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Neurospora from natural populations: Toward the population biology of a haploid eukaryote
EXPERIMENTAL
MYCOLOGY
12, 91-131 (1988)
REVIEW eurospora from Natural Populations: Toward the Populati Biology of a Haploid Eukaryote DAVID D. PERKINS AND BARBARA C. TURNER Department
of Biological
Sciences,
Stanford
Accepted for publication
University,
Stanford,
California
94305
December 4, 1987
PERKINS, D. D., AND TURNER, B. C. 1988. Neurospora from natural populations: Toward the population biology of a haploid eukaryote. Experimental Mycology 12, 91-131. Natural populations of the ascomycete Neurospora have been sampled systematically throughout much of the world, and the haploid strains from colonies in nature have been characterized genetically in the laboratory. Our findings are described in the context of a broader review of wild-collected strains, their uses, and their significance for population genetics. Visible Neurospora colonies found on recently burned vegetation are usually unique in genotype. More than three-fourths are pure strains originating from a single ascospore; the remainder can be purified. Thus, despite the potential for clonal propagation, these colonies provide effective population samples comparable to those collected for higher plants and animals. Over 3900 isolates from burned substrates have been analyzed from over 500 collection sites, mostly from tropical and subtropical regions. These strains have been assigned to five species-four heterothallic species with eight-spored asci and one pseudohomothallic species with four-spored asci. Each species has a unique pattern of distribution, but each overlaps with all the others in one or another part of its range. All of these species are similar in vegetative morphology, with orange or yellow-orange conidia. All have two homologous mating types, but the different species are reproductively isolated from one another. Fertility in crosses with reference strains has provided a reliable and convenient criterion for species classification of heterothallic strains. The species of a newly obtained haploid strain is determined by finding a tester strain with which it is fully fertile and produces predominantly viable ascospores. Viable ascospores are extremely rare for most interspecific combinations, but genes can nevertheless be transferred by matings among all but one of the nonhomothallic species. Abundant but mostly inviable ascopores are produced by some interspecific combinations. Karyotypes, karyogamy, and meiotic chromosome behavior are similar for all the known Neurospora species. There are seven chromosomes and a single terminal nucleolus organizer. This pattern also applies to the five eight-spored homothallic Neurospora lines that were designated by their discoverers as different species on the basis of ascospore morphology. These homothallic lines all lack orange pigment and are devoid of conidia. They were obtained by enrichment from soil samples and would not have been obtained by our collecting methods, which rely on visibility in the field. Examination of wild-collected strains of N. crassa and N. intermedia has revealed a wealth of intraspecific genetic variation. Genetic polymorphism of isozymes in local populations is comparable to that in outbreeding higher animals and plants. DNA restriction fragment length polymorphisms are also abundant, as are differences at vegetative (heterokaryon) incompatibility loci and recessive genes that adversely affect one or more stages of the sexual diplophase. Chromosomally located factors, called Spore killer, act in the sexual phase to produce meiotic drive. The four Spore-killer-sensitive ascospores in every ascus are killed in crosses of sensitive x killer, but all eight ascospores remain viable in crosses of killer x killer and sensitive x sensitive. Mitochondrial genomes of wild strains differ in both length mutations and nucleotide substitutions. Many isolates contain mitochondrial plasmids. A few strains have been found to undergo senescence following insertion of a foreign element into mitochondrial DNA. o 1988 Academic PZSS, I~C.
91 0147-5975188 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved
92
PERKINS TABLE
OF
AND
TURNER
CONTENTS
Abstract I. Introduction II. Characteristics relevant to collecting and identification III. Collecting methods A. Visible colonies on burned substrates B. Visible colonies on unburned substrates C. Cultures from soil samples IV. Laboratory culture and preservation V. Determination of species A. Rationale for diagnostic criteria B . Laboratory procedure C. Effectiveness of species tester strains and the testing procedure D. Mixed samples VI. Scope of the collections VII. Species in nature A. Abundance and distribution B. Substrate preference C. Possible subspecific categories D. Dispersal E. Retention of the sexual phase in homothallic species VIII. Genetic variation within natural populations A. Categories of variation B. Studies permitting evaluation of genetic polymorphism within populations 1. Protein polymorphism 2. Vegetative incompatibility 3. Recessive genes expressed during the sexual phase 4. Resistance to killing by Spore killer C. Spore killers D. Genes specifying ribosomal RNA E. Restriction fragment length polymorphisms F. Genes controlling recombination G. Chromosome rearrangements H. Mitochondrial variants I. Variants of other types IX. Transfer of genes from one species to another X. Conclusion: Neurosuora in nonulation and evolutionary genetics Dedication and Acknowledgments References
I. INTRODUCTION
Well over 5000 papers have been published on research with Neurospora since the genus was described in 1927 by Shear and Dodge. Most of these appeared after 1941, when Beadle and Tatum used Neurospora to obtain the first nutritional mutants. Most of the publications concern experimental work in the laboratory and have employed strains of Neurospora crassa that were derived from only five isolates originally collected in Louisiana. In contrast to the extensive knowledge
91 92 93 95 9.5 96 96 97 97 97 99 101 102 103 105 105 107 108 109 110 111 111 111 113 114 11.5 116 116 118 119 119 119 120 122
122 123 123 124
from laboratory experiments, very little was known until recently about Neurosporu in nature. The distribution, systematic status, ecology, or genetics of natural populations had not been investigated, and the few natural isolates that were available had been acquired hit or miss. We have tried to remedy this situation by devising effective collecting methods for the conidiating species and using them to sample Neurospora populations in several parts of the world. In the laboratory, we have classified the collected strains and carried out genetic analyses. In this review,
Neurospora
FROM
the main findings regarding occurrence and variability are summarized, laboratory investigations for which they have proved useful are described, and some of the unanswered questions are discussed. Many of the findings have not previously been published . We have shown that natural populations of Neurospora can be studied in such a way as to provide comparisons with the diploid higher eukaryotes with which population geneticists have been largely concerned. Neurospora is truly eukaryotic, and it undergoes a moderate amount of differentiation. Chromosome organization, meiosis, and genetic recombination are basically similar to those of plants and animals. Each strain of single-ascospore origin can be defined as an individual organism. Populations of heterothallic Neurospora species are polymorphic for vegetative incompatibility genes that prevent heterokaryon formation. Therefore haploid clones persist as distinct individuals. Thus, Neurospora is a useful organism for investigating the consequences of a predominantly haploid life cycle and for making comparisons with organisms whose life cycle is predominantly diploid. Significant differences are to be expected between fungi and the plant and animal kingdoms because of the great phylogenetic distance and because of the very different genome sizes and life styles. On the other hand, numerous genetic similarities exist between outbreeding populations of Neurospora and those of plants and animals For example, the outbreeding Neurospora species possess a high level of genetic polymorphism. Meiotic drive systems are present in nature. Natural populations contain numerous recessive alleles that act only in the sexual phase; these alleles are comparable to recessive lethal mutations in diploid organisms. In spite of the advances reviewed here, knowledge of the population genetics and ecology of Neurospora and of fungi in general is extremely limited compared to that of animals and plants. It is hoped that this
93
NATURE
review will identify areas of fungal population biology where research is most neede and where it is now feasible. The beginnings of our work on collected Neurospora strains were rep by Perkins et al. (1976), who desc methods for collecting and testing ne lates, outlined criteria for determining species of newly acquired haploid cultures, traced the derivation of authent ties-tester strains, and summari based on population samples first 6 years of our investigation. present review covers findings during entire B-year period since our study began, and is based on over 4000 isolates more than 650 sites. Developments in odology are outlined, and numerous uses of our wild-collected strains by ourselves and by other workers are described. Sources of background i~ormatiQ~ are given in Table I. II.
CHARACTERISTICS
COLLECTING
AND
RELEVANT
TQ
IDENTIFICATION
The genus Neurospora is defined as having grooved ascospores (Shear an c&s y 1927). This trait distinguishes it m its closest relatives Sordaria (smooth ascospores) and Gelasinospora (pitte spores) (see Moreau, 1953; Figs. Dutta et al., 1981). Neurospora inchdes and heterothallic, pseudohomothallic, mothallic species. Characteristics w distinguish the three groups are list Table 2. Large orange conidiating colonies on recently burned substrates key out almost invariably as heterothallic or pse~dob~~othallic Neurospora species. Vegetative c onies or cultures of these species are re ily distinguished visually from homotba~~i~ Neurospora and from closely related genera such as Sordaria. Only the nonherothallic Neurospora species produce conidia, and only these species are orange or yellow because of carotenoids in macroconidia and mycelia. The color is often vivid. T
94
PERKINS
Sources of Background Species Heterothallic N. crassa
AND TURNER
TABLE 1 Information on the Established Neurospora Reference
Type of information
species
N. discreta N. intermedia
N. sitophila Pseudohomothallic N. tetrasperma
Shear and Dodge (1927) Perkins and Barry (1977)“, Perkins (1979) Perkins et a/. (1982)” Krumlauf and Marzluf (1980), Perkins (1985) Perkins and Raju (1986) Raju (1980) Tai (1935) Perkins et al. (1976) Shew (1978) Turner (1987) Shear and Dodge (1927) Fincham (1951) Perkins et al. (1976)
Description Cytogenetics, rearrangements
Shear and Dodge (1927) Howe (1963a, b)
Description Markers, reference strains, methodology Linkage groups E variant with g-spored asci Molecular systematics
Markers and maps Genome organization Description Chromosomes Description Characteristics, reference strains Markers, reference strains Ecotypes Description Markers Reference strains
species
Howe and Haysman (1966) Calhoun and Howe (1968) Natvig ef al. (1987) Homothallic General
Species
species
N. africana
N. dodgei N. galapagosensis N. lineolata N. terricola
Frederick et al. (1969) Austin et al. (1974) Raju (1978) Mahoney et al. (1969) Sands (1982), Arnold (1983), Arnold and Howe (1985) Nelson et al. (1964) Nelson and Backus (1968) Mahoney et al. (1969) Frederick et al. (1969) Gochenaur and Backus (1962) Howe and Page (1963) Nelson and Backus (1968)
Key Ascospore morphology Chromosomes, ascus development Description Genetics using self-sterile mutants Description Perithecial development Description Description Description Absence of conidia Perithecial development, chromosome number
Note. Nearly 6000 available wild-type and mutant strains of Neurospora are listed in stock lists published periodically by the Fungal Genetics Stock Center (University of Kansas Medical Center, Department of Microbiology, Kansas City, KS 66103). Several strains formerly designated as Neurospora species are now considered of dubious validity or have been revised. N. phoenix has been transferred to the Xylariaceae and renamed XyZaria phoenix (Jong and Davis, 1973). N. toroi (Tai, 1935) is indistinguishable from N. tetrasperma (Metzenberg and Ahlgren, 1971; Perkins et al., 1976). Another four-spored form named N. erythea by Shear and Dodge (1927) is not known from living material. The differences in perithecial and ascospore size on which its status depends now appear to be an insufficient basis for distinguishing N. erythea from N. tetrasperma (see Perkins et al., 1976). An eight-spored Pyrenomycete described as Anixiella sublineata by Furuya and Udagawa (1976) has been reclassified as Neurospora sublineata because its ascospores are grooved (von Arx, 1981a). The strain is homothallic (D. D. Perkins, unpublished observations). The anamorph (vegetative stage) of the conidiating Neurospora species, formerly classified as Monilia Pers., has been renamed Chrysonilia (von Arx, 1981b). n Introductory sections of these comprehensive reviews describe and document the life cycle and characteristics of Neurospora, the history of Neurospora research, and sources of stocks and information.
Neurospora
FROM
TABLE
Characteristics
Distinguishing
the Heterothahic,
NATURE
2
Pseudohomothallic,
and Homothallic
Neurospora
Species
Homothallic Heterothallic (N. crassa, N. intermedia, N. sitophila, N. discreta)
Pseudohomothallic (N.
tetrasperma)
Eight-spored asci Each ascospore A or a
Four-spored asci Each ascospore typically (A + 4
Macro- and microconidia present Orange or yellow-orange mycelia and conidia”
Macro- and microconidia present Orange mycelia and conidia
(N. terricoiaa, N. dodgei, N. galapagosensis, N. africana, N. lineolata)
Eight-spored asci No mating type; cultures of single-ascospore origin self-fertile No macro- or microconidia Grey-brown mycelia; no carotenoids apparent
Note. For a key based on crossing behavior, see Perkins et al. (1976); for a key based on traditional morphology, see Frederick et al. (1969). The five homothallic taxons were distinguished and described as species on the basis of ascospore morphology, using strains obtained from soil samples. u All collected N. crassa and N. sitophila strains, most N. discreta strains, and N. intermedia strains from burned vegetation have orange color, often called salmon orange. The only yellow-orange strains found in nature were the N. discreta from Kirbyville (Perkins and Raju,1986) and some Neurospora intermedia from unburned substrate (Turner, 1987).
macroconidia are usually abundant, powdery, and readily airborne. Growth of the nonhomothallic species is remarkably rapid: linear rates can exceed 5 mm/h (Ryan et al., 1943; Perkins and Pollard, 1986). Ascospore dormancy is broken by heatshock in all the Neurospora species. HI.
COLLECTING
METHODS
We have collected Neurospora in the field as visible colonies, predominantly from burned vegetation but also from unburned substrates. Other investigators have sometimes recovered Neurospora from soil samples brought to the laboratory, as described in Section C below. Only conidiating species are obtained when visible colonies are sampled. The homothallic species, which are aconidiate, have been obtained only from heat-treated soil samples, presumably from the activation of dormant ascospores. There are reasons to expect that Neurospora might also be obtained from airborne ascospores (see Dispersal, Section
VII.D), but this method has not been use for collecting. A. Visible Colonies on Burned ~~~strat~s Neurospora colonies appear as pow orange patches on burned vegetation in moist tropical or subtropical area minetimes as soon as 7 days after a fire. e have concentrated chiefly on burned substrates because each colony from such a substrate, when fresh and undisturbed, usually re sents a genetically unique, sexually derived haploid line that originated from a single heat-activated ascospore. These cQlo~ie~ provide a sample of the gene pool of the local breeding population. Some isolates are mixtures, but the majority of heterothallic strains from recently burned substrates have only a single component. dence of purity and of uniqueness comes from scoring mating type and other traits (See Section V), Whenever possible, 7 to 10 discrete colonies are sampled at each local site. iti-
96
PERKINS
AND
TURNER
mum distance between collection sites may on nonburned substrates such as corncobs, sugarcane bagasse, filter mud from sugarvary from over 10 km to less than 1 km, cane refineries, or oncham (ontjam, a Jadepending upon the abundance of Neurosporu and upon available transport. (It is vanese food). (See Tables 7 and 9.) These not clear how large an area is covered by a are discussed under Substrate Preference (Section VI1.B). The collecting technique is “local” breeding population.) Materials for collecting consist of small similar to that for burned substrates. On at least some unburned substrates, (40 x 11.5 mm) sterile envelopes, each containing a wooden toothpick and small piece separate colonies are likely to have origiof filter paper. Each Neurospora colony is nated from conidia rather than from freshly sampled by transferring conidia with the activated ascospores, and the possibility exists that they represent vegetative derivtoothpick so as to make an orange smudge atives of the same clone. Thus, though culon the filter paper strip, which is then tures from such a source are still informasealed into its envelope. Identifying details tive, they cannot be used in the same way are written on the envelope. American Hospital Supply No. 25658 “Tomac” self- as samples from burned substrates to make seal glassine needle envelopes have been inferences about genetic variability and polymorphism. For this reason samples used. After assembly, these are sterilized by autoclaving, dried, and bundled 20 to a from unburned substrates have consisted of fewer isolates per site than those from bag in 4 x 6-inch presterilized polyethylene bags (Falcon Plastics No. 5018) sealed with burned substrates. plastic tape. Envelopes for a day’s collecting can C. Cultures from Soil Samples readily be carried in a pocket. After use, the An alternative strategy for sampling the sealed collecting envelopes are mailed to ascospore population has been employed in the laboratory for subculture and analysis. Up to 40 can be enclosed in a small airmail several laboratories. Soil samples are proletter envelope. Permits for shipment into cessed so that colonies are obtained selecthe United States are obtained from the tively from heat activation of ascospores. This method, called “soil pasteurization,” U.S. Department of Agriculture (Plant results in the recovery of strains that belong Quarantine Division, Hyattsville, MD 20782). to various Neurospora species as well as Colonies can be recognized as Neurosporepresentatives of other genera whose asra with high efficiency under field condicospores also remain dormant until heat activated. (See Mahoney et al., 1969; Fredertions. Mistaking unwanted fungi for Neuick et al., 1969; Maheshwari and Antony, rospora is not a significant problem when sampling is done visually. Only rarely is a 1974; Palanivelu and Maheshwari, 1979.) fungus other than Neurospora picked up Soil sampling has the advantage of conwhen orange colonies are sampled. Even tributing strains that are atypical in appearthen, it is usually not a matter of mistaken ance or that do not conidiate. It has been identity but rather that an unlikely prospect the source of all the known homothallic was picked as a last resort in conditions Neurospora strains. The method does not where a burn was too old and no typical require prior burning and its use is not limNeurospora could be found. ited to a short period following a fire. On the other hand, there is no immediate asB. Visible Colonies on surance that Neurospora is present when Unburned Substrates soil samples are taken in the field, so it is Neurospora may also be found growing impossible to know how extensive the sam-
Neurospora
pling should be before collection is terminated. IV.
FROM
in an area
LABORATORY CULTURE AND PRESERVATION
In the laboratory, the collecting envelopes are placed at - 20°C for 24 h to ensure destruction of mites and mite eggs that might be present (Subden and Threlkeld, 1966). Conidia are then transferred by needle from the filter paper to a slant of minimal medium (Vogel, 1964) containing chloramphenicol (0.2 mg/ml, autoclaved in the medium) to inhibit possible bacterial contaminants. A second transfer is made to leave behind possible fungal contaminants. (Unless inhibited, Neurospora outgrows all contaminants on solidified medium.) One third-stage transfer is frozen at - 20°C and conidia from another third-stage transfer are preserved permanently in suspended animation on anhydrous silica gel (Perkins, 1977b). By making permanent silica-gel stocks promptly, following only the briefest growth, it is hoped to retain original wild genotypes unchanged by mutation and selection. After species identifications have been made (Section V), strains representing each species found at each locality have been deposited in the Fungal Genetics Stock Center (FGSC) ’ (Department of Microbiology, University of Kansas Medical Center, Kansas City, KS 66103). Where available, opposite mating types have been included for each species. These strains are included in the FGSC stock lists (Fungal Genetics Stock Center, 1986). (For intensely sampled areas, not all of the sites are represented in FGSC for N. intermedia, the most abundant species.) Thus for many sites where one species predominates, only one or two strains are sent to FGSC while an additional five to eight are not deposited. 1 Abbreviations used: FGSC Fungal Genetics Stock Center; RFLP, restriction fragment length polymorphisms; OR, Oak Ridge (laboratory strain).
NATURE
The nondeposited strains are available from silica-gel stocks at Stanford University. Where purity of the Neurospora culture must be assured, successive subcultures are grown up from single conidia. The strains deposited in FGSC have not bee purified in this way, but a variety of studies indicate that most of the deposited cultures are pure (see, for example, Spieth, 1975). V.
DETERMINATION
A. Rationale for Diagnostic
OF
SPECIES
Criteria
We feel that lack of a fertility barrier is the most fundamental basis for concludi that two heterothallic strains belong to t same species. In practice, fertility tests have proved to be the only reliable criterion, and they are by far the most convenient. Our diagnostic crossing procedure was used successfully to make species signments for 99% of all isolates collec before 1983. (The tests have not been completed for all of the isolates from th 1985 period of intensive collecting.) all the isolates were assigned to one or another of the four previously established conidiating species: N. crassu, N. sitophila, N. intermedia, or N. tetras~erma~ The procedure also revealed the existe of a rare new species, N. discreta, whit reproductively isolated from the others (Perkins and Raju, 1986). Although the heterothallic species ten to differ somewhat in size and mo~h~logy of ascospores (Table 3; see also Table 3 of Perkins et al., 1976), these differences are only averages. It would be difficult or impossible to assign strains reliably to the heterothallic species either by vegetative morphology or by ascospore size There is too much variation wit and there is overlap between species. ascospore measurements requi be two parents to make the cr duces the ascospores. Since the isolates are haploid, it is necessary to
PERKINS
98
AND TURNER
TABLE 3 Diagnostic Characteristics of Interspecific N. crassa N. N. N. N. N.
crassa intermedia sitophila discreta tetraspermab
+
N.
intermedia
W +
N. sitophila
R W +
Crosses” N.
discreta
R R R +
N.
tetraspermab
R R R R +c
+ , Fully fertile. Perithecia with beaks. Ascospores are abundant, with >50% black and viable. In N. because strains containing Spore killer-l are common, test crosses that are Sk-lK x Sk-l’ can be expected; these produce asci with four black: four aborted ascospores, classed here as +. W, “White.” Perithecia are full size and often produce and eject ascospores. Very few ascospores are fully pigmented and viable, but these few make introgression relatively easy. R, Perithecia are absent or rudimentary with no ascospores. Occasional exceptions are found where a few viable ascospores can be recovered from interspecific combinations. See Section IX. D Similar results are obtained regardless of which species is used as female (protoperithecial) parent. b Homokaryotic A or a strains are necessarily employed as N. tetrasperma species testers and for interspecific crosses. c Asci four-spored. Note. sitophila,
choose partners for them. In the initial work, we tried intercrossing all isolates from the same site, but the process is very time-consuming and is fruitless unless both mating types of a species are found at the same site. It also means that each cross involves two unknowns instead of one. For these reasons, all heterothalhc species assignments have been made on the basis of fertility in crosses to strains established as species testers. The crossing behavior of different combinations of species is summarized in Table 4. Diagnosis of wild-collected strains of the pseudohomothallic (secondarily homothallit) N. tetrasperma is made on a different basis. N. tetrasperma cultures are usually A + a heterokaryons, and under appropriate conditions each culture will produce perithecia containing four-spored asci in which each large ascospore contains both A and a nuclei. The four-spored asci categorize the strain as a pseudohomothallic without the need for a test cross. Occasionally, small homokaryotic ascospores are produced. If a N. tetrasperma strain is homokaryotic when collected, or if the A and a components of a heterokaryon are separated experimentally, each homokaryotic culture crosses only with testers of the
opposite mating type. The homokaryotic strains thus behave as though they were heterothallic strains of a single mating type, except that the crosses produce fourspored asci. A and a components have been resolved and test crossed for only a few of the many four-spored pseudohomothallic strains that have been collected. Until crossing tests have been made, the possibility must be considered that not all the pseudohomothallic isolates belong to the same species, N. tetrasperma. However, Natvig et al. (1987) have obtained molecular evidence that four-spored isolates from widely diverse origins are all closely related, consistent with their belonging to a single species or species complex. Attempts to employ protein polymorphisms as taxonomic criteria for distinguishing species (see, for example, Reddy , 1973) have not been successful, nor have measures of overall DNA homology based on Cot curves provided a practical criterion for diagnosis at the species level (see Dutta et al., 1981). Comparison of cloned uniquesequence DNA fragments by Southern blotting seems promising as an indicator of relatedness between species (Natvig et al., 1987). This method has been developed for
Neurospora
TABLE 4 Commonly Found Accessory Characteristics of the Five Conidiating N.
crassa
N.
sitophila
N. intermedia
N. discreta
N. tetrasperma
9
FROM NATURE
Species of Neurosporaa
Typical ascospores 29 x 1.5 km, more pointed than N. intermedia. Carotenoids almost always orange. Typical ascospores 23-24 x 14 pm, more pointed than N. intermedia. Carotenoids almost always orange. Typical ascospores 27 x 17 pm, visibly more rounded than the other species. Strains with golden-yellow (saffron) carotenoids and very large conidia (>12 pm diameter) always prove to be N. intermedia, but most N. intermedia cultures are orange and have conidia in the smaller size range. The first population discovered, from Kirbyville, Texas, differs from other Neurospora species in ascospore morphology and vegetative traits, but these differences are not found in other populations that appear to be N. discreta on the basis of fertility tests. The N. discreta isolates from Kirbyville, but not those from elsewhere, produce ascospores that are ornamented with dot-like pits in the ribs between confluent parallel grooves. Unlike the others, the Kirbyville strains are yellowish rather than orange, and large empty barren protoperithecia (‘“false perithecia”) are found abundantly in unfertilized haploid cultures. Isolates from sources other than Kirbyville are infertile as female parents on synthetic crossing medium unless sucrose is replaced by filter paper as a carbon source. Until other populations are more adequately represented, traits characteristic of tbe species cannot be listed with confidence. Typical ascospores 34 x 15-16 Frn, each giving rise to a self-fertile A + a culture. Occasionally smaller A or a ascospores are produced in asci having more than four spores. Most cultures conidiate poorly at 25°C whether heterokaryotic or homokaryotic, with the protoperithecia in homokaryotic cultures becoming brown and enlarged but without beaks or spores. At 34°C false perithecia do not appear and conidiation is better. Carotenoids orange.
a Except for the large heterokaryotic ascospores of N. tetrasperma, which occur in four-spored asci, these traits are more variable and less reliable for distinguishing species than are the fertility characteristics in Table 3. Ascospore measurements are taken from Table 3 of Perkins et al. (1976).
the purpose of defining phylogenetic relationships. It cannot be expected to substitute for the convenient and economical crossing tests that we have used in everyday practice for assigning unknown isolates to species, as described in the following section. B. Laboratory
Procedure
Synthetic crossing medium (Westergaard and Mitchell, 1947) has been used for test crosses, with 1% sucrose and 1.5% agar unless otherwise indicated. Tester strains containing the gene fr (fluffy) are convenient because conidia are absent. The fluffy testers are inoculated to slants in 12 x 75 mm culture tubes; these are ready to fertilize after 4 days at 25°C. If nonfluffy strains are used as testers, they are inoculated to larger slants (13 x 100 mm) to avoid ob-
struction by conidia; these are ready to fertilize at 5 days. Test slants can then be held at 5°C for at least 2 weeks before being fertilized. In early stages of the work, while experience was being gained with species tester stocks, tests were made between testers in all combinations, collected isolates were crossed to set of testers (see Table 5 and Section V.C). The full set of tester strains is still e with some wild isolates that are di diagnose, but a species assignment can often be made with confidence on the basis of observing full fertility with the first or second tester that is used. Our present procedure is to cross wil collected strains first to N. crassa testers of both mating types. If the sample came from a region with a great predominance of AJ.
