taxonomic diversity and interactions of insect-associated ascomycetes

Taxonomic diversity and interactionsof insect-associated ascomycetes

MEREDITH BLACKWELL and KEVIN JONESDepartment of Plant Biology, Louisiana State University, Baton Rouge, LA 70803, USA

Received 25 July 1996; accepted 18 August 1996

Many ascomycetes are associated with insects to form symbioses. The fungi are necrotrophic and

biotrophic parasites, endosymbionts, insect-dispersed forms, and other obligate associates thatprovide nourishment for insects. Diversity among these fungi can be categorized in several di�erentways: taxonomic diversity, variety of interaction types occurring within a fungal lineage, and number

of fungal species. Previously our inability to produce well supported phylogenetic hypotheses hasobscured these views of diversity. Over the past 5 years our knowledge of insect-associated fungi hasbeen improved by the use of DNA sequence analysis. Such studies have revealed that ascomycetes in

almost all major clades are associated intimately with insects. Of particular interest has been thesorting out of relationships of taxa with convergent morphologies, unique characters, and lostcharacters, including those associated with sexual reproduction. Within some fungal groups the

types of interactions with insects are diverse, and eventually phylogenetic analysis will help to tracethe evolutionary development of symbioses. Molecular studies also contribute to our understandingof the number of species which may vary according to species concepts used in their study.

Keywords: ascomycetes; insects; mites; symbiosis; interactions; rDNA.

Introduction

Insects and fungi are closely associated in most environments, and many types of inter-action that have arisen repeatedly have been observed among diverse taxa of these groupsof organisms. The interactions may be categorized roughly as necrotrophic and biotrophicparasitism, dispersal and fertilization associations, gardening symbioses, andendosymbioses (Batra, 1979; Bultman and White, 1988; Pirozynski and Hawksworth,1988; Samson et al., 1988; Wilding et al., 1989; Carroll and Wicklow, 1992). The inter-actions sometimes are extremely complex and may involve other organisms in addition tofungi and insects (Blackwell et al., 1986; Price, 1991).

Our knowledge of the diversity (the number of taxonomic groups of insect fungi, thevariety of interactions occurring within each fungal lineage and their histories, and numberof fungal species) has been improved by the use of phylogenetic analysis of molecularcharacters.

Methods and materials

The techniques used for DNA extraction, polymerase chain reaction, sequencing, and dataanalysis have been published elsewhere (Spatafora and Blackwell, 1993, 1994a, b; Cassarand Blackwell, 1996; Jones and Blackwell, 1996), and some information is given in the®gure legends. DNA sequences used in the analyses and their sources are shown in Table 1.Although some new data are discussed, they will be published in detail elsewhere.

0960-3115 Ó 1997 Chapman & Hall

Biodiversity and Conservation 6, 689±699 (1997)

Table 1. List of taxa and references to GenBank accession numbers or references to published 18SrDNA sequences used in analyses

Ambrosiella brunnea U40423Ambrosiella ferruginea U40016Ambrosiella gnathotrichi U40015

Ambrosiella hartigii U40017, U40054Ambrosiella ips U40018Ambrosiella macrospora U40019

Ambrosiella sulcati U40020Ambrosiella sulfurea U40021Ambrosiella xylebori U40422

Ambrosiozyma platypodis L36984Aphysiostroma stercorarium U32398Ascosphaerica apis M83264Athelia bombacina M55638

Aureodasidium pullulans M55639Candida albicans X53497Cephaloascus fragrans U20355

Ceratocystis adiposa Hausner et al. (1993b)Ceratocystis coerulescens Hausner et al. (1993b)Ceratocystis fagacearum Hausner et al. (1993b)

Ceratocystis ®mbriata U32418Ceratocystis moniliformis Hausner et al. (1993b)Ceratocystis paradoxa Hausner et al. (1993b)Ceratocystis radicicola U36987

Ceratocystis virescens U32419Cercophora septentrionalis U32400Chaetomium globosum U20355

Claviceps paspali U32401Coccidioideus immitis U32401Cudonia confusa Z30240

Daldinia concentrica U32402Diaporthe phaseolarum L36985Diatrype disciformis U32403

Eleutherascus peruvianus U63553Glomerella cingulata U48427Gyromitra esculenta Z30238Halosphaeriopsis mediosetigera U32420

