Barbara C. Turner

Neurospora from natural populations: Toward the population biology of a haploid eukaryote

Abstract 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 produced meiotic drive. The four Spore-killer-sensitive ascospores in every ascus are killed in crosses of sensitive × killer, but all eight ascospores remain viable in crosses of killer × killer and sensitive × 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.

STRAINS OF NEUROSPORA COLLECTED FROM NATURE

The fungus Neurospora is genetically and biochemically one of the most studied eukaryotic microorganisms. However, little is known of natural populations, and there has been little systematic collecting or study to provide information on population genetics or ecology. This study was undertaken with the hope that investigations of Neurospora from nature might contribute significantly to evolutionary biology in several ways. It seemed of interest to examine some of the tenets of population genetics using an organism whose life cycle and life style are radically different from those of the diploid organisms conventionally considered. It was hoped specifically to gather information on the role and significance of sexual reproduction, heterokaryosis, and vegetative incompatibility, on genic and chromosomal variability within and between Mendelian populations, on reproductive isolation, and on systematic relationships. The studies reported here show that it is feasible to find and observe Neurospora in the field, and that populations can readily be sampled by obtaining cultures that have originated from single ascospores. The results indicate clearly that Neurospora can contribute information of value to evolutionary biology. At the same time, an evolutionary perspective promises to enrich laboratory investigations of the fungi. Researches on nutritional and regulatory variants, variations in chromosome structure and behavior, heterokaryon compatibility, and the sexual mating types, have been largely concerned in the past with mechanisms, and have given little

Meiotic Drive in Neurospora and Other Fungi

When a gene complex called Spore killer is heterozygous, ascospores representing two of the four products of each meiosis are killed. Only those that receive the killer complex survive. This article reviews what is known of the mode of action of the Neurospora Spore killers, their chromosomal basis, and their occurrence in nature. Similar genes or gene complexes have been found in other fungi. An example from Podospora anserina is analyzed.

Two ecotypes of Neurospora intermedia

Neurospora intermedia is found on burned substrates in all tropical and subtropical areas that have been sampled in both Eastern and Western Hemispheres. Cultures from burned substrates are salmon orange and are virtually indistinguishable in appearance from typical strains ofN. crassa and N. sitophila. A second, distinct N. intermedia ecotype is found almost exclusively on nonburned substrates and only in the Eastern Hemisphere. This type is saffron yellow rather than salmon orange, with conidia that are significantly larger than those of the other type. The two ecotypes are conspecific on the basis of intercross fertility, although strains of the yellow type show reduced fertility both among themselves and in crosses with orange strains. The visible differences between the types are due to multiple genes which are not tightly linked.

Neurospora strains incorporating fluffy, and their use as testers.

We have used fluffy (fl) strains extensively as female parents in mating-type tests and for a variety of other applications where high fertility and absence of conidia are advantageous.

Cytogenetics of an intrachromosomal transposition in Neurospora

Knowledge of intrachromosomal transpositions has until now been primarily cytological and has been limited to Drosophila and to humans, in both of which segmental shifts can be recognized by altered banding patterns. There has been little genetic information. In this study, we describe the genetic and cytogenetic properties of a transposition in Neurospora crassa. In Tp(IR→IL)T54M94, a 20 map unit segment of linkage group I has been excised from its normal position and inserted near the centromere in the opposite arm, in inverted order. In crosses heterozygous for the transposition, about one-fifth of surviving progeny are duplications carrying the transposed segment in both positions. These result from crossing over in the interstitial region. There is no corresponding class of progeny duplicated for the interstitial segment. The duplication strains are barren in test crosses. A complementary deficiency class is represented by unpigmented, inviable ascospores. Extent of the duplication was determined by duplication-coverage tests. Orientation of the transposed segment was determined using Tp x Tp crosses heterozygous for markers inside and outside the transposed segment, and position of the insertion relative to the centromere was established using quasi-ordered half-tetrads from crosses x Spore killer. Quelling was observed in the primary transformants that were used to introduce a critical marker into the transposed segment by repeat-induced point mutation (RIP).

Main features of Spore killer systems in Neurospora.

