some aspects of bipolar heterothallism in fomes cajanderi karst · 2020. 4. 2. · heterothallism...
TRANSCRIPT
Some aspects of bipolar heterothallismin Fomes cajanderi Karst
Item Type text; Thesis-Reproduction (electronic)
Authors Neuhauser, K. Sieglind
Publisher The University of Arizona.
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SOME ASPECTS OF BIPOLAR HETEROTHALLISM
IN FOMES CAJANDERI KARST.
by
Karen S ie g l in d e Neuhauser
A T h e s is S ubm itted to th e F a c u l ty o f t h e
DEPARTMENT OF PLANT PATHOLOGY
I n P a r t i a l F u l f i l l m e n t o f th e R equirem ents For th e D egree o f
MASTER OF SCIENCE
I n the. G raduate C o lle g e
THE UNIVERSITY OF ARIZONA
1 9 7 0
V
STATEMENT BY AUTHOR
T his t h e s i s has been s u b m it te d in p a r t i a l f u l f i l l m e n t o f r e q u i r e ments f o r an advanced d e g re e a t The U n i v e r s i t y o f A rizona and i s d e p o s i te d in th e U n iv e r s i t y L ib r a r y to be made a v a i l a b l e to bo rro w ers under r u l e s o f th e L ib r a r y .
B r i e f q u o ta t io n s from t h i s t h e s i s a r e a l lo w a b le w i th o u t s p e c i a l p e rm is s io n , p ro v id ed t h a t a c c u r a t e acknowledgment o f s o u rc e i s made. R eques ts f o r p e rm is s io n f o r ex tended q u o ta t i o n from or r e p r o d u c t io n o f t h i s m a n u sc r ip t in whole o r i n p a r t may be g ra n te d by th e head o f th e m ajor d ep a r tm e n t o r th e Dean o f th e G raduate C o lleg e when i n h i s ju d g ment th e p roposed u se o f th e m a t e r i a l i s in th e i n t e r e s t s o f s c h o l a r s h ip . In a l l o th e r i n s t a n c e s , however, p e rm is s io n must be o b ta in e d from th e a u t h o r .
APPROVAL BY THESIS DIRECTOR
This t h e s i s has been approved on th e d a t e shown below:
P r o f e s s o r o f P la n t P a th o lo g y
ACKNOWLEDGMENTS
The a u th o r w ishes to e x p re s s h e r d e e p e s t g r a t i t u d e f o r th e i n
v a l u a b le gu idance and counsel o f Dr. R o b e r t L» G i lb e r t s o n , P r o f e s s o r o f
P la n t P a th o lo g y , U n iv e r s i t y o f A r iz o n a , u nder whose d i r e c t i o n . t h i s s tu d y
was c o n d u c ted . Thanks a r e a l s o ex tended to C l i f f A, H a r r i s f o r h i s con
t i n u i n g i n t e r e s t and a d v ic e , and to Miss A dela Saucedo f o r h e r h e lp i n
p r e p a r i n g th e m a n u s c r ip t .
TABLE OF : CONTENTS
Page
LIST OF ILLUSTRATIONS . . . . . . . . . . . . . . . v
LIST OF TABLES . . . . . ....................... v i
ABSTRACT . . ................................ v i i
INTRODUCTION............................................................. ............................................................. 1
REVIEW OF RELATED STUDIES . . , . . . . . . . . . . . . . . . . . 3
MATERIALS AND METHODS ............................ 5
RESULTS. . , . .......................................... 10
C o n f irm a tio n o f B ip o la r N a tu re of. F. c a j a n d e r i . . . . 10G en e tic I s o l a t i o n o f F. c a j a n d e r i and F. rpseu.s . , , . 13G eo g rap h ic a l D i s t r i b u t i o n .13E s t im a t io n o f th e Number o f A lle lom orphs , ................... 19D ik a r y o t i c F r u i t i n g . . . . . . .................... - • • • « • • 22M onokaryotic F r u i t i n g ......................... . . . . . . . . . . . 24P a i r i n g R e a c t io n s . . ............................ 26
DISCUSSION . . . . . . . . . . . . . . . . . . ...................................... 36
SUMMARY • . . . . •• . . . . . . . . . . . . . . . . . . . . . . . 40
LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . 41
LIST OF ILLUSTRATIONS.
F ig u re Page
1. R e s u l t s o f p a i r i n g 19 s i n g l e sp o re i s o l a t e s , o b ta in e d froms to c k d ik a ry o n RLG 7942, in a l l p o s s i b l e co m b in a tio n s , , . . 11
2. R e s u l t s as i n F ig . 1, r e a r r a n g e d t o show th e two c o m p a tib i l i t y g ro u p s , a t y p i c a l b i p o l a r p a t t e r n . . . ............................ 12
3 . R e s u l t s o f p a i r i n g 8 s i n g l e s p o re i s o l a t e s , from F. r oseuss to c k d ik a ry o n RLG-6954, w i th two monokaryons o f F. c a j a n d e r i (RLG-79.42) , c a r r y i n g c o m p a t ib le m a tin g f a c t o r s , A ~ and"A^"' r e s p e c t i v e l y ............................ 14
4 . R e s u l t s o f m a ting th e 36 m onokaryo tic i s o l a t e s i n th e samplei n a l l p o s s i b l e c o m b in a tio n s ................................................... 15
5 . Map showing th e d i s t r i b u t i o n o f th e 25 a l le lo m o rp h s found inth e sample . . . . . . . . . . . . . . . . . . . . . . . . . 18
60 T y p ic a l D ik a r y o t i c f r u i t i n g i n an (Ag x Ag) d ik a ry o n . . . . 23
7. M onokaryotic f r u i t i n g i n Ag (ME-34) 25
8 . M onokaryotic f r u i t i n g i n d u c t io n i n two in c o m p a t ib le p a i r i n g s . 27
9. Two s e t s o f m a tings betw een s i n g l e - s p o r e i s o l a t e s o b ta in e dfrom a s i n g l e s to c k d ik a ry o n . , . . . . , .................................. 28
10. M atings o f s i n g l e - s p o r e i s o l a t e s o f s to c k d ik a ry o n MD-350 . 29
11. In c o m p a t ib le m a tings . . . . . , , . . . , . . . . . . . . , 30
12. M atings o f s i n g l e - s p o r e i s o l a t e s o f s to c k d ik a ry o n MD-14 . . 32
13. Di-mon p a i r i n g s . . . . . . . . . . . . . . . . . . 35
v
LIST OF TABLES
T able / Page
I M ating f a c t o r s found i n th e sample . 16
I I Comparison o f th e f r e q u e n c ie s o f r e p l i c a t i o n s i n p a i r i n g s o fi s o l a t e s from w i t h i n and betw een th e E a s te r n and W este rn c o n i f e r o u s f o r e s t r e g i o n s ................................ , . . . . , , 20
ABSTRACT
- ' Fomes e a j a n d e r i K a r s t . i s a w id e ly d i s t r i b u t e d member o f th e
P o ly p o race ae w hich c a u se s a c u b i c a l brown r o t on c o n i f e r lo g s and stumps*
I t was p r e v io u s l y r e p o r t e d to be h e t e r o t h a l l i c , b i p o l a r and to p o s se s s
m u l t i p l e a l le lo m o rp h s a t th e locus f o r h e t e r o th a l l i s m *
In t h i s r e s e a r c h , s t u d i e s o f the. s e x u a l i t y o f t h i s fungus were
c a r r i e d o u t . T h i r t y - s i x r e p r e s e n t a t i v e m onokaryons, i s o l a t e d from 18
d i k a r y o t i c s to c k s t h a t r e p r e s e n t e d the N orth A m e r i c a n .d i s t r i b u t i o n o f
th e s p e c i e s , were used* P a i r i n g s betw een th e s e 36 i s o l a t e s were made*
From th e r e s u l t s i t was p o s s i b l e to e s t i m a t e th e number o f m u l t i p l e
a l le lo m o rp h s a t th e locus f o r h e t e r o t h a l l i s m , and ro u g h ly d e te rm in e th e
randomness o f t h e i r d i s t r i b u t i o n . M onokaryotic and d i k a r y o t i c . f r u i t i n g ,
and p a i r i n g r e a c t i o n s were a l s o s tu d ie d * In th e l a t t e r c a t e g o r y , th e
b e h a v io r o f di-m on p a i r i n g s was o f s p e c i a l i n t e r e s t * D ik a r y o t i z a t io n
o f th e component monokaryon was found to occu r i n t h i s w ork , b u t had
p r e v io u s l y been r e p o r t e d n o t to occur f o r t h i s sp e c ie s*
v i i
INTRODUCTION
Fomes c a j a n d e r i K a r s t , i s a h e t e r o t h a l l i c 3 b i p o l a r b a s id io m y c e te
b e lo n g in g to th e f a m i ly P o ly p o ra c e a e , This s p e c ie s i s c i rc u m b o re a l in
th e c o n i f e r o u s f o r e s t r e g io n s o f th e n o r th e r n te m p e ra te zone. I t i s
commonly found on c o n i f e r logs and s tu m p s , in which i t c au ses a c u b i c a l
brown r o t .
The s p e c ie s was f i r s t d e m o n s tra te d to be b i p o l a r and to p o s se s s
m u l t i p l e a l le lo m o rp h s a t th e locus f o r h e t e r o t h a l l i s m over t h i r y y e a rs
ago by Mounce and Macrae (1937) as an a n c i l l a r y f e a t u r e o f t h e i r i n v e s t i
g a t i o n o f th e g e n e t i c d i f f e r e n c e s betw een t h i s s p e c ie s and th e c l o s e l y
r e l a t e d F, r oseus (A lb, and Schw, ex F r , ) K a r s t , W ith th e e x c e p t io n o f
some work on p a i r i n g r e a c t i o n s o f g e n e t i c a l l y d i s t i n c t m y c e l ia (Adams
and R o th , 1967), how ever, v e ry l i t t l e has been done s i n c e . B ip o la r s e x u
a l i t y , i n g e n e r a l , seems to have been l a r g e l y ig n o re d i n r e c e n t y e a r s .
