Species and Speciation in Mushrooms Author(S): Ronald H

Home , Basidiocarp, Clamp connection, Lentinus, Omphalotus olivascens, Pleurotus djamor, Pleurotus populinus

Species and Speciation in Mushrooms Author(s): Ronald H. Petersen and Karen W. Hughes Source: BioScience, Vol. 49, No. 6 (June 1999), pp. 440-452 Published by: University of California Press on behalf of the American Institute of Biological Sciences Stable URL: http://www.jstor.org/stable/10.1525/bisi.1999.49.6.440 . Accessed: 13/07/2011 12:47

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http://www.jstor.org Species and Speciation in Mushrooms Development of a species concept poses difficulties

Ronald H. Petersenand KarenW. Hughes

It is really laughable to see what Classification of the major groups differentideas are prominent in vari- For these the of fungi is based on aspects of their ous naturalists' minds when they fungi, sexual reproductive cycle. Thus, as- speak of species; in some, resem- the ed- blance and should comycetes (including highly is everything descent of species concept ible truffles and morels as well as little weight-in some, resemblance seems to for and Cre- be grounded in a Neurospora, widely used in genetic go nothing, meiotic in a ation the reigning idea-In some, studies) produce spores descentis the key,-in some, steril- demonstration of sac called an ascus. Basidiomycetes ity an unfailingtest, with othersit is (the mushrooms and their relatives) not worth a farthing.It all comes, I phenetic cohesiveness, produce meiotic products from a believe, from trying to define the common microscopic club-shaped structure indefinable.(Letter from Darwinto evolutionary called a basidium. The zygomycetes Hooker, 24 December 1856; Dar- (black bread molds) form sexual win descent, and reproductive 1887, p. 88) spores as an elaborately ornamented isolation where possible zygosporangium. Fungi that do not is the processby which reproduce sexually are placed in an Speciationa cohesive of genetically group artificial group, the Deuteromycetes individuals di- interbreeding to whether two groups are different ("imperfect fungi"). In discussing into two or more verges genetically species or just locally adapted but species concepts and speciation pro- distinct of individuals. A groups req- interbreeding groups within a spe- cesses in this article, we emphasize uisite in this is a hiatus in process cies (Figure 1). Because speciation is the basidiomycete fungi. between the two gene exchange a process that occurs over time, there This hiatus allows emerging groups. is uncertainty concerning the exact accumulate Speciesconcepts in fungi each group to genetic point in this process when a popula- differences the natural through pro- tion becomes a species. "Species concepts" are the philo- cesses of muta- evolution, including Fungi pose special problems in sophical criteria through which and drift. When tion, selection, genetic determining species status because investigators communicate their defi- two groups have become sufficiently little is known about what consti- nition of the term "species." Con- are as different, they recognized spe- tutes an individual; the extent of versely, species concepts are not the cies. In fruit- many cases, however, variation within populations is taxonomic characters used to distin- differences are body phenotypic largely unexamined; fungi often have guish a particular species from other subtle and there is as disagreement complex, varied life cycles, each stage species. Implicit in most species con- Ronald H. Petersen(e-mail: repete@utk. of which may be subject to different cepts is the idea that two species are edu) and Karen W. Hughes (e-mail: evolutionary pressures; and repro- genetically distinct from each other [email protected]) are professors of duction (sexual and asexual) is ex- and are reproductively isolated, ei- at the of Botany University Tennessee, tremely complex and may affect evo- ther by intrinsic reproductive barri- Knoxville, TN 37996-1100. After many in that we ers or of Petersenhas lutionary patterns ways (i.e., genetic incompatibility) years morphotaxonomy, do not understand. For extrinsic factors, such as ecology investigated mating systems in mush- yet many by rooms for over a decade. is a fungi, the sexual stage of their life of the organism or geographical sepa- Hughes of geneticist with interests in the evolution cycle is unknown and, in fact, may ration. Each group organisms for and biogeography of populations. ? not exist. Many species of Penicil- poses special problems investiga- 1999 American Institute of Biological lium, from which penicillin is de- tors, and species concepts that have Sciences. rived, are amorig such fungi. been developed for one group of or-