100
PERKINS
AND TURNER
TABLE 5 Strains Used as Species Testers in Diagnostic Crosses Strain designation
FGSC No.
Origin and characteristics
1. Preferred testers (species reference strains) Neurospora crassa Highly fertile, aconidiate fl&fi strains essentially 4317 fI’A 4347 coisogenic with the standard OR laboratory wild types. frpa jlp originated spontaneously from 74A x 73a, and was backcrossed recurrently to OR or OR-related strains. Neurospora intermedia 3416 Conidiating f, isolates from N. intermedia P13A x P17a from Shp-1A 3417 Taiwan, selected for fertility and uniform growth Shp-la (Shew, 1978). Neurospora sitophila jl; Sk-I” A 4762 These contain J&ii allele P1012, which arose 4163 spontaneously in N. sitophila, and the Spore killer jl; Sk-lK a allele Sk-IK. From third recurrent backcross to P8085 and P8086. Neurospora discreta 3228 P851 collected near Kirbyville, Texas. P8127 is from P851A 4th recurrent backcross to P851 of Kirbyville-1 a P8127a 4378 (P846) (Perkins and Raju, 1986). Neurospora tetrasperma 1270 Homokaryotic f,,‘s from A + a strain 87 of Dodge (Howe, 1963a). 85A 85a 1271 2. Supplementary testers and strains used previously Neurospora crassa Wild-collected N. intermedia strains from Florida. P420A 2316 1940 P405a jlufi (allele P) introgressed from N. crassa by seven 5798 ;it recurrent backcrosses to Shp-1A and Shp-la. (Expected 5799 to replace Shp-1A and Shp-la after more extensive testing.) Neurospora sitophila 2216 Conidiating Sk-l” strains previously used as reference P8085A testers (Perkins et aZ., 1976). P8086a 2217 jl’; Sk-l’ A 4887 These contain jlufi allele P, which was introgressed from N. crassa by five recurrent backcrosses jl’; Sk-l’ a 4888 to N. sitophila. They are sensitive to Sk-l”. P2443A 5940 Conidiating Sk-l’ strains from Tahiti. P2444a 5941 Note. For further information, see Perkins et al. (1976) and Perkins and Raju (1986). The supplementary strains have been authenticated in crosses to the species reference strains. They have been used in areas where some strains are infertile with the reference strains or produce a high percentage of immature ascospores with them. Testers P420 and P405 are sometimes more fertile than the Shp testers when crossed with N. intermedia isolates from the western hemisphere. Members of the two sets are conspecific on the basis of intercrosses (Perkins et al., 1976). The N. crassa and N. intermedia strains listed here are all sensitive to killing by Spore killer-2 (Sk-su) and Spore killer-3 (Sk-3u). Spore killer strains are rare in these species. In N. sitophila both Sk-lK killer and Sk-l’ sensitive strains are common (Turner and Perkins, 1979). Both Sk-I” killer and Sk-l’ testers are therefore listed.
Neurospora
FROM
each strain is then usually crossed also to a N. intermedia tester of appropriate mating type. The N. intermedia testers can be fertilized as soon as perithecia are seen in one of the N. crassa tests. For samples from some regions it is more efficient to await maturation of the N. crassa tests and to treat different outcomes separately: 1. If the test cross with N. crassa produced at least 90% black ascospores, the isolate is scored as N. CF-ussuand not tested further. 2. If the test cross with N. crassa produced large perithecia either having no spores or having eight-spored asci with predominantly defective ascospores, the isolate is tentatively scored as N. intermedia and is crossed to a N. intermedia tester (or testers). Recently this procedure has been modified for bright yellow strains from unburned substrates. More than 100 of these strains have been fertile enough with N. intermedia to be diagnosed as conspecific, but these crosses tend to make few perithecia and few ascospores (Turner, 1987). Over 50 additional yellow strains have been diagnosed on the basis of their cross to the N. crassa testers, substrate preference, color, and giant conidia. 3. If a test cross with the N. crassa tester as female produced no reaction better than rudimentary perithecia, but the culture being tested behaved as self-fertile and by itself produced perithecia with four-spored asci, it is tentatively scored as N. tetrasperma. (Confirmation requires separation into homokaryotic A and a components and testing the components with singlemating-type N. tetrasperma testers. This has rarely been done.) 4, If the test cross with N. crassa was infertile (made no reaction better than rudimentary perithecia) and the tested culture was not a pseudohomothallic strain with four-spored asci, the unknown is crossed to N. sitophila. If the test cross to N. sitophila is also infertile, the isolate is then crossed intermedia,
NATURE
to N. discreta and to single-mating-ty testers of N. tetrasperma. C. Effectiveness of Species Tester Strains and the Testing Procedure
The standard reference strains in current use are listed in Table 5. All have shown to be conspecific with the strai which the original species descriptions were based (Perkins et al., 1976). mentary strains that have been u special purposes are also liste aconidiate mutant “fluffy” whenever possible because th nidia makes it much easier to see peri and ejected ascospores. Strains containing fl also show enhanced fertility as female parents. All of the species that we have co11 are closely enough related so that a phogenetic reaction triggering perit~eci~l growth can take place when i~terspec~e crosses are attempted between representative isolates of opposite mating type. This is true of some but not all strains even between the least reactive pairs of species. 0n this basis, individual isolates can be assigned to one of the two mating type a, as originally defined in N. crassa. N. crassa fluffy testers are fertilize discreta, by N. sitophila, or by si~~~~mating-type cultures of N. tetrasperma~ perithecia may be very rudimentary. ertheless, in the majority of cases a reredos can be seen with one mating type but not with the other. Intraspecific and interspecific crosses in all combinations are compared in Table 3. Except for N. crassa x N. intermedia, which typically produces 1 to 10% viable ascospores, ail cornbi~at~Q~s of interspecific crosses either produce no ascospores or produce ascospores that are almost all (>99%) inviable. Inviable ascospores are easily recognized becaus are usually hyaline; they will be refe as white, in contrast to the intens that is characteristic of normal, viable ascospores.
102
PERKINS
AND
For the great majority of heterothallic strains, species assignment is clear and straightforward, based on a fertile cross with >90% black ascospores. However, there are important exceptions that should be noted by anyone attempting to identify newly collected strains: 1. Newly acquired strains that are bona tide N. sitophila may produce either 95 or 50% black ascospores when crossed with a given N. sitophila tester. Those that are like the tester strains in their Spore killer genotype (see VII1.C) produce 95%, and those that are unlike produce 50%. Occurrence of tests with 50% black ascospores does not affect species diagnosis because the N. sitophila testers do not produce even 1% black ascospores with other species. 2. The N. discreta group has two parts. The standard reference strains from Texas, others from the United States, and one from Guatemala make ~90% black ascospores when intercrossed. Those from other countries make about 10% black ascospores when crossed to the reference strains and ~90% black in the few fertile crosses obtained within the group. However, a discrepancy in reciprocal crosses raises the possibility that the defective ascospores may be caused by some sort of incompatibility not sufficient to warrant dividing the group into separate species. This problem is under study. Another problem illustrated by the N. discreta group is that not all strains have the same requirements for successful crossing. Many do not produce protoperithecia on our standard crossing medium (Westergaard and Mitchell, 1947) with 1% sucrose but do so when filter paper is supplied and the sugar is omitted. 3. N. intermedia and N. crassa are the closest pair among the known species. Usually they are easily distinguished, but strains from certain regions (India (particularly the state of Andra Pradesh), Pakistan, Thailand, and Malaya (Penang)) cross unusually well with the testers from both species. Diagnosis can be completed by using
TURNER
clearly identified local testers. Other strains cross poorly with the standard testers from both species, but most of these can also be resolved by crosses to local testers. In these most difficult cases the possible presence of chromosome rearrangements must be investigated. A reciprocal translocation, for example, would result in 50% defective ascospores in a cross to a tester of the same species (see Section VII1.G). 4. Some collected strains do not make a fertile cross (sometimes not even a rudimentary reaction) with certain testers from a species yet make a fully fertile cross with others from the same species. Many strains from the three African countries-Congo, Gabon, and Ivory Coast-are still unidentified, partly because they were collected more recently, but mainly because they have proven to be among the most demanding to work with. The majority are probably N. intermedia-they gave a reaction typical of N. intermedia when crossed to the N. crassa testers but did not make fertile crosses to the standard N. intermedia testers or to identified N. intermedia strains from Africa. Others did not cross to any testers. When crosses were attempted between a sampling of the latter and various strains from the N. discreta group (using filter paper as the carbon source in the crossing medium), some of them made fertile crosses and were thus diagnosed as N. discreta even though they had not evoked any reaction in attempted crosses with the standard N. discreta testers under standard crossing conditions. D. Mixed Samples
Of the mixed samples that have been detected, most were recognized because they crossed or reacted to testers of both mating types but did not make four-spored asci in a self-cross. Among the cultures that have given a reliable mating-type reaction to one of the standard heterothallic testers, the overall incidence of a cross to the other
Neurospora
FROM
mating type by the same culture is 12%. It must be assumed that there is a similar frequency of samples containing two strains of the same mating type. So we estimate that among 100 original cultures, 12 are mixed mating type, 12 are mixed but of the same mating type, and 76% are completely pure. The 12% estimate for mixtures containing both mating types applies both to burned and to unburned substrates, as well as to each species taken separately. However, these pooled totals include many collections that were made under unfavorable conditions, from disturbed sites and from very old burns. The frequency of mixed cultures varies greatly from region to region and site to site, depending on age of the colonies and on collecting conditions that are beyond our control. For some regions, very few of the cultures were mixed. Only 14 samples (in more than 4000) have been found to contain a mixture of species. Because our protocol takes advantage of the predominance of N. intermedia or N. crassa in nature by crossing to those species first and because the first successful test cross ends the procedure for that strain, there is little probability of detecting a trace of a less common (and less interfertile) species of the same mating type in a sample that is predominantly N. intermedia or N. crassa. However, even when a cross to another species is successful, the presence of N. tetrasperma as a second component is revealed by the production of fourspored asci in the original culture tube. In the tables that follow, strains collected as mixed species are listed according to the components that have been subcultured and isolated. Mixtures of both mating types of the same species are counted as one strain if not separated or as two if separated. All mixtures of N. intermedia A and a (as judged by N. crassa crosses) were used to tabulate the proportion of mixed cultures, but for areas with many N. intermedia samples, some were not further tested. Among all countries, a total of 112 N. in-
NATURE
termedia mixed cultures were set aside an are not included here. VI.
SCOPE
OF
THE
COLLECTIONS
Over 4000 cultures have been co and characterized since 1968, ori~uat~~g from five continents and from oceanic islands. The wild-collected isolates are summarized according to geographical origin in Tables 6-9. Results of our collections from burned substrates are given in Table 6, an the results from unburned substrates in Table 7. Collections in Tables 6 and 7 were made either by the first au&or or in his presence by others whose assistance is credited under Acknowledgments. Strains collected and donated by other workups have also been put through our standar testing procedures, with the results shown in Tables 8 and 9. A few of these isolates were obtained by heat processing ples (e.g., Mahoney et al., 1969; wari and Antony, 1974). Our strains foun through 1974 were listed in Table 4 of kins et al. (1976). These strains are incl here in Tables 6 and 7. Results wi sent to us by individuals who bad colle them prior to 1975 comprised part 5 of Perkins et al. (1976) and are incl here in Tables 8 and 9. The 1976 table included strains obtained from estab~i culture collections; these are not repeate here. In cases of uncertainty about s identification, we have tried to list strains in such a way as to give the most useful overall picture. Sources of uncertainty were explained in Section V. Except for the probable N. intermedia strains from Africa scribed in Section V, we have tried to li “undetermined” to strains that are really candidates for belonging to the N. discreta group or to some unknown rare species. The identification of a or N. intermedia strai pending on the degree of is not as certain as we would require if tbe
47
488
Totals
2181
150
6i 177 34 6 35 23 124 107 42 18 0 171 93 11 0 418 4 38 140 26 11 14 12 163 60
240
N. intermedia
312
113
; 1 0 17 0 76 0 23 0 0 46 0 8 0 0 0 13 0 0 0 0 5 0
0 1 0
370
40
0 0 0 1 7 12 1 51 6 15 0 102 2 9 8 0 0 8 0 1 8 2 3 86 0 4 4
317
137
8 0
8 47
8: 0 2 0 0 4
0 0 1 3 0 7 0 5 21 0 5 1 0 0
N. tetrasperma
Vegetation
No. of strains
Burned
N. sitophila
from
TABLE6 Collected
N. crama
Strains
44
8
8 0 0 0 4 0
: 0 0 7 0 17 0 0
ii 0 0 0 0 0 0 0
0 0
N. dim-eta groupa
Note. The 3406 isolates in this table were collected by D. D. Perkins or by accompanying colleagues between August 1968 and April 1986. a See text Section V.C.2. b See text Section V.C.4 regarding undetermined strains from Africa (Congo, Gabon, and Ivory Coast). For other countries, testing has been exhaustive, undetermined strains have been crossed successfully to some species tester, but with ascospores that were mostly or entirely defective.
: 23 1 31 14
31 1 14 23 21 6 6 16 16 27 9 39 1 30 20 12 1 63 2 8 12
Australia Bangladesh Borneo Brazil Congo Gabon Guam Haiti Hawaii India Indonesia Ivory Coast Japan Malaya (Malaysia) Malaya (Penang) New Zealand Pakistan Papua New Guinea Philippines Ponape Puerto Rico Rota Singapore Tahiti-Moorea Taiwan Thailand Truk United States (continental)
SOlKCC
No. of collection sites
Neurosporu
and most of the
182
2
4
: 0 85
:
46 8
0 0
Undetermined6
Neurospora
FROM NATURE
TABLE 7 Strains Collected from Nonbumed
Substrate No. of strains
Source
Substrate
Australia Brazil Congo Gabon Haiti Indonesia
Ivory Coast Japan Malaya Papua New Guinea Philippines
Filter mud Plywood Corncob Corncob Corncob Corncob Ontjom Temple offerings (rice, cake) Corncob Corncob Corncob Corncob Corncob
N. intermedia
N.
crassa
N. sitophila
11 3 19 3 1 15 26
-
-
13 1 7 6 53 5
-
-
1
1 1
-
-
Undetermined* 1 3 1 7 -
-I Note. The 178 isolates in this table were collected by D. D. Perkins between August 1968 and April 1986. u Most of the strains in this column are persistently sick and infertile but are probably N. intermedia as judged either by yellow color or by presence of a few defective ascospores in crosses with the very fertile N. crassa testers. From 5 to 10% of strains collected from unburned substrates are contaminated or infected, but in most cases healthy subcultures were obtained by careful transfer or by plating a few conidia. Six cultures from Papua New Guinea were growing on corncobs that had been partially burned (so it is not obvious whether they belong here or in Table 6). All have an unusual pale color, suggesting that they are all the same species; they make mostly defective ascospores with N. crassa, indicating that they are N. intermedia. Three make fertile crosses with N. intermedia testers, but the other three do not and are therefore included with the undetermined strains.
strains were to be used as examples of the species. VII.
A. Abundance
SPECIES IN NATURE
and Distribution
The search for Neurospora on burned substrates has met with great success in the moist tropics but with very little success in Mediterranean climates and in cooler temperate climates. Though the search has been less exhaustive in the temperate zones, it seems clear the Neurospora is not commonly found in areas where the warm season is dry and where winters are cold with extensive freezing. The following generalizations about species distribution ignore some very small collections from isolated sites. N. intermedia is the most abundant species on burned substrates. As shown in Table 6, it is the predominant species in many geographic
areas scattered around the globe. The other species show individual patterns of clistribution, though all overlap with each other. N. tetrasperma and N. sitophila show the greatest divergence in ecological preference, though both are abundant in Tahiti, and individual samples of both are found throughout the world. In areas farthest from the equator (e.g., New Zeal North Carolina), N. tetrasperma is th dominant species from burned substrates. This suggests adaptation to survive the cold season. N. sitophila seems least adapted to cold, since extensive collections have been obtained only from tropical areas (Maiti, Tahiti, Ivory Coast). It occurs in temperate regions mainly as a contaminant on food or on laboratory medium. In fact it is t ties of Neurospora commonly fo such substrates in North America. N. crassa, the only species of Ne~~Q~~~-
106
PERKINS
Contributed
AND TURNER
TABLE 8 Strains from Burned Substrate
No. of Source
Contributor
-
Australia
D. E. Shaw”
Brazil
M. Bjdrkman G. J. Samuels
Costa Rica Gabon Guyana
9 1 1 12
R. 0. N. G.
L. Metzenberg Ezavin C. Franklin J. Samuels
Species N. N. N. N. N. N. N. N.
intermedia tetrasperma intermedia intermedia crassa discreta tetrasperma crassa
No. of strains
group
Undetermined N. N. N. N. N.
discreta intermedia crassa sitophila tetrasperma
Undetermined Honduras India
K. S. Hsu N. B. Raju
Malaya Mexico
C. C. Ho R. L. Metzenberg
New Zealand
R. E. Beever
N. N. N. N. N.
intermedia intermedia crassa sitophila tetrasperma
Undetermined
Papua New Guinea Tonga U.S.A.
3 1 1
D. E. Shawb L. S. Olive F. J. Doe
Vanuatu
1 3
N. H. Horowitzc E. C. H. McKenzied
Venezuela
1
G. J. Samuels
Totals n Includes strains b Includes strains c Strain collected d Transmitted by
N. N. N. N. N. N. N. N. N. N. N. N.
intermedia tetrasperma discreta intermedia sitophila intermedia tetrasperma crassa sitophila intermedia crassa sitophila
group
59
239
collected by R. Borch, C. D. Jones, J. O’Rourke, collected by A. van Noaren and J. R. Walsh. by W. Scott. R. E. Beever.
ru known to many scientists and students, seems to be more restricted in range than the species already mentioned. It is frequent in samples from the Gulf of Mexico area, the Caribbean, Africa, and India. Both mating types of N. discreta were found in only a single small population in Texas, but single isolates of N. discreta are
31 1 10 1 11 2 1 61 1 1 14 4 3 8 4 3 7 19 1 2 3 2 23 1 7 3 3 2 1 2 1 5 1
and R. Grattige.
known from two other areas in the Western hemisphere (Florida, Guatemala). The other strains listed as “N. discreta group” in Table 6 cross to the N. discreta from Texas and make abundant ascospores, but the ascospores are predominantly defective and inviable. Further study is needed to determine whether this reflects a divergence
Neurospora FROM
Contributed Source
Australia
TABLE 9 Strains from Nonburned
Contributor
Substrate
D. E. A. Catcheside D. E. Shaw”
Crumpets Bee pollen basket Filter mud Bread
T. Angelb China
NATURE
Corncob
India
Z. J. Sheng’ (T. C. Sheng) Y. N. Ming R. Maheshwarid
Palau Taiwan
R. A. Lewin H. W. Li
Turkey
A. Sazci
United States
F. M. Turner H. M. Luscombe M. Revenaugh
Dying sponge Bagasse Fiber Filter mud Lab/hospital contaminants Coffee grounds Tortilla Bread Nut shell Coffee grounds Lab contaminant
M. Schechtman 0. C. Yoder
Unknown Soil
Substrate Species N. N. N. N. N. N. N. N. N. N. N. N. N. N. N. N.
intermedia intermedia intermedia intermedia sitophila intermedia sitophila intermedia intermedia tetrasperma sitophila intermedia intermedia intermedia intermedia sitophila
N. N. N. N. N. N.
sitophila sitophila sitophila sitophiia sitophila sitophila
Total
No. of strains
1 2 f 1 I 2 93
a Includes strains collected by R. Birch, G. Wilson, A. G. Hayes, D. R. Strong, L. Tilley, E. 0. Maynard, R. 0. Spalding, P. R. Downs, C. D. Jones, P. English, and A. I. Linedate. b Transmitted by D. G. Catcheside. ’ Collected by members of the Genetics Society of China at the request of Z. J. Sheng and transported from China by N. H. Giles. d Tncludes strains collected by P. Palanivelu.