Herpomyces ectobiae Unpublisheda

Hirsutella thompsonii U32406Hypocrea lutea U32407

Hypocrea pallida U32408Hypocrea schweinitzii L36986Hypocrella sp U32409

Inermisia aggregata Z30241Kathistes analemmoides Unpublisheda

Kathistes calyculata Unpublisheda

Laodelphax striatellus yeast Unpublisheda

Leucostoma persoonii M83259Melanospora fallax U47842

690 Blackwell and Jones

Results and discussion

Taxonomic distribution of insect fungi

Studies of developmental morphology, life histories, and physiology have contributed toour understanding and recognition of many groups of ascomycetes; however, because ofstrong selection for certain features involved with an insect-associated life style, use ofmorphological characters in systematics of these particular ascomycetes has beenespecially di�cult. We will discuss fungi with convergent morphologies (ophiostomatoidfungi), divergent morphologies (Hypocreales), autapomorphies (Laboulbeniales), reducedmorphologies (gut symbionts), and absence of characters (asexual fungi), all of which aretaxonomically problematical without use of molecular characters. For example studiesusing molecular characters have shown that some previously recognized taxonomic groupsare not monophyletic, but based on convergent morphological characters. The net e�ecthas been to recognize the occurrence of new insect-associated ascomycete lineages. Othergroups encompass greater morphological variation than previously had been recognized.

Ophiostomatoid fungi. The ophiostomatoid morphology is characterized by evanescent asciwith passively discharged sticky ascospores that collect at the tip of a long-necked

Table 1. (Continuted)

Melanospora zamiae Unpublisheda

Microascus trigonosporus L36987

Morchella esculenta L36986Nectia haematococca U32413Neocosmospora vasinfecta U32414

Neurospora crassa X04971Nilaparvata lugens yeast Unpublisheda

Ophiostoma piliferum U20377

Ophiostoma ulmi U83261Petriella setifera U32421Pichia ambrosiae U63039Pichia scolyti U32421

Pleospora rudis U00975Protomyces inouyei D11377Pyxidiophora sp. 1 Unpublisheda

Pyxidiophora sp. 2 Unpublisheda

Pyxidiophora CBS 253.81 Unpublisheda

Rickia passalina Unpublisheda

Rhytidhysteron rufulum U20506Saccharomyces cerevisiae J01353Sogatella furcifera yeast Unpublisheda

Spathularia ¯avida Z30293Subbaromyces splendens U63552Symbiotaphrina buchnerii U26205Symbiotaphrina kochii U26206

Talaromyces ¯avus M83262Taphrina deformans U20376

aUnpublished and aligned sequences available from M. Blackwell.

Insect-associated ascomycetes 691

perithecium (Malloch and Blackwell, 1992, 1993). In the past many genera with thismorphologywere classi®ed in theOphiostomatales, and species ofOphiostoma,Ceratocystis,Pyxidiophora,Kathistes and Subbaromyces have all been included in the order at one time oranother (Blackwell and Spatafora, 1994). A number of observations have helped to establishthat species of these genera are probably dispersed solely by insects, and, in some cases, theirphoretic mites (Malloch and Blackwell, 1992). Cain and Weresub (1957) and, later, Cain(1972), suggested that ophiostomatoid characters might have been selected for the dispersaladvantage they convey. Since that time, in addition tomorphological characters, a wealth ofphysiological and ecological information has been obtained to support the separation ofOphiostoma and Ceratocystis, the best studied genera of ophiostomatoid fungi (see Spata-fora andBlackwell, 1994b). Analysis ofmolecular characters supports convergent evolution.Although few mycologists who study these fungi were surprised that species of ophiosto-matoid genera did not comprise a monophyletic group, the degree of divergence amongspecies of the assemblage was unexpected.

The tree diagram (Fig. 1) shows that the ophiostomatoid taxa included in the analysisare not monophyletic. Ophiostoma and Ceratocystis are perithecial ascomycetes, with,Ophiostoma as a subclade distinct from Ceratocystis. Furthermore, the analysis supportsexclusion of Kathistes, Pyxidiophora and Subbaromyces from the main body of perithecialascomycetes, a conclusion certainly not arrived at using morphological characters. Thecurrent view suggests that ophiostomatoid species can be placed not only in theOphiostomatales (Ophiostoma), but also in the Microascales (Ceratocystis) and severallineages that are poorly resolved but which may reside among the paraphyletic lo-culoascomycete and discomycete lineages (Kathistes, Pyxidiophora and Subbaromyces) (seebelow). Thus, we have a much reduced Ophiostomatales, but a greater overall taxonomicdiversity of insect-dispersed forms (Hausner et al., 1992, 1993a, b; Blackwell and Spata-fora, 1994; Spatafora and Blackwell, 1994b).