Main features of Spore killer systems in Neurospora. This mini review is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol34/iss1/19

Strains for studying Spore killer elements in four Neurospora species

This note gives a comprehensive list of stocks useful for working with Spore killer elements, including reference strains for use as testers, genetically marked derivatives, and strains sensitive and resistant to killing by Sk-1K, Sk-2K, and . Geographical site of origin is indicted for the various killer alleles. Many of the strains are newly deposited in FGSC. Some are listed also under other categories. Updated versions of the list will appear in future FGSC Stock Lists (Part IV, Special Purpose Stocks). Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol40/iss1/30 Strains for studying Spore killer elements in four Neurospora species B.C. Turner and D.D. Perkins Department of Biological Sciences, Stanford University, Stanford CA 94305-5020 This note gives a comprehensive list of stocks useful for working with Spore killer elements, including reference strains for use as testers, genetically marked derivatives, and strains sensitive and resistant to killing by Sk-1K, Sk-2K, and Sk-3K. Geographical site of origin is indicted for the various killer alleles. Many of the strains are newly deposited in FGSC. Some are listed also under other categories. Updated versions of the list will appear in future FGSC Stock Lists (Part IV, Special Purpose Stocks). Characteristics of chromosomally located Spore killer elements have been summarized by Turner et al. (Am. Nat. 137:416-429, 1991; Fungal Genet. Newsl. 34:59-62, 1987) and will only be summarized briefly here. In crosses heterozygous for killer and sensitive (SkK x SkS), four ascospores are usually killed in each 8-spored ascus and the survivors are SkK. All eight ascospores survive in crosses homozygous for the same killer element. Killer elements have been found in natural populations of N. intermedia (Sk-2K and Sk-3K, both rare) and N. sitophila (Sk1K, common). Sk-2K and Sk-3K have been introgressed from N. intermedia into N. crassa for convenience of genetic analysis. Both are haplotypes-presumed gene complexes in a centromerespanning segment of linkage group III within which meiotic recombination is repressed in the killer/sensitive heterozygotes. Sk-2K and Sk-3K are similar in behavior except that each is sensitive to killing by the other. The two also differ in their reaction to genes conferring resistance to killing. Genes conferring resistance to Sk-2K or to Sk-3K are present in some populations of N. intermedia. Resistance to Sk-2K is found at low frequency throughout the range of N. crassa. The resistance genes, symbolized r(Sk)-2 and r(Sk)-3, have been mapped in wild type sequence in N. crassa. They are linked to loci within the killer complex. Map relations in N. crassa are shown in Figure 1. Linkage relations of Sk-1K are not known, nor is it known whether this killer element is associated with a complex. Sk-2K strains have been found only in N. intermedia, and only in four localities: Brunei (B), Java (J), Papua New Guinea (P), and Sabah (SA). Sk-3K is known solely from Papua New Guinea. All commonly used N. crassa laboratory wild-type strains and their derivatives are sensitive to killing both by Sk-2K and by Sk-3K. Strains containing the aconidiate mutation fluffy (fl) are conveniently used a female parents in test crosses for scoring killer vs. sensitive. The fl testers are highly fertile, and because conidia are absent, ascospores ejected to the sides of the tube can be seen clearly. With N. crassa, tests are made by fertilizing the testers on 10 x 75 mm slants of synthetic cross medium with 1% sucrose and examining shot ascospores after 10 days at 25 C. With N. intermedia, N. sitophila, and N. discreta, tests are best made on 13 x 100 mm slants using synthetic cross medium with filter paper as sole carbon source. If this medium is employed, stocks without the fluffy mutation Published by New Prairie Press, 2017 can be used, because few conidia are produced. Standard stocks of N. tetrasperma are also satisfactory as testers because they make few conidia at 25 C, even on sucrose medium. Table 1. Strains for identification and study of Spore-killers in Neurospora Species and Origin of FGSC No. Comment genotype allele* A a Neurospora crassa Sk-2K B 6648 6647 10th backcross to N. crassa, mixed background Sk-2K B 3114 3115 10th backcross to N. crassa, inbred to OR wild type cum Sk-2K acr-7 B 7432 Sk-2K acr-7 B 6930 10th backcross to N. crassa Sk-2K acr-7 leu-1 his-7 B 7373 Sk-2K acr-2 leu-1 his-7 B 7387 7388 Sk-2K acr-2 leu-1 B 7375 7374 Sk-2K acr-2 his-7 B 7376 7377 Sk-2K leu-1 B 7371 Sk-2K his-7 B 7378 Sk-2K phe-2 dow B 4538 4539 Sk-2K dow B 4260 4261 Sk-2K; fl B 3297 3298 9th backcross to N. crassa Sk-2K P 7368 7367 12th backcross to N. crassa Sk-2K acr-2 P 7385 7386 Sk-2K J 7369 7370 12th backcross to N. crassa cum Sk-2K acr-2 J 7383 7384 Sk-2K acr-2 J 6928 6929 15th backcross to N. crassa Sk-2K J 7392 7393 Used for testing N. crassa from India Sk-2S Sk-3S fl@ 6682 6683 flP (RL) testers r(Sk-2)-1 2222 Iowa-1, LA (P527) r(Sk-2)-1 cum 7379 7380 cum r(Sk-2)-1 acr-7 7389 r(Sk-2)-2 7398 Derived from N. crassa P2604, Georgetown, Malaya Sk-3K P 3577 3578 10th backcross to N. crassa cum Sk-3K P 7382 7381 cum Sk-3K his-7 P 7390 7391 Sk-3K acr-2 P 7077 Sk-3K acr-7 P 6931 6932 15th backcross to N. crassa Sk-3K fl P 3579 3580 10th backcross to N. crassa Sk-2K Sk-3S fl@ 6682 6683 flP (RL) testers r(Sk-3) 7395 6th backcross to N. crassa cum r(Sk-3) 7396 6th backcross to N. crassa cum r(Sk-3) leu-1 7394 9th backcross to N. crassa r(Sk-3) acr-7 ser-1 7397 6th backcross to N. crassa Neurospora intermedia Sk-2K B 7401 7402 3rd and 4th backcross to Taipei background Sk-2K P 7429 3rd backcross to Taipei background Sk-2K J 7399 7400 f1 of Tjiawi-2d (P162) x Taipei-1c (P13) http://newprairiepress.org/fgr/vol40/iss1/30 DOI: 10.4148/1941-4765.1421 Sk-2K SA 7426 Menggatal, Sabah (P3126) r(Sk-2) 1832 1833 Townsville-1b (P113), Townsville-1 (P112) Sk-3K P 3193 3194 Derived from Rouna-1 (P32) r(Sk-3) 6595 5123 Tahiti (P2427, P2421) Sk-2S Sk-3S@ 3416 3417 Shew wild types (Taipei background) Sk-2S Sk-3S fl@ 5798 5799 7th backcross of flP from N. crassa to Shew wild types Species and Origin of FGSC No. Comment genotype allele* A a Neurospora sitophila Sk-1K 2216 2217 Derived from Dodges Arlington stocks Sk-1K; fl 4762 4763 fl P(1012) from Whitehouse N. sitophila, 3rd backcross to Dodge stocks Sk-1S 5940 5941 Tahiti (P2443, P2444) Sk-1S; fl 4887 4888 5th backcross of flP from N. crassa to Panama VP203 or derivative Neurospora tetrasperma (See Raju and Perkins 1991 Genetics 129: 25-37. E: 8-spored ascus.) Sk-2K acr-2 J 6934 6935 8th-9th backcross to N. tetrasperma Sk-2K acr-2; E J 6936 6937 4th backcross to N. tetrasperma Sk-3K acr-7 P 6938 6939 7th-8th backcross to N. tetrasperma Sk-3K acr-7; E P 6940 6941 8th backcross to N. tetrasperma Sk-2S 127

