arabinosum, tre- cum, acidum citricum, ethanolum et propane-\,l-diolum halosum, cellobiosum, salicinum, arbutinum, glycerolum, {infirme, variabilitre). Non assimilantur D-sorbosum, erythritolum, ribitolum, xylitolum {variabilitre), D- D-ribosum, L-arabinosum, D-arabinosum, D-rhamnosum, arabinitolum {variabilitre), D-glucitolum, D-mannitolum, melibiosum, lactosum, raffinosum, inulinum, amylum gluconolactonum, 2-keto-D-gluconatum, D-gluconatum solubile, erythritolum, D-arabinitolum, galactitolum, inosito- {variabilitre), acidum succinicum, acidum citricum, ethano- lum, D-glucuronatum, DL-acidum lacticum, methanolum, lum et propane-\,2-diolum {infirme, variabilitre). Non butano-2,5-diolum, acidum quinicum, D-glucaratum et assimilantur D-rhamnosum, sucrosum, maltosum, methyl D-galactonatum. Assimilantur ethylaminum, D-lysinum, a-D-glucosidum, melibiosum, lactosum, raffinosum, melezi- cadaverinum et glucosaminum {variabilitre). Non assimi- tosum, inulinum, amylum solubile, galactitolum, inositolum, lantur kali nitratum, sodii nitritum, creatinum, creatininum, D-glucuronatum, DL-acidum lacticum, methanolum, imidazolum et D-tryptophanum. Amylum non formatur. butano-2,3-diolum, acidum quinicum, D-glucaratum et Biotinum externum ad crescentiam necessarium est {variabi- D-galactonatum. Assimilantur ethylaminum, D-lysinum, litre). In temperatura 30 °C crescit neque 35 °C. Non crescit cadaverinum et glucosaminum {variabilitre). Non assimilan- in medio 10 |ig ml~^ cycloheximido addito. Typus: NRRL tur kali nitratum, sodii nitritum, creatinum, creatininum, Y-27566^ (=CBS 9830^), désignât stirpem typicum. Isolata imidazolum et D-tryptophanum. Amylum non formatur. a He coleopterorum {Tritoma sp.; Erotylidae), Athens, GA, Biotinum externum ad crescentiam necessarium est. USA, depositata in Collectione Culturarum (NRRL), Peoria, Augmentum non fiunt in temperatura 40 °C. Crescit in IL, USA. medio 100 |xg ml~' cycloheximido addito. Typus: NRRL Y-27584'^ ( = CBS 9831"^), désignât stirpem typicum. Isolata Description of Candida cliicl

2422 International Journal of Systematic and Evolutionary Microbiology 54 Expansion of the Candida tanzawaensis clade

After 7 days growth in YM broth at 25 °C, cells are globose occurring in Ganoderma basidiocarps from Vermont to to oval (2-6 X 2-8 |im), and occur singly, in pairs or in Louisiana, USA). short chains (Fig. 2i). Pseudohyphae may be present. After 7 days on YM agar at 25 °C, colonies are white to cream- After 7 days growth in YM broth at 25 °C, cells are globose coloured, smooth and butyrous with slightly wrinkled edge. to subglobose (2-6 x 2-6 um), mostly globose, and occur After 10 days Dalmau plate culture on corn meal agar at singly or in short chains (Fig. 2j). Pseudohyphae may be 25 °C, pseudohyphae and septate hyphae may be present. present. After 7 days on YM agar at 25 °C, colonies are Aerobic growth is white, shiny, smooth and slightly fiizzy. white to cream-coloured with very pale-pinkish from No ascospores produced after 6 weeks at 17 °C from indi- centre to edge, and smooth to slightly wrinkled centre vidual strains on YM agar or strains crossed in all com- with mycelial edge. After 10 days Dalmau plate culture on binations on half-strength cornmeal agar. See Table 2 for a corn meal agar at 25 °C, pseudohyphae are present. Septate summary of physiological and other characteristics. hyphae may be present. Aerobic growth is white, shiny and smooth with filamentous margin. No ascospores produced The type strain is NRRL Y-27584'^ ( =CBS 9831"^). after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half-strength Latin diagnosis of Candida bolitotheri Suh et cornmeal agar. See Table 2 for a summary of physiological Blackwell sp. nov. and other characteristics. In medio liquido dextrosum et peptonum et extractum The type strain is NRRL Y-27587'^ ( = CBS 9832^^). levidinis continente post 1 dies ad 25 °C cellulae vegetativae globosae aut subglobosae (2-6 x 2-6 ^m), singulae vel binae. Pseudohyphae fiunt. Cultura in agaro extramalti et faecis Latin diagnosis of Candida atakaporum Suh et continente post 1 dies ad 25 °C, albida, teres, margine ciliata. Blackwell sp. nov. In agaro farina Zeae may dis confecto post 10 dies ad 25 °C, pseudohyphae fiunt, hyphae verae fiunt. Ascosporae non In medio liquido dextrosum et peptonum et extractum fiunt. Glucosum, galactosum et trehalosum fermentantur. levidinis continente post 7 dies ad 25 °C cellulae vegetativae Maltosum, methyl a-D-glucosidum, sucrosum, melibiosum, subglobosae aut cylindratae (3-5 x 5-9 |im), singulae vel lactosum, cellobiosum, melezitosum, raffinosum, inulinum, binae. Pseudohyphae