smriE SONOMA COUNTY HYPOGEOU s FUNGI

by Russell Kent Heblack

A thesis submitted to

Sonoma State College in partial fulfillment of the requirements for the degree of

fr.ASTER OF ARTS

in

Biology

J~~ph (. Powell, Chairman

ehrfs K. Xj'e-Idsen I

· Harry D'. Thier s I ii

SOME SONOMA COUNTY HYPOGEOUS FUNGI A thesis by Russell Kent Heblack

ABSTRACT Purpose of the study: None of the early mycologists who explored the Califor­ nia hypogeous fungal flora recorded the occurrence of these fungi in Sonoma County. This study sought to determine which species of hypogeous fungi are present in Sonoma County and with which woody plants they are associated. Procedure: Field research was done during the two years beginning in December 1975. The· method was to locate stands of woody plants and dig under them in search of hypogeous fungi. Once found, the fungi were identified and pre­ served. Notes were made regarding suspected mycorrhizal associates. Findings: Eighteen species of fungi were found. These fungi are representatives of two classes, Ascomycetes: Balsamia magnata, Genabea cerebriformis, Genea arenaria, Genea gardneri, Genea harknessii, Hydnobolites californicus, Hydnotrya ellipsospora, Tuber californicum, Tuber ~­ didum, and Basidiomycetes: Alpova fulvus, Alpova guerc­ icola, Gautieria graveolens, Gymnomyces ferruginascens, Hymenogaster parksii, Hysterangium se~arabile, Leuco­ gaster rubescens, ~elanogaster parksii, Melanogaster tuberiformis. Some collections, representatives of the genera Rhizopogon, Kartellia, llymenogaster, Balsamia, and Genea, were only identifiable to genera. These may represent undescribed species. Two Alpova species, Alpova fulvus Trappe & Heblack sp. nov. ined. and Alpova guercicola Trappe & Heblack sp. nov. ined., were new to science. All of the fungi found were new distribution records for Sonoma County, and some were found with new woody plant associates. Conclusions: This study has shown that the Sonoma County hypogeous fungal flora is diverse. Within this diverse flora, two iii

new species were found and described. It is suspected that additional undescribed species will be discovered by fUture researchers.

Chairperson: ..,- · ~ . ~ - ,, ,, Dr. tjose''H: · Powe11 M.A. Program: Biology Sonoma State College lV

ACKNOWLEDGEMENTS

I would like to give sincere thanks to my major professor, Dr. Joseph H. Powell, and my committee members, Drs. Chris K. Kjeldsen and Harry D. Thiers, for their unselfish effort throughout this project. I also wish to thank Mrs. Ellen Thiers for her help with the latin diagnoses, and Dr . .James M. Trappe for his help in identifying collections and the loan of herbarium specimens.

R.K.H. v

TABLE OF CONTENTS Page ABSTRACT ...... ii ACKNOWLEDGEMENTS ...... iv LIST OF FIGURES ...... vii LIST OF TABLES ...... viii INTRODUCTION ...... 1 LITERATURE REVIEW ...... 2 Evolution 2 Basidiomycete Phylogeny ...... 6 Ascomycete Phylogeny 8 Ascomycete Taxonomy ...... 9 Basidiomycete Taxonomy 10 Habitat ...... 12 Reproduction ...... 13 Nutrition ...... 15 Collection ...... 16 Spore Dispersal ...... 17 Culturing Attempts ...... 18 Reagents and Stains ...... 19 Economic Use ...... 20 METHODS AND MATERIALS ...... 22 Collection ...... 22 Field Notes ...... 22 Preservation ...... 23 vi

Page Reagents and Stains ...... 23 Microtechnique ...... 25 RESULTS ...... 26 Class Basidiomycetes ...... 26 Class Ascomycetes ...... 27 Probable Mycorrhizal Associates ...... 27 Keys to the Species of Fungi ...... 29 New Species Descriptions ...... JJ DISCUSSION ...... 37 Taxa Identification ...... 37 Probable Mycorrhizal Associates 40 CONCLUSIONS ...... 4J

SUMMARY e I e e e e e e e e e e e e e e e e e e a e e a e e e e a e e e e e e a e e e a e e e e e e 44 APPENDIX ...... 45 LITERATURE CITED ...... 47 vii

LIST OF FIGURES Figure Page 1. Hydnotrya ellipsospora: spores x 14JO .•...... 52 2. Genabea cerebriformis: spores x 1110 ...... 52 J. Genea arenaria: spores x 1180 ...... 53 4. Genea sp. (#43): spores x 1420 ...... 53 5. Genea gardneri: spores x 1320 ...... 54 6. Genea harknessii: spores x 1540 ...... 54 7. Balsamia magnata: spores x 1850 ...... 55 8. Balsamia sp. (#47): spores x 1350 ...... 55 9. Hydnobolites californicus: spores x 1550 ...... 56 10. Tuber californicum: spores x 1000 ...... 56 11. Tuber candidium: spores x 1000 ...... 57 12. Hysterangium separabile: spores x 1670 ...... 57 lJ. Gymnomyces ferruginascens: spores x 1570 ...... •. 58 14. Martellia sp. (#99): spores x 1500 ...... 58 15. Leucogaster rubescens: spores x 1420 ...... 59 16. Gautieria graveolens: spores x 1680 ...... 59 17. Hymenogaster parksii: spores x 2100 ...... 60 18. Hymenogaster sp. (#90): spores x 1440 ....•...... 60 1?. Rhizopogon sp. (#38): spores x 1300 ...... 61 20. Alpova fulvus: spores x 1560 ...... ••...... 61 21. Alpova guercicola: spores x 1240 ...... 62 22. l\Ielanogaster tuberiformis: spores x 2000 ...... 62 23. Melanogaster parksii: spores x 3100 ...... 63 viii

LIST OF TABLES

Table Page 1. Probable mycorrhizal associates ..•..••...... 42 1

INTRODUCTION

Truffles and false truffles, hypogeous members of Classes Ascomyceteae and Basidiomyceteae, are relatively little known fungi. Few mycologists collect them and even fewer are well acquainted with their taxonomy(Trappe, 1975). Early in this century there were several workers, centered around the S.F. Bay area, who were quite active in Alameda, Napa, Santa Clara, and Marin Counties. None of these workers (Harkness, 1899; Gilkey, 1916; Parks, 1921) recorded the occurrence of hypogeous fungi in Sonoma County, California. Though these workers reported the occurrence of many hypogeous fungi, they were often vague concerning the woody plant associates of the fungi. It is now known (Trappe, 1974} that most hypogeous rnacromycetes are probably rnycorrhizal. The purpose of this research was to discover which hypogeous fungi occur in Sonoma Co. and with which woody plants they are associated. The research consisted of sampling species of trees and shrubs for hypogeous macro­ mycete associates. This sampling was carried out during the two years beginning in December, 1975. This report demon­ strated the presence of hypogeous fungi in Sonoma Co., and the inventory will undoubtedly expand with further studies. Other workers are urged to continue the search in the county. 2

REVIEW OF THE LITERATURE

Evolution There is widespread agreement among mycologists (Thiers, 1971; Heim, 1971) that discussions on the evo­ lution of fungi are speculative, without fossil evidence, and rest on logic, not fact. All of the arguments concerning the origin of hypo­ geous Basidiomycetes are elaborated by workers who are primarily agaricologists like Singer (1962) who are trying to unravel the phylogeny of the Agaricales. These workers often mention the evolution of hypogeous forms because these fungi frequently occupy the spotlight in the argument as to whether evolution has proceeded from agaricoid to gastromycetaceous forms or vice versa. The central argument is agreed upon (Smith, 1971; Singer, 1971; Heim, 1971) and it is best elaborated by Singer (1962): "The three logically possible, and actually inportant theories of today, two of them (2 and J) defended or favored by a group of systematists, are the following: 1. Derivation of the Agaricales from the Gastromycetes and from the Aphyllophorales.

