Journal of General Microbiology (1989, 131, 2359-2365.
Printed in Great Britain
2359
Hybridization of Ririzopus Species
By M . A . A . SCHIPPER,' W . G A U G E R 2 A N D H . V A N D E N E N D E 3 *
Centraalbureau voor Schimmelcultures, PO Box 273, 3740 AG, Baarn, Holland
School of Biological Sciences, The University of Nebraska- Lincoln, Lincoln,
Nebraska 68588, USA
Department of Plant Physiology, University of Amsterdam, Kruislaan 318,
1098 SM Amsterdam, Holland
(Received I I March 1985)
~
~
~~~
~~~
The progeny resulting from crosses between Rhizopus microsporus and R . rhizopodiformis were
analysed for a number of morphological characters and the ability to produce either zygospores
or azygospores. The interspecific crosses resulted in relatively stable cultures exhibiting
intermediate morphology and producing only azygospores. The results suggest that the progeny
are diploid or aneuploid, which leads to heterozygosity, particularly with respect to mating type.
INTRODUCTION
In taxonomic studies, sexual isolation of a group of organisms is an important criterion in
delimiting a species. The boundaries set by the ability of individuals to mate are usually well
defined because the mating process is normally very specific. This specificity may be due to one
or more recognition steps, either of a long-range type (e.g. sex pheromones) or of a short-range
type (cellular contact reactions). In many instances, however, due to a low mating specificity,
crossings can occur between individuals which, on the basis of other characteristics, are
considered to belong to different species. For such cases it is helpful to extend the criterion of
sexual isolation by stating that a mating is only successful if it gives rise to genetically stable and
fertile progeny.
In the fungi of the order Mucorales both long-range and short-range sexual interactions are
operative, but their specificity is remarkably low. A number of sex pheromones, governing, inter
alia, zygophore production, have been described, and there is considerable evidence that the
same compounds are produced by, and exert their action in, several groups of fungi, which on
the basis of morphological and physiological characters are considered to belong to different
species (for reviews see Jones et al., 1981; van den Ende, 1984).
On the other hand, very little is known of the interactions that take place on cellular contact.
The phenomenon of 'interspecific' reactions, documented originally by Burgeff (1924) and
Blakeslee & Cartledge (1927), implies that this type of interaction also has a low specificity, but
generally it is restricted to the formation of intricate gametangia-like structures, without ensuing
fusion and zygospore formation. There are, however, notable exceptions, One of these is the
mating between Rhizopus microsporus Van Tieghem and R . rhizopodiformis Cohn. These species
are distinguished by a number of morphological and physiological characters : in R . microsporus
the sporangiospores are angular-ellipsoidal, striated, and have a length of approximately 8 pm,
while in R.rhizopodiforrnis the sporangiospores are globose-subglobose, are minutely spiny and
have a length of about 5 pm (cf. Fig. 1). In R . microsporus the columella is subglobose-conical,
and in R . rhizopodiformis it is pyriform (especially the larger ones). A further distinction is that
growth of R. rnicrosporus is completely suppressed at 50 "C,while R . rhizopodiformis grows well
at this temperature. The species have been described in detail by Schipper & Stalpers (1980).
0001-2558 0 1985 SGM
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M . A . A . SCHIPPER, W . G A U G E R A N D H . V A N D E N E N D E
When mycelia of these species are crossed, they. regularly produce either zygospores
(connected with two suspensors derived from mycelium of either mating type) or azygospores
(borne on one suspensor originating from one of either parent mycelia). The latter are
considered to be the result of some irregular way of selfing (Schipper, 1978, 1979; Stalpers &
Schipper, 1980; Schipper & Stalpers, 1980). Apparently, there is only a slight sexual barrier, if
any, between these species, and the aim of this study was to determine whether the resulting
(a)zygospores are fertile and give rise to progeny which are sufficiently stable and fertile that the
status of R. microsporus and R . rhizopodiformis as different species should be reconsidered. Some
strains were included for comparison which are intermediate in morphology between R .
microsporus and R . rhizopodijormis and are tentatively designated as R. chinensis (Schipper &
Stalpers, 1980).
