Beetles and their yeast associates from basidiocarp habitats (NSF proposal DEB-0417180)
Introduction / Participants / Basidiocarps as habitat / Insects / Yeasts / Methods / Publications / Literature cited / Proposal I / Proposal II/ Mycology at LSU
This material is based upon work supported by the National Science Foundation under Grant No. 0072741.  Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
Project Summary
WHAT IS THE INTELLECTUAL MERIT OF THE PROPOSED ACTIVITY? Symbionts play important roles in the ecology and evolution of their hosts. To investigate potential symbiotic associations we sampled yeasts from a largely unexplored habitat, the gut of beetles. About 650 isolates were obtained from the gut of primarily basidiocarp-feeding (mycophagous) and other beetles in 27 families from the southeastern USA and Barro Colorado Island, Panama. Yeasts were characterized by their LSU rDNA sequences and 100 morphological and metabolic traits. These isolates represent almost 200 species of undescribed yeasts, about 30% more than the 700 previously known yeast species. Specific beetle--gut yeast associations have been discovered, the basis of which is not yet understood. Bayesian analysis estimates that resampling of the same habitats would greatly increase the numbers of species from the specialized habitat. Such a discovery would be equal to about half the currently known species from all habitats of the Earth. In a continuing study we propose to collect in previously sampled localities and extend species discovery to South Africa. The collecting would be done in the context of several questions to maximize the scientific value of the study.
  • Do many yeasts remain to be discovered in the sampled habitat? The primary objective of this study is yeast species discovery. A well-supported model used to analyze species acquisition data indicated that approximately 1.5--2 times as many taxa remain to be discovered in the previously sampled habitats and localities. Literature reports suggest that many insects in targeted groups at previously sampled localities remain to be collected, requiring recollection to improve knowledge of yeasts, mycophagus beetles, and macrofungi. New taxa would be described.
  • Are changes in yeast mycota correlated with changes in nutritional mode of certain beetles? The Cerylonid beetle series (CS) comprises eight families of Superfamily Cucujoidea. Most species are mycophagous, feeding on fungi or fungus-altered plant matter, but there have been dramatic trophic shifts within the group. In the process of looking for new taxa, the yeasts from CS beetles would be examined and compared to determine if patterns of yeast distribution are related to the trophic mode of the beetle hosts.
  • What influences associations between beetles and gut yeasts?  Beetle sampling strategy during discovery of new taxa would be designed to examine the effect of locality, fungus, and beetle taxon on yeasts. Erotylidae (Cucujoidea) and Tenebrionidae, the most common beetles in our collections, would be sampled from certain basidiocarps (Ganodermataceae), and used to compare yeasts in common habitats at distant localities. Additionally, yeasts from different beetles in the same basidiocarp would be compared for information on mode of yeast dispersal.
  • What is the basis of the beetle/gut yeast associations?  A value-added component of our work would be an investigation of the beetle/gut associations in which the beetles would be cured of yeasts in order to focus on vitamin and enzyme exchange for carbon source as suggested by preliminary observations.

    • WHAT ARE THE BROADER IMPACTS OF THE PROPOSED ACTIVITY? (1) The PIs have a strong record of graduate and undergraduate education. Students would continue to have opportunities to do research and to be authors on presentations at national meetings and publications in peer-reviewed journals. (2) Research results would be disseminated in presentations, in widely recognized peer-reviewed journals, and on the Internet. We would continue to develop and update our yeast database, including interactive keys and photographs of the organisms involved in the tritrophic relationships between gut yeasts, mycophagous beetles, and basidiomycetes. (3) We would submit an article to a popular scientific publication to highlight this complex and apparently widespread aspect of life on Earth. (4) The results from the study of the organismal assemblage could serve as the basis to expand hypotheses on the evolution of megadiverse groups (e.g., beetles and fungi).
    Introduction: Beetles and their yeast endosymbionts from basidiocarp habitats
    Four years ago we justified a proposal to isolate yeasts from the gut of basidiocarp-feeding beetles with the prediction that we would discover more than 50 undescribed yeast species. Our results surpassed even our high expectations, and significant findings from the previous project follow:
  • More than 650 yeast isolates were cultured from the digestive tract of over 90% of the beetles dissected. Many of the yeasts were localized in gastric caecae (Fig. 1, left). Many of these beetles from 27 families depicted in the graph (Fig. 2, below) were from basidiocarp habitats (Suh and Blackwell 2004, in press). Most were true yeasts (Ascomycota: Saccharomycetes), but some were basidiomycete yeasts (Tremellales).
  • The 650 yeasts were divided into 290 genotypes, representing almost 200 undescribed taxa.
  • Insect-associated yeasts were distributed in clusters throughout the yeast phylogenetic tree (Fig. 3, below, see four shaded terminal clades of a reduced data set). Several previously unknown insect-associated yeast clades were discovered or expanded. The Candida tanzawaensis clade in the tree has over 16 new taxa associated exclusively with insects in one lepidopteran and 11 coleopteran families.
  • Yeast/beetle specificity was common, especially between yeasts and erotylid, tenebrionid, or passalid beetles. Species-specific yeast/beetle associations were observed across broad geographical ranges and multiple developmental stages for individual beetle species.
  • We identified wide-ranging yeast genotypes associated with certain beetle species (e.g., from Vermont to Louisiana and Pennsylvania to Louisiana) using ITS rDNA markers.
  • There was little overlap of yeast or insect taxa between the USA and Panama, although certain basidiomycetes (e.g., Tinctoporella epimiltinus, Hexagona hydnoides) were common substrates in both regions.
