Molecular Ecology (2003) 12,3137-3145 doi:10.1046/j.l365-294X.2003.01973.x Wood ingestion by passalid beetles in the presence of xylose-fermenting gut yeasts SUNG-OUI SUH,* CHRISTOPHER J. MARSHALL,t JOSEPH V. McHUGHJ and MEREDITH BLACKWELL* *Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 USA, ^Department of Entomology, Smithsonian Institution, National Museum of Natural Science, Washington, D.C., USA, ?^Department of Entomology, University of Georgia, Athens, GA 30602 USA Abstract During a survey of insect gut micro-organisms, we consistently isolated Pichia stipitis-like yeasts (Fungi: Ascomycota, Saccharomycetes) from the wood-ingesting beetles, Odonto- taenius disjunctus and Verres stembergianus (Cole?ptera: Passalidae). The yeasts were iso- lated from passalid beetles over a wide area, including the eastern and midwestern USA and Panama. Phylogenetic analyses of the nuclear encoded small and large subunit rRNA gene (rDNA) sequences distinguished a well-supported clade consisting of the passalid yeasts and Pichia stipitis, P. segobiensis, Candida shehatae and C. ergatensis. Members of this clade have the ability to ferment and assimilate xylose or to hydrolyse xylan, major components of the polysaccharide, hemicellulose. Sexual reproduction was present in the passalid isolates but was rare among the gut yeasts of other beetles to which they were com- pared. Minor genetic and phenotypic variation among some of the passalid yeasts was detected using markers from the internal transcribed spacer region of the rDNA repeat unit, morphology, and in vitro metabolic tests. The consistent association of xylose-fermenting yeasts of almost identical genotypes with passalid beetles across a broad geographical distribution, suggests a significant symbiotic association. Keywords: Enteroramus dimorphus, evolution, LSU rDNA, SSU rDNA, symbiosis, wood decomposition Received 7 April 2003; revision received 1 July 2003; accepted 30 July 2003 Introduction Numerous associations between invertebrate animals and endosymbiotic micro-organisms have been described over the past century (B?chner 1965; Nardon & Grenier 1989). Invertebrates rely on microbes for various metabolic functions, including synthesis of amino acids, vitamins, lipids, sterols and pheromones, degradation of nutritional substrates, and detoxification of inhibitory compounds. A continuum of insect-fungus symbiotic associations exists in terrestrial ecosystems. These range from casual inter- actions in a shared habitat to strict obligate endosymbioses. Martin (1987) emphasized the contributions of microbial enzymes to the survival of various insects: anobiid beetles are able to live in cigarette packs because the tobacco is detoxified by yeast-like fungi (Dowd 1989,1991), siricid Correspondence: Meredith Blackwell. Fax: 01 225578 2597; E-mail: mblackwell@lsu.edu wood wasps acquire fungal enzymes to degrade woody plant parts (Gilbertson 1984), and termites use cultivated fungi or gut symbionts to break down their cellulosic food- stuffs. Microbes profoundly affect the abilities of insects to utilize intractable nutritional resources and occupy habitats that otherwise would be unavailable to them. The present work grew from a need to obtain yeasts for comparison with the gut-inhabiting yeasts of fungus- feeding beetles. We investigated passalid beetles (Cole- ?ptera: Passalidae) for gut yeasts because: (i) passahds inhabit the same woody substrates used by certain basidiomycete fungi and the beetles that eat those fungi; (ii) fungi assist insects and other animal hosts by degrading the complex polysaccharides (cellulose and hemicelluloses) and phe- nylpropane polymers (lignin) that comprise the secondary cell walls of woody plants; (iii) passalid beetles are sub- social, they live in colonies of related individuals with over- lapping generations, which could promote associations with symbionts; and (iv) yeast-like fungi of undetermined ? 