sufficient to warrant splitting the group into two or more species. In areas where several Neurosporu species are present, two or even three species may be represented among the 10 colonies sampled from the same burned site. This diversity was seen most strikingly in Haiti. Interpreting the observed distribution is greatly complicated by the possibility that human activities have altered the distribution in recent times. Most sites have been in or near areas of agriculture and human habitation where Neurospora could have been introduced with crop plants from another part of the world. Perhaps molecular markers in chromosomal or mitochondrial DNA can be used to trace lineages and to indicate
centers of origin. It seems obvious that humans have played some role in dist~b~ti~g Neurospora and providing conditions for its growth, but the importance of the& role remains to be defined. B. Substrate Preference Burned substrates. At sites where vegetation has been burned or scorched and where humidity and temperature are favorable, the conidiating Neurospora species may be found growing on the remnants of many different plant species. ~ucc~e~ts and monocotyledonous plants with pithy stems and sugary sap seem to be especially good substrates, supporting prolific sws-
108
PERKINS
AND
tained growth of the fungus. Burned wood can provide a good substrate if it is freshly cut and uncured, but dried dead wood is a relatively poor substrate. Because Neurospora is so catholic in its tastes, we have not made any special effort to identify the species of plants serving as substrates. Instead, our records describe the substrate in general terms such as burned grass, tree trunk, or succulent. Nonburned substrates. A particular ecotype of N. intermedia is characterized by yellowish carotenoids and large conidia. This ecotype is almost never found on burned substrates, but it accounts for almost all samples taken from unburned substrates in tropical parts of the eastern hemisphere (Turner, 1987). The common substrates are cooked maize cobs, onchom, and waste products of sugar cane processing. Maize corncobs support prolific growth. Cobs bearing Neurospora are commonly found in tropical marketplaces, where they have been discarded after the kernels have been eaten from the cooked ear. It is not known whether the Neurospora on corncobs originates from ascospores or conidia, or whether the inoculum is airborne or possibly insect borne. Samples from the same cob appear to be from one clone, but sometimes both mating types are found on nearby cobs. Onchom (ontjom) is prepared for human consumption in West Java by cultivating Neurospora on soybean or peanut solids (see Shurtleff and Aoyagi, 1979). Numerous samples from onchom have been identified as N. intermedia (Ho et al., 1973; Perkins et al., 1976; Ho, 1986). In onchom production, the producers deliberately inoculate each batch of cakes with conidia from the previous culture. Both mating types and a variety of genotypes affecting visible traits have been recovered from the same onchom cake. Neurospora colonies were found conidiating profusely on filter mud from sugar
TURNER
cane refineries at numerous sites in Australia (Shaw and Robertson, 1980). The colonies may originate from ascospores activated by heat during the refining process, but inoculation might also involve conidia already in the environment. Even a small transfer of a single colony from filter mud often contains strains of both mating types. In temperate climates, Neurospora has been reported mainly in particular manmade habitats where suitable substrates are heated but not burned. Until the introduction of chemicals to retard fungal growth, Neurospora was a serious problem for bakeries in Europe and North America. Activated ascospores are probably responsible for infestation of bakeries (see Shear and Dodge, 1927; Moreau-Froment, 1956). Neurospora has also been found in plywood factories, lumberyards, and on substrates such as nutshells (see Tables 7 and 9). Strains from bakeries have usually been identified as N. sitophila or assumed to belong to this species. However, most of the strains collected by Yassin (1980) from bakeries and related sources in England were N. crassa. In contrast, a bakery in Germany yielded only N. sitophila. Because grain is imported, origins of the bakery contaminations are uncertain. C. Possible Subspecific
Categories
There is no indication that the yellow ecotype of N. intermedia is reproductively isolated from the orange strains of N. intermedia that are found on burned substrates. Crosses involving a strain of the yellow ecotype are typically less fertile than those of orange x orange, but the fertility is as poor for yellow X yellow as for yellow X orange (Turner, 1987). Thus there seems to be no reason for according subspecitic taxonomic status to the yellow strains. Compared to crosses between strains from the same continent, there is a slight elevation in the frequency of aborted asco-
Neurospora
FROM
spores in crosses between N. crassa from India and N. CY~SSUfrom North America. Likewise, strains classified as N. intermedia from Asia and from North America are more likely to be fully fertile with other strains from the same hemisphere. The decrease in fertility is neither complete nor consistent, however, and it does not seem to warrant formal systematic recognition. Of all the recognized species, N. interme&a and N. crussa seem closest. Chromosome pairing at pachytene is nearly perfect, and intercrosses may result in 5 or 10% viable ascospores. Classification of N. intermedia and N. crassa as subspecies would be a conceivable option. For reasons stated by Perkins et aZ. (1976), we prefer to retain the present classification as separate, though closely related, species. D. Dispersal
Putting aside the question of human influence, we are quite ignorant of natural dispersal mechanisms. What is the role of conidia? Of ascospores? Of insects? Of the atmosphere? Microconidia can be dismissed as vegetative propagules because they show low viability, are extremely fragile, and do not become airborne. Macroconidia, in contrast, are far less fragile and readily become airborne. Macroconidia are produced in very large numbers, and they are small enough to assure aerial transport over considerable distances because of the low fallout rate. They would seem to be ideal propagules for rapid exploitation of newly available resources under conditions calling for a strategy involving r-selection (density-independent selection for rapid proliferation, allowing exploitation of substrate in advance of potential competitors in an uncrowded environment) . But conidia are highly vulnerable to killing by ultraviolet light (see Schroeder, 1970). Dormancy of macroconidia is broken by moisture, and nondormant conidia are relatively short lived (Fa-
NATURE
B
hey et al., 1978). These properties would seem to limit the role of conidia in dispersal over long distances. Macroconidia are absent in all of the known homothallic Neurospora species, and they are present in all of the nonhomothallic species. This difference suggests that macroconidia play a role in cross fertilization similar to that of windblown pollen in plants such as maize. Is it possible that fertilization is a more fundamental role of macroconidia than dispersal? Honeybees (Apis mellifera) ia from N. intermedia blooms from sugar refineries (Shaw an 1980). Corbiculae (pollen baskets) were found to be filled exclusively with orange macroconidia, just as though the fungus spores were pollen. It seems unlikely fertilization of Neurospora by bees have any significant impact, however, considering the abundance of conidia transported in the air. Ascospores are larger than macroconidia but not so large as to preclude their airborne. Ascospores, with their black walls, are capable of remaini mant and viable for months or years until activated by heat or chemicals. They are far more resistant than macroconidia to k by uv light (K. C. Atwood, personal communieation). These features and others discussed below suggest the possibility that airborne ascospores play an important role in dispersal, especially over long distances. Ascospores are the only pr es produced by homothallic specie urospora (Mahoney et al., 1969; Frederick et ai., 1969; Howe and Page, 1963). Aspects of spore ejection and aerial persal of ascospores have been reviewe Ingold (197 I), Pedgley (1982)) (1973), and Tryon (1971). The following considerations suggest t a search for airborne Neurospora ascospores might be rewarding: (a) A gists who determine the incidence and fungus spores in air samples re
110
PERKINS
AND
presence of ascospores (see Gregory, 1973). Unfortunately, published records lump the ascopores of many genera. We have found no report sufficiently detailed to identify Neurospora. (b) Airborne ascospores are known to be the agents of dispersal and infection of the plant pathogen Venturia inaequalis, an Ascomycete with sexual structures similar to those of Neurospora (Hirst and Stedman, 1961a, b). Neurospora ascospores (-18 x 25 pm) are twice as large as those of Venturia, but spores that are even larger are commonly found in air samples (Gregory, 1973). (c) The trajectory of ascospores shot from Neurospora perithecia is high enough for them to be caught up in turbulent air currents (Ingold, 1971). Elongation and grooved ornamentation are expected to slow the fall of an ascospore relative to a smooth sphere of the same volume (Gregory, 1973). (d) The colonies that grow up in burned areas appear from our observations to be randomly spaced rather than clustered as might be expected if ascospores remained in the immediate vicinity of the perithecia of origin. It would be of considerable interest to know whether ascospores play the dispersal role we have suggested and to know the distances involved. Ability to obtain ascospores from the air might also provide a less biased population sample than do the methods now available. Existing programs that monitor air to obtain information on allergens and plant pathogens could perhaps also provide information and samples of living ascospores. Because Neurospora ascospores are grooved, visual recognition should be possible. Alternatively, spore samples from the air could be suspended, heatshocked, and plated, following a protocol similar to those used for recovering Neurospora from soil. Small-scale independent collection programs should also be feasible, because efftcient and inexpensive devices are available for collecting airborne particles the size of ascospores (see, for ex-
TURNER
ample, Edmonds, Lacey, 1964).
1972;
Gregory
and
E. Retention of the Sexual Phase in Homothallic Species
In the homothallic Neurospora species, each strain that originates from a single haploid ascospore is capable of carrying out the complete life cycle, including the sexual (teleomorphic) phase. Karyogamy, meiosis, and ascus development in the selfed crosses of the homothallic Neurospora species are cytologically indistinguishable from what is seen in crosses between strains of opposite mating types in a heterothallic species such as N. crassa (Raju, 1978). Yet because there is only a single haploid parent, the sexual cycle would appear to play no role in generating diversity by recombination of genetic differences. A strong case can be made (Section VIII.B.3) that the sexual cycle would be expected to disappear if it did not confer a significant advantage on the organism, resulting in its maintenance by natural selection. Since the homothallic Neurospora species retain a robust sexual phase, the question arises, what selective advantage does sexuality confer on them? It has been proposed that the original function of recombination in primitive organisms was repair of genomic damage (Dougherty, 1955; Maynard Smith, 1978), and that recombinational repair continues to be a major selective advantage that accounts for the prevalence of sexuality and meiosis in eukaryotes (Bernstein et al., 1985). Without judging the merits of the repair hypothesis, we suggest here yet another advantage of the sexual cycle that might account for retention of the perithecial phase by the homothallic species of Neurospora. In Neurospora,
the only long-lived propagules are the sexually generated ascospores, which are resistant to environmental insults and are capable of prolonged
Neurospora
FROM
dormancy under conditions unfavorable for vegetative growth. All known homothallic species are completely devoid of conidia, leaving ascospores as the only propagules. Could not the overriding advantage of the sexual phase in this special situation be its ability to produce dormant resistant spores? In Chaetomium as in Neurospora, conidia are present in heterothallic and absent in homothallic forms. But the correlation between conidiation and heterothallism is not universal, and conidia are present in both heterothallic and homothallic species of some other ascomycete genera such as Nectria and Gibberella (see Miiller, 1981; Nelson et al., 1983). VIII.
GENETIC NATURAL
A. Categories
VARIATION POPULATIONS
WITHIN
of Variation
Many classes of variants have been found in strains from nature (Table 10). The investigators who identified these variants have had diverse motivations. Most often tbey were interested in the intrinsic properties of the variant itself and its potential usefulness. However, some of the work has been directed at the organism and population levels. Several studies have been undertaken specifically to evaluate genetic polymorphism of chromosomal genes within populations. Noteworthy are investigation of protein polymorphisms, vegetative incompatibility, and recessive genes expressed in the sexual diplophase. These will be considered in more detail in Section B below. Another category of chromosomal variants consists of the segregationdistorting Spore killer genes, whose distribution and characteristics raise questions at the population, cellular, and molecular levels. These are considered in Section C. Later sections describe other categories of chromosomal variants. For example, restriction fragment length polymorphisms (RFLPs) have been found with high fre-
NATURE
119
quency in N. crassa. Investigations of wildcollected strains have concerned not only the nuclear but also the mitochondrial nome. Unanticipated results include the discovery of mitochondrial plasmid share no sequence homology with chondrial DNA and that may be transmits ted horizontally across species, and the discovery that wild strains from some areas can undergo senescence as a result of sequences inserted into mitochondrial D Mitochondrial variants are discusse Section H. B. Studies Permitting Evaluation Genetic Polymorphism within Populations
Q!
A revolution in thinking occu years ago when it was realized that populations of diploid higher organisms such as man and Drosophila carry multiple alleles at a large fraction of their loci (see Lewontin, 1973; Ayala, 1984 for reviews). The frequency of these genetic polymorphisms was too high to be explaine occurrence of new mutations. One esis proposed that the polymorp isted because heterozygotes were at a selective advantage. We can now ask whether Ne~~o§~5 with its haploid vegetative phase, res bles diploids in having high genie vat-i ty, or whether Neurospora populations are genetically more uniform than dip plants and animals, perhaps as a consequence of haploidy. Because we sample 7 to 10 colonies at each collection site we possess the material to address this question. The answer is clear. Local po~~lat~~~~ the heterothallic species N. crassa an intermedia contain a remarkably amount of genie variability-a level comparable to what is found in diploid higher organisms. Such polymorphism is co~mo for electrophoretically disti~g~isl~able isozymes, vegetative incompatibility, and
112
PERKINS
Genetic Variants Originating Variant Chromosomal genome Proteins Amylase Aryl-sulfatase P-Glucosidases
Locus
-
AND TURNER TABLE 10 in Wild-collected
Neurospora
Species”
Strains References
ars -
sit tet era, int
Cellulase
-
int
Esterases
-
int, tet
Ornithine transcarbamylase Leucyl-tRNA synthetase Invertase Nitrate reductase Perithecial protein Protease (extracellular) Nitrogen regulation
arg-12
era
Reddy and Threlkeld (1971a, b, 1972) Metzenberg and Ahlgren (1971) Mahadevan and Eberhart (1964), Eberhart and Madden (1973) Eberhart and Beck (1969), Eberhart et al. (1977) Reddy and Threlkeld (1972), Reddy (1973). Sagarra (1973), Egashira (1986) Grindle and Davis (1966, 1970)
leu-5
era
Beauchamp et al. (1977)
inv (= mig)b nit-4 pts-1
era int 8 SPP. era
Yu et al. (1971) Blakely and Srb (1962) Nasrallah and Srb (1973, 1977) Hanson and Marzluf (1975)
nit-2
sit
Grove and Marzluf (1981) Whitehouse (1942, p. 57) Horowitz et al. (1961a), Horowitz and Fling (1953), Rtiegg et al. (1982) Horowitz et al. (1961b)
(=pink)
Tyrosinase
T
era, int
Tyrosinase (regulatory) Various proteins
KY-2
sit
-
era, int, sit
Nutritional requirements Threonine Thiamine Vegetative incompatibility her loci
int
Chang et al. (1962), Reddy and Threlkeld (1971a, b, 1972) Spieth (1975)
-
era era
Perkins et al. (1976, p. 303) Leslie and Raju (1985)
het-c, -d, -e het-5-het-10
era era
Perkins (197.5), Mylyk (1975, 1976), Leslie (1987) Newmeyer (1970)
era
Perkins and Borkman (1978)
era, int
Al-Saqur and Smith (1980)
era
Wallace (1970), see Perkins
Suppressor to1 Morphology Morphology scat Resistance to toxic agents Surfactant resistant Triphenyltet tetrazolium chloride Canavanine cnr Meiosis, recombination, ascus development Sexual-phase recessives Meiosis impaired mei-l Recombination ret-I, -3 repression Synaptic sequence ss
et al. (1982)
era
Perkins (1960), see Perkins et al. (1982)
era
Leslie and Raju (1985)
era era
Smith (1975) Catcheside (1975)
era
Catcheside (1981)
Neurospora FROM NATURE TABLE Variant
Locus
Dominance of peak alleles Eight-spored ascus Spore killer Spore killer Resistance to Sk
lo--Continued
pk
sit, tet
Srb and Basl (1972)
E
tet
Dodge (1939) Dodge et al. (19%)
Sk-l, Sk-2, Sk-3 r(Sk-2), r(Sk-3)
sit, int int, era
Turner and Perkins (1979) Turner (1977), Campbell and Turner (1987)
int, era SPP.
zeta-eta
4
-
era, int, sit
Senescence
-
int
Mitochondrial plasmids
-
era, int, tet
-
int
Virus-like
particles
References
Species”
Restriction fragment length polymorphisms Nontranscribed NO 5 SPP. spacers, rDNA Sequences flanking era SS RNA genes Chromosome rearrangements, structural polymorphisms Satellite sat era Translocations Tandem duplication Mitochondrial genome Mitochondrial DNA variants
113
Russell et a/ (1984), Perkins et al. (1986a) Metzenberg et al. (1984, 1985) Perkins et al. (1986a), Newmeyer et aE. (1987) Perkins et al. (1976 and unpublished) Selker and Stevens (1987) Mannella et al. (1979), Collins and Lambowitz (1983), Taylor et al. (1986, 1987), Natvig and Jackson (1986) Rieck et al. (1982), Griffiths and Bertrand (1984), Bertrand et al. (1985, 1986), Bertrand (1986), Court ef a[. (1987) Stahl et al. (1982), Coihns and Lambowitz, (1983), Nargang et al. (1984), Natvig et al. (1984) Taylor et al. (1985), Lambowitz ez al. (1986), Nargang (1985, 1986), Pande and Nargang (1986) Tuveson and Peterson (1972)
era, N. crassa; int, N. intermedia; sit, N. sitophila; tet, N. tetrasperma. ’ See White et al. (1985) regarding allelism of inv and mig. a
recessive genes affecting the sexual phase. Genes conferring resistance to killing by Spore killer are also polymorphic in some populations. 1. Protein polymorphisms. Electrophoretically detectable allozyme variation was surveyed by Spieth (1975) in N. intermedia populations from Malaya, Papua New Guinea, Australia, and Florida. General proteins, acid phosphatases, and esterases were examined in 4 to 11 strains from each of 19 local sites in the four regions. His sur-
vey showed that genetic variation is present at a high level, and the pattern of variation within and between populations resembles that in mouse, man, and Dr~sQp~i~~~ A majority of the variation in the species is found in the local population, just as is true for outbreeding diploids . Theoretical interpretation of such striking variability in a predominantly h organism is complicated by Neuro brief but important sexual phase, during which application of diploid selection t
114
PERKINS
AND
ries is appropriate. However, Spieth (1975) suggests that “haplophase selection ought to dominate the behavior of some, if not many, allozyme loci.” In addressing the question of how selection could act during the vegetative haplophase to maintain polymorphisms, he presents a model based on environmental heterogeneity which evokes gene flow among neighboring populations that occupy shifting multiple adaptive niches. Spieth concludes that regardless of the mechanisms involved, the Neurosporu results show that “high intrapopulational genetic variation at the molecular level is not an exclusive property of species with life cycles that constrain the effects of selection almost entirely to the diplophase.” 2. Vegetative incompatibility. Differences in genes that determine vegetative (heterokaryon) incompatibility were studied by Mylyk (1976) in local populations of N. CY~SSUfrom different localities in Louisiana. As found for protein polymorphisms, an unexpectedly hir;h level of variability exists within each population. The large number of differences at vegetative incompatibility loci effectively precludes heterokaryon formation between N. crassa strains in nature. Vegetative incompatibility in N. crassa is determined by genes at numerous het (“heterokaryon incompatibility”) loci (Garnjobst, 1953; Holloway, 1955; Mylyk, 1975). An allelic difference at any one het locus is sufficient to evoke the incompatibility reaction. Without special techniques it would be very laborious to perform a precise genetic analysis because crosses involving wild strains and laboratory testers are likely to segregate for different alleles at several loci, all with similar phenotypes. This problem can be circumvented by crossing the strains under study to testers that contain duplication-producing rearrangements. Among the progeny from each such cross, one defined chromosome segment is present in duplicate, and the rest of the genome is haploid (see Perkins and
TURNER
Barry, 1977; Perkins, 1975). If a het locus i, included in the duplicated segment and i: heterozygous, a nonlethal incompatibilit! reaction occurs in every cell. Thus, the lo cus can be recognized because the incom patibility difference makes the culture sick with characteristic abnormal growth am morphology. Heterozygous duplications were used ir this way by Mylyk (1975) to detect 6 of the 10 het loci that have been mapped in Neurospora. Mylyk (1976) also used the method to test five isolates from each of three wild populations for differences at 5 het loci. He found that incompatibility differences were so numerous that no two strains of the same population were capable of forming heterokaryons with each other. Furthermore, each population was polymorphic for at least 3 of the 5 tested incompatibility loci. The results essentially rule out a role for heterokaryosis in natural populations of outbreeding species such as N. crassa. Because each culture has a virtually unique set of alleles for vegetative incompatibility, colonies remain genetically distinct monokaryotic individuals, and the vegetative unit that is exposed to natural selection is a discrete homokaryotic haploid genotype, not a heterokaryon. This feature of vegetative incompatibility has important consequences for the genetic organization and evolution of Neurospora populations (Spieth, 1975). Numerous other hypotheses have been suggested regarding the significance of vegetative incompatibility. Perhaps it protects from infectious agents (Caten, 1972; Bremermann, 1980; Hamilton, 1982) or from invasion by inferior genotypes (Hart1 et al., 1975). Its role might be to promote the initiation of sexual reproduction (Butcher, 1968; F. J. Doe, unpublished observations), perhaps by the trigger of a protease-mediated injury reaction as suggested for Podospora (see Labarere and Bernet, 1979 for references). Vegetative incompat-
Neurospora
FROM
ibility might, of course, play more than one of these roles. Polymorphism for bet genes is by no means limited to N. crussa but appears to be widespread. For example, populations of several ascomycetes that are important plant pathogens have been shown to contain vegetative incompatibility differences at numerous loci. Among the best analyzed are Cochliobotus heterostrophus (Bipolaris maydis
= Helminthosporium
maydis)
(Leach and Yoder, 1983), the cause of corn leaf blight; Gibberella fujikuroi (F’usarium moniliforme) (Puhalla and Spieth, 1985), an important pathogen of maize, rice, sugar cane, and sorghum; Endothia parasitica (Anagnostakis, 1977,1982,1983), the chestnut blight pathogen; and Ophiostoma (Ceratocystis) ulmi (Brasier, 1983a, b), the cause of Dutch elm disease. Vegetative incompatibility has hindered control of Endothia by restricting transfer of a hypovirulence agent. Transfer of a cytoplasmic factor infecting Ophiostoma is also limited by vegetative incompatibility differences in the fungus. The high specificity of self-nonself recognition that is shown by vegetative incompatibility systems is suggestive of the specificity shown by some genotypes governing susceptibility and virulence in plant hosts and fungal pathogens. It remains to be seen whether the mechanisms underlying vegetative incompatibility and pathogenicity share some common features. Vegetative incompatibility bears on plant pathology in yet another respect. In certain asexually reproducing pathogenic fungi, formae speciales and races, defined by host specificity, are congruent with vegetative incompatibility groups (Puhalla, 1985; Puhalla and Eiummel, 1983). Conventional genetic analysis is not possible in many of the pathogens. Vegetative incompatibility in Neurospora may serve as a model. 3. Recessive genes expressed during the sexual phase, A systematic search by Les-
lie and Raju (1985) revealed that most of the
NATURE
115
N. crassa strains obtained from nature carry one or more recessive genes that are expressed only in the sexual diplophase and that are detrimental to fertility when they are homozygous . Laboratory strains were constructed as testers containing selective markers crossover suppressors such that survi f, progeny from test crosses would receive most of their genome from the tested parent (Leslie, 1985). The tester strains wer crossed to isolates from N. crassa wi1 populations, and several f, progeny from each test cross were then backcrossed i dividually to the wild-collected parent a to a laboratory strain as control. The presence of one or more phase-specific recessive genes in a wild isolate was indicated if some abnormality of the perithecial phase was seen in some of the backcrosses to the wild parent (i.e., those in which the gene was homozygous) but not in the corresponding controls (where the gene remained heterozygous). Ninety-nine isolates were analyzed from 26 natural populations. Eighty of the strains contained among them 106 recessive genes that were expressed in the sexu a majority of the mutants, meiosis cus development were blocked the remainder, ascospore maturation was significantly impaired. The rn~ta~ts are thus equivalent to recessive le semilethals in higher diploid orga Intercrosses between 1200 m all showed positive complementat~o~. Clearly, many different loci must be involved. Presumably, this load of deleterious mutations is tolerated only because outbreeding is extensive. These results confirm that meiosis and sporogenesis are under complex and finetuned genetic control, and that mutation in any of a multitude of genes can impair fertility. This implies that the sexual part of the life cycle would quickly be by mutation if it were not mai diligent selection.
116
PERKINS
AND
4. Resistance to killing by Spore killer. In the study of Spore killer systems described below in Section C, we found wild strains that are resistant to killing but that are not themselves killers (Turner, 1977). There are four expected phenotypes for resistance to killing-resistant to both Sk-2K and Sk-3K, resistant to neither, or singly resistant. Resistance is determined by a gene or by two closely linked genes in linkage group III. Strains collected before 1977 have been analyzed. All four phenotypes were found in Australia, Papua New Guinea, Indonesia, and Malaya. There was very little clustering within sites. For example, among 70 strains from eight sites in Australia, each of the four phenotypes was represented at six or seven of the sites. Conclusions on intrapopulation variation. Whenever genetic variability has been carefully assayed in N. crassa or N. intermedia a very high level has been found within the local population. The genetic polymorphism is comparable in level and pattern to that in outbreeding diploid eukaryotes. It seems clear that these heterothallic Neurospora species are strict outbreeders. C. Spore Killers A mechanism which affects the genetic composition of succeeding sexual generations by inequalities of chromosome transmission is termed meiotic drive or transmission-ratio distortion. “Driven” genes contribute disproportionately to the resulting gene pool, regardless of their selective advantage for the individual organism (reviewed by Zimmering et al., 1970; Crow, 1979). Spore killer strains of Neurospora have provided a well-documented example of meiotic drive in fungi. Three distinct Spore killer factors have been obtained independently in Neurospora from different wild sources. The factors are chromosomal, and they have been
TURNER
symbolized Sk-lK, Sk-2K, and Sk-3K. Characteristics of the Spore killer systems are outlined below. Sk-2K, which is used as an example, originated in N. interrnedia and was introgressed into N. crassa for ease of study. Unreferenced information is from Turner and Perkins (1979), Raju (1979), or Turner et al. (1987). When a Spore killer strain is crossed by Spore killer (Sk-2K X Sk-2K), all eight ascospores survive. When a Spore killer strain is crossed with a normal laboratory strain or with any other strain that is sensitive to killing (Sk-2K X Sk-2’), eight ascospores are formed in each ascus, but four of the spores degenerate. The survivors all carry the killer allele-it is the normal wild type that is killed. Killing does not depend on which strain is used as female and which as fertilizing parent-reciprocal crosses are alike. Sk-2K shows 100% first-division segregation. No difference between Sk-2K and Sk-2’ has been detected in the vegetative phase. Heterokaryons containing nuclei of both types in the same cytoplasm are vigorous and healthy. Likewise, meiosis and ascus differentiation are normal in heterozygous crosses where killing will occur. Abnormalities are seen only after Sk-2’ nuclei are sequestered by being enclosed in an ascospore wall. The prospective lethality is not irreversible until this time, because Sk-2’ nuclei which would otherwise die are rescued if an Sk-2K nucleus or chromosome is enclosed within the same ascospore wall. When Sk-2K is crossed with a non-killer strain, crossing over is virtually eliminated in a region at least 30 units long which extends to both sides of the III centromere. Marker sequence and crossing over frequencies are normal in Sk-2K x Sk-2K (Campbell and Turner, 1987). Sk-3K resembles Sk-2K in its mode of killing, its map location, and its effect on recombination. But Sk-3K differs from Sk-2K in specificity of killing and resistance. Sk-
Neurospora
FROM
3K does not kill itself but is sensitive to killing by Sk-2K, and vice versa. In crosses between Sk-2K and Sk-3K, all ascospores abort in most of the asci. Both Sk-2K and Sk-3K are rare. They have been identified only in eastern hemisphere populations of N. intermedia. Sk-2K has been found four times, and Sk-3K once. Laboratory strains and most of 1400 tested wild-collected isolates of N. intermedia and N. crassa are sensitive to both Sk-2K and Sk-3K, but neutral (resistant) strains that neither kill nor are killed are also found in nature (Turner, 1977). Strains resistant to Sk-2K, to Sk-3K, or to both are common in N. intermedia in the eastern hemisphere. Though no Spore killer has been found in N. crassa, the only resistant isolate of any kind that has been found in the western hemisphere is a N. crassa strain resistant to Sk-2K. Resistance segregates as a single chromosomal gene, r(Sk-2)-l, in IIIL. A gene from N. intermedia conferring resistance to Sk-3K maps in the same region. Recombination of markers in the III centromere region is not affected by the genes conferring resistance to killing-it is blocked in Sk-2K x r(Sk-2)-l but not in Sk2’ X r(Sk-2)-I, and similarly for r(Sk-3)-l with Sk-3K (Campbell and Turner, 1987). Both Sk-lK and Sk-IS are widely distributed in wild-collected N. sitophila, but the large samples from Haiti and the Ivory Coast are ail Sk-IS. Sk-lK and Sk-IS are sometimes found in the same geographical region, and in Tahiti they were found together in the same local population. No resistant non-killers have been identified. Attempts to introgress Sk-lK into N. crassa have failed. The chromosomal location of Sk-lK is unknown, as is its effect on recombination. In addition to their theoretical interest, Spore killers have had practical applications in the laboratory, enabling the efficiency of tetrad analysis to be increased significantly (Perkins et al., 1986b).
