male transmission of linear plasmids and mitochondrial dna ... · male transmission of linear...

Copyright 0 1992 by the Genetics Society of America

Male Transmission of Linear Plasmids and Mitochondrial DNA in the Fungus Neurospora

Xiao Yang and Anthony J. F. Griffiths

Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 124 Manuscript received February 3, 1993 Accepted for publication April 9, 1993

ABSTRACT One of the general rules of heredity is that in anisogamous matings genetic elements in organelles

are inherited maternally. Nevertheless, there are cases of paternal transmission, both as rare exceptions, and as regular modes of inheritance. We report two new cases of paternal transmission in crosses of the model fungus Neurospora. First, we show leakage of a linear plasmid from males, the first case in fungi and the second in eukaryotes. Transmission frequencies ranged from 1 % to 15% in different crosses, but some crosses showed no detectable male transmission. Second, we show leakage of male mitochondrial DNA (mtDNA), the second case in fungi. Some of the resulting progeny have only the male mtDNA type, but some are heteroplasmons. Heteroplasmons show novel restriction fragments attributable to recombination or rearrangement. Heteroplasmy of mtDNA through male transmission has not been reported previously in any eukaryote. In addition we have shown paternal leakage of circular mitochondrial plasmids, supporting another reported case. In a male bearing a linear and a circular plasmid, these plasmidsand the mtDNA are transmitted in different combinations. These results show a potential for mitochondrial segregation and assortment during the sexual cycle in anisogamous fungi, pointing to more potential avenues for novel associations between genomic compartments, and between genomic and extragenomic elements.

T HE experiments to be described concern maternal and paternal inheritance of mitochondrial

genetic elements in the fungus Neurospora. Although the results relate specifically to Neurospora and to other anisogamous organisms, they also raise issues about the general mechanisms of uniparental inheritance.

Maternal inheritance of organelle DNA and its as- sociated phenotypes is one the general rules of the genetics of anisogamous organisms (GILLHAM 1978). However, as with all rules there are exceptions, and these exceptions fall into two broad classes. First, there are cases of paternal leakage of organellar elements in systems showing predominantly maternal inheritance. Some examples are the paternal leakage of mitochondrial DNA (mtDNA) in mice (GYLLENSTEN et al. 1991) and in pine trees (WAGNER et al. 1991), and of circular DNA plasmids in Neurospora (MAY and TAYLOR 1989). Second, there are cases in which paternal inheritance of organelle elements is a regular mode of transmission. Some examples are paternal inheritance of plastids in Daucus (BOBLENZ, NOTHAN- GEL and METZKAFF 1990), of mtDNA in rapeseed (ERICKSON and KEMBLE 1990), of chloroplast DNA (cpDNA) in Douglas fir (NEALE, WHEELER and AL- LARD 1986), and of a linear mitochondrial plasmid in Brassica (ERICKSON, KEMBLE and SWANSON 1989).

The first case of maternal inheritance in Neuro- spora was the demonstration that the slow growth

Genetics 134: 1055-1062 (August, 1993)

phenotype “poky” was transmitted to virtually all the progeny of crosses of poky females to normal males (MITCHELL and MITCHELL 1952). The few non-poky progeny were attributed not to male transmission but to the establishment of rare protoperithecia by the fertilizing cells. Such sporadic exceptions are regularly observed in crosses of other mitochondrial mutants in Neurospora (our personal observations). However, male transmission of a mitochondrial circular plasmid has been demonstrated in Neurospora (MAY and TAY- LOR 1989). That experiment used female strains bearing the mutation per, which gives orange perithecia, in order to demonstrate the lack of contaminating perithecia from the male, which would be black. The experiment raised the possibility of the male transmission of other mitochondrial elements in Neurospora, so we designed experiments to detect the transmission of linear plasmids and mtDNA.

The 8.6-kb Neurospora linear mitochondrial plasmid “kalilo” is found only in Hawaiian natural isolates (GRIFFITHS and BERTRAND 1984; BERTRAND, CHAN and GRIFFITHS 1985; MYERS, GRIFFITHS and BER- TRAND 1989). Kalilo DNA (kalDNA) replicates auton- omously, reaching copy numbers far in excess of mtDNA, so it was a good candidate for exploring male transmission. The plasmid also can kill cells by insert- ing into mtDNA, but this property was not central to the experimental design. In most kalilo strains there is also a circular plasmid Hanalei-2 (Han-2, BER-

1056 X. Yang and A. J. F. Griffiths

TRAND, CHAN and GRIFFITHS 1985; YANG and GRIF- FITHS 1993), and such two-plasmid strains were used as males in crosses to females lacking these plasmids. Large numbers of progeny were tested by dot blotting using kalDNA and Han-2 probes. Subsequent analyses used a probe that detects a dimorphism in mtDNA. We found many cases of paternal leakage of the plasmids and mtDNA. Furthermore, the entry of mtDNA establishes heteroplasmons in which recombination and segregation of mtDNA can be detected.