  • We collected over 2500 beetles, primarily members of the superfamily Cucujoidea and Tenebrionoidea. Of 40 species of Erotylidae (Cucujoidea) collected in Panama, 30 had not been reported from Panama. Because almost all dissected beetles in these groups bore yeasts, untapped beetle diversity lends support to high estimates of undiscovered yeasts.
  • Many of the Panamanian beetles could be identified only to genus, and many new species are expected. This taxonomic work, however, should be done within the context of broad scale revisions of monophyletic groups such as those to be done by the PEET project (see Division of Labor and Similar Research, below). Much of the relevant type material resides in European museums.
    The discovery of almost 200 undescribed yeasts gains greater significance with the realization that fewer than 700 species of yeasts have been described previously from all of the Earth’s habitats. Our study exceeded expectations to the extent that our success delayed completion of the project, and a one-year no-cost extension was needed because of the requirements for formally describing so many new yeast species. Based on the statistical prediction of many more yeasts to come, we propose a continuation of the project in which the sampling strategy would allow us to discover more taxa within the conceptual framework outlined below for a continuation of the beetle/gut yeast study. 
    CONCEPTUAL ISSUES

    • •ARE MANY MORE YEASTS UNDISCOVERED IN PREVIOUSLY COLLECTED AREAS? The primary objective of this study remains species discovery. To obtain estimates of the number of species yet to be discovered in this unique ecosystem, our colleague David Pollock (see letter of support) applied a Bayesian analysis using Poisson, multiple rates of discovery with adjustable frequencies and Gamma-distributed rates of species discovery to our data (Pollock and Larkin, submitted to Genetics). The model indicates that, using our previous methods, 60% of the species present remain undiscovered in the habitat. Recollection would reveal not only more species from the same localities, but also would test the model of species discovery. Many unsampled beetle hosts that exist in previously collected localities and additional adjacent collecting sites are readily available to increase our diversity sampling further.
    •ARE CHANGES IN YEAST MYCOTA CORRELATED WITH CHANGES IN NUTRITIONAL RESOURCES OF BEETLES? The Cerylonid Beetle Series (CS) consists of eight derived families, about half of all known species of the superfamily Cucujoidea.  Although cucujoid beetles tend to feed on fungi and on fungus-altered plant material, beetles within several CS groups have undergone trophic changes to feeding on myxomycetes, plants, and insect prey.  Within some CS clades there has been a shift away from a free-living lifestyle to ectoparasitism of wood-boring insects or inquilinity with social insects. We would compare the yeasts across both monophyletic groups within the CS and across trophic modes.  Tracking these changes would help us to maximize species discovery in continuing studies. This work would be coordinated with McHugh’s recently funded PEET project (see Division of Labor and Similar Research, below).
    •WHAT INFLUENCES BEETLES/GUT YEAST ASSOCIATIONS?  We would target species of the polypore family (Ganodermataceae) and basidiocarp-feeding erotylid beetles to compare yeasts at distant localities. The sampling strategy would maximize species discovery, and in addition examine the effect of fungus host and beetle taxon on yeast associates with attention to physiological profiles. In addition to erotylids, other beetles would be present for comparison of yeasts in common basidiocarp habitats. Comparisons would be made between South Africa and our New World sites (southeastern USA and Panama) regions with minimal recent contact.
    •WHAT IS THE BASIS FOR THE YEAST/BEETLE INTERACTIONS? A value-added component will be investigation of the basis of interactions based on our observations: 1) repeated associations between particular yeasts and beetles indicating specificity and universality, 2) high number of colony-forming units in many isolations, 3) failure to isolate the yeasts from the surrounding habitat, 4) localization of certain yeasts in anterior midgut caecae, and 5) vertical transmission of certain yeasts. Together these observations provide strong evidence that many yeasts are neither transients or food yeasts acquired from the habitat. 
    RESEARCH PLAN
    We propose to continue the study of beetle gut yeasts, because the successful project resulted in unexpectedly high levels of species discovery. In addition to continuing species discovery (Objective 1), the next phase of the study (2004-2007) would provide data on changes in yeast/beetle interactions with changes in trophic conditions and beetle phylogeny (Objectives 2 and 3); information on the interactions between the yeasts and beetles would come from Objective 4. (For methods see Management Plan, below).

    OBJECTIVE 1: COLLECT ADDITIONAL YEASTS AND TEST HYPOTHESIS THAT MANY YEASTS REMAIN UNDISCOVERED IN THE PREVIOUSLY COLLECTED LOCALITIES.

    OBJECTIVE 2:  EXAMINE IF CHANGES IN YEAST MYCOTA ARE CORRELATED WITH TRANSITIONS IN NUTRITIONAL RESOURCES OR TROPHIC MODES OF BEETLES. OBJECTIVE 3: CLARIFY INFLUENCES AFFECTING THE ASSOCIATIONS BETWEEN BEETLES AND GUT YEASTS. OBJECTIVE 4:  INVESTIGATE THE INTERACTIONS BETWEEN YEASTS AND BEETLES (ADDED VALUE STUDY THAT IS PLANNED AS A PART OF RESEARCH BY STUDENTS).