2. Derivation of the Agaricales from the Gastromycetes alone. J. Derivation of the Agaricales from the Aphyllophor­ ales alone." 3

Which group the Agaricales were derived from does not con­ cern this paper; the importance of the above theories is that their elaboration by the three main antagonists, Heim (1948, 1971), Singer (1958, 1962, 1971), Smith (1971, 197J) causes some insight into the evolution of hypogeous forms. Singer and Smith (1960) become the allied proponents of the "Derivation of the Agaricales from the Gastromycetes alone" school, while Heim (1948, 1971) advocates the "Derivation of the Agaricales from the Aphyllophorales alone" theory. The argument centers around whether the Russulaceae evolved via "progressive evolution" (Singer and Smith, 1960) from such hypogeous genera as Gymnomyces and Martellia or vice versa. The arguments become irrational; Singer (1958) states: "Heim and others have drawn rash conclusions from incomplete facts and erroneously believed to have demon­ strated the correctness of their views whereas, in reality, they have only added - valid or questionable: this is a controversial question - new arguments in favor of their own predilected way of looking at the whole problem." Smith (1973) changes camps and states the possible mechanism of evolution: "It is true that if the basidiocarp of a mush­ room encounters unfavorable conditions of moisture or temp­ erature it will remain in the unexpanded stage (button stage) and either dry out, be eaten by larvae, or if it is retarded simply by cold, the synchronization of the devel­ opmental processes will be interrupted to the extent that 4

the hymenium (which is still protected from drying out) will continue to mature even though the stipe and pileus do not expand. This, in a nutshell, is the beginning of a secotioid Gastromycete. Since the pileus does not expand, it is difficult to ascertain at exactly what stage (or how long it will take in terms of evolution) for a race to develop which will have lost all ability to discharge spores from its basidia. We know it has happened because we have many species in which this capacity no longer is present. The 'primitive' secotioid gastromycete, following this line of reasoning, will thus resemble in gross morphology the unex­ panded button of a member of the Agaricales, but will pro­ duce ma tu re spores." Smith thinks unfavorable moisture and temperature conditions are the environmental factors sel­ ecting for evolution towards gastromycete forms. Thiers (1976, personal communication) agrees with this but adds that another probable selection factor is the more effi­ cient spore dissemination of forms which have lost the ability to discharge their spores. He stated that there is probably a tremendous loss of spores in the epieous fungi utilizing wind dissemination because many spores do not land on favorable substrates and thus never germinate. Thiers contrasted this with the hypogeous forms whose spores are not randomly cast into the wind, but instead are disper­ sed by mycophagist animals who deposit the spores on a substrate usually favorable for germination. Thus unfavor- 5

able moisture and more effective spore dissemination are thought to be the causative forces for evolution proceeding from agaricoid to hypogeous forms. Lange (1956) and Hawker (1954) who have written hypogeous fungus floras of, res­ pectively, Denmark and Great Britian, say little of evo­ lution other than that they both agree the hypogeous gast­ romycetes are best considere_d forms which have degenerated from various epigeous groups. Trappe (1975) agrees also with this line of reasoning and feels that there is a poss­ ible analogy between the evolution of hypogeous Basidiomy­ cetes and hypogeous Ascomycetes. He cites the developmental continuum that can be traced from Geopora with forcibly discharged spores to genera of the Tuberales with solid glebae and indehiscent asci. He states that what constitutes a primitive state in the hypogeous Basidiomycetes is pres­ ently a matter of conjecture. With this qualification, he speculates that hypogeous taxa such as the Melanogastraceae, which have glebal chambers generally lacking a well-defined hymenial palisade, may represent a structurally retrogress­ ive but ecologically progressive evolution from families such as the Hymenogastraceae, which have a well organized hymenium. Trappe reasons that the solid, spore-filled gleba of the Melanogastraceae is perhaps better adapted for spore dispersal through feeding by insects or larger animals. These same selection factors are thought to have been oper­ ative in the Tuberales. 6

Trappe (1971) states that evolution in the Tuberales has been simultaneously progressive and retrogressive. Progressive trends are towards increasing specialization of habitat, means of dispersal (by insects and animals) and mycorrhizal association. The accompanying retrogressive trends include simplification of ascocarp morphology, loss of spore discharge mechanisms, and change from cylindrical asci in a hymenium to rounded asci borne in unorganized fertile tissue.

Basidiomycete Phylogen~

Hawker (1954) states that the hypogeous Basidiomycetes are almost certainly a group of forms with diverse origins and few interrelationships. This has not been met with general agreement; lange (1956) states that while these fungi are a very heterogeneous group, some genera are obvio­ usly related to genera in the Agaricales, and he cites examples that are in agreement with Smith (197J). Some of the relationships are very clear, such as those (Thiers and Smith, 1969) between the Russulaceae and the Astrogastrace­ ous series. These relationships are based on spore char­ acters and anatomical features. For example the above mentioned Astrogastraceous series is composed of members sharing the following (Smith, 1973) features: pale to hya­ line spores which are typically globose to broadly elliptic and either having a coating of amylaceous material over the 7

surface or the ornamentation amyloid at least in part. The Russulaceae, in comparison, have spores varying from broadly elliptic to globose and having amyloid ornamentation. Other connections of hypogeous forms with the Agaricales are not so certain; Thiers and Trappe (1969) state that it is tempting to speculate that the gastroboletes represent a transition to a hypogeous and gastromycetoid habit after having arisen from epigeous boletoid ancestors. Smith(197J) is also cautious on this group, but he does think that Gastroboletus, Truncocolumella, and Rhizopogon are probably related; consequently he puts them all in one family, the Rhizopogonaceae. Lange (1956) and Hawker (1956) agree with the probable relationships between the Boletaceae and Rhizopogon. The Cortinariaceae (Smith, 1973) has several hypogeous or secotioid representatives: Thaxterogaster, Gautieria, and Hymenogaster share the same spore morphology and are thus placed in this family. The genus Cortinarius itself has several truly hypogeous species (Thiers and Smith, 1969). Smith (1973) states that Leucogaster and Octavianina are hypogeous genera with no known relationships or affinities with other taxa. Perhaps the major problem confronting phylogenetic studies of the hypogeous Basidio­ mycetes is that(Smith, 1971) adequate studies of their div­ ersity have yet to be done for any area, including Europe. \ Smith states that this makes it extremely difficult to sort out the relationships between the various taxa. 8

Ascomycete Phylogeny

Despite the lack of fossil evidence and knowledge of developmental sequences, most authors agree that the Pezizales and Tuberales are closely related and that the latter are derived from the former. Generally the epigeous habit and the presence of forcibly discharged spores in the Pezizales are contrasted with the hypogeous habit and the lack of a mechanism for forcible spore discharge in the Tuberales (Kimbrough, 1970). Travpe (1971) states that the development of a rational phylogenetic scheme for the order entails recognition of the earlier cited evolutionary fea­ tures; these are the progressive trends towards increasing specialization of habitat, means of dispersal by insects and animals and mycorrhizal association. Accompanying retro­ gressive characters are simplification of ascocarp morph­ ology, loss of spore discharge mechanisms, and change from cylindrical asci in a hymenium to rounded asci borne in unorganized fertile tissue. Because selection for spore discharge is no longer operative, those Tuberales which are farthest evolved from the hypothetical culpate Pezizales ancestors are considered to be the most advanced. The simplest forms (Hydnotrya and Hydnocystis) are culpate or wholly spherical with a central cavity lined with asci and paraphyses. The hymenium becomes convoluted into labyrinthine chambers in higher forms 9

(Genea, Hydnotrya). The paraphyses tips often form an extra layer of tissue over the hymenium (Genea, Petchiomyces), and in more advanced forms completely fill the chambers of the ascocarp with tissue (Tuber), (Kimbrough, 1970; Gilkey, 1939). To the above morphological progression Trappe (1971) adds some refinements concerning asci and spore anatomy. These trends reflect the movement away from forceful spore discharge: (a) Spores small, smooth, hyaline v.s. large, ornamented, colored. (b) Asci thin-walled, stipitate, eight-spored v.s. thick-walled, astipitate, one to five­ spored. All of the above evolutionary trends are incorporated into current taxonomic systems which attempt to be as nat­ ural as possible.