440 BioScience Vol. 49 No. 6

University of California Press is collaborating with JSTOR to digitize, preserve, and extend access to BioScience ® www.jstor.org ganisms may not be workable for another group, although numerous Sexual attempts have been made to find A species concepts that are broadly Species applicable across kingdoms (Carson incompatibitySpecies 1985, Mayden 1997). Numerous species concepts exist, all of which 9 have their proponents and detrac- A tors (see Mayden 1997). The three Subspecies Subspecies B species concepts that are most appli- aI cable to fungi are the "morphological" species concept (i.e., differences in morphology reflect underlying RaceHRace~or A orRcB im- genetic differences and can thus Sibling Species A B ply a genetic hiatus between two Sibling Species groups), the "phylogenetic" species concept (i.e., populations may be grouped and ranked as species only Interbreedingpopulation if they share a common evolutionary lineage, usually expressed as a terminal node on a phylogenetic tree), and Genetic Divergence - the "biological" species concept (i.e., 0 the ability of individuals in two groups to interbreed). Figure 1. Interbreedingpopulations may diverge genetically over time until speciation occurs. Sexual occur to The incompatibility may quickly, leading sibling species morphological species concept. with little genetic divergence; may accompany the gradual genetic divergence; or In this concept, characters (pheno- may not develop, even between populations judged sufficiently divergent to be types)of individualorganisms are com- designated species. pared,and similarindividuals are designated as a species. Inherentin this construction is an assumed genetic are appropriateand then lasting only used-is the primarycriterion defin- hiatusbetween dissimilarorganisms. a short time. The result of these prob- inga species(Mayden 1997). In prac- Decisions about similarity and dis- lems has been an intuitive classifica- tice, however,these groupsmay or similarity of characters, of course, tion, with each taxonomist grouping maynot reflectthe evolutionaryhis- are left to the taxonomist. Tradi- and ranking specimens in his or her tory of the species. tionally, characters used to identify own way. mushrooms and their relatives have Other characters have recently The phylogeneticspecies concept. been taken from the macro- and mi- been added to the repertoireof traits The phylogenetic species concept cromorphology of the basidioma (i.e., used to estimate species status of (Hennig1966), by contrast,requires the fruitingbody or mushroom).It is fungi. Fungi are often potent pro- that speciesrepresent monophyletic little wonder, then, that mushroom ducers of secondary metabolites (in- groups(i.e., the productof a single systematicshas been informedby the deed, many drugs and antibiotics are evolutionarylineage or the terminal morphological species concept (see derived from fungi). Analysis of sec- taxa on a cladogramestimating an Smith 1968, Clemenqon1977). ondary metabolites has identified evolutionarylineage). Although cla- The use of morphology to deter- relationships between morphologi- dogramsare often DNA based,they minespecies boundariesin fungi has, cally divergent groups of fungi (Gill can be developedfrom any available however, proven inadequatefor sev- and Steglich 1987, Whalley and data, includingmorphological data. eral reasons: all charactersare based Edwards 1987). In addition, electro- Most modernphylogenetic species on only one part of the life cycle, the phoretic analysis of isozymes is now conceptssupport the idea that spe- fruitbody; the mushroom fruitbody being used to evaluate population cies lie on the boundarybetween is relatively simple, so limited char- structure, as are some DNA-based interbreedingpopulations and repro- acters are available for separating techniques, such as restriction frag- ductivelyisolated, genetically diver- species; fungi of divergent evolution- ment length polymorphism (RFLP) gent taxa (Davis 1996). In this re- ary lineages may share similar mor- analysis. When such additional char- spect phylogeneticand biological phological characters as a conse- acters are treated as indicators of species concepts agree, but in the quence of convergent evolution; and, genetic differences(in much the same phylogeneticspecies concept the ter- finally, the breadth of infrataxon way that morphological characters minaltaxa may or maynot retainthe phenotypic plasticity in mushrooms have been used), the species concept ability to interbreed.The phyloge- is largely unknown. In part, this in- is termed the "phenetic species con- netic approachto conceptualizing adequacy arises because most fungal cept." In this concept, overall phe- "species"has been criticized because fruitbodies are ephemeral, forming notypic similarity-including mor- it mayrank well-defined populations only when environmental conditions phology and any other characters as species and because it fails to

June 1999 441 recognize that taxa can be para- Species terminology. Existing termi- of the mushroomArmillaria bulbosa phyletic (Brummitt 1997). Neverthe- nology for fungal infraspecific ranks covered several hectares, with all less, in basidiomycete systematics, is complex because it is derived from fruitbodies belonging to the same some investigators argue for a phy- fungal genetics and studies with fun- individual (Smith et al. 1992). In logenetic species concept. Hibbett et gal pathogens. Brasier and Rayner contrast, we were able to identify al. (1995), for example, argued for (1987) have examined terminology five different individuals of the coral recognition of morphologically dis- and concepts associated with infra- fungus Clavicoronapyxidata within tinct but interfertile lineages of the specific groupings and have suggested a 10 m2 plot, some represented by shiitake mushroom as a means of simplified terminology. In their more than one fruitbody. The largest understanding character evolution, scheme, the primary hierarchical sub- individual of Amanita francheti in a biogeography, and distribution of divisions are races and subspecies, 300 m2 plot spanned 25 m2, but for genetic variation. with or without associated repro- Suilluspungens, the largestindividual ductive isolation. Additional quali- spanned 300 m2 (Brunset al. 1998). The biological species concept. The fying terms would describe the na- Kay and Vilgalys (1992) found indi- biological species concept recognizes ture of the subdivision (e.g., serotype viduals of Pleurotus ostreatus to be reproductive isolation as the critical and pathotype). The term "sibling quite small, often with more than element in speciation. As Mayr species" is reserved for morphologi- one individualoccupying a singlepiece (1942), Dobzhansky (1951), and oth- cally similar or identical populations. of rottenwood. Forexperimental pur- ers presented this species concept, all Sibling species are ranked below true poses, however,it is likelythat collec- other character suites were subordi- species in this hierarchical system. tions (or mycelia)separated by several nated to the ability (or potential abil- miles representdifferent individuals; in studies that ity) to interbreed. Thus, this spe- Mushroom biology many showing widely cies concept, if two populations are separated individuals have different able to interbreed, then, regardless Fungal life forms are unique in many mating-typealleles confirm this as- of other variation (i.e., morphologi- ways, and comprehending their biol- sumption. cal, physiological, ecological, molecu- ogy is important to understanding lar), they constitute a shared gene pool speciation patterns and processes. The mushroom life cycle. As tradi- and belong to one (biological) species. Among the important features of tionally presented,the mushroomlife The biological species concept has fungi are the identification of a fun- cycle begins at the sexual spore stage met with criticisms that are aimed gal individual, the complexity of the (Figure2A). These spores, borne on mainly at the primacy of the inter- mushroom life cycle, the importance specialized organs termed basidia, breeding requirement (Donoghue of clonal reproduction, and the struc- are called basidiospores and are nor- 1985, Bock 1986, Templeton 1989). ture of fungal populations. mally the result of meiosis, either This concept is perhaps more appli- directly or with one of several varia- cable to animals than to plants, for The mushroom individual. Just as tions in pre- or post-meiotic nuclear which good morphological species species comprise populations, so behavior. Whereas basidiospores of exist in nature that still retain an populations comprise individuals. saprophytic mushrooms (i.e., mush- ability to interbreed. The genetic hia- Species concepts in mushrooms rest, rooms that obtain nutrition by de- tus between two such plant species in part, on identifying individuals grading nonliving organic material) may be due to a variety of factors, within a "group." This identifica- are usually easily germinatedon un- including geographic separation, tion is the first step in evaluating defined agar media, basidiosporesof ecology, pollinator preferences, and whether a genetic hiatus exists be- ectomycorrhizal mushrooms (i.e., post-reproductive barriers, such as tween two groups and, therefore, mushroomsin a symbiotic relation- seed inviability. whether these groups can be classi- ship with plant roots) are notori- In determining species boundaries fied as populations, races, or species. ously more difficult to germinate, in mushrooms, all available data There is usually little doubt over although some progress has been ought to be considered, but as we what is meant by an individual mite, made (Fries 1978). Under appropri- show in this article, mating studies redwood tree, or bacterium, even ate conditions, basidiosporesimbibe have proven among the most en- when considering clonal reproduc- water, autolyse their spore wall, and lightening (e.g., Boidin 1986, Stenlid tion, but fungal individuals are less extrude a "germ tube" (Figure 2B) and Karlsson 1991, Vilgalys 1991, discrete (Anderson and Kohn 1995). that soon begins to produce exog- Petersen and Hughes 1993, 1998, With the filamentous vegetative or- enous enzymes to digest the sur- Petersen 1995a, 1995b, Hallenberg ganism existing within an opaque rounding substrate and elongates into et al. 1996). Mushrooms have mat- substrate (e.g., wood or soil) and a hypha (i.e., a single thallus fila- ing systems that are designed to en- thus hidden from view, there can ment). The hypha in turn grows and courage outbreeding, and strong re- only be conjecture about whether all branches into an aggregate of hy- productive barriers often accompany the mushroom fruitbodies of one phae called a mycelium (Figure 2C). differentiation into species. Thus, the species within a few feet of one an- The mycelium that results from the biological species concept (Mayr other are being produced by a single germination of a single basidiospore 1942) provides a good starting point mycelium or by more than one. For perpetuates the nuclear number and for a philosophical species concept example, nuclear and mitochondrial mating type (i.e., equating to "sex," in mushrooms. markers showed that an individual for the purposes of this article) of the

