Beetles and their yeast endosymbionts from basidiocarp habitats (NSF proposal 0072741)
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
This study will provide baseline knowledge on newly discovered endosymbiotic associations between saccharomycetalean yeasts and basidiocarp-dwelling beetles. We believe that these beetles may use fungi to eat fungi. Basidiocarps to be examined are the spore-containing structures of agarics and polypores. Our preliminary study indicates specificity in some of the yeast-beetle associations, although presumably nonsymbiotic yeasts with less specificity also may be present. Until now, yeasts and yeast-like fungi were known only from the gut of planthoppers (Homoptera) and three families of beetles (Coleoptera): (Anobiidae, Cerambycidae, and Scolytidae). Our preliminary study has increased the number of beetle families known to harbor endosymbionts to five additional families (Ciidae, Erotylidae, Nitidulidae, Scarabaeidae, Tenebrionidae).
    The sampling strategy will emphasize a range of localities and basidiocarp habitats that are likely to produce the greatest diversity; and the sampling strategy also will allow us to acquire specimens to  address contextual issuesincluding species specificity, yeast metabolism, biogeographical variation, competition, population biology, evolution of symbiotic associations, beetle substrate expansion, and evolutionary radiations.
    We will collect and characterize species of yeasts and associated fungus-feeding beetles from along an east-west transect along the gulf coastal plain of the United States and in the vicinity of Xalapa, Vera Cruz, Mexico, and Barro Colorado Island, Panama. We will make cultures and extract DNA from the yeasts for identification. Beetles will be examined for site of endosymbiont localization and identified. Basidiocarp specimens will serve as vouchers for the insect collections and yeast isolations. All specimens and yeast cultures and residual DNA will be available for other researchers. Specimens will be accessioned into the Louisiana State University Herbarium and University of Georgia Arthropod Collection or for those collected in Mexico and Panama in the designated collections of the institutions.
    Electronic relational databases will link all organisms from a collection and include collection information, such as basidiomycete species and substrate (e.g., mycorrhizal host or saprobic substrate), beetle species, and yeast species. These records and associated data including yeast DNA sequences and culture repository, will be posted on an LSU server. The software will allow for rapid single query in parallel across multiple processors. We will supply checklists, descriptions, and interactive keys for the beetles and associated yeasts. The data will be of unusual quality because of the linked organismal associations. The tradition of both PIs of involving a diverse group of undergraduate and graduate students and postdoctoral researchers in their research will continue.
Introduction: Beetles and their yeast endosymbionts from basidiocarp habitats
Over the last century the recognition of the presence of endosymbionts in a variety of arthropods has become well established. Intense interest in the prokaryotic rickettsial endosymbionts, widespread among insects (van Meer, Witteveldt, and Stouthamer, 1999), has led to the discovery that the rickettsia may induce sterility of the host. Thus, speciation or increased rates of speciation have been attributed to the presence of the symbionts (Shoemaker, Katju, and Jaenike, 1999). By contrast, although there were a number of early reports of fungal endosymbionts of insects, few of them were substantiated (Buchner, 1965). Until now, yeasts and yeast-like fungi have been known from the gut only of a small group of planthoppers (Homoptera) and of three families of beetles (Coleoptera: Anobiidae, Cerambycidae, and Scolytidae) (Table 1).  We believe that eukaryotic endosymbionts play an equally important role in insect evolution, and the example of Symbiotaphrina in detoxification of food resources for anobiid beetles (Dowd, 1989, 1991) supports this contention. Another system, although not endosymbiotic, the cactophilic yeast-Drosophila associations studied by Starmer and his colleagues (Starmer et al., 1991), provides another example of the influence that yeasts have in ameliorating the nutritional resources of insects.
 
Table 1.  Previously known  species of yeasts and yeast-like  endosymbionts in insects*
Host insect /Fungus species
Homoptera
     Nilaparvata lugens/undescribed hypocreatean fungus
     Sogatella furcifera/undescribed hypocrealean fungus
     Laodelphax striatellus/undescribed hypocrealean fungus
Anobiidae
     Lasioderma serricorne/ Symbiotaphrina kochii
     Sitodrepa paniceae/ Symbiotaphrina buchneri
     Ernobius abietis/ Candida karawaiewii
     Ernobius mollis/ Candida ernobii
     Xestobium plumbeum/ Candida xestobii
Cerambycidae
     Rhagium inquisitor/ Candida rhagii
     Rhagium  mordax/ Candida tenuis
     Rhagium bifasciatum/ Candida tenuis
     Rhagium sycophanta/ Candida tenuis
     Gaurotes virginea/ Candida rhagii
     Leptura rubra/ Candida tenuis
     Leptura maculicornis/ C. shehataevar. insectosa
     Leptura cerambyciformis/ C. shehataevar. insectosa
     Leptura sanguinolenta/ Candida sp.
     Phoracantha semipunctata/ Candida tenuis
     Microplophorus magellanicus/ Candida tenuisand Rhodotorula glutinis
     Grammicosum flavofasciatum/ Candida parapsilosisvar. intermedia
     Ergates faber/ Candida ergatensis
Scolytidae
     Dendroctonus monticolae/ Candida nitratophila
*The list has been modified from that of Nardon & Grenier (1989).

Recently we have discovered a species-rich association between gut-associated endosymbiotic yeasts and basidiocarp-inhabiting beetles; the beetles presumably use fungi to eat fungi.  Beetles in the families we examined with this habitat routinely harbor distinctive yeasts. Placed in the context of previous knowledge of endosymbiotic yeasts in only three families of beetles (Table 1), our finding of endosymbionts in five out of the six beetle families examined randomly (Table 2) portends the success of the project.