NATURE
117
Chromosomally based Spore killing systems are known in two other Pyrenomycetes. Two unlinked ascospore-abortion genes in Podospora anserina (Padieu and Bernet, 1967) behave exactly as would be expected if their mechanism of killing were the same as for Sk-2K and Sk-3K in Nernrospora. A majority of wild isolates of 6. J% jikuroi (F. moniliforme) contain a Spore killer allele (Kathariou and Spieth, 1982). The fungal Spore killers resemble meiotic drive systems in higher plants and animals, which are typically manifested postmeiotitally by differential survival of spores or gametes (see Zimmering et al., 1970 review). The Spore killer phenomenon raises some evolutionary questions for w it may be difficult to obtain answers. Is it possible that today’s predominant normal wild type in each species is carrying a killer allele that was once a newly minority type but that has since eliminate the previously ubiquitous sensitive allele‘? Could it be that Spore killers play a role in promoting genetic isolation between incipient species, as might be expected if ent, mutually sensitive, Spore killer genes similar to Sk-2K and Sk-3K had beco fixed during the differentiation of nascent sibling species, with each population carrying one of the killers but not the otbe~? It was possible to return and res one site where Spore killer strains we found during an earlier visit. In 1968, bo Sk-2K and Sk-3K were represented alone six isolates collected from bnr vegetation at a site near Rouna Falls, ua New Guinea. Not a single killer strain was found in a sample of over 100 isolates colle years later from the same site. Killer were also absent from samples taken elsewhere in New Guinea in 1983. Thus it seems that Sk-2K and Sk-3K are not replacing their sensitive counterparts in nature; apparently the killer alleles are being in check by counterselection or resistance.
118 D. Genes Specifying
PERKINS
Ribosomal
AND
RNA
Wild-collected strains have been used extensively in studying the gene families that specify ribosomal RNA in Neurospora. As in other eukaryotes, the 17S, 5.8S, and 25s components of cytoplasmic ribosomes are processed from a larger precursor molecule which is coded by a family of tandemly repeated chromosomal genes that specify ribosomal RNA. The repeats are separated by nontranscribed DNA segments (Free et al., 1979; Cox and Peden, 1979). The number of repeats is around 200 in N. crassa, but ranges from approximately 155 to 265 in different strains (Rodland and Russell, 1983a, b). The ribosomal RNA genes are located in the nucleolus organizer region (Perkins et al., 1986a) at the left end of linkage group V (Barry and Perkins, 1969). The sequence organization of these repeats has been examined by Russell et al. (1984) in strains from different geographical regions, including representatives of five species. Restriction enzyme digests were probed using cloned segments of known constitution. Transcribed portions of the ribosomal DNA appear to be invariant. In contrast, the nontranscribed spacer segments are variable from strain to strain, although they are homogeneous within each individual strain. The spacer variation between N. crassa isolated from different geographical regions is as great as that between different species. It has not been determined whether similar variability in nontranscribed DNA spacers is found among the individuals of a single local population. Genes speciJLing 5.7 RNA.
differs having demly widely
Neurospora
from most other eukaryotes in not its 5s gene family clustered or tanrepeated. Instead, the 5s genes are dispersed (Free et al., 1979; Selker et al., 1981). There are about 100 5s genes per genome. Restriction fragment length polymorphisms (RFLPs) have been used to map the individual 5s genes at loci scat-
TURNER
tered through six different chromosomes (Metzenberg et al., 1984, 1985). Although a majority of the 5s genes code for a 5s RNA sequence called alpha, several alternate sequences also exist (called beta, gamma, etc.). Similar 5s RNA heterogeneity is also found within different Neurospora species and in related genera (Selker et al., 1985a). Only one of 23 EcoRl restriction fragments from an Oak Ridge strain of N. crassu, cloned on the basis of positive hybridization with 5s RNA, contains more than one 5s RNA gene. The exceptional clone contains a pair of 5s genes or pseudogenes, “zeta” and “eta,” embedded in a short direct tandem duplication in linkage group I (Selker and Stevens, 1985). Unlike Neurospora 5s RNA genes generally, the zeta and eta genes are heavily methylated. However, many strains of N. crassa and strains of N. tetrasperma, N. sitophila, and N. intermedia have one instead of two copies of the homologous DNA, and when there is a single copy it is unmethylated (Selker and Stevens, 1987). It has been suggested that families of small genes such as 5s RNA genes may be more functional or better preserved in Neurospora in a dispersed rather than a tandemly repeated arrangement (Selker et al., 1985b). E. U. Selker and R. L. Metzenberg (personal communication) have surveyed wildcollected N. crassa strains from seven widely separated wild populations and have compared them to a standard laboratory strain. All the strains show differences in the size of the restriction fragments that contain their 5s genes. This means either that the genes are in different chromosomal locations in the different wild strains or that the chromosomal regions flanking the individual 5s genes have diverged in DNA sequence. The latter hypothesis was supported by experiments in which regions flanking 5s genes in the standard laboratory strain were used to clone homologous seg-
Neurospora
FROM
ments from a different wild strain. Each of the new clones was found to contain a 5S genein the sameposition as in the standard, indicating that deletion or insertion of 5s genes is not occurring at detectable frequency (Morzycka-Wroblewska et al., 1985).
NATURE
to detect RFLPs among wild strains representing several species. This method can apparently provide a far more sensitive measureof genetic relatednessthan is sible with isozyme electrophoresi DNA:DNA solution hybridization. F. Genes Controlling
E. Restriction Fragment Length Polymorphisms
11
Re~om~i~atio~
In N. crassa, the recombination cy in specific map intervals varies tally in crosses of different parenting. D. 6. Catchesideand his associateshave shown that this variation is due to a series of ret genesthat precisely control meiotic recombination in specific chromosome regions to which they are not linked (sum ries in Catcheside, 1977; Fincham et al., 1979).Local control of recombination also involves a cis-acting element cog (recognition), which is believed to r for initiating recombinatio joining structural gene (see 1986).Different alleles of these regulatory geneswere discoveredin existing laboratory stocks and presumablyoriginated in different wild ancestors.The probable or&i of someof them can be traced back throug pedigrees to two original collections in Louisiana (Catcheside,1975;Newmeyer et al., 1987).All three known alleles of re have been shown to be present in w collected stocks (D. G. Catcheside, personal communication). Recombination can also be aftecte variants of a different type called ss aptic sequence),which have beenfoxed at or nearthe nit-2 locus in wild-colIecte~ and laboratory strains of N. crassa. nation in the nit-2 locus is reduce alleles are heterozygous (Ca 19sa>.
Genetic polymorphisms that can be detected by electrophoreticdifferencesat the protein level are abundant (Spieth, 1975). DNA-level variants are expectedto be even more abundant.This expectation has been confirmed (Metzenberget al., 1984; Natvig et al., 1987). For example, although the wild-collected strain Mauriceville-lc A is completely fertile with the laboratory wild type Oak Ridge a and is phenotypically indistinguishablefrom the inbred Oak Ridge strains, it is nevertheless differentiated from Oak Ridge by thousands of nucleotides throughout the chromosomes, as demonstratedby differencesin sites subject to cleavageby restriction endonucleases. The first use of RFLPs to map chromosomal genes in Neurospora was with cloned DNA segments containing the 5s RNA genes(Metzenberget al., 1984,1985). The RFLP method has since been widely used for genetic mapping, e.g., by Berlin and Yanofsky (1985),Orbach et al. (1986), Akins and Lambowitz (1985),and Stewart and Vollmer (1986). RFLPs in ribosomal RNA gene spacershave provided markers for analyzing deamplification of ribosomal RNA genes (Rodland and Russell, 1983a) and for analyzing a recombinationally generated translocation (Perkins et al., 1986a). Another application of restriction analysis is to provide information on phylogenet- 6. Chromosome Rearrangements ic relationships. Natvig et al. (1987)have Structural variants suchas translocatio~s used cloned single-copy nuclear DNA sequencesfrom a N. crassa reference strain recombine meiotically with standard sein combination with Southernhybridization quenceto produce deficiencies, which are
120
PERKINS
AND
detected in our screening method by the presence of inviable white ascospores in predictable numbers and patterns (Perkins and Barry, 1977). Reciprocal translocations are especially easy to detect in Neurospora, and hundreds have been obtained following mutagenesis in the laboratory. Only four reciprocal translocations have been identified among all our wild-collected isolates, however (Perkins et al., 1976 and unpublished). Each of these translocations was present in a population that also included the standard sequence. Additional rearrangements, in other wild isolates, may have gone undetected, because even when crosses to a tester are fertile enough to determine species, other causes of ascospore abortion are often present that would mask the additional abortion caused by a chromosome rearrangement. Among the more recent collections many crosses with aborted ascospores remain to be analyzed. No paracentric inversions have been detected either in wild strains or among rearrangements originating in the laboratory. Perhaps this reflects our inability to recognize them (see Perkins and Barry, 1977). Chromosome complements of all 10 Neurospora species appear to be similar, judging from pachytene karyotypes observed after staining with hematoxylin (Raju, 1978, 1980). Pachytene morphology has been examined in detail only for N. crassa and N. intermedia and for the intercross between them, using aceto-orcein. The two species do not differ by any obvious chromosome rearrangements, judging from pairing of homologs at pachytene in the interspecific cross (Perkins et al., 1976). There is no evidence that chromosome rearrangements are responsible for the fertility barriers between species. A small nucleolus satellite, found in some laboratory strains of N. crussa, has provided a cytological marker for genetic mapping of the nucleolus organizer (Barry and Perkins, 1969) and the genes that specify ribosomal RNA (Perkins et al., 1986a). The
TURNER
satellite has been traced to material col lected in Louisiana in 1943 (Newmeyer ei al., 1987). Satellites have not been seen in N. crassa from other sources or in other Neurospora species. However, only a few wild-collected strains have been examined cytologically in such a way that a satellite would be revealed; critical examination is too laborious to allow a broad survey. H. Mitochondrial
Variants
Extranuclear genetic determinants are transmitted maternally in N. crassa (Mitchell and Mitchell, 1952; Manella et al., 1979). It is reasonable to assume that maternal transmission is the rule in the other heterothallic species where fertilization also occurs via a trichogyne, and that maternal transmission occurs even in N. tetrasperma when homokaryotic strains of opposite mating types are used as male and female parents. (G. Bistis [personal communication] has observed that protoperithecia of homkaryotic A and a strains of N. tetrasperma produce trichogynes which orient their growth toward cells of the opposite mating type, just as though the species were heterothallic.) Mitochondrial DNA. Strain differences are not limited to the chromosomal genome. Initially, two types of mitochondrial DNA were identified by restriction analysis of laboratory strains (Bernard et al., 1976). Their probable origins were traced by pedigree to ancestral strains from two different wild sources (Mannella et al., 1979). Additional structural variants have since been identified in wild-collected strains. There is much variability, consisting in part of nucleotide substitution but predominantly of insertions and deletions (Collins and Lambowitz, 1983; Taylor et al., 1986). Taylor et al. (1986) have characterized mitochondrial DNA in N. crassa strains from different parts of the world. Length mutations are frequent and are correlated with geographic distribution. Site changes are also present
Neurospora
FROM
but are infrequent. Because of the length mutations, mitochondrial DNA may range from 60 to 78 kb in different isolates. The insertions or deletions are typically located outside the region that encodes ribosomal or transfer RNA. Introns in the gene encoding cytochrome oxidase subunit 1 are commonly involved, and as many as four introns may be present (Collins and Lambowitz, 1983). Senescence. Neurospora strains from most wild sources are potentially immortal and will continue growing indefinitely. A systematic survey of wild-collected strains revealed that certain N. intermedia strains from Hawaii were exceptional in showing a limited duration of vegetative growth (Rieck et al., 1982; Griffiths and Bertrand, 1984). Upon the onset of aging, cytochromes become abnormal and mitochondrial DNA is seen to be altered by insertion of a 9-kb DNA segment (kal DNA) into the mitochondrial chromosome. Each inserted kal DNA sequence is flanked by long homologous inverted repeats of mitochondrial DNA (Dasgupta et al., 1987). The altered mitochondria accumulate during growth, leading to respiratory deficiency and death (Bertrand et al., 1985). Deletion or partial deletion of the kal insertion may change the pattern of aging (Myers and Griffiths: 1985). The kal DNA sequences are initially associated with the nucleus. However, kal DNA is transmitted to progeny only through the female parent and never through the male (Bertrand et al., 1986; Bertrand, 1986). A N. crassa strain from India contains a structurally different linear plasmid which, like kal, induces senescence by inserting in mitochondria (Court et al., 1987). Plasmids. Circular 3.6- to 5.2-kb plasmids were first found in the mitochondria of wild N. crassa and N. intermedia strains which were being screened for differences in mitochondrial DNA (Collins et al., 1981; Stohl et al., 1982). Mitochondrial plasmids were also found in wild-collected N. tet-
NATURE
121
(Natvig et al., 1984). The plasmids show little or no sequence ho with Neurospora mitochondrial However, weak homology betwe mid and mitochondrial DNAs has tected in one exceptional instance by Pa et al. (1987). No biological function has been identified for the plasmids. Codon usage and conserved sequence elements of the best studied plasmid resemble those of Group I mitochondrial DNA introns (Nargang et al., 1984), but none of the Neurospora plasmids has been found integrated in mitochondrial DNA of normal strains (Collins et al., 1981; Stohl et al., 1982; Narga~g et al., 1984; Nargang, 1986). For reviews, see Nargang (1985), Kinsey (19856, and Lambowitz et al. (1986). A survey of many Neurospora strains by S. J. Vollmer (personal communication) has shown that plasmids are cornrno~~~ present and that more than one class of plasmid may coexist in the same strain. These plasmids were inherited maternally and were not transmitted from the male (fertilizing) parent in reciprocal crosses, as had also been shown for other plas Collins et al. (1981) and Stohl et aE. ( Plasmids in different Neurospora s may show sequence homology wi other, while different plasmids i species may not (Natvig et al., presence of identical or hornol~g~~s plasmids in three species suggests that mission can sometimes occur ind dently of the host mitochondria (Taylor et al., 1985). J. W. Taylor and G. May (personal communication, 1987) have dejected paternally derived plasmid DNA but significant fraction of progen imental crosses between strains crassa. Since mitochondria are deriv from the maternal parent, the pla thus transmitted independently of chondria. Akins et al. (1986) impaired mutant de mid-bearing strains. rasperma
122
PERKINS
AND
mids in the mutants have acquired inserts of mitochondrial DNA; mitochondria containing an altered plasmid outcompete normally functioning mitochondria (i.e., they have become suppressive). Also, mitochondrial DNAs have acquired plasmid sequences in at least some of the mutants. I. Variants
of Other Types
Many variants that have been recovered from the wild-collected strains are not related in any direct way to questions of population variability or evolution but have been of interest for other reasons. Numerous chromosomal loci have been identified using allelic differences that originated from wild material rather than as mutations in the laboratory. Examples in addition to those described above are cnr, mei-1, pts, sar, scat, T, and tet (see Perkins et al., 1982). Variants from some of the wildcollected strains produce altered proteins that have been used in biochemical studies. One N. intermedia isolate from Javanese oncham (Tjisarua, P760) resembles the per1 mutant of N. crassa, which makes perithecial walls devoid of black pigment when the strain is used as female parent. The diverse uses to which a single wildcollected isolate may be put are illustrated by the strain Mauriceville-lc A. This strain of N. crassa (P538, FGSC No. 2225) was collected in 1972 in Texas. It is identical in appearance to the standard Oak Ridge (OR) laboratory strains, and intercrosses between the two are highly fertile. Pachytene pairing is good in the intercross of Mauriceville with OR, so the chromosomes are apparently isosequential. Yet, genetic differences present in Mauriceville-lc A have had important applications: (a) A new lea-5 allele from the Mauriceville strain results in altered mitochondrial leucyl-tRNA synthetase (Beauchamp et al., 1977). (b) The Mauriceville strain contains a 3.6-kb circular plasmid which has been completely se-
TURNER
quenced and subjected to intensive study (Collins et al., 1981; Nargang et al., 1984; Akins et al., 1986). The plasmid was discovered during a survey of mitochondrial DNA polymorphisms (Collins and Lambowitz, 1983). (c) Chromosomal DNA of the Mauriceville strain differs from the OR standards by nucleotide differences at a very large number of sites. This led to its choice as one parent in the development of a method for mapping cloned DNA fragments using restriction fragment length polymorphisms as genetic markers (Metzenberg et al., 1984, 1985; Section E, above). (d) A strain derived from Mauriceville-lc has been used as a transformation host to demonstrate the existence of a signal at the zeta-eta locus, causing DNA methylation de nova (Selker et al., 1987). IX.
TRANSFER SPECIES
OF TO
GENES
FROM
ONE
ANOTHER
We have used reproductive isolation as the primary criterion for distinguishing Neurospora species. That isolation is not always complete, however-at least not in the laboratory. While N. discreta has never produced viable ascospores in an interspecific cross, other combinations of the nonhomothallic species have all produced at least occasional hybrid progeny when just the right strains were selected as parents, The ability to produce hybrid progeny does not, of course, invalidate the species status of the parents, whether for fungi, plants, or animals. In flowering plants, for example, crossing and introgression can occur even between distant taxons (Anderson, 1949). Once an fi is obtained from a cross between different Neurospora species, successive backcrosses are typically more fertile, and desired markers can readily be put into the genetic background of a recurrent backcross parent. Progeny from crosses between Neurospora species were first obtained by Dodge
123
Neurospora FROM NATURE
(1928). Many genes have since been transferred for various purposes. Both naturally occurring differences and mutant alleles of laboratory origin have been introgressed. Transfers from N. sitophila to N. crassa (and vice versa) have been the most extensive (Dodge, 1931; Finchman, 1951; ScottEmuakpor, 1965; Srb and Jarolmen, 1967; Mishra and Threlkeld, 1967; Grindle and Davis, 1970; Srb and Basl, 1972; Perkins, 1977a). Genes have also been introgressed between N. intermedia and N. crassa (Horowitz et al., 1961a, b; Blakely and Srb, 1962; Srb and Jarolmen, 1967; Turner and Perkins, 1979), between N. tetrasperma and N. sitophila (Dodge, 1928), and between N. tetrasperma and N. crassa (Johnson and Srb, 1962; Howe and Haysman, 1966; Metzenberg and Ahlgren, 1971, 1973). For some purposes, two steps have been used, as by Metzenberg and Ahlgren (1973), in taking mating type from N. tetrasperma through N. intermedia into N. crassa. Srb (1958) transferred a cytoplasmic mutation SG from N. crassa into N. sitophila and N. intermedia by recurrent crosses using SG as protoperithecial parent. Similarly, Reich and Luck (1966) transferred the mitochondrial genome from N. crassa into N. sitophila, taking advantage of the fact that mitochondria are transmitted only through the female line. Transient hyphal anastomoses have been used to transfer the kal senescence element, mitochondrial DNA, and a mitochondrial plasmid from N. intermedia into N. crassa (D. A. Court and H. Bertrand, cited by Bertrand, 1986; Cheng et al., 1987). Little is known about hybridization and gene transfer between Neurospora species in nature. Opportunities certainly exist, because isolates belonging to different species are frequently found growing in close proximity at the same site. The presence of identical or similar plasmids in strains of different species suggests that horizontal transfer is a natural occurence (Taylor et al., 1985).
X. CONCLUSION:
NEUROSPORAIN
POPULATION EVOLUTIONARY
AND GENETICS
Aspects of Neurospora in relation to the broader aspects of population genetics an evolutionary biology have been discusse from different viewpoints by Spieth (1975) Leslie (1981), Leslie and Raju (1982), Natvig et al. (1984), Taylor et al. (19 and Taylor (1986). The fungi are now considered a separate eukaryote kingdom (Whittaker, 1969). Nuclei are predominantly haploid in the fungi, genomes are small, and lifestyles are vastly different from those of the diploi and plants with which population genetics as a discipline has been largely conc~r~ed~ Fungal populations should yield sig~~c~~t new information and ideas. Yet the fungi have been largely ignored in the literature of population and evolutionary genetics, chiefly because essential information on fungal populations has been lacking. N. crassa is the most studied ~ete~ot~a~lit euascomycete species, with ~urner~~s advantageous features for population analysis. The genus Neurospora includes species with diverse breeding systemsheterothallic outbreeders, the homothallic N. tetrasperma, and horn~tba~~ lit inbreeders. As a typical Pyre~omyce~c, Neurospora represents a subgroup of euascomycetes that includes many important plant pathogens as well as saprophytes with diverse morphologies and adaptations. We are hopeful that the Neuro work reviewed here will encourage studies in the genetics of fungal popul and will contribute to an increased a~pr~c~~ ation of the potential cont~but~ons of fungi. DEDICATION
AND ACKNOWL~~~ME~T~
B. 0. Dodge first recognized the advantages ofNew demonstrated its usefulness for ex~e~irnent~ research, and made intra- and interspee& crosses for comparison. This paper is dedicated to his memory on rospora,
124
PERKINS
AND TURNER
the 60th anniversary of the description of Neurospora (Shear and Dodge, 1927) (see Robbins, 1962; Beadle, 1973; Lindegren, 1973). We are grateful for help from many persons who have participated in collecting or have made collecting possible. The help of individuals who aided in collecting before 1975 has been acknowledged by Perkins et al. (1976). We are indebted to many persons for help since that time. In Brazil: Joao Lticio de Azevedo and Jose Branco de Miranda Filho (Piracicaba). In Papua New Guinea: James Croft and Karl Karenga (Lae); Allen Allison (Watt); James Nasa (Bayer River); D. Kari, Alain Ross, Dickson Bailand, and Charles Yowana (Port Moresby). In Australia: Dorothy E. Shaw (Brisbane). In New Zealand: Ross E. Beever (Aukland). In India: R. Maheshwari, P. V. Balasubramanyam, and P. Palanivelu (Bangalore); K. Dharmalingam, P. Gunasekaran, and Kalaga Ramakrishnan (Madurai); T. V. Damodaran and K. M. Marimuthu (Madras). In Thailand: Visut Bamai, Charoenwitt Hankaew, and Yingsak Swasdipanich (Bangkok). In Malaya: Adenan Jaafer and Tan Sai Tee (Penang); Ho Coy Choke, Fong Foo Woon, and Toh Yow Pong (Kuala Lumpur). In Singapore: Nga Been Hen. In Borneo: Chuah Ching Geh (Kuching); Ghazally Ismael and John Beaman (Kota Kinabalu). In Haiti: Nancy Edling and Gary Edling. The 1985 collecting trip in West Africa was arranged by George Rizet, Denise Marcou, and Pascal Lissouba. Collecting in the Ivory Coast was aided by Gilles Bezancon and D. Le Pierres (Adiopodoume); F. Anthony (Divo); J. Louam (Man); K. M. Miezan (Bouake); in the Congo by Evelyne Gamier-Sillam (Dimonika); and in Gabon by Paul Posso, Olivier Ezavin, Fabienne Beurel, and Christophe Le Houcq (Makokou). We are also deeply grateful for strains collected independently and given to us by the persons named in Tables 8 and 9. Allan Wheals, University of Bath, United Kingdom, kindly sent isolates collected from bakeries (primarily in England) by M. S. B. M. Yassin. Ann S. Fairfield has carried out and helped to improve laboratory procedures. We also thank Virginia C. Pollard, Adam M. Richman, and Joseph L. Campbell for assistance in the laboratory. Dorothy Newmeyer has made many valuable suggestions during preparation of the manuscript. We are grateful to Philip T. Spieth, John W. Taylor, John F. Leslie, and Eric U. Selker for discussion and comments. This review was drafted during a visit by the senior author to the Department of Physiology, Carlsberg Laboratories, Copenhagen, Denmark; the hospitality of Diter von Wettstein and his colleagues is deeply appreciated. This work was supported by Public Health Service Research Grant AI-01462 and Research Career Award K6-GM-4899. The Fungal Genetics Stock Center,
whose holdings include many of the strains reportec here, is supported by National Science Foundatior Grant BSR-8546273. Field work of G. J. Samuelr (DSIR Plant Sciences Division, Auckland, New Zealand) in South America was supported by Grant No. 2736-83 to him from the National Geographic Society and by Project Flora Amazonica. Collecting and writing by DDP in 1983-1985 was carried out during tenure of a Guggenheim Foundation Fellowship. Note added in proof. Following molecular cloning of the A mating type allele of N. crussa (Volhner and Yanofsky, 1986), DNA sequences have been identified that are unique to each of the two mating types, A and a, and are present as a single copy per haploid genome (Glass ef al., 1988). Using these as probes, Glass et al. have shown that each of the homothallic species listed in Tables 1 and 2 retains sequences homologous to one or both of the mating type alleles N. terricola hybridizes with both A- and a-specific sequences, N. galapagosensis only to a-, and the others only to A-specific probes. An additional homothallic isolate, D301 from Dominica, West Indies, provisionally designated N. dominicana (L. H. Huang, personal communication), also contains sequences homologous to the N. crassa A mating type but not to a. REFERENCES
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128
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AND TURNER
Variation
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629
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MYCOLOGY
12, 91-131 (1988)
REVIEW eurospora from Natural Populations: Toward the Populati Biology of a Haploid Eukaryote DAVID D. PERKINS AND BARBARA C. TURNER Department
of Biological
Sciences,
Stanford
Accepted for publication
University,
Stanford,
California
94305
December 4, 1987
PERKINS, D. D., AND TURNER, B. C. 1988. Neurospora from natural populations: Toward the population biology of a haploid eukaryote. Experimental Mycology 12, 91-131. Natural populations of the ascomycete Neurospora have been sampled systematically throughout much of the world, and the haploid strains from colonies in nature have been characterized genetically in the laboratory. Our findings are described in the context of a broader review of wild-collected strains, their uses, and their significance for population genetics. Visible Neurospora colonies found on recently burned vegetation are usually unique in genotype. More than three-fourths are pure strains originating from a single ascospore; the remainder can be purified. Thus, despite the potential for clonal propagation, these colonies provide effective population samples comparable to those collected for higher plants and animals. Over 3900 isolates from burned substrates have been analyzed from over 500 collection sites, mostly from tropical and subtropical regions. These strains have been assigned to five species-four heterothallic species with eight-spored asci and one pseudohomothallic species with four-spored asci. Each species has a unique pattern of distribution, but each overlaps with all the others in one or another part of its range. All of these species are similar in vegetative morphology, with orange or yellow-orange conidia. All have two homologous mating types, but the different species are reproductively isolated from one another. Fertility in crosses with reference strains has provided a reliable and convenient criterion for species classification of heterothallic strains. The species of a newly obtained haploid strain is determined by finding a tester strain with which it is fully fertile and produces predominantly viable ascospores. Viable ascospores are extremely rare for most interspecific combinations, but genes can nevertheless be transferred by matings among all but one of the nonhomothallic species. Abundant but mostly inviable ascopores are produced by some interspecific combinations. Karyotypes, karyogamy, and meiotic chromosome behavior are similar for all the known Neurospora species. There are seven chromosomes and a single terminal nucleolus organizer. This pattern also applies to the five eight-spored homothallic Neurospora lines that were designated by their discoverers as different species on the basis of ascospore morphology. These homothallic lines all lack orange pigment and are devoid of conidia. They were obtained by enrichment from soil samples and would not have been obtained by our collecting methods, which rely on visibility in the field. Examination of wild-collected strains of N. crassa and N. intermedia has revealed a wealth of intraspecific genetic variation. Genetic polymorphism of isozymes in local populations is comparable to that in outbreeding higher animals and plants. DNA restriction fragment length polymorphisms are also abundant, as are differences at vegetative (heterokaryon) incompatibility loci and recessive genes that adversely affect one or more stages of the sexual diplophase. Chromosomally located factors, called Spore killer, act in the sexual phase to produce meiotic drive. The four Spore-killer-sensitive ascospores in every ascus are killed in crosses of sensitive x killer, but all eight ascospores remain viable in crosses of killer x killer and sensitive x sensitive. Mitochondrial genomes of wild strains differ in both length mutations and nucleotide substitutions. Many isolates contain mitochondrial plasmids. A few strains have been found to undergo senescence following insertion of a foreign element into mitochondrial DNA. o 1988 Academic PZSS, I~C.