2003 Blackwell Publishing Ltd 3138 S.-O. SUH ET AL. taxonomic placement had been reported previously from passalids (Lichtwardt et al. 1999). Odontotaenius disjunctus (lUiger) is broadly distributed in eastern North America from Ontario and Quebec southwards to Lake Okeechobee, Florida (Schuster 1994). The beetles may grow to a length of 3.5 cm; they develop rapidly by maturing from egg to adult in several months. They have subsocial behaviour with adults tunnelling into moist rotten logs and stumps that have been decayed for several years by white rot fungi in temperate regions (Gray 1946; Schuster & Schuster 1997) or within 3-4 months in the tropics (Rodriquez & Zorrilla 1986). Adult beetles tear and masticate the ingested wood to a fine grain. First-instar larvae presumably require wood that is first processed by the adults (Pearse et al. 1936; Gray 1946; Schuster & Schuster 1985), and both adults and larvae feed on the masticated wood plastered on the walls of the tunnels by the adults. Several earlier studies reported gut micro-organisms to be absent (Pearse et al. 1936; Gray 1946; Schuster & Schuster 1985), but because both adults and larvae ingest faeces, predigestion of the faecal pellets by microbial action has been postulated to occur and compared to an 'external rumen.' Details of the process by which the wood ingested by O. disjunctus is degraded are not clear, but it is unlikely that Trichomycetes (Fungi: Zygomycota) and an unclassi- fied fungus noted as regular inhabitants of the gut (Heymons & Heymons 1934; Lichtwardt et al. 1999) are involved. Analysis of the chemical composition of passalid faecal pellets from the field and under laboratory conditions showed that available nitrogen increased with time (Rodriquez 1985; Rodriquez & Zorrilla 1986). Behavioural observations of many species of passalids indicated that O. disjunctus is characteristic of the family as a whole (Schuster & Schuster 1997). Here we report the consistent isolation of closely related yeasts (Fungi: Ascomycota, Saccharomycetes) from O. dis- junctus in the eastern USA and Kansas and from a second passalid species with a similar life history. Verres sternber- gianus, from Panama. Phylogenetic analysis groups the undescribed yeasts in a well-supported clade with four other yeast taxa, including Pichia stipitis. The ability of P. stipitis and a few other yeasts to ferment xylose has led to intense interest in their use in biotechnology to produce ethanol from wastes with high xylose content (van Dijken et al. 1986; Jeffries & Kurtzman 1994; Biely & Kremnicky 1998; Winkelhausen & Kuzmanova 1998; Jeffries & Jin 2000; Ward & Singh 2002). Methods and materials Host beetles Yeasts were isolated from the gut and external surface of specimens of Odontotaenius disjunctus (Passalidae: Procxilini) collected from rotten wood in Pennsylvania and several different localities in Georgia, South Carolina and Louisiana, and from Verres sternbergianus (Passalidae: Proculini) from Barro Colorado Island, Panama (Table 1). Vouchers were deposited at the Georgia Museum of Natural History, Collection of Arthropods. Yeast isolation and culture Beetles collected from decaying logs were frozen until dead and submerged in 95% ethanol for 1-2 min to disinfect their surfaces. The alcohol wash was followed by a 0.7% saline rinse; the rinse liquid was plated on acidified YM agar (Difco YM broth, 2% plain agar, adjusted to pH 3.5 with HCl) as a negative control. Forceps, dissecting needles and minute insect pins were used to dissect the beetles on sterile microscope slides under a dissecting microscope. The beetle gut was removed aseptically, cut into pieces and transferred to tubes containing 0.7% saline. Gut segments were crushed in the saline solution with a pipette tip and streaked with a loop onto the surface of