Ascomycete Taxonomy The taxonomy of the Tuberales is in flux. If one views the literature in a time sequence from old to new, he will see a definite and pronounced trend from "lumping" to "splitting". Thus Gilkey (1916, 1939) places the entire order in one family, Tuberaceae. In 1954 she split the rrub­ eraceae into three families: Geneaceae, Tuberaceae, and Terfeziaceae (Gilkey, 1954). Trappe (1969) listed five fam­ ilies, having split the Tuberaceae into three separate elements: Tuberaceae, Hydnotryaceae, and Balsamiaceae. 10

Later, Trappe (1971) split the Terfeziaceae into two families, the Terfeziaceae and the Carbomycetaceae. Thus there are currently six families in the order: Balsamiaceae, Carbornycetaceae, Geneaceae, Hydnotryaceae, Terfeziaceae, and Tuberaceae. All of these familial groupings reflect varying degrees of retrogression from iorceful spore discharge mechanisms. The most primitive being the Hydnotryaceae, and the most advanced being the Tuberaceae. There is no current up to date monograph on the order; the last one published being Gilkey's 1954 monograph of North American Tuberales. Dr. Trappe is currently gathering data for a world mono­ graph of the order (Trappe, 1977, personal communication). Forcible spore discharge establishes the line between the Pezizales and the tuberales. Using this criterion, Burdsall (1968) relegated Geopora, formerly in the Tuber­ ales, to the Pezizales. Geopora fruits underground, a typical Tuberales habit, but its spores are forcibly dis­ charged, and it has operculate asci. In addition to the Pezizales and the Tuberales, a third order, Eurotiales, is represented in the hypogeous Ascomycetes by Elaphomyces (Trappe, 1969).

Basidiomycete Taxonomy

The taxonomy of the false truffles is described by Smith (1965) as : "fraught w:th difficulties caused on the 11

one hand by inadequate information on basic features of the supposedly well known species, and on the other by a lack of agreement as to species concerts in the various groups." These problems are compounded by the lack of a standard; there is no reference point, such as Singer's The Agaricales

in Modern Taxo~, to set guidelines for workers. Con­ sequently, taxonomic treatments of hypogeous Basidiomycetes are fragmentary and unnatural. Zeller (1949) exemplifies the state of the art in his preface to some Gasteromycete keys: "It is realized that there are several ways to build keys. One method is to follow strictly along lines of phylogeny, while another is merely an implement of plant identification. The keys pres­ ented below are primarily for plant identification without particular emphasis on relationships." Although unspoken, the keys in the floras of Lange (1956) and Hawker (1954) echo the same sentiments. These are in direct contrast to Smith's (1973) "Key to Families of Hymenogastrales". Smith defines this order as: "Containing Gasteromycetes in which the hymenium is relatively persistent to maturity whether they are tuber-like, or have a columella or stipe-columella or a stipe." He further states that these fungi "connect up to the families of the Agaricales, and it is this approach to their systematics that is. used here." Thus Smith has constructed a natural system of classification. 12

Habitat

The habitats of hypogeous Ascomycetes and Basidio­ mycetes are generally the same (Trappe, 1974). Parks (1921) states that the two groups are frequently found growing together. Hypogeous fungi are typically found near mycorrhial roots in forests, groves, and shrub areas. The fungi reach maximum development in species and numbers near the interface between humus and mineral soil; it is here that mycorrhizae develop most profusely (Trappe, 1974). A few species fruit more deeply, and sometimes occur several inches under the mineral soil (Gilkey, 1916). Certain microhabitats appear to be particularily favored by some species. Hydnotrya variiformis fruits pro­ fusely in half-buried, very rotten conifer logs. Hymeno­ gaster alnicola, an apparent mycorrhizal associate of Alnus spp., occurs in soils so boggy that one would hardly expect to find such organisms (Trappe, 1974). Gilkey (1916) states that Tuber candidium seems to prefer a heavy clay soil, but that it also grows in leaf mold and sand. Hawker (1954) states that the edible truffles of France occur only on light calcareous soils and are never found in clays or in acid soils; that the majority of British species occur in greatest abundance in slightly alkaline soils, but there are also a number of exceptions to this rule. Fogel (1976) reports that a 1967 study by Ceruti et al. showed that soil lJ

pH was not important, but availability of mycorrhizal hosts and soil moisture were.

Reproduction

Authors agree that soil moisture and temperature control sporocarp production. Fogel (1976), in a quantitat­ ive study of sporocarp production in a western Oregon Doug­ las fir stand, found the productivity curve to be bimodal as a result of temperature and moisture effects, with peaks in May-June and October. Trappe (1974) states that every kind of seasonal occurrence is represented among the hypogeous fungi; in temperate regions there is usually a flush of fruiting in spring and autumn, and in the higher mountains the most abundant fruiting occurs in the summer. The length of the fruiting cycle depends on the spec­ ies, but some generalizations can be made concerning the two classes: Trappe (1974) and Hawker (1954) state that the Tuberales and Elaphomyces spp. are slow to mature and usu­ ally take several months or even the greater part of a year to mature. The Basidiomycetes, by contrast, are relatively ephemeral; their development is rapid and the mature fruit bodies are quick to decay. Fogel (1976) found there is no difference in the time of fruiting of Ascomycetes and Basidiomycetes, however there are definite spring and fall fruiting spp. in each class. In this study Fogel found 14

Hysterangium separabile and H. crassum are opportunistic, i.e. spp. fruiting whenever conditions were suitable. Reproductive activities at the cellular level are poorly known and ignored by most authors. Gilkey (1939) assumes that, as in other Ascomycetes, the mycelium is hap­ loid, and that nuclear fusion occurs during the formation of asci. She reports the origin of asci from croziers in Genea, Piersonia, Tuber, Pachyphloeus, and other genera. The developmental morphology of the false truffles is equally little-known because of the difficulty of procuring young stages of the sporocarps. Zeller's (19J9) monograph on the development of Alpova is the only complete treatment of hypogeous Basidiomycete sporocarp development. On the origin go basidia and basidiospores, Zeller states: "The basidium at first is a clavate binucleate cell produced from a clamp connection or terminal hypha composed of binuc­ lea te cells. The two nuclei unite to form a large fusion nucleus which divides two or three times to form four or eight nuclear bodies. By this time the basidium is usually turbinate or very much broadened ... Around the periphery of this broadened tip bud-like knobs appear and the whole divided nuclear mass accumulates at the tip of the basidium ... The necks of the projections, sterigmata, now become somewhat narrowed and the plastic nuclei migrate into them." 15