    TAXONOMIC BREADTH
    Yeasts.— The taxonomic emphasis of this proposal is ascomycete yeasts (Saccharomycetes), a derived order of about 700 previously known species. Collections and inventories of associated beetles and macrofungi would also be made. Over the four years of the previous study we isolated about 650 yeasts. In phylogenetic analyses the yeasts occurred in clusters across the phylogenetic tree of all Saccharomycetes, and 15 new basidiomycete yeasts also were discovered. Beetles.— We collected more than 2500 beetles. Of those dissected about 90% had yeasts. The beetles represented two of the four coleopteran suborders and 27 families (Fig. 4, above), many belonging to the superfamilies Cucujoidea and Tenebrionoidea. More than 200 basidiocarps in about 50 species served as beetle habitats.  Basidiomycota.— The basidiomycetes include groups informally known as agarics, boletes, puffballs, and polypores and other wood-decaying basidiomycetes that can be placed in about 20 clades (Moncalvo et al. 2002). Tripartite associations.— The data would be used to test a new model of how to predict species accumulation (Objective 1) and test hypotheses of yeast/beetle/basidiomycete correlation (see Objectives 2 and 3). In addition to acquiring taxa from the three diverse groups, our added value data would link them in a specific association (Objective 4). The proposed study avoids being too broad because we would direct our investigations to particular locations, habitats and taxa. Estimates of new taxa.— A minimum of 600 more yeast taxa, many of which would be undescribed are expected, based on the Pollock estimate derived from our previous results. We are less clear about the number of beetle new taxa to be discovered, but coleopteran fauna associated with fungi and decaying vegetation is, in general, poorly studied. McHugh has observations to back up this statement in the two groups that he studies most.  For example, in the basidiomycete-feeding family Erotylidae, the genus Lybanodes comprised one species until with revision by Skelley et al. (1997), it grow to include six newly discovered species from Central and South America. The small myxomycete-feeding family Sphindidae was represented by a single species in the Gulf and Caribbean region before 1990.  Taxonomic studies by McHugh and colleagues (McHugh, 1990, 1993; McHugh and Lewis, 2004) increased the known diversity of this group in the Gulf Coast region by expanding the ranges of some existing North American taxa and describing 13 new species and two new genera from the region. Collecting in the southeastern USA has yielded 17 new species for McHugh. The high numbers that we expected at Barro Colorado Nature Monument based on the results of a large trapping project primarily involving phytophagous insects (Windsor, personal communication) were fulfilled. Basidiomycetes, while not the main focus of the study, could provide new species. Along the Gulf Coastal Plain where several resident mycologists previously studied wood-decaying basidiomycetes, Blackwell and R. L. Gilbertson described about fifteen aphyllophoralean species, including two large polypores that harbor beetles; more are possible. Species concepts.— Species number estimates rest upon the species concept used. Generally, we use a concept roughly equivalent to a phylogenetic concept for the organisms we study. The current yeast standard is based upon morphological and metabolic characters and base position differences of the D1/D2 region  of 26S rDNA. A criterion of more than four base pair differences (Fig. 4, above) coincided roughly with a phylogenetic concept proposed by Kurtzman and Robnett (1998), and our concept is slightly more conservative than that of other current yeast taxonomists. The concept is more conservative when one considers that the D1/D2 loop sequence indicator almost certainly underestimates variation. Beetle morphospecies concepts approach a phylogenetic concept. We primarily are interested in identifications based on morphological characters for the basidiomycetes and rely on the literature established by basidiomycete systematists, but if undescribed species are encountered we would arrange loans to experts in the group.
    GEOGRAPHIC AND ECOLOGICAL SCALE
    1. The Southeastern USA. We are committed to recollecting in this region, and emphasize that the justification for this study is (1) the poor state of knowledge of the yeasts in association with beetles and basidiocarps at all localities, including this one. (2) More collecting would be effective because we would be able to collect year-round with a laboratory at hand. A lab base is required most of the year for medium preparation, sterile dissection, isolation, and eventual identification. Knowledge of weather conditions and presence of ephemeral basidiocarp fruitings would be available. (3) Basidiomycete diversity is high. For many agarics the mycota is rich in temperate species, as well as those once thought to be indigenous to southern Mexican, Costa Rica, and Puerto Rico (Ronald Peterson, see letter of support; new and unusual agarics have been discovered by visiting mycologists hosted by us over the past. Blackwell and R.L. Gilbertson have collected about 200 species of wood-rotting fungi along the Gulf Coastal Plain, fifteen of which were new species of polypores and corticioid basidiomycetes <http://lsb380.plbio.lsu.edu/wood-rotting%20fungi>. The basidiomycetes include components of tropical regions such as Tinctoporellus epimiltinus, Ganoderma colossum, Hexagonia hydnoides, and Laetiporus persicinus. In fact the shared mycota of the Gulf Coast and Neotropics, first suggested to us that we include Barro Colorado Nature Monument in the survey. (4) The prediction that supposes we have 60% more yeasts to collect under the conditions we used in the southeastern USA and in Panama requires recollection as an integral part of this study. (5) In addition habitat destruction and deterioration has occurred in the southeastern USA (USGS FS-019-00 and USGS FS-018-00, 2000, Sierra Club 1998 <http://www.pirg.org/reports/enviro/wildlife/index.htm>. There are no LTER sites in the biotically diverse forests of the southeastern USA, so we would continue to depend on a variety of collecting sites familiar to us throughout the region. Lands owned by Louisiana State University and the University of Georgia offer diverse, easily accessible, secure collecting sites encompassing all southern forest types, and it is here that we would concentrate our efforts. The southern Appalachians “Mycoblitz” will be held in August 2004, in Ashville, NC. Suh, Blackwell, and a student plan to attend to participate in this ATBI (see letters of support from Ronald Petersen and Karen Hughes).