Hypocreales. Hypocreales provides a very di�erent example of changes in a higher taxon,one in which the concept of a monophyletic group has been enlarged by the discovery ofincreased morphological variation. The traditional concept of Hypocreales includesspecies with light-coloured ascospores and perithecium and phialidic anamorphs. Al-though several earlier workers considered Clavicipitaceae distant from Hypocreales,Rogerson (1970) recognized their close relationship and this has been borne out by studiesemploying molecular characters. Spatafora and Blackwell (1993) placed members of thetwo orders in a monophyletic group. In addition the type of Melanospora, with darkspores, is included. Subsequent work applying sequence analysis to other DNA regionssupports uniting Hypocreales, Clavicipitaceae, and Melanospora in Hypocreales (Rehnerand Samuels, 1995).

New ®ndings indicate that several more genera should be placed in the Hypocreaceae±Clavicipitaceae±Melanosporaceae lineage, increasing the morphological diversity of thegroup to the extreme. Newly discovered members of the lineage include a termite parasite,Cordycepioideus, and yeast-like endosymbionts of leafhoppers. Cordycepioideus has amassive stroma similar to that of Cordyceps but has been considered hypocrealean on thebasis of ascospore morphology and purported apical paraphyses in centrum development(Sti¯er, 1941; Blackwell and Gilbertson, 1981); in addition the ascospores become verydark at maturity (Blackwell and Gilbertson, 1984; Ochiel, 1995). In our preliminary workCordycepioideus has been found to occur within Clavicipitaceae in analyses of both small


Figure 1. Strict consensus of 36 trees of 1373 steps each obtained using PAUP 3.1.1 (Swo�ord, 1993)using the heuristic search option and 50 stepwise addition replications. Characters from 61 taxa arefrom the nuclear encoded small subunit ribosomal DNA (1134 base positions from the 5¢ end, 395phylogenetically informative positions). Characters are unordered and unweighted. Atheliabombacina was designated as an outgroup taxon. Tree statistics for the 36 trees are: consistency index(0.483), rentension index (0.697), rescaled consistency index (0.337), and homoplasy index (0.517).

Results from 500 bootstrap resamplings are shown for branches that received > 50% support. Taxain boldface are associated with insects.


and large subunit ribosomal DNA. The yeast-like leafhopper symbionts also group amongpyrenomycetes (Noda et al., 1995) with clavicipitaceous ascomycetes. Both of these resultswill be reported in detail elsewhere, but we mention them here to point out that previoustaxonomic range of anamorphs, ascospore colour, centrum development, and thallusmorphology in the Hypocreaceae±Clavicipitaceae±Melanosporaceae clade has beenexpanded further.

Saccharomycetales. Phylogenetic studies of saccharomycetalean yeasts, many of which areassociated with insects, indicate that the order is monophyletic although some traditionaltaxa within the order are not (Kurtzman and Robnett, 1994). Many yeasts haveassociations with insects which feed on and disperse them. Although our sample of speciesis small, we have been interested in ruling out the possibility that yeast-like endosymbiontsof anobiid beetles and ophiostomatoid associates of scolytid beetles were not related. Ourresults indicate that similar associations arose independently among these distantly relatedfungi and, in addition, that closely related yeasts have evolved relationships with beetles indi�erent families.

Laboulbeniales. Laboulbeniales is placed as a well supported sister group to Pyxidiophora;however, other close relationships of these ascomycetes are not known. Although speciesof Pyxidiophora, Kathistes, and Subbaromyces all have distinctive long-necked peritheciacomprised of a single layer of wall cells, they do not appear to be closely related. Thebranches that unite Subbaromyces with the Laboulbeniales±Pyxidiophora clade andKathistes with the pyrenomycetes in some analyses (Fig. 1) receive no bootstrap supportand are dependent upon the other taxa included in the analysis.