442 BioScience Vol. 49 No. 6 L

2n n

H77

F "

Figure2. Mushroomlife cycle. n, haploidmonokaryon state ("haplont");n + n, dikaryonstate; 2n, diplont state. Basidiospores(A) undergogermination (B); germ tubes branchinto germlingmycelium (C). Monokaryonhyphae anastomose (D) to producedikaryon hyphae (E) with clamp connections.Monokaryon hyphae can produce asexual spores (F); dikaryonhyphae can produceasexual sporeswith clamp connections(G). Under appropriateconditions, the dikaryoticmycelium produces mushroom basidiomata (H; the individualon the righthas upturnedpileus, revealing lamellae). A cross-sectionof the lamella(I) shows sterilecells (cystidia)and basidia.Nuclear fusion occurswithin the young basidia(J; the lower basidiumhas two nuclei, and in the upperbasidium the nuclei havefused). Meiosis takesplace in the basidialinitial (K),ultimately resulting in a maturebasidium with sterigmataand four haploid monokaryonbasidiospores (L). basidiospore. system is termed "tetrapolar" (or "bipolar" or "unifactorial"). (For a The most common sexual system "bifactorial," for the two mating- summary of the molecular basis for in mushrooms is governed by two type genes). A minority of mush- mating-type genes, see Casselton and unlinked mating-type genes, each rooms, by contrast, have only one Kiies 1994.) In bifactorial mating with multiple allelic forms. Such a mating-type gene (a system called systems, individuals with different