acidified YM agar plates. Plates were incubated at 25 ?C, and after 3 days single colonies were streaked for purification. This procedure was replicated one or more times. Cultures were maintained on YM agar. A culture established from the original type material of Enteroramus dimorphus (KS-42-W2) from the gut of O. disjunctus collected in Kansas (Lichtwardt et al. 1999), was compared with the other yeasts isolated in this study. Morphological observations and metabolic tests comprising the yeast 'standard description', were performed on the passalid yeasts listed in Table 1, according to established methods (Kurtzman & Fell 1998; Barnett etal. 2000). The cultures established in this study have been deposited in the Agricultural Research Service (ARS) Culture Collection (NRRL Y-27547-Y-27555). DNA methods Yeast cells were harvested from agar cultures, and the nucleic acids were extracted and purified following the procedures of Lee & Taylor (1990). The primer sets NSl- NS8, LS1-LR5 and ITS5-ITS4 were used for amplifying SSU and LSU rRNA genes (rDNA), and 5.8S rDNA and internal transcribed spacer (ITS) sequences (White et al. 1990; Hausner etal. 1993), respectively, using the polymerase chain reaction (PCR). PCR products were purified using a DNA purification kit (Bio-Rad Laboratories). The purified double-stranded PCR products were used as templates for sequencing with an ABl PRISM^" BigDye Terminator Cycle sequencing kit, version 2 (PE Applied Biosystems). The complete sequences of SSU rDNA, 5.8S rDNA including ITS and the D1/D2 region of the LSU rDNA were sequenced with the primers NSl, NS2, 18H, NS8, ITSl, ITS4, LSI and LR3 using an ABI ? 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 3137-3145 GUT YEASTS OF PASSALID BEETLES 3139 Table 1 Yeasts isolated from passalid beetles and GenBank DNA accession numbers GenBank rDNA accession no. DNA group* Yeast isolate no. Host and other information LSU SSU ITS/5.8S PASSl BG 01-5-4-2-1 GA 012-1-1 BG 02-7-16-1 BG 02-2-11-6-5 BG 02-4-1-3-1 BG 03-3-25-1-3 BG 03-3-25-l-5t BG 02-7-14-003-1-1 BG 02-7-14-003-2-1 PASS5 KS-42-W2 Odontotaenius disjunctus; Passalidae Burke Co., Shell Bluff, G A (March 22,2001) (NRRL Y-27547) Odontotaenius disjunctus; Passalidae Clarke Co., Athens, GA (July 9,2001) (NRRL Y-27548) Odontotaenius disjunctus; Passalidae Orangeburg Co., SC (August 13,1999) Yeast isolate provided by C. E. Beard (NRRL Y-27549) Odontotaenius disjunctus; Passalidae East Baton Rouge Parish, Baton Rouge, LA (February 11, 2002) (NRRL Y-27550) Odontotaenius disjunctus; Passalidae East Baton Rouge Parish, Baton Rouge, LA (April 3,2002) (NRRL Y-27551) Odontotaenius disjunctus; Passalidae Oxford, PA, in red oak log (20 March 2003) (NRRL Y-27554) Odontotaenius disjunctus; Passalidae Oxford, PA, in red oak log (20 March 2003) (NRRL Y-27555) Verres sternhergianus; Passalidae Barro Colorado Island, Panama (July 14,2002) (NRRL Y-27552) Verres sternhergianus; Passalidae Barro Colorado Island, Panama (July 14,2002) (NRRL Y-27553) Odontotaenius disjunctus; Passalidae Douglas Co. KS (September 19,1997) (NRRL Y-27535) AY227721 AY227898 AY227901 AY227720 AY227897 AY227900 AY227723 AY227724 AY227725 AY325109 AY325110 AY227726 AY227727 AY227903 AY227904 AY227905 AY325111 AY325112 AY227906 AY227907 AY227722 AY227899 AY227902 *DNA group based on the sequence of D1/D2 region in LSU rDNA. tMetabolic tests not performed. PRISM 377 Automated DNA sequencer (PE Applied Biosystems). GenBank accession numbers for DNA sequenced from passalid beetles in this study are listed in Table 1. In addi- tion to the new yeast sequences in Table 1, several other yeasts were sequenced in this study [Candida ambrosiae (NRRL YB-1316), Candida tanzawaensis (NRRL Y-17324), Candida ernobii (acquired as Candida karawaiewii) (ATCC 22994), Candida xestobii (ATCC 24001), Symbiotaphrina buch- neri (CBS 420.63), and Symbiotaphrina kochii (CBS 