T h e re fo re , i n l i g h t o f th e need f o r f u r t h e r i n v e s t i g a t i o n , th e
p r im a ry p u rp o se o f t h i s s tu d y was to e l u c i d a t e i n g r e a t e r d e t a i l v a r io u s
a s p e c t s o f th e s e x u a l i t y o f F, c a j ander i . S in c e clamp c o n n e c t io n s form
r e g u l a r l y i n th e d ik a ry o n and f r u i t i n g can be induced i n th e l a b o r a to r y ,
t h i s fungus was c o n s id e re d i d e a l f o r s tu d y as a r e p r e s e n t a t i v e b i p o l a r
s p e c i e s . Those a r e a s w hich were s tu d i e d in c lu d e ;
1) C o n f irm a tio n o f i t s b i p o l a r n a t u r e and g e n e t i c d i s t i n c t i o n from F, ro s e u s
2) G eo g rap h ic a l d i s t r i b u t i o n o f th e a l le lo m o rp h s
, E s t im a t io n o f th e number o f a l le lo m o rp h s a t th e lo c u s fo r h e t e r o t h a l l i s m
D ik a ry o t i c and M onokaryotlc f r u i t i n g
P a i r i n g R e a c t io n s
REVIEW. OF RELATED STUDIES
H e te r o t h a l l i s m i n th e Hymenomycetes was f i r s t r e p o r t e d by Ben- .
saude (1917) and Kniep (1920) w orking in d e p e n d e n t ly w i th Co p r ln us fim en-
t a r i u s and Sch izophy llum commune5 r e s p e c t i v e l y . Both s p e c ie s were o f
th e ty p e term ed t e t r a p o l a r by Kniep and had c o m p a t i b i l i t y f a c t o r s a t two
in d e p e n d e n t ly s e g r e g a t in g l o c i . B i p o l a r i t y , p o s s e s s in g c o m p a t i b i l i t y
f a c t o r s a t a s i n g l e locus o n ly , was f i r s t r e p o r t e d by V andendries (19 2 3 ) .
Many members o f th e Hymenomycetes and G asterom ycetes a r e now known to be
h e t e r o t h a l l i c » The r e l a t i v e d i s t r i b u t i o n i n th e s e groups i s a p p ro x im a te
ly 55% h e t e r o t h a l l i c and t e t r a p o l a r , 35% h e t e r o t h a l l i c b i p o l a r and 10%
h o m o th a l l ie (W hitehouse, 1949a).
These f i g u r e s may be m is le a d in g , however. A nonrandom f a c t o r ,
f a v o r in g h e t e r o t h a l l i s m i s r e f l e c t e d i n th e a v a i l a b l e d a t a . Being e a s i e r
to work w i th , th e f l e s h y p o ly p o re s and a g a r i c s , which a r e m o s t ly h e t e r b -
t h a l l i c , tend to be p r e f e r r e d e x p e r im e n ta l o rgan ism s. S tu d ie s o f th e se
s p e c ie s p redom ina te i n t h e l i t e r a t u r e . More r e c e n t l y , a d e t a i l e d s tu d y
o f th e r e s u p i n a t e Hymenomycetes (B o id in and B an q u e t in , 1965) has shown
th e f re q u e n c y o f h o m o th a l l ism among a sample o f 150 s p e c ie s in t h i s
group to be 26%. This i s a much h ig h e r f i g u r e th a n t h a t based on d a ta
ta k e n p r i m a r i l y from th e s o u rc e s j u s t m en tioned . W hether o r n o t t h i s i s
t r u e o f o t h e r , l e s s w e l l known groups rem ains to be s e e n , b u t th e q u e s
t i o n c e r a i n l y d e s e rv e s c l a r i f i c a t i o n .
M u l t ip le a l le lo m o rp h s a t th e l o c i f o r h e t e r o t h a l l i s m were f i r s t
d is c o v e re d in S. c ommune (K niep; 1920, 1922), S h o r t ly t h e r e a f t e r .
Vandendries (1923) and o th e r s r e p o r t e d " m u l t ip le a l le lo m o rp h s h e t e r o t h a l l i s m "
i n s e v e r a l b i p o l a r s p e c i e s . I t i s now commonly th o u g h t t h a t a l l h e t e r o -
t h a l l i c s p e c ie s i n th e Hymenomycetes p o s s e s s m u l t i p l e f a c t o r s a t t h e i r
c o m p a t i b i l i t y l o c i , '
W hitehouse (1949b), u s in g th e a v a i l a b l e d a t a , e s t im a te d th e num
b e r o f a l le lo m o rp h s a t th e l o c i f o r h e t e r o t h a l l i s m in th e h e t e r o t h a l l i c
Hymenomycetes as a whole to be o f th e o rd e r o f m agnitude o f 100 p e r l o c u s .
R ap e r* K ronge lb , and B a x te r (1958 ) , thorough ly i n v e s t i g a t i n g a l a r g e w o r ld
wide sam ple o f i s o l a t e s o f IS, commune, ■ o b ta in e d e s t i m a t e s f o r th e number
o f a l le lo m o rp h s a t each lo c u s w hich w ere in e s s e n t i a l agreem ent w i th
W hitehouse , No com parable s tu d y d e a l in g w i th a b i p o l a r s p e c ie s had as
y e t been u n d e r ta k e n p r i o r to th e work r e p o r t e d h e r e . I t sh o u ld a l s o be
n o te d t h a t th e a n a l y s i s o f th e d a ta o f Raper e t a l , d i f f e r e d from t h a t o f
W hitehouse i n t h a t th e y used a method deve loped by W righ t (1937) and p r e
v i o u s l y a p p l i e d to e s t i m a t i o n o f the . number o f s e l f - s t e r i l i t y a l l e l e s
i n c l o v e r by Bateman (1 947 ) ,
T here has been some c o n t ro v e r s y i n th e p a s t ov e r th e s i g n i f i c a n c e
. o f th e g e o g ra p h ic a l d i s t r i b u t i o n o f c o m p a t i b i l i t y f a c t o r s , W hitehouse
(1949b) f e l t t h a t t h e i r d i s t r i b u t i o n must be homogeneous th ro u g h o u t th e
p o p u la t io n due to th e ex trem e e f f i c i e n c y o f sp o re d i s p e r s a l in the Hy
menomycetes . In d e e d , Raper e t a l , (1958) found no e v id e n c e to th e con
t r a r y i n t h e i r a n a l y s i s o f a l a r g e w o r ld -w id e sample o f a l le lo m o rp h s o f
S, commune. The to p i c u n d o u b te d ly d e s e rv e s more i n t e n s i v e s t a t i s t i c a l
and e c o l o g i c a l s tu d y b u t , as i t has been p o in te d o u t b e f o r e , th e en o rm ity
o f such an a n a l y s i s i s a lm o s t p r o h i b i t i v e . Some s im p le o b s e r v a t io n s on
th e s u b j e c t have , n e v e r t h e l e s s , been made in t h i s s tu d y .
MATERIALS AND METHODS
E ig h te e n s to c k d ik a ry o n s were used in th e g r e a t e r p a r t o f t h i s
s tu d y , f i f t e e n o f w hich were o b ta in e d from th e F o r e s t D ise a s e L a b o ra to ry ,
U. S . F o r e s t S e r v ic e , B e l t s v i l i e , M aryland, and 3 o f which were o b ta in e d
from specim ens i n The U n i v e r s i t y o f A rizo n a M yco lo g ica l Herbarium , These
i s o l a t e s r e p r e s e n t e d th e N orth American ran g e o f th e s p e c i e s .
The fo l lo w in g c u l t u r e s o f F, c a j a n d e r i were o b ta in e d from th e
F o r e s t D ise a se L a b o ra to ry , U* S, F o r e s t S e r v ic e , B e l t s v i l l e , M aryland,
Mad,. LP~66~ ~ ro t i s o l a t e from lo d g e p o le p in e ( P inus con- t o r t a D o ug l,) pulpwood, su b m it te d by Thilmany Paper C o , , to F o r e s t P ro d u c ts L a b o ra to ry , M adison, W ise , , r e c e iv e d A p r i l 6, 1969,
MD~14~~rot i s o l a t e from c r e o s o te d " s o u th e r n y e l lo w p in e " u t i l i t y p o le i n s e r v i c e i n Ohio, c o l l e c t e d by Vaughn, i s o l a t e d a t FPL by C a th e r in e G, Duncan, R e e d , , A p r i l 8 , 1954.
ME~34~S~~sporophore t i s s u e i s o l a t e from sp o ro p h o re on s u b f lo o r and m o is t a t s i t e o f shower le a k i n home, N orvato , C a l i f , , c o l l , C, S. Moses, i s o l a t e d by W,E, E sy ln , A p r i l 29, 1961. Madison FLL i s o l a t e ,
Aho 61~23~*-rot i s o l a t e from D o u g la s - f i r lo g a t 72*,W alla W alla Ranger D i s t r i c t , U m a t i l l a N a t l , F o r e s t ,O re g , , c o l l . P au l E, Aho, A ugust 3 , 1961.
M D -183--rot i s o l a t e , " s . y , p , f* t e s t s t a k e t r e a t e d w i th 4 .5 lb . f u e l o i l , i n ground t e s t i n D o u g la s - f i r p l a n t a t C o r v a l l i s , O r e g . , i s o l a t e d C a th e r in e G. Duncan,J u l y 9, 1965, Madison i s o l a t e ,
. M D -206--rot i s o l a t e , " s . y . p . " t e s t s t a k e t r e a t e d w i th 5 .1 lb . f u e l o i l , i n ground t e s t a t M adison, W is e . , i s o l . C a th e r in e G, Duncan, S e p t . , 1956. Madison i s o l a t e .
F lo -2 4 3 -A ~ ~ ro t i s o l a t e , r e d sp ru c e (P i c e a rubens S a r g . ) , Mbosehead Lake, M e,, c o l l . P au l V. Mook, summer 1949,
5
6
MD~350~~rot i s o l a t e , w e s te rn re d c e d a r (Thuja p l i - ,c a t a Donn) te le p h o n e p o le i n s e r v i c e 9 y e a r s a t Ann A rb o r , M ic h ig a n , , i s o l , C a th e r in e G. Duncan, Aug,4 , 1959, Madison i s o l a t e .
Mad, 529~~rot i s o l a t e , w e s te rn l a r c h (L a r ix o c c i d e n t a l i s N u t t , ) b o a rd , c o l l , C, J , Humphrey, June 14, 1920, Madison i s o l a t e ,
RLG~2942~S~~sporophore t i s s u e i s o l a t e , on Douglas f i r ,Tub S p r in g , C h ir ic a h u a M ts , , Coronado Nat, F o r e s t , C ochise County, A r i z , , c o l l , RLG J u ly 25, 1968,
FP 70798~S~~sporophore t i s s u e i s o l a t e , on Douglas f i r . E lm s, W ash ,, c o l l , K e i th R, Shead, f a l l 1963,
FP 71244-S— r o t i s o l a t e , c h e r r y , N ic o le t N a t l , F o r e s t , W i s e , , c o l l , Roland C, L o ren z , A ug,, 1936,
FP 103788-Sp^rvpolysporous i s o l a t e , p in e , n o r th woods. P la n t I n d u s t r y S t a t i o n , B e l t s v i l l e , Md,, c o l l . H azel H, McKay, e t a l , Dec, 17, 1952,
FP 104132~ S p --p o ly sp o ro u s i s o l a t e , down p in e lo g , E n to mology P ic n ic g ro u n d , Agr, Res, C t r , , B e l t s v i l l e , Md,, c o l l , F rances F, Lombard & H azel H, McKay, June 27, 1956.