    2. Barro Colorado Island (BCI) and Barro Colorado Nature Monument (BCNM) (Smithsonian Tropical Research Institute, STRI), Panama.  This Smithsonian laboratory site is not endangered; it is, however, an example of a lowland moist tropical forest, many of which are under stress. This site is a seasonal tropical moist forest with annual rainfall more than 2600 mm rainfall/year.  The collection sites are in stands of secondary forests ranging in age from recently cut-over forest to 500 year old-growth forest. BCI fills our need both for a lush habitat and for a laboratory for efficient yeast isolation. BCI also provides some species of basidiomycetes that also occur in Louisiana bottomland hardwood forests <http://lsb380.plbio.lsu.edu/wood-rotting%20fungi>. As we mentioned above, recollection would allow us to test the prediction of 60% more yeasts to be discovered with continued collecting.  Correlated with this prediction, we estimate that fewer than half the species of erotylids known to occur at BCI have been collected by us. Erotylidae is one of the largest beetle families with a basidiocarp-linked life style. Donald Windsor, STRI, has studied the beetles of Barro Colorado Nature Monument for many years, and he has offered his invaluable service to us (see letter of support). STRI personnel help to obtain the necessary collecting and exportation permits once funding has been secured as has been done before.
    3. South Africa (based in Pretoria). In the third year of funding we would collect in South Africa. Prof. J. P. van der Walt and his colleagues, studied the South African yeast mycota for over forty years and described 120 species of yeasts. The collection he established now holds over 4000 cultures of 450 species, mainly from South Africa, making this region relatively well known for yeasts. The collections, however, are largely from soil and plant materials, not usually insects. This situation would be a test of our findings that the yeasts we discovered in the gut of beetles are not often present in the environment outside the gut. Collecting in South Africa additionally would complement the collecting regions already planned for study by PEET participants, McHugh, Miller, Whiting, and their students. We would use phylogenies they produce to compare gut yeasts bracketing trophic shifts between mycophagy, myxomycophagy, phytophagy, and predation, as well as those involved with shifts between free-living lifestyles and inquilinity or parasitism (Objective 2). Objective 3 depends on South African collections of erotylids from Ganodermataceae to compare gut yeasts from the same tribes and families that we collect in North America to identify factors that influence the occurrence of yeasts in particular beetles and basidiocarps. We would have logistic support for collecting and studying the organisms in South Africa provided by Michael Wingfield and Brenda Wingfield, Forestry and Agricultural Biotechnology Institute, University of Pretoria (see letter of support).
    URGENCY
    Our ignorance on the topic of beetle gut yeasts is worldwide but includes the Gulf Costal Plain and Barro Colorado, Panama, in the Western Hemisphere and southern Africa (see also collecting regions, outlined immediately above). The yeast/insect assemblage may have profound effects on the ability of beetles to occupy particular niches and to utilize specific nutritional resources. Current research topics include hypotheses of rapid radiation and megadiversity. Some hypotheses are aimed at explaining evolutionary radiations of beetles based on the availability of nutritional resources. Such studies could be better informed with more information on the possibility that yeasts modify insect food resources. The discovery of these yeasts is important for yeast systematics and evolution, because large new clades have been discovered in association with insects. The progress made by yeast workers toward a phylogenetic classification over the past six years has been phenomenal, and our results on yeasts from an understudied habitat would be a valuable contribution toward yeast classification. Our study, therefore, might help to clarify the extent and nature of a symbiotic relationship that may be have an important role in the radiation of one of the most successful clades of life on Earth, the Coleoptera.
    MANAGEMENT PLAN
    Division of Labor.  We functioned well during the earlier study, and the management plan would continue in the manner that was successful. During the previous funding period many operations were performed almost simultaneously. For example collection and identification of beetles and basidiocarps, isolation of yeasts, characterization of yeasts using cultural methods, and DNA sequencing were done as soon as collections were made when possible. The bottleneck came with the task of yeast characterization. PIs Blackwell, McHugh, and Suh were all involved in collecting beetles from basidiocarps. We collected together whenever possible, and each collector was responsible for ensuring a good voucher specimen for each kind of collection. Because we collected all year long in the southeastern USA, we could not always collect together.  Beetles and basidiocarp vouchers would be sent to LSU; alcohol preserved beetle specimens would go back to McHugh at Georgia for identification after dissection. Beetles would be collected in 200- proof ethyl alcohol, so that the PEET students could use them for DNA extraction. Suh, Blackwell, and LSU undergraduate students would dissect beetles and establish cultures. Suh would be primarily responsible for the culture of yeasts and would characterize them with the help of undergraduate researchers with DNA sequences (650 bp LSU rDNA) and other standard techniques. Excess DNA and cultures would be stored at ?120C in the LSU Frozen Tissue Collection (see letter of support, below).  Other cultures would be maintained in Blackwell’s lab until their permanent deposition at NRRL or CBS.  Blackwell and undergraduates would sort basidiocarps and put them in zip lock bags for drying at 60C. She would identify the specimens or send them to specialists for identification. Basidiomycete specimens would be deposited in the LSU Mycological Herbarium (Blackwell advises on fungi) or other appropriate collection, except when compliance with collecting permits requires other arrangements. McHugh and colleagues would identify beetles and place them in the University of Georgia Collection of Arthropods for US specimens, MIUP and STRI Synoptic Insect Collection for Panama specimens, or other collections required by collecting future permits. Specialists would be alerted if we find basidiomycetes or insects that we feel would be of particular interest to them. The study would complement work done by the McHugh PEET group (see Similar Research, below).