Symbiotaphrina. A second lineage is placed outside recognized taxonomic groups. Twospecies of Symbiotaphrina, yeast-like endosymbionts that detoxify the food of anobiidbeetles (Dowd, 1991) previously were considered to be saccharomycetalean yeasts orrelatives of Taphrina (KuÈ hlwein and Jurzitza, 1961; Gams and von Arx, 1980). Theplacement of the two species of the genus outside either of these groups is stronglysupported, but their placement, perhaps among loculoascomycetes or discomycetes, isdependent on taxon sampling and is not supported (Jones and Blackwell, 1996; Noda andKodama, 1996). It is interesting to note that endosymbionts with yeast-like morphologiesnow are known in three lineages. In addition to Symbiotaphrina these include the leaf-hopper endosymbionts of Hypocreales discussed above and several saccharomycetaleanyeasts also associated with anobiid beetles (see below).

The position of Symbiotaphrina, as with the Laboulbeniales±Pyxidiophora clade andKathistes and Subbaromyces is not satisfactorily resolved, but the result has been toexpand the diversity of insect fungi outside of the main pyrenomycete and yeast groups, toa region of the tree that until recently was not known to be rich in insect-associated fungi,but to contain only a few parasites of insects.

Diversity of interactions within taxa

A number of ascomycete lineages are known to have close associations with insects, andthese apparently have arisen over and over again. It is of interest, however, that the mostbasal of the ascomycetes (Taphrina, Protomyces, Schizosaccharomyces, Saitoella andPneumocystis) are not known to be associated with insects, although the complete lifecycles in nature of most of these fungi are not known fully. Within other higher fungal


taxa certain groups have few members with insect interactions, while others are notable fortheir ability to interact, not only with insects, but with other animals and plants as well.The present situation of incomplete taxon sampling among these groups prevents completetracking of interactions within clades across the spectrum of arthropod-associatedascomycetes, but eventually phylogenetic studies with increased breadth of taxon samplingshould enable us to do this.

The Hypocreaceae±Clavicipitaceae±Melanosporaceae clade in its expanded versioncontains plant endophytes and parasites, insect parasites and endosymbionts, myco-parasites, and insect-dispersed species (Spatafora and Blackwell, 1993). It is of interest thatamong these ascomycetes certain types of associations have arisen on more than oneoccasion. For example mycoparasites occur among species of Cordyceps and hypo-creaceous groups that are not closely related within the larger clade; insect pathogens alsooccur among Cordyceps and several di�erent hypocreaceous and nectriaceous groups.Plant pathogens occur in several di�erent lineages. However, because the phylogeny is notyet complete, more speci®c appraisals about the host associations of the basal members ofeach lineage currently cannot be made.

As presented here, Microascales comprises fungi that lack forcible ascospore discharge.Of these clades Microascaceae (represented by Microascus and Petriella), a group thatincludes some mammalian parasites, has species with ophiostomatoid morphology,apparently dispersed by insects; however, no other types of associations with insects areknown. Conversely, the related clade containing Ceratocystis and asexual species has veryclose associations with scolytid bark beetles and dispersal often is by ascospores that adhereto the body surfaces of the beetles; in some of these associations the fungi help to overcomethe defences of the tree host of the beetles (Schowalter and Filip, 1993). From theseassociations independent lineages of mycangial beetle symbionts appear to have arisenindependently on several occasions (see below). Very similar associations with scolytidbeetles also occur among many fungi in the more distantly related Ophiostomatales, anddi�erent lineages of mycangial associates are even more common among these species.

Saccharomycetales has many species that are closely associated with a variety of insectsin their habitats. These interactions often involve feeding on the yeasts, and, concurrently,dispersal may occur. In this regard particular attention has been paid to Drosophila speciesand cactophilic yeasts (Pha� and Starmer, 1987; Starmer et al., 1988), bark beetle galleries(Callaham and Shifrine, 1960; Bridges et al., 1984; Leufven and Nehls, 1986), and slime¯ux yeasts (Batra, 1979). As we mentioned earlier, some yeasts are endosymbionts ofanobiid beetles, the same family of beetles that are associated with Symbiotaphrina.

Reconstruction of the evolutionary histories of ascomycete-insect associations occurs inthe lineages excluded from the main body of perithecial forms. Many taxa of ascomycetesfrom these groups remain to be sampled. For example, even for the strongly supportedPyxidiophora-Laboulbeniales clade the closest sister group to this clade is not known, sothere is no information on the basal condition of this group that contains mycoparasites aswell as insect parasites (Blackwell, 1994).