Table 2. Yeast isolates from gut of beetles usually can be grouped by 26S rDNA sequences.
Host beetle ex host fungus, yeast group by place*, isolate number**, 26S rDNA***
Erotylidae
 Unidentified ex Amanita sp., RLM
    98-8-25-1-2   E1
    98-8-25-1-4  E1
    98-8-25-1-5  E1
 Unidentified ex Amanita sp., SF 98-9-24-2-1  E1
 Unidentified ex  Amanita sp., SF 99-6-12-1-1  E2
 Megalodacne fasciata ex Ganoderma applanatum,MP
    99-8-11-1-1  E3
    99-8-11-1-2-1  E3
    99-8-11-1-2-3  E3
    99-8-11-1-3  E3
    99-8-11-1-2-2  E4
    99-8-11-1-2-4  E4
    99-8-11-1-4-1  E5
    99-8-11-1-4-2  E6
Tenebrionidae
 Neomida bicornis ex Fomitella supina,BS
    98-8-18-2  T1
 Neomida bicornis ex Fomitella supina,SF
    98-12-9-1-1  T1
    98-12-9-1-3  T1
 Neomida bicornis ex Fomitella supina,HA
    99-2-5-2-1-1   T1
    99-2-5-2-1-2  T1
    99-2-5-2-2-1  T1
 Neomida bicornis ex Fomitella supina,SF
    99-3-22-1-1  T1
    99-3-22-1-2  T1
    99-3-22-1-3  T1
    99-3-22-1-4  T1
 Neomida bicornis ex Fomitella supina,SF
    98-12-9-1-2  T2		  Neomida bicornis ex Fomitella supina, SF
    98-9-24-3-1   T3
    98-9-24-3-2  T3
 Platydema sp. ex Decayed polypore,  DT 98-9-2-2-4  T4
 Diaperis nigronotata ex Inonotus ludovicianus,LSU
    99-8-18-1-1  T5
    99-8-18-1-3-1  T6
    99-8-18-1-3-2  T6
    99-8-18-1-6  T6
    99-8-18-1-4-1  T7
    99-8-18-1-4-2  T7
Nitidulidae
 Carpophilus sp. ex Pisolithus tinctorius,LSU
    98-8-14-1-2-1  N1
    98-8-14-1-3-3  N1
    98-8-14-1-4-3  N1
 Carpophilus sp.  ex Pisolithus tinctorius,LSU
    98-8-14-1-3-1  N2
 Carpophilus sp.  ex Pisolithus tinctorius,LSU
    98-8-14-1-3-2  N3
    98-8-14-1-4-2  N3
 Carpophilus sp.  ex Pisolithus tinctorius,LSU
    98-8-14-1-4-1  N4
Ciidae
 cf.Ceracis curtus ex Fomitella supina,SF
    98-12-9-2-1   C1
    98-12-9-2-2-1  C1
    98-12-9-2-2-2  C1
 cf.Ceracis curtus ex Fomitella supina,HA
    99-2-5-7-1-1  C2*T1
    99-2-5-7-1-2  C2
    99-2-5-7-1-3  C2
Scarabaeidae
     Unidentified sp. ex Boletus sp., RLM 98-8-5-1-1  S1*N1
Some of the yeast isolates discovered in the preliminary study.  Color coding of beetle families corresponds to that used in Fig. 1.  * LSU (Louisiana State University campus, Baton Rouge, LA), RLM (Rural Life Museum, Baton Rouge, LA), HA (Hilltop Arboretum, Baton Rouge, LA), DT (downtown, Baton Rouge, LA),  BS (Bluebonnet Swamp, Baton Rouge, LA). SF (St. Francisville, LA; 30mi north of Baton Rouge ), MP (Memorial Park, Athens, GA).  **First three digits indicate date of collection.  *** The yeasts in each group have identical sequences of 26S rDNA (D1/D2 region; about 600bp).
We propose a study with the primary goal of discovering beetles and endosymbiotic yeasts in macroscopic fruiting bodies of basidiomycetes. The basidiocarp habitat is targeted because it is an exceptionally productive habitat that previously has gone unsampled. We still do not know if mycophagy is a common factor in the endosymbiont distribution throughout a diverse group of six beetle families or if beetle endosymbionts are much more widespread in other beetles than has been known. We will concentrate on the beetles from basidiocarps in our collecting, but in order not to overlook a potential source of endosymbionts, we also will examine beetle species of some of the sister taxa or other close relatives that are not mycophagous to determine the limits of the endosymbiotic associations within the lineages.

We will collect, identify, and name species of yeasts and associated basidiocarp-feeding beetles from the northern Gulf of Mexico, the vicinity of Xalapa, Veracruz, Mexico, and Barro Colorado Nature Monument, Panama. We will make cultures and extract DNA from the yeasts. Yeasts will be identified using standard and molecular methods. Basidiomycete hosts and beetles will be identified and named, and collections will serve as vouchers for the yeast isolations. Basidiocarp condition (fresh, decayed) will be recorded. Specimens will be accessioned into collections as described in the management plan below.  Electronic databases will link all organisms from a collection: basidiomycete substrate (e.g., mycorrhizal host or saprobic substrate), basidiomycete, beetle, and yeast species; other data including aligned yeast DNA sequences and place of deposition of yeast cultures. Links will be provided for some kinds of information. Data will be posted on a server at LSU. The data will be of unusual quality because not only poorly known organisms will be listed, but their associations with each other will be linked. Our sampling strategy will emphasize a range of localities and basidiocarp habitats that are likely to produce the high species diversity for beetles and yeasts within our collecting areas. The sampling strategy will allow us to acquire baseline information with which, at a later time, we will test hypotheses of symbiont specificity, biogeographical and population variation, competition, phylogenetics, evolution of symbiotic associations, beetle and yeast evolutionary radiations, and niche expansion.