MATERIALS AND METHODS

Strains and culturing methods. Standard protocols for culturing and crossing Neurospora were used throughout (DAVIS and DFSERRFS 1970).

The Neurospora intermedia strains used were Fungal Ge- netics Stock Center (FGSC, Department of Microbiology, University of Kansas Medical School, Kansas City) numbers 1766A (Tapei-lc from Taiwan), 3977a (Beijing from China), 3983a (Harbin from China), and 3991A (Boading from China), per4364a (an Indian natural isolate bearing a mutation per causing orange perithecia, donated by Dr David Jacobson, Botany Department, Michigan State Uni- versity), and C4a, C12A and C14A (kalDNA and Han-2 plasmid-bearing ascospore cultures from a cross of the Kalilo strain FGSC 5014a, Hanalei-lg from Hawaii, by1766A as male).

The Neurospora crassa strains used were FGSC strains 2499A (a Maranhar strain, Aarey-le from India; COURT et al. 1991), 3212A (Ravenswood-1 from Louisiana, USA), 3309A and 33 1 Oa (bearing the orange perithecial mutation per-I, allele number PBJl), 4558A and 4559a (also per-1 but allele UG1837), and three strains, 3-7a, 5-2A and 5-4a, that are prototrophic kalDNA- and Han-2-bearing progeny from a cross of the N . crassa kalilo strain HB9006a (GRIF- FITHS et al. 1990) by standard Oak Ridge wild-type 74- OR23-1A. The N. crassa per mutations were originally isolated from mutagenized strains of genetic background St. Lawrence, one of the progenitors of the Oak Ridge stand- ards.

Dot blots: Small mycelial cultures were macerated in DNA isolation buffer (0.1 M LiCI, 10 mM EDTA pH 8, 10 mM Tris-HCI, pH 8, 0.5% SDS), and 0.5-ml aliqouts were loaded into the wells. The filters were hybridized separately by kalDNA and Han-2 probes. The kalilo probe was a cloned XbaI fragment 3 from the terminal inverted repeat, and the Han-2 probe was the entire plasmid cut from low melting point agarose gels and linearized by restriction digestion (YANG and GRIFFITHS 1993).

DNA isolation and analysis: All the techniques used were described in YANC and GRIFFITHS 1993. The 1-kb calibration ladder was from Bethesda Research Laborato- ries.

RESULTS

We analyzed four crosses of N. intermedia and six of N . crassa for male transmission of the linear plasmid kalDNA, and the circular plasmid Han-2. Six of the crosses used a female parent bearing a female-acting mutation per that makes the perithecia orange. This enabled cross plates to be examined for the presence of black perithecia, which would have indicated that

0 J

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Hanalei-2

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kalilo-X3 FIGURE 1.-Demonstration of male transmission of plasmids

using dot blot analysis. A single dot blot membrane shows examples of positives and negatives. Arrows show cultures with the Han-2 but not the kalilo plasmid; all other positives show both. The strains were 95 ascospore isolates from the cross 3309per X 3-7 (kal, Han- 2). plus a positive control (bottom right). The probes were prepared from a circular plasmid Hanalei-2 and a linear plasmid kalilo.

conidia from either the purported male, or from a contaminant, might have established a colony that acted as a female parent. In reality, no black perithecia were observed in such crosses.