443 June 1999 alleles at both loci will mate. The annelids; and symbiont availability. to form individual monokaryotic presence of multiple alleles for mat- Conventional wisdom, based pre- mycelial colonies. Monokaryon in- ing type within a species encourages sumably on in vitro experience, ocula of the same putative morpho- outcrossing at the expense of self- says that monokaryon mycelia are species are paired (i.e., placed near crossing between individuals in a more fragile or ephemeral than each other on nutrient agar) and population and thus acts in opposi- dikaryotic mycelia of the same "spe- allowed to grow together. The con- tion to evolutionaryforces that might cies" and, thus, that dikaryotic tis- tact zone is then examined for the fragment a population. sue has a selective advantage in a presence of viable dikaryotic hyphae, The mycelium from a typical ba- given substrate. which are indicative of mating (Fig- sidiospore is often termed a "mono- It is the dikaryotic mycelium that, ure 3b). karyon," which reflects the single under appropriate physiological and nucleus per hyphal compartment biochemical conditions, produces Clonal reproduction. Clonal repro- (usually called a cell, although nar- hyphal ganglia and subsequent basi- duction occurs commonly in fungi. row holes in the transverse septa of diome primordia and mushrooms Asexual spores give the mushroom the hypha allow cytoplasmic conti- (Figure 2H). The mushroom is nor- mycelium a means of dissemination nuity and even nuclear migration). mally composed of dikaryotic hy- separate from the sexual life cycle The mycelium at this stage is typi- phae that differentiate into cap with its meiotic basidiospores cally haploid (aswas its meiotic basid- (pileus), gills (lamellae), and stem (Anderson and Kohn 1995). In fact, iospore;Figure 2C). Thismonokaryon (stipe) tissues. In mushrooms and mushroom production is unneces- (haploid)mycelium, if isolated at this their relatives, any one or a combi- sary for dissemination (although ben- time, acts as a gametic generation nation of these tissues can be re- eficial for gene recombination) if a and can be used in mating experi- duced or missing, but ordinarily the clonal state is adequate for perpetu- ments of various types: among sib- basidiospore-producing tissue, the ation of the organism. Clonal repro- ling isolates from a single fruitbody, "hymenium," covers the surface of duction has important evolutionary among individuals from different the lamellae (Figure 21) and is com- implications. If the monokaryon hy- populations of a given species, or posed of microscopic hyphal tips in a pha forms asexual spores, the myce- between individualsclassified as dif- palisade. These hyphal tips often lium has a means of extending and ferent morphological species within comprise sterile elements, called widening its availability to an ap- a genus. cystidia, and "fertile," club-shaped propriate mate. This reproductive Although the mechanistic details basidia. Within the basidia, karyo- shortcut might be an advantage when of mating systems in mushrooms gamy, or nuclear fusion, occurs (i.e., a fungus is colonizing new territory. continue to be elucidated, it is clear the paired haploid nuclei of the If the dikaryon hypha forms asexual that two monokaryon hyphae of dikaryon fuse into a diploid (Figure spores, then the dikaryon genotype sexuallycompatible mating types can 2J); fusion is then followed by meio- is perpetuated unchanged (Ander- anastomose and proceed through sis (Figure 2K). During late meiosis, son and Kohn 1995). This means of plasmogamy (Figure 2D). In occa- the basidia produce narrowly curved dissemination could be an advan- sional groups (e.g., Armillaria), conical outgrowths called sterigmata. tage in perpetuating rare gene com- karyogamy soon follows to produce Four sterigmata typically form per binations that allow colonization of a diploid mycelium, but, typically, basidium, reflecting the four prod- new ecological niches; it also has donor nuclei pair but do not fuse. ucts of meiosis. The tips of the sterig- implications for speciation processes. Nuclear division, followed by nuclear mata are "blown out" into spores It is no surprise, therefore, that many migration, may take place; the result (Figure 2L) into which the post-mei- fungi produce asexual spores through will be a proliferating "dikaryon" otic nuclei migrate; the spores are a variety of unusual and unique pro- (from the two nuclei per cell; Figures subsequently ejected forcibly from cesses. Moreover, the mechanisms 2E and 3a). The dikaryotic condi- the sterigmata. The basidiospores of their production provide phenetic tion is accomplished by the forma- (Figure 2A), which are also techni- information that can be used to group tion of "clamp connections" at the cally known as "meiospores" because fungi into taxonomic units. transverse septa in the majority of they are the result of meiosis, complete Asexual propagules are termed mushroom taxa (Figure 3a). These the sexual life cycle. In rare cases, "conidia," with additional technical hyphal connections allow nuclei to segregation of nuclei into spores may terms used for particular modes of migrate between two cells, maintain- vary within a single morphospecies. production. In mushrooms, conidia ing the dikaryotic condition. Di- For example, Mycena and Trogia are typically the result of fragmenta- karyotic mycelia are assumed to be species occur in two- and four-spored tion of a hypha; therefore, they are the major vegetative unit of the indi- forms, with two nuclei (n + n) or one called "arthroconidia" or "arthro- vidual mushroom fungus. nucleus (n), respectively, per basid- spores." Such asexual spores are The growth of dikaryon mycelia iospore (Kihhnerand Lamoure 1958, rarely produced as part of the mush- appears to be limited only by a vari- Corner 1991). This nuclear varia- room itself but, when produced, are ety of micro- and macro-ecological tion would presumably be reflected almost always formed by the vegeta- factors, such as appropriate sub- in the nuclear condition of the single- tive mycelium, whether monokaryon strate or host availability; substrate basidiospore mycelium. (Figure 2F) or dikaryon (Figure 2G). moisture; substrate preparation by For mating studies, single basid- Because the vegetative mycelium is other organisms, from bacteria to iospores are collected and germinated not normally seen in nature (being

444 BioScience Vol. 49 No. 6 immersedin the woody, leafy, or soil anamorphicmorphology is extremely which gene flow can occur between substrate, and microscopic as well), varied, and the anamorph is often populations:through asexual hyphal asexual spore production is usually found (i.e., isolated in culture) with- fusion and through spore produc- discoveredonly when the mushroom out the teleomorph. Many workers tion. In many species, hyphal fusion is cultured in vitro. As more and have spent many years using conven- is prevented by a genetic system of moremushroom species are cultured, tional means (e.g., light and electron vegetative incompatibility, which the number of species in which microscopy or growth media en- acts to protect an individual geno- asexual reproductionis known to oc- hancement) to determine which type (Carlile 1987, Worrall 1997); cur continuesto grow. Certainmush- anamorphs and which teleomorphs thus, the primarymechanism of gene room groups (e.g., Flammulina, represent alternate stages of a single flow between populations is sexual Collybia sensu stricto, Melanotus, species.Newer techniques(e.g., DNA and asexual spore production. There Psathyrella, and the related genus sequencing and fingerprinting)hold has been considerable debate about Polyporus) are well known for the the probabilityof confirmingor eluci- long-distancespore dispersalin fungi productionof asexualspores, but oth- dating these anamorph-teleomorph (Vilgalys and Sun 1994b), but iso- ers are only now being discovered. relationships. Conversely, in other zyme and molecular evidence from In some mushroom groups, much fungi-mushrooms and their rela- many mushroom disjunct popula- of the fruitbody (basidiome) is con- tives-asexual reproductionhas been tions suggests that long-distance verted into asexual propagules. The overlooked for taxonomic character spore dispersal does not occur at a most famous is the genus Astero- suites. Most of the known anamor- rate sufficient to prevent population phora, whose basidiomata form on phic stages have not even received divergence (Gordon and Petersen basidiomataof other mushroomsand "form taxon" status or rank. 1997, Methven et al. 1997). When undergo an almost complete conver- Needless to say, an understanding gene flow cannot be measured di- sion to thick-walled conidia (often of the entire life cycle of each mush- rectly, divergenceis inferredby find- termed"chlamydospores"; McMeekin room taxon is invaluable before one ing reproductivelyisolated subgroups 1991). Species of the parasitic genus can be confident about its taxonomy (i.e., biological species)within a mor- Squamanita cause distorted growth and phylogeneticplacement. Conidia phological species or well-differenti- of their mushroom host and produce can be used in pairing experiments, ated morphologicalsubgroups within asexual spores on the surface of the just like single-basidiospore isolates a biological species. host carcass and on their own stipes can. Thus, in addition to perpetuat- (Redheadet al. 1994). In Flammulina ing the organism asexually, appro- Morphologicalspecies versus stratosa, a newly identified species priate single-conidial mycelia can species:Examples from New Zealand, abortive fruit- form dikaryons and reinitiate the biological bodies also convert into small balls sexual life cycle. A number of recent studies allow of brown, thick-walled chlamydo- morphological species concepts in spores (Redhead et al. 1998). DNA The mushroom population. Locally fungi to be comparedwith biological sequence data show that F. stratosa adapted populations are the first species concepts. The use of a bio- is highly divergent from all other stage in divergenceleading to specia- logical species concept must be tem- Flammulina species that we have tion. Although several studies have pered by the large number of mush- examined. Its restricted island habi- examined population structure in room species that have not yet been tat and genetic divergence suggest plant pathogenic fungi (Anderson established in culture, much less in that chlamydosporeproduction may and Kohn 1995 and references monokaryon culture. Ectomycor- have contributed to invasion of the therein), only a few have attempted rhizalmushroom taxa, includingsuch New Zealandisland habitat and rapid to characterizethe relationships be- large genera as Cortinarius, Lac- adaptive evolution, a possibility that tween individual mushrooms and tarius, and Russula, are notorious we are currentlyinvestigating. their populations in any detail (Kay for resisting domestication. Further- Clonal reproduction can be ac- and Vilgalys 1992, Smithet al. 1994, more, even if two fungi are able to complished in at least one other way. Anderson and Kohn 1995, Kerrigan mate in culture, there may be barri- Certainstrains of Volvariellabomby- et al. 1995, Gardesand Bruns 1996). ers in nature to mating or to the cina (Chiu 1993) and Xerula radicata The paucity of such studies is due in survival of hybrid offspring (Brasier (Petersen and Methven 1994) form part to the difficulty of collecting a 1987). Mating studies, however, pro- fruitbodies composed of mono- sufficient sample size in an organism vide some "ground truth" in that if karyon hyphae. Basidia do not un- that produces fruitbodies ephemer- two taxa can mate, they are geneti- dergo meiosis and therefore produce ally and in part to the complexity of cally related. If they cannot mate, spores of only a single mating type. the mushroom individual. they are genetically separate species. Asexually reproducing stages are Gene flow, or the movement of termed the "anamorph"; the mei- genes between populations, deter- Biological species within morpho- otic, sexual stage is termed the mines if and how fast populations logical species. The "classic" case of "teleomorph"; and the entire life can evolve into separate species using crossing studies to identify spe- cycle, with all possible stages, is (Carlile 1987). High gene flow fa- cies can be found in studies of termed the "holomorph." vors population coherence, whereas Armillaria, the honey mushroom. For In fungal groups in which teleo- low gene flow favors population di- many years, fastidious taxonomists morphs form ascospores (ascomycetes), vergence. There are two methods by used small differences in basidiome