Table 3. The closest known taxa in a clade with newly discovered yeast species.
Yeast

T1
T4 (rare)
T5
T6
T7
N1
N2 (rare)
N3 (rare)
N4
C1
C2
E1
E2
E3
E4
E5
E6
E7
E8
S1
 

 Closest known taxon (bp difference of D1/D2)

Candida tazawaensis (36)
Candida tazawaensis (36)
Candida tazawaensis (40)
Candida galacta (70)
Candida tazawaensis (9)
Candida xestobii (1)
Pichia  (Kodamaea) ohmeri (1)
Debaryomyces occidentalis (21)
Zygoascus hellenicus (0)
Arxula adeninivorans (29)
Candida tazawaensis (36)
Candida kruisii (32)
Candida kruisii (31)
Candida tazawaensis (42)
Candida tazawaensis (34)
Debaryomyces hansenii  var. fabryi (2)
Candida tazawaensis  (29)
Candida xestobii (9)
Candida tazawaensis (9)
Candida xestobii (1)
Preliminary results
Our preliminary results are intriguing. Using the techniques to be described in the methods of the management plan below, we already have increased the number of coleopteran families known to harbor endosymbiotic yeasts from three to eight (Table 2) by looking at members of the first six beetle families we encountered. Many of the taxa are undescribed species of Saccharomycetales (Table 3, Fig. 1) that appear to have strict associations with particular groups of mycophagous beetles. We chose basidiocarps of agarics, boletes, puffballs, and polypores as habitats in which large numbers of taxonomically diverse beetles could be collected repeatedly at different localities; discovery of new species of yeasts in the gut of beetles feeding in these habitats far exceeded our expectations. In fact of the six beetle families randomly sampled, only one (Staphylinidae: Bolitocharini) lacked yeast associates! The beetles were collected from basidiocarps (Amanita sp., Boletus sp., Inonotus ludovicianus, Ganoderma lucidum, Fomitella supina, and Pisolithus tinctorius) in southern Louisiana and Athens, Georgia. The target basidiocarps occur widely and are relatively easy to spot in the field; they harbor a diverse group of beetles. Beetles have been found repeatedly at different localities in the eight months of the year in which we sampled. The data have lead us to the following conclusions:

Figure 1.  Identification of yeasts using PAUP*  (Swofford, 1999) (parsimony option, default settings, gaps coded as missing data, 1000 bootstrap replications).  Analysis provides a means of identification of selected yeasts discovered in our preliminary study. The database of Kurtzman and Robnett (1998) is the basis for comparison of new taxa, a standard to which yeast specialists adhere. Color coding is the same as in Table 2; bold type face indicates a previously known endosymbiont (Table1).  Resolved terminal branches distinguish species. In the case of T1 and C2, metabolic tests distinguished strains from different beetles.

Justification of the project
Basidiocarps long have been known to provide resources for beetles and other insects, but the discovery of the association between the mycophagus beetles and endosymbiotic yeasts was unsuspected. Independently of the preliminary study reported here, McHugh and his colleagues (1997) already had discovered gut endosymbionts in the erotylid, Megalodacne heros. The yeast isolation study has extended the number of hosts to include members of five of six coleopteran families and shown that unique yeasts are present in the associations. We believe that the number of species to be discovered, including yeasts and the poorly known beetles of this habitat, will be high.  In addition to the discovery of new species, the project will be performed in a context that will provide information on evolutionary processes, especially in light of the interest in nutritional resource of beetles in relation to speciation (e.g., Farrell, 1998).  It is important to understand the role of the unexpected symbionts in enabling beetles to expand their resources, perhaps depending upon acquired fungal enzymes for detoxification or degradation of the substrates as noted in the introduction. The survey proposed here is the essential first step toward investigating many evolutionary questions.
Taxonomic breadth
The study is extremely broad taxonomically, even if only yeasts were to be considered; however, in addition to the saccharomycetalean yeasts [Saccharomycetales (4-5 families)] and basidiomycetous yeasts [Tremellales (one family)], the beetles with which they are associated [as many as 20-25 families], and to some extent, the basidiocarps in which the beetles feed [Agaricaceae, Bolitaceae, “gasteromycetes,” Aphyllophorales], also will receive our attention. The study avoids being too broad, however, because we will limit our investigations primarily to the basidiocarp habitat with the exception of determining if nonmycophagous sister taxa of mycophagous beetles also harbor endosymbiotic yeasts.  A brief taxonomic discussion of three components follows.  There also is a fourth component, the plant with which the basidiomycete is associated.
Table 4.  What is a yeast? Yeasts and yeast-like fungi associated with insects.  Groups collected in the preliminary study are indicated by bold type face. See also, Table 1.