Large numbers of ascospore-derived cultures were tested for male mitochondrial plasmids by dot blotting. Many clear positive signals were obtained, as shown in the sample in Figure 1. Strains showing a positive signal will be termed positives. All the positives showed Han-2 DNA, and most but not all of these also showed kalDNA. In the figure, two strains with Han-2 but no kalDNA are arrowed. The results from the ten crosses are summarized in Table 1. In five crosses no male elements were detected, but the remaining five showed many cases of paternally derived elements. The frequencies of transmission ranged from 1 % to a surprising 17%. The summmar- ies of cross 1766 X C4 are from three separate exper-

Mitochondrial Paternal Transmission 1057

TABLE 1

Transmission of a linear plasmid (kalilo) and a circular plasmid (Han-2) from the male parent to progeny in crosses of

N. intermedia and N. crmsa

Kalilo No. of No. of Hanalei-2 No. of

Female Kalilo

male ascospores positives" positives' Hanalei-2

N . intermedia 1766 c 4 1531 29 29 399 1 c 4 672 12 12 4364 per C 12 190 0 0 4364 per C14 190 0 0

2499 3-7 480 5 5 3212 3-7 480 0 0 4558 per 5-4 190 0 0 4559 per 5-2 190 2 2 3309 per 3-7 190 28 32 3310 per 5-2 190 0 0

The numbers of cultures testing positive in dot blots.

N . crassa

iments individually showing 31382, 251959 and 1/ 190 positives per ascospore. All other cross results are from single experiments.

The results suggest male transmission of plasmids in some crosses. Perithecia from males are unlikely: the crosses all used vigorous fertile females, ascospores were collected early, and some of the cases of male transmission were from crosses using per females. Furthermore, the mating pheromone of the female (BISTIS 1983) inhibits germination of male conidia, making growth into a protoperitheciating colony unlikely. Another unlikely possibility is that male conidia in the crossing tube were isolated along with ascospores and survive the germination heat shock. There is no precedent for such a process, but we tested the survival of conidia from two sources; conidial samples from the precise cross tubes showing the presumptive male transmission, and conidia from fresh cultures of strains used as males. No conidia survived at the heat exposures used, 40 min at 60". Furthermore, the positive cultures did not show mixtures of mating types, nor an excess of cultures with the mating type of the male parent, both of which would have been expected from such male contamination (data not shown).

A restriction analysis of DNA preparations from mitochondria of selected progeny from the cross 3309 X 3-7 is shown in Figure 2. Panel A shows an ethidium bromide-stained gel of EcoRV digests of the two parents, female (F) and male (M), a 1-kb ladder L, four ascospore isolates showing no plasmids from the male, acting as negative controls (lanes 1-4), four showing kalDNA and Han-2 (lanes 5-8), and the four showing Han-2 only (lanes 9-1 2). Panel B shows hybridization with the terminal kalilo fragment XbaI-3. (The signal in lane L is from part of the vector that has homology with one of the fragments used to make the ladder.)

Panel C shows hybridization by the Han-2 plasmid. EcoRV cuts Han-2 twice, giving fragments of 4.3 and 0.7 kb, (YANG and GRIFFITHS 1993), but the 0.7-kb fragment is not visible on these gels. In this figure, El and E2 label the left and right terminal EcoRV fragments of kalDNA and J marks junction fragments with mtDNA. Also shown in panel D are restriction maps of Han-2 and kalDNA. The EcoRV digest pat- tern of the female parent is that of mtDNA alone, whereas the male shows extra bands characteristic of the free and inserted kalilo plasmid, and the Han-2 plasmid. The eight positive strains show Han-2, and four of these also show kalDNA. This and similar analyses confirmed the presence of plasmids from the male parent in a total of 54 strains that were classified as positives in the dot blot analysis. An interesting exception was in the case of the cross of the Maranhar strain 2499 (COURT et al. 199 1) as female to the Kalilo strain 3-7. In this case, when positive strains were grown for detailed DNA analysis, four of the five positives showed the Han-2 and kalDNA bands but no maranhar plasmid band: the other positive showed maranhar plasmids but no Han-2 or kalDNA. Since all these strains showed as strong double positives on the dot blots, it is likely that cytoplasmic segregation of the two mitochondrial types had occurred during growth of the culture for DNA isolation.