June 1999 445 characters to describe "species" of species, found mostly west of the the southern Appalachian taxon Armillaria. As might be expected, Mississippi and usually fruiting on could be linked to Quercus (oak) opinions about such practices dif- aspen (Populus spp.) was named litter, whereas the more widespread fered with the philosophy and taxo- Pleurotus populinus. Furthermore, northern collections seem to prefer nomic experience of the user-in the biological species P. ostreatus conifer debris as a fruiting substrate short, whether the user was a exhibits geographical morphologi- (Gordon and Petersen 1997). "lumper" or a "splitter." There was, cal variation. Whereas European P. The relationship between Ompha- of course, no overarching authority ostreatus basidiomata usually have lotus illudens and Omphalotus to render binding judgments. Over gray, charcoal gray, blue gray, or subilludens is another example of time, however, most mushroom tax- dark gray-black caps (Figure 4e), diverging biological species. O. onomists agreed that there was only those from eastern North America illudens (called the "Jack-O-Lantern one species of Armillaria, A. mellea, are off-white to tan to brown. So far, mushroom" because its basidiomata but that it varied morphologically, the only American collections of P. are brilliant orange and biolumines- depending on substrate, geographic ostreatus exhibiting European col- cent; Figure 4c) is distributed in the location, and other factors. ors have been made from woody eastern United States from Georgia In the 1970s, however, the mating Lupinus shrubs on the northern Cali- northward and is commonly collected system of A. "mellea" was eluci- fornia coast. on hardwood stumps in the southern dated, paving the way for inter-col- In both the Armillaria and Pleuro- Appalachians. O. subilludens, which lection crossing experiments-and, tus examples, once mating studies fruits on dead palmetto in Florida, is therefore, for other crossing experi- pointed toward enlarged species only doubtfully separable from O. ments, such as inter-substrate and numbers, delineation of the appro- illudens based on morphology. inter-geographic (Hintikka 1973, priate taxa was relatively straight- Analysis of the internal transcribed Ullrich and Anderson 1978). The forward. Mating studies can also spacer (ITS) region of the rRNA gene first revelation was that there were reveal the presence of "sibling spe- by restriction analysis gave an esti- at least five "biological species" of cies" (i.e., populations whose basidi- mated sequence difference of 7.35% Armillaria in Scandinavia (Korhonen ome morphology appears identical (Hughes and Petersen 1998). This 1978); intercollection crossing ex- but that sort into two or more "bio- level of divergence is high-higher periments revealed at least 10 such logical species"). One such situation than that observed between the geo- "intersterility groups" in northeast- has been discovered in Xerompha- graphically separated morphologi- ern North America (Korhonen 1978, lina campanella (Johnson 1997). cal species Omphalotus olearius from Anderson and Ullrich 1979). With Over 80 cultured collections of X. Europe and Omphalotus olivascens discovery of this genetic complexity, "campanella" from all over the North from California (for comparison, mycologists re-examined Armillaria Temperate Zone were found to fall European and eastern North Ameri- morphology and ecology. In the fol- into two intersterility groups. Mush- can P. pulmonarius collections dif- lowing years, each "biological spe- rooms of the two groups differed fer by 0.58% and collections of P. cies" was matched to its basidiomata morphologically only in the pigmen- ostreatus by 1.41%; data from Vil- and substrate (Guillaumin et al. tation of individual sterile cells on galys 1994a). Crossing studies be- 1991). The majority of intersterility the stipe. A similar situation occurs tween O. illudens and O. subilludens groups were found to represent mor- in Lentinula, the genus of the popu- showed a reduced ability to inter- phological species that had previ- lar shiitake mushrooms. Until re- breed (only 4 of 24 crosses produced ously been named but were histori- cently, the common species of sub- hybrid mycelia in vitro; Petersen and cally overlooked. Current studies tropical America was known as Hughes 1998). The combination of involve the molecular characteriza- Lentinula boryana, but recent stud- reduced ability to intercross in vitro, tion of these genetic-taxonomic units ies (Petersen et al. 1998) show that different substrate preference, and and their placement on a phylogeny another species with almost identi- sequence divergence strongly sug- (Anderson and Stasovski 1992). cal basidiomata fruits along the Gulf gests that these species do not ex- Much the same story applies to Coast from east Texas to central change genes in nature despite the Pleurotus, the genus of "oyster mush- Florida and in Puerto Rico and Cuba potential to do so. In this case, there- rooms" now popular in supermar- in the Caribbean. This species (now fore, both mating studies and mo- kets. For many years, most oyster called Lentinula raphanica) is sexu- lecular analysis were keys to under- mushrooms were identified as Pleuro- ally incompatible with collections of standing that O. illudens and O. tus ostreatus (usually without refer- L. boryana from Costa Rica and subilludens are different species, de- ence to keys or descriptions), which tropical Mexico. The most striking spite morphological similarities. was assumed to fruit throughout the difference between the two species is Numerous other examples of bio- North Temperate Zone. Vilgalys and the shape of the sterile cells that logical species within morphospecies coworkers (1993), however, identi- form on the edge of the lamellae. are presented by Brasier (1987) for fied three "intersterility groups" (i.e., Finally, mating tests with Marasmius all fungal groups, not just mush- biological species) in North America androsaceus clearly reveal two taxa, rooms. Brasier classified the pro- by mating haploid monokaryon iso- one in Europe and northeastern cesses leading to divergence as geo- lates. Two of these already bore names: North America, the other from the graphical (i.e., allopatric), as P. ostreatus and Pleurotus pulmo- southern Appalachian Mountains. sympatric speciation in response to narius. A previously undescribed From voucher herbarium specimens, ecological influence, and as sympat-