ASCOMYCOTA
    Basal ascomycetes (archiascomycetes) --many yeast forms, but none yet known in arthropod associations
   Hemiascomycetes (true yeasts, encompassing Saccaromycetales)
    Euascomycetes (ascohymenial or filamentous ascomycetes)
          Perithecial ascomycetes (Ceratocystis, homopteran hypocreaceous symbiont, Ophiostoma)
          Perithecial outliers (Kathistes, Pyxidiophora, Symbiotaphrina)
BASIDIOMYCETES
   Tremellales 
Yeasts  The terms “yeast” and “yeast-like fungus” encompass nearly one thousand described taxa, and may include even basidiomycetes. Fungi that reproduce somatically by budding or cell fission are yeasts. Yeasts generally can be distinguished from yeast-like fungi by the absence of a fruiting body to contain ascospores in sexual reproduction (Alexopoulos, Mims, and Blackwell, 1996; Kurtzman and Fell, 1998). The endosymbionts reported in the preliminary study mostly are species of true ascomycetous yeasts (Saccharomycetales) (Table 4). The homopteran yeast-like endosymbionts (cf. Table 1) are members of Hypocreales (Blackwell and Jones, 1997). In addition to saccharomycetalean yeasts, anobiid beetles also harbor species of Symbiotaphrina, yeast-like members of a poorly resolved discomycete-loculoascomycete euascomycete clade (Jones and Blackwell, 1996; Jones, Dowd, and Blackwell, 1999).  Although we expect mostly saccharomycetalean yeasts, we will not overlook any derived yeast-like ascomycetes should they be present. Tremellalean basidiomycetous yeasts, although probably not endosymbionts, often have been reported from mushrooms (Prillinger et al., 1987), and besides the ascomycetes they are expected among the nonsymbiotic yeasts in the habitats of the beetles.
Table 5.  Many beetle families are expected in basidiocarps at the proposed collecting sites; those discovered to have endosymbionts in the preliminary study are shown in bold type face. (*) indicates other reports of endosymbiosis. Taxonomic list: Superfamily: Family Classification in part, after Lawrence and Britton (1991), Lawrence and Newton (1982) ), and papers in Pakaluk and Slipinski (1995).

Staphylinoidea: Ptiliidae, Leiodidae, Staphylinidae
Eucinetoidea: Eucinetidae, Clambidae
Scarabaeoidea:  Geotrupidae
Dryopoidea: Ptilodactylidae
Dermestoidea: Derodontidae, Dermestidae
Bostrichoidea: Anobiidae
Cleroidea: Trogossitidae
Cucujoidea: Nitidulidae, Cryptophagidae, Erotylidae, Phalacridae, Corylophidae, Endomychidae, Lathridiidae
Tenebrionoidea: Tetratomidae, Ciidae, Melandryidae, Colydiidae, Tenebrionidae
Curculionoidea: Anthribidae, Scolytidae*, Platypodidae
Mycophagous beetles  Lawrence (1989), knowing only about endosymbionts associated with two beetle families, estimated that fully half of all coleopterous families are primarily mycophagous or dependent upon plant material that has been altered by fungal action.  However, more strictly speaking, about twenty five families of beetles (Table 5) are expected to be associated with basidiocarps at the chosen collecting localities. In the preliminary study, only two beetle species (both Staphylinidae: Bolitocharini) among members of the six families sampled did not yield endosymbiotic yeasts.  Basidiomycetes   Although yeasts and beetles are the primary focus of the study, new species of agarics and Aphyllophorales could be discovered in the proposed collecting localities. The groups we will examine for the presence of beetles are numerous (Table 6). The literature points out that mycophagous insects encounter a number of problems in their life histories. Many of the agarics are too ephemeral and some of the polypores, too hard and dry.  However, beetles manage to inhabit the basidiocarps of both types, sometimes in very large numbers. In fact over 257 arthropod species were reported from 2660 basidiocarps of one polypore in an extensive study in eastern Canada (see Gilbertson, 1984, for this and other interesting numbers). It is doubtful that there is much specificity of beetles for the basidiocarps, especially in the case of the more ephemeral agarics, because the predictability of occurrence is so low. However, there are groups of basidiocarps, perhaps sorted by hardness, for which a beetle, especially a ciid, may show specificity. These groups are not necessarily closely related, but they are composed of tissues of a common hardness such as the grouping of Phellinus, Inonotus, Melanoporia, Cyclomyces, Phaeolus (Lawrence, 1973). It is interesting that ciids and tenebrionids that came from the same polypore in our study harbored yeasts that were very closely related.
Table 6.  Families of basidiomycetes* to be targeted for beetle collection.  Habitats sampled in the preliminary study are indicated by bold type face.

Agarics: Agaricaceae, Amanitaceae, Bolbitiaceae, Coprinaceae, Cortinariaceae, Fistulinaceae, Hygrophoraceae, Paxillaceae, Pluteaceae, Russulaceae, Schizophyllaceae, Strophariaceae, Tricholomataceae, etc.
Gasteromycetes: Lycoperdales, Nidulariales, Sclerodermatales, Tulostomatales, etc.
Boletes: Boletaceae,
Aphyllophorales (mostly “polypores”): Bondarzewiaceae, Cantharellaceae,Coniophoraceae, Ganodermataceae, Gomphiaceae, Hericiaceae, Hymenochaetaceae, Polyporaceae, Sparrasidaceae, Steriaceae, etc.
*Classification after Donk (1964), Gilbertson and Ryvarden (1986), and Singer (1986), but see Hibbett et al. (1997) for a developing phylogenetic approach.