250.77)], and their GenBank accession numbers are listed in the reference data below. LSU rDNA sequences were used to distinguish yeast genotypes (Kurtzman & Robnett 1998); in this study PASSl and PASS5 are named using the first four letters of the beetle family and a unique number (Table 1). Data analysis DNA sequences were aligned with sequences obtained from GenBank using the multialignment program CLUSTAL X (Thompson et al. 1997). The newly sequenced yeasts were analysed with LSU and SSU rDNA sequences of other yeasts and fungi obtained from GenBank. GenBank accession numbers of SSU and LSU rDNA sequences, respectively, were as follows: Arxula adeninivorans (AB018123; U40094), Aureobasidium pullulans (M55639; AF050239), Brettanomyces naardenensis (X85110; U76200), Candida albicans (M60302; AF156536), Candida ambrosiae (AY227712; AY013716), Candida ergatensis (AB013524; U45746), Candida insectamans (AB013518; U45791), Candida insedorum (AB013565; U45753), Candida intermedia (AB013571; U44809), Candida ernobii (acquired as Candida karawaiewii) (AY227714; U94921), ? 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 3137-3145 3140 S.-O. SUH ET AL. Candida kruisii (AB013543; U45718), Candida lyxosophila (AB013522; U76204), Candida odintsovae (AB054570; U70182), Candida parapsilosis (AB013588; AF485969), Candida shehatae var. insectosa (AB013584; U45773), Candida rhagii (AB018172; U45729), Candida tennis (AB013516; U45774), Candida tanzawaensis (AY227713; U44811), Candida xestobii (AY227715; U45707), Chromocleista malaciiitea (D88323; AB000621), Dipodascus albidus (X69840; U40081), Galadomy?s geotrichum (X69842; U40118), Hamigera avellanea (D14406; AF454075), Hanseniaspora uvamm (X69844; U84229), Hypocrea lutea (D14407; U00739), Kluyveromy?s polysporus (X83825; U68548), Lodderomyces elongisporus (X78600; U45763), Neurospora crassa (X04971; U40124), Pachysolen tannophilus (AF132030; U76346), Pichia an?mala (AB054562; AF330115), Pichia guilliermondii (AB013587; AF374616), Pichia segobiensis (AB054288; U45742), Pichia stipitis (AB054280; U45741), Protomyces inouyei (D11377; U84344), Saccharomyces cerevisiae (Z75578; J01355), Saccharomycopsis capsularis (X69847; U40082), Stephanoascus farinosus (AB000660; U40132), Symbiotaphrina buchneri (AY227716 AY227718), Symbiota- phrina kochii (AY227717; AY227719), Taphrina deformans (U00971; AF492038), Williopsis saturnus var. mrakii (Y11318; U94929), yeast-like symbiont of Laodelphax striatellus (AF267232; AF267235), yeast-like symbiont of Sogatella fur- cifera (AF267234; AF267237), yeast-like symbiont o? Nilaparvata lugens (AF267233; AF267236), Zygoascus hdlenicus (AF294751; U40125), Zygosaccharomyces rouxii (X90758; U72163). The alignments were optimized visually, and ambiguous regions were excluded from the analyses. Protomyces inouyei and Taphrina deformans, determined to be basal ascomy- cetes in previous phylogenetic studies, were designated as out group taxa. Maximum parsimony analyses were performed using PAUP 4.0bl0 (Swofford 2002). Heuristic tree searches were executed using the tree bisection-reconnection branch swapping algorithm with random sequence analysis. Boot- strap values of the most parsimonious tree were obtained from 1000 replications. Base-pair differences in a gene were counted using BLAST 2 sequences (Tatusova & Madden 1999) or from a manually aligned sequence database. Results Yeast cultures PASSl yeasts (Table 1) were isolated from the gut and external surface of about 22 adult passalid beetles {Odonto- taenius disjunctus and Verres sternbergianus) and purified on YM agar. More than 100 colony-forming units were obtained from the gut of every beetle, except for a single anomalous individual from which no yeast was isolated. Although other yeast species were not cultured from the passalids, we cannot rule out the presence of yeasts that would not grow under our