446 BioScience Vol. 49 No. 6 ric speciation with unknown cause. cantly sexually intercompatible. Simi- For some groups, two causes could larly, using morphology, isozyme be identified. Of the examples given analyses, and mating compatibility, by Brasier (1987), only 5 were geo- McCleneghan (1996) was able to jus- graphical, 33 were ecological, and tify only a single species within the 11 were probable sympatric specia- Pholiota alnicola complex (Figure 4d), tion with unknown cause. in which seven taxa had originally Such examples are satisfying in nuclei. been described (Smith and Hesler that they reveal the underlying popu- 1968). Similar studies with the lation structure and genetics of spe- Pholiota spumosa complex reduced ciation the number of from 11 to 2. processes. However, descrip- ii species tions of "new taxa" that are based Chang and Mills (1992) showed that on behavior or without mating (with clam several morphotaxa in the Psilocybe small differences in morphology) do subaeruginosa complex could be sub- not make the tasks of identifying sumed under significantly fewer bio- species and estimating biodiversity logical taxa, and that enzyme electro- easier for the "clients" of taxonomy phoretic patterns were consistent and (e.g., foresters, plant pathologists, and confirmatory with this conclusion. ecologists). It may well be that, for the purposes of assessing biodiversity, spe- Allopatricspeciation: Physical cies concepts based on distinct mor- barriersto phological characters will be preferred. interbreeding For many mushroom species, popu- Morphological variability within bio- lations on different continents or logical species. Crossing studies can, separated by other geographical bar- as already described, lead to more riers may be completely interfertile. accurate taxonomic conclusions by John Raper, one of the early promot- increasing the number of recognized Figure3. Fungalhyphae and typical pair- ers of the biological species concept in other ing experiment.(a) Basidiomycetehy- in showed that world- species. However, situations, connection and mushrooms, crossing studies have led to decreased pha showing clamp wide collections of dikaryoncondition. 1000x magnifica- Schizophyllum numbers of putative taxa. tion. with commune were intercompatible in For (b) Pairing experiments example, Pleurotus diamor monokaryonmycelium. Contact zone spite of morphological variation fruits along the Gulf Coast into myceliumis morecongested than donor (Raper and Flexer 1971). Collections Florida and extends somewhat up mycelium. of Auriscalpiumvulgare (tooth fun- the Atlantic Coastal Plain. It also gus) from Asia, North and Central fruits throughout eastern Asia and nature were damped when fruiting America, and Scandinavia were all Southeast Asia and in parts of New took place under more or less stan- intercompatible, with minimal mor- Zealand, southern Australia, and dard conditions. This result indicates phological variation of the fruitbody subtropical North, Central, and that some substrate or edaphic fac- and spores (Petersen 1994). World- South America. It has also been re- tors may maintain the natural vari- wide collections of Panellus stypticus ported from super- and sub-Saharan ants. Crossing experiments showed (Figure 4b) were found to be com- Africa. Basidiomata occur in several that the natural color and texture patible (Macrae 1937, Petersen and rather distinct morphological stat- forms result from nongenetic pheno- Bermudes 1992a, 1992b), as were ures and colors. Caps vary from typic plasticity and are not indica- collections of Clavicorona pyxidata bright pink (Figure Sa) to off-white tive of discrete genetic units. (coral fungus; Figure 4f) from China, (Figure 5b) to sallow olive to tan McCleneghan and Hughes (1998) Scandinavia, and eastern North (Figure Sc) and from smooth to rather further confirmed the high genetic America (Wu et al. 1995) and world- hairy. These color variants and tex- similarity of the color variants by wide collections of P. pulmonarius tures were previously given separate RFLP mapping of the rRNA ITS re- (Petersen and Hughes 1993, Petersen species names. Names such as gion. As might be expected from the 1995b). In Omphalotus, as discussed "Pleurotus opuntiae" refer to the Armillaria experience, at least one above, the morphological species O. New Zealand grey fibrillose form, taxonomist (Corner 1981) had cor- olearius from Europe, O. subilludens "Pleurotus salmoneostramineus" to rectly described most of the variants from Florida, and O. olivascens from the pink northeast Asian basidio- as infra-specific units within P. California were completely interfer- mata, and "Plerotus ostreatoroseus" djamor as it fruited in Southeast Asia. tile. In all of these cases, morphologi- to the rosy fruitbodies from tropical Another example of decreased cal differentiation based on geo- America. taxon number comes from studies of graphically disjunct separation could By fruiting cultures of upward of Collybia by Vilgalys (1991), who be concluded. 60 collections from all the locations showed that even though European When fungi from different conti- where P. djamor fruits except Af- and eastern North American basidi- nents are able to interbreed in vitro, rica, Nicholl (1996) found that the omata of C. dryophila exhibit differ- three explanations are possible: long- color and texture variations noted in ent morphologies, they are signifi- distance distribution of viable spores