fiunt. Cultura in agaro extramalti et amylum solubile et D-xylosum non fermentantur. Assimi- faecis continente post 7 dies ad 25 °C, albida, butyrosa, lantur glucosum, galactosum, D-sorbosum (variabilitre), margine ciliata. In agaro farina Zeae may dis confecto post 10 D-glucosaminum, D-ribosum {variabilitre), D-xylosum, dies ad 25 °C, pseudohyphae et hyphae verae fiunt. Ascosporae trehalosum, cellobiosum, salicinum, arbutinum, glycerolum, non fiunt. Glucosum et trehalosum {lente) fermentantur. erythritolum, ribitolum, xylitolum, D-glucitolum, D- Galactosum, maltosum, methyl a-D-glucosidum, sucrosum, mannitolum, gluconolactonum, 2-keto-D-gluconatum, melibiosum, lactosum, cellobiosum, melezitosum, raffinosum, D-gluconatum {lente, infirme), acidum succinicum, acidum inulinum, amylum solubile et D-xylosum non fermentantur. citricum et ethanolum. Non assimilantur L-arabinosum, Assimilantur glucosum, galactosum, D-glucosaminum {lente), D-arabinosum, D-rhamnosum, sucrosum, maltosum, methyl D-xylosum, D-arabinosum {infirmé), sucrosum, maltosum, a-D-glucosidum, melibiosum, lactosum, raffinosum, melezi- trehalosum, methyl a-D-glucosidum, cellobiosum, salicinum, tosum, inulinum, amylum solubile, D-arabinitolum, galacti- arbutinum, melezitosum, amylum solubile {infirmé), glycer- tolum, inositolum, D-glucuronatum, Di.-acidum lacticum, olum, ribitolum, D-glucitolum, D-mannitolum, gluconolacto- methanolum, propane-l,2-diolum, butano-2,3-diolum, num, 2-keto-D-gluconatum, D-gluconatum {infirme), acidum quinicum, D-glucaratum et D-galactonatum. Assimi- DL-acidum lacticum {lente), acidum succinicum {lente), lantur ethylaminum, D-lysinum, cadaverinum et gluco- acidum citricum {lente), ethanolum et propane-1,2-diolum saminum. Non assimilantur kali nitratum, sodii nitritum, {infirme). Non assimilantur D-sorbosum, D-ribosum, L- creatinum, creatininum, imidazolum et D-tryptophanum. arabinosum, D-rhamnosum, melibiosum, lactosum, raffino- Amylum non formatur. Biotinum externum ad crescentiam sum, inulinum, erythritolum, xylitolum, D-arabinitolum, necessarium est. Augmentum non fiunt in temperatura galactitolum, inositolum, D-glucuronatum, methanolum, 45 °C. Variabilitre in medio 10 |ig ml~' cycloheximido butano-2,3-diolum, acidum quinicum, D-glucaratum et D- addito, non crescit in medio 100 |xg ml~\ Typus: NRRL galactonatum. Assimilantur ethylaminum, D-lysinum, cada- Y-27587^ (=CBS 9832^), désignât stirpem typicum. Isolata verinum, glucosaminum {infirme) et D-tryptophanum {lente). a He coleopterorum {Bolitotherus cornutus; Tenebrionidae), Non assimilantur kali nitratum, sodii nitritum, creatinum, Athens, GA, USA, depositata in Collectione Culturarum creatininum et imidazolum. Amylum non formatur. Biotinum (NRRL), Peoria, IL, USA. externum ad crescentiam necessarium est. Augmentum non fiunt in temperatura 40 °C. Non crescit in medio 10 |ig ml~^ cycloheximido addito. Typus: NRRL Y-27570'^ ( = CBS Description of Candida boiitotheri Suh & 9833^), désignât stirpem typicum. Isolata a He coleopterorum Blackwell sp. nov. {Triplax festiva; Erotylidae), Baton Rouge, LA, USA, Candida bolitotheri (bo.li.to.the'ri. N.L. n. gen. bolitotheri depositata in Collectione Culturarum (NRRL), Peoria, IL, is named for the coleopteran host, Bolitotherus cornutus. USA. http://ijs.sgmjournals.org 2423 S.-O. Suh, J. V. McHugh and M. Blackwell

Description of Candida atakaporum Suh & Colorado Island, Panama, depositata in Collectione Blaclcwell sp. nov. Culturarum (NRRL), Peoria, IL, USA. Candida atakaporum (a.ta.ka.po'rum. N.L. m. gen. ataka- porum commemorates the Atakapa, native Americans of Description of Candida panamericana Suh & the northwestern coast of the Gulf of Mexico and their Blackwell sp. nov. unique language). Candida panamericana (pan.a.mer.i.can'a. N.L. f. adj. After 7 days growth in YM broth at 25 °C, cells are sub- panamericana to call attention to its broad distribution globose, ellipsoidal or cylindrical (3-5 x 5-9 |im), and occur spanning the regions from Louisiana to Panama). singly, in pairs or in short chains (Fig. 2k). Pseudohyphae After 7 days growth in YM broth at 25 °C, cells are globose and septate hyphae may be present. After 7 days on YM to oval (3-6-25 x 3-75-7 |xm), mostly subglobose, and agar at 25 °C, colonies are off-white with membranous occur singly, in pairs or in short chains (Fig. 21). Some margin and butyrous with small filamentous spots. After cells form clusters. Pseudohyphae are present. After 7 days 10 days Dalmau plate culture on corn meal agar at 25 °C, on YM agar at 25 °C, colonies are white to off-white pseudohyphae and septate hyphae are