Number of species expected in the survey is difficult to estimate; however, previously observed patterns provide some basis for discussion. We know that there are undescribed species of the yeasts and beetles within the southeastern United States, and more new species are expected from the tropical regions we would visit.  Yeasts:  Of the beetle-associated yeasts discovered to date, more than ten species appear to be undescribed (Tables 2, 3; Fig. 1).  Based on the preliminary study that has barely scratched the surface, the vastly expanded geographical region and greater beetle diversity, could produce over 50 new species. One problem with such an estimate is that, although we suspect more beetle groups that the five families in three superfamilies thus far discovered will have yeast associates, we are not certain of this. However, should additional yeast-harboring beetle groups be discovered, the number easily could exceed 50.  Beetles:  The coleopteran fauna associated with fungi and decaying vegetation is, in general, poorly studied taxonomically. 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 had a single known species until it was revised by Skelley et al. (1997), when it grew to include six newly discovered species from Central and South America. The small myxomycete-feeding family Sphindidae was represented by a single species in gulf and Caribbean region before 1990.  Taxonomic studies by McHugh and colleagues (McHugh, 1990, 1993; McHugh and Lewis, 1999) have increased the known diversity of this group in the circum-gulf region by expanding the known ranges of some existing North American taxa and describing 13 new species and two new genera from the region. Collecting in the southeastern U.S. has yielded 17 new species for McHugh. High numbers are expected at Barro Colorado Nature Monument based on the results of a large trapping project primarily involving phytophagous insects (Windsor, personal communication, 1999). Basidiomycetes:  Basidiomycetes, while not the main focus of the study, could provide new species. For example even along the coastal plain of the U.S., Blackwell has reported new aphyllophoralean species from the gulf coastal plain, including two species of polypores that harbor beetles and more are possible.
    To some extent species numbers rest upon the species concept that will be used. Generally, we will use a concept that is roughly equivalent to a phylogenetic concept for the organisms we will study.  The current yeast standardis based upon morphological and metabolic characters and base position differences of the D1/D2 region of 26S rDNA (see Table 3). The criterion of base pair differences actually coincides well with a phylogenetic concept (Kurtzman and Robnett, 1998). The beetle morphospecies concept that will be used also approaches a phylogenetic concept. We primarily are interested in identifications of basidiomycetes based on morphological characters that have been established by basidiomycete systematists. Voucher specimens will be available for detailed study whenever they might be needed.
Scientific context
The study primarily is one in which we will discover new species and reveal hidden associations of fungi in the gut of beetles. But, the study also is rich in contextual issues, and a large number of evolutionary, phylogenetic, and biogeographical questions could be addressed using the material to be collected in the proposed study.
Urgency
This study has intellectual urgency. An unexpected organismal assemblage has been discovered, and it is important to recognize its extent, and also discover the effect of yeasts on their insect associates. We suspect that the yeasts may have profound effects on the ability of the beetles to occupy a particular niche and depend on a specific nutritional resource. Among current research topics are hypotheses of rapid radiations and areas of megadiversity. Some of the hypotheses are aimed specifically 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.  Additionally, a newly discovered clade of yeasts is basal to all yeasts but the Dipodascaceae and sister to the remainder of the Saccharomycetaceae (Fig. 1). This finding is important for studies of yeast systematics and evolution, and more such groups still could be unknown. The progress made by yeast workers toward a phylogenetic classification over the past five years has been phenomenal, and our results on yeasts from an unstudied habitat would be a contribution toward the yeast classification.
Project management plan and methods
Collecting localities This survey could be conducted in several of many localities to establish the extent of endosymbiosis in beetles.  However, our choice of collecting sites was ruled somewhat by a need to work where we conveniently can establish pure cultures of yeasts and where the basidiomycete hosts and plants are known to some extent to provide comparative data for future studies. Outside of this we were drawn to localities where the mushroom and beetle diversity was expected to be high, maximizing the possibility of the discovery of a diverse group of yeasts and beetles. We have chosen to study the fungus/beetles/basidiocarp assemblages at three localities along the northern gulf coastal plain of the United States, the region around Xalapa, Veracruz, Mexico, and Barro Colorado Nature Monument, Panama. These localities provide somewhat contiguous coverage ofthe northern and western gulf with extension into the tropics. Each site will be collected at least three times during the duration of the funding period (Table 5).  A Magellan Trailblazer XL GPS receiver is available to record precise locality data, and collection coordinates can linked to our databases on the web site or transfered to GIS units at either university for mapping.