METHODS
Strains. The strains used were R . microsporus CBS 699.68, mating type plus (mt'), CBS 700.68 (mt-), R .
rhizopodiformis CBS 343.29, 607.73, 712.73, 196.77, 536.80, 608.81, 609.81 and 610.81 (all mt+),R . chinensis
CBS 261.28, 294.31, 289.71, 258.79, 537.80 (all mt+)and CBS 262.28, 388.34 and 631.82 (all mt+).Also included
were R . chinensis CBS 344.29 (no mating reaction observed and 394.34 (mt+),both able to produce azygospores
under certain conditions of age or culturing. An adenine-requiring mutant of R . microsporus (CBS 700.68, Ade-,
also designated as U N B 302( - ) adel ; Gauger, 1985) was also used.
Cultivation. The routine medium for morphological and growth studies was malt agar (MEA), prepared with
malt extract from a brewery diluted till it contained 4% (w/v) sugars. For mating experiments YEA medium was
used, consisting of yeast extract (Difco), 4 g 1-' ;malt extract (Difco), 10 g 1-' ; glucose, 4 g 1-* ; pH 7.3. The cultures
were grown in 90mm dishes containing 15ml medium at 30°C. The growth/temperature relations were
established by measuring radial growth. Sample preparation for scanning electron microscopy was according to
Samson et al. (1979).
Isolation of zygosporrs and azygospores. Zygospores or azygospores were isolated and purified as described by
Gauger (1985) and deposited on 2% (w/v) water agar. Sections of the water agar containing the spores were cut out
and placed on sterile filter discs (12 mm diameter) in a shell vial (65 x 18 mm) which contained a rolled-up 9 cm
filter paper for support. Care was taken to keep the spores moist. The vials were capped with plastic or stainless
steel closures.
Germination of'zygosporesand azygospores. Vials containing zygospores or azygospores were inspected weekly
until germinations were observed. If a spore produced a germ sporangium, it was transferred, using fine forceps, to
a YEA plate and the spores were spread over the surface. Single germinating germ spores were subsequently
transferred to YEA slants. If a germinating zygospore or azygospore produced mycelium, the filter disc was
transferred to a sterile disc and the mycelium subsequently transferred to a YEA slant (Gauger, 1985). As this
study progressed, it was found that some zygospores and azygospores could be induced to undergo mycelial
germination if the agar plug was transferred directly to a YEA plate. A modification of this procedure was
developed in which the agar plug was suspended in 5 ml glucose-asparagine medium (Gauger, 1977) with 1% (w/v)
agar, which had been melted and cooled to 45 "C. After a brief, brisk shaking, the mixture was poured over the
surface of a YEA plate. Zygospores or azygospores suspended in the overlay could be easily observed, and when
seen to germinate, were transferred to YEA slants.
Test ,for auxotrophy. Adenine auxotrophy was assessed by inoculating 3 ml liquid glucose-asparagine medium
(Gauger, 1977) with vegetative spores of the strain in question. If no growth occurred after 24 h, 0.1 mg adenine
was added, and the response determined 12-18 h later. Alternatively, the spores were seeded on the surface of
glucose-asparagine plates, overlaid with 5 ml medium, with or without 1.0 mg added adenine.
RESULTS
To determine the extent of mating specificity, all available wild-type strains were crossed
(Table 1). R. microsporus mated successfully with each of the compatible strains available. The
mating ability of the R. chinensis strains was much poorer. Even the best partner, CBS 262.28
(mt+),which is closest in morphology to R . microsporus, had very low mating ability. The
zygospores obtained from all combinations were similar : they were reddish brown, stellate,
approximately 90 pm in diameter, and borne on two suspensors of unequal size. Indications of
bisexual mating ability were never seen in this group. Azygospores were not observed to occur
spontaneously nor to be induced by the mating process (as has been found in some crosses with
other species, e.g. Absidia blakesleeana, A . corymbifera, Rhizomucor pusillus; Schipper, 1976 and
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Hybridization in Rhizopus
Table 1. Mating reaction between strains of' R. microsporus, R . rhizopodiformis and R . chinensis
Production of (a)zygospores was assessed on YEA at 30 "C. Z, zygospore production; AZ, azygospore
production; -, no reaction observed. For brevity, 'CBS' is omitted from the strain numbers.