91 0147-5975188 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved
92
PERKINS TABLE
OF
AND
TURNER
CONTENTS
Abstract I. Introduction II. Characteristics relevant to collecting and identification III. Collecting methods A. Visible colonies on burned substrates B. Visible colonies on unburned substrates C. Cultures from soil samples IV. Laboratory culture and preservation V. Determination of species A. Rationale for diagnostic criteria B . Laboratory procedure C. Effectiveness of species tester strains and the testing procedure D. Mixed samples VI. Scope of the collections VII. Species in nature A. Abundance and distribution B. Substrate preference C. Possible subspecific categories D. Dispersal E. Retention of the sexual phase in homothallic species VIII. Genetic variation within natural populations A. Categories of variation B. Studies permitting evaluation of genetic polymorphism within populations 1. Protein polymorphism 2. Vegetative incompatibility 3. Recessive genes expressed during the sexual phase 4. Resistance to killing by Spore killer C. Spore killers D. Genes specifying ribosomal RNA E. Restriction fragment length polymorphisms F. Genes controlling recombination G. Chromosome rearrangements H. Mitochondrial variants I. Variants of other types IX. Transfer of genes from one species to another X. Conclusion: Neurosuora in nonulation and evolutionary genetics Dedication and Acknowledgments References
I. INTRODUCTION
Well over 5000 papers have been published on research with Neurospora since the genus was described in 1927 by Shear and Dodge. Most of these appeared after 1941, when Beadle and Tatum used Neurospora to obtain the first nutritional mutants. Most of the publications concern experimental work in the laboratory and have employed strains of Neurospora crassa that were derived from only five isolates originally collected in Louisiana. In contrast to the extensive knowledge
91 92 93 95 9.5 96 96 97 97 97 99 101 102 103 105 105 107 108 109 110 111 111 111 113 114 11.5 116 116 118 119 119 119 120 122
122 123 123 124
from laboratory experiments, very little was known until recently about Neurosporu in nature. The distribution, systematic status, ecology, or genetics of natural populations had not been investigated, and the few natural isolates that were available had been acquired hit or miss. We have tried to remedy this situation by devising effective collecting methods for the conidiating species and using them to sample Neurospora populations in several parts of the world. In the laboratory, we have classified the collected strains and carried out genetic analyses. In this review,
Neurospora
FROM
the main findings regarding occurrence and variability are summarized, laboratory investigations for which they have proved useful are described, and some of the unanswered questions are discussed. Many of the findings have not previously been published . We have shown that natural populations of Neurospora can be studied in such a way as to provide comparisons with the diploid higher eukaryotes with which population geneticists have been largely concerned. Neurospora is truly eukaryotic, and it undergoes a moderate amount of differentiation. Chromosome organization, meiosis, and genetic recombination are basically similar to those of plants and animals. Each strain of single-ascospore origin can be defined as an individual organism. Populations of heterothallic Neurospora species are polymorphic for vegetative incompatibility genes that prevent heterokaryon formation. Therefore haploid clones persist as distinct individuals. Thus, Neurospora is a useful organism for investigating the consequences of a predominantly haploid life cycle and for making comparisons with organisms whose life cycle is predominantly diploid. Significant differences are to be expected between fungi and the plant and animal kingdoms because of the great phylogenetic distance and because of the very different genome sizes and life styles. On the other hand, numerous genetic similarities exist between outbreeding populations of Neurospora and those of plants and animals For example, the outbreeding Neurospora species possess a high level of genetic polymorphism. Meiotic drive systems are present in nature. Natural populations contain numerous recessive alleles that act only in the sexual phase; these alleles are comparable to recessive lethal mutations in diploid organisms. In spite of the advances reviewed here, knowledge of the population genetics and ecology of Neurospora and of fungi in general is extremely limited compared to that of animals and plants. It is hoped that this
93
NATURE
review will identify areas of fungal population biology where research is most neede and where it is now feasible. The beginnings of our work on collected Neurospora strains were rep by Perkins et al. (1976), who desc methods for collecting and testing ne lates, outlined criteria for determining species of newly acquired haploid cultures, traced the derivation of authent ties-tester strains, and summari based on population samples first 6 years of our investigation. present review covers findings during entire B-year period since our study began, and is based on over 4000 isolates more than 650 sites. Developments in odology are outlined, and numerous uses of our wild-collected strains by ourselves and by other workers are described. Sources of background i~ormatiQ~ are given in Table I. II.
CHARACTERISTICS
COLLECTING
AND
RELEVANT
TQ
IDENTIFICATION
The genus Neurospora is defined as having grooved ascospores (Shear an c&s y 1927). This trait distinguishes it m its closest relatives Sordaria (smooth ascospores) and Gelasinospora (pitte spores) (see Moreau, 1953; Figs. Dutta et al., 1981). Neurospora inchdes and heterothallic, pseudohomothallic, mothallic species. Characteristics w distinguish the three groups are list Table 2. Large orange conidiating colonies on recently burned substrates key out almost invariably as heterothallic or pse~dob~~othallic Neurospora species. Vegetative c onies or cultures of these species are re ily distinguished visually from homotba~~i~ Neurospora and from closely related genera such as Sordaria. Only the nonherothallic Neurospora species produce conidia, and only these species are orange or yellow because of carotenoids in macroconidia and mycelia. The color is often vivid. T
94
PERKINS
Sources of Background Species Heterothallic N. crassa
AND TURNER
TABLE 1 Information on the Established Neurospora Reference
Type of information
species
N. discreta N. intermedia
N. sitophila Pseudohomothallic N. tetrasperma
Shear and Dodge (1927) Perkins and Barry (1977)“, Perkins (1979) Perkins et a/. (1982)” Krumlauf and Marzluf (1980), Perkins (1985) Perkins and Raju (1986) Raju (1980) Tai (1935) Perkins et al. (1976) Shew (1978) Turner (1987) Shear and Dodge (1927) Fincham (1951) Perkins et al. (1976)
Description Cytogenetics, rearrangements
Shear and Dodge (1927) Howe (1963a, b)
Description Markers, reference strains, methodology Linkage groups E variant with g-spored asci Molecular systematics
Markers and maps Genome organization Description Chromosomes Description Characteristics, reference strains Markers, reference strains Ecotypes Description Markers Reference strains
species
Howe and Haysman (1966) Calhoun and Howe (1968) Natvig ef al. (1987) Homothallic General
Species
species
N. africana
N. dodgei N. galapagosensis N. lineolata N. terricola
Frederick et al. (1969) Austin et al. (1974) Raju (1978) Mahoney et al. (1969) Sands (1982), Arnold (1983), Arnold and Howe (1985) Nelson et al. (1964) Nelson and Backus (1968) Mahoney et al. (1969) Frederick et al. (1969) Gochenaur and Backus (1962) Howe and Page (1963) Nelson and Backus (1968)
Key Ascospore morphology Chromosomes, ascus development Description Genetics using self-sterile mutants Description Perithecial development Description Description Description Absence of conidia Perithecial development, chromosome number
Note. Nearly 6000 available wild-type and mutant strains of Neurospora are listed in stock lists published periodically by the Fungal Genetics Stock Center (University of Kansas Medical Center, Department of Microbiology, Kansas City, KS 66103). Several strains formerly designated as Neurospora species are now considered of dubious validity or have been revised. N. phoenix has been transferred to the Xylariaceae and renamed XyZaria phoenix (Jong and Davis, 1973). N. toroi (Tai, 1935) is indistinguishable from N. tetrasperma (Metzenberg and Ahlgren, 1971; Perkins et al., 1976). Another four-spored form named N. erythea by Shear and Dodge (1927) is not known from living material. The differences in perithecial and ascospore size on which its status depends now appear to be an insufficient basis for distinguishing N. erythea from N. tetrasperma (see Perkins et al., 1976). An eight-spored Pyrenomycete described as Anixiella sublineata by Furuya and Udagawa (1976) has been reclassified as Neurospora sublineata because its ascospores are grooved (von Arx, 1981a). The strain is homothallic (D. D. Perkins, unpublished observations). The anamorph (vegetative stage) of the conidiating Neurospora species, formerly classified as Monilia Pers., has been renamed Chrysonilia (von Arx, 1981b). n Introductory sections of these comprehensive reviews describe and document the life cycle and characteristics of Neurospora, the history of Neurospora research, and sources of stocks and information.
Neurospora
FROM
TABLE
Characteristics
Distinguishing
the Heterothahic,
NATURE
2
Pseudohomothallic,
and Homothallic
Neurospora
Species
Homothallic Heterothallic (N. crassa, N. intermedia, N. sitophila, N. discreta)
Pseudohomothallic (N.
tetrasperma)
Eight-spored asci Each ascospore A or a
Four-spored asci Each ascospore typically (A + 4
Macro- and microconidia present Orange or yellow-orange mycelia and conidia”
Macro- and microconidia present Orange mycelia and conidia
(N. terricoiaa, N. dodgei, N. galapagosensis, N. africana, N. lineolata)
Eight-spored asci No mating type; cultures of single-ascospore origin self-fertile No macro- or microconidia Grey-brown mycelia; no carotenoids apparent
Note. For a key based on crossing behavior, see Perkins et al. (1976); for a key based on traditional morphology, see Frederick et al. (1969). The five homothallic taxons were distinguished and described as species on the basis of ascospore morphology, using strains obtained from soil samples. u All collected N. crassa and N. sitophila strains, most N. discreta strains, and N. intermedia strains from burned vegetation have orange color, often called salmon orange. The only yellow-orange strains found in nature were the N. discreta from Kirbyville (Perkins and Raju,1986) and some Neurospora intermedia from unburned substrate (Turner, 1987).
macroconidia are usually abundant, powdery, and readily airborne. Growth of the nonhomothallic species is remarkably rapid: linear rates can exceed 5 mm/h (Ryan et al., 1943; Perkins and Pollard, 1986). Ascospore dormancy is broken by heatshock in all the Neurospora species. HI.
COLLECTING
METHODS
We have collected Neurospora in the field as visible colonies, predominantly from burned vegetation but also from unburned substrates. Other investigators have sometimes recovered Neurospora from soil samples brought to the laboratory, as described in Section C below. Only conidiating species are obtained when visible colonies are sampled. The homothallic species, which are aconidiate, have been obtained only from heat-treated soil samples, presumably from the activation of dormant ascospores. There are reasons to expect that Neurospora might also be obtained from airborne ascospores (see Dispersal, Section
VII.D), but this method has not been use for collecting. A. Visible Colonies on Burned ~~~strat~s Neurospora colonies appear as pow orange patches on burned vegetation in moist tropical or subtropical area minetimes as soon as 7 days after a fire. e have concentrated chiefly on burned substrates because each colony from such a substrate, when fresh and undisturbed, usually re sents a genetically unique, sexually derived haploid line that originated from a single heat-activated ascospore. These cQlo~ie~ provide a sample of the gene pool of the local breeding population. Some isolates are mixtures, but the majority of heterothallic strains from recently burned substrates have only a single component. dence of purity and of uniqueness comes from scoring mating type and other traits (See Section V), Whenever possible, 7 to 10 discrete colonies are sampled at each local site. iti-
96
PERKINS
AND
TURNER
mum distance between collection sites may on nonburned substrates such as corncobs, sugarcane bagasse, filter mud from sugarvary from over 10 km to less than 1 km, cane refineries, or oncham (ontjam, a Jadepending upon the abundance of Neurosporu and upon available transport. (It is vanese food). (See Tables 7 and 9.) These not clear how large an area is covered by a are discussed under Substrate Preference (Section VI1.B). The collecting technique is “local” breeding population.) Materials for collecting consist of small similar to that for burned substrates. On at least some unburned substrates, (40 x 11.5 mm) sterile envelopes, each containing a wooden toothpick and small piece separate colonies are likely to have origiof filter paper. Each Neurospora colony is nated from conidia rather than from freshly sampled by transferring conidia with the activated ascospores, and the possibility exists that they represent vegetative derivtoothpick so as to make an orange smudge atives of the same clone. Thus, though culon the filter paper strip, which is then tures from such a source are still informasealed into its envelope. Identifying details tive, they cannot be used in the same way are written on the envelope. American Hospital Supply No. 25658 “Tomac” self- as samples from burned substrates to make seal glassine needle envelopes have been inferences about genetic variability and polymorphism. For this reason samples used. After assembly, these are sterilized by autoclaving, dried, and bundled 20 to a from unburned substrates have consisted of fewer isolates per site than those from bag in 4 x 6-inch presterilized polyethylene bags (Falcon Plastics No. 5018) sealed with burned substrates. plastic tape. Envelopes for a day’s collecting can C. Cultures from Soil Samples readily be carried in a pocket. After use, the An alternative strategy for sampling the sealed collecting envelopes are mailed to ascospore population has been employed in the laboratory for subculture and analysis. Up to 40 can be enclosed in a small airmail several laboratories. Soil samples are proletter envelope. Permits for shipment into cessed so that colonies are obtained selecthe United States are obtained from the tively from heat activation of ascospores. This method, called “soil pasteurization,” U.S. Department of Agriculture (Plant results in the recovery of strains that belong Quarantine Division, Hyattsville, MD 20782). to various Neurospora species as well as Colonies can be recognized as Neurosporepresentatives of other genera whose asra with high efficiency under field condicospores also remain dormant until heat activated. (See Mahoney et al., 1969; Fredertions. Mistaking unwanted fungi for Neuick et al., 1969; Maheshwari and Antony, rospora is not a significant problem when sampling is done visually. Only rarely is a 1974; Palanivelu and Maheshwari, 1979.) fungus other than Neurospora picked up Soil sampling has the advantage of conwhen orange colonies are sampled. Even tributing strains that are atypical in appearthen, it is usually not a matter of mistaken ance or that do not conidiate. It has been identity but rather that an unlikely prospect the source of all the known homothallic was picked as a last resort in conditions Neurospora strains. The method does not where a burn was too old and no typical require prior burning and its use is not limNeurospora could be found. ited to a short period following a fire. On the other hand, there is no immediate asB. Visible Colonies on surance that Neurospora is present when Unburned Substrates soil samples are taken in the field, so it is Neurospora may also be found growing impossible to know how extensive the sam-
Neurospora
pling should be before collection is terminated. IV.
FROM
in an area
LABORATORY CULTURE AND PRESERVATION
In the laboratory, the collecting envelopes are placed at - 20°C for 24 h to ensure destruction of mites and mite eggs that might be present (Subden and Threlkeld, 1966). Conidia are then transferred by needle from the filter paper to a slant of minimal medium (Vogel, 1964) containing chloramphenicol (0.2 mg/ml, autoclaved in the medium) to inhibit possible bacterial contaminants. A second transfer is made to leave behind possible fungal contaminants. (Unless inhibited, Neurospora outgrows all contaminants on solidified medium.) One third-stage transfer is frozen at - 20°C and conidia from another third-stage transfer are preserved permanently in suspended animation on anhydrous silica gel (Perkins, 1977b). By making permanent silica-gel stocks promptly, following only the briefest growth, it is hoped to retain original wild genotypes unchanged by mutation and selection. After species identifications have been made (Section V), strains representing each species found at each locality have been deposited in the Fungal Genetics Stock Center (FGSC) ’ (Department of Microbiology, University of Kansas Medical Center, Kansas City, KS 66103). Where available, opposite mating types have been included for each species. These strains are included in the FGSC stock lists (Fungal Genetics Stock Center, 1986). (For intensely sampled areas, not all of the sites are represented in FGSC for N. intermedia, the most abundant species.) Thus for many sites where one species predominates, only one or two strains are sent to FGSC while an additional five to eight are not deposited. 1 Abbreviations used: FGSC Fungal Genetics Stock Center; RFLP, restriction fragment length polymorphisms; OR, Oak Ridge (laboratory strain).
NATURE
The nondeposited strains are available from silica-gel stocks at Stanford University. Where purity of the Neurospora culture must be assured, successive subcultures are grown up from single conidia. The strains deposited in FGSC have not bee purified in this way, but a variety of studies indicate that most of the deposited cultures are pure (see, for example, Spieth, 1975). V.
DETERMINATION
A. Rationale for Diagnostic
OF
SPECIES
Criteria
We feel that lack of a fertility barrier is the most fundamental basis for concludi that two heterothallic strains belong to t same species. In practice, fertility tests have proved to be the only reliable criterion, and they are by far the most convenient. Our diagnostic crossing procedure was used successfully to make species signments for 99% of all isolates collec before 1983. (The tests have not been completed for all of the isolates from th 1985 period of intensive collecting.) all the isolates were assigned to one or another of the four previously established conidiating species: N. crassu, N. sitophila, N. intermedia, or N. tetras~erma~ The procedure also revealed the existe of a rare new species, N. discreta, whit reproductively isolated from the others (Perkins and Raju, 1986). Although the heterothallic species ten to differ somewhat in size and mo~h~logy of ascospores (Table 3; see also Table 3 of Perkins et al., 1976), these differences are only averages. It would be difficult or impossible to assign strains reliably to the heterothallic species either by vegetative morphology or by ascospore size There is too much variation wit and there is overlap between species. ascospore measurements requi be two parents to make the cr duces the ascospores. Since the isolates are haploid, it is necessary to
PERKINS
98
AND TURNER
TABLE 3 Diagnostic Characteristics of Interspecific N. crassa N. N. N. N. N.
crassa intermedia sitophila discreta tetraspermab
+
N.
intermedia
W +
N. sitophila
R W +
Crosses” N.
discreta
R R R +
N.
tetraspermab
R R R R +c
+ , Fully fertile. Perithecia with beaks. Ascospores are abundant, with >50% black and viable. In N. because strains containing Spore killer-l are common, test crosses that are Sk-lK x Sk-l’ can be expected; these produce asci with four black: four aborted ascospores, classed here as +. W, “White.” Perithecia are full size and often produce and eject ascospores. Very few ascospores are fully pigmented and viable, but these few make introgression relatively easy. R, Perithecia are absent or rudimentary with no ascospores. Occasional exceptions are found where a few viable ascospores can be recovered from interspecific combinations. See Section IX. D Similar results are obtained regardless of which species is used as female (protoperithecial) parent. b Homokaryotic A or a strains are necessarily employed as N. tetrasperma species testers and for interspecific crosses. c Asci four-spored. Note. sitophila,
choose partners for them. In the initial work, we tried intercrossing all isolates from the same site, but the process is very time-consuming and is fruitless unless both mating types of a species are found at the same site. It also means that each cross involves two unknowns instead of one. For these reasons, all heterothalhc species assignments have been made on the basis of fertility in crosses to strains established as species testers. The crossing behavior of different combinations of species is summarized in Table 4. Diagnosis of wild-collected strains of the pseudohomothallic (secondarily homothallit) N. tetrasperma is made on a different basis. N. tetrasperma cultures are usually A + a heterokaryons, and under appropriate conditions each culture will produce perithecia containing four-spored asci in which each large ascospore contains both A and a nuclei. The four-spored asci categorize the strain as a pseudohomothallic without the need for a test cross. Occasionally, small homokaryotic ascospores are produced. If a N. tetrasperma strain is homokaryotic when collected, or if the A and a components of a heterokaryon are separated experimentally, each homokaryotic culture crosses only with testers of the
opposite mating type. The homokaryotic strains thus behave as though they were heterothallic strains of a single mating type, except that the crosses produce fourspored asci. A and a components have been resolved and test crossed for only a few of the many four-spored pseudohomothallic strains that have been collected. Until crossing tests have been made, the possibility must be considered that not all the pseudohomothallic isolates belong to the same species, N. tetrasperma. However, Natvig et al. (1987) have obtained molecular evidence that four-spored isolates from widely diverse origins are all closely related, consistent with their belonging to a single species or species complex. Attempts to employ protein polymorphisms as taxonomic criteria for distinguishing species (see, for example, Reddy , 1973) have not been successful, nor have measures of overall DNA homology based on Cot curves provided a practical criterion for diagnosis at the species level (see Dutta et al., 1981). Comparison of cloned uniquesequence DNA fragments by Southern blotting seems promising as an indicator of relatedness between species (Natvig et al., 1987). This method has been developed for
Neurospora
TABLE 4 Commonly Found Accessory Characteristics of the Five Conidiating N.
crassa
N.
sitophila
N. intermedia
N. discreta
N. tetrasperma
9
FROM NATURE
Species of Neurosporaa
Typical ascospores 29 x 1.5 km, more pointed than N. intermedia. Carotenoids almost always orange. Typical ascospores 23-24 x 14 pm, more pointed than N. intermedia. Carotenoids almost always orange. Typical ascospores 27 x 17 pm, visibly more rounded than the other species. Strains with golden-yellow (saffron) carotenoids and very large conidia (>12 pm diameter) always prove to be N. intermedia, but most N. intermedia cultures are orange and have conidia in the smaller size range. The first population discovered, from Kirbyville, Texas, differs from other Neurospora species in ascospore morphology and vegetative traits, but these differences are not found in other populations that appear to be N. discreta on the basis of fertility tests. The N. discreta isolates from Kirbyville, but not those from elsewhere, produce ascospores that are ornamented with dot-like pits in the ribs between confluent parallel grooves. Unlike the others, the Kirbyville strains are yellowish rather than orange, and large empty barren protoperithecia (‘“false perithecia”) are found abundantly in unfertilized haploid cultures. Isolates from sources other than Kirbyville are infertile as female parents on synthetic crossing medium unless sucrose is replaced by filter paper as a carbon source. Until other populations are more adequately represented, traits characteristic of tbe species cannot be listed with confidence. Typical ascospores 34 x 15-16 Frn, each giving rise to a self-fertile A + a culture. Occasionally smaller A or a ascospores are produced in asci having more than four spores. Most cultures conidiate poorly at 25°C whether heterokaryotic or homokaryotic, with the protoperithecia in homokaryotic cultures becoming brown and enlarged but without beaks or spores. At 34°C false perithecia do not appear and conidiation is better. Carotenoids orange.
a Except for the large heterokaryotic ascospores of N. tetrasperma, which occur in four-spored asci, these traits are more variable and less reliable for distinguishing species than are the fertility characteristics in Table 3. Ascospore measurements are taken from Table 3 of Perkins et al. (1976).
the purpose of defining phylogenetic relationships. It cannot be expected to substitute for the convenient and economical crossing tests that we have used in everyday practice for assigning unknown isolates to species, as described in the following section. B. Laboratory
Procedure
Synthetic crossing medium (Westergaard and Mitchell, 1947) has been used for test crosses, with 1% sucrose and 1.5% agar unless otherwise indicated. Tester strains containing the gene fr (fluffy) are convenient because conidia are absent. The fluffy testers are inoculated to slants in 12 x 75 mm culture tubes; these are ready to fertilize after 4 days at 25°C. If nonfluffy strains are used as testers, they are inoculated to larger slants (13 x 100 mm) to avoid ob-
struction by conidia; these are ready to fertilize at 5 days. Test slants can then be held at 5°C for at least 2 weeks before being fertilized. In early stages of the work, while experience was being gained with species tester stocks, tests were made between testers in all combinations, collected isolates were crossed to set of testers (see Table 5 and Section V.C). The full set of tester strains is still e with some wild isolates that are di diagnose, but a species assignment can often be made with confidence on the basis of observing full fertility with the first or second tester that is used. Our present procedure is to cross wil collected strains first to N. crassa testers of both mating types. If the sample came from a region with a great predominance of AJ.