FP 10447-’Sp~^polysporous i s o l a t e , p in e . Log Cabin wood. P l a n t I n d u s t r y S t a t i o n , B e l t s v i l l e , Md,, c o l l . Ross W, D avidson , e t a l . O ct. 31, 1956,
The fo l lo w in g c u l t u r e s o f Fomes e a j a n d e r i K a r s t , were o b ta in e d
from th e U n iv e r s i t y o f A rizona M yco log ica l H erbarium .
PDK-100-sporophore t i s s u e i s o l a t e , on p o n d e ro sa p in e , n e a r Shuway, Coconino County, A r i z , , c o l l , P. D.K een er ,
RLG-6696-spo ro p h o re t i s s u e i s o l a t e , on b la c k sp ru ce( P ic e a ma r i a n a ( M i l l . ) B ,X ,P , ) , W h i te c o u r t , A lb e r ta , Canada, c o l l . R. L, G i lb e r t s o n , A ugust 21, 1966,
RLG -8165-sporophore t i s s u e i s o l a t e , on Douglas f i r , 16 S p r in g s , Sacram ento M ts . , L in c o ln N a t11 F o r e s t , O tero County, New Mexico, c o l l . R . L , G i lb e r t s o n , August 23, 1968.
. ' . ' . ’ . . ' ; . . ' . ‘ " . 7 '
The i s o l a t e o f . Fomes ro se u s 'A lb . e t Schw:. ex F r , ) K a rs t , was
o b ta in e d from th e fo l lo w in g specim en, 1RLG-6954~sporophore t i s s u e i s o l a t e , on q uak ing aspen
(Populus t r emul o i des M ich x ,) , P a r a d is e P a rk , F o r t Apache I n d ia n R e s e r v a t io n , W hite M ts , , Apache County, A r i z . , c o l l . R. L, G i lb e r t s o n , J u l y 16, 1967.
A l l c u l t u r e s were grown oil 1.5% m a l t e x t r a c t medium (N ob les , 1965)
a t room te m p e ra tu re .
I s o l a t e s were made from h e rb a r iu m specim ens in th e fo l lo w in g
manner* The b a s id i o c a r p was b roken in two and th e newly exposed s u r f a c e
flam ed. Sm all c u b e s , a p p ro x im a te ly 2-3 mm?, were removed from th e
i n t e r i o r , f lam ed , and p la c e d on a g a r s l a n t s . E x te n s iv e mycelium d e v e l -/ " ; " : .
oped in I -2 weeks.
I t was found t h a t , w i th most i s o l a t e s , a d ik a ry o n grown in a
p e t r i d i s h f r u i t e d i n 3 -4 w eeks. I n v e r s io n o f th e p e t r i d i s h r e s u l t e d
i n a s p o re p r i n t b e in g d e p o s i t e d on th e l i d , A n o n - q u a n t i t a t i v e d i l u t i o n
method was used to o b t a i n s i n g l e sp o re i s o l a t e s , A d rop o f s t e r i l e , d i s
t i l l e d w a te r was p la c e d on th e sp o re p r i n t , A loop o f th e r e s u l t i n g
sp o re su s p e n s io n was removed and in t ro d u c e d i n t o a tu b e 1/8 f u l l o f s t e r
i l e d i s t i l l e d w a te r . Th is g e n e r a l l y p roduced a s u i t a b l e c o n c e n t r a t i o n
o f s p o r e s . Three tu b e s o f m e lted a g a r each r e c e iv e d a loop o f th e d i l u t e
s p o re su s p e n s io n and w ere poured i n t o empty s t e r i l e p e t r i d i s h e s . Germin
a t i o n o c c u r re d in 3 -4 d ay s . To e n s u re t h a t o n ly one s p o re a t a tim e was
p ic k ed up, th e p l a t e s w ere viewed under a d i s s e c t i n g scope w h i le removing
i n d i v i d u a l g e rm in a t in g s p o re s in s m a l l b lo c k s o f a g a r w i th a s c a l p e l .
The b lo c k s w ere p la c e d on a g a r s l a n t s , An e x t e n s iv e m onokaryo tic m ycel
ium deve loped w i t h i n 14 d a y s , Wet mounts i n p h lo x in e o r c o t to n b lu e were
u s u a l l y made o f each s i n g l e sp o re i s o l a t e to c o n f irm , by th e absence o f
clamp c o n n e c t io n s , i t s m onokaryo tic c o n d i t i o n b e f o r e use in f u r t h e r work.
S to ck c u l t u r e RLG 7942 was used to c o n f i rm th e b i p o l a r n a t u r e o f
Fv c a j a n d e r i and i t s g e n e t i c i s o l a t i o n from F> r o s e u s , N in e teen mono-
mar yons w ere o b ta in e d and p a i r e d in a l l p o s s i b l e co m b in a tio n s (F ig , 1 ) ,
These and a l l su b se q u e n t p a i r i n g s were made in 60 x 15 mm p l a s t i c p e t r i
d i s h e s , " o v e r - f i l l e d " to p r e v e n t p rem a tu re d r y in g . Three to fo u r days
a f t e r th e two m y c e lia had met ( a p p ro x im a te ly 2 w eek s ) , a wet mount o f
hyp.hae. from th e i n t e r f a c e was made. The p r e s e n c e o f clamp c o n n e c t io n s
i n d i c a t e d a c o m p a tib le mating, and was d e s ig n a te d by a " f " s i g n , and th e
absence o f c lam ps, o r an in c o m p a t ib le m a tin g , was d e s ig n a t e d by a
s ig n . E xcep t f o r RLG 7942, o n ly 6 to 9 monokaryons w ere i s o l a t e d from
each d i k a r y o t i c s to c k . P a i r i n g s w ere made betw een th e s e 6-9 monokaryons
to d i s t i n g u i s h th e m a tin g ty p e s o f each s to c k , A s e r i e s o f 36 r e p r e s e n
t a t i v e monokaryons was th e n o b ta in e d . I t was th e n n e c e s s a r y to d e te rm in e
i f t h e r e was any d u p l i c a t i o n o f a l le lo m o rp h s in th e sam ple . To t h i s end,
r e p r e s e n t a t i v e monokaryons from e v e ry o th e r s to c k d ik a ry o n , and th e o u t
comes s c o re d "+" o r " - " a s was a p p ro p r ia te *
A s e r i e s o f di-m on p a i r i n g s was made in th e c o u r s e o f th e i n v e s t i
g a t io n o f p a i r i n g r e a c t i o n s . S tock d ik a ry o n FP 103788 was p a i r e d w i th
each o f th e r e p r e s e n t a t i v e monokaryons o b ta in e d from th e o th e r 17 s to c k
d ik a ry o n s . The p a i r i n g s w ere made in sm a l l p l a s t i c p e t r i d i s h e s , and
in c u b a te d a t room te m p e ra tu re f o r 3 w eeks. At th e end o f t h i s p e r io d ,
hyphae from n e a r th e m y c e l ia l m arg in o f th e m onokaryo tic component most
d i s t a n t from th e d i k a r y o t i c mycelium was examined f o r th e p re s e n c e o f
clamp, c o n n e c t io n s . This was done to p re c lu d e th e p o s s i b i l i t y o f h av in g
9
in v a d in g d i k a r y o t i c hyphae o r i g i n a t i n g from th e component d ik a ry o n p r e s e n t
i n th e p o r t i o n o f th e mycelium examined. F u r th e r , t a k in g hyphae from th e
m y c e l ia l m arg in in s u re d th e e x a m in a t io n o n ly o f grow th o c c u r r in g a f t e r
p o s s i b l e d i k a r y o t i z a t i o n , and, hence , o n ly o f t h a t p a r t o f th e o r i g i n a l
"monokaryon" e x p ec ted to have c lam p s , i f d i k a r y o t i z a t i o n had o c c u r re d .
An i n v e s t i g a t i o n o f p o s s i b l e f r u i t i n g in d u c t io n by a s p e c ie s o f
P e n ic i I l i u m was c a r r i e d ou t in th e fo l lo w in g manner. S t e r i l e d i s c s o f
c e l lo p h a n e were p la c e d on th e s u r f a c e o f m a lt e x t r a c t medium in p e t r i
d i s h e s , The Pen i c i I l i um sp , was a l low ed to grow on th e c e l lo p h a n e u n t i l
i t had co v e re d a c i r c l e a p p ro x im a te ly 1 in c h in d ia m e te r . The d i s c and
fu n g a l hyphae were th e n c a r e f u l l y removed and F, c a j a n d e r i was a l low ed to
grow d i r e c t l y on th e a g a r . The p l a t e s were th e n checked r e g u l a r l y f o r
th e p ro d u c t io n o f f r u i t i n g s t r u c t u r e s , The i s o l a t e s used in t h i s e x p e r i
ment were o b ta in e d from f r e s h f r u i t i n g b o d ie s on a dead Douglas f i r ( P seu -
d o tsu g a m e n z ie s i i (M irb , ) F ranco) stump on Mount Lemmon, Coronado N a t io n a l
F o re s t , . Pima County, A rizo n a , They were p a r t o f a l i n e a r s e r i e s o f 14
i s o l a t e s (ML-A th ro u g h ML^N) made from f r u i t i n g b o d ie s ta k e n a t one f o o t
i n t e r v a l s on th e stump, a l l o f which had p r e v io u s l y f a i l e d to f r u i t .
RESULTS
Con f i r m a t i o n o f B ip o la r N atu re o f F, c a j a n d e r l
D e te rm in a t io n o f th e type o f h e t e r o t h a l l i s m t h a t a g iven Hymeno-
mycete s p e c ie s may p o s s e s s i s accom plished by a s e t o f p a i r i n g s . When
s e v e r a l monokaryons d e r iv e d from s i n g l e sp o re i s o l a t e s o f th e same s to c k
d ik a ry o n a r e p a i r e d in a l l p o s s i b l e c o m b in a t io n s , th e p a t t e r n o f compat
i b l e and in c o m p a t ib le m a tings t h a t i s o b se rved i n d i c a t e s w he the r th e
s p e c ie s i s b i p o l a r o r t e t r a p o l a r » H o m o th a ll ie s p e c i e s , o f c o u r s e , do n o t
r e q u i r e t h i s . A b i p o l a r p a t t e r n i s one in w hich a l l o f th e monokaryons
f a l l i n t o two groups on th e b a s i s o f t h e i r s e x u a l b e h a v io r . Each member
o f a group i s in c o m p a t ib le w i th a l l o th e r members o f th e same group, and
co m p a t ib le w i th a l l members o f th e o th e r group . T h is , o f c o u r s e , i s w hat
would be e x p ec ted from th e s e g r e g a t io n o f c o m p a t i b i l i t y f a c t o r s a t a s i n g l e
lo c u s d u r in g m e io s is and b a s id i o s p o r e fo rm a t io n i n th e d ik a ry o n .