Nutrition

Hypogeous fungi are believed to derive their nutrition from mycorrhizal associations. Trappe (1974) states that nearly all hypogeous fungi are presumed to be mycorrhizal. Experimental evidence (Trappe, 1969) for their mycorrhizal habit is scanty because of the general failure of growing them in pure culture. This difficulty in itself is strong evidence that they are not saprophytes. Moreover, they are mostly obligate associates with mycorrhizae. As proof of mycorrhizal association, Zak (1973) states that the sporo­ carps of hypogeous fungi are often connected to mycorrhizae via rhizomorphs or mycelia. More specifically, the myco­ rrhizae formed by hypogeous Ascomycetes and Basidiomycetes (Trappe, 1977) are ectomycorrhizae. Peyronel et al. (1969) have defined "ectomycorrhiza" as a mycorrhiza where the fungus develops only outside the root cells as a Hartig net. Among the many genera of woody plants with ectomyco­ rrhizae which Meyer (1973) cites, are the following exam­ ples: Pinus, Pseudotsuga, Lithocarpus, Quercus, and Arbutus. In citing the woody associates of hypogeous fungi, early workers (Zeller and Dodge, 1936; Gilkey, 1954) were freq­ uently vague, often simply stating: "Habitat: under trees and shrubs." Other workers such as Smith and Smith ( 1973) are primarily concerned with taxonomy and are similarly 16

vague in their statements concerning associated woody plants. Trappe has tabulated the known mycorrhizal assoc­ iates of (1962) Basidiomycetes and, (1969) Ascomycetes. He has stated (Trappe, 1977, personal communication) that these tabulations are far from complete, and it is not at all unusual to find new associations simply because so little work has been done in this area. Some hypogeous fungi have wide host ranges (Trappe, 1974), for example, Melanogaster ambiguus and Tuber puberulum associate with trees of sev­ eral genera and families. Apparent narrow host specificity is displayed by other hypogeous fungi. Trappe (1974) cites the following examples of narrow host specificity: Alpova cinamomeus with Alnus spp. and Tuber melanosporum with Quercus spp.

Collecting

The methods of collecting both classes (Parks, 1921) of hypogeous fungi were the same. Harkness (1899) stated that experience teaches the collector which habitats are likely to yield specimens. Parks (1921) stated: " ... the collectors must go at the work more or .less blindly until experience has been gained." Trappe (1974) gives the most complete dis­ course on collecting. He cites the small pits from rodent excavations in the forest floor as one of the better clues denoting the presence of hypogeous fungi. Once the collector 17

has picked a likely spot, he should use a garden cultivator to rake aside the humus, all the while, closely watching for specimens. The interface between the humus and the mineral soil should be closely examined for fruit, and having reached mineral soil, the collector will often find it worthwhile to rake several inches deeper. After collecting all the specimens uncovered at the site, the collector should take notes on their fresh color and odor, on whether latex is exuded from a cut surface, and whether the speci­ mens change color when cut or bruised, or upon application of chemicals.

Spore Dispersal

Because most hypogeous fungi lack forcible spore dis­ charge mechanisms, they must depend on mycophagy or physical movement, i.e. rainwater, for spore dispersal (Fogel, 1976). The reported mycophagists include small mammals, deer, pigs, arthropods, baboons, bears, and slugs (Fogel and Peck, 1975; Fogel, 1976). As the animals defecate within the ecosystem, they spread the viable spores (Maser, Trappe, and Nussbaum, unpublished data). It is now common knowledge that many small mammals depend to a large degree on hypogeous fungi as a food source. A study by Stienecker (1977) showed that, during some months, the diet of Western gray squirrels is 18 as much as ninety percent hypogeous fungi, by volume. The spores of the ingested fungi are passed through the digest­ ive tract and are excreted without morphological change (Trappe and Maser 1976). Spores of Glomus macrocarpus (Endo­ gonaceae) were germinated by Trappe and Maser (1976) after passage through a rodent digestive tract. Spores of ecto­ mycorrhizal fungi are very difficult to germinate in the laboratory. Determination of final proof of animals as the spore vectors of truffles and false truffles awaits more sophist­ icated germination proceedures or a demonstrably reliable vital staining technique (Trappe and Maser, 1976).

Culturing Attempts

Trappe (1977) states that many mycorrhizal fungi grow poorly or not at all in pure culture methods tried so far. Hypogeous Basidiomycetes would be relatively easy to isolate by tissue culture techniques (Trappe, 1974) could they be counted on to grow on defined media in pure culture. Many Rhizopogon species do well on potato glucose agar or on various synthetic media; by contrast, Martellia species rarely have grown on the media so far tried (Trappe, 1974). Rhizopogon colossus has been successfully cultured and utilized as the mycobiont in pure culture synthesis of Douglas fir mycorrhizae (Trappe, 1967). 19

Isolation of the Tuberales is much more difficult. Trappe (1974) states that most species have openings from the surface to the interior; the result being that the inner tissue usually harbors abundant bacteria and microfungi that rapidly overwhelm the tissue from which a culture is attempted. Single-spore isolation has been successful in at least one case, but a variety of species have not been tried, and no general principles have been established. The imperfect Ascomycete Cenococcum graniforme is unquestionably related (Trappe, 1969) to the genus Elapho­ myces. Q. graniforme has been utilized extensively in pure culture synthesis of mycorrhizae, but its relative, Elaphomyces granulatus, only grows very slowly when iso­ lated in liquid culture (Trappe, 1977).

Reagents and Stains

In the study of hypogeous fungi, reagents and stains are commonly used to rehydrate dried specimens, to test for reactions used as taxonomic characters, and to make micro­ scopic features more visible. Those most commonly used are: 1) KOH in 2-3% aqueous solution is used to rehydrate dried specimens (Korf, 1973); color changes resulting from KOH are used as taxonomic features (Smith and Zeller 1966); KOH is used as a general mounting medium. Trappe and Guzman (1971) state that KOH must be used with caution when working 20

with Genea species due to the tendency of the spore orna­ mentation to dissolve in KOH. Misidentification may result if mounts are not also made in other media. 2) Melzer's reagent is used as a general differential stain and to test

for amyloid reactions (Smith and Smith, 1973). J) 10% Feso4 is used to test for color changes used as taxonomic features (Smith and Zeller, 1966). 4) Cotton blue-lactic acid sol­ ution is used as a general differential stain and, taxono­ mically, as a test for cyanophilic spore staining; spores which fail to turn blue when heated in the solution are termed cyanophobic (Korf 1973).

Economic Use of Hypogeous Fungi

Hypogeous fungi of both classes have been used as food -items in many parts of the world. Trappe (1971) states that truffles, Terfezia and Tirmania spp., were served at the table of the Pharaoh Cheops, discussed by Theophrastus and Pliny the Elder, and imported from Africa by the Romans. Modern epicures favor (Singer 1;61) the black, or Perigord truffle, Tuber melanosporum and Tuber griseum (=I· mag­ natum), the white Piedmont truffle. In 1976 1· griseum was selling for $600/kg fresh weight (Trappe, 1977). Truffle culture has been undertaken in France for the last hundred years (Ainsworth, 1971). Trappe (1?77) suggests that the edible qualities of mycorrhizal fungi can be a 21

valid and important criterion in selection of mycorrhizal fungi for nursery inoculation. Hypogeous fungi of other taxa are utilized in diff­ erent cultures. Zeller and Dodge (1936) report that the natives of Chakrata, India, cook and eat Melanogaster durissimus. In 1902, Lumholtz reported that the tribes of Guachochic, Mexico, used Melanogaster umbrinigleba as food (Trappe and Guzman, 1971). Harkness (1899) states that all of the California spp. are edible and would be esteemed as a luxury were it not for their rarity. Conversely, Gilkey (1916) states that the Tuber spp. of California lack the marked odors of the European truffles and consequently are useless as condi­ ments; she predicted that our species will not prove to have any great economic importance.

METHODS AND MATERIALS

Collection

Hypogeous fungi apparently are only found as mycorrhiz­ al associates of woody plants (see section on habitat). The most successful collecting occurred in areas which were not disturbed (particularily by overgrazing), had sparse under­ story v-egetation (mainly lack of grasses), and a thin to medium layer of duff. These conditions were often found in 22

parks and in suburban areas being held for development. Annadel, Mt. Hood and Sugarloaf parks were all found to have a variety of excellent collecting habitats. As the collector gained experience it was found that there are abundant clues to the presence of hypogeous fungi; those conditions cited above are the best clues. Frequently rodent excavations are excellent clues. Shallow depressions or holes, but not deeper burrows, often indicate foraging by rodents; they also indicate the depth at which the fungi occur. Occasionally, small mounds in the forest floor, sometimes accompanied by cracks in the surrounding soil, yield the expanding sporocarps. The collections were unearthed by scraping away the duff layer with a garden cultivator. The interface between the duff layer and the mineral soil is where most species fruit. Digging deeper into the soil (up to 8 cm) yielded occasional collections of Genea, Hydnobolites or Tuber. If a sporocarp was found in a given location, digging in all directions from the find would often yield more sporocarps, as many species fruit gregariously.