Species diversity

Characters derived from DNA sequences, especially when used in conjunction with geo-graphic or host distribution data, provide a means with which to discover genetic diversityamong closely related fungi that have little apparent morphological distinction. Amonginsect-associated fungi Ambrosiella provides an example of the way in which cryptic


diversity can be discovered and a phylogenetic species concept applied. Our work withasexual species of Ambrosiella revealed that the genus is polyphyletic (Cassar andBlackwell, 1996); one group of species occurs in a clade with Ceratocystis and the other,with species of Ophiostoma (Fig. 2). Although the Ceratocystis clade relatives appearedmonophyletic in our earlier studies, only two species of Ceratocystis had been included.When additional species of Ceratocystis were added to the taxa being analysed and a morevariable DNA region was studied, the Ambrosiella subclade was dispersed (Fig. 3). Thisobservation indicated that the species of Ambrosiella within the Ceratocystis clade werenot monophyletic but were derived independently from di�erent ancestral species ofCeratocystis.

Genetic variation among Ambrosiella ferruginea isolates of the Ceratocystis clade wasdescribed more fully by restriction analysis of the intergenic spacer region (IGS) of theribosomal DNA repeat unit of twenty isolates from each of three sympatric beetle hosts.Restriction patterns of the IGS from isolates of a particular beetle host can bedistinguished from those of other beetles by this method. Because observed A. ferrugineagenetic diversity correlated with beetle host di�erences, we considered the possibility that amonophyletic lineage of all A. ferruginea isolates might have diverged in association with

Figure 2. Strict consensus of 24 trees each of 631 steps obtained with PAUP 3.1.1 (Swo�ord, 1993)using the heuristic search option and 100 stepwise addition replications. Characters from the nuclear

encoded small subunit ribosomal DNA (801 base positions from the 5¢ end). Characters are un-ordered and unweighted. Taphrina deformans was designated as an outgroup taxon. Tree statisticsfor the 24 trees are: consistency index (0.782), rentension index (0.834), rescaled consistency index

(0.652), and homoplasy index (0.218). Results from 1000 bootstrap resamplings are shown forbranches that received > 50% support.


di�erent beetle species. However, a second possibility has not been ruled out in subsequentstudies, and the three asexual lineages, all known as A. ferruginea, may have arisenindependently from within a sexual ancestral population.

Multiple divergences from sexual populations would correspond more closely to studiesindicating that the repeated divergence of asexual fungi from ancestral sexual populationsmay be a common mode of speciation of conidial fungi in Trichocomaceae (LoBuglioet al., 1993; Berbee et al., 1995; Geiser et al., 1996). If this is so, by a phylogenetic speciesconcept the three A. ferruginea lineages, which are closely related and morphologicallyvery similar, would each have a phylogenetic history that is not connected directly to theothers. Recognition of each lineage would call for dividing Ambrosiella ferruginea intothree species corresponding with the host beetle. This certainly is not a move we wouldcare to make until isolates from more host beetles from throughout the wide geographicalrange, along with genetic variation of inferred sexual progenitors, can be studied inten-sively. However, if the scenario is correct, large numbers of unrecognized asexual speciesmay lurk within described taxa.

Acknowledgements

The authors thank Sung-Oui Suh and Joseph W. Spatafora for access to unpublished dataand analyses. Hiroaki Noda was helpful in providing extracted DNA of intracellularsymbionts of leafhoppers, and Gerald Ochiel and Harry Evans supplied cultures ofCordycepioideus. We gratefully acknowledge the ®nancial support of the National ScienceFoundation (DEB-9208027) and the Louisiana Educational Quality Support Fund toM. Blackwell.

Figure 3. Broader taxon sampling to include additional species of Ceratocystis divides the

Ceratocystis-related Ambrosiella clade (see Fig. 2). Characters are from 276 base positions from the 5¢end of the nuclear encoded large subunit ribosomal DNA and are unpublished or from Hausner et al.(1993b) (Ceratocystis moniliformis, C. paradoxa, C. radicicola). An exhaustive search with tree-bi-

section-reconnection branch-swapping produced a single most parsimonious tree of 57 steps inlength. Characters are unordered and unweighted, and Kernia pachypleura (Hausner et al., 1993b)was designated as an outgroup taxon. Tree statistics for the trees are: consistency index (0.877),

rentension index (0.833), rescaled consistency index (0.731), and homoplasy index (0.123). Resultsfrom 500 bootstrap resamplings are shown for branches that received > 50% support. Although fewbranches are supported, those showing polyphyly within the Ambrosiella clade do receive support.


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