R. microsporus
R . rhizopodiformis
P
R . microsporus
700.68
R. chinensis
262.28
388.34
63 1.82
z
z
z
z
z
z
z
z
z
-
z
z
-
z
-
z
-
R. chinensis*
A
f
z
-
z
z
z
\
z
z
&
z
z
z
z
z
z
z
z
z
z
z
AZ
-
* CBS 394.34 produces azygospores in old
slant cultures.
Table 2. Characteristics ojprogeny of' the cross
R. microsporus CBS 700.68 (mt-) x R. rhizopodiformis CBS 536.80 (mt+)
Parents
I
Progeny*
536.80 (+)
700.68 ( - )
f
A
1646(2)1
1646(2)2
1646(4)6
1646(4)1
1646(6)1
1646(6)2
1646(8)1
I646(9)1
>
Colonies
A zygospores
Columellae (largest)
grey
grey
yellow
yellow
grey
-
yellow
pyriform
subgloboseconical
pyriformellipsoidal
ellipsoidal
subgloboseconical
Sporangiospores :
Max. diam.
Shape
Ornamentation
Growth at 50 "C
conicalgloboseellipsoidal
5 Clm
globose
spiny
good
7.5-8-9 pm
angular
ridges
none
6 pm
subglobose
mixt
good
6 Pm
subglobose
mixt
good
7-5 ym
angular
ridges
poor
6 pm
subglobose
mix?
good
-
-
+
+
+
* Explanation of numbering: zygospores resulting from the mating CBS 536.80 x 700.68 were designated 1646;
in parentheses is the number given to the germinating zygospore; e.g. 1646(2);the last number refers to the single
germ spore/mycelial tip isolate, e.g. 1646(2)1.
t Mix: sporangiospore ornamentation a mixture of weakly defined 'ridges' and 'spines' - see Fig. 1 (c).
unpublished work) with the exception of R . chinensis CBS 344.29 and 394.34. In CBS 344.29,
azygospores were induced by crossing with R. microsporus CBS700.68 (mt-) or CBS 699.68
(mt+),which suggests a bisexual nature. On the other hand, CBS 394.34 produced azygospores
only in combination with R. chinensis CBS 388.34. Both strains also produced azygospores
spontaneously in old slants.
The next question was, could the true zygospores, produced in various combinations, produce
stable progeny? Firstly, zygotes of the 'intraspecific' combination R. microsporus CBS 699.68
(mt+) x R. microsporus CBS 700.68 (mt-) were examined. They germinated after a relatively
short period (approx. 4 weeks). From each of 25 germinations, 10 single-germ sporangiospores
were cultured. The resulting cultures resembled the parents in morphology. Their mating type
was established by crossing them with the parent strains. From each germ sporangium either
only mt+cultures, only mt- cultures, or both, were derived. However, a number of cultures could
not be designated as mt+ or mt-, because they produced numerous azygospores spontaneously.
When these were germinated and the resulting mycelia subcultured repeatedly via single
vegetative spore isolations, the azygosporic condition disappeared gradually, resulting in typical
mt+ or mt- cultures after two years. A likely explanation for this phenomenon is that these
azygosporic cultures were diploid or aneuploid with respect to mating type. Subsequent mitotic
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M . A. A . SCHIPPER, W . G A U G E R A N D H . V A N D E N E N D E
Fig. 1. Sporangiospores of (u)R. microsporus CBS 700.68; (h) R. rhizopodifbrmis CBS 536.80; (c) strain
1646(6)2, an azygosporic isolate from a cross between CBS 700.68 and CBS 536.80 (see Table 2). Bar,
2 pm.
reduction could be expected to yield haploid cultures with normal morphology and sexual
characteristics.