    Timetable for the Study. This study is designed for four years, somewhat longer than usual in the competition. This duration, however, is necessary to accomplish the cohesive objectives outlined above and is realistic, based on our recent experience with this kind of study.
    Yr 1. Collection in USA (year-round) and Panama (summer), DNA sequencing and other characterization of yeasts collected to date; data (except sequences) made available by pdf on web site or in database; distribution of yeasts and other identified specimens; value-added interaction studies.
    Yr 2. Collection in USA (year-round) and Panama (summer), DNA sequencing and other characterization of yeasts collected to date; data (except sequences) made available by pdf on web site or in database; distribution of yeasts and other identified specimens; valid publication of new species; value-added interaction studies.
    Yr 3. Collection in USA (year-round) and South Africa, DNA sequencing and other characterization of yeasts collected to date; data (except sequences) made available by pdf on web site or in database; distribution of yeasts and other identified specimens; publication; valid publication of new species; value-added interactions studies.
    Yr 4. Completion of characterizations, analyses, continuations of IDs, database; valid publication of new species; distribution of all cultures, identified specimens, and sequences; use phylogenetic hypotheses of CS beetles generated by PEET project to interpret evolutionary hypotheses of transitions; value-added interaction studies.

    PROTOCOLS
    Collecting (see also specific localities at Geographic and Ecological Scale, above). We would proceed as we have in the past few years of the previous funding period. Blackwell would continue to handle permits, and she has been granted “courtesy letters” from the USDA (APHIS) for importation of nonpathogenic cultures as required. Other permits (collecting, export, and import) would continue to be requested by Blackwell, a successful process with the help of the STRI staff for Panama (see letter of support from Donald Windsor). They do not initiate requests until trips and travel plans have been finalized. Michael Wingfield in South Africa has hosted many foreign visitors, including from the USA and has always been successful in securing collecting and exportation permits (see letter of support). Collecting would be done in the general regions described and justified above (see Geographic and Ecological Scale).  A Magellan Trailblazer XL GPS receiver would be used to record precise locality data, and collection coordinates would be linked to our databases on the web site or transferred to GIS units at either university for mapping. One unit is available at Georgia and another is budgeted for LSU.  Tied to a discussion on collecting is the question estimating the completeness of the biotic survey (see Conceptual Issues 1) by using other statistical methods cited above. Resampling and standardizing our search efforts would be attempted, but this may be difficult because the basidiocarps often are ephemeral and unpredictable in the environment. We would attempt exhaustive searches within a set area to standardize the searching effort. Basidiomycete collection.— Basidiocarps would be hand collected from appropriate sites, and all basidiocarps encountered would be examined for the presence of beetles. We would try to search exhaustively but would be certain to include geographically-broad ranging, common species with long-lived basidiocarps as we have done before; this would provide some measure of comparison of the yeasts and beetles in different localities if we can have basidiocarps as a common factor. Beetle collection.— Collections of targeted beetles would be made repeatedly at different localities and months. Because we are focusing primarily on the beetle fauna associated with basidiomycetes, direct collecting from basidiocarps provides the required host link for the third and fourth components of our chain: yeast-beetle-basidiomycete fungus-substrate/host). In cases where trophic transitions are examined, other hosts will be documented and included in the database as well. Beetles must be kept alive until dissection. Yeast isolation and culture.— Our protocols on the web site provide cookbook methods for the isolation of yeasts from the gut of beetles and their culture, and these have been tested repeatedly by us and others. Once removed from the basidiocarp, beetles are surface disinfected by submerging in 95% ethanol for 1-2 min. to disinfect the surface. The alcohol wash is followed by a 0.7% saline (NaCl) 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 are used to dissect the beetles on sterile microscope slides under a dissecting microscope. The beetle gut is removed aseptically, cut into pieces, and transferred to tubes containing 0.7% saline.  Gut segments are crushed in the saline solution with a pipette tip and streaked with a loop onto the surface of acidified YM agar plates. Plates are incubated at 25°C, and after three days single colonies are streaked for purification. This procedure is carried out two or more times. Cultures are maintained on YM agar. While we mainly target yeasts that grow in culture in this proposal, we are aware that unculturable (by our methods) yeasts may be present, and we would continue to clone the LSU rDNA gene from beetles in four families without finding unculturable ascomycete yeasts. Morphological observations and metabolic tests comprising the yeast “standard description,” are made according to established methods (Kurtzman and Fell 1998; Barnett et al. 2000). The cultures from this study would be deposited in the Agricultural Research Service (ARS) Culture Collection and Centraalbureau voor Schimmelcultures (CBS), Fungal Biodiversity Center, Utrecht, The Netherlands. Characterization and identification of yeasts.— We would characterize the cultivated yeasts in two ways. The observations and tests for standard descriptions (Yarrow, 1998) include observations on carbon assimilation, fermentation tests, nitrogen assimilation, growth under certain conditions, morphological observations, and a number of other tests, including crosses to determine sexual competence.  