100
PERKINS
AND TURNER
TABLE 5 Strains Used as Species Testers in Diagnostic Crosses Strain designation
FGSC No.
Origin and characteristics
1. Preferred testers (species reference strains) Neurospora crassa Highly fertile, aconidiate fl&fi strains essentially 4317 fI’A 4347 coisogenic with the standard OR laboratory wild types. frpa jlp originated spontaneously from 74A x 73a, and was backcrossed recurrently to OR or OR-related strains. Neurospora intermedia 3416 Conidiating f, isolates from N. intermedia P13A x P17a from Shp-1A 3417 Taiwan, selected for fertility and uniform growth Shp-la (Shew, 1978). Neurospora sitophila jl; Sk-I” A 4762 These contain J&ii allele P1012, which arose 4163 spontaneously in N. sitophila, and the Spore killer jl; Sk-lK a allele Sk-IK. From third recurrent backcross to P8085 and P8086. Neurospora discreta 3228 P851 collected near Kirbyville, Texas. P8127 is from P851A 4th recurrent backcross to P851 of Kirbyville-1 a P8127a 4378 (P846) (Perkins and Raju, 1986). Neurospora tetrasperma 1270 Homokaryotic f,,‘s from A + a strain 87 of Dodge (Howe, 1963a). 85A 85a 1271 2. Supplementary testers and strains used previously Neurospora crassa Wild-collected N. intermedia strains from Florida. P420A 2316 1940 P405a jlufi (allele P) introgressed from N. crassa by seven 5798 ;it recurrent backcrosses to Shp-1A and Shp-la. (Expected 5799 to replace Shp-1A and Shp-la after more extensive testing.) Neurospora sitophila 2216 Conidiating Sk-l” strains previously used as reference P8085A testers (Perkins et aZ., 1976). P8086a 2217 jl’; Sk-l’ A 4887 These contain jlufi allele P, which was introgressed from N. crassa by five recurrent backcrosses jl’; Sk-l’ a 4888 to N. sitophila. They are sensitive to Sk-l”. P2443A 5940 Conidiating Sk-l’ strains from Tahiti. P2444a 5941 Note. For further information, see Perkins et al. (1976) and Perkins and Raju (1986). The supplementary strains have been authenticated in crosses to the species reference strains. They have been used in areas where some strains are infertile with the reference strains or produce a high percentage of immature ascospores with them. Testers P420 and P405 are sometimes more fertile than the Shp testers when crossed with N. intermedia isolates from the western hemisphere. Members of the two sets are conspecific on the basis of intercrosses (Perkins et al., 1976). The N. crassa and N. intermedia strains listed here are all sensitive to killing by Spore killer-2 (Sk-su) and Spore killer-3 (Sk-3u). Spore killer strains are rare in these species. In N. sitophila both Sk-lK killer and Sk-l’ sensitive strains are common (Turner and Perkins, 1979). Both Sk-I” killer and Sk-l’ testers are therefore listed.
Neurospora
FROM
each strain is then usually crossed also to a N. intermedia tester of appropriate mating type. The N. intermedia testers can be fertilized as soon as perithecia are seen in one of the N. crassa tests. For samples from some regions it is more efficient to await maturation of the N. crassa tests and to treat different outcomes separately: 1. If the test cross with N. crassa produced at least 90% black ascospores, the isolate is scored as N. CF-ussuand not tested further. 2. If the test cross with N. crassa produced large perithecia either having no spores or having eight-spored asci with predominantly defective ascospores, the isolate is tentatively scored as N. intermedia and is crossed to a N. intermedia tester (or testers). Recently this procedure has been modified for bright yellow strains from unburned substrates. More than 100 of these strains have been fertile enough with N. intermedia to be diagnosed as conspecific, but these crosses tend to make few perithecia and few ascospores (Turner, 1987). Over 50 additional yellow strains have been diagnosed on the basis of their cross to the N. crassa testers, substrate preference, color, and giant conidia. 3. If a test cross with the N. crassa tester as female produced no reaction better than rudimentary perithecia, but the culture being tested behaved as self-fertile and by itself produced perithecia with four-spored asci, it is tentatively scored as N. tetrasperma. (Confirmation requires separation into homokaryotic A and a components and testing the components with singlemating-type N. tetrasperma testers. This has rarely been done.) 4, If the test cross with N. crassa was infertile (made no reaction better than rudimentary perithecia) and the tested culture was not a pseudohomothallic strain with four-spored asci, the unknown is crossed to N. sitophila. If the test cross to N. sitophila is also infertile, the isolate is then crossed intermedia,
NATURE
to N. discreta and to single-mating-ty testers of N. tetrasperma. C. Effectiveness of Species Tester Strains and the Testing Procedure
The standard reference strains in current use are listed in Table 5. All have shown to be conspecific with the strai which the original species descriptions were based (Perkins et al., 1976). mentary strains that have been u special purposes are also liste aconidiate mutant “fluffy” whenever possible because th nidia makes it much easier to see peri and ejected ascospores. Strains containing fl also show enhanced fertility as female parents. All of the species that we have co11 are closely enough related so that a phogenetic reaction triggering perit~eci~l growth can take place when i~terspec~e crosses are attempted between representative isolates of opposite mating type. This is true of some but not all strains even between the least reactive pairs of species. 0n this basis, individual isolates can be assigned to one of the two mating type a, as originally defined in N. crassa. N. crassa fluffy testers are fertilize discreta, by N. sitophila, or by si~~~~mating-type cultures of N. tetrasperma~ perithecia may be very rudimentary. ertheless, in the majority of cases a reredos can be seen with one mating type but not with the other. Intraspecific and interspecific crosses in all combinations are compared in Table 3. Except for N. crassa x N. intermedia, which typically produces 1 to 10% viable ascospores, ail cornbi~at~Q~s of interspecific crosses either produce no ascospores or produce ascospores that are almost all (>99%) inviable. Inviable ascospores are easily recognized becaus are usually hyaline; they will be refe as white, in contrast to the intens that is characteristic of normal, viable ascospores.
102
PERKINS
AND
For the great majority of heterothallic strains, species assignment is clear and straightforward, based on a fertile cross with >90% black ascospores. However, there are important exceptions that should be noted by anyone attempting to identify newly collected strains: 1. Newly acquired strains that are bona tide N. sitophila may produce either 95 or 50% black ascospores when crossed with a given N. sitophila tester. Those that are like the tester strains in their Spore killer genotype (see VII1.C) produce 95%, and those that are unlike produce 50%. Occurrence of tests with 50% black ascospores does not affect species diagnosis because the N. sitophila testers do not produce even 1% black ascospores with other species. 2. The N. discreta group has two parts. The standard reference strains from Texas, others from the United States, and one from Guatemala make ~90% black ascospores when intercrossed. Those from other countries make about 10% black ascospores when crossed to the reference strains and ~90% black in the few fertile crosses obtained within the group. However, a discrepancy in reciprocal crosses raises the possibility that the defective ascospores may be caused by some sort of incompatibility not sufficient to warrant dividing the group into separate species. This problem is under study. Another problem illustrated by the N. discreta group is that not all strains have the same requirements for successful crossing. Many do not produce protoperithecia on our standard crossing medium (Westergaard and Mitchell, 1947) with 1% sucrose but do so when filter paper is supplied and the sugar is omitted. 3. N. intermedia and N. crassa are the closest pair among the known species. Usually they are easily distinguished, but strains from certain regions (India (particularly the state of Andra Pradesh), Pakistan, Thailand, and Malaya (Penang)) cross unusually well with the testers from both species. Diagnosis can be completed by using
TURNER
clearly identified local testers. Other strains cross poorly with the standard testers from both species, but most of these can also be resolved by crosses to local testers. In these most difficult cases the possible presence of chromosome rearrangements must be investigated. A reciprocal translocation, for example, would result in 50% defective ascospores in a cross to a tester of the same species (see Section VII1.G). 4. Some collected strains do not make a fertile cross (sometimes not even a rudimentary reaction) with certain testers from a species yet make a fully fertile cross with others from the same species. Many strains from the three African countries-Congo, Gabon, and Ivory Coast-are still unidentified, partly because they were collected more recently, but mainly because they have proven to be among the most demanding to work with. The majority are probably N. intermedia-they gave a reaction typical of N. intermedia when crossed to the N. crassa testers but did not make fertile crosses to the standard N. intermedia testers or to identified N. intermedia strains from Africa. Others did not cross to any testers. When crosses were attempted between a sampling of the latter and various strains from the N. discreta group (using filter paper as the carbon source in the crossing medium), some of them made fertile crosses and were thus diagnosed as N. discreta even though they had not evoked any reaction in attempted crosses with the standard N. discreta testers under standard crossing conditions. D. Mixed Samples
Of the mixed samples that have been detected, most were recognized because they crossed or reacted to testers of both mating types but did not make four-spored asci in a self-cross. Among the cultures that have given a reliable mating-type reaction to one of the standard heterothallic testers, the overall incidence of a cross to the other
Neurospora
FROM
mating type by the same culture is 12%. It must be assumed that there is a similar frequency of samples containing two strains of the same mating type. So we estimate that among 100 original cultures, 12 are mixed mating type, 12 are mixed but of the same mating type, and 76% are completely pure. The 12% estimate for mixtures containing both mating types applies both to burned and to unburned substrates, as well as to each species taken separately. However, these pooled totals include many collections that were made under unfavorable conditions, from disturbed sites and from very old burns. The frequency of mixed cultures varies greatly from region to region and site to site, depending on age of the colonies and on collecting conditions that are beyond our control. For some regions, very few of the cultures were mixed. Only 14 samples (in more than 4000) have been found to contain a mixture of species. Because our protocol takes advantage of the predominance of N. intermedia or N. crassa in nature by crossing to those species first and because the first successful test cross ends the procedure for that strain, there is little probability of detecting a trace of a less common (and less interfertile) species of the same mating type in a sample that is predominantly N. intermedia or N. crassa. However, even when a cross to another species is successful, the presence of N. tetrasperma as a second component is revealed by the production of fourspored asci in the original culture tube. In the tables that follow, strains collected as mixed species are listed according to the components that have been subcultured and isolated. Mixtures of both mating types of the same species are counted as one strain if not separated or as two if separated. All mixtures of N. intermedia A and a (as judged by N. crassa crosses) were used to tabulate the proportion of mixed cultures, but for areas with many N. intermedia samples, some were not further tested. Among all countries, a total of 112 N. in-
NATURE
termedia mixed cultures were set aside an are not included here. VI.
SCOPE
OF
THE
COLLECTIONS
Over 4000 cultures have been co and characterized since 1968, ori~uat~~g from five continents and from oceanic islands. The wild-collected isolates are summarized according to geographical origin in Tables 6-9. Results of our collections from burned substrates are given in Table 6, an the results from unburned substrates in Table 7. Collections in Tables 6 and 7 were made either by the first au&or or in his presence by others whose assistance is credited under Acknowledgments. Strains collected and donated by other workups have also been put through our standar testing procedures, with the results shown in Tables 8 and 9. A few of these isolates were obtained by heat processing ples (e.g., Mahoney et al., 1969; wari and Antony, 1974). Our strains foun through 1974 were listed in Table 4 of kins et al. (1976). These strains are incl here in Tables 6 and 7. Results wi sent to us by individuals who bad colle them prior to 1975 comprised part 5 of Perkins et al. (1976) and are incl here in Tables 8 and 9. The 1976 table included strains obtained from estab~i culture collections; these are not repeate here. In cases of uncertainty about s identification, we have tried to list strains in such a way as to give the most useful overall picture. Sources of uncertainty were explained in Section V. Except for the probable N. intermedia strains from Africa scribed in Section V, we have tried to li “undetermined” to strains that are really candidates for belonging to the N. discreta group or to some unknown rare species. The identification of a or N. intermedia strai pending on the degree of is not as certain as we would require if tbe
47
488
Totals
2181
150
6i 177 34 6 35 23 124 107 42 18 0 171 93 11 0 418 4 38 140 26 11 14 12 163 60
240
N. intermedia
312
113
; 1 0 17 0 76 0 23 0 0 46 0 8 0 0 0 13 0 0 0 0 5 0
0 1 0
370
40
0 0 0 1 7 12 1 51 6 15 0 102 2 9 8 0 0 8 0 1 8 2 3 86 0 4 4
317
137
8 0
8 47
8: 0 2 0 0 4
0 0 1 3 0 7 0 5 21 0 5 1 0 0
N. tetrasperma
Vegetation
No. of strains
Burned
N. sitophila
from
TABLE6 Collected
N. crama
Strains
44
8
8 0 0 0 4 0
: 0 0 7 0 17 0 0
ii 0 0 0 0 0 0 0
0 0
N. dim-eta groupa
Note. The 3406 isolates in this table were collected by D. D. Perkins or by accompanying colleagues between August 1968 and April 1986. a See text Section V.C.2. b See text Section V.C.4 regarding undetermined strains from Africa (Congo, Gabon, and Ivory Coast). For other countries, testing has been exhaustive, undetermined strains have been crossed successfully to some species tester, but with ascospores that were mostly or entirely defective.
: 23 1 31 14
31 1 14 23 21 6 6 16 16 27 9 39 1 30 20 12 1 63 2 8 12
Australia Bangladesh Borneo Brazil Congo Gabon Guam Haiti Hawaii India Indonesia Ivory Coast Japan Malaya (Malaysia) Malaya (Penang) New Zealand Pakistan Papua New Guinea Philippines Ponape Puerto Rico Rota Singapore Tahiti-Moorea Taiwan Thailand Truk United States (continental)
SOlKCC
No. of collection sites
Neurosporu
and most of the
182
2
4
: 0 85
:
46 8
0 0
Undetermined6
Neurospora
FROM NATURE
TABLE 7 Strains Collected from Nonbumed
Substrate No. of strains
Source
Substrate
Australia Brazil Congo Gabon Haiti Indonesia
Ivory Coast Japan Malaya Papua New Guinea Philippines
Filter mud Plywood Corncob Corncob Corncob Corncob Ontjom Temple offerings (rice, cake) Corncob Corncob Corncob Corncob Corncob
N. intermedia
N.
crassa
N. sitophila
11 3 19 3 1 15 26
-
-
13 1 7 6 53 5
-
-
1
1 1
-
-
Undetermined* 1 3 1 7 -
-I Note. The 178 isolates in this table were collected by D. D. Perkins between August 1968 and April 1986. u Most of the strains in this column are persistently sick and infertile but are probably N. intermedia as judged either by yellow color or by presence of a few defective ascospores in crosses with the very fertile N. crassa testers. From 5 to 10% of strains collected from unburned substrates are contaminated or infected, but in most cases healthy subcultures were obtained by careful transfer or by plating a few conidia. Six cultures from Papua New Guinea were growing on corncobs that had been partially burned (so it is not obvious whether they belong here or in Table 6). All have an unusual pale color, suggesting that they are all the same species; they make mostly defective ascospores with N. crassa, indicating that they are N. intermedia. Three make fertile crosses with N. intermedia testers, but the other three do not and are therefore included with the undetermined strains.
strains were to be used as examples of the species. VII.
A. Abundance
SPECIES IN NATURE
and Distribution
The search for Neurospora on burned substrates has met with great success in the moist tropics but with very little success in Mediterranean climates and in cooler temperate climates. Though the search has been less exhaustive in the temperate zones, it seems clear the Neurospora is not commonly found in areas where the warm season is dry and where winters are cold with extensive freezing. The following generalizations about species distribution ignore some very small collections from isolated sites. N. intermedia is the most abundant species on burned substrates. As shown in Table 6, it is the predominant species in many geographic
areas scattered around the globe. The other species show individual patterns of clistribution, though all overlap with each other. N. tetrasperma and N. sitophila show the greatest divergence in ecological preference, though both are abundant in Tahiti, and individual samples of both are found throughout the world. In areas farthest from the equator (e.g., New Zeal North Carolina), N. tetrasperma is th dominant species from burned substrates. This suggests adaptation to survive the cold season. N. sitophila seems least adapted to cold, since extensive collections have been obtained only from tropical areas (Maiti, Tahiti, Ivory Coast). It occurs in temperate regions mainly as a contaminant on food or on laboratory medium. In fact it is t ties of Neurospora commonly fo such substrates in North America. N. crassa, the only species of Ne~~Q~~~-
106
PERKINS
Contributed
AND TURNER
TABLE 8 Strains from Burned Substrate
No. of Source
Contributor
-
Australia
D. E. Shaw”
Brazil
M. Bjdrkman G. J. Samuels
Costa Rica Gabon Guyana
9 1 1 12
R. 0. N. G.
L. Metzenberg Ezavin C. Franklin J. Samuels
Species N. N. N. N. N. N. N. N.
intermedia tetrasperma intermedia intermedia crassa discreta tetrasperma crassa
No. of strains
group
Undetermined N. N. N. N. N.
discreta intermedia crassa sitophila tetrasperma
Undetermined Honduras India
K. S. Hsu N. B. Raju
Malaya Mexico
C. C. Ho R. L. Metzenberg
New Zealand
R. E. Beever
N. N. N. N. N.
intermedia intermedia crassa sitophila tetrasperma
Undetermined
Papua New Guinea Tonga U.S.A.
3 1 1
D. E. Shawb L. S. Olive F. J. Doe
Vanuatu
1 3
N. H. Horowitzc E. C. H. McKenzied
Venezuela
1
G. J. Samuels
Totals n Includes strains b Includes strains c Strain collected d Transmitted by
N. N. N. N. N. N. N. N. N. N. N. N.
intermedia tetrasperma discreta intermedia sitophila intermedia tetrasperma crassa sitophila intermedia crassa sitophila
group
59
239
collected by R. Borch, C. D. Jones, J. O’Rourke, collected by A. van Noaren and J. R. Walsh. by W. Scott. R. E. Beever.
ru known to many scientists and students, seems to be more restricted in range than the species already mentioned. It is frequent in samples from the Gulf of Mexico area, the Caribbean, Africa, and India. Both mating types of N. discreta were found in only a single small population in Texas, but single isolates of N. discreta are
31 1 10 1 11 2 1 61 1 1 14 4 3 8 4 3 7 19 1 2 3 2 23 1 7 3 3 2 1 2 1 5 1
and R. Grattige.
known from two other areas in the Western hemisphere (Florida, Guatemala). The other strains listed as “N. discreta group” in Table 6 cross to the N. discreta from Texas and make abundant ascospores, but the ascospores are predominantly defective and inviable. Further study is needed to determine whether this reflects a divergence
Neurospora FROM
Contributed Source
Australia
TABLE 9 Strains from Nonburned
Contributor
Substrate
D. E. A. Catcheside D. E. Shaw”
Crumpets Bee pollen basket Filter mud Bread
T. Angelb China
NATURE
Corncob
India
Z. J. Sheng’ (T. C. Sheng) Y. N. Ming R. Maheshwarid
Palau Taiwan
R. A. Lewin H. W. Li
Turkey
A. Sazci
United States
F. M. Turner H. M. Luscombe M. Revenaugh
Dying sponge Bagasse Fiber Filter mud Lab/hospital contaminants Coffee grounds Tortilla Bread Nut shell Coffee grounds Lab contaminant
M. Schechtman 0. C. Yoder
Unknown Soil
Substrate Species N. N. N. N. N. N. N. N. N. N. N. N. N. N. N. N.
intermedia intermedia intermedia intermedia sitophila intermedia sitophila intermedia intermedia tetrasperma sitophila intermedia intermedia intermedia intermedia sitophila
N. N. N. N. N. N.
sitophila sitophila sitophila sitophiia sitophila sitophila
Total
No. of strains
1 2 f 1 I 2 93
a Includes strains collected by R. Birch, G. Wilson, A. G. Hayes, D. R. Strong, L. Tilley, E. 0. Maynard, R. 0. Spalding, P. R. Downs, C. D. Jones, P. English, and A. I. Linedate. b Transmitted by D. G. Catcheside. ’ Collected by members of the Genetics Society of China at the request of Z. J. Sheng and transported from China by N. H. Giles. d Tncludes strains collected by P. Palanivelu.