Analysis of two additional loci in Neurospora crassa related to Spore killer-2

Two new loci found in one strain of Neurospora crassa (P2604) collected in Malaya are related to the meiotic drive system Spore killer Sk-2. Sk-2 was found in Neurospora intermedia and introgressed into N. crassa. P2604 showed high resistance to killing when crossed to Sk-2. This resistance was found to be linked to, but not allelic to, resistance locus r(Sk-2) on LGIIIL. Analysis showed that the high resistance phenotype of P2604 requires resistance alleles at two different loci on LGIIIR. Strains carrying a resistance allele at only the proximal or the distal locus, respectively, were obtained and intercrossed. Highly resistant strains were obtained by rejoining the two genes. The proximal locus alone confers a low level of resistance. This locus was named pr(Sk-2) for partial resistance to Sk-2. The distal locus was named mod(pr) because its only known phenotype is to modify pr(Sk-2).

Genetically determined round ascospores

Genetically determined round ascospores Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This research note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol19/iss1/6 Borry,E. G., D. Newmeyer, D. D. Perkins and B.C. Turner. Genetically determined rwnd ascorprer in N. crasm. In addition to the dominant gene 5 Round spore, discovered by M. B. Mitchell (1966 Neurorpora Newl. 10x5 ), two other genotypes ore now known to result in round spores. Because of their potential interest for studier of morphogeneris, these will be reported briefly, together with mne new cbrerwtions on R. a. Round spores frcm cot-2 x cot-2 (Newmeyer): All arcapares are round when cot-2 (colonial-temperature-sensitive, RlD36; Gorniobst and Tatm967 Genetics 57:579) is homozygcws. Ascorpores are~ormal in heterozygcu crwes. Ar noted by Gum