The appearance of large amounts of the inserted form of kalDNA in progeny (fragments labeled J in Figure 2) suggested that the plasmid entered as a passenger within a mitochondrion, because generat- ing high levels of kalDNA insert usually takes many subcultures (BERTRAND, CHAN and GRIFFITHS 1985). This idea was explored using a cross 1766 X C4, which showed a mtDNA restriction fragment length polymorphism (RFLP) for enzyme EcoRV. Represent- ative results are shown in Figure 3. The male shows a 10.2-kb fragment, which is subdivided by one cut into 7.5- and 2.7-kb fragments in the female. Eight selected positives are shown. The Southern analysis using the male 10.2-kb EcoRV fragment 2 as probe (panel B) shows that the male marker is in four progeny cultures. However, two individuals (1 and 3) show both the male and female markers. This result supports the idea that mitochondria from the male can enter the ascogenous tissues. Panels C and D show that all the strains with male mtDNA also contain both plasmids. All the 29 positives from cross 1766 x C4 were analyzed, and the study revealed 5 strains showing the male morph only, 19 showing only the female morph, and five showing both. Other progeny showed weaker signals that seemed to correspond to the male morph, but these disappeared upon subculture. However, we feel that the total of 10 out of 29 probably represents a minimum frequency for detectable male mtDNA transmission in this cross. Cross 3991 X C4 also

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X . Yang a n d A. J. F. Griffiths

B C

F M L 1 2 3 4 5 6 7 89101112 F M L 1 2 3 4 5 6 7 8 9101112

&OR V kalilo-X3 Hanalei-2 FIGURE 2.-Restriction analysis of DNA from mitochondria of representative ascospores from the cross 3309 X 3-7, which showed male

transmission of plasmids. (A) Ethidium bromide-stained gel of EcoRV digests of the two parents, female (F) and male (M), a I-kb ladder L, four ascospores showing no plasmids from the male (lanes 1-4), four showing kalDNA and Han-2 (lanes 5-8). and four showing Han-2 only (lanes 9-12). (B) Hybridbation with the terminal kalDNA fragment Xbal-3. T h e signal in lane L is from part of the vector which has homology with one of the fragments used to make the ladder. (C) Hybridi7ation by the Han-2 plasmid. Bands El and E2 are the left and right terminal EcoRV fragments. T h e label J marks junction fragments with mtDNA. (D) Restriction maps of Han-2 and kalDNA. Note: the small EcoRV fragment of Hanalei-2 is detectable but is not visible on the gels shown in this study.

showed the same RFLP and the 12 positive progeny were all tested: nine showed the male morph, one the female, and two were mixtures. Therefore, these analyses showed that mtDNA is transmitted from the male along with the plasmids, and mtDNA mixtures are detectable in some cases. In addition, the analysis of the positives in these crosses also reveals the processes of mitochondrial segregation and assortment taking place in the sexual cycle, leading to new combinations of mtDNA and plasmids.

The cultures that showed both RFLP markers (for example, 1 and 3 in Figure 3) presumably originated as heteroplasmic ascospores produced by entry of male mtDNA into ascogenous tissue. The behaviour of such heteroplasmons was studied further by testing conidial isolates of strain 3. Restriction digests of DNA from ten conidial isolates are shown in Figure 4. Most of these isolates show not only both parental mtDNA morphs (10.2 kb and 7.5 + 2.7 kb, panel C), but the ethidium bromide-stained gel (panel A) also shows a

range of extra bands (arrowed). These extra bands do not hybridize to the kalilo terminal fragment probe XbaI-3 (panel B), suggesting that they are not junction fragments from kalDNA insertions, but are mtDNA recombinants or rearrangements.

The demonstration of male transmission still leaves the route of transmission uncertain. The most obvious route is the normal contact point of the spermatium, the trichgyne. However, it is also possible, although unprecedented, that conidia from the male could an- astamose with the mycelium of the female parent, and mitochondria enter ascogenous tissue through the “back door.” This is difficult to rule out experimen- tally. We have tried to reconstruct a reasonable fac- simile of the scenario by co-inoculating fresh conidia of both parents (1 766 and C4) onto minimal medium, allowing the strains to grow up together in the tube, reisolating conidia of the mating type of the 1766 parent, and testing for the presence of plasmids by dot blotting. No plasmids were detected in a total of

A

Mitochondrial Paternal Transmission

B C 1059

D

F M L 1 2 3 4 5 6 7 8 F M L 1 2 3 4 5 6 7 8 F M L 1 2 3 4 5 6 7 8 F M L 1 2 3 4 5 6 7 8

, - ”’”, :p

EcoR V ECOR V-2 of C4 kalilo-X3 Hanalei-2 FIGURE 3.-Restriction analysis of DNA from mitochondria of representative ascospores from the cross 1766 X C4, which showed male

transmission of mtDNA. (A) Ethidium bromide-stained gel of EcoRV digests of the two parents F and M, eight representative ascospores, and a 1-kb ladder L. (B) Hybridization by a probe consisting of a gel-isolated EcoRV fragment 2 of strain C4, which reveals an RFLP in the parents. (C) Hybridization by kalilo probe Xbai-3. (D) Hybridization by a probe consisting of linearized gelderived Han-2 plasmid. The marginal numbers show the size in kilobases of the fragments that constitute the RFLP.