sufficient to warrant splitting the group into two or more species. In areas where several Neurosporu species are present, two or even three species may be represented among the 10 colonies sampled from the same burned site. This diversity was seen most strikingly in Haiti. Interpreting the observed distribution is greatly complicated by the possibility that human activities have altered the distribution in recent times. Most sites have been in or near areas of agriculture and human habitation where Neurospora could have been introduced with crop plants from another part of the world. Perhaps molecular markers in chromosomal or mitochondrial DNA can be used to trace lineages and to indicate
centers of origin. It seems obvious that humans have played some role in dist~b~ti~g Neurospora and providing conditions for its growth, but the importance of the& role remains to be defined. B. Substrate Preference Burned substrates. At sites where vegetation has been burned or scorched and where humidity and temperature are favorable, the conidiating Neurospora species may be found growing on the remnants of many different plant species. ~ucc~e~ts and monocotyledonous plants with pithy stems and sugary sap seem to be especially good substrates, supporting prolific sws-
108
PERKINS
AND
tained growth of the fungus. Burned wood can provide a good substrate if it is freshly cut and uncured, but dried dead wood is a relatively poor substrate. Because Neurospora is so catholic in its tastes, we have not made any special effort to identify the species of plants serving as substrates. Instead, our records describe the substrate in general terms such as burned grass, tree trunk, or succulent. Nonburned substrates. A particular ecotype of N. intermedia is characterized by yellowish carotenoids and large conidia. This ecotype is almost never found on burned substrates, but it accounts for almost all samples taken from unburned substrates in tropical parts of the eastern hemisphere (Turner, 1987). The common substrates are cooked maize cobs, onchom, and waste products of sugar cane processing. Maize corncobs support prolific growth. Cobs bearing Neurospora are commonly found in tropical marketplaces, where they have been discarded after the kernels have been eaten from the cooked ear. It is not known whether the Neurospora on corncobs originates from ascospores or conidia, or whether the inoculum is airborne or possibly insect borne. Samples from the same cob appear to be from one clone, but sometimes both mating types are found on nearby cobs. Onchom (ontjom) is prepared for human consumption in West Java by cultivating Neurospora on soybean or peanut solids (see Shurtleff and Aoyagi, 1979). Numerous samples from onchom have been identified as N. intermedia (Ho et al., 1973; Perkins et al., 1976; Ho, 1986). In onchom production, the producers deliberately inoculate each batch of cakes with conidia from the previous culture. Both mating types and a variety of genotypes affecting visible traits have been recovered from the same onchom cake. Neurospora colonies were found conidiating profusely on filter mud from sugar
TURNER
cane refineries at numerous sites in Australia (Shaw and Robertson, 1980). The colonies may originate from ascospores activated by heat during the refining process, but inoculation might also involve conidia already in the environment. Even a small transfer of a single colony from filter mud often contains strains of both mating types. In temperate climates, Neurospora has been reported mainly in particular manmade habitats where suitable substrates are heated but not burned. Until the introduction of chemicals to retard fungal growth, Neurospora was a serious problem for bakeries in Europe and North America. Activated ascospores are probably responsible for infestation of bakeries (see Shear and Dodge, 1927; Moreau-Froment, 1956). Neurospora has also been found in plywood factories, lumberyards, and on substrates such as nutshells (see Tables 7 and 9). Strains from bakeries have usually been identified as N. sitophila or assumed to belong to this species. However, most of the strains collected by Yassin (1980) from bakeries and related sources in England were N. crassa. In contrast, a bakery in Germany yielded only N. sitophila. Because grain is imported, origins of the bakery contaminations are uncertain. C. Possible Subspecific
Categories
There is no indication that the yellow ecotype of N. intermedia is reproductively isolated from the orange strains of N. intermedia that are found on burned substrates. Crosses involving a strain of the yellow ecotype are typically less fertile than those of orange x orange, but the fertility is as poor for yellow X yellow as for yellow X orange (Turner, 1987). Thus there seems to be no reason for according subspecitic taxonomic status to the yellow strains. Compared to crosses between strains from the same continent, there is a slight elevation in the frequency of aborted asco-
Neurospora
FROM
spores in crosses between N. crassa from India and N. CY~SSUfrom North America. Likewise, strains classified as N. intermedia from Asia and from North America are more likely to be fully fertile with other strains from the same hemisphere. The decrease in fertility is neither complete nor consistent, however, and it does not seem to warrant formal systematic recognition. Of all the recognized species, N. interme&a and N. crussa seem closest. Chromosome pairing at pachytene is nearly perfect, and intercrosses may result in 5 or 10% viable ascospores. Classification of N. intermedia and N. crassa as subspecies would be a conceivable option. For reasons stated by Perkins et aZ. (1976), we prefer to retain the present classification as separate, though closely related, species. D. Dispersal
Putting aside the question of human influence, we are quite ignorant of natural dispersal mechanisms. What is the role of conidia? Of ascospores? Of insects? Of the atmosphere? Microconidia can be dismissed as vegetative propagules because they show low viability, are extremely fragile, and do not become airborne. Macroconidia, in contrast, are far less fragile and readily become airborne. Macroconidia are produced in very large numbers, and they are small enough to assure aerial transport over considerable distances because of the low fallout rate. They would seem to be ideal propagules for rapid exploitation of newly available resources under conditions calling for a strategy involving r-selection (density-independent selection for rapid proliferation, allowing exploitation of substrate in advance of potential competitors in an uncrowded environment) . But conidia are highly vulnerable to killing by ultraviolet light (see Schroeder, 1970). Dormancy of macroconidia is broken by moisture, and nondormant conidia are relatively short lived (Fa-
NATURE
B
hey et al., 1978). These properties would seem to limit the role of conidia in dispersal over long distances. Macroconidia are absent in all of the known homothallic Neurospora species, and they are present in all of the nonhomothallic species. This difference suggests that macroconidia play a role in cross fertilization similar to that of windblown pollen in plants such as maize. Is it possible that fertilization is a more fundamental role of macroconidia than dispersal? Honeybees (Apis mellifera) ia from N. intermedia blooms from sugar refineries (Shaw an 1980). Corbiculae (pollen baskets) were found to be filled exclusively with orange macroconidia, just as though the fungus spores were pollen. It seems unlikely fertilization of Neurospora by bees have any significant impact, however, considering the abundance of conidia transported in the air. Ascospores are larger than macroconidia but not so large as to preclude their airborne. Ascospores, with their black walls, are capable of remaini mant and viable for months or years until activated by heat or chemicals. They are far more resistant than macroconidia to k by uv light (K. C. Atwood, personal communieation). These features and others discussed below suggest the possibility that airborne ascospores play an important role in dispersal, especially over long distances. Ascospores are the only pr es produced by homothallic specie urospora (Mahoney et al., 1969; Frederick et ai., 1969; Howe and Page, 1963). Aspects of spore ejection and aerial persal of ascospores have been reviewe Ingold (197 I), Pedgley (1982)) (1973), and Tryon (1971). The following considerations suggest t a search for airborne Neurospora ascospores might be rewarding: (a) A gists who determine the incidence and fungus spores in air samples re
110
PERKINS
AND
presence of ascospores (see Gregory, 1973). Unfortunately, published records lump the ascopores of many genera. We have found no report sufficiently detailed to identify Neurospora. (b) Airborne ascospores are known to be the agents of dispersal and infection of the plant pathogen Venturia inaequalis, an Ascomycete with sexual structures similar to those of Neurospora (Hirst and Stedman, 1961a, b). Neurospora ascospores (-18 x 25 pm) are twice as large as those of Venturia, but spores that are even larger are commonly found in air samples (Gregory, 1973). (c) The trajectory of ascospores shot from Neurospora perithecia is high enough for them to be caught up in turbulent air currents (Ingold, 1971). Elongation and grooved ornamentation are expected to slow the fall of an ascospore relative to a smooth sphere of the same volume (Gregory, 1973). (d) The colonies that grow up in burned areas appear from our observations to be randomly spaced rather than clustered as might be expected if ascospores remained in the immediate vicinity of the perithecia of origin. It would be of considerable interest to know whether ascospores play the dispersal role we have suggested and to know the distances involved. Ability to obtain ascospores from the air might also provide a less biased population sample than do the methods now available. Existing programs that monitor air to obtain information on allergens and plant pathogens could perhaps also provide information and samples of living ascospores. Because Neurospora ascospores are grooved, visual recognition should be possible. Alternatively, spore samples from the air could be suspended, heatshocked, and plated, following a protocol similar to those used for recovering Neurospora from soil. Small-scale independent collection programs should also be feasible, because efftcient and inexpensive devices are available for collecting airborne particles the size of ascospores (see, for ex-
TURNER
ample, Edmonds, Lacey, 1964).
1972;
Gregory
and
E. Retention of the Sexual Phase in Homothallic Species
In the homothallic Neurospora species, each strain that originates from a single haploid ascospore is capable of carrying out the complete life cycle, including the sexual (teleomorphic) phase. Karyogamy, meiosis, and ascus development in the selfed crosses of the homothallic Neurospora species are cytologically indistinguishable from what is seen in crosses between strains of opposite mating types in a heterothallic species such as N. crassa (Raju, 1978). Yet because there is only a single haploid parent, the sexual cycle would appear to play no role in generating diversity by recombination of genetic differences. A strong case can be made (Section VIII.B.3) that the sexual cycle would be expected to disappear if it did not confer a significant advantage on the organism, resulting in its maintenance by natural selection. Since the homothallic Neurospora species retain a robust sexual phase, the question arises, what selective advantage does sexuality confer on them? It has been proposed that the original function of recombination in primitive organisms was repair of genomic damage (Dougherty, 1955; Maynard Smith, 1978), and that recombinational repair continues to be a major selective advantage that accounts for the prevalence of sexuality and meiosis in eukaryotes (Bernstein et al., 1985). Without judging the merits of the repair hypothesis, we suggest here yet another advantage of the sexual cycle that might account for retention of the perithecial phase by the homothallic species of Neurospora. In Neurospora,
the only long-lived propagules are the sexually generated ascospores, which are resistant to environmental insults and are capable of prolonged
Neurospora
FROM
dormancy under conditions unfavorable for vegetative growth. All known homothallic species are completely devoid of conidia, leaving ascospores as the only propagules. Could not the overriding advantage of the sexual phase in this special situation be its ability to produce dormant resistant spores? In Chaetomium as in Neurospora, conidia are present in heterothallic and absent in homothallic forms. But the correlation between conidiation and heterothallism is not universal, and conidia are present in both heterothallic and homothallic species of some other ascomycete genera such as Nectria and Gibberella (see Miiller, 1981; Nelson et al., 1983). VIII.
GENETIC NATURAL
A. Categories
VARIATION POPULATIONS
WITHIN
of Variation
Many classes of variants have been found in strains from nature (Table 10). The investigators who identified these variants have had diverse motivations. Most often tbey were interested in the intrinsic properties of the variant itself and its potential usefulness. However, some of the work has been directed at the organism and population levels. Several studies have been undertaken specifically to evaluate genetic polymorphism of chromosomal genes within populations. Noteworthy are investigation of protein polymorphisms, vegetative incompatibility, and recessive genes expressed in the sexual diplophase. These will be considered in more detail in Section B below. Another category of chromosomal variants consists of the segregationdistorting Spore killer genes, whose distribution and characteristics raise questions at the population, cellular, and molecular levels. These are considered in Section C. Later sections describe other categories of chromosomal variants. For example, restriction fragment length polymorphisms (RFLPs) have been found with high fre-
NATURE
119
quency in N. crassa. Investigations of wildcollected strains have concerned not only the nuclear but also the mitochondrial nome. Unanticipated results include the discovery of mitochondrial plasmid share no sequence homology with chondrial DNA and that may be transmits ted horizontally across species, and the discovery that wild strains from some areas can undergo senescence as a result of sequences inserted into mitochondrial D Mitochondrial variants are discusse Section H. B. Studies Permitting Evaluation Genetic Polymorphism within Populations
Q!
A revolution in thinking occu years ago when it was realized that populations of diploid higher organisms such as man and Drosophila carry multiple alleles at a large fraction of their loci (see Lewontin, 1973; Ayala, 1984 for reviews). The frequency of these genetic polymorphisms was too high to be explaine occurrence of new mutations. One esis proposed that the polymorp isted because heterozygotes were at a selective advantage. We can now ask whether Ne~~o§~5 with its haploid vegetative phase, res bles diploids in having high genie vat-i ty, or whether Neurospora populations are genetically more uniform than dip plants and animals, perhaps as a consequence of haploidy. Because we sample 7 to 10 colonies at each collection site we possess the material to address this question. The answer is clear. Local po~~lat~~~~ the heterothallic species N. crassa an intermedia contain a remarkably amount of genie variability-a level comparable to what is found in diploid higher organisms. Such polymorphism is co~mo for electrophoretically disti~g~isl~able isozymes, vegetative incompatibility, and
112
PERKINS
Genetic Variants Originating Variant Chromosomal genome Proteins Amylase Aryl-sulfatase P-Glucosidases
Locus
-
AND TURNER TABLE 10 in Wild-collected
Neurospora
Species”
Strains References
ars -
sit tet era, int
Cellulase
-
int
Esterases
-
int, tet
Ornithine transcarbamylase Leucyl-tRNA synthetase Invertase Nitrate reductase Perithecial protein Protease (extracellular) Nitrogen regulation
arg-12
era
Reddy and Threlkeld (1971a, b, 1972) Metzenberg and Ahlgren (1971) Mahadevan and Eberhart (1964), Eberhart and Madden (1973) Eberhart and Beck (1969), Eberhart et al. (1977) Reddy and Threlkeld (1972), Reddy (1973). Sagarra (1973), Egashira (1986) Grindle and Davis (1966, 1970)
leu-5
era
Beauchamp et al. (1977)
inv (= mig)b nit-4 pts-1
era int 8 SPP. era
Yu et al. (1971) Blakely and Srb (1962) Nasrallah and Srb (1973, 1977) Hanson and Marzluf (1975)
nit-2
sit
Grove and Marzluf (1981) Whitehouse (1942, p. 57) Horowitz et al. (1961a), Horowitz and Fling (1953), Rtiegg et al. (1982) Horowitz et al. (1961b)
(=pink)
Tyrosinase
T
era, int
Tyrosinase (regulatory) Various proteins
KY-2
sit
-
era, int, sit
Nutritional requirements Threonine Thiamine Vegetative incompatibility her loci
int
Chang et al. (1962), Reddy and Threlkeld (1971a, b, 1972) Spieth (1975)
-
era era
Perkins et al. (1976, p. 303) Leslie and Raju (1985)
het-c, -d, -e het-5-het-10
era era
Perkins (197.5), Mylyk (1975, 1976), Leslie (1987) Newmeyer (1970)
era
Perkins and Borkman (1978)
era, int
Al-Saqur and Smith (1980)
era
Wallace (1970), see Perkins
Suppressor to1 Morphology Morphology scat Resistance to toxic agents Surfactant resistant Triphenyltet tetrazolium chloride Canavanine cnr Meiosis, recombination, ascus development Sexual-phase recessives Meiosis impaired mei-l Recombination ret-I, -3 repression Synaptic sequence ss
et al. (1982)
era
Perkins (1960), see Perkins et al. (1982)
era
Leslie and Raju (1985)
era era
Smith (1975) Catcheside (1975)
era
Catcheside (1981)
Neurospora FROM NATURE TABLE Variant
Locus
Dominance of peak alleles Eight-spored ascus Spore killer Spore killer Resistance to Sk
lo--Continued
pk
sit, tet
Srb and Basl (1972)
E
tet
Dodge (1939) Dodge et al. (19%)
Sk-l, Sk-2, Sk-3 r(Sk-2), r(Sk-3)
sit, int int, era
Turner and Perkins (1979) Turner (1977), Campbell and Turner (1987)
int, era SPP.
zeta-eta
4
-
era, int, sit
Senescence
-
int
Mitochondrial plasmids
-
era, int, tet
-
int
Virus-like
particles
References
Species”
Restriction fragment length polymorphisms Nontranscribed NO 5 SPP. spacers, rDNA Sequences flanking era SS RNA genes Chromosome rearrangements, structural polymorphisms Satellite sat era Translocations Tandem duplication Mitochondrial genome Mitochondrial DNA variants
113
Russell et a/ (1984), Perkins et al. (1986a) Metzenberg et al. (1984, 1985) Perkins et al. (1986a), Newmeyer et aE. (1987) Perkins et al. (1976 and unpublished) Selker and Stevens (1987) Mannella et al. (1979), Collins and Lambowitz (1983), Taylor et al. (1986, 1987), Natvig and Jackson (1986) Rieck et al. (1982), Griffiths and Bertrand (1984), Bertrand et al. (1985, 1986), Bertrand (1986), Court ef a[. (1987) Stahl et al. (1982), Coihns and Lambowitz, (1983), Nargang et al. (1984), Natvig et al. (1984) Taylor et al. (1985), Lambowitz ez al. (1986), Nargang (1985, 1986), Pande and Nargang (1986) Tuveson and Peterson (1972)
era, N. crassa; int, N. intermedia; sit, N. sitophila; tet, N. tetrasperma. ’ See White et al. (1985) regarding allelism of inv and mig. a
recessive genes affecting the sexual phase. Genes conferring resistance to killing by Spore killer are also polymorphic in some populations. 1. Protein polymorphisms. Electrophoretically detectable allozyme variation was surveyed by Spieth (1975) in N. intermedia populations from Malaya, Papua New Guinea, Australia, and Florida. General proteins, acid phosphatases, and esterases were examined in 4 to 11 strains from each of 19 local sites in the four regions. His sur-
vey showed that genetic variation is present at a high level, and the pattern of variation within and between populations resembles that in mouse, man, and Dr~sQp~i~~~ A majority of the variation in the species is found in the local population, just as is true for outbreeding diploids . Theoretical interpretation of such striking variability in a predominantly h organism is complicated by Neuro brief but important sexual phase, during which application of diploid selection t
114
PERKINS
AND
ries is appropriate. However, Spieth (1975) suggests that “haplophase selection ought to dominate the behavior of some, if not many, allozyme loci.” In addressing the question of how selection could act during the vegetative haplophase to maintain polymorphisms, he presents a model based on environmental heterogeneity which evokes gene flow among neighboring populations that occupy shifting multiple adaptive niches. Spieth concludes that regardless of the mechanisms involved, the Neurosporu results show that “high intrapopulational genetic variation at the molecular level is not an exclusive property of species with life cycles that constrain the effects of selection almost entirely to the diplophase.” 2. Vegetative incompatibility. Differences in genes that determine vegetative (heterokaryon) incompatibility were studied by Mylyk (1976) in local populations of N. CY~SSUfrom different localities in Louisiana. As found for protein polymorphisms, an unexpectedly hir;h level of variability exists within each population. The large number of differences at vegetative incompatibility loci effectively precludes heterokaryon formation between N. crassa strains in nature. Vegetative incompatibility in N. crassa is determined by genes at numerous het (“heterokaryon incompatibility”) loci (Garnjobst, 1953; Holloway, 1955; Mylyk, 1975). An allelic difference at any one het locus is sufficient to evoke the incompatibility reaction. Without special techniques it would be very laborious to perform a precise genetic analysis because crosses involving wild strains and laboratory testers are likely to segregate for different alleles at several loci, all with similar phenotypes. This problem can be circumvented by crossing the strains under study to testers that contain duplication-producing rearrangements. Among the progeny from each such cross, one defined chromosome segment is present in duplicate, and the rest of the genome is haploid (see Perkins and
TURNER
Barry, 1977; Perkins, 1975). If a het locus i, included in the duplicated segment and i: heterozygous, a nonlethal incompatibilit! reaction occurs in every cell. Thus, the lo cus can be recognized because the incom patibility difference makes the culture sick with characteristic abnormal growth am morphology. Heterozygous duplications were used ir this way by Mylyk (1975) to detect 6 of the 10 het loci that have been mapped in Neurospora. Mylyk (1976) also used the method to test five isolates from each of three wild populations for differences at 5 het loci. He found that incompatibility differences were so numerous that no two strains of the same population were capable of forming heterokaryons with each other. Furthermore, each population was polymorphic for at least 3 of the 5 tested incompatibility loci. The results essentially rule out a role for heterokaryosis in natural populations of outbreeding species such as N. crassa. Because each culture has a virtually unique set of alleles for vegetative incompatibility, colonies remain genetically distinct monokaryotic individuals, and the vegetative unit that is exposed to natural selection is a discrete homokaryotic haploid genotype, not a heterokaryon. This feature of vegetative incompatibility has important consequences for the genetic organization and evolution of Neurospora populations (Spieth, 1975). Numerous other hypotheses have been suggested regarding the significance of vegetative incompatibility. Perhaps it protects from infectious agents (Caten, 1972; Bremermann, 1980; Hamilton, 1982) or from invasion by inferior genotypes (Hart1 et al., 1975). Its role might be to promote the initiation of sexual reproduction (Butcher, 1968; F. J. Doe, unpublished observations), perhaps by the trigger of a protease-mediated injury reaction as suggested for Podospora (see Labarere and Bernet, 1979 for references). Vegetative incompat-
Neurospora
FROM
ibility might, of course, play more than one of these roles. Polymorphism for bet genes is by no means limited to N. crussa but appears to be widespread. For example, populations of several ascomycetes that are important plant pathogens have been shown to contain vegetative incompatibility differences at numerous loci. Among the best analyzed are Cochliobotus heterostrophus (Bipolaris maydis
= Helminthosporium
maydis)
(Leach and Yoder, 1983), the cause of corn leaf blight; Gibberella fujikuroi (F’usarium moniliforme) (Puhalla and Spieth, 1985), an important pathogen of maize, rice, sugar cane, and sorghum; Endothia parasitica (Anagnostakis, 1977,1982,1983), the chestnut blight pathogen; and Ophiostoma (Ceratocystis) ulmi (Brasier, 1983a, b), the cause of Dutch elm disease. Vegetative incompatibility has hindered control of Endothia by restricting transfer of a hypovirulence agent. Transfer of a cytoplasmic factor infecting Ophiostoma is also limited by vegetative incompatibility differences in the fungus. The high specificity of self-nonself recognition that is shown by vegetative incompatibility systems is suggestive of the specificity shown by some genotypes governing susceptibility and virulence in plant hosts and fungal pathogens. It remains to be seen whether the mechanisms underlying vegetative incompatibility and pathogenicity share some common features. Vegetative incompatibility bears on plant pathology in yet another respect. In certain asexually reproducing pathogenic fungi, formae speciales and races, defined by host specificity, are congruent with vegetative incompatibility groups (Puhalla, 1985; Puhalla and Eiummel, 1983). Conventional genetic analysis is not possible in many of the pathogens. Vegetative incompatibility in Neurospora may serve as a model. 3. Recessive genes expressed during the sexual phase, A systematic search by Les-
lie and Raju (1985) revealed that most of the
NATURE
115
N. crassa strains obtained from nature carry one or more recessive genes that are expressed only in the sexual diplophase and that are detrimental to fertility when they are homozygous . Laboratory strains were constructed as testers containing selective markers crossover suppressors such that survi f, progeny from test crosses would receive most of their genome from the tested parent (Leslie, 1985). The tester strains wer crossed to isolates from N. crassa wi1 populations, and several f, progeny from each test cross were then backcrossed i dividually to the wild-collected parent a to a laboratory strain as control. The presence of one or more phase-specific recessive genes in a wild isolate was indicated if some abnormality of the perithecial phase was seen in some of the backcrosses to the wild parent (i.e., those in which the gene was homozygous) but not in the corresponding controls (where the gene remained heterozygous). Ninety-nine isolates were analyzed from 26 natural populations. Eighty of the strains contained among them 106 recessive genes that were expressed in the sexu a majority of the mutants, meiosis cus development were blocked the remainder, ascospore maturation was significantly impaired. The rn~ta~ts are thus equivalent to recessive le semilethals in higher diploid orga Intercrosses between 1200 m all showed positive complementat~o~. Clearly, many different loci must be involved. Presumably, this load of deleterious mutations is tolerated only because outbreeding is extensive. These results confirm that meiosis and sporogenesis are under complex and finetuned genetic control, and that mutation in any of a multitude of genes can impair fertility. This implies that the sexual part of the life cycle would quickly be by mutation if it were not mai diligent selection.
116
PERKINS
AND
4. Resistance to killing by Spore killer. In the study of Spore killer systems described below in Section C, we found wild strains that are resistant to killing but that are not themselves killers (Turner, 1977). There are four expected phenotypes for resistance to killing-resistant to both Sk-2K and Sk-3K, resistant to neither, or singly resistant. Resistance is determined by a gene or by two closely linked genes in linkage group III. Strains collected before 1977 have been analyzed. All four phenotypes were found in Australia, Papua New Guinea, Indonesia, and Malaya. There was very little clustering within sites. For example, among 70 strains from eight sites in Australia, each of the four phenotypes was represented at six or seven of the sites. Conclusions on intrapopulation variation. Whenever genetic variability has been carefully assayed in N. crassa or N. intermedia a very high level has been found within the local population. The genetic polymorphism is comparable in level and pattern to that in outbreeding diploid eukaryotes. It seems clear that these heterothallic Neurospora species are strict outbreeders. C. Spore Killers A mechanism which affects the genetic composition of succeeding sexual generations by inequalities of chromosome transmission is termed meiotic drive or transmission-ratio distortion. “Driven” genes contribute disproportionately to the resulting gene pool, regardless of their selective advantage for the individual organism (reviewed by Zimmering et al., 1970; Crow, 1979). Spore killer strains of Neurospora have provided a well-documented example of meiotic drive in fungi. Three distinct Spore killer factors have been obtained independently in Neurospora from different wild sources. The factors are chromosomal, and they have been
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symbolized Sk-lK, Sk-2K, and Sk-3K. Characteristics of the Spore killer systems are outlined below. Sk-2K, which is used as an example, originated in N. interrnedia and was introgressed into N. crassa for ease of study. Unreferenced information is from Turner and Perkins (1979), Raju (1979), or Turner et al. (1987). When a Spore killer strain is crossed by Spore killer (Sk-2K X Sk-2K), all eight ascospores survive. When a Spore killer strain is crossed with a normal laboratory strain or with any other strain that is sensitive to killing (Sk-2K X Sk-2’), eight ascospores are formed in each ascus, but four of the spores degenerate. The survivors all carry the killer allele-it is the normal wild type that is killed. Killing does not depend on which strain is used as female and which as fertilizing parent-reciprocal crosses are alike. Sk-2K shows 100% first-division segregation. No difference between Sk-2K and Sk-2’ has been detected in the vegetative phase. Heterokaryons containing nuclei of both types in the same cytoplasm are vigorous and healthy. Likewise, meiosis and ascus differentiation are normal in heterozygous crosses where killing will occur. Abnormalities are seen only after Sk-2’ nuclei are sequestered by being enclosed in an ascospore wall. The prospective lethality is not irreversible until this time, because Sk-2’ nuclei which would otherwise die are rescued if an Sk-2K nucleus or chromosome is enclosed within the same ascospore wall. When Sk-2K is crossed with a non-killer strain, crossing over is virtually eliminated in a region at least 30 units long which extends to both sides of the III centromere. Marker sequence and crossing over frequencies are normal in Sk-2K x Sk-2K (Campbell and Turner, 1987). Sk-3K resembles Sk-2K in its mode of killing, its map location, and its effect on recombination. But Sk-3K differs from Sk-2K in specificity of killing and resistance. Sk-
Neurospora
FROM
3K does not kill itself but is sensitive to killing by Sk-2K, and vice versa. In crosses between Sk-2K and Sk-3K, all ascospores abort in most of the asci. Both Sk-2K and Sk-3K are rare. They have been identified only in eastern hemisphere populations of N. intermedia. Sk-2K has been found four times, and Sk-3K once. Laboratory strains and most of 1400 tested wild-collected isolates of N. intermedia and N. crassa are sensitive to both Sk-2K and Sk-3K, but neutral (resistant) strains that neither kill nor are killed are also found in nature (Turner, 1977). Strains resistant to Sk-2K, to Sk-3K, or to both are common in N. intermedia in the eastern hemisphere. Though no Spore killer has been found in N. crassa, the only resistant isolate of any kind that has been found in the western hemisphere is a N. crassa strain resistant to Sk-2K. Resistance segregates as a single chromosomal gene, r(Sk-2)-l, in IIIL. A gene from N. intermedia conferring resistance to Sk-3K maps in the same region. Recombination of markers in the III centromere region is not affected by the genes conferring resistance to killing-it is blocked in Sk-2K x r(Sk-2)-l but not in Sk2’ X r(Sk-2)-I, and similarly for r(Sk-3)-l with Sk-3K (Campbell and Turner, 1987). Both Sk-lK and Sk-IS are widely distributed in wild-collected N. sitophila, but the large samples from Haiti and the Ivory Coast are ail Sk-IS. Sk-lK and Sk-IS are sometimes found in the same geographical region, and in Tahiti they were found together in the same local population. No resistant non-killers have been identified. Attempts to introgress Sk-lK into N. crassa have failed. The chromosomal location of Sk-lK is unknown, as is its effect on recombination. In addition to their theoretical interest, Spore killers have had practical applications in the laboratory, enabling the efficiency of tetrad analysis to be increased significantly (Perkins et al., 1986b).