culture conditions or that were present in low population numbers. The PASSl and PASS5 cultures were used as a source of genomic DNA for sequencing. Approximately 1730 base pairs (bp) of SSU rDNA representing most of the gene; about 600 bp of the LSU rDNA gene, including the variable D1/D2 region; and about 600 bp of the ITS and 5.8S region were obtained from PCR products of the passalid yeasts. Relationships of the yeasts Phylogenetic analysis using a combined LSU and SSU rDNA sequence database of the eight passalid isolates and a diverse group of other yeasts chosen from among all described species of yeasts, resulted in a single most parsimonious tree (Fig. 1). The passalid isolates formed a well-supported clade with two closely related taxa (Pichia stipitis and P. segobiensis), hereafter called the P. stipitis clade. Candida shehatae was a sister taxon to the P. stipitis clade, and C. ergatensis was sister to the P. stipitis and C. shehatae clade and the basal member of the lineage (bold typeface. Fig. 1). The other relationships obtained were consistent with results from previous studies (Kurtzman & Robnett 1998). Genetic and metabolic similarity among passalid yeast isolates Two passalid sequences were distinguished by comparison of LSU rDNA (PASSl, PASS5, Table 2), following a convention used in the study of yeasts (Kurtzman & Robnett 1995,1998). Other PASS isolates from Panama exist, but these are from passalid beetles with a different habit, and they will be discussed in a future study. The PASSl and PASS5 LSU rDNA sequences varied by only 1 bp from each other, as did their SSU rDNA sequences. ITS sequences revealed greater variation (Table 2). The PASSl ITS sequ- ences from the USA (Pennsylvania BG 03-3-25-1-3, BG 03- 3-25-1-5; Georgia BG 01-5-4-2-1, GA 012-1-1; South Carolina BG 02-7-16-1; Louisiana BG 02-2-11-6-5, BG 02-4- 1-3-1) were identical, but varied by 1 bp from the Panama PASSl ITS sequences from Verres sternbergianus (BG 02-7- 14-003-1-1, BG 02-7-14-003-2-1). The PASS5 (Kansas KS-42- W2) ITS sequence varied by 1 bp from the USA PASSl isolates and by 2 bp from the Panamanian PASSl isolate. Pichia stipitis and P. segobiensis were more variable, differing from the passalid isolates by 6-7 hp and 16- 18 bp, respectively (Table 2). BLAST searches did not reveal other similar sequences deposited in GenBank or collected by us. Besides the DNA sequences, approximately 20 morpho- logical and 80 physiological traits were compared in vitro for the PASSl isolates to other clade members. The PASSl and PASS5 isolates varied in relatively minor details when compared to the reference cultures of P. stipitis and ? 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 3137-3145 I? lapnnna aerormans i. .. 1 Protomycesinouyei | Archiascomycetes 0) % rE- o o I? n 3 UJ O o 5? ?-E- p j? Hamigera avellanea Chromocleista malachitea Neurospora crassa Hypocrea l?tea 52r Laodelphax striatellus YLS (planthopper) Sogatella furcifera YLS (planthopper) Nilaparvata lugens YLS (planthopper) I Neur H I?"M T-J?2^ , I 1001 I? Symbiotaphrina buchneri (anobiid beetle) '?Symbiotaphrina /cocft/7 (anoblId beetle) Aureobasidium pullulans 83i Arxula adeninivorans ? Stephanoascus farinosus ? Zygoascus liellenicus 100 I Candida intermedia' ? Brettanomyces naardenensis' Dipodascus albidus - Galactomyces geotrichum GUT YEASTS OF PASSALID BEETLES 3141 Fig. 1 Single most parsimonious tree obtained from combined LSU and SSU rDNA sequence data. The xylose-fermenting clade, including yeasts isolated from pas- salid beetles, appears in bold typeface, and subphyla of ascomycetes are indicated on branches. Yeasts that ferment xylose are indicated by an asterisk (*); note that five species outside of the Pichia stipitis clade possess the rare trait. Tree length = 3589; consistency index = 0.4266; homoplasy index = 0.5734; retention index = 0.6450; rescaled consistency index = 0.2751. Num- bers on tree branches indicate the percentages of bootstrap samplings derived from 1000 samples that supported the internal branches by 50% or higher. ? Sacciiaromycopsis capsularis - Zygosaccharomyces rouxii ? Saccttaromyces cerevisiae ? Kluyveromyces polysporus ? Hanseniaspora uvarum ? Candida ernobii (anobiid beetle) Pachysolen tannoptiilus' Pictiia an?mala I 981?