Gulf Coastal Plain and southeastern United States.  Our home region is rich in agarics and polypores and offer the decided advantage of our near constant presence and intimate knowledge of the local conditions necessary to discover the most ephemeral basidiocarps; in many cases we know of trees with heart and root rots, and we can observe the large beetle-ridden conks daily during their fruiting season. The entire region is undercollected and even the basidiomycetes are not well known.  For example the gulf coastal plain recently has provided about fifteen new species of Aphyllophorales, including two new polypores that harbor beetles. An ongoing check list of the wood-decaying basidiomycetes of the gulf coastal plain and their substrates is available at <http://lsb380.plbio.lsu.edu/wood-rotting%20fungi>. Species typical of more tropical regions found along the northern gulf coast include Tinctoporellus epimiltinus, Ganoderma colossum, and Laetiporus perscinus. New and unusual species of agarics in several groups have been routinely discovered by visiting mycologists (G. Mueller, T. Baroni, R. Vilgalys, personal communications, 1999). In many agaric groups the mycota is rich in temperate species as well as species once thought to be indigenous only to southern Mexican, Costa Rica, and Puerto Rico (R. Peterson, personal communication, 1999). There is no LTER site in this biotically diverse region of the country, so we will depend on a variety of collecting sites familiar to us. 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 will concentrate our efforts. In addition we commonly collect in a variety of other sites throughout the southeast. For example Blackwell also has had profitable experience in collecting along back roads along a “transect” roughly corresponding to Interstate Highway 10 between east Texas and Jacksonville, FL. McHugh is in a similar position to observe basidiocarps and insects in the southeast. The fact that the mycota of the gulf coast is similar in some components to neotropical areas, first suggested to us that we include the more- or-less contiguous tropical regions in our geographically based survey.  Instituto de Ecologia, Xalapa, Veracruz, Mexico.   Louisiana State University recently signed a collaborative research agreement with the Instituto de Ecologia, and collaborations are only now being arranged. There are many mycological projects underway in the tropical forests of the Xalapa region, including biodiversity studies of ascomycetes and basidiomycetes and of biological control of plants and insects. The institute also boasts an excellent fungal herbarium. The internationally acclaimed mycologist, Dr. Gaston Guzman, is based at the institute. In addition to having a place where to make yeast cultures, we will have the advantage of working with collaborators who know both the basidiomycetes and the region. The first set of specimens will be retained by the institute, and yeast cultures will be returned to them if they desire them. Collecting permits will be secured with the help of personnel from the Instituto de Ecologia, and we will abide by them. Blackwell will apply for USDA, APHIS permits for importation of cultures. Shipping of study material will be from Veracruz, and costs are budgeted. Barro Colorado Nature Monument (BCNM) (Smithsonian Tropical Research Institute, STRI), Panama. This site is a seasonal tropical moist forest. Annual rainfall is somewhat more than 2600 mm rainfall/year.  The collection sites we would have access to are in stands of secondary forests ranging in age from recently cut-over forest to 500 year old-growth forest. We will access on data from the stands that include identified trees and shrubs (Julie Denslow, personal communication). Another colleague at STRI, Donald Windsor, has studied the beetles of Barro Colorado Nature Monument for many years, and he has offered his invaluable service to us. STRI personnel will help us to obtain the necessary collecting and exportation permits by which we will abide once funding has been secured. Blackwell will apply for USDA, APHIS permits for importation of cultures. Shipping of study material will be from STRI, and costs are budgeted.
Similar research  We are not aware of other studies of endosymbiotic yeasts or of any yeasts associated with beetles in basidiocarp habitats. The closest studies are those of William T. Starmer,  André Lachance, Herman J. Phaff, and their colleagues, who are studying yeasts from several beetle and Drosophila habitats, including senescent flowers (W. T. Starmer and M.-A. LaChance, personal communication, 1999). Our study will complement this work rather than conflict with it.  As this proposal was being completed, a student of Brian Farrell contacted us to investigate the possibility of working with the yeast-like endosymbionts of anobiid beetles. We have offered advice to him on some aspects of the project and plan to collaborate should he wish to do so; he is interested in questions similar to our contextual issues, but with phytophagous beetles.  Mycologists more generally interested in yeast systematics include Junta Sugiyama, emeritus professor at Tokyo University, and Cletus Kurtzman, ARS-USDA at Peoria, co-editor of the yeast compendium recently revised in 1998. More recently, Rytas Vilgalys, Duke University, has become interested in yeasts as part of the Great Smokies (Discover Life in America) ATBI; however, his work will be a high throughput study including non-symbiotic yeasts (personal communication, 1999). We are all committed to sharing data, especially important when new species are discovered. Another collaborator, Hiroaki Noda, has studied endosymbiotic yeasts of homopterans but now is in another area of research.
     Several entomologists study beetles from basidiocarps, including some in the families we will emphasize; some of them are collaborators of CoPI McHugh: Quentin Wheeler (Cornell University), James S. Ashe (University of Kansas), and Richard Leschen (LandCare, New Zealand). In addition Richard Brown (Mississippi State University) and Christopher Carlton (Louisiana State University), entomologists familiar with beetles of the southern United States, have agreed to help when needed. Brian Farrell and his student, mentioned above, are interested in similar questions concerning phytophagous beetles. We welcome collaboration with any other entomologists who could provide live beetles for dissection, and we will try to make more contacts.
Time frame  During the three years of the funding period we would do a number of operations almost simultaneously. For example collection and identification of beetles and basidiocarps, isolation of yeasts, and characterization of yeasts using cultural methods, DNA sequencing, and database updates will all be done as near the time of collection as possible; in other words, we will have to keep up with everything. The primary time-dependent components are date of collecting at a given place and print publication. Good collecting is dependent largely on rainfall and to some extent temperature for all basidiomycete species and additionally to season for most mycorrhizal species. We would arrange collecting trips for optimum collecting conditions, but also for times when we are not teaching classes (Table 7).  Print publication must come after data collection, and although we plan to produce manuscripts periodically they necessarily will lag somewhat. We plan to have most of the research reports completed and submitted for publication within the last six months of funding by the latest.
 
Table 7.  Projected collecting schedule.

           Summer   Autumn   Winter   Spring
Year 2000         12               13               13            12
Year 2001         13               12               12            13
Year 2002           1                 1                -               -
1=Southeastern U.S.; 2=Xalapa; 3=STRI
The division of labor is clear cut.  PIs (Blackwell and McHugh) and the postdoctoral associate (Suh) will all be involved in collecting beetles from basidiocarps. We will all collect together whenever possible. Each collector will be responsible for ensuring a good voucher specimen for collections. On some short local trips we may collect separately, but then live beetles and basidiocarp vouchers will be shipped to Blackwell in Baton Rouge and beetle specimens will be shipped to McHugh in Athens. Blackwell and Suh will receive beetles for culture of yeasts. they will be responsible for identification (including identification by DNA sequencing), storage, and eventual culture deposition in one of two public collections for the yeasts; they also will be responsible for identification and deposition of the basidiomycete specimens at the Louisiana State University Mycological Herbarium or appropriate collections at the foreign cooperating institutions. McHugh will be responsible for beetle identifications and curation in the University of Georgia Collection of Arthropods or appropriate collections at the foreign cooperating institutions. Specialists will be alerted if we find basidiomycetes or insects that we feel will be of particular interest to them. Data will be posted at LSU, and print publications will be prepared by all participants singly or as collaborators as is appropriate.