Field Notes

Field notes were taken on the following characters: 1) size and shape of the sporocarps 2) consistency 3) color of peridium, gleba, and, if present, rhizomorphs 4) odor 23

5) depth in ground 6) associated woody plants 7) presence or absence of rhizomorphs b) color changes due to handling, exposure to the atmosphere, or application of chemicals 9) date and location. The collections were then placed in small paper bags and assigned a permanent collection number. Upon return to the lab, the collections were prepared for preservation.

Preservation

Before drying, the sporocarps were sectioned into halves or quarters. This procedure guaranteed the ease of later sectioning attempts in that fruit-bodies often become very hard when dried, and unless there is a suitable sect­ ioning surface such as that provided by previous sectioning, the worker is force~ to employ "heroic" techniques, e.g. hammer and chisel. Collections were dried in a standard circulating herb­ arium drier at approximately 35 degrees centigrade. After drying, the specimens were placed in boxes and stored for later study. The collections used in this study will be retained in the author's personal herbarium.

Reagents and Stains

Color changes of the gleba and/or peridium in response 24

to the application of potassium hydroxide and ferrous sulfate are important in determining Rhizopogon taxonomy. These reagents were utilized on fresh collections, in the field or laboratory, before the specimens were dried. The degree, or lack of, color change was recorded in the field notes. The following reagents and stains were used: 1) Potassium hydroxide (KOH); 2.5% solution was used as a general mounting medium, to rehydrate sections from dried specimens, and to test for color chane es of the gleba and peridium of fresh Rhizopogon collections. This reagent should not be used in the study of Genea spp. as it dissol­ ves the spore ornamentation, upon which much Genea taxonomy is based.

2) Ferrous sulfate (Fe304 ); 10% solution is used in the determination of Rhizopogon taxonomy as noted above.

J) ~elzer's reagent; made to the standard of Smith and Smith (1973), this reagent was used in rehydrating dried specimens (particularily Genea), as a general differential stain, and to test for the following reactions: a) Amyloid: this is the development of blue, violet, or violet black color in hyphae or spores that signals an amyloid reaction. b) Dextrinoid: a reddish brown, vinaceous red or dark red color change in hyphae or spores. c) Inamyloid: no change or a simple yellowing due to the color of the reagent. 4) Cotton blue in 85% lactic acid; this solution is used as 25 a general differential stain and, taxonomically, as a test for cyanophilic spore staining; spores failing to turn blue when heated in the solution are termed cyanophobic.

Micro technique

Sections to be mounted were made by freehand using a sharp, single edged razor blade. This method is suitable for both fresh and dried material although, depending on the species being sectioned, the firmer consistency of dried material often yields better sections. As characters of the peridial anatomy are frequent taxonomic criteria, radial sections which incorporated the gleba or hymenium were most frequently made. Dry sections were rehydrated with 2.5% KOH, or in the case of Genea spp., with Melzer's reagent. KOH solution was used as the standard mounting medium, except for Genea spp. where cotton-blue or Melzer's was used. Cotton blue or Melzer's were used as general differential stains and to test for, respectively, cyanophilous or amyloid reactions when the taxonomic keys called for such tests. Microscopic observations were made with a Leitz SM-LUX compound binocular microscope at powers of 40x, lOOx, and lOOOx. Measurements of anatomical and reproductive struct­ ures were made at 1000x with the oil immersion lens.

Photomicrographs were made by use of the above micro- 26

scope and an attached Orthomat camera. The film used was Kodak Plus-X pan for black and white prints.

RESULTS

A total of twenty three different taxa were found; twelve were members of Class Basidiomycetes, and eleven were members of Class Ascomycetes. Two of the Basidiomycetes, both in the genus Alpova, were new and are described in a later portion of this paper. The following is a list, by class, of the fungi found in this research. Those marked by an asterisk presented identification problems and are discussed in a later section of this paper. The collection sites and dates are listed in appendix I.

Class Basidiomycetes

Alpova fulvus Trappe & Heblack sp. nov. ined. Alpova guercicola Trappe & Heblack sp. nov. ined. Gautieria graveolens Vittadini Qymnomyces ferruginascens Singer & Smith *Hymenogaster Vittadini sp. Hymenogaster parksii Zeller & Dodge Hysterangium separabile Zeller Leucogaster rubescens Zeller & Dodge *Martellia Mattirolo sp. 27

*Melanogaster Qarksii Zeller & Dodge Melanogaster tuberiformis Corda *Rhizopogon Fries & Nordholm sp.

Class Ascomycetes

Balsamia magnata Harkness *Balsamia Vittadini sp. Genabea cerebriformis (Harkness) Trappe Genea arenaria Harkness Genea gardneri Gilkey Genea harknessii Gilkey Genea Vittadini sp. Hydnobolites californicus Fischer Hydnotrya ellipsospora Gilkey Tuber californicum Harkness Tuber candidum Harkness

Probable Mycorrhizal Associates

The following list compiles the woody associate(s) of each hypogeous fungus found. The collection number of the fungus precedes the name of its woody associate(s). In cases 28

where a fungus was found more than once under a given ass­ ociate, e.g. Alpova guercicola was collected five times under Quercus agrifolia Nee, it is compiled as only having been found once. The fungi and their associates are listed by fungal class.

Class Basidiomycetes

Alpova fulvus #11 Arbutus menziesii Pursh., #58 Arctosta­ phylos Adans. sp. Alpova guercicola #22 Quercus agrifolia Nee Gautieria graveolens #60 Quercus dumosa Nutt.

Gymnomyces ferruginascens #24 Arctostaphylos sp., #76 ~· du-

~ Hymenogaster sp. #90 Lithocarpus densiflora (H.&A.) Rehd.

Hymenogaster parksii #29 ~· agrifolia Hysterangium separabile #23 Arctostaphylos sp., #26 g. agrifolia, #68 L. densiflora, #87 A· menziesii Leucogaster rubescens #19 Pseudotsuga menziesii (Mirb.) Franco

Martellia sp. #99 ~- agrifolia, #55 Arctostaphylos sp. Melanogaster parksii #45 g. agrifolia, #53 g. kelloggii Newb. Melanogaster tuberiformis # JO Pinus muricata D. Don., #46 Arctostaphylos sp. Rhizopogon sp. #17 P. menziesii, #21 P. muricata, #25 A. 29

menziesii, #38 g. agrifolia, #61 1· densiflora, #70 g. dumosa, #80 Arctostaphylos sp. #85 Pinus ponderosa Dougl. ex. P. & C. Lawson

Class Ascomycetes

Balsamia magnata #66 Ceanothus L. sp., #81 Arctostaphylos sp., #1 Pinus sabiniana Dougl. Balsamia sp. #47 Arctostaphylos sp.