present. Aerobic in colour, smooth, glistening and flat with filamentous growth is off-white and fuzzy. No ascospores produced after margin. After 10 days Dalmau plate culture on corn meal 6 weeks at 17 °C from the single strain on YM agar or agar at 25 °C, pseudohyphae are present. Septate hyphae half-strength cornmeal agar. See Table 2 for a summary of may be present. Aerobic growth is white, shiny and smooth physiological and other characteristics. with filamentous margin. No ascospores produced after 6 weeks at 17 °C from individual strains on YM agar or The type strain is NRRL Y-27570'^ ( = CBS 9833'^). strains crossed in all combinations on half-strength cornmeal agar. See Table 2 for a summary of physiological and other characteristics. Latin diagnosis of Candida panamericana Suh et Blackwell sp. nov. The type strain is NRRL Y-27567'^ ( = CBS 9834*^). In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae Latin diagnosis of Candida bribrorum Suh et globosae aut ovoidae (3-6-25 x 3-75-7 |im), plerumque Blackwell sp. nov. subglobosae, singulae vel binae. Pseudohyphae fiunt. Cultura In medio liquido dextrosum et peptonum et extractum in agaro extramalti et faecis continente post 7 dies ad 25 °C, levidinis continente post 7 dies ad 25 °C cellulae vegetativae albida, teres, margine ciliata. In agaro farina Zeae maydis globosae aut ellipsoideae (3-8 x 3-8 |im), plerumque sub- confecto post 10 dies ad 25 °C, pseudohyphae fiunt, hyphae globosae, singulae vel binae. Pseudohyphae fiunt. Cultura in verae fiunt. Ascosporae non fiunt. Glucosum, galactosum agaro extramalti et faecis continente post 7 dies ad 25 °C, (lente), trehalosum et cellobiosum fermentantur. Maltosum, albida, butyrosa, margine ciliata. In agaro farina Zeae methyl a-D-glucosidum, sucrosum, melibiosum, lactosum, maydis confecto post 10 dies ad 25 °C, pseudohyphae et melezitosum, raffinosum, inulinum, amylum solubile et hyphae verae fiunt. Ascosporae non fiunt. Glucosum, D-xylosum non fermentantur. Assimilantur glucosum, galactosum (variabilitre), maltosum [infirme, variabilitre) galactosum, D-glucosaminum, D-xylosum, sucrosum, et trehalosum fermentantur. Methyl a-D-glucosidum, maltosum, trehalosum, methyl a-D-glucosidum, cellobiosum, sucrosum, melibiosum, lactosum, cellobiosum, melezitosum, salicinum, arbutinum, melezitosum, glycerolum, ribitolum, raffinosum, inulinum, amylum solubile et D-xylosum D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D- non fermentantur. Assimilantur glucosum, galactosum, gluconatum, D-gluconatum, acidum succinicum et acidum D-sorbosum {variabilitre), D-glucosaminum, D-ribosum citricum. Non assimilantur D-sorbosum, D-ribosum, {variabilitre), D-xylosum, "L-arabinosum {variabilitre), D- L-arabinosum, D-arabinosum, D-rhamnosum, melibiosum, arabinosum {variabilitre), sucrosum, maltosum, trehalosum, lactosum, raffinosum, inulinum, amylum solubile, ery- methyl a-D-glucosidum, cellobiosum, salicinum, arbutinum, thritolum, xylitolum, D-arabinitolum, galactitolum, inosito- melezitosum, glycerolum, erythritolum, ribitolum, xylitolum, lum, D-glucuronatum, DL-acidum lacticum, methanolum, D-glucitolum, D-mannitolum, gluconolactonum, 2-keto-D- ethanolum, propane-l,2-diolum, butano-2,3-diolum, acidum gluconatum, D-gluconatum, acidum succinicum, acidum quinicum et D-glucaratum. Assimilantur ethylaminum, D- citricum, ethanolum et propane-\,2-diolum {variabilitre). lysinum, cadaverinum et glucosaminum (variabilitre). Non Non assimilantur D-rhamnosum, melibiosum, lactosum, assimilantur kali nitratum, sodii nitritum, creatinum, raffinosum, inulinum, amylum solubile, D-arabinitolum, creatininum, imidazolum et D-tryptophanum. Amylum non galactitolum, inositolum, D-glucuronatum, DL-acidum lacti- formatur. Vitaminae externae ad crescentiam necessaria non cum, methanolum, butano-2,3-diolum, acidum quinicum, sunt. Augmentum non fiunt in temperatura 40 °C. Non crescit D-glucaratum etD-galactonatum. Assimilantur ethylaminum, in medio 10 |ig ml~' cycloheximido addito. Typus: NRRL Y- D-lysinum, cadaverinum et glucosaminum {variabilitre). Non 27567^ ( = CBS 9834^), désignât stirpem typicum. Isolata a He assimilantur kali nitratum, sodii nitritum, creatinum, coleopterorum {Mycotretus interstitialis; Erotylidae), Barro creatininum, imidazolum et D-tryptophanum. Amylum non