June 1999 447 For example, Flammulina velutipes (Figure 4a) has a pan-North Tem- perate distribution, and collections from all geographical areas are interfertile (Ron Petersen and Karen Part of a `?AL Hughes, unpublished data). rRNA gene repeat was sequenced, and sequence differences were identi- IS fied. One set of sequence differences was uniquely European, whereas another set was uniquely North Ameri- can. Clearly, these two populations have not exchanged genes for a long time (Methven et al. 1997). P. pulmonarius and P. ostreatus .v:...... also show continental-specific se- differences and Sun ...... Zir...... quence (Vilgalys io 1994a, Petersen 1995b). And Chi- Alm- nese and North American populations of Clavicorona pyxidata differ ...... in rRNA gene sequences (Ed Lickey, University of Tennessee, personal communication) and in laccase (a lignin-degrading enzyme) electro- phoretic patterns (Wu et al. 1995). Panellus stypticus from New Zealand shows significant sequence divergence from European and North American P. stypticus populations (Jin et al. 1998) but easily mates with them (Petersen and Bermudes 1992a, 1992b). A further example of divergence: fruitbodies of P. stypticus are bioluminescent only in eastern North America and nowhere else in its worldwide distribution (Petersen and Bermudes 1992a). An interesting question concerns how long fungi on different continents have been separated without evolving barriers to gene exchange. Some information can be derived from biogeographical patterns in higher plants. Plant taxa with widely disjunct distributions in temperate Figure4. Basidiomataof mushroomsand relatedfungi. (a) Flammulinavelutipes, 0.5x regions of the Northern Hemisphere magnification.(b) Panellus stypticus, 0.8x magnification.(c) Omphalotus illudens, have long been noted (Gray 1846). 0.3x magnification.(d) Pholiota alnicola, 0.5x magnification.(e) Pleurotusostreatus These distributions are thought to from 0.2x Clavicorona 0.5x Europe, magnification.(f) pyxidata, magnification. have been derived from a widespread mixed deciduous forest in the early allows ongoing gene flow between For a numberof intercompatible to mid-Tertiary Period (Graham thecontinents, vectors mediate trans- taxa, thereis good evidencethat al- 1993 and references therein). Dur- fer of fungi from one geographical lopatric speciation is in progress. ing that time, a much warmer climate areato another,or no currentinter- Unique alleles are not shared be- allowed plants to spread northward, continentalgene flow occursbut relic tween intercontinentalpopulations, and plants migrated between Europe intercompatibilitypersists. In the last and even within continentsbarriers and North America via the now sun- case,populations on differentconti- suchas mountainranges can prevent dered landmass of Euramerica. Fol- nents may representancient distri- geneflow. Forthese taxa, therefore, lowing the Tertiary Period, the con- butions(i.e., distributionsthat were neitherlong-distance spore dispersal tinents separated farther and colder established before the continents nor vector-mediateddispersal ap- climates pushed plant species' ranges separated)and may be in the process pears to be an effective means of farther south. If fungi followed the of allopatricspeciation. sharinggenes betweenpopulations. same distribution pattern, then ex-