The r e s u l t s o f th e 171 p a i r i n g s o f 'n i n e t e e n monokaryons, from
d i k a r y o t i c s to c k RLG 7942, i n a l l p o s s i b l e c o m b in a t io n s , were a t y p i c a l
b i p o l a r p a t t e r n (F ig . 1 ) , more e a s i l y s een a f t e r r e a r r a n g e m e n t i n t o th e
two c o m p a t i b i l i t y groups (F ig . 2 ) . The a l le lo m o rp h s r e p r e s e n t e d by th e
two groups were d e s ig n a te d 1 and 2, r e s p e c t i v e l y . A l l p a i r s o f a l l e l o
morphs s i m i l a r l y d i s t i n g u i s h e d in l a t e r work were d e s ig n a t e d 3, 4; 5 , 6 ;
e t c . , i n a l i k e manner. These d a t a con firm ed what Mounce and Macrae (1937)
had p r e v io u s l y r e p o r t e d , t h a t Fomes c a j a n d e r i was a h e t e r o t h a l l i c , b i p o l a r
s p e c i e s .
' Y ' Y-V ' . 10 ' - Y
11
10
F ig . 1. R e s u l t s o f p a i r i n g 19 s i n g l e sp o re i s o l a t e , o b ta in e d from s to c k d ik a ry o n RLG-7942, in a l l p o s s i b l e c o m b in a t io n s .
12
A, A,
A,
A
>r )
r/ 3 4 5 8 12 15 15 16 17 \S /9 2 6 7 9 \0 II 14
/ \ — — — —4- + 4- 4 4 4 43 —\ ——— -— — ——4- 4" 4- 4 4 4 44 — ———— — — — — 4- 4- 4- 4 4 4 45 — — — — *— ' — — — + 4- 4 4 4 4 48 — — — — ------ — — — — 4- 4~ 4 4 4 4 4
j /Z —4- 4* 4 4 4 4 4< /3 — — — — — — — — — •— 4- 4" 4 4 4 4 4
/5 — — — — — — — — 4- 4- 4 4 4 4 4/6 — ■ — — — — ------- — — — 4- 4- 4 4 4 4 4/7 — — --- — — — — \ — 4- 4- 4 4 4 4 4IQ \ — 4- 4- 4 4 4 4 419 — — — — — — — — \ 4- 4“ 4 4™4 4 4
r Z -f- 4- 4- -V 4- 4- 4- + 4- 4- + + \6 + 4* 4- 4- 4- 4* 4- 4- 4- 4- 4- 4- —\ — — — — —7 4- 4" 4- 4- 4" 4- 4- 4- 4- 4- 4- 4- — —\ ————
< 9 4- 4- 4- 4 4- 4- 4- 4- 4- 4" 4* 4- —— \ — ——/O -f 4- 4- 4~ 4- 4- 4- 4- 4- 4- 4- 4- — — — —\ ---
II 4- 4- 4- 4" 4- 4- 4- 4- 4- 4* 4- 4- ——— — — —V 14 + 4- 4- 4r 4- 4* 4- 4- 4- 4" 4- 4" — ——
F ig . 2. R e s u l t s as in F ig . 1, r e a r r a n g e d to show th e two c o m p a t i b i l i t y g ro u p s , a t y p i c a l b i p o l a r p a t t e r n .
13
G en e tic I s o l a t i o n of F„ c a i a n d e r i and F. r o s e u s
The g e n e t i c i s o l a t i o n o f F, c a j a n d e r i and F, ro s e u s was a l s o i n
v e s t i g a t e d by Mounce and Macrae (1 9 3 7 ) , s i n c e the two s p e c ie s a re q u i t e
s i m i l a r m o rp h o lo g ic a l ly and t h e i r d i s t i n c t i o n on t h i s l e v e l had been some
what i n c o n c lu s iv e . T h e i r work was r e p e a te d in t h i s s tu d y w i th e x a c t ly
th e same r e s u l t s . R e p r e s e n ta t i v e and A2 monokaryons o f F, c a j a n d e r i
w ere each p a i r e d w i th e i g h t m onokaryo tic i s o l a t e s o b ta in e d from s i n g l e
s p o re s o f F, ro s e u s s to c k d ik a ry o n RLG-6954, A l l p a i r i n g s were incom pat
i b l e (F ig , 3 ) , S ince com ple te i n t e r f e r t i l i t y i s th e r u l e between mono
k aryons r e p r e s e n t i n g d i f f e r e n t m a tin g ty p e s from d i f f e r e n t i s o l a t e s o f
th e same s p e c i e s , and some i n t e r f e r t i l i t y would be found even i f . b o t h
c o m p a t i b i l i t y f a c t o r s were d u p l i c a t e d in th e i s o l a t e i d e n t i f i e d as F,
r o s e u s i t i s r e a s o n a b le to assum e, as d id Mounce and M acrae, t h a t d a t a such
as th e s e r e p r e s e n t p a i r i n g s betw een monokaryons o f two d i f f e r e n t s p e c i e s ,
In view o f t h i s , th e f a c t t h a t t h e r e was a t l e a s t p a r t i a l i n t e r f e r t i l i t y- , - " N - . ' ■
i n a l l su b se q u e n t s e t s o f p a i r i n g s made betw een monokaryons i s o l a t e d from
d i f f e r e n t d i k a r y o t i c s t o c k s , i d e n t i f i e d as F, c a j a n d e r i , was s u f f i c i e n t
p ro o f t h a t no e r r o r s I n i d e n t i f i c a t i o n had been made.
G eo g rap h ica l D i s t r i b u t i o n
As p r e v io u s l y s t a t e d , to d e te rm in e th e number o f c o m p a t i b i l i t y
f a c t o r s p r e s e n t in th e sam ple , i t was n e c e s s a r y to p a i r th e 36 monokary
o t i c i s o l a t e s in a l l p o s s i b l e c o m b in a t io n s , When th e 630 p a i r i n g s n e c e s
s a ry , to accom plish , t h i s were exam ined, n in e t e e n in c o m p a t ib le p a i r i n g s , o r
f a c t o r r e p l i c a t i o n s , were observed (F ig , 4 ) , Out o f a p o s s i b l e maximum o f
36, 25 s e p a r a t e and c o m p a tib le a l le lo m o rp h s were found (T ab le 1 ) , Of
14
F ig . 3
/ Z 3 4 5 6 7 8A — — — — --- — — "
& — " —" ■— — —
R e s u l t s o f p a i r i n g 8 s i n g l e sp o re i s o l a t e s , . from. F. r oseus s to c k d ik a ry o n RLG~6954 w ith two monokaryons o f F. c a j a h d e r i (RLG~7942), c a r r y i n g co m p a tib le m a ting f a c t o r s , and Ag, r e s p e c t i v e l y .
15
F ig u re
/ 7 J 4 5 6 7 8 9 /o // /2 13 /4 IS Z6 I? IS Z920 2/ 22232< 2526272829303/ 32333*3S 36
z \ 4 4 4 4 4 4 4 4 4 4 4 4 + 4 f 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
? \ -t- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 + 4 4 4 4 4 4 —4 4 4 4 4 4 4
3 -f 4 4 4 4 f 4 4 4 4 4 4 4 4 t 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
+ + 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 + 4 4 4 4 t 4
5 -f 4- 4 4 f- 4 4 4 —4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 46 4- 4 4 \ 4 4 4 —4 4 4 4 4 4 4 - 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 47 + 4- 4 4 4 4 \ 4 -j- 4 4 4 4 4 4 4 4 4 4 4 4 4 —4 4 4 4 4 4 4 4 4 4 4 4
g + 4 4 4 4 k 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 -f* 4 4 4 4 4 4 4 4
9 + 4- 4 4 4 4 4 4 \ 4 t 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
/O+ 4 4 4 4 - 4- 4 \ 4 4 4 + 4 4 4 - 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
// +4 4 4 4 4 4 4- 4 4 \ 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 T 4 4 4 4 4 + 4
/2 + 4 4 4 - 4 4 4 4 4 \ 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
/3 t 4 4 4 4 4 4 4 4 4 4 4 \ 4 4 4 4 4 4 4 4 + 4 4 4 4 4 4 4 4 4 4 4 4 4
/f + 4 4 4 4 4 4 4 4 4 4 4 \ 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 + 4 f 4 4/5 4 4 4 4 4 4 4 4 4 4 4 4 4 \ 4 4 - 4 —4 4 4 4 4 4 4 4 4 - + 4 4 4 -/6 +• 4 4 4 4 + 4 4 4 4 4 4 4 4 \ + T 4 4 4 - 4 4 4 4 4 4 4 4 4 4 4 - 4 4
17 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 \ 4 4|4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4/8 •f 4 4 4 4 - 4 4 4 - 4 4 4 4 4 4 \ 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
/9 f 4 4 4 4 4 4 4 4 4 4 4 4 4 - 4 4 4 \ 4 4 4 4 4 4 4 4 - + 4 4 4 -20 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
V4 4 4 4 4 4 4 4 4 4 + 4 4 4 4
2/ + 4 4 4 4 4 4 4 4 4 4 4 4 - 4 4 4 - + 4 4 4 4 4 4 4 - 4 4 4 4
22 -f- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 —-r 4 4 4-) \ 4 4 4 4 4 4 4 4 4 4 4 - 4 4
23 +■ 4 4 4 4 4 4 4 4 + 4 4 4 4 4 4 4 4 4 r 4 + \ 4 4 4 4 4 4- 4 4 4 4 4 4
24 -h 4 4 4 4 4 - 4 4 t 4 + 4 4 4 4 4 4 + 4 4 4 \ 4 4 4 4 + 4 - 4 4 - 4 425 + 4 4 • 4 4 4 4 4 + 4 4 4 4 4 4 4 + + 4 4 4 4 + \ 4 4 4 4 + 4 4 4 + 4-26 + 4 4 4 4 + + 4 4 4 4 4 4 + 4 4 4 - t 4 + 4 4 4 \ 4 4 4 4 T 4 4 4 4 -27 + 4 4 4 4 4 4 4 + + 4 4 4 + 4 4 4 4 4. 4 4 4 4 4 4 \ 4 4 4 4 4 4 4 +23 t 4 4 4 4 4 4 4 + t- 4 4 + 4- 4 4 4 + 4 -r~ 4 4 •r \ 4 4 4 4 4 4 +29 4 - 4 4 4 4 4 4 + 4 4 t- f t 4 * 4 4 4 4j4 4 4- 4 4 4 -h \ 4 - 4 4 4 4
30 + 4 4 4 + 4 4 4 + 4 4 + + 4 4 4 4 4 4 4 4 4 4 4 + 4 \ 4 4 4 4 4 431 -t- 4 4 4 4 4 4 4 + 4 4 4 4 - 4 r - 4 + 4 4 4 -t 4 4 4 + \ 4 4 4 -32 4- 4 + 4 4 4 4 4 4 4 4 4 4 4 + *+* + 4 4 + + 4 4 4 4 4 4 - 4 \ 4 4 4 +33 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 r 4 4 4 4 4 4 4 4 4 + + 4 + t 4 434 + 4 4 4 4 4 4 4 4 4 4 4 4 4 4 - 4 -r 4 + 4 - 4 4 4 + 4 4 4 4 4 4 l + +35 4- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 -t- 4 4 4 4 4 t 4 -t- 4 4 4 -t- 4 4 4 4 f 436 4 4 4 4 + 4 4 4 4 4 4 4 + 4 - 4 4 + - - 4 + 4 4 4 4 4 4- 4 4 4 4 4
4. R e s u l t s o f m a ting th e 36 m onokaryo tic i s o l a t e s in th e sample in a l l p o s s i b l e c o m b in a t io n s .