NATURE
117
Chromosomally based Spore killing systems are known in two other Pyrenomycetes. Two unlinked ascospore-abortion genes in Podospora anserina (Padieu and Bernet, 1967) behave exactly as would be expected if their mechanism of killing were the same as for Sk-2K and Sk-3K in Nernrospora. A majority of wild isolates of 6. J% jikuroi (F. moniliforme) contain a Spore killer allele (Kathariou and Spieth, 1982). The fungal Spore killers resemble meiotic drive systems in higher plants and animals, which are typically manifested postmeiotitally by differential survival of spores or gametes (see Zimmering et al., 1970 review). The Spore killer phenomenon raises some evolutionary questions for w it may be difficult to obtain answers. Is it possible that today’s predominant normal wild type in each species is carrying a killer allele that was once a newly minority type but that has since eliminate the previously ubiquitous sensitive allele‘? Could it be that Spore killers play a role in promoting genetic isolation between incipient species, as might be expected if ent, mutually sensitive, Spore killer genes similar to Sk-2K and Sk-3K had beco fixed during the differentiation of nascent sibling species, with each population carrying one of the killers but not the otbe~? It was possible to return and res one site where Spore killer strains we found during an earlier visit. In 1968, bo Sk-2K and Sk-3K were represented alone six isolates collected from bnr vegetation at a site near Rouna Falls, ua New Guinea. Not a single killer strain was found in a sample of over 100 isolates colle years later from the same site. Killer were also absent from samples taken elsewhere in New Guinea in 1983. Thus it seems that Sk-2K and Sk-3K are not replacing their sensitive counterparts in nature; apparently the killer alleles are being in check by counterselection or resistance.
118 D. Genes Specifying
PERKINS
Ribosomal
AND
RNA
Wild-collected strains have been used extensively in studying the gene families that specify ribosomal RNA in Neurospora. As in other eukaryotes, the 17S, 5.8S, and 25s components of cytoplasmic ribosomes are processed from a larger precursor molecule which is coded by a family of tandemly repeated chromosomal genes that specify ribosomal RNA. The repeats are separated by nontranscribed DNA segments (Free et al., 1979; Cox and Peden, 1979). The number of repeats is around 200 in N. crassa, but ranges from approximately 155 to 265 in different strains (Rodland and Russell, 1983a, b). The ribosomal RNA genes are located in the nucleolus organizer region (Perkins et al., 1986a) at the left end of linkage group V (Barry and Perkins, 1969). The sequence organization of these repeats has been examined by Russell et al. (1984) in strains from different geographical regions, including representatives of five species. Restriction enzyme digests were probed using cloned segments of known constitution. Transcribed portions of the ribosomal DNA appear to be invariant. In contrast, the nontranscribed spacer segments are variable from strain to strain, although they are homogeneous within each individual strain. The spacer variation between N. crassa isolated from different geographical regions is as great as that between different species. It has not been determined whether similar variability in nontranscribed DNA spacers is found among the individuals of a single local population. Genes speciJLing 5.7 RNA.
differs having demly widely
Neurospora
from most other eukaryotes in not its 5s gene family clustered or tanrepeated. Instead, the 5s genes are dispersed (Free et al., 1979; Selker et al., 1981). There are about 100 5s genes per genome. Restriction fragment length polymorphisms (RFLPs) have been used to map the individual 5s genes at loci scat-
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tered through six different chromosomes (Metzenberg et al., 1984, 1985). Although a majority of the 5s genes code for a 5s RNA sequence called alpha, several alternate sequences also exist (called beta, gamma, etc.). Similar 5s RNA heterogeneity is also found within different Neurospora species and in related genera (Selker et al., 1985a). Only one of 23 EcoRl restriction fragments from an Oak Ridge strain of N. crassu, cloned on the basis of positive hybridization with 5s RNA, contains more than one 5s RNA gene. The exceptional clone contains a pair of 5s genes or pseudogenes, “zeta” and “eta,” embedded in a short direct tandem duplication in linkage group I (Selker and Stevens, 1985). Unlike Neurospora 5s RNA genes generally, the zeta and eta genes are heavily methylated. However, many strains of N. crassa and strains of N. tetrasperma, N. sitophila, and N. intermedia have one instead of two copies of the homologous DNA, and when there is a single copy it is unmethylated (Selker and Stevens, 1987). It has been suggested that families of small genes such as 5s RNA genes may be more functional or better preserved in Neurospora in a dispersed rather than a tandemly repeated arrangement (Selker et al., 1985b). E. U. Selker and R. L. Metzenberg (personal communication) have surveyed wildcollected N. crassa strains from seven widely separated wild populations and have compared them to a standard laboratory strain. All the strains show differences in the size of the restriction fragments that contain their 5s genes. This means either that the genes are in different chromosomal locations in the different wild strains or that the chromosomal regions flanking the individual 5s genes have diverged in DNA sequence. The latter hypothesis was supported by experiments in which regions flanking 5s genes in the standard laboratory strain were used to clone homologous seg-
Neurospora
FROM
ments from a different wild strain. Each of the new clones was found to contain a 5S genein the sameposition as in the standard, indicating that deletion or insertion of 5s genes is not occurring at detectable frequency (Morzycka-Wroblewska et al., 1985).
NATURE
to detect RFLPs among wild strains representing several species. This method can apparently provide a far more sensitive measureof genetic relatednessthan is sible with isozyme electrophoresi DNA:DNA solution hybridization. F. Genes Controlling
E. Restriction Fragment Length Polymorphisms
11
Re~om~i~atio~
In N. crassa, the recombination cy in specific map intervals varies tally in crosses of different parenting. D. 6. Catchesideand his associateshave shown that this variation is due to a series of ret genesthat precisely control meiotic recombination in specific chromosome regions to which they are not linked (sum ries in Catcheside, 1977; Fincham et al., 1979).Local control of recombination also involves a cis-acting element cog (recognition), which is believed to r for initiating recombinatio joining structural gene (see 1986).Different alleles of these regulatory geneswere discoveredin existing laboratory stocks and presumablyoriginated in different wild ancestors.The probable or&i of someof them can be traced back throug pedigrees to two original collections in Louisiana (Catcheside,1975;Newmeyer et al., 1987).All three known alleles of re have been shown to be present in w collected stocks (D. G. Catcheside, personal communication). Recombination can also be aftecte variants of a different type called ss aptic sequence),which have beenfoxed at or nearthe nit-2 locus in wild-colIecte~ and laboratory strains of N. crassa. nation in the nit-2 locus is reduce alleles are heterozygous (Ca 19sa>.
Genetic polymorphisms that can be detected by electrophoreticdifferencesat the protein level are abundant (Spieth, 1975). DNA-level variants are expectedto be even more abundant.This expectation has been confirmed (Metzenberget al., 1984; Natvig et al., 1987). For example, although the wild-collected strain Mauriceville-lc A is completely fertile with the laboratory wild type Oak Ridge a and is phenotypically indistinguishablefrom the inbred Oak Ridge strains, it is nevertheless differentiated from Oak Ridge by thousands of nucleotides throughout the chromosomes, as demonstratedby differencesin sites subject to cleavageby restriction endonucleases. The first use of RFLPs to map chromosomal genes in Neurospora was with cloned DNA segments containing the 5s RNA genes(Metzenberget al., 1984,1985). The RFLP method has since been widely used for genetic mapping, e.g., by Berlin and Yanofsky (1985),Orbach et al. (1986), Akins and Lambowitz (1985),and Stewart and Vollmer (1986). RFLPs in ribosomal RNA gene spacershave provided markers for analyzing deamplification of ribosomal RNA genes (Rodland and Russell, 1983a) and for analyzing a recombinationally generated translocation (Perkins et al., 1986a). Another application of restriction analysis is to provide information on phylogenet- 6. Chromosome Rearrangements ic relationships. Natvig et al. (1987)have Structural variants suchas translocatio~s used cloned single-copy nuclear DNA sequencesfrom a N. crassa reference strain recombine meiotically with standard sein combination with Southernhybridization quenceto produce deficiencies, which are
120
PERKINS
AND
detected in our screening method by the presence of inviable white ascospores in predictable numbers and patterns (Perkins and Barry, 1977). Reciprocal translocations are especially easy to detect in Neurospora, and hundreds have been obtained following mutagenesis in the laboratory. Only four reciprocal translocations have been identified among all our wild-collected isolates, however (Perkins et al., 1976 and unpublished). Each of these translocations was present in a population that also included the standard sequence. Additional rearrangements, in other wild isolates, may have gone undetected, because even when crosses to a tester are fertile enough to determine species, other causes of ascospore abortion are often present that would mask the additional abortion caused by a chromosome rearrangement. Among the more recent collections many crosses with aborted ascospores remain to be analyzed. No paracentric inversions have been detected either in wild strains or among rearrangements originating in the laboratory. Perhaps this reflects our inability to recognize them (see Perkins and Barry, 1977). Chromosome complements of all 10 Neurospora species appear to be similar, judging from pachytene karyotypes observed after staining with hematoxylin (Raju, 1978, 1980). Pachytene morphology has been examined in detail only for N. crassa and N. intermedia and for the intercross between them, using aceto-orcein. The two species do not differ by any obvious chromosome rearrangements, judging from pairing of homologs at pachytene in the interspecific cross (Perkins et al., 1976). There is no evidence that chromosome rearrangements are responsible for the fertility barriers between species. A small nucleolus satellite, found in some laboratory strains of N. crussa, has provided a cytological marker for genetic mapping of the nucleolus organizer (Barry and Perkins, 1969) and the genes that specify ribosomal RNA (Perkins et al., 1986a). The
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satellite has been traced to material col lected in Louisiana in 1943 (Newmeyer ei al., 1987). Satellites have not been seen in N. crassa from other sources or in other Neurospora species. However, only a few wild-collected strains have been examined cytologically in such a way that a satellite would be revealed; critical examination is too laborious to allow a broad survey. H. Mitochondrial
Variants
Extranuclear genetic determinants are transmitted maternally in N. crassa (Mitchell and Mitchell, 1952; Manella et al., 1979). It is reasonable to assume that maternal transmission is the rule in the other heterothallic species where fertilization also occurs via a trichogyne, and that maternal transmission occurs even in N. tetrasperma when homokaryotic strains of opposite mating types are used as male and female parents. (G. Bistis [personal communication] has observed that protoperithecia of homkaryotic A and a strains of N. tetrasperma produce trichogynes which orient their growth toward cells of the opposite mating type, just as though the species were heterothallic.) Mitochondrial DNA. Strain differences are not limited to the chromosomal genome. Initially, two types of mitochondrial DNA were identified by restriction analysis of laboratory strains (Bernard et al., 1976). Their probable origins were traced by pedigree to ancestral strains from two different wild sources (Mannella et al., 1979). Additional structural variants have since been identified in wild-collected strains. There is much variability, consisting in part of nucleotide substitution but predominantly of insertions and deletions (Collins and Lambowitz, 1983; Taylor et al., 1986). Taylor et al. (1986) have characterized mitochondrial DNA in N. crassa strains from different parts of the world. Length mutations are frequent and are correlated with geographic distribution. Site changes are also present
Neurospora
FROM
but are infrequent. Because of the length mutations, mitochondrial DNA may range from 60 to 78 kb in different isolates. The insertions or deletions are typically located outside the region that encodes ribosomal or transfer RNA. Introns in the gene encoding cytochrome oxidase subunit 1 are commonly involved, and as many as four introns may be present (Collins and Lambowitz, 1983). Senescence. Neurospora strains from most wild sources are potentially immortal and will continue growing indefinitely. A systematic survey of wild-collected strains revealed that certain N. intermedia strains from Hawaii were exceptional in showing a limited duration of vegetative growth (Rieck et al., 1982; Griffiths and Bertrand, 1984). Upon the onset of aging, cytochromes become abnormal and mitochondrial DNA is seen to be altered by insertion of a 9-kb DNA segment (kal DNA) into the mitochondrial chromosome. Each inserted kal DNA sequence is flanked by long homologous inverted repeats of mitochondrial DNA (Dasgupta et al., 1987). The altered mitochondria accumulate during growth, leading to respiratory deficiency and death (Bertrand et al., 1985). Deletion or partial deletion of the kal insertion may change the pattern of aging (Myers and Griffiths: 1985). The kal DNA sequences are initially associated with the nucleus. However, kal DNA is transmitted to progeny only through the female parent and never through the male (Bertrand et al., 1986; Bertrand, 1986). A N. crassa strain from India contains a structurally different linear plasmid which, like kal, induces senescence by inserting in mitochondria (Court et al., 1987). Plasmids. Circular 3.6- to 5.2-kb plasmids were first found in the mitochondria of wild N. crassa and N. intermedia strains which were being screened for differences in mitochondrial DNA (Collins et al., 1981; Stohl et al., 1982). Mitochondrial plasmids were also found in wild-collected N. tet-
NATURE
121
(Natvig et al., 1984). The plasmids show little or no sequence ho with Neurospora mitochondrial However, weak homology betwe mid and mitochondrial DNAs has tected in one exceptional instance by Pa et al. (1987). No biological function has been identified for the plasmids. Codon usage and conserved sequence elements of the best studied plasmid resemble those of Group I mitochondrial DNA introns (Nargang et al., 1984), but none of the Neurospora plasmids has been found integrated in mitochondrial DNA of normal strains (Collins et al., 1981; Stohl et al., 1982; Narga~g et al., 1984; Nargang, 1986). For reviews, see Nargang (1985), Kinsey (19856, and Lambowitz et al. (1986). A survey of many Neurospora strains by S. J. Vollmer (personal communication) has shown that plasmids are cornrno~~~ present and that more than one class of plasmid may coexist in the same strain. These plasmids were inherited maternally and were not transmitted from the male (fertilizing) parent in reciprocal crosses, as had also been shown for other plas Collins et al. (1981) and Stohl et aE. ( Plasmids in different Neurospora s may show sequence homology wi other, while different plasmids i species may not (Natvig et al., presence of identical or hornol~g~~s plasmids in three species suggests that mission can sometimes occur ind dently of the host mitochondria (Taylor et al., 1985). J. W. Taylor and G. May (personal communication, 1987) have dejected paternally derived plasmid DNA but significant fraction of progen imental crosses between strains crassa. Since mitochondria are deriv from the maternal parent, the pla thus transmitted independently of chondria. Akins et al. (1986) impaired mutant de mid-bearing strains. rasperma
122
PERKINS
AND
mids in the mutants have acquired inserts of mitochondrial DNA; mitochondria containing an altered plasmid outcompete normally functioning mitochondria (i.e., they have become suppressive). Also, mitochondrial DNAs have acquired plasmid sequences in at least some of the mutants. I. Variants
of Other Types
Many variants that have been recovered from the wild-collected strains are not related in any direct way to questions of population variability or evolution but have been of interest for other reasons. Numerous chromosomal loci have been identified using allelic differences that originated from wild material rather than as mutations in the laboratory. Examples in addition to those described above are cnr, mei-1, pts, sar, scat, T, and tet (see Perkins et al., 1982). Variants from some of the wildcollected strains produce altered proteins that have been used in biochemical studies. One N. intermedia isolate from Javanese oncham (Tjisarua, P760) resembles the per1 mutant of N. crassa, which makes perithecial walls devoid of black pigment when the strain is used as female parent. The diverse uses to which a single wildcollected isolate may be put are illustrated by the strain Mauriceville-lc A. This strain of N. crassa (P538, FGSC No. 2225) was collected in 1972 in Texas. It is identical in appearance to the standard Oak Ridge (OR) laboratory strains, and intercrosses between the two are highly fertile. Pachytene pairing is good in the intercross of Mauriceville with OR, so the chromosomes are apparently isosequential. Yet, genetic differences present in Mauriceville-lc A have had important applications: (a) A new lea-5 allele from the Mauriceville strain results in altered mitochondrial leucyl-tRNA synthetase (Beauchamp et al., 1977). (b) The Mauriceville strain contains a 3.6-kb circular plasmid which has been completely se-
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quenced and subjected to intensive study (Collins et al., 1981; Nargang et al., 1984; Akins et al., 1986). The plasmid was discovered during a survey of mitochondrial DNA polymorphisms (Collins and Lambowitz, 1983). (c) Chromosomal DNA of the Mauriceville strain differs from the OR standards by nucleotide differences at a very large number of sites. This led to its choice as one parent in the development of a method for mapping cloned DNA fragments using restriction fragment length polymorphisms as genetic markers (Metzenberg et al., 1984, 1985; Section E, above). (d) A strain derived from Mauriceville-lc has been used as a transformation host to demonstrate the existence of a signal at the zeta-eta locus, causing DNA methylation de nova (Selker et al., 1987). IX.
TRANSFER SPECIES
OF TO
GENES
FROM
ONE
ANOTHER
We have used reproductive isolation as the primary criterion for distinguishing Neurospora species. That isolation is not always complete, however-at least not in the laboratory. While N. discreta has never produced viable ascospores in an interspecific cross, other combinations of the nonhomothallic species have all produced at least occasional hybrid progeny when just the right strains were selected as parents, The ability to produce hybrid progeny does not, of course, invalidate the species status of the parents, whether for fungi, plants, or animals. In flowering plants, for example, crossing and introgression can occur even between distant taxons (Anderson, 1949). Once an fi is obtained from a cross between different Neurospora species, successive backcrosses are typically more fertile, and desired markers can readily be put into the genetic background of a recurrent backcross parent. Progeny from crosses between Neurospora species were first obtained by Dodge
123
Neurospora FROM NATURE
(1928). Many genes have since been transferred for various purposes. Both naturally occurring differences and mutant alleles of laboratory origin have been introgressed. Transfers from N. sitophila to N. crassa (and vice versa) have been the most extensive (Dodge, 1931; Finchman, 1951; ScottEmuakpor, 1965; Srb and Jarolmen, 1967; Mishra and Threlkeld, 1967; Grindle and Davis, 1970; Srb and Basl, 1972; Perkins, 1977a). Genes have also been introgressed between N. intermedia and N. crassa (Horowitz et al., 1961a, b; Blakely and Srb, 1962; Srb and Jarolmen, 1967; Turner and Perkins, 1979), between N. tetrasperma and N. sitophila (Dodge, 1928), and between N. tetrasperma and N. crassa (Johnson and Srb, 1962; Howe and Haysman, 1966; Metzenberg and Ahlgren, 1971, 1973). For some purposes, two steps have been used, as by Metzenberg and Ahlgren (1973), in taking mating type from N. tetrasperma through N. intermedia into N. crassa. Srb (1958) transferred a cytoplasmic mutation SG from N. crassa into N. sitophila and N. intermedia by recurrent crosses using SG as protoperithecial parent. Similarly, Reich and Luck (1966) transferred the mitochondrial genome from N. crassa into N. sitophila, taking advantage of the fact that mitochondria are transmitted only through the female line. Transient hyphal anastomoses have been used to transfer the kal senescence element, mitochondrial DNA, and a mitochondrial plasmid from N. intermedia into N. crassa (D. A. Court and H. Bertrand, cited by Bertrand, 1986; Cheng et al., 1987). Little is known about hybridization and gene transfer between Neurospora species in nature. Opportunities certainly exist, because isolates belonging to different species are frequently found growing in close proximity at the same site. The presence of identical or similar plasmids in strains of different species suggests that horizontal transfer is a natural occurence (Taylor et al., 1985).
X. CONCLUSION:
NEUROSPORAIN
POPULATION EVOLUTIONARY
AND GENETICS
Aspects of Neurospora in relation to the broader aspects of population genetics an evolutionary biology have been discusse from different viewpoints by Spieth (1975) Leslie (1981), Leslie and Raju (1982), Natvig et al. (1984), Taylor et al. (19 and Taylor (1986). The fungi are now considered a separate eukaryote kingdom (Whittaker, 1969). Nuclei are predominantly haploid in the fungi, genomes are small, and lifestyles are vastly different from those of the diploi and plants with which population genetics as a discipline has been largely conc~r~ed~ Fungal populations should yield sig~~c~~t new information and ideas. Yet the fungi have been largely ignored in the literature of population and evolutionary genetics, chiefly because essential information on fungal populations has been lacking. N. crassa is the most studied ~ete~ot~a~lit euascomycete species, with ~urner~~s advantageous features for population analysis. The genus Neurospora includes species with diverse breeding systemsheterothallic outbreeders, the homothallic N. tetrasperma, and horn~tba~~ lit inbreeders. As a typical Pyre~omyce~c, Neurospora represents a subgroup of euascomycetes that includes many important plant pathogens as well as saprophytes with diverse morphologies and adaptations. We are hopeful that the Neuro work reviewed here will encourage studies in the genetics of fungal popul and will contribute to an increased a~pr~c~~ ation of the potential cont~but~ons of fungi. DEDICATION
AND ACKNOWL~~~ME~T~
B. 0. Dodge first recognized the advantages ofNew demonstrated its usefulness for ex~e~irnent~ research, and made intra- and interspee& crosses for comparison. This paper is dedicated to his memory on rospora,
124
PERKINS
AND TURNER
the 60th anniversary of the description of Neurospora (Shear and Dodge, 1927) (see Robbins, 1962; Beadle, 1973; Lindegren, 1973). We are grateful for help from many persons who have participated in collecting or have made collecting possible. The help of individuals who aided in collecting before 1975 has been acknowledged by Perkins et al. (1976). We are indebted to many persons for help since that time. In Brazil: Joao Lticio de Azevedo and Jose Branco de Miranda Filho (Piracicaba). In Papua New Guinea: James Croft and Karl Karenga (Lae); Allen Allison (Watt); James Nasa (Bayer River); D. Kari, Alain Ross, Dickson Bailand, and Charles Yowana (Port Moresby). In Australia: Dorothy E. Shaw (Brisbane). In New Zealand: Ross E. Beever (Aukland). In India: R. Maheshwari, P. V. Balasubramanyam, and P. Palanivelu (Bangalore); K. Dharmalingam, P. Gunasekaran, and Kalaga Ramakrishnan (Madurai); T. V. Damodaran and K. M. Marimuthu (Madras). In Thailand: Visut Bamai, Charoenwitt Hankaew, and Yingsak Swasdipanich (Bangkok). In Malaya: Adenan Jaafer and Tan Sai Tee (Penang); Ho Coy Choke, Fong Foo Woon, and Toh Yow Pong (Kuala Lumpur). In Singapore: Nga Been Hen. In Borneo: Chuah Ching Geh (Kuching); Ghazally Ismael and John Beaman (Kota Kinabalu). In Haiti: Nancy Edling and Gary Edling. The 1985 collecting trip in West Africa was arranged by George Rizet, Denise Marcou, and Pascal Lissouba. Collecting in the Ivory Coast was aided by Gilles Bezancon and D. Le Pierres (Adiopodoume); F. Anthony (Divo); J. Louam (Man); K. M. Miezan (Bouake); in the Congo by Evelyne Gamier-Sillam (Dimonika); and in Gabon by Paul Posso, Olivier Ezavin, Fabienne Beurel, and Christophe Le Houcq (Makokou). We are also deeply grateful for strains collected independently and given to us by the persons named in Tables 8 and 9. Allan Wheals, University of Bath, United Kingdom, kindly sent isolates collected from bakeries (primarily in England) by M. S. B. M. Yassin. Ann S. Fairfield has carried out and helped to improve laboratory procedures. We also thank Virginia C. Pollard, Adam M. Richman, and Joseph L. Campbell for assistance in the laboratory. Dorothy Newmeyer has made many valuable suggestions during preparation of the manuscript. We are grateful to Philip T. Spieth, John W. Taylor, John F. Leslie, and Eric U. Selker for discussion and comments. This review was drafted during a visit by the senior author to the Department of Physiology, Carlsberg Laboratories, Copenhagen, Denmark; the hospitality of Diter von Wettstein and his colleagues is deeply appreciated. This work was supported by Public Health Service Research Grant AI-01462 and Research Career Award K6-GM-4899. The Fungal Genetics Stock Center,
whose holdings include many of the strains reportec here, is supported by National Science Foundatior Grant BSR-8546273. Field work of G. J. Samuelr (DSIR Plant Sciences Division, Auckland, New Zealand) in South America was supported by Grant No. 2736-83 to him from the National Geographic Society and by Project Flora Amazonica. Collecting and writing by DDP in 1983-1985 was carried out during tenure of a Guggenheim Foundation Fellowship. Note added in proof. Following molecular cloning of the A mating type allele of N. crussa (Volhner and Yanofsky, 1986), DNA sequences have been identified that are unique to each of the two mating types, A and a, and are present as a single copy per haploid genome (Glass ef al., 1988). Using these as probes, Glass et al. have shown that each of the homothallic species listed in Tables 1 and 2 retains sequences homologous to one or both of the mating type alleles N. terricola hybridizes with both A- and a-specific sequences, N. galapagosensis only to a-, and the others only to A-specific probes. An additional homothallic isolate, D301 from Dominica, West Indies, provisionally designated N. dominicana (L. H. Huang, personal communication), also contains sequences homologous to the N. crassa A mating type but not to a. REFERENCES
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