     Tied to the time frame and number of collecting trips is the problem of estimating the completeness of the survey. We will use an estimation of local richness by extrapolation of species accumulation curves (Colwell and Coddington, 1994).  We probably will have trouble with getting meaningful diversity estimates for sites that are sampled only four times and at different seasons (Mexico, Panama), but for some sites along the gulf coastal plain, we plan to get several samplings in each season of each year.  Even in the cases when we would be able to resample a site extensively, we should point out that standardizing our search effort may be difficult because the basidiocarps often are unpredictable in the environment. We will attempt exhaustive searches within a set area, so we can standardize the searching effort.
Methods The methods that we will use are basically those that already have served to obtain the preliminary results reported above:   Basidiomycete collection  Basidiocarps will be hand collected from appropriate sites where all basidiocarps will be examined for the presence of beetles. We will try to search exhaustively but will be certain to include geographically-broad ranging, common species with long-lived basidiocarps as we have done before; this will provide some measure of comparison of the yeasts and beetles in different localities if we can have basidiocarp as a common factor. This method also will help in evaluating the completeness of the collecting by affording the opportunity for repeated resampling.  Beetle collection  The collections for target beetles have been repeatedly performed at different localities and months. Because we are focusing on the beetle fauna associated with basidiomycetes, direct collecting from the basidiocarp will provide the required host link for the third and fourth components of our chain: yeast-beetle-basidiomycete-fungus substrate/host). Beetles must be kept alive until dissection. Yeast isolation  Basidiocarps will be brought to the laboratory for examination or rearing of beetles.  Once removed from the basidiocarp, the beetles are held on filter paper in a culture dish for several days, so that the gut is cleared partially and surface debris is lost. Beetles are frozen before a 95% alcohol wash for surface sterilization, dissection, and removal of the gut for culturing on acidified yeast-malt agar; the acidified medium largely inhibits bacterial growth.  An essential step in the procedure is the plating of the saline rinse after the alcohol wash as a control for surface microorganisms. Only on one occasion has a yeast appeared in a control. Examination of the colonies streaked on agar is performed from day 1, and all of colonies that have different morphologies are purified. A test of this method has shown that usually a single yeast and seldom more than two are isolated from an individual beetle. Yeast colonies are then grown in pure culture to provide inoculum for morphological and metabolic characterization and for DNA extraction. Cultures will be overlain with sterile mineral oil for medium term storage. We are targeting only yeasts that grow in culture in this proposal, and although we do not have evidence that unculturable yeasts are present, we are aware of that possibility.  Characterization and identification of yeasts  We will characterize the yeasts we discover in two ways. First, the prescribed "standard description" for yeasts (Yarrow, 1998) (Table 8) will be used to provide morphological and metabolic data for different isolates. 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.
    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 bp) for all described yeast species (over 600) has been made available to us, and we will continue to use this method to compare the species we isolate with previously described species (Kurtzman and Robnett, 1998). Recent updates have been sent to us by Kurtzman and Robnett, and our sequences to date have been sent to them for addition to the complete data base.
    The isolates in the preliminary study were tested for assimilation of 19 major carbon compounds to provide information to supplement the sequence data for identification. This method has provided us with information to separate closely related yeast strains. Morphological observations will be made at two months after the initial culturing to allow enough time for possible spore formation. When appropriate cultures of non-sporulating yeasts are available, we will attempt to establish mating competence by mixing cultures. Because we were interested in yeast enzymatic activity that might be of use in the beetle habitat, we investigated the capacity of the cultures to degrade a variety of substrates (Untereiner and Malloch, 1999).  One test, the difference in lipid degradation also distinguished closely related strains in one case.
DNA sequencing  Sequences will be obtained for all yeasts isolated in the study for approximately 600 bp from the 5’ end of 26S rDNA (D1/D2 region).  We have have used readily available primers for the PCR reactions and sequencing (Kurtzman and Robnett, 1998). After initial screening one or two isolates of each group will be used to determine the entire 18S rDNA sequence (Suh and Blackwell, 1999). The species of previously known yeast-like fungi in Coleoptera, Symbiotaphrina kochii CBS 588.33 and CBS 250.77, Symbiotaphrina buchneri CBS 420.63, Candida karawaiewii ATCC 22994, C. xestobii ATCC 24001, C. rhagii NRRL Y-2596, and C. tenuis NRRL Y-2597, were sequenced in these regions as well and will provide a conserved region for comparison to ensure that we do not have contaminating DNA, often a problem when endosymbiotic systems are studied.
    The purified double stranded PCR products were used directly as templates for sequencing with an ABI PRISM™  Dye Terminator Cycle sequencing kit. The DNA sequences will be determined by an ABI PRISM 310 Genetic Analyzer. The 26S rDNA sequences will be aligned in the database of more than 600 D1/D2 sequences of almost all yeasts in Saccharomycetales (Kurtzman and Robnett, 1998, and unpublished results) using Clustal W (Thompson et al., 1994). Phylogenetic analysis is designed for identification, but the same process also allows for discovery of close relatives, and informs subsequent sampling. We use the parsimony options in PAUP* (Swofford, 1999) to compare the yeast sequences.