16
Table I . M ating f a c t o r s found i n th e sam ple.
S tock I s o l a t e A lle lom orph L o c a t io n
RLG-7942 1 A i A rizona2 : A 2
RLG-8165 3 a 3 New Mexico4 a 4
F l o - 243-A 5 A5 Maine6 A 6
PDK-100 7 a 7 A rizo n a8 A8
Aho 71-23 9 a 9 Oregon10 a6
ME-34 . 11 A10 C a l i f o r n i a12 a 5
RLG-6696 13 A11 A lb e r t a , Canada14 A 12
MD-206 15 a 13 W isconsin16 A14
MAD.529 17 A15 W iscons in18 H
MD-350 19 a 13 M ichigan20 A16
MD-14 21 a 13 Ohio22 A14
FP-70798 23 a 17 W ashington24 a 7
F P -104407 25 A18 Maryland26 A19
FP -71244 27 a20 W isconsin28 A21
MAD.IP-6 29 A2 W isconsin30 A22
MD-183 31 A13 Oregon32 A23
F P -104132 33 A24 Maryland34 . A14
F P -103788 35 A25 Maryland36 A13
■ ' 17
t h e s e , one o c c u r r e d ' f i v e t im e s , two o c c u r re d t h r e e t im e s , t h r e e o c c u r re d
tw ic e , and th e rem a in in g 19 o n ly once.
The g e o g ra p h ic a l o r i g i n s o f th e i s o l a t e s which c a r r i e d r e p l i c a t e d
c o m p a t i b i l i t y f a c t o r s were as f o l l o w s :
O ccu rr in g 5 t im e s ;
Wiscons in-M ichigan-O hio- 'O regon-M ary land
O ccu rr in g 3 t i m e s :
M aine-O regon-W isconsin
W isconsin -O hiq rM ary land
O ccu rr in g tw ic e ;
A rizona-W ash ing ton
M a in e - C a l i f o r n ia
A rizo n a -W isco n s in
There was no obvious d e p a r t u r e , e i t h e r i n th e above l i s t i n g o r
th e accompanying map (F ig , 5 ) , from e x p e c t a t i o n based on th e a ssum ption
o f random d i s t r i b u t i o n o f m a ting f a c t o r s . A t e s t f o r randomness was,
n e v e r t h e l e s s , made (R a p e r , e t a l , , 1958),
The 36 m onokaryo tic i s o l a t e s f e l l n a t u r a l l y i n t o 2 groups t h a t r e
f l e c t e d a s i g n i f i c a n t d i s c o n t i n u i t y i n th e s p e c ie s d i s t r i b u t i o n ; nam ely,
th e e a s t e r n and w e s te rn c o n i fe r o u s f o r e s t r e g io n s . A lthough th e s e two
r e g io n s a r e a c t u a l l y c o n t in u o u s f a r t h e r n o r t h , th e c e n t r a l p l a in s s e p a r
a t e s /fhem f o r much of. t h e i r l e n g th . In term s o f e f f e c t i v e sp o re d i s p e r
s a l , th e o v e r r id i n g r e a s o n f o r i n i t i a l l y h y p o th e s iz in g random d i s t r i b u t i o n ,
a s i g n i f i c a n t e c o l o g i c a l b a r r i e r would seem to be p r e s e n te d by th e p l a i n s
r e g io n . The p roposed g roup ing i s th u s j u s t i f i e d .
th e sam ple.- They a re grouped 00
th e d i s t r i b u t i o n o f th e 25 a l le lo m o rp h s found i nwhich th e y o r i g i n a l l y o c c u r re d i n th e s to c k d ik a ry o n s .F ig . 5 . Map showing
i n th e p a i r s i n
19
The r a t i o o f th e number o f r e p l i c a t i o n s to th e t o t a l number o f
p a i r i n g s o f i s o l a t e s from w i th i n a r e g io n was compared to i’th e analogous
r a t i o f o r p a i r i n g s between i s o l a t e s from d i f f e r e n t r e g io n s (T ab le 2 ) ,
There was no d i f f e r e n c e i n th e r a t i o s a t th e 5% l e v e l o f s i g n i f i c a n c e .
T h e re fo re , th e d a t a d id n o t c o n t r a d i c t th e a ssu m p tio n o f random d i s t r i
b u t io n .
E s t im a t io n o f th e Number o f A lle lom orphs
Knowing th e f re q u e n c y o f f a c t o r r e p l i c a t i o n i n the. sam ple , i t i s
p o s s i b l e to e s t i m a t e th e number o f f a c t o r s in th e p o p u la t io n .
The g e n e r a l n a t u r e o f th e p rob lem can be s t a t e d as fo l lo w s :
g iv e n a s e r i e s o f n f a c t o r s p r e s e n t i n th e p o p u la t io n , and an experim en
t a l p ro c e d u re o f random p a i r i n g , w hat i s th e p r o b a b i l i t y o f g e t t i n g an
i d e n t i c a l p a i r ? This has been shown to be
p-1 ~f- n 2. * ■
where p i s th e p r o b a b i l i t y o f r e p l i c a t i o n , and p i s th e v a r i a n c e o f th e
f a c t o r f r e q u e n c i e s (W right and Dobzhansky, 1 9 41 )e The r a t i o o f r e p l i c a
t i o n s to the t o t a l number o f p a i r s i s e q u a l to p fo r a s u f f i c i e n t l y l a r g e
2sample and an e s t im a te o f n i s th u s p o s s i b l e , i f p i s known.
I n th e p r e s e n t c a s e , s in c e t h e r e i s no e v id e n c e to th e c o n t r a r y ,
i t i s assumed t h a t th e c o m p a t i b i l i t y f a c t o r s a re p r e s e n t in eq u a l f r e q u e n c y .
F u r th e r , as has a l r e a d y been shown, th e d a t a do n o t c o n t r a d i c t a h y p o th
e s i s o f random d i s t r i b u t i o n o f f a c t o r s . Under th e s e two a s su m p tio n s ,
W r ig h t 's e q u a t i o n becomes
V - ■ ■ V..v : ;•: - ' ' . n - - ■ - 1 •
and a f i r s t a p p ro x im a tio n e s t im a te o f n can be made.
20
T able I I . Comparison o f th e f r e q u e n c ie s o f r e p l i c a t i o n s i n p a i r i n g s
o f i s o l a t e s from w i t h i n and betw een th e E a s t e r n and W este rn
c o n i f e r o u s f o r e s t r e g io n s .
I n t r a - r e g i o n a l
No. o f No. o fRegion p a i r i n g s : r e p l i c a t i o n s
E a s t 160 10West 120 J .
T o ta l 280 11P,= 11
280
I n t e r - r e g i o n a l
E as t-W est 380 p a i r i n g s 8 r e p l i c a t i o n s
P ^ P g a t 5% l e v e l o f s i g n i f i c a n c e
' '■ ■ ■. 21 For a sequence o f s t a t i s t i c a l l y in d e p en d en t e x p e r im en ts hav ing
two p o s s i b l e outcom es, one o f w hich has a low p r o b a b i l i t y , p, o f o c c u r
r i n g , th e p r o b a b i l i t y t h a t t h i s e v e n t w i l l o ccu r K t im es o u t o f N e x p e r i
ments i s
pcse k-
kT .
where ~Np and i s m odera te in m agn itude . That i s , th e number o f t im es
t h a t th e im probab le e v e n t o ccu rs i s P o is so n d i s t r i b u t e d ( F e l l e r , 1957),
In t h i s s tu d y , a sequence o f s t a t i s t i c a l l y in d e p e n d e n t p a i r i n g s
was made. For each p a i r i n g t h e r e were two p o s s i b l e ou tcom es , c o m p a t ib i l
i t y o r i n c o m p a t i b i l i t y ; and th e p r o b a b i l i t y o f an in c o m p a t ib le outcome
was s m a l l (p==19/630) „ The number o f e v e n ts , N, was 630, and ~Np~19 was
moderate* Thus th e c o n d i t io n s a r e s a t i s f i e d and th e number o f r e p l i c a
t i o n s i s P o is s o n d i s t r i b u t e d .
This means t h a t , as o th e r s have n o te d (Bateman 1947, S tevens 1942),
th e u se o f th e s ta n d a rd e r r o r to compute c o n f id e n c e l i m i t s i s i n c o r r e c t
h e r e . The f i d u c i a l l i m i t s employed by th e s e a u th o r s a r e th e a p p r o p r i a t e
o n e , The e r r o r o f e s t i m a t i o n i s l a r g e as th e c a l c u l a t i o n s fo r t h i s c a s e
d e m o n s t ra te :
p « l« 19 w i th 5% l i m i t s o f 11 ,61 and 29 ,51 n 630 ~630 630
n~33 w i th 5% l i m i t s o f 54 and 21,
I t sh o u ld be n o te d t h a t th e c o n c e p ts o f random p a i r i n g and s t a t i s
t i c a l independence a r e somewhat s t r a i n e d h e re s in c e t h e r e i s an e lem en t o f
p r e d i c t a b i l i t y in h e r e n t i n th e e x p e r im e n ta l p ro c e d u re . T hat i s , once a
r e p l i c a t i o n i s o b se rv e d , th e outcomes o f a c e r t a i n number o f o th e r p a i r
in g s become p r e d i c t a b l e e v e n t s , However, s in c e t h i s la c k o f independence
i s a f u n c t i o n o f th e o rd e r i n which th e p a i r s a r e exam ined, i t i s con
v e n i e n t to c o n s id e r a l l p a i r i n g s as though th e y were in d e p e n d e n t . Any
a t te m p t to e l im i n a t e t h i s non-random ness would be e x p e r im e n ta l ly p r o h ib
i t i v e ,
D ik a r y o t i c F r u i t i n g
The s to c k d ik a ry o n s used in t h i s s tu d y were q u i t e v a r i a b l e i n
t h e i r v e g e t a t i v e c h a r a c t e r i s t i c s , This v a r i a b i l i t y was n o t r e s t r i c t e d
to v e g e t a t i v e g ro w th , however. A lthough d i s t i n c t , w e l l p igm ented p o re
s u r f a c e s d eve loped in a l l b u t two c a s e s , th e e n t i r e , f r u i t i n g s t r u c t u r e s
v a r i e d from f l a t , i r r e g u l a r " p a tc h e s " to a lm o s t s p h e r i c a l o u tg ro w th s ,
d e v e lo p in g a t th e p e r ip h e r y o f th e m y c e l ia l mat (F ig , 6) , S tocks MAD-
LP6 and FP 71244, were a t y p i c a l in t h a t the pore s t r u c t u r e had d e g e n e ra te d
to s m a l l , c i r c u l a r , c r u s t - l i k e a r e a s o f exposed hymenium. There was
l i t t l e o r no p ig m e n ta t io n in e i t h e r c a s e , b u t FP 71244 produced numerous
d r o p l e t s o f t h i c k p in k i s h e x u d a te i n th e v i c i n i t y o f th e f r u i t i n g a r e a .