2424 International Journal of Systematic and Evolutionary Microbiology 54 Expansion of the Candida tanzawaensis clade formatur. Biotinum externum ad crescentiam necessarium cornmeal agar. See Table 2 for a summary of physiological est. Augmentum non fiunt in temperatura 40 °C. Infirme and other characteristics. crescitin medio 10 |xg ml~^ cycloheximido addito, variabilitre in 100 \ignir\ Typus: NRRL Y-27572^ ( = CBS 9835^), The type strain is NRRL Y-27572'^ ( = CBS 9835'^). désignât stirpem typicum. Isolata a He coleopterorum (Pselaphacus sp.; Erotylidae), Barro Colorado Island, Latin diagnosis of Candida maxii Suh et Panama, depositata in Collectione Culturarum (NRRL), Blackwell sp. nov. Peoría, IL, USA. In medio liquido dextrosum et peptonum et extractum levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut subglobosae (2-6-25 x 3-75-6-25 |im), singulae Description of Candida bribrorum Suh & vel binae. Pseudohyphae et hyphae verae fiunt. Cultura in Blacltwell sp. nov. agaro extramalti et faecis continente post 7 dies ad 25 °C, Candida bribrorum (bri.bro'rum. N.L. m. gen. bribrorum albida, margine ciliata. In agaro farina Zeae maydis confecto to commemorate the Bribri, a small group of indigenous post 10 dies ad 25 °C, pseudohyphae et hyphae verae fiunt. people of northern Panama, who apply ecological principles Ascosporae non fiunt. Glucosum {lente), trehalosum {infirme) and recognize symbiotic relationships). et cellobiosum {infirme) fermentantur. Galactosum, malto- sum, methyl a-n-glucosidum, sucrosum, melibiosum, lacto- After 7 days growth in YM broth at 25 °C, cells are globose sum, melezitosum, raffinosum, inulinum, amylum solubile et to ellipsoidal (3-8 x 3-8 ^m), mostly subglobose, and occur D-xylosum non fermentantur. Assimilantur glucosum, galac- singly, in pairs or in short chains (Fig. 3a). Pseudohyphae tosum, D-glucosaminum {lente), D-ribosum, D-arabinosum are present. After 7 days on YM agar at 25 °C, colonies {infirme), sucrosum, maltosum, trehalosum, methyl oc-D- are white to cream in colour, butyrous and smooth sur- glucosidum, cellobiosum, salicinum, arbutinum, melezitosum, face with slightly fuzzy spots. After 10 days Dalmau plate glycerolum {lente), erythritolum, ribitolum, xylitolum culture on corn meal agar at 25 °C, pseudohyphae and {infirme), D-glucitolum, D-mannitolum, gluconolactonum septate hyphae are present. Aerobic growth is white to {lente), 2-keto-D-gluconatum, D-gluconatum, acidum succi- cream-coloured with fuzzy margin. No ascospores pro- nicum, acidum citricum et ethanolum. Non assimilantur duced after 6 weeks at 17 °C from individual strains on YM D-sorbosum, D-xylosum, L-arabinosum, D-rhamnosum, meli- agar or strains crossed in all combinations on half-strength biosum, lactosum, raffinosum, inulinum, amylum solubile, D-arabinitolum, galactitolum, inositolum, D-glucuronatum, DL-acidum lacticum, methanolum, propane-l,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 |ig ml~^ cycloheximido addito. Typus: NRRL Y-27588'^ ( = CBS 9836^), désignât stirpem typicum. Isolata a He coleopterorum (Tenebrionidae), Barro Colorado Island, Panama, deposi- tata in Collectione Culturarum (NRRL), Peoría, IL, USA. (d) ^ <> (e) 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 (2-5-6-25 x 3-75-6-25 |im). Pseudohyphae and septate hyphae are present (Fig. 3b, c). After 7 days Fig. 3. Budding yeast cells, pseudohyphae and septate hyphae on YM agar at 25 °C, colonies are white in colour, ridged, of the novel species, (a) C. bribrorum NRRL Y-27572^; (b, c) glistening and flat with filamentous margin. After 10 days C. maxii NRRL Y-27588^; (d) yeast cells of C. anneliseae Dalmau plate culture on corn meal agar at 25 °C, pseudo- NRRL ¥-27563"^; (e) C. taliae NRRL Y-27589"^. (a-c) Seven hyphae and septate hyphae are present. Aerobic growth is days, YM broth, 25 °C; (d, e) 7 days, half-strength cornmeal white, shiny and smooth with filamentous margin. No agar, 25 °C. Bars, 5 urn. ascospores produced after 6 weeks at 17 °C from individual http://ijs.sgmjournals.org 2425 S.-O. Suh, J. V. McHugh and M. Blackwell

Strains on YM agar or strains crossed in all combinations plate culture on corn meal agar at 25 °C, pseudohyphae are on half-strength cornmeal agar. See Table 2 for a summary present. Septate hyphae may be present. Aerobic growth is of physiological and other characteristics. white, shiny, smooth, and the growth consistent throughout mycelium with filamentous-like periphery. No ascospores The type strain is NRRL Y-27588'^ ( = CBS 9836"^). produced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half- Latin diagnosis of Candida anneliseae Suh et strength cornmeal agar. See Table 2 for a summary of Blaclcwell sp. nov. physiological and other characteristics. In medio liquido dextrosum et peptonum et extractum The type strain is NRRL Y-27563'^ ( = CBS 9837"^). levidinis continente post 7 dies ad 25 °C cellulae vegetativae globosae aut ellipsoideae (1-25-7 