160 conidia, so simple proximity of C4 to mycelium of 1766 did not lead to a detectable transfer of mitochondrial plasmids.

DISCUSSION

Using dot blot analyses of large numbers of progeny, we have shown that mitochondrial plasmids from male parents can be detected in the progeny of Neu- rospora crosses. Transmission frequencies are generally in the 1-2% range, but in one cross the frequencies were in the 10-20% range. In crosses showing an RFLP in mtDNA, we have shown that some progeny acquiring male plasmids also contain the male RFLP morph. In some cases mixtures of male and female mtDNA morphs were observed, presumably representing hetroplasmons. In these heteroplasmons gen- erated by male mtDNA entry, new restriction fragments were observed that suggested recombination of mtDNA.

Overall, a likely general explanation for our results is that mitochondria from males occasionally enter the ascus, probably through the trichogyne. Once present,

several different events could ensue. First, the male mitochondria could persist together with the female mitochondria, constituting a heteroplasmon. The maternal and paternal mitochondria could fuse and en- gage in recombination or other rearrangement to generate the array of new restriction fragments shown in Figure 4. Second, rapid cytoplasmic segregation could occur, resulting in the two parental mtDNA types being found among the ascospore progeny con- taining plasmids from the male. Third, plasmids could jump from paternal to maternal mitochondria, fol- lowed by cytoplasmic segregation of mitochondria. Transfer of the two different plasmids must be some- what independent because four isolates were found with Han-2 but no kalDNA. Precedents exist for all these processes. Some examples are MANNELLA and LAMBOWITZ (1978) for recombination and cytoplasmic segregation of mtDNA, COLLINS and SAVILLE (1 990) and GRIFFITHS et al. ( 1 990) for plasmid j u m p ing, VESTVABER and SCHATZ (1989) for DNA entry into mitochondria, and NACY, TOROK and MALICA ( 1 98 1 ) for mtDNA rearrangement in heteroplasmons.


A B C

1 2 3 4 5 6 7 8 9 1 0 F M L 1 2 3 4 5 6 7 8 9 1 0 F M L 1 2 3 4 5 6 7 8 9 1 0 F M L

- 10.2

- 7.5

El

E2

EcoR V kalileX3 ECOR V-2 Of C4

- 2.7

FIGURE 4.-Restriction analysis of DNA from mitochondria of conidial isolates of a strain that originally contained a mixture of both male and female parental mtDNAs. (A) Ethidium bromide-stained gel of EcoRV digests of parental strains F and M, 10 representative conidial isolates of the heteroplasmic culture #3 from the cross 1766 X C4 (see Figure 3), and a 1-kb ladder L. The arrowheads show some of the new restriction fragments attributed to recombination or rearrangement. (B) Hybridization of a kalilo-specific probe. (C) Hybridization of the EcoRV fragment that detects the RFLP with a 10.2-kb male fragment, and 7.5- and 2.7-kb fragments in the female.

It is also possible that plasmids could travel down the trichogyne independent of mitochondrial transmission.

These results presented are the first evidence of male leakage of mtDNA in fungi showing predominantly maternal inheritance of mitochondria and as- sociated phenotypes. The only other report of any kind of male mtDNA transmission in anisogamous fungi is of exclusively male transmission in Allomyces (BORKHARDT and OLSON 1983). However, in that case the male inheritance is clearly programmed. Further- more the relative contributions of male and female cytoplasms are not as unequal as in conidium-trichogyne systems.

The paternal leakage of linear plasmids is the first case in fungi, and the second in eukaryotes generally. (ERICKSON, KEMBLE and SWANSON (1989) showed male transmission of linear plasmids in Brassica plants.) We have confirmed the paternal leakage of circular plasmids first reported by MAY and TAYLOR