There were s e v e r a l d i k a r y o t i c i s o l a t e s on hand t h a t n ev er f r u i t e d ,
b u t f r u i t i n g a b i l i t y d id n o t ap p ea r to be r e l a t e d to t im e i n c u l t u r e . I n
f a c t , two s to c k s used i n t h i s s tu d y t h a t f r u i t e d most r e a d i l y , MAD 529 and
FP 71244, had been in c u l t u r e s in c e 1920 and 1936, o r f o r p e r io d s o f 49
and 33 y e a r s r e s p e c t i v e l y .
S e v e ra l i s o l a t e s t h a t had a t f i r s t f a i l e d to f r u i t were n o t i c e d
to do so when c o n tam in a ted w i th an u n i d e n t i f i e d P e n ic i I l i u m s p , . This l e d
to c o n s i d e r a t i o n o f th e p o s s i b i l i t y t h a t th e co n tam in a n t induced d ik a ry o
t i c f r u i t i n g in F, c a j a n d e r i , a p o s s i b i l i t y s u g g e s te d by th e r e c e n t r e p o r t
o f a s i m i l a r phenomenon in v o lv in g m onokaryo tic f r u i t i n g in S, commune
23
F ig u re 6. T y p ic a l D ik a ry o t ic f r u i t i n g in an (Ag x A^) d ik a ry o n .
' 24
(Leonard and R aper, 1969). The c e l lo p h a n e d i s c te c h n iq u e was employed
h e re . A l l fo u r i s o l a t e s ( M L ~ C , . M L - E , & ML~F) used in t h i s ca se
had p r e v io u s l y n ev e r f r u i t e d . Only one, ML-C, d id f r u i t a f t e r b e ing
grown on th e p r e t r e a t e d medium. These r e s u l t s were so in c o n c lu s iv e t h a t ,
due to tim e l i m i t a t i o n s , th e s u b j e c t was p u rsu ed no f u r t h e r . However,
i t was o b se rv ed t h a t , i n th e one c a s e where f r u i t i n g d id o c c u r , i t oc
c u r r e d i n a s h o r t p e r io d o f t im e , abou t t h r e e weeks, and t h a t g e n e r a l .
v e g e t a t i v e growth was more v ig o ro u s ,
'Monokaryot i c •F r u i t i n g
Two o f th e 36 monokaryons o b ta in e d d u r in g th e c o u r s e o f t h i s
i n v e s t i g a t i o n were found to p roduce m onokaryo tic f r u i t i n g s t r u c t u r e s ,
They were i s o l a t e s 12 (Ag) and 23 (Apy), In b o th c a s e s , s u b c u l t u r i n g
d id n o t a t t e n u a t e t h i s a b i l i t y , and a l l t r a n s f e r s p ro m p tly f r u i t e d a f t e r
s u f f i c i e n t m y c e l ia l developm ent, o r ab o u t one week. The o th e r 34 mono
k a ryons were n e v e r n o te d to f r u i t s p o n ta n e o u s ly a f t e r , i n some c a s e s ,
o ver a y e a r i n c u l t u r e .
Of g r e a t i n t e r e s t was th e f a c t t h a t b o th f r u t i n g i s o l a t e s p ro
duced c o m p le te ly s t e r i l e f r u i t i n g s t r u c t u r e s . R epea ted a t te m p ts were
made to f in d b a s i d i a o r b a s id io s p o r e s in s e c t i o n s o f m onokaryo tic f r u i t
in g b o d ie s o f v a r io u s a g e s , and th e r e n e v e r appea red to be even a v e s t i
g i a l hymenium p r e s e n t . F u r th e r , i n v e r s i o n o f s e v e r a l p l a t e s o f b o th
monokaryons f o r up to th r e e weeks f a i l e d to r e s u l t in th e fo rm a t io n o f
a sp o re p r i n t .
I s o l a t e 12 always p roduced c o p io u s amounts o f a t h i c k p in k i s h
ex u d a te on th e p o re s u r f a c e , s i m i l a r to t h a t produced by s to c k d ik a ry o n FP
71244 which was a l s o abnormal in i t s f r u i t i n g b e h a v io r (F ig , 7 ) ,
F ig u re 7. M onokaryotic f r u i t i n g in A5 (ME-34). - Note drops o f e x u d a te .
26
A second s i t u a t i o n where m onokaryo tic f r u i t i n g was n o te d to o ccu r
was i n some p a i r i n g s o f in c o m p a t ib le i s o l a t e s , These p a i r i n g s in v o lv ed
monokaryons t h a t had n o t f r u i t e d a lo n e , and b o th members o f th e p a i r w ere
always a f f e c t e d (F ig , 8) , These f r u i t i n g s t r u c t u r e s a l s o appeared to be
q u i t e s t e r i l e .
P a i r in g Reac t i o n s
D uring th e c o u r s e o f t h i s i n v e s t i g a t i o n i t was n o te d t h a t t h e r e
were v a r io u s m acroscop ic changes i n th e a p p ea ran ce o f m y c e l ia in bo th
c o m p a tib le and in c o m p a t ib le p a i r i n g s . Th is made i t p o s s i b l e to i d e n t i f y
c o m p a tib le and in c o m p a t ib le m a tings by v i s u a l i n s p e c t i o n i n many c a s e s .
However, th e s e changes were seldom obv ious b e fo re th e second week o f
in c u b a t io n . In c o m p a tib le p a i r i n g s were found to be o f two t y p e s ; 1~
m a c r o s c o p ic a l ly in c o m p a t ib le , and 2- m a c r o s c o p ic a l ly c o m p a t ib le . The
form er was c h a r a c t e r i z e d by th e c l a s s i c a l f e a t u r e s o f such p a i r i n g s , ob
se rv e d by o th e r w orkers in s e v e r a l s p e c ie s (Adams and R oth , 1967;
V e r r a l , 1937)0 A d i s t i n c t l i n e o f .d e m a r c a t io n fo rm ed .be tw een the
two m y c e l ia , accompanied by d i s c o l o r a t i o n o f th e medium below th e l i n e
and changes i n growth and p ig m e n ta t io n o f th e im m ed ia te ly a d j a c e n t hyphae
o f b o th m y c e l ia (F ig , 8 , 10, 11), E x p re s s io n o f the a v e r s io n r e a c t i o n
was s t r o n g e r w i th t im e . The second ty p e , however, d i s p la y e d l i t t l e o r
no d i s c o l o r a t i o n o f th e medium and th e hyphae o f th e two m y c e l ia ap p ea red
to in t e r m in g le f r e e l y w i th .n o s ig n s o f a v e r s io n o r changes i n g ro ss
m orphology. R epeated m ic ro sc o p ic e x a m in a t io n of. hyphae from th e i n t e r f a c e
r e g io n s o f p a i r i n g s o f t h i s ty p e as much as 30 days o ld f a i l e d to r e v e a l
th e p re s e n c e o f clamp c o n n e c t io n s , I t was, t h e r e f o r e , n e c e s s a r y to
27
b.F ig u re 8 . M onokaryotic f r u i t i n g in d u c t io n in two in c o m p a tib le p a i r i n g s -
a . (L e f t ) A-£3 (MD-206) x (MD-350); ( r i g h t ) MD-183-4 x MD- 183-6, two s i n g l e spo re i s o l a t e s o f s to c k d ik a ry o n MD-183.b. Obverse o f above, showing d i s c o l o r a t i o n o f medium along i n t e r f a c e .
28
l Z 3 5 6 7 83 — — \ \ --- - --- 4-4 — — \ --- --- +
Ou
1 2 3 4 5 6 7 8 92 — — 4- — — 4- — —
3 — \ 4" — — 4- — —
b.F ig . 9. Two s e t s o f m a tings between s i n g l e - s p o r e i s o l a t e s o b ta in e d from a
s i n g l e s to c k d ik a ry o n . “ An in c o m p a t ib le p a i r i n g t h a t was macro- s c o p i c a l l y c o m p a tib le o ccu rred in each s e t , w i th o u t c o n t r a d i c t i o n , a) HD-14-3x7 was m a c ro s c o p ic a l ly c o m p a t ib le ; in 4x7 th e r e was an a v e r s io n r e a c t i o n , b) MD 350-2x5 was m a c ro s c o p ic a l ly c o m p a tib le ; an a v e r s io n r e a c t i o n o ccu rred in 3x5.
MD 350 - pa>red monoVaruons- to p rou3-covrxpa.tib\eYna.Vxnq5 > b o t t o m - corr.paVvblB nrk&A.\r»gs
F igu re 10. M atings o f s i n g l e - s p o r e i s o l a t e s o f s to c k d ik a ry o n MD-350.
30
F ig u re 11. In co m p a tib le m a tin g s - ( to p ) Ag (F lo-253-A ) x A, (A ho-61 -23 );(bottom ) two p l a t e s o f A5 (F lo-243-A ) x A5 (ME-34) component i sf r u i t i n g and the o th e r i s n o t . The i r r e g u l a r m argin o f th e A5(ME-34) i s o l a t e r e s u l t e d in an i r r e g u l a r l i n e o f d em arca tio n .