x 1-25-7 |im), singulae vel Latin diagnosis of Candida taliae Suh et binae. Pseudohyphae fiunt Cultura in agaro extramalti et Blackwell sp. nov. faecis continente post 7 dies ad 25 °C, albida, teres, margine ciliata. In agaro farina Zeae may dis confecto post 10 dies In medio liquido dextrosum et peptonum et extractum ad 25 °C, pseudohyphae fiunt, hyphae verae fiunt. Ascosporae levidinis continente post 7 dies ad 25 °C cellulae vegetativae non fiunt. Glucosum et trehalosum {lente, infirme) fermen- globosae aut ovoidae (2-5-5 x 3-75-6-25 |im), plerumque tantur. Galactosum, maltosum, methyl a-D-glucosidum, ellipsoideae, singulae vel binae. Pseudohyphae et hyphae sucrosum, melibiosum, lactosum, cellobiosum, melezitosum, verae fiunt. Cultura in agaro extramalti et faecis continente raffinosum, inulinum, amylum solubile et D-xylosum non post 7 dies ad 25 °C, albida, butyrosa, teres, margine ciliata. fermentantur. Assimilantur glucosum, galactosum {vari- In agaro farina Zeae maydis confecto post 10 dies ad 25 °C, abilitre), D-sorbosum {variabilitre), D-glucosaminum, pseudohyphae fiunt, hyphae verae non fiunt. Ascosporae D-ribosum {variabilitre), D-xylosum, D-arabinosum, sucro- non fiunt. Glucosum, galactosum {lente) trehalosum {lente) sum, maltosum, trehalosum, methyl a-V)-glucosidum, cello- et cellobiosum {lente) fermentantur. Maltosum, methyl biosum, salicinum, arbutinum, melezitosum, glycerolum, a-D-glucosidum, sucrosum, melibiosum, lactosum, melezito- erythritolum, ribitolum, xylitolum, D-arabinitolum {vari- sum, raffinosum, inulinum, amylum solubile et D-xylosum abilitre), D-glucitolum, D-mannitolum, gluconolactonum, non fermentantur. Assimilantur glucosum, galactosum, 2-keto-D-gluconatum, D-gluconatum, acidum succinicum, D-glucosaminum, D-xylosum {lente), D-arabinosum, sucro- acidum citricum, ethanolum et propane-l,2-diolum sum, maltosum, trehalosum, methyl a-D-glucosidum, cello- {infirme, variabilitre). Non assimilantur L-arabinosum, D- biosum, salicinum, arbutinum, melezitosum, glycerolum, rhamnosum, melibiosum, lactosum, raffinosum, inulinum, erythritolum, ribitolum, xylitolum {lente), D-glucitolum, D- amylum solubile, galactitolum, inositolum, D-glucuronatum, mannitolum, gluconolactonum {lente), 2-keto-D-gluconatum, DL-acidum lacticum, methanolum, butano-2,3-diolum, D-gluconatum, acidum succinicum, acidum citricum et acidum quinicum, D-glucaratum et D-galactonatum. ethanolum. Non assimilantur D-sorbosum, D-ribosum, Assimilantur ethylaminum, D-lysinum, cadaverinum et L-arabinosum, D-rhamnosum, melibiosum, lactosum, raffi- glucosaminum {variabilitre). Non assimilantur kali nitratum, nosum, inulinum, amylum solubile, D-arabinitolum, galacti- sodii nitritum, creatinum, creatininum, imidazolum et tolum, inositolum, D-glucuronatum, DL-acidum lacticum, D-tryptophanum. Amylum non formatur. Biotinum externum methanolum, propane-\,2-diolum, butano-2,3-diolum, ad crescentiam necessarium est. Augmentum non fiunt in acidum quinicum, D-glucaratum et D-galactonatum. temperatura 40 °C. Variabilitre in medio 10 ^g ml~^ Assimilantur ethylaminum, D-lysinum, cadaverinum et cycloheximido addito, non crescit in medio 100 |ig ml~\ glucosaminum {infirme). Non assimilantur kali nitratum, Typus: NRRL Y-27563'^ {=CBS 9837'^), désignât stirpem sodii nitritum, creatinum, creatininum, imidazolum et D- typicum. Isolata a He coleopterorum {Megalodacne fasciata; tryptophanum. Amylum non formatur. Biotinum externum Erotylidae), Athens, GA, USA, depositata in Collectione ad crescentiam necessarium est. Augmentum non fiunt in Culturarum (NRRL), Peoria, IL, USA. temperatura 40 °C. Non crescit in medio 10 |ig ml~' cycloheximido addito. Typus: NRRL Y-27589'^ ( = CBS Description of Candida anneliseae Suh & 9838^), désignât stirpem typicum. Isolata a He coleopterorum Blackwell sp. nov. (Tenebrionidae), Barro Colorado Island, Panama, deposi- tata in Collectione Culturarum (NRRL), Peoria, IL, USA. Candida anneliseae (an.ne.lis.e'ae N.L. n. gen. anneliseae for Annelise Berler, whose grandparents generously support Description of Candida taliae Suh & Blackwell the Mycological Society of America). sp. nov. After 7 days growth in YM broth at 25 °C, cells are globose Candida taliae (tal'i.ae. N.L. n. gen. taliae for Talia Berler, to ellipsoid ( 1 -25-7 x 1-25-7 |xm), and occur singly, in pairs whose grandparents generously support the Mycological or in chains (Fig. 3d). Pseudohyphae may be present. After Society of America). 7 days on YM agar at 25 °C, colonies are white to cream- coloured, pale-pinkish from centre to edge and smooth After 7 days growth in YM broth at 25 °C, cells are globose with slightly winkled mycelial edge. After 10 days Dalmau to oval (2-5-5 X 3-75-6-25 |im), mostly ellipsoidal, and