(1 989). If our general hypothesis of plasmids entering in mitochondria is correct, plasmid transmission is an indicator of mitochondrial transmission. This being the case, we were surprised at the high transmission percentages obtained in the crosses showing paternal leakage. Previous studies on kalilo strains have shown only maternal transmission of senescence and of kalDNA. GRIFFITHS and BERTRAND (1 984) showed that the senescence phenotype was not transmitted from male parents in crosses using six different males. However, only 20 progeny were analyzed in each cross. BERTRAND et al. (1986) showed lack of transmission of plasmids from the male, but analyzed only one octad. Nevertheless, it seems likely that entry of male mitochondrial elements is a regular event, and must be considered in any case of apparent patrocli- nous cytoplasmic inheritance. The variability in transmission percentage might reflect a genetic regulation of the process, based on either nuclear or cytoplasmic determinants. Certainly the interaction between

Mitochondrial Paternal Transmission 1061

strains 3309 and 3-7 seems to be a favorable one .for the promotion of male entry. Also, the crosses with no paternal transmission could be explained by parental genotypes not conducive to transmission. All the N. crassa per strains are in a St. Lawrence background, and all of the N. crassa strains used as males are of Oak Ridge nuclear background. Therefore, the different male transmission frequencies within this group of crosses are unlikely to be due to broad differences of genetic background. However; the St. Lawrence strains had been mutagenized, so this is a possible source of the variation.

The predominantly maternal mode of transmission of organelles in anisogamous matings of fungi could have one of two basic mechanisms. First, there might be an active exclusion or destruction of paternal elements. There is evidence for this in plants (KUROIWA and HORI 1986; MOGENSEN 1988) but not in anisogamous fungi. However the situation in seemingly isogamous fungi might be relevant. It has been shown in Coprinus cinereus (MAY and TAYLOR 1988) and in Agaricus bitorquis (HINTZ, ANDERSON and HORGEN 1988) that in mating confrontations, although there is reciprocal nuclear migration, mitochondria do not migrate and the mitochondria transmitted sexually are those of the nuclear recipient parent. In the genus Armillaria, even though mitochondria migrate across mycelial confrontations in crosses on Petri dishes, in nature clones have only one mtDNA type, again suggesting uniparental inheritance (SMITH et al. 1990). These three Basidiomycete examples suggest the existence of a mitochondrial destruction or exclusion mechanism. A second explanation of maternal inheritance in anisogamous matings is the unequal contri- bution of cytoplasms, resulting in a ratio of female to male mitochondria approaching 1 : O . It is possible that a few paternal mitochondria enter the trichogyne in all Neurospora crosses. The travel of male nuclei down trichogynes might lead to further loss of male mitochondria, either by dilution or by mating-type or heterokaryon incompatibility (the latter representing another type of exclusion). The present data do not support either model strongly, but if the variation between crosses does have a genetic basis, then clearly the effect is not simply one of passive dilution. Fur- thermore, when the data from all fungi are considered together, it must be concluded that the evidence overall is in favor of exclusion.

We have not tested paternal mtDNA transmission from plasmid-free males. (This could be done by selecting for an optional intron; COLLINS and LAM- BOWITZ 1983). Nevertheless we feel justified in enter- taining the conjecture that plasmids can mobilize mitochondria for entry through the male. KAWANO et al. (1 991) have shown that in isogamous matings in Physarum, mitochondrial inheritance is normally un-

iparental, but when one of the mating partners pos- sesses a plasmid, mitochondria fuse and recombine, and the plasmid is transmitted to all resulting mitochondria. Hence there is precedence for plasmids affecting mitochondrial transmission. This would support the model of HICKEY and ROSE (1988) which proposes that it is advantageous for a parasitic element to promote such transmission, because this prevents it from becoming trapped in a vertical hereditary line- age. Whether this is true or not, the results show that male transmission opens up new opportunities for associations between mitochondrial elements, and between mitochondrial elements and the nuclear ge- nome. These new associations also could have potential adaptive value. In an analysis of mtDNA of natural isolates of N. crassa, TAYLOR, SMOLICH and MAY (1986) found that there were many different combinations of length mutations which could not be fitted to a simple phylogenetic tree. mtDNA recombination via male transmission could provide an explanation for much of this kind of variation. In addition, the existence of male transmission and assortment of several mitochondrial elements makes it possible to as- semble a variety of new mitochondrial combinations for experimental purposes in cases in which effective heterokaryotic association is prevented by cell incompatibility.

This study was supported by grant A6599 of the Natural Sciences and Engineering Research Council of Canada. We thank DAVID JACOBSON for donating strains, the Fungal Genetics Stock center for their continuing cooperation, and FONS DEBETS and MYRON SMITH for reviewing the manuscript. In addition we thank the anonymous reviewers for several useful suggestions.

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