Yeast taxonomists have moved rapidly to the use of molecular methods, not necessarily for phylogenetic studies, but as another method of identification. A data base of sequences (600-650 bp) for more than 650 described yeast species is available, and we would continue to use this method for rapid comparison the species we isolate with previously described species (Kurtzman and Robnett, 1998). Study of interactions.— Beetles would be reared in lab colonies, and we have been successful in rearing for passalid beetles for at least nine  months in a previous experiment. As a first beetles would be cured of yeasts using methods that could be compared for effectiveness. Protocols involving higher growth temperatures and antibiotics known to inhibit ascomycete growth are available. Also, it may be possible to eliminate yeasts by aseptic treatment of the beetles, eliminating the yeasts in the life cycle as the larvae hatch from the egg, a known source of yeast acquisition in some of the beetles were have observed. Methods are described fully by Vega and Dowd (2004) and references therein. DNA Methods.— A cell suspension with 1 loopful of yeast cells in 50 µl of autoclaved water is boiled at 95°C for 5 min. and 2 µl of supernatant after centrifugation for 1 min is used directly for the polymerase chain reaction (PCR) to amplify the D1/D2 loop of LSU rDNA (about 600 bp). For amplifying other genes longer than 1kb, nucleic acids are extracted and purified following the procedures of Lee and Taylor (1990). The primer sets NS1-NS8, LS1-LR5, and ITS5-ITS4 are used for amplifying SSU and LSU rRNA genes (rDNA), and 5.8S rDNA and internal transcribed spacer (ITS) sequences (White et al. 1990; Hausner et al. 1993), respectively, using the polymerase chain reaction (PCR). PCR products are purified using a DNA purification kit (Bio-Rad Laboratories, Hercules, CA). The purified double-stranded PCR products are used as templates for sequencing with an ABI PRISM™ BigDye Terminator Cycle sequencing kit, version 2 (PE Applied Biosystems, Foster City, CA). The complete sequence of SSU rDNA, 5.8S rDNA including ITS, and the D1/D2 region of the lsu rDNA are sequenced with the primers NS1, NS2, 18H, NS8, ITS1, ITS4, LS1, and LR3 using an ABI PRISM 377 Automated DNA sequencer (PE Applied Biosystems, Foster City, CA) (Suh et al. 2001, 2003). Sequences are submitted to GenBank at the time manuscripts are submitted. Unique sequences of the D1/D2 loop (650 bp) of the LSU rDNA sequences distinguish yeast genotypes (Kurtzman and Robnett 1998), and these are named using the first four letters of the beetle family and a unique number. Data Analysis.— We would not routinely acquire sequences from several genes for phylogenetic purposes. We do, however, use LSU and SSU rDNA sequences for screening and identification and other sequences for specific reasons.  The sequences would be aligned in a database made available by Cletus Kurtzman and augmented with our sequences. “Type” culture sequences of all known yeasts in culture are available (Kurtzman and Robnett 1998). Because the database is dense, we can use phylogenetic analysis with parsimony criteria and BLAST searches to identify new isolates or near relatives. DNA sequences would be aligned with other sequences obtained from GenBank or the Kurtzman database using the multialignment program Clustal X (Thompson et al. 1997). The alignments would be optimized visually, and ambiguous regions, excluded from analyses. Maximum parsimony analyses would be performed using PAUP 4.0b10 (Swofford 2002). Heuristic tree searches are executed using the tree bisection-reconnection branch swapping algorithm with random sequence analysis. Bootstrap values would be obtained from 1000 replications. Base pair differences in a gene would be counted using Blast 2 sequences (Tatusova and Madden 1999) or from a manually aligned sequence database. The gene trees probably would not be robust phylogenetic hypotheses, but other genes can be acquired (see above, Fig. 3, and related topic, Species concept).
    Deposition of Cultures and Specimens. We would comply with all regulations required by collecting permits (see above, Response to comments on previous proposal), and all materials would be available in public collections through loans or by other arrangements to qualified researchers and databases. Cultures.— Purified, identified, and characterized and lyophilized types specimens cultures would be deposited in public culture collections. Cletus Kurtzman [see letter of support] would accept our cultures at the USDA collection, Peoria, Illinois (NRRL), to supplement this large specialist collection of yeasts. We would provide information on the cultures for inclusion in the fifth edition of the Yeasts: A taxonomic study, under revision by Kurtzman and Fell. Duplicate sets of cultures would be sent to the Centraalbureau voor Schimmelcultures (CBS), Fungal Biodiversity Center, Utrecht, The Netherlands, which has a large holding of yeasts and expert yeast taxonomists as curators. In addition our data would be included in the CBS database [see letter of support from Director, Pedro Crous].  Both NRRL and CBS collections are supported by their governments and do not accept funds to add our collections or for databasing.  Basidiomycetes.— Specimens (including splits and duplicates from other countries whenever possible) would be accessioned in the LSU Herbaria (LSU-M), currently housing collections of Neotropical wood-decaying basidiomycetes. The new herbarium complex allows almost unlimited deposit of specimens. We would place specimens at PMA or other herbaria as required (see Supporting documents). An associate curator position is provided to the LSU herbaria, but hourly wages would be used for additional curatorial help with specimens from this study at LSU or elsewhere. Beetles.— Specimens would be deposited at the University of Georgia Collection of Arthropods where McHugh is curator and at MIUP (see letter of support). The University of Georgia Collection of Arthropods (UGCA) has holdings comprising more than 1.2 million insect specimens. Approximately 95% of the specimens are from the southeastern United States, and more than 80% have been determined to species. Hourly wages are requested to help with specimen preparation, photography, database work, and curation.  Extracted DNA.— DNA and some cultures would continue to be accessible in the Genetic Resources Collection, Natural Science Museum, LSU (see letter of support from Robb Brumfield, Director).  Extracted DNA from beetles collected in the study would be used in the PEET project and would be available after publication of PEET results.