2426 International Journal of Systematic and Evolutionary Microbiology 54 Expansion of the Candida tanzawaensis clade occur singly, in pairs or in short chains (Fig. 3e). Pseudo- Host specificity of yeasts and dispersal in the hyphae and septate hyphae are present. After 7 days on CT clade YM agar at 25 °C, colonies are off-white, butyrous and Our data reveal several examples of highly specific asso- mostly smooth with bumpy areas in centre. After 10 days ciations between certain beetles and yeasts. The same yeast Dalmau plate culture on corn meal agar at 25 °C, pseudo- was associated repeatedly with Neomida bicornis (Tenebrio- hyphae are present; septate hyphae are absent. Aerobic nidae); this yeast (Tenel of C. choctaworum) was isolated growth is white, shiny and smooth with filamentous from the beetle gut at least eight times from five different margin. No ascospores produced after 6 weeks at 17 °C localities in southern Louisiana over a 5-year period fi-om the single strain on YM agar or half-strength cornmeal (Table 1). Another specialized association occurred between agar. See Table 2 for a summary of physiological and other Bolitotherus cornutus (Tenebrionidae) and Tenel 1 of C. characteristics. bolitotheri in all samples examined from Vermont to The type strain is NRRL Y-27589'^ {=CBS 9838"^). southwestern Louisiana (Table 1). 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, Gut yeasts close to Candida ambrosias Erot38 (C. bribrorum) has been found in both a pupa Kurtzman (Table l) and an adult of Megalodacne audouini, and Mela2 (C. anneliseae) was isolated from larvae and adults of the After 7 days growth in YM broth at 25 °C, cells are globose same melandreid beetle species. Our indirect evidence, to ellipsoidal (2-5-5 x 2-5-6-25 |J,m), and occur singly, in therefore, supports a view that certain yeasts are present pairs or in chains. Pseudohyphae are present. After 7 days during the entire life cycle of their beetle hosts and that on YM agar at 25 °C, colonies are white to cream-coloured, parental transmission occurs early in successive genera- rough and partly powdery with wrinkled mycelial edge. tions (Suh & Blackwell, 2004). Previous studies have After 10 days Dalmau plate culture on corn meal agar shown maternal transmission of yeasts to insects, includ- at 25 °C, pseudohyphae are present. Aerobic growth is ing examples such as the yeast-like endosymbionts of white, dull, dry, powdery; consistent growth throughout anobiid beetles that are transmitted to their offspring by mycelium with branch-like periphery. No ascospores pro- contamination of the egg shell (Jurzitza, 1979). duced after 6 weeks at 17 °C from individual strains on YM agar or strains crossed in all combinations on half- Several beetle species in the Tenebrionidae are associated strength cornmeal agar. See Table 2 for a summary of with closely related yeasts. An example of a close asso- physiological and other characteristics. ciation is Tene4 (C. anneliseae), isolated from species of Diaperis, Neomida, Platydema, Alobates and several un- identified taxa. These yeasts have identical D1/D2 geno- CT clade: a major clade of gut yeasts from types and small, but consistent, differences in physiological basidiocarp-feeding beetles traits to distinguish among them. Other CT clade yeasts The repeated isolation of C. tanzawaensis-Wkt yeasts from also show evidence of past host-switching among distantly beetles first attracted our attention to these fungi. As related beetles. For example, isolates of C. anneliseae (Mela2, mentioned above, about 30 % of all 650 yeast isolates from Hist5 and Ciid5) are identical to Tene4 in D1/D2 geno- our ongoing study are members of the clade. Except for a type, although the beetle hosts are not closely related and few isolates we placed in C. ambrosiae, none had been belong to four different families spread throughout a broad described previously (Fig. 1). Most of the 16 novel CT clade geographical range in our sampling. We will need to members appear to be common associates of basidiocarp- investigate the possibility of more variable DNA markers feeding beetles, and some of the yeasts have broad to explain transmission among phylogenetically distinct geographical distributions. The 22-year interval between hosts. We believe that these beetles most likely acquired the isolation of C. tanzawaensis and its description, and their yeast associates comparatively recently, perhaps by the absence of any additional reports in the intervening relatively rare host-switching within a common habitat. 