^y \?SS?H~? ' H 751 h 581 92[~~l_ Candida odintsovae Williopsis saturnus var. mrakii mi Pichia gu/ffiermond//(cerambycid beetle) ?? Candida xestobii (anoblld beetle) 100 r Candida tenuis' (cerambycid beetle) ? Candida insectorum I? Candida liruisii ?I 100 r Candida tanzawaensis I I ? Candida ambrosias ' Candida rhagii (cerambycid beetle) 96|? Lodderomyces elongisporus 1 STTLC 56 I C andida parapsilosis (cerambycid beetle) andida albicans j Candida insectamans ?T"? Candida lyxosophila' Candida shehatae var. insectosa* (cerambycid beetle) Pichia segobiensis* Pichia stipitis* Pass5* (passalid beetle) 95 looi passi* (passalid beetle) ? Candida ergatensis (cerambycid beetle) sor fPi LJlp ? 50 changes P. segobiensis discussed in the literature (Kurtzman & Fell 1998; Barnett et al. 2000), such as ability or rate of ferment- ation and assimilation of several carbon compounds and growth at certain temperatures (Table 3). Most members of the clade, including PASSI and PASS5 isolates, fermented and assimilated xylose (Kurtzman 1990; Jeffries & Kurtzman 1994), albeit often delayed until about 10 days after inocu- lation. The exception was the basal member, C. ergatensis, as reported in the literature (Kurtzman & Fell 1998; Barnett et al. 2000). However, this species, along with P. stipitis, has been reported to hydrolyse xylan, a component of hemicel- lulose composed of D-xylose residues (Biely & Kremnicky 1998). In addition most of the clade members synthesize a wide range of vitamins (myo-inositol, pantothenate, thia- mine, pyrodoxine, niacine), but only PASSI isolates pro- duced small amounts of biotin in vitro. Again, C. ergatensis was the outlier producing fewer vitamins, requiring both biotin and thiamine for growth (Kurtzman & Fell 1998; Barnett et al. 2000). Two morphological differences were detected among clade members. The symbiotic filamentous growth that marked the PASS5 isolate in situ was absent in culture, and the PASSI isolates varied in production of pseudohyphae or hyphae in culture. The other members of the clade all form pseudohyphae or true hyphae in culture. Sexual reproduction occurred in the passahd isolates, P. stipitis and ? 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 3137-3145 3142 S.-O. SUH ET AL. Table 2 Comparison of LSU rDNA (577 bp) (bold figures) and ITS/5.8S rDNA (567 bp) bp differences in rl^SiA gene of yeasts from passalid beetles and closest relatives Locality Yeast isolates 1 2 3 4 5 6 7 8 9 10 11 12 PASSl USA (GA) BG 01-5-4-2-1 1 ? 0 0 0 0 0 0 6 16 GA 012-1-1 2 0 ? 0 0 0 0 0 6 16 USA (SO BG 02-7-16-1 3 0 0 ? 0 0 0 0 6 16 USA (LA) BG 02-2-11-6-5 4 0 0 0 ? 0 0 0 6 16 BG 02-4-1-3-1 5 0 0 0 0 ? 0 0 6 16 USA (PA) BG 03-3-25-1-3 6 0 0 0 0 0 ? 0 6 16 BG 03-3-25-1-5 7 0 0 0 0 0 0 ? 6 16 Panama (BCD BG 02-7-14-003-1-1 8 0 0 0 0 0 0 0 ? 0 2 7 17 BG 02-7-14-003-2-1 9 0 0 0 0 0 0 0 0 - 2 7 17 PASS5 USA (KS) KS-42-W2 10 1 1 1 1 1 1 1 1 1 ? 7 17 France Pkhia stipitis (JCM 10742) 11 1 1 1 1 1 1 1 1 1 1 ? 