' - . ■ 31
co n c lu d e t h a t th e s e p a i r i n g s were a l s o in c o m p a t ib le . This f in d in g was
f u r t h e r s t r e n g th e n e d by. th e f a c t t h a t , i n two c a s e s , th e s e p a i r i n g s
f i t t e d w i th o u t c o n t r a d i c t i o n in t o b i p o l a r m a ting p a t t e r n s . They o c c u r re d
in th e c o u rse o f p a i r i n g s e v e r a l s i n g l e sp o re i s o l a t e s from two s to c k
d ik a ry o n s (MD-14 and MD-350) in o rd e r to d i s t i n g u i s h th e two m a ting ty p e s
i n each (F ig , 9 & 12 ) , T h e i r a p p a re n t i n c o m p a t i b i l i t y c o u ld be checked
a g a i n s t th e o th e r outcomes in th e r e s u l t a n t b i p o l a r p a t t e r n s , I t sh o u ld
a l s o be n o te d t h a t d i s c o l o r a t i o n does n o t always o ccu r i n a v e r s io n r e a c
t i o n s i n o th e r s p e c ie s as n o te d in Femes p i n i by Roth (1 9 5 2 ) , .
U n less th e m acroscop ic i n c o m p a t i b i l i t y r e a c t i o n was q u i t e s t r o n g ,
a wet mount o f hyphae was s t i l l made f o r c o n f i r m a t io n . Th is was due to
t h e f a c t t h a t , w hereas most m onokaryo tic i s o l a t e s o f F, c a j a n d e r i had
c o l o r l e s s hyphae r e g a r d l e s s o f th e p ig m e n ta t io n o f th e p a r e n t d ik a ry o n ,
many d i k a r y o t i c s to c k s deve loped p in k o r brown m y e e lia a n d /o r caused d i s ^
c o l o r a t i o n o f th e medium. The c o m p a t ib le p a i r i n g o f two unpigm ented mono-
k aryons o f t e n r e s u l t e d in th e fo rm a t io n o f a p igm ented o r pigm ent p ro d u c
in g d ik a ry o n s growing a t th e i n t e r f a c e . T h is s i t u a t i o n had th e ap p ea ran ce
o f a weak i n c o m p a t i b i l i t y r e a c t i o n .
I t was im p o ss ib le to c o n s id e r f r u i t i n g s t r u c t u r e p ro d u c t io n as a
c r i t e r i o n f o r d e t e r m in a t io n o f i n t e r f e r t i l i t y . The two monokaryons t h a t
r e g u l a r l y produced m onokaryo tic f r u i t i n g s t r u c t u r e s i n c u l t u r e u s u a l l y
a l s o d id so i n th e c o u rs e o f a p a i r i n g , co m p a tib le o r n o t (F ig , . 11),
F u r th e rm o re , p a i r i n g in c o m p a t ib le monokaryons, t h a t n o rm a l ly d id n o t form
m onokaryo tic f r u i t i n g s t r u c t u r e s , sometimes induced b o th members o f th e
p a i r to do so . This r e s u l t e d in th e s u p e r f i c i a l l y c o n t r a d i c t o r y j u x t a p o s i
t i o n o f a c l e a r , h e a v i ly d i s c o l o r e d , l i n e o f d e m a rc a t io n and l a r g e a r e a s
32
p a i r e d m o n o k a ru o n S # "bop rouu- coyrxpoAxbXe mf\a\inc^3 •, b o ^ o m - Vnc.ompo*ife\e rnoJbir^y
F ig u re 12. M atings of s i n g l e - s p o r e i s o l a t e s o f s to c k d ik a ry o n MD-14.“ The l a s t p l a t e in th e bottom row i s m a c ro s c o p ic a l ly c o m p a tib le .
o f p in k pore s u r f a c e on th e same p l a t e . To add to th e c o n fu s io n , com
p a t i b l e m a tings were n o t always fo llo w ed by d i k a r y o t i c f r u i t i n g on th e "
p l a t e . Thus, t h i s c h a r a c t e r i s t i c was v a l u e l e s s as a m acroscop ic i n d i c a
t o r o f co m p a tib le p a i r i n g s .
Com patib le m a t in g s , on th e o th e r hand, cou ld sometimes be recog--
n iz e d by th e g ro ss morphology o f th e "component m onokaryon-d ikaryon-
component inonokaryon" complex. In th e s e c a s e s , wedges o f th e more v i g o r
ous d i k a r y o t i c mycelium growing o u t from th e e a r l i e s t p o i n t o f c o n t a c t o f
th e component m y c e l ia , occup ied th e i n t e r f a c e r e g io n . This was o n ly ob
v io u s when th e r a t e o f growth or mat ap p ea ran ce o f th e d ik a ry o n d i f f e r e d
s i g n i f i c a n t l y from th o s e o f i t s com ponents. The phenomenon was most o f t e n
n o te d when one o f th e component monokaryons had a v e ry slow growth r a t e .
This c r e a t e d a s i t u a t i o n i n which th e new ly formed d ik a ry o n had more med
ium a v a i l a b l e f o r unhampered grow th , a l lo w in g d i f f e r e n c e s in g ro s s morph
o logy to be c l e a r l y e x p re s s e d . U n f o r tu n a te ly , th e more f r e q u e n t c a s e , a
p a i r i n g o f two r e l a t i v e l y v ig o ro u s monokaryons, le d to th e p l a t e b e in g
a l m o s t .e n t i r e l y covered by m onokaryo tic t i s s u e b e fo re s i g n i f i c a n t grow th
o f th e d ik a ry o n co u ld o c c u r , . Any o th e rw is e v i s i b l e d i f f e r e n c e s in morph
o logy were th u s o b scu red .
A f t e r i d e n t i f i c a t i o n o f th e a l le lo m o rp h s i n th e sam ple, th e d i
karyon c o n s t i t u e n t o f th e di-mon p a i r i n g s was found to c a r r y a l le lo m o rp h s
A33 and A25. Due to r e p l i c a t i o n s w i t h i n th e sam ple , p a i r i n g (A^g X A^^)
w i th each o f th e f i r s t 34 m onokaryo tic i s o l a t e s im p l ie d t h a t s e v e r a l r e
dundan t p a i r i n g s were made.
E xam ina tion o f hyphae n e a r th e m y c e l ia l m arg in o f th e component .
monokaryon, a f t e r t h r e e weeks o f grow th , r e v e a le d th e p re se n c e o f clamps
34
in a l l b u t fo u r p a i r i n g s (F ig , 13 ) , Three d ik a r y o t i z e d monokaryons p r o
duced abn o rm a l, c o n to r t e d clam ps. Of th e t h r e e > two were X A25) X
Ag p a i r i n g s , and one was an (A^g X Agg) X Ag p a i r i n g . S ince Ag was found
t h r e e t im es i n th e sam ple , a t h i r d p a i r i n g o f th e same ty p e was a v a i l a b l e
f o r com parison . This p a i r i n g d id n o t ap p e a r to be d ik a r y o t i z e d a t a l l ,
a l th o u g h an i n o r d i n a t e number o f pseudoclam ps were o b se rv e d . In f a c t , i t
was p o s s i b l e s e v e r a l t im es to o b se rv e pseudoclam ps a t s e v e r a l c o n s e c u t iv e
s e p t a a lo n g a hypha in t h i s p a i r i n g . In th e c a s e o f th e (A^g X Agg) X Ag
p a i r i n g , t h e r e was, s i m i l a r l y , a second p a i r i n g o f th e same ty p e in th e
s e r i e s . Th is one was d ik a r y o t i z e d , and produced q u i t e norm al clamp
c o n n e c t io n s .
I n a l l b u t two o f th e di-mon p a i r i n g s , l i n e s o f d e m a rc a t io n were
e v id e n t a t th e i n t e r f a c e , though th e s t r e n g t h o f th e r e a c t i o n was v a r i a b l e
For b o th p a i r i n g s i n w hich t h e r e was no a v e r s io n , th e m onokaryo tic compon
e n t was d i k a r y o t i z e d .
Of th e f o u r p a i r i n g s i n which d i k a r y o t i z a t i o n f a i l e d to o c c u r , one
(A^g X A25) X Ag, has a l r e a d y been m en tioned . The o th e r t h r e e , in v o lv in g
monokaryons c a r r y i n g an Ag, Ag and Agg a l le lo m o rp h , r e s p e c t i v e l y , s h a re d
w i th th e form er one common f e a t u r e , ex trem e d e s i c c a t i o n o f th e medium on
w hich th e m y c e l ia were grow ing. Thus, th e ad v e rse e f f e c t o f env ironm en t,
low h u m id ity i n t h i s c a s e , on clamp c o n n e c t io n in c id e n c e , as p r e v io u s ly
m e n tio n ed , c an n o t be d is c o u n te d h e r e , The s i g n i f i c a n c e o f th e s e fo u r p a i r
in g s in i n t e r p r e t i n g d a t a shou ld be c o r r e s p o n d in g ly low in l i g h t o f t h i s
knowledge.
35
F ig u re 13. Di-mon p a i r i n g s . " The upper mycelium on each p l a t e i s th e d ik a ry o n (A 3 X A25) . The s t r e n g t h o f th e a v e r s io n r e a c t i o n i s v a r i a b l e . The monokaryon component were (from r i g h t to l e f t ) : A24i A%3(MD- 14) upper row; A^4 (FP 104132); A21 lower row. A l l were d ik a ry o - t i z e d .
DISCUSSION
An e s t i m a t e o f 33 f o r th e number o f a l le lo m o rp h s in th e p o p u la r
t i o n may seem low in com parison to th e an a lo g o u s v a lu e s f o r s e v e r a l t e t r a -
p o l a r s p e c ie s (E g g e rs to n , 1953; Raper e t a l „ , 1958; W hitehouse , 1 9 4 9 b ) .
The o u tb re e d in g p o t e n t i a l , however, d i f f e r s l i t t l e from t h a t o f S, commune,
f o r b o th l o c i o f which n was much g r e a t e r . The v a lu e f o r S. commune was
98.15%, and f o r JF. c a j a n d e r i 96.97%. In o th e r w ords, f o r n=33, th e f r a c
t i o n o f c o m p a tib le m a tin g s b u t o f th e t o t a l number o f p o s s i b le . c o m b i n a t i o n
f o r F. c a j a n d e r i i s ;
1-1= 1- 1= 0 .9697
A t e t r a p o l a r m a ting system , a l th o u g h r e d u c in g th e maximum in b r e e d
in g l e v e l to 25%, a l s o d e c r e a s e s th e p o s s i b l e number o f f e r t i l e m a tings
w i th i n th e whole p o p u la t i o n f o r an av e ra g e n a t b o th l o c i , r e l a t i v e to
"the same f r a c t i o n i n a b i p o l a r sy stem f o r th e same v a lu e o f n. Thus, to
a c h ie v e an a p p ro x im a te ly e q u a l o u tb re e d in g e f f i c i e n c y , th e v a lu e s o f n a t
b o th l o c i must be somewhat g r e a t e r f o r t e t r a p o l a r fo rm s . I t shou ld be
n o te d t h a t b i p o l a r system s s u p p o r t a n . i n h e r e n t maximum in b re e d in g l e v e l of
50%, N e v e r th e le s s , i n term s o f th e c r i t i c a l p a ra m e te r , o u tb re e d in g e f f i
c i e n c y , i t i s s een t h a t a b i p o l a r s p e c i e s would be e x p e c te d to have few er
a l le lo m o rp h s a t th e s i n g l e lo cu s f o r h e t e r o t h a l l i s m th a n a com parable
t e t r a p o l a r form, s in c e fewer a re n e c e s s a r y to re a c h th e same l e v e l o f
o u tb re e d in g e f f i c i e n c y ,
■ '' 36 i ; : ...