21 years since the description (Nakase etal, 1988), contrasts with the common occurrence of the novel CT yeasts and Diversification and resolution of taxa within the C. ambrosiae in the specialized gut habitat in which we CT clade have found them thus far. The discovery of so many novel species of undescribed yeasts suggests that the microbial Identification of yeast taxa based on morphological and flora of the beetle gut is distinctive and has not been well physiological characteristics has been problematic and often examined previously. We do not know the basis of the leads to misidentification (Kurtzman & Phaff, 1987; Price apparent widespread relationship between these organi- et al, 1978). For this reason molecular methods increas- sms, but it must be significant to beetles or yeasts or both ingly are being used to identify yeasts; additionally, molec- because the associations are so common. In one case an ular markers, especially DNA sequences, have become the unsuspected genetic resource that might be tied to such a standard for the description of yeast species. The variable function has been suggested for a yeast associate of certain D1/D2 sequence of the LSU rRNA gene (about 600 bp) has wood-ingesting beetles (Suh et al, 2003). been determined for currently recognized ascomycete yeasts http://ijs.sgmjournals.org 2427 S.-O. Suh, J. V. McHugh and M. Blackwell

(Kurtzman & Robnett, 1995, 1997, 1998; Suh & Blackwell, Jones, K. G. & Blackwell, M. (1996). Ribosomal DNA sequence 2004). In addition to helping to delimit species, com- analysis places the yeast-like genus Symbiotaphrina within filamen- parisons of the D1/D2 sequences also are a useful tool tous ascomycetes. Mycologia 88, 212-218. for rapid identification of yeasts and detection of novel Jones, K. G., Dowd, P. F. & Blackwell, M. (1999). Polyphyletic species (e.g. Kurtzman, 2000) with a few exceptions among origins of yeast-like endocytobionts from anobiid and cerambycid closely related species, such as Saccharomyces bayanus and beetles. Mycol Res 103, 542-546. Saccharomyces pastorianus (Kurtzman & Robnett, 1998). Jurzitza, G. (1979). The fungi symbiotic with anobiid beetles. In Insect-Fungus Symbiosis: Nutrition, Mutualism, and Commensalism, In our study, SSU rRNA gene sequences, as expected, did pp. 65-76. Edited by L. R. Batra. New York: Wiley. not provide enough variation to distinguish all species in Kurtzman, C. P. (2000). Four new yeasts in the Pichia anómala clade. the CT clade. Identical sequences were determined for Int J Syst Evol Microbiol 50, 395-404. about 1750 bp of the SSU rRNA gene for three pairs of Kurtzman, C. P. (2001). Six new anamorphic ascomycetous yeasts taxa: C. tanzawaensis and C. ambrosiae, C. guaymorum and near Candida tanzawaensis. FEMS Yeast Res 1, 177-185. C. bokatorum, and C. maxii and C. anneliseae. The more Kurtzman, C. P. & Phaff, H. J. (1987). Molecular . In The variable D1/D2 loop sequences of the LSU rRNA gene, Yeasts, vol. 1, Biology of Yeasts, pp. 63-94. Edited by A. H. Rose & which sometimes may be too variable to align among I. S. Harrison. London: Academic Press. phylogenetically distant groups when comparing a broad Kurtzman, C. P. & Robnett, C. J. (1995). Molecular relationships range of yeasts, were useful in distinguishing CT clade among hyphal ascomycetous yeasts and yeastlike taxa. Can } Bot 73, S824-S830. yeasts (Suh et al., 2004). The differences in D1/D2 sequences, varying fi-om 6 to more than 60 bp between Kurtzman, C. P. & Robnett, C. J. (1997). Identification of chnically important ascomycetous yeasts based on nucleotide divergence in species and less than 3 bp for multiple isolates of a species, the 5' end of the large subunit (26S) ribosomal DNA gene. / Clin revealed some close relationships. For example, the strains Microbiol 35, 1216-1223. Endo2 and Erot24 in C. emberorum are only 3 bp different Kurtzman, C. P. & Robnett, C. J. (1998). Identification and in D1/D2 region, and there are only minor differences phylogeny of ascomycetous yeasts from analysis of nuclear large within the species of C. panamericana and C. bribrorum in subunit (26S) ribosomal DNA partial sequences. Antonie Van that region (Table 1). The closely related CT clade yeasts Leeuwenhoek 73, 331-371. additionally were supported by other taxonomic characters, Lee, S. B. & Taylor, J. W. (1990). Isolation of DNA from fungal and more than 80 physiological tests have been useful in mycelia and single spores. In PCR Protocols - a Guide to Methods and distinguishing the taxa (Table 2). In addition to identifica- Applications, pp. 