448 BioScience Vol. 49 No. 6 tant membersof one biologicalspe- Figure5. Basidiomataof Pleurotusdjamor. cieson two continentsmay have been (a) Pink form from Asia. (b) Off-white separatedsince the mid-TertiaryPe- pan-tropicalform. (c) Olive-tanform from riod but may not have yet evolved New Zealand. Approximately0.Sx mag- crossingbarriers, even in the face of nification. Photographs:David Nicholl. time and divergingmorphology. Evenwithin continents, there ap- that we have studied, there is no pearto be barriersto geneexchange evidenceof eitherlong-distance spore leadingto speciation.For example, dispersal or vector-mediateddis- the wet West Coast of the United persal. There are some examples, Statesis geographicallydiverse and however,of recenthuman-mediated isolated from the eastern United introductionof fungi into new geo- Statesby the Cascade Mountains and graphical areas. Because of these the Rocky Mountains. P. pulmo- examples,the occurrenceof a mush- nariusisolates from the West Coast room species far from its first re- havea seriesof uniquealleles that are ported range raises questions,not not sharedwith isolatesfrom east of onlyabout its accurateidentification, the Rocky Mountains (Petersen butalso about long-distance dispersal 1995b).McCleneghan (1996) found a versusvectored introduction. geneticallyisolated population of P. The"button mushroom" of super- spumosajust south of PugetSound, markets,Agaricus bisporus, is one notfar from the southernlimit of the suchhuman-mediated introduction. glacialice shield.Likewise, there are This species has been domesticated more species of Flammulinafrom for over a century,first in Europe northernCalifornia to BritishCo- (primarilyFrance), then in eastern lumbiathan in any otherpart of the North America,and, most recently, world (Ron Petersen and Karen in westernNorth America.In Cali- Hughes,unpublished data). Two rare fornia, Kerriganet al. (1995) were species of Xeromphalinaare also ableto distinguishtwo strainsbased found in the area (Johnson1997). on RFLPpatterns. One strain,usu- One is Xeromphalina orickiana, ally found fruiting in lawns, was whichfruits on redwood.The other, similar to the commercialstrains unnamedspecies has been found in originallydomesticated in Europe, only two locations: the Olympic whereasthe otherstrain, fruiting at Mountainsand southern coastal Brit- the marginsof native woods, was ish Columbia. unique and probably endemic to In the face of physical barriers California.Other populations of A. over long time periods, and even bisporusendemic to theUnited States morphologicaldifferentiation, why and Canadahave now been identi- is it that reproductivebarriers may fied, but thereis stronggenetic evi- not have evolvedbetween intercon- dence that isolates of A. bisporus tinentalpopulations? According to with the EuropeanRFLP patterns Mayr's(1942) model of allopatric enough that the mating system is have probablyescaped from com- speciation,two thingsmust happen retainedunchanged, even when mor- mercialproduction sources and from for speciationto occur:an isolated phological (and other) differentia- home compost piles (with current population must be restructured tion occurs. In essentiallyall cases dispersalby 18-wheelers). (throughmutation, selection, or ge- we have studied involving geneti- Similarly,collections of F. velu- neticrearrangement) so that it sur- callydivergent populations of a single tipes from Chile and Argentina,all vives in its new environment (see biological species, the populations sexuallycompatible with their North also Carson 1985), and reproductive. were isolated from each other by TemperateZone counterparts,have isolating mechanisms must evolve some vicarianceevent (e.g., inter- RFLPpatterns matching the RFLP that protect the reorganized gene continentalor intermountainsepa- patternsof Europeanisolates of F. pool. In Mayr's view, reproductive ration). When strong reproductive velutipes but not those of North isolating mechanisms "arise as an barriershave been observed,they Americanisolates (Methvenet al. incidental byproduct of genetic di- have, as discussedabove, occurred 1997). Presumably,when European vergence in isolated populations" underconditions of relativesympa- immigration and commercial inter- (Mayr 1970). Fungi, however, have try(e.g., sibling species of Xerompha- course with these Patagonian areas unique mating systems with strong lina) or between geneticallydiver- developed, along with the people and selection for outcrossing, and it may gent species. introduced trees came associated be that within geographical regions fungi. the gene pool is large enough and Distributionsthat suggest recent dis- Recently, Pleurotopsis longinqua selection for outcrossing strong persal.For many of thefungal groups was shown to be sexually inter-

June 1999 449 compatible throughout its dis- be unwise because it limits definition the authors of this article disagree. junctional range from Australia-New of species to phenetic analysis, even Ultimately, the determination of spe- Zealand and Chilean and Argentine when additional data are available. cies status depends on the best judg- Patagonia to the Pacific Northwest of We suggest that for the fungi, the ment of the experienced investigator North America. The Australian-New species concept should be based on a based on all available data. Zealand populations showed signifi- demonstration of phenetic cohesive- more basidiome common cantly morphological ness, evolutionary descent, Acknowledgments variation than those from other loca- and reproductive isolation where tions, but RFLP patterns and rRNA possible. The concept of using a va- We thank the following students and gene ITS sequences differed little, riety of species concepts within a alumni of our labs whose work is suggesting that the species may have group is not new (Mishler and cited herein: Scott Gordon, Ed been recently introduced into one or Donoghue 1992, Claridge et al. Lickey, Coleman McCleneghan, more of its locations (Petersen 1992, 1997), and it may be necessary to Laura McGhee (Threshold Scholar), Petersen and McCleneghan 1995, capture the complexity of speciation and David Nicholl. Photographs of Hughes et al. 1998). patterns in nature. the Pleurotus djamor group were In this article, we have argued taken by David Nicholl. The photo- Conclusions that mating studies have been espe- graph of Pleurotus ostreatus was cially helpful in elucidating specia- taken by Dr. I. Krisai-Greilhuber. Drs. Considerable time and effort has been tion patterns in the mushroom fungi, Scott Redhead and Andy Methven col- spent trying to codify species con- a point others have also noted laborated on the Flammulina research cepts that are "universal" (i.e., (Brasier 1997 and references therein). project. We also thank four anony- broadly applicable to all forms of It is now a "given" that various char- mous reviewers for their comments. life), without resolution (Hull 1997). acter suites (e.g., morphology, eco- Research was supported, in part, by Suggesting a universal species con- logical preference, physiology, bio- National Science Foundation grant cept for mushrooms is a particularly chemistry, and molecular biology) 95-21526 (PEET program) to R. H. difficult exercise. Most investigators change and diverge at different rates P. and by Hesler Endowment Fund would agree that a species should be with little predictability. Through awards to K. W. H. the result of an evolutionary process processes qf selection, it may be that and should represent a cohesive in- sexual recognition, compatibility, References cited terbreeding group that is reproduc- and interfertility are among the last tively isolated from other cohesive suites to diverge in allopatric situa- Anderson JB, Kohn LM. 1995. Clonality in interbreeding groups. The use of a tions, and that speciation is far from soilborne plant-pathogenic fungi. Annual in Review of Phytopathology 33: 369-391. morphological species concept may abrupt. Thus, our studies, when Anderson Stasovski E. 1992. Molecular be JB, hampered by an inadequate suite strong reproductive barriers are phylogeny of Northern Hemisphere spe- of morphological characters and a present among allopatric groups, cies of Armillaria. Mycologia 84: 505- considerable degree of convergent they are accompanied by phenotypic 516. evolution for mushroom and and differences that Anderson JB, Ullrich RC. 1979. 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