Secondly, the progeny of the cross of R . rnicrosporus CBS 700.68 (mt-) x R . rhizopodiforrnis
CBS 536.80 (rnt+) were investigated. Zygospores obtained from this mating germinated much
less frequently than those produced from the mating described above: after two months, only a
few germinating zygospores were obtained. One of ten germinated zygospores produced a germ
sporangium, the others produced mycelium only. As is shown in Table 2, the majority of the
resulting cultures produced azygospores, and with the exception of one culture, had
morphological characteristics intermediate between those of the parents (Figs 1 and 2).
However, these features did not appear to be stable. After subculturing for two years (three
successive mass-inoculations) none of the cultures was uniform in appearance. Rather, they
were 'blotchy' with dark- and light-coloured parts. There seemed to be a connection between
profuse sporulation, pyriform columellae and small azygospores on the one hand, and poor
sporulation, ellipsoidal columellae and larger azygospores on the other. The azygosporic
condition was maintained. There was a strong correlation between the yellow pigmentation of
the mycelium and the ability to produce azygospores. When the cultures of the progeny were
mated with the parent cultures, more and larger (a)zygospores were formed in crosses with R .
microsporus CBS 699.68 (rnt+) than with R . microsporus CBS 700.68 (mt-). The abundant
formation of chlamydospores prevented the distinction of azygospores from putative zygotes.
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Hybridization in R hizopus
2363
Fig. 2. Morphological characteristics of parents and progeny in the mating 16-16, ( ( I ) Columella and
sporangiospores of R . riii=opodjforniisCBS 536.80; ( h )columella and sporangiospores of R . niicrosporus
CBS 700.68; ( c )Lygospore; ( d )columella and sporangiospores of progeny: ( c )a/!gospores of progeny.
One of the azygosporic isolates (1646(6)2; Table 2) was subcultured through single vegetative
spore isolations. All of the resulting subcultures produced azygosporic mycelia. Surprisingly, the
azygospores, when spread on water agar at 20°C, germinated directly. producing hyphae on
which sporangia later formed. On germination, spores from these sporangia again produced
azygosporic mycelia, morphologically similar to the original 1646(6)2 culture. No sign of
reversion of the azygosporic condition to the normal heterothallic condition was observed.
From another cross, R . microsporus CBS 700.68 Ade- (mt-) x R . rhizopodiformis CBS 536.80
(mt+),five zygospores germinated with viable mycelium (out of abundant initial but abortive
germinations) and the resulting mycelia were propagated via mycelial tips. Three of them had
the morphology and mating type of the mt- parent (two of them being Ade-), whereas two
cultures were azygosporic, of intermediate morphology, and likewise prototrophic. One of the
latter was propagated via single vegetative spores and via germination of azygospores. The
following characteristics proved to be stable for five generations : colonies yellow to cinnamon;
colonies irregular in outline ; sporangia production varying from rich to poor; columellae and
sporangiospores of intermediate type ; azygospores numerous ; good growth on glucoseasparagine medium at 50°C. A few exceptions with short spines on columellae and large
sporangiospores reverted after some generations to the above morphology. From one such
culture azygospores were germinated, resulting in 16 isolates, 12 of which were azygosporic and
showed the stable intermediate morphology. However, four of these had intermediate
morphology, but had lost the ability to produce azygospores. They mated successfully with R .
rhizopodijormis CBS 536.80 (mt+)and with R . chinensis CBS 261.28, 294.3 1 and 289.7 1 (mt’), to
which they were similar in morphology. Zygospores from the mating with CBS536.80,
subsequently germinated, produced mycelia which had the typical R . microsporrrs morphology
(mt-), did not grow at 50°C and, significantly, were auxotrophic with respect to adenine.