    Electronic Products. At the beginning of the previous funding period we posted information on the project (the submitted proposal, methods, datasets available by pdf) on our laboratory web site <http://lsb380.plbio.lsu.edu/beetlebellyfolder/beetlebellyeast.home>. More recently we developed a web site on the USDA-ARS Systematic Botany and Mycology server <http://nt.ars-grin.gov/SBMLWeb/Home.cfm>. The database now is about 75% complete. The USDA site has a promise of permanency and other fungal databases reside there. Experienced personnel offer support maintaining the materials posted. Fees are not charged for use of the server or assistance in building the database (see letter of support from Amy Rossman). Data on yeasts, originally entered into Excel files, were added directly to Microsoft Access tables, and ColdFusion was used to manipulate the data in the database. ColdFusion uses CFML markup tags similar to HTML tags.  In addition to HTML and CFML codes, JavaScript was used.  The yeast pages are almost entirely database driven facilitated by David Farr at the USDA lab. New fields, characters, and character states can be added at will. The front matter includes the rationale for the study, an introduction of the participants, yeasts, beetles, and basidiomycete hosts, and a basic picture glossary for yeast morphology. The main features of the site are the descriptions of yeasts, yeast photographs, names of insect and basidiocarp habitats, and interactive identification tools.  Almost 200 yeasts are entered with more to come. The yeasts are mostly unnamed at this time but descriptions are underway. Designation of yeasts is by insect host family and unique LSU rDNA group pending valid publication as new species. The database details the cultural and microscopic observations from 100 metabolic and other tests, totaling more than 20,000 characters with 3-6 states for each character. Links will be made to 751 new DNA sequences pending GenBank release. The complete host information we are incorporating makes this database unique. In addition to the data we have developed, data on yeast cultures also would be entered at no cost into the CBS Yeast Database containing almost all known yeasts. The permanent CBS database includes 4500 strains and 700 yeast species [see letter of support from director, Pedro Crous]. In addition Blackwell would work with Astrid Ferrer (see letter of support) to produce a web-based checklist of BCI wood-decaying basidiomycetes. Ferrer, a native of Colombia and current University of Illinois postdoctoral associate, has unpublished collections from her PhD project at SUNY, College of Environmental & Forest Biology, Syracuse.
    Training Opportunities. Our interest in training and education is documented in “Results from Prior NSF Support,” in our Biographical Sketches, and lab web sites. Students work side by side with the PIs. Some students use research time merely to learn modern techniques; others are truly excited about independent research and assume their own projects and publish or present their work (see Results from Prior NSF Support, above). We have a good record of applying for Research Experiences for Undergraduates (REU) support of exceptionastudents. Two students collected at BCI in 2002, supported by REU funds. We include research results in teaching, and we would train a diverse group of graduate and undergraduate students and postdoctoral associates in our laboratories. The yeast study would enhance the education component of the McHugh, Miller, and Whiting PEET grant (see below, Similar Research). The work also would broaden the outlook of beetle systematists and mycologists, adding the new dimension of symbiosis to their individual ways of thinking.
    Similar Research. PEET participants.— Joseph McHugh, PI on this proposal, Kelly Miller, and Michael Whiting were funded by a PEET grant [Building taxonomic expertise for Cucujoidea (Coleoptera): Monographic and phylogenetic research in the Cerylonid Series], and we would coordinate the two studies to maximum benefit of both for synergistic and cost effective research. The PEET project would provide robust phylogenetic hypotheses and a diverse sampling of cucujoid beetles for the yeast study. The PEET study would benefit from the addition of South African and Panamanian fieldwork and material. Yeast researchers.— Several yeast taxonomists study these organisms from nature in terrestrial environments. Cletus Kurtzman (see letter of support), coeditor of the 4th edition of the essential yeast compendium (1998), has wide interests in the group and its habitats. The closest studies to ours are those of André Lachance and William T. Starmer and their colleagues, who are studying yeasts from several insect habitats, including senescent flowers. Our work would complement theirs, rather than conflict with it (see letter of support). We are committed to continuing to share our data before publication, an important activity when different workers are discovering new species at rapid rates.
    REFERENCES CITED
  • Andrews, FG (2002) 95. Latridiidae Erichson 1842.  Pp. 395-398.  In American beetles, vol. 2 (eds. Arnett RH Jr., Thomas MC, Skelley PE, Frank JH) CRC Press, Boca Raton, Florida.
  • Barnett JA, Payne RW, Yarrow D (2000) Yeasts: characteristics and identification, 3rd edn. Cambridge University Press, Cambridge, UK.
  • Christen JA, Nakamura M (2003) Sequential stopping rules for species accumulation. Journal of Agricultural, Biological and Environmental Statistics 8: 184-195.
  • Colwell RK, Coddington JA (1994) Estimating terrestrial biodiversity through extrapolation. Philosophical Transactions of the Royal Society of London B 345: 101-118.
  • Hausner G, Reid J, Klassen GR (1993) On the subdivision of Ceratocystis s.l., based on partial ribosomal DNA sequences. Canadian Journal of Botany 71: 52-63.
  • Kurtzman CP, Fell JW (1998) The yeasts, a taxonomic study, 4th edn. Elsevier, Amsterdam, The Netherlands.
  • Kurtzman CP, Robnett CJ (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek International Journal of General and Molecular Microbiology 73: 331-371.