Genabea cerebriformis #5 ~· dumosa, #8 A. menziesii, #15 P. menziesii Genea arenaria #49 g. agrifolia Genea gardneri #4 g. dumosa, #69 P. menziesii Genea harknessii #41 g. aerifolia Hydnobolites californicus #3 P. menziesii, #10 g. agrifolia Hydnotrya ellipsospora #86 g. agrifolia Tuber californicum #62 P. menziesii Tuber candidum #2 g. kelloggii

Key to the Species of Hypogeous Fungi Found in this Study

A. Spores borne in asci ... Class Ascomycetes AA. Spores borne on basidia (the basidia someti1::ies auto­ lysing by maturity) ... Class Basidiomycetes

CLASS ASCOMYCETES JO

1. Asci elongate, borne primarily or entirely among para­ physes in a hymenial palisade ... 2 1. Asci globose to elongate, scattered in glebal tissue ... 7 2. Hymenium enclosed by an epithelium of pseudoparenchyma formed by growth of paraphysal tips ... 3 2. Pseudoparenchymatic epithelium lacking ... Hydnotrya ellipsospora - fig. 1 3. Spores globose and spinose in both Melzer's reagent and KOH; mycelial tuft lacking ... Genabea cerebriformis - fig. 2 3. Spores ellipsoid to rarely subglobose, never globose; spore ornamentation verrucose to papillose; if some­ what spinose in Melzer's solution, then papillose in KOH; mycelial tuft present ... 4 4. Spore ornaments mostly narrower than 2u, asci thin walled; spores 27-30 x 21-25 u; sporocarp surface with an erratically dispersed tomentum ... Genea arenaria - fig. J 4. Spore ornaments often 2-4(-5)u broad ... 5 5. Paraphyses irregularly inflated and constricted; warts on spores not exceeding 3.5u dia ... Genea sp. #43 - fig. 4 5. Paraphyses evenly cylindric or slightly swollen ... 6 6. Warts on spores mostly 3-10u broad ... Genea gardneri - fig. 5 31

6. Warts on spores mostly narrower than Ju ... Genea harknessii - fig. 6 7. Spores smooth to minutely ornamented ... 8 7. Spores prominently ornamented, subglobose to globose ... 9 8. Spores almost always less than twice as long as wide, globose-ellipsoid •.. Balsamia magnata - fig. 7 8. Spores mostly two or more times as long as broad, frequently narrow-ellipsoid ... Balsamia sp. #47 - fig. 8 9. Ascocarp cavities empty or nearly so .•• Hydnobolites californicus - fig. 9 9. Ascocarp cavities filled with hyphae ... 10 10. Spores alveolate ... Tuber californicum - fig. 10

10. Spores spinose ... Tuber candidum - fig. 11

CLASS BASIDIOR'lYCETES

1 . Columella absent ... 8 1. Columella present (may be much reduced or absent) ... 2 2. Columella distinct and 9ften branching; peridium readily cracking and becoming free from the gleba when sporocarp is compressed; gleba olive green ..• Hyster­ angium separabile - fig. 12 2. Peridium not as above; columella much reduced or absent ... J J. Spore ornamentation amyloid ... 4 32

J. Spore ornamentation inamyloid ... 5 4. Mediostratum of tramal plates containing sphaerocysts; peridium poorly developed; spores 12-14(18) x 9-12(15)u ... Gymnomyces ferruginascens - fig. lJ 4. Mediostratum of tramal plates lacking sphaerocysts; peridium well developed; spores globose and spinose .•. Martellia sp. #99 - fig. 14 5. Spores enclosed in a gelatinous sheath, globose to oblong, 11-15u dia. including sheath •.. Leucogaster rubescens - fig. 15 5. Not as above ... 6 6. Spores longitudinally grooved; peridium absent or poorly developed ... Gautieria graveolens - fig. 6 6. Not as above ... 7 7. Columella rudimentary or absent; peridium present; spores mostly 20u or more long ... Hymenogaster parksii - fig. 17 7. As above except spores mostly less than 20u long ... Hymenogaster sp. #90 - fig. 18 8. Prominent hymenium lining glebal chambers ... Rhizopogon sp. JJS - fig. 19 8. Hymenium absent ... 9 9. Basidiospores hyaline to dilute yellow or brown ... 10 9. Basidiospores strongly brown to purplish black ... 11 10. Base of spores rounded; peridium subcutis of subpar­ allel hyphae ... Alpova fulvus - fig. 20 33

10. Base of spores truncate-cupped; subcutis hyphal cells irregularly inflated ... Alpova guercicola - fig. 21 11. Spores 10-11.5 x 5.5-7u ... Melanogaster tuberiformis - fig. 22 11. Spores 8-9.6 x 6.7-7,7u •.. Melanogaster parksii - fig. 23

New Species

Two new species of Alpova are described in this section. Colors in quotation marks are designated by page, column, and line, e.g. "light brown 5F6", in Kornerup & Wanscher (1967). Collections are deposited in the Mycolog­ ical Herbarium of Oregon State University (OSC).

ALPOVA QUERCICOLA Trappe & Heblack sp. nov. ined. Fructificationes 5-35 mm latae, subglobosae, applanat­ ae, elongatae vel irregulares. Peridium plus minusve 0.7 mm crassum, cinnamomeum vel fusco-brunneum, coactum, cellulis inflatis numerosis. Gleba gelatinosa, loculis repletis, venis albis. Sporae 7.5-10(-11) x 5-7.5(-10) um, plerumque late obovoideae, leves, basi truncatae, dilute griseo-vir­ idae, poris nullis, leptodermae vel crassitunicatae. Holo­ typus: California, Sonoma Co., Heblack 27. ETYMOLOGY : Latin, quercicola ("dweller of oaks"), referring to the oak woodland habitat of the species. J4

BASIDIOCARPS 5-35 mm broad, subglobose to flattened, elongate or irregular with lobes and furrows. PERIDIUM plus­ minus 0.7 mm thick, felty, "light brown 5F6" to "dark brown ?HJ", slowly staining darker brown as handled, spotted with light brown droplets of exudate; when dried, dull brown; rhizomorphs concolorous, coarse, sparse near base or absent; basal mycelium brilliant yellow, massed in soil near sporo­ carps. GLEBA rubbery-gelatinous, the gel-filled chambers separated by white veins, the chamber contents in youth translucent light yellow, at maturity becoming dark brownish black, when dried golden yellow to "dark brown 7H1". ODOR mild in young specimens and reminiscent of cooked artichokes to strongly acetylenic at maturity. KOH on dried peridium instantly brownish black; Feso4 on dried peridium slowly dark brown. SPORES 7.5-10(-11) x 5-7.5(-lO)um, mostly broadly obovoid but a few subangular, truncate-cupped, the walls thin to 1.0 um thick, smooth; in KOH light greyish green singly, pale grayish yellow in mass; in Melzer's reagent light orange singly, reddish brown in mass; in cotton blue weakly cyanophilic. BASIDIA borne among gelatinizing hyphae that fill the chambers, the basidia gelatinizing, indistinct at early stages of development and autolysed by maturity, 4-8 spored. PERIDIUM: epicutis a tangle of appressed to emergent hyphae 2.5-5 um diameter, with thin pale yellow walls and dark 35 orange contents that diffuse into the fluid of KOH mounts, the end cells often inflated or versiform, clamp connections occasional. Subcutis reviving to 400-800 um thick, of inter­ woven, subhyaline to yellow hyphae 2.5-7 um diameter at septa with most cells irregularly inflated at maturity to

5-25 um, the walls of the hyphae progressively more gelat­ inized near the gleba. GLEBA: tramal walls 100-150 um thick, the mediostratum of subparallel, hyaline, highly gelatin­ ized, poorly reviving hyphae. Distribution, habitat, and season: known only from the type collection, hypogeous in oak woodlands, a probable mycorrhizal associate of Quercus agrifolia Nee. January and Febuary. Collections examined: HOLOTYPE: CALIFORNIA, Sonoma Co.,

Sebastopol, on slope above High School Rd., 500 m south of jct. Occidental Rd. and High School Rd., Heblack 27, (OSC). PARATYPES: CALIFORNIA, Sonoma Co., Sebastopol, near holo­ type, under Quercus agrifolia Nee, 16 January 1977, Heblack 22 (OSC); same location as holotype; 8 Febuary 1976, Heblack 65 (OSC). Alpova guercicola has a peridium similar to that of Alpova nauseosus (Coker & Couch) Trappe, but the two species are differentiated by several anatomical features, most obviously, the spores of A. guercicola average broader and thicker-walled than those of A. nauseosus; the spores of A. guercicola are truncate, while those of A· nauseosus are not. 36