' ' " . 37
The b e h a v io r o f th e m onokafyo tic i s o l a t e s w i th r e s p e c t to f r u i t
in g was. somewhat s i m i l a r to t h a t o f L e n z i t e s t r a b e a P e r s , ex F r , , and
Tyromyces undosus M urr, , b o th b i p o l a r 5 h e t e r o t h a l l i c s p e c i e s , In th e s e
c a s e s , monokaryons e i t h e r d id o r d id n o t f r u i t , and i f f r u i t i n g d id o ccu r
i t d id so r e g u l a r l y and p r e d i c t a b l y ( B a r n e t t and L i l l y , 1947; Brotzman
and G i lb e r t s o n , 1967), However, i n c o n t r a s t to F, c a j a n d e r i , bo th th e s e
s p e c ie s do produce b a s i d i a and b a s l d i o s p o r e s ,
The com ple te s t e r i l i t y o f th e m onokaryo tic f r u i t i n g s t r u c t u r e s ,
w h i le u n u s u a l , i s thought to be m ere ly an ex trem e c a se o f th e more common
s i t u a t i o n , i n which f r u i t i n g b o d ie s o f monokaryons p roduce fewer and l e s s
v i a b l e sp o re s th a n th e d ik a ry o n . This i s a l s o in acc o rd w i th the p re v a
l e n t c o n ce p t o f m onokaryo tic f r u i t i n g as an a t t r i b u t e o f l i t t l e o r no b io
l o g i c a l s i g n i f i c a n c e in n a t u r e (R ap e r , 1960),
The a p p a re n t i n d u c t io n o f m onokaryo tic f r u i t i n g d u r in g th e c o u r s e
o f c e r t a i n in c o m p a t ib le p a i r i n g s i s b e l i e v e d to be th e f i r s t i n c i d e n t o f
i t s k in d to be r e p o r t e d . That th e phenomenon occu rs a t a l l has i n t e r e s t
in g r a m i f i c a t i o n s w i th r e s p e c t to c u r r e n t id e a s c o n c e rn in g th e r e l a t i o n
s h ip betw een c o m p a t i b i l i t y f a c t o r s and f r u i t i n g body developm ent. The
s e x u a l p ro c e s s in B a s id io m y c e te s , a l th o u g h p o o r ly u n d e rs to o d due to i t s
c o m p le x i ty , has been assumed to p roceed v i a th e r e c i p r o c a l r e g u l a t i o n o f
c o n s e c u t iv e s t e p s m ed ia ted th ro u g h th e cy to p lasm a n d /o r by d i f f u s a b l e
s u b s ta n c e s . I t has a l s o been shown t h a t gen ic c o n t ro l , o f f r u i t i n g i s a t
l e a s t p a r t l y in d e p en d en t o f th e c o m p a t i b i l i t y f a c t o r lo c u s (Leonard and
Raper, 1969; R aper, 1960), Thus, th e in d u c t io n o f m onokaryo tic f r u i t i n g
d u r in g an in c o m p a t ib le p a i r in g , would seem to i n d i c a t e t h a t th e c r i t i c a l
d i s r u p t i o n in th e s e x u a l sequence due to i n c o m p a t i b i l i t y may o ccu r a t
38
more th a n one p o i n t , and, in some c a s e s , n o t b e fo re a c e r t a i n amount o f
r e c i p r o c i t y , in d e p en d en t o f th e c o m p a t i b i l i t y f a c t o r ( s ) p r e s e n t , has
ta k e n p la c e between th e two m y c e l ia .
I t has p r e v io u s l y been r e p o r t e d t h a t d i k a r y o t i z a t i o n o f th e com
ponen t monokaryon o f a di~mon p a i r i n g d id n o t o ccu r i n F, c a j a n d e r i (Adams
and R o th , 1967), T h e i r c o n c lu s io n s w ere based on t h e i r f a i l u r e to o b se rv e
clamp c o n n e c t io n s in hyphae ta k e n from th e m onokaryo tic s i d e o f th e i n t e r
f a c e , From th e symmetry o f th e growth p a t t e r n o f such p a i r i n g s , i t i s
p ro b a b le t h a t th e y were exam ining t i s s u e w hich was th e r e s u l t o f grow th
o c c u r r in g p r i o r to th e m ee tin g o f th e two m y c e lia . F o rm ation o f clamps .
would n o t be e x p ec ted i n t h a t p o r t i o n o f th e ,fmonokaryonf! mycelium, even
i f d i k a r y o t i z a t i o n had ta k e n p la c e . In th e p r e s e n t s tu d y , hyphae from
n e a r th e m y c e l i a l m arg in was examined. These hyphae, formed fo r the . most
p a r t a f t e r th e m e e tin g o f th e two m y c e l ia , would be e x p e c te d to form clamps
had d i k a r y o t i z a t i o n o c c u r re d . Clamps w ere found, and i t now can be s a id
t h a t d i k a r y o t i z a t i o n o f monokaryons by d ik a ry o n s in F c a j a n d e r i n o t o n ly
can o c c u r , b u t seems to be th e g e n e r a l r u l e . The f a c t t h a t an a p p a re n t
a v e r s io n r e a c t i o n o c c u r re d i n most c a s e s i s n o t i r r e c o n b i l a b l e w i th th e
phenomenon o f d i k a r y o t i z a t i o n , s in c e , i n most c a s e s , two d ik a ry o n s s h a r in g
o n ly one c o m p a t i b i l i t y f a c t o r were p roduced and a v e r s io n would be e x p e c te d .
In th e fo u r c a s e s where an (A^g X A25) X A13 p a i r i n g was made, d i k a r y o t i
z a t i o n o c c u r r e d , thus p ro d u c in g two d ik a ry o n s w i th t h e same c o m p a t i b i l i t y
f a c t o r s on th e same p l a t e . However, d e f i n i t e a v e r s io n r e a c t i o n s were
n o te d i n a l l fo u r p a i r i n g s . I f a v e r s io n i s indeed an in d ex o f d i f f e r e n c e s
i n g e n e t i c make-up, th e n i t must be conc luded t h a t genes o th e r th an th o s e
a t th e lo c u s fo r h e t e r o t h a l l i s m a r e in v o lv e d . This i s n o t c o n t r a d i c t e d
39 .
by th e e v id en ce h e r e , s i n c e th e fo u r i s o l a t e s w h i l e hav ing a com
p a t i b i l i t y f a c t o r in common, came from e n t i r e l y d i f f e r e n t s o u r c e s , and
u n d o u b te d ly d i f f e r e d in many o th e r r e s p e c t s g e n e t i c a l l y .
SUMMARY
I n t h i s s tu d y some a s p e c t s o f th e s e x u a l i t y o f Fomes c a j a n d e r i
K a r s t , have been i n v e s t i g a t e d . The r e s u l t s o b ta in e d may p ro v id e th e
b a s i c in fo rm a t io n n e c e s s a r y to con d u c t f u r t h e r i n v e s t i g a t i o n s w i th t h i s
organ ism . The f in d in g s a r e ;
1) The m u l t i p l e - a l l e lo m o r p h s a t th e lo c u s f o r h e t e r o t h a l l i s m
to be randomly d i s t r i b u t e d i n th e N orth American p o p u la t io n
o f th e s p e c i e s , The number o f a l le lo m o rp h s i s e s t im a te d to
be 33,
2) Monokaryons can produce f r u i t i n g s t r u c u t r e s , b u t th e s e a r e
c o m p le te ly s t e r i l e ,
3) The in d u c t io n o f f r u i t i n g i n monokaryons, and p o s s i b ly d i -
k a ry o n s , o ccu rs i n a manner, t h a t i n d i c a t e s th e e x i s t e n c e o f
complex p h y s i o l o g i c a l and g e n e t i c mechanisms g o vern ing th e
sequence o f e v e n ts i n th e s e x u a l c y c l e . F u r th e rm o re , th e
use o f f r u i t i n g in d u c t io n as a means o f s tu d y in g th e s e mech
anism s s u g g e s ts i t s e l f as a f r u i t f u l app ro ach to th e problem ,
4) There a r e m acroscop ic c h a r a c t e r i s t i c s o f b o th co m p a tib le and
in c o m p a t ib le p a i r i n g s o f monokaryons t h a t g e n e r a l l y a l lo w
them to be d i s t i n g u i s h e d by v i s u a l i n s p e c t i o n ,
5) D i k a r y o t i z a t i o n o f monokaryons by d ik a ry o n s can occur in t h i s
s p e c i e s , a p p a r e n t ly w i th h ig h f re q u e n c y , th u s p ro v id in g an
a d d i t i o n a l p o s s i b l e mechanism f o r p ro d u c in g new recom binan t
ty p e s in th e f i e l d ,
, - 40 ..
l it e r a t u r e c it e d
Adams5 D„ and L» Roth (19 6 7 ) . D em arcation l i n e s i n p a i r e d c u l t u r e s o fFomes c a j a n d e r i as a b a s i s f o r d e t e c t i n g g e n e t i c a l l y d i s t i n c tm y c e l ia . Can* J o u r . B o t . 4 5 ( 9 ) , 1583-90.
B a r n e t t , H. L. and V. G. L i l l y (1947 ) . P ro d u c t io n o f h a p lo id and d i p l o i d f r u i t i n g b o d ie s o f L e n z i te s t r a b e a in c u l t u r e . Proc* W. Va. Acad. S c i . , 34 -39 .
Bateman, A. J . (1947 ) . The number o f s - a l l e l e s in a p o p u la t io n . Na-s. t u r e , London, 160, 337.
Bensaude, M. (1 917 ) . Sur la s e x u a l i t e chez l e s Champignons Basidiom y-c e t e s . Co R. Acad. S c i . , P a r i s , 165, 286-9 .
B o id in , J . and P. L an q u e tin (1965)» H e te rb b a s id o m y ce te s s a p ro p h y te s e t Homobasidiomycetes r e s u p in e s . X. N o v e lle s donnees su r la p o l a r i t e d i t e s e x u e l l e s . Revue de M ycologie , Tome X X X (l-2),3 - 16.
Brotzman, H. G. and R. L. G i lb e r t s o n (1 967 ) . Tyromyces undosus in North A m erica. M ycopatho log ia e t M ycologia A p p l ic a ta 33, 33-42 .
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