18 Spain Pichia segobiensis (JCM 10740) 12 2 2 2 2 2 2 2 2 2 1 2 PASSl and PASS5 designations are based on unique LSU rDNA sequences. Table 3 Variable traits* of PASSl, PASS5, Pichia stipitis and P. segobiensis among about 100 traits tested Variable trait PASSl t PASS5 Pichia stipitis^ Pichia segobiensis^ Morphology Pseudohyphae? +/- - Fermentation Maltose D D Cellobiose - - Starch - - Carbon assimilation L-Sorbose - - L-Arabinose +/D D D-Arabinose +/D + L-Rhamnose +/- - a-Methyl-D-glucoside + + Maltose + + Lactose - + Soluble starch +/D D Erythritol +/D - L-Arabinitol +/D/W - D-Gluconate +/D + DL-Lactate D/W D Propane 1,2 diol +/D/W W Nitrogen assimilation D-Glucosamine W - Vitamin requirements W/O PABA +/W + W/O Biotin w/- w W/O Biotin, Thiamin w/- - Growth temperature at 35 ?C + - at40?C - - +/D D/- D/- D/- +/- +/D +/D +/- D/- D/- D/- D/- D D/- D +/- *Variable reactions: +, positive reaction; -, negative reaction; D, delayed positive reaction; W, weak positive reaction. tData obtained from eight PASSl isolates listed in Table 1. JData from Barnett et al. (2000). ?Observation on corn meal agar after 7 da (Kurtzman &: Fell 1998). ? 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 3137-3145 GUT YEASTS OF PASSALID BEETLES 3143 P. segobiensis, all of which usually produced two hat-shaped ascospores per ascus. Candida shehatae and C. ergatensis are asexual yeasts. Discussion Symbiotic passalid yeasts Yeasts sometimes have been considered conspecific if they share identical or similar sequences for about 600 bp in the D1/D2 loop region of the LSU rDNA (Kurtzman & Robnett 1998), and, using this conservative measure, PASSl, PASS5 and Pichia stipitis might be considered conspecific by some yeast systematists. The chance determination that the PASS5 isolate (KS-42-W2), previously described as Enteroramus dimorphus (Lichtwardt et al. 1999), is a yeast shows the utility of a dense DNA database for identifica- tion of yeasts if not for routine species delimitation. The morphology and physiology of the passalid isolates and P. stipitis also are similar, and consistent with small differences sometimes observed among isolates of a single taxon. The PASSl and PASS5 isolates have not been dis- tinguished as species distinct from P. stipitis, because without greater geographical sampling and additional genomic markers, we do not know if the differences detected among the isolates (Tables 2 and 3) are significant. There is, however, the possibility that certain yeast popula- tions have become isolated in association with the passalids. Interestingly, the capacity of fungi to reproduce sexu- ally is usually correlated with their degree of symbiosis and method of transmission. Sexual reproduction by the passalid-gut yeast isolates implies that their biology is not strictly tied to that of their coleopteran hosts and that they are not simply clonally propagated by vertical transmis- sion. Thus, if xylose-fermenting yeasts help to improve the fitness of the beetles, it may be necessary for the beetles to repeatedly co-opt these yeasts from the environment. Of course, given the subsocial behaviour of passalid beetles with requisite intergenerational transfer of gut fauna, associations lasting more than a generation are likely. Fur- thermore, as has happened with termite endosymbionts, association with yeasts may have played a fundamental role in the evolution of subsocial behaviour, a characteristic of all Passalidae. Wood-decay and the importance of microbes This is the first specific report of a widespread association of passalid beetles with a known endosymbiotic organism. Earlier researchers suggested microbes were involved in passalid digestion, but such micro-organisms were not identified in these studies (Pearse et al. 1936; Gray 1946; Schuster & Schuster 1985). The isolation of other yeasts belonging to the P. stipitis dade from different wood-ingesting beetles and their habitats, indicates that Passalidae are not the only beetles to make use of yeasts having the rare ability to ferment and assimilate xylose (Fig. 1) (Martin 1987; Nardon & Grenier 1989). In the yeast pathway xylose is converted to xylitol, and then, xylulose; conversion of xylulose is by the pentose phosphate pathway to fructose- 6-phosphate to provide a substrate for oxidation or fermentation (Jeffries & Jin 2000; Jackson & Nicolson 2002). Xylose is usually not found as a soluble sugar in