ALPOVA FULVUS Trappe & Heblack sp. nov. ined. Fructificationes 20-50 mm latae, subglobosae vel irreg­ ulares. Peridium plus minusve 0.2 mm crassum, glabrum, bru­ nneoaurantiacae, cellulis inflatis nullis. Gleba gelatinosa, loculis repletis, venis albis. Sporae 7.5-10(-12.5) x 5-7.5 (-10) um, plerumque late ellipsoideae vel obovoideae, leves, porosis nullis, dilute fulvae. Holotypus: California, Sonoma Co., Heblack 11 (OSC). ETYMOLOGY: Latin fulvus ("tawny") referring to the color of fresh basidiocarps. BASIDIOCARPS 20-50 mm subglobose to somewhat irregular. PERIDIUM plus-minus O. 2 mm thick, glabrous, "brownish orange 6D5" as fresh, dull brown mottled with yellowish brown as dried; "dark brown 7F2" rhizomorphs often interconnecting sporocarps. GLEBA gelatinous with gel-filled chambers sep­ arated by white veins, the chamber contents dark brown, when dried, blackish brown. ODOR mildly acetylenic. KOH on dried peridium quickly black; Feso4 slowly dark brown. SPORES 7.5-10(-12.5) x 5-7.5(-10) um, mostly broadly ellipsoid to obovoid, in some specimens a few fusiform, reniform, subangular, globose, etc., rounded, not truncate - cupped, walls smooth and thin to 1.0 um thick; in Melzer's reagent yellowish orange singly, dark orange in mass; in cotton blue, acyanophilic. BASIDIA borne among gelatinizing hyphac stuffing the chambers, autolysing by maturity. PERIDIUM: epicutis 25-75 um thick, of subparallel 37

appressed hyphae 2-7.5 um diam with thin, hyaline to dark orange walls, clamp connections scattered. Subcutis 60-300 um thick, of highly gelatinized, light yellow to hyaline, subparallel, hyphae. GLEBA: tramal hyphae 1-5 um diam, hya­ line, highly gelatinized, subparallel in the mediostratum.

Distribution, habitat and season: known only from the type collection, hypogeous in broadleaf evergreen forest, probably forming mycorrhizae with Arbutus menziesii Pursh. May. Collection examined: HOLOTYPE: CALIFORNIA, Sonoma Co., Santa Rosa, on hillside above Los Alamos Rd. 500 m north of jct. Los Alamos Rd. and Futura Way, Heblack 11 (OSC). Alpova fulvus most closely resembles Alpova mollis (Lloyd) Trappe but differs in several anatomical features. The most pronounced are the signifjcantly lareer spores of A. fulvus which have a rounded base unlike the short-cupped bases characteristic of the basidiospores of A. mollis.

DISCUSSION

Taxa Identification

In the list of fungi found, those marked with an aster­ isk, except Melanogaster parksii, were not possible to identify to species. It is suspected that these represent undescribed species. This is to be expected in that the 38 hypogeous macromycetes are a little-known group, and no previous research has been done in Sonoma County. M. parksii presented an interesting problem: my coll­ ections of this fungus keyed out very easily using the

Zeller and Dodge (1~J6) monograph on the genus. As a matter of routine, I wished to compare my collections with herb­ arium specimens, so I borrowed the type, H. E. Parks no. 136, from the U.C. Berkeley herbarium. I had been told (Trappe, 1977, personal communication) that the Zeller and Dodge key is not dependable as it is based on spore size, a highly variable character in this genus. Out of curiosity, I tried keying out H. E. Parks no.136 in the monograph; the same monograph which cites H.E. Parks no. 136 as type mat­ erial for fil· parksii. No. 136 has spores averaging 11.9 x 8.5 u and keys very readily to M. interrnedius (Berkeley) Zeller & Dodge. Consequently, although my collections key neatly to M· parksii, I do not know how accurate these det­ erminations are. The other taxa marked with asterisks could not be

~eyed to species. The following is a discussion of how these fungi differ from the known species:

Hy~enogaster #90, with spores averaging 18 x 11 u, keys out in Smith & Smith (1973) to: "Not staining blue. A num­ ber of species as yet not critically restudied will key out here but are not included since some may require new com­ binations." 39

Rhizopogon proved to be the most difficult genus. None of the collections made in this study fit the species dis­ criptions. The following two collections illustrate the trend: Rhizopogon #35 keys (Smith and Zeller, 1966) to Rhizopogon sepelibilis Smith, but does not fit the species description because its spores are too small, its epicutis hyphae are too narrow and not cystidial, and its epicutis lacks greatly inflated cells. Rhizopogon #71 keys to R. subsalmonius var. persicinus Smith, but fails to fit the species for these reasons: its basidiocarps are too large; it has a definite field mushroom odor rather than no odor; KOH on pale areas of the peridium gives a strong purple reaction rather than no reaction; its spores average too

broad; Feso4 gives no reaction near the gleba rather than the dark olive reaction of the discription.

Martellia was another difficult genus. For example my collection nos. 55 and J9 key to (Smith & Smith, 1973) M· scissilis (Zeller & Dodge) Singer & Smith, but they do not fit the species description for the following reasons: their spines, on the spores, are too long and too narrow; their hymenial cystidia are not abundant, and they lacked a strong witch hazel odor.

Balsamia #47 does not fit Gilkey's (1954) species due to its spores being too long and too narrow. Using Trappe's 40

keys to the Tuberales (Trappe, unpubl. ms.), #47 does not fit the description of~· vulgaris Vitt. due to the peridial cells of #47 being elongate rather than isodiametric in radial section. In addition, the spores of #47 are not frequently J0-45 u long, as are those of B. vulgaris.

Genea #43 keys using Trappe's keys, to Genea sphaerica Tul., but this determination cannot be regarded as concrete because the apical portion of the single ascocarp in this collection is missing. There is a couplet in the key regard­ ing the anatomy of the apical orifice hyphae; in this coup­ let the only alternative to Q. sphaerica is Petchiornyces kraspedostoma Gilkey, which has considerably smaller spores than #4J. Upon com~aring #4J with syntype material of G. sphaerica, there is good agreement microscopically, but the syntype contrasts macroscopically in that it is much smaller in gross morphology, has finer verrucae, and its peridium is much darker colored.

Probable Mycorrhizal Associates

On the following page is a graphical representation of the probable mycorrhizal associates of the fungi found in this study. Comparison of this data with reports in the scanty literature gives varying results. The following are typical examples: 41

Balsamia magnata: the only mention of the woody assoc­ iates of this fungus is Gilkey's (1954) statement that the habitat of the synonymous Pseudobalsamia magnata (Hark.) Gilkey, is "forests".

Tuber californicum: Trappe, (1969) in agreement with this paper, reports this species only in association with P. menziesii.

Genabea cerebriformis: Fogel (1976) and Trappe (1 :;69) report this species being associated with P. menziesii, as does this study, but there is no mention of it associating with Arctostaphylos sp. or~· dumosa as in this report.