282-287. Edited by M. A. Innis, D. H. Gelfand, I. I. tion of CT clade species using BLAST searches and phylo- Sninsky & T. I. White. San Diego, CA: Academic Press. genetic analyses based on DNA, identification was possible Nakase, T., Itoh, M., Takematsu, A. & Komagata, K. (1988). Candida using physiological traits. tanzawaensis, a new species of yeast isolated from moss collected in lapan. Trans Mycol Soc Jpn 29, 331-338. Nardon, P. & Grenier, A. M. (1989). Endosymbiosis in Coleóptera: biological, biochemical, and genetic aspects. In Insect Endocytobiosis: ACKNOWLEDGEMENTS Morphology, Physiology, Genetics, Evolution, pp. 175-216. Edited by W. Schwemmler & G. Gassner. Boca Raton, EL: CRC Press. We thank Louisiana State University undergraduate students Christine Noda, H. & Kodama, K. (1996). Phylogenetic position of yeastlike Ackerman, Katie Brillhart, Cennet Erbil, Nhu Nguyen, Ebony Spikes endosymbionts of anobiid beetles. Appl Environ Microbiol 62, 162-167. and Amy Whittington for their skilled assistance in all phases of this Noda, H. & Omura, T. (1992). Purification of yeast-like symbiotes of study. Dr Donald Windsor, Dr Annette Aiello, Ms Oris Acevedo and planthoppers. / Invertebr Pathol 59, 104-105. Ms Maria Leone graciously provided information and helped with logistics for collecting at the Smithsonian Tropical Research Institute, Noda, H., Nakashima, N. & Koizumi, M. (1995). Phylogenetic Barro Colorado Island, Panama. Drs lean Euzéby and Richard Warga position of yeast-like symbiotes of rice planthoppers based on partial gave expert advice on Latin names and descriptions. We acknowledge 18S rDNA sequences. Insect Biochem Mol Biol 25, 639-646. the curators and culture collections that preserve the germ plasm Price, C. W., Fuson, G. B. & Phaff, H. J. (1978). Genome comparison derived from and used in our studies: NRRL, CBS and ATCC. Use of in yeast systematics: delimination of species within the genera the GenBank public database also is acknowledged. The work was Schwanniomyces, Saccharomyces, Debaryomyces and Pichia. Microbiol supported by the National Science Foundation, Biodiversity Surveys Rev 42, 161-193. and Inventories Program (DEB-0072741) and by the Louisiana State Suh, S.-O. & Blackwell, M. (2004). The beetle gut as a habitat for University Boyd Professor Fund. new species of yeasts. In Insect Fungal Associations: Ecology and Evolution, (in press). Edited by F. E. Vega & M. Blackwell. New York: Oxford University Press. Suh, S.-O., Noda, H. & Blackwell, M. (2001). Insect symbiosis: REFERENCES derivation of yeast-like endosymbionts within an entomopathogenic Barnett, J. A., Payne, R. W. & Yarrow, D. (2000). Yeasts: filamentous Uneage. Mol Biol Evol 18, 995-1000. Characteristics and Identification, 3rd edn. Cambridge: Cambridge Suh, S.-O., Marshall, C. J., McHugh, J. V. & Blackwell, M. (2003). University Press. Wood ingestion by passalid beetles in the presence of xylose- Hausner, G., Reid, J. & Klassen, G. R. (1993). On the subdivision of fermenting gut yeasts. Mol Ecol 12, 3137-3145. Ceratocystis s.l., based on partial ribosomal DNA sequences. Can ] Bot Suh, S.-O., McHugh, J. V. & Blackwell, M. (2004). Metschnikowia 71, 52-63. chrysoperlae sp. nov., Candida picachoensis sp. nov. and Candida

2428 International Journal of Systematic and Evolutionary Microbiology 54 Expansion of the Candida tanzawaensis clade pimensis sp. nov., isolated from the green lacewings Chrysoperla strategies for multiple sequence alignment aided by quality analysis comanche and Chrysoperla carnea (Neuroptera: Chrysopidae). Int tools. Nucleic Acids Res 25, 4876^882. J Syst Evol Microbiol 54, 1883-1890. White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990). Amplification and Swofford, D. L (2002). PAUP*. Phylogenetk Analysis Using direct sequencing of fungal ribosomal RNA genes for phylogenetics. Parsimony (*and Other Methods), version 4.0blO. Sunderland, MA: In PCR Protocols - a Guide to Methods and Applications, Sinauer Associates. pp. 315-322. Edited by M. A. Innis, D. H. Gelfand, J. J. Sninsky Tatusova, T. A. & Madden, T. L. (1999). BLAST 2 Sequences, a new & T. J. White. San Diego, CA: Academic Press. tool for comparing protein and nucleotide sequences. FEMS Yarrow, D. (1998). Methods for the isolation, maintenance and Microbiol Lett 174, 247-250. identification of yeasts. In The Yeasts, a Taxonomic Study, 4th edn, Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & pp. 77-100. Edited by C. P. Kurtzman & J. W. Fell. Amsterdam: Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible Elsevier.

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