nature, unlike sucrose and fructose, so the insect gut offers a place in which hemicellulose can be broken down for assimilation (Jeffries & Jin 2000). Some wood decay may be required before the invasion of adult passalids. Early in the wood decay process, bac- teria, some of which fix nitrogen, and fungi, including yeasts and stain fungi, invade fallen timber. For example stain fungi help to remove extractives such as phenolics that can inhibit fungal enzymes. Later in the succession, wood- decaying basidiomycetes contribute their decay enzymes to the process. In the relatively early stages of the white rot process (Alexopoulos et al. 1996), lignin-cellulose bonds are cleaved to expose unprotected hemicellulose that is vulnerable to fungal enzymes (Eriksson et al. 1990; Blanchette 1991). Our findings do not preclude the possibility that gut micro-organisms in addition to the PASSl and PASS5 yeasts may be involved in the digestion of ingested wood in passalids; for example we have not attempted to isolate bacteria, nor are we certain that other eukaryotes are absent. Several trichomycetes are known to partition them- selves within the gut of passalids and they could also be involved (Lichtwardt et al. 1999, 2001). Beetle invasions into woody substrates may have been facilitated by their association with yeasts. The association of passalids from distant localities with yeasts of such high genetic similarity suggests a significant commensal or symbiotic relationship. Acknowledgements We are grateful to Drs Merlin M. White and Robert W. Lichtwardt for supplying the culture of the PASS5 isolate, Enteroramus dimorphus. Drs Eddie Beard and Will Reeves, Clemson University, provided the yeast cultures isolated from South Carolina, and Dr Douglas Tallamy, University of Delaware, collected several bee- tles. Nhu Nguyen collected numerous passalid beetles from the Baton Rouge region, and undergraduates Will Hampton and James Robertson participated in the collection of the Panamanian passalids. We especially appreciate the untiring commitment of current undergraduate students Christine Ackerman, Katie Brill- hart, Cennet Erbil, Nhu Nguyen, and Amy Whittington, and those of the past, including Doan Dang, Ebony Spikes, Rebecca Sweany, and John Williams, who helped in all phases of the work from col- lection of beetles and culturing to sequencing of yeast isolates. Dr Jack Schuster was helpful early in the study bv answering passalid ? 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 3137-3145 3144 S.-O. SUH ET AL. questions and showing interest in our findings. We also benefited from informative and encouraging discussions with Drs Robert Blanchette, University of Minnesota, and Thomas W. Jeffries, USDA Forest Service, Forest Products Laboratory. The manu- script was improved by editorial comments by Drs Thomas W. Jeffries, Robert W. Lichtwardt and Merhn M. White. 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White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols - a Guide to Methods and Applications (eds Innis MA, Gelfand DH, Sninsky JJ, White TJ), pp. 315-322. Academic Press, San Diego, CA. Winkelhausen E, Kuzmanova S (1998) Microbial conversion of D- xylose to xylitol. Journal of Fermentation and Bioengineering, 86, 24-37. The authors of this study are interested in the systematics, ecology, and evolution of fungi and beetles and their interactions. This study by Suh, senior postdoctoral associate, and Blackwell is part of an ongoing study in the Blackwell laboratory on fungi and their interactions with insects. Together with McHugh, a cucujoid beetle systematist, the mycologists are investigating the bio- diversity of yeasts associated with mushroom-feeding and other beetles. Marshall brings his expertise in passalid beetle biology and systematics to the study. ? 2003 Blackwell Publishing Ltd, Molecular Ecology, 12, 3137-3145