The varying degrees of agreement on the mycorrhizal associates of the fungi found in this research and those reported by earlier workers is due to the fact that scant attention has been given to these fungi, and most of the work has been primarily concerned with the fungal taxonomy. 'l'he literature on mycorrhizal associates is far from com­ plete. The fact that this study lists three previously unrecorded associates of Balsamia magnata, not to mention many other new associations, shows that much baseline work remains to be done in this area. 42

TABLE I

PROBABLE MYCORRHIZAL ASSOCIATES

,_, l.r· ..-- !l> !l> It-' 11:: !'"d ''1: l ftj .c ci • I~ • • • • • • • • CD i3 ct IP :s;- s ~ m § .~ CD 0 CD ~ CD ~ 0 0 ~ g ::s 'i g 'i [g I~ Cll ~ C/l i ·~ I-' I-' I-' I-' ~ tll I-' 0 CD i3 CD IH ID ~ CD s CJl CD en 11- ~ m l~ 6 ·~ ::s I-' ~ 0 I-' ~ I-' Pl C/l Cf.I !:'! I-' ~ '1 ID I-' I-' ID ~ "d I-' 0 Pl • CD Cf.I CIJ I-' I-' ~ BASIDIOIVIYCETES • A.Lpova !"U.lVUS x x A.lPova quercicola x Gaut1er1a ~raveolens x Gymnomvces ferru~inascens x x ttymenoP-aster sn. !t'-JV x Hymeno~aster parks11 x Hysteran~ium separabile x xx x Leuco~aster rubescens x Martellia sn. x x Melanoraster narks11 x x Melano.a-aster tuber1form1s x x lilil.ZOPO:CZOn sp. x x x x :x x x x

ASCOJ',IYCETES Balsamia ma.~ata x x x Balsarnia ;;4 7 x Genabea cerebr1form1s x x x Gene a arenaria x Genea .r:rardneri x x Gene a harknessii x Genea so. tt43 x Hvdnobolites californicus x x Hvdnotrva ellipsosnora x Tuber californicum x Tuber candidum x 43

CONCLUSIONS

This research shows that Sonoma County has a diverse hypogeous fungal flora. All of the eighteen species of fungi identified are new distribution records for the county. Five of the taxa were identifiable to genus only: some of these probably represent undescribed species. It is highly pro­ bable that future research will discover undescribed taxa in addition to the two new Alpova species reported in this thesis. Comparison of probable mycorrhizal associations with those recorded in the literature reveals many new fungal - woody plant associations. This is to be expected in that little work has been done on the partners involved in ecto­ mycorrhizal associations. 44

SUMMARY A. In the two years beginning in December 1975, hypo­ geous macromycetes were collected from a variety of habitats in Sonoma County, California. Fungal - woody plant associations were recorded, and the fungi, when possible, were identified to species. B. Eighteen species of fungi were identified. They were representatives of the two classes, Ascomycetes: Balsamia magnata, Genabea cerebriformis, Genea arenaria, Genea harknessii, Hydnobolites californicus, Hydnotrya ellipso­ spora, Tuber californicum, Tuber candidum, and Basidio­ mycetes: Alpova fulvus, Alpova guercicola, Gautieria graveolens, Gymnomyces ferruginascens, Hymenogaster parksii, Hysterangi um separabile, Leucogastcr rubescens, l't.elanogaster tuberiformis. Some collections, representatives of the genera Rhizopogon, Martellia, Hymenogaster, Balsamia, and Genea, were only identifiable to genera. These may represent new species. The two Alpova species are newly described. All are new distribution records for Sonoma County. Comparison with the literature revealed new fungal - woody plant associations which may be mycorrhizal. C. A wide variety of hypogeous fungi are represented in the Sonoma County flora. Those only identifiable to genus may represent new species, and further studies will undoubtedly reveal additional new taxa. APPENDIX

The following data: collection number, date of collect­ ion, and location, refer to the fungi listed in the same order on pp. 28-JO. All collection sites, unless otherwise noted, are located in Sonoma Co., California.

Number Date Location 11 5-2-76 Los Alamos Rd., Santa Rosa 58 4-4-77 Sugarloaf State Park 22 1-16-77 Highschool Rd., Sebastopol 60 4-4-77 Sugarloaf State Park 24 1-23-77 Los Alamos Rd., Santa Rosa 76 12-6-77 Los Alamos Rd., Santa Rosa 90 12-18-77 Silala Lane, Sebastopol 29 1-26-77 Watson School, Bodega 23 1-23-77 Los Alamos Rd., Santa Rosa 26 1-23-77 Los Alamos Rd., Santa Rosa 68 11-13-77 Bohemian Highway, Freestone 87 12-18-77 Bohemian Highway, Freestone 19 1-4-76 Salt Point State Park 99 1-21-78 Trenton-Healdsburg Rd., Winsor 55 J-28-77 Oakmont 45 J-7-77 Trenton-Healdsburg Rd., Winsor 53 J-28-77 Oakmont JO 1-2;i-77 Point Reys St. Pk. Marin Co. 46 J-20-77 Lake Ralphine, Santa Rosa 17 4-20-76 Annadel State Park, Santa Rosa 21 1-4-76 Salt Point State Park 25 1-23-77 Los Alamos Rd., Santa Rosa JS 1-J0-77 Coleman Valley Rd., Occidental 61 4-5-77 Armstrong Grove, Guerneville 70 11-25-77 Kelley Rd., Cloverdale 80 1-6-77 Pine Flat Rd., Healdsburg 85 1-6-77 Pine Flat Rd., Healdsburg 66 11-11-77 Silala Lane, Sebastopol 1 2-5-76 Pine Flat Rd., Healdsburg 47 J-20-77 Lake Ralphine, Santa Rosa 5 4-8-76 Hood Mtn. St. Park, Santa Rosa 8 4-27-76 Annadel St. Park, Santa Rosa 15 4-20-76 Annadel St. Park, Santa Rosa 49 4-20-77 Lake Ralphine, Santa Rosa l-1-6

(Appendix, cont.) Number Date Location 4 4-8-76 Hood Mtn. State Park, Santa Rosa 63 4-18-77 Annadel State Park, Santa Rosa 41 3-7-77 Trenton - Healdsburg Rd., Winsor 43 3-7-77 Trenton - Healdsburg Rd., Winsor 3 4-8-76 Hood Mtn. State Park, Santa Rosa 10 4-27-76 Annadel State Park, Santa Rosa 86 12-12-77 Osborne Preserve, Penngrove 62 4-18-77 Annadel State Park, Santa Rosa 2 4-17-76 Oakmont 47

LITERATURE CITED

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Fig. 1. Hydnotrya ellipsospora spores x 1430

Fig. 2. Genabea cerebriformis spores x 1110 5J

Fig. J. Genea arenaria spores x 1180

Fig. 4. Genea sp. (#4J) spores x 1420 54

Fig. 5. Genea gardneri spores x 1320

, ! / .... ., 4 '· ' . 11{ ~ -~ ~· .. ./ . , .,, ~ .. • '{,. . • .. . • • .. \- , • ,,.

t. lt~· ; "'.,,,,, '

~ ...... ,. • ·-.. ~- , '"' .... ' - • . .. (r;"'

~ . ~ .,. ; •. . . '> .. l-a 1" . r - ··- "'"" >- • -· Fig. 6. Genea harknessii spores x 1540 55

Fig. 7. Balsamia magnata spores x 1850

Fig .. ;8. Balsamia sp. (#47) spores x 1350 56

Fig. 9. Hydnobolites californicus spores x 1550

Fig. 10. Tuber californicum spores x 1000 57

Fig. 11. Tuber candidium spores x 1000

Fig. 12. Hysterangium separabile spores x 1670 58

Fig. 13. Gymnomyces ferruginascens spores x 1570

Fig. 14. Martellia sp. (#99) spores x 1500 59

Fig. 15. Leucogaster rubescens spores x 1420

Fig. 16. Gautieria graveolens spores x 1680 60

0

Fig. 17. Hymenogaster parksii spores x 2100

Fig. 18. Hymenogaster sp {#90) spores x 1440 61

Fig. 19. Rhizopogon sp. (#38) spores x 1300

Fig. 20. Alpova f'ulvus spores x 1560 62

Fig. 21. Alpova guercicola spores x 1240

Fig. 22. Melanogaster tuberiformis spores x 2000 63

Fig. 23. Melanogaster parksii spores x 3100