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2364
M. A. A. SCHIPPER, W. GAUGER A N D H. VAN D E N ENDE
DISCUSSION
The results presented in this paper can be explained by assuming that incomplete homology
between the chromosomes of the two parents R . microsporus and R . rhizopodiformis leads to
defective meioses, the products of which are either diploid or aneuploid. Such cultures are
intermediate in morphology between the two parents and in addition are intermediate in mating
type, i.e. they are azygosporic. The wild-type allele of the adel locus is dominant to the mutant
locus. Mitotic reduction may lead to cultures which have a normal appearance and mating type.
One may expect that only certain chromosome combinations are viable, since only the typical R .
rnicrosporus morphology reappeared, next to intermediate forms. From the literature on nonmucoraceous fungi it is known that diploids in normally haploid fungi are often unstable and,
through consecutive losses of chromosomes, pass through various aneuploid stages before finally
reverting to haploids.
Frequently, recombinants of the two original genotypes appear in the haploid progeny (Kafer,
1961). However, in our study, recombination was not observed, suggesting that the homology
between the parental chromosomes was very low, which is reinforced by the observed low
viability of the majority of the resulting isolates. The conclusion therefore seems warranted that
there is considerable sexual isolation between R . microsporus and R . rhizopodiformis, and that
their ability to conjugate through various stages of the mating process up to nuclear fusion is only
due to a low specificity of the mechanism by which the two thalli interact.
Generally, cultures obtained from the crosses between R . microsporus and R . rhizopodiformis
showed considerable stability. This suggests that diploids or aneuploids do not haploidize easily.
In this respect it is interesting that a recently described Rhizopus species, R . azygosporus, isolated
from tempeh in Indonesia (Yuan & Jong, 1984), produces azygospores only and shows a striking
morphological resemblance to the azygosporic strains in the progeny of R . microsporus and R .
rhizopodijo rmis.
There is a distinction between azygosporic strains which are heterozygous with respect to
mating type due to diploidy or aneuploidy, as described above (cf. also Gauger, 1975), and
strains which are azygosporic because they are heterokaryotic. The latter are unstable in the
sense that single vegetative spore isolates rarely give rise to heterokaryons (Newsham & Gauger,
1985).
An additional feature of the azygosporic strains was an enhanced yellow pigmentation. On the
basis of the UV-absorption spectrum of mycelial hexane extracts (A,, 430,450 and 480 nm), we
tentatively ascribe this to the accumulation of P-carotene (cf. Table 2). In this respect, diploid or
aneuploid mycelia resemble those which are heterokaryotic, and contain nuclei of two mating
types (e.g. Gauger et al., 1980; Gauger, 1985). Thus, the presence within one thallus of two
different mating-type loci, which are evidently to some extent both expressed, leads to enhanced
carotene production. This is reminiscent of the well-established fact that in several mucoraceous
fungi, and especially in Blakeslea trispora, sex pheromones, which are produced by the
cooperation of two mycelia of opposite mating type, stimulate carotenogenesis (reviewed by van
den Ende, 1984). One might expect therefore, that heterokaryons or dikaryons, in which two
mating type loci are operative, would exhibit enhanced sex-pheromone production, leading to
stimulated carotenoid synthesis. However, preliminary attempts to isolate sex pheromones
(trisporate derivatives) from azygosporic strains derived from R . microsporus x R . rhizopodijormis gave ambiguous results. This may be due to insufficient physiological similarity between the
Rhizopus species and the species Blakeslea trispora and Mucor mucedo, in which the trisporate
derivatives are clearly operative. A continued analysis of the relatively stable azygosporic
cultures described in this paper might contribute to a better understanding of sexual physiology
in these species.
This work was supported by a visitor's grant from the Netherlands Organization of Pure Research (ZWO) to
W.G. The help of Drs G. Marsman and A. van der Does is gratefully acknowledged.
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Hybridization in Rhizopus
2365
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