  • Lawrence JF (1991) Coleoptera. Pp. 144-658.  In Immature insects, vol. 2 (ed. Stehr FW) Kendall/Hunt, Dubuque, IA.
  • Lee SB, Taylor JW (1990) Isolation of DNA from fungal mycelia and single spores. Pp. 282-287. In PCR Protocols-A Guide to Methods and Applications (eds. Innis MA, Gelfand DH, Sninsky JJ, White TJ) Academic Press, San Diego, California.
  • McHugh JV (1990) Carinisphindus, a new genus and three new species of neotropical Sphindidae (Coleoptera: Clavicornia). The Coleopterists’ Bulletin 44: 307-322.
  • McHugh, JV (1993) A revision of the world Eurysphindinae with a review of classification and phylogeny in Sphindidae. Systematic Entomology 18: 57-92.
  • McHugh JV, Lewis CN (2004) Three new species of Carinisphindus (Coleoptera: Sphindidae) from Bahamas, Florida and Puerto Rico. Coleopterists’ Bulletin, in press.
  • Moncalvo JM, Vilgalys R, Redhead SA, Johnson JE, James JY, Aime MC, Hofstetter V, Verduin SJW, Larsson E, Baroni TJ, Thorn RG, Jacobsson S, Clémençon H, Miller OK Jr. (2002) One hundred and seventeen clades of euagarics. Molecular Phylogenetics and Evolution 23: 357-400.
  • Pakaluk J (1984) Natural history and evolution of Lycoperdina ferruginea (Coleoptera: Endomychidae) with descriptions of immature stages.  Proceedings of the Entomological Society of Washington 86: 312-325.
  • Philips TK, Ivie MA (2002) 90. Bothrideridae Erichson 1845.  Pp. 358-362.  In: American beetles, vol. 2 (eds. Arnett RH Jr., Thomas MC, Skelley PE, Frank JH) CRC Press, Boca Raton, Florida.
  • Skelley PE, Leschen RAB, McHugh JV (1997) A revision of Lybanodes Gorham, 1888 (Coleoptera: Erotylidae: Tritominae). Annales Zoologici 47: 3348.
  • Skelley PE, Leschen RAB (2002) 92. Endomychidae Leach 1815.  Pp. 366-370. In American beetles, vol. 2 (eds. Arnett RH Jr., Thomas MC, Skelley PE, Frank JH) CRC Press, Boca Raton, Florida.
  • Schmit JP, Murphy JF, Mueller GM (1999) Macrofungal diversity of a temperate oak forest: a test of species richness estimators. Canadian Journal of Botany 77: 1014-1023.
  • Shen TJ, Chao A, Lin CF (2003) Predicting the number of new species in further taxonomic sampling. Ecology 84: 798-804.
  • Suh S-O, Blackwell M (2004) Beetles as hosts for undescribed yeasts. In Insect Fungal Associations: Ecology and Evolution (eds. Vega FE, Blackwell M) Oxford University Press, NY, in press.
  • Suh S-O, Kurtzman CP, Blackwell M (2001) The status of Endomyces scopularum  --a filamentous fungus and two yeasts. Mycologia 93:316-321.
  • Suh S-O, Marshall C, McHugh JV, Blackwell M (2003) Wood ingestion by passalid beetles in the presence of xylose-fermenting gut yeasts. Molecular Ecology 12:3137-3145.
  • Suh S-O, McHugh JV, Pollock D, Blackwell M (0000) Budding yeasts from the gut of basidiocarp feeding beetles have astonishing biodiversity levels. Submitted to Science.
  • Suh S-O, White MM, Nguyen NH, Blackwell M (2004) The identification of Enteroramus dimorphus: a xylose-fermenting yeast attached to the gut of beetles, Mycologia, in press.
  • Swofford DL (2002) PAUP. Phylogenetic Analysis Using Parsimony (*and Other Methods), version 4.0b10. Sinauer Associates, Sunderland, Massachusetts.
  • Tatusova TA, Madden TL (1999) Blast 2 sequences - a new tool for comparing protein and nucleotide sequences. FEMS Microbiology Letters 174: 247-250.
  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tool. Nucleic Acids Research 25: 4876-4882.
  • Vandenberg NJ (2002) 93. Coccinellidae Latreille 1807.  Pp. 371-389. In American beetles., vol. 2 (eds. Arnett RH, Jr., Thomas MC, Skelley PE, Frank JH) CRC Press, Boca Raton, Florida.
  • Vega FE, Dowd PF (2004) The role of yeasts as insect endosymbionts. In Insect Fungal Associations: Ecology and Evolution (eds. Vega FE, Blackwell M) Oxford University Press, NY, in press.
  • White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pp. 315-322. In PCR protocols--a guide to methods and applications (eds. Innis MA, Gelfand DH, Sninsky JJ, White TJ) Academic Press, San Diego, California.
  • Yarrow, D (1998) Methods for the isolation, maintenance and identification of yeasts. Pp. 77-100. In The yeasts, a taxonomic study. 4th Ed.  (eds. Kurtzman CP, Fell JW) Elsevier, Amsterdam.
  • Zhang N, Suh S-O, Blackwell M (2003) Microorganisms in the gut of beetles: evidence from molecular cloning. Journal of Invertebrate Pathology 84: 226-233.

    • Introduction / Participants / Basidiocarps as habitat / Insects / Yeasts / Methods / Publications / Literature cited / Proposal I / Proposal II/ Mycology at LSU
    Return to Mycology at LSU
    Last update: 13 June 2000
    Questions?