Electronic Products  A relational database will be available from a Department of Biological Sciences server, and data on yeasts and beetles can become available almost immediately after identification or in some cases by collection and isolate numbers. The database eventually will link all collections (yeasts, beetles, basidiocarps or other habitat data, date and locality, collector, isolation medium, storage, accession in culture collections, sequences, standard description, etc). Links can be established to the GenBank database where the sequences will be banked, the collections where cultures will be stored, and TreeBase, and our sequence data will be incorporated with those of Kurtzman and Robnett (1998).  We also will supply checklists, descriptions, and interactive keys for the beetles and associated yeasts.
     The main relational database software that we will be using is Microsoft's SQL Server 7.0.  This software is made to handle enormous amounts of data and distribute them to the users and also datawarehousing. The other software that we will be using is HyperSQL, developed by the Northwest Alliance for Computational Science and Engineering. Ms Sherry Pittam (message attached), Database Specialist, based at Oregon State University in the Northwest Alliance for Computational Science and Engineering, part of the Metacenter Regional Alliances program sponsored by the National Science Foundation, already has been helpful and has offered to be of additional assistance when we begin to develop the databases. Of special interest to us is software that was developed as part of two NSF grants. HyperSQL software works with Unix databases Sybase and Oracle but data from a variety of programs (Paradox, DBase, Foxpro, Access, or FileMakerPro) can be uploaded easily. This will act as an intermediate software, communicating between our SQL Server database and the internet. The HyperSQL will construct the web-based query interfaces to our SQL Server database. The user will fill out the forms on the web, then this will generate a query file consisting of "query form", "results screen", and the "browse screens".  The query form is where the user will enter input and submit there query. In the results screen, the SQL Server will be accessed for the database information and HyperSQL will have automatically generated SQL queries from the user's input and send it  to the browser's window. The Browse Screen will have hyperlinked results, where the user can select and get images or access to other files.  The selected links can also generate additional calls to our SQL Server. The data transmission and presentation protocols will be TCP/IP, HTTP, and HTML. The University of Georgia Collection of Arthropods uses Paradox 8 for the collection records.  The Louisiana State University Herbarium currently uses a Botanical Research and Herbarium Management System (BRAMS) written in FoxPro. These systems are comparable with the HyperSQL software.
     The server that we will use will be a rather powerful one, since the SQL software (see below) uses a lot of memory and hard drive space. We have requested a Dell Power Edge 4300 with (2) Pentium III 600mhz processors and (6) 18GB SCSI hard drives with RAID 5 running on them. The dual processor will take advantage of SQL Server's "Parallel Query Execution."  This allows the SQL Server to execute a single query in parallel across multiple processors very quickly. The operating system will be Windows NT, since we have existing servers running NT, and using Microsoft's Internet Information (Web) Server. They should coexist and function properly, and we can have them backing up one another and distributing the SQL load evenly.
Deposition of cultures and specimens  Requirements for material to remain at foreign collecting localities always will be obliged. In addition we will try to accommodate specialists of the collected material and other products of the study by providing loans. Unless there are other requirements to be met we will proceed in the following manner:  Once the cultures have been purified, identified or described as necessary, they will be deposited public culture collections.  Cletus Kurtzman [see letter] will accept type cultures at Peoria (ARS) and these will supplement his large specialist collection of the group.  Cultures, including non-type material, also will be sent to the American Type Culture Collection (ATCC) [see letter from S.-C. Jong]. Kurtzman and Jong both have declined a monetary contribution for depositing the cultures. Basidiomycete vouchers will be accessioned into the LSU Mycological Herbarium (LSU-M) of which Blackwell is curator. A new herbarium complex for plants, lichens, and fungi with a total of 6624 square feet of space, 3305 of which is for compactor storage, will be available Spring 2001 and will allow almost unlimited accession of specimens by Blackwell. An associate curator position is provided to the combined herbaria. The most important collections are of neotropical wood-decaying basidiomycetes in the Bernard Lowy Collection, which the new collections should complement.  Beetle specimens will be deposited at the University of Georgia Collection of Arthropods of which McHugh is curator. The University of Georgia Collection of Arthropods (UGCA) has holdings comprising more than 700,000 insect specimens. Approximately 95% of the specimens are from the southeastern United States and more than 80% have been determined to species. The regular personnel of the UGCA includes Dr. Joseph McHugh, Curator, and Dr. Cecil Smith, Associate Curator/Collections Manager.  Extracted DNA will be stored in the LSU Frozen Tissue Collection, Natural Science Museum. A message from the curator Frederick Sheldon is attached.  Other products  In addition to electronic inventories and associated materials, other products will be DNA sequences contributed to GenBank, yeast phylogenetic trees contributed to TreeBase, and whole genomic DNA and frozen cultures available from the LSU Frozen Tissue Collection. The sequence data we acquire also will be used to expand the over 600 species alignment of Kurtzman and Robnett (1998); in addition results from the standard morphological observations and culture tests (Kurtzman and Fell, 1998) will be used to characterize the yeasts will be available on the web site. Cultures will be available from us for a time, and will be permanately available from ATCC or the ARS collection.  The PIs are committed to graduate and undergraduate education, and research materials often are adapted in teaching <http://lsb380.plbio.lsu.edu/Home.html>. We will continue to train a diverse group of graduate and undergradadute students and postdocs in our laboratories (see previous funding for Blackwell and biographical sketches for both PIs).
[Letters of support were sent to the program director.]
References
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?