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The genus Leucosporidium in Southern British Columbia, an area of temperate climate 1981

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THE GENUS LEUCOSPORIDIUM IN SOUTHERN BRITISH COLUMBIA, AN AREA OF TEMPERATE CLIMATE by RICHARD CHARLES SUMMERBELL Bachelor of Science A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department, of Botany) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA 5 August 1981 c Richard C h a r l e s Summerbell, 1981 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of BOTANY The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 AUG. 31, 1981 Date i i ABSTRACT A s e a r c h f o r members of the genus Leucospor i d i u m ( U s t i l a g i n a c e a e ) i n and near s o u t h e r n B r i t i s h Columbia has y i e l d e d 147 i s o l a t e s of L. s c o t t i i , and a s i n g l e i s o l a t e of an u n d e s c r i b e d s p e c i e s w i t h apparent a f f i n i t i e s i n the genus. L. s c o t t i i was p r i m a r i l y found on d e c a y i n g marine v e g e t a t i o n and d r i f t w o o d , but i s o l a t e s were a l s o o b t a i n e d from stream foam, snow, a d e c a y i n g t u r n i p r o o t , bark mulch, and r a i n - d e r i v e d stem f l o w over the t r u n k of a l i v i n g t r e e . The s p e c i e s predominated i n l a b o r a t o r y i n c u b a t i o n s of marine a l g a l m a t e r i a l s c o l l e c t e d i n the w i n t e r , s p r i n g , and l a t e autumn. The m a j o r i t y of i s o l a t e s o b t a i n e d d i r e c t l y from n a t u r a l s u b s t r a t e s were a l s o found d u r i n g p e r i o d s of c o l d s e a s o n a l c o n d i t i o n s . I t i s suggested t h a t low temperature i s c h a r a c t e r i s t i c of L. s c o t t i i h a b i t a t s . L o c a l l y - o b t a i n e d L. s c o t t i i s t r a i n s are a l l h e t e r o t h a l l i c , and are c o m p l e t e l y i n t e r f e r t i l e w i t h mating t e s t s t r a i n s o r i g i n a t i n g from the s o u t h e r n hemisphere. M o r p h o l o g i c a l and known p h y s i o l o g i c a l and b i o c h e m i c a l c h a r a c t e r s of the l o c a l s t r a i n s are s i m i l a r t o those p r e v i o u s l y d e s c r i b e d f o r the s p e c i e s . However, mating s t u d i e s of l o c a l i s o l a t e s have r e v e a l e d t h a t the t e t r a p o l a r i n c o m p a t i b i l i t y system of L. s c o t t i i i s d i s t i n g u i s h e d by the p o s s e s s i o n of m u l t i p l e a l l e l e s a t the A l o c u s . S i m i l a r m o d i f i c a t i o n s of the t e t r a p o l a r system a r e .known i n T r e m e l l a and o t h e r h e t e r o b a s i d i o m y c e t e genera, but have not p r e v i o u s l y been r e p o r t e d i n s p e c i e s of the L e u c o s p o r i d i u m - R h o d o s p o r i d i u m group. The s t r a i n thought t o belong t o an u n d e s c r i b e d s p e c i e s of L e u c o s p o r i d i u m was o b t a i n e d from a sample of f i l a m e n t o u s green a l g a e growing i n a v e r n a l pond near a peat bog. The fungus i s h o m o t h a l l i c , and i s m o r p h o l o g i c a l l y and p h y s i o l o g i c a l l y d i s t i n c t from o t h e r L e u c o s p o r i d i u m and Rhodosporidium s p e c i e s . Of the s i x known s p e c i e s of L e u c o s p o r i d i u m , f i v e (L. a n t a r c t icum, L. f r i q idum, L. ge l i d u m , L. n i v a l i s , and L. s t o k e s i i ) were not found d u r i n g the c o u r s e of the p r e s e n t s t u d y . A l l f i v e s p e c i e s a re o b l i g a t e l y p s y c h r o p h i 1 i c and may not be a b l e to remain e s t a b l i s h e d w i t h i n the study a r e a . However, a survey of l o c a l L. s c o t t i i i s o l a t e s showed t h a t 5 out of 147 i s o l a t e s (3.4%) were o b l i g a t e l y p s y c h r o p h i 1 i c . i v TABLE OF CONTENTS ABSTRACT i i L I S T OF TABLES v i L I S T OF FIGURES v i i ACKNOWLEDGEMENTS v i i i I . I n t r o d u c t i o n 1 I I . M a t e r i a l s and Methods 14 C o l l e c t i o n of samples 14 E n r i c h m e n t i n c u b a t i o n s 18 S t r e a k i n g of samples 19 S e l e c t i o n o f y e a s t s f o r f u r t h e r t e s t i n g 20 S t o r a g e of y e a s t s t o c k c u l t u r e s 21 D i a z o n i u m b l u e B s t a i n i n g 21 B a s i d i o m y c e t o u s y e a s t c o l o n y m o r p h o l o g y 22 I n o s i t o l a s s i m i l a t i o n 23 C r o s s i n g 25 E x t r a c e l l u l a r s t a r c h t e s t 26 F e r m e n t a t i o n of g l u c o s e 27 D e t e c t i o n o f p s y c h r o p h i 1 i c s t r a i n s 27 I I I . R e s u l t s 29 D e s c r i p t i o n o f L e u c o s p o r i d i u m s t r a i n s examined 29 D i s t r i b u t i o n o f L. s c o t t i i w i t h i n s t u d y a r e a 31 P s y c h r o p h i l i c s t r a i n s 41 M a t i n g g e n e t i c s 41 IV. D i s c u s s i o n 45 V R e f e r e n c e s 63 v i LIST OF TABLES TABLE I : I s o l a t i o n of L. s c o t t i i from v a r i o u s environments i n and near s o u t h e r n B r i t i s h Columbia 35 TABLE I I : I s o l a t i o n of L. s c o t t i i from the marine environment i n and near s o u t h e r n B r i t i s h Columbia 36 TABLE I I I : I s o l a t i o n of L. s c o t t i i from f r e s h w a t e r h a b i t a t s i n s o u t h e r n B r i t i s h Columbia 38 TABLE IV: I s o l a t i o n of L. s c o t t i i from t e r r e s t r i a l h a b i t a t s i n s o u t h e r n B r i t i s h Columbia 39 TABLE V: I s o l a t i o n of L. s c o t t i i from samples c o l l e c t e d i n s o u t h e r n B r i t i s h Columbia 40 TABLE V I : M a t i n g of l o c a l i s o l a t e s of L. s c o t t i i 43 TABLE V I I : R e s u l t s of c r o s s i n g A.B, and A.B 2 s t r a i n s 44 L I S T OF FIGURES g u r e I : The l i f e c y c l e of L e u c o s p o r i d i u m s c o t t i i ACKNOWLEGEMENTS T h i s t h e s i s would not have been p o s s i b l e w i t h o u t the guidance, t i r e l e s s e f f o r t s , and support of my s u p e r v i s i n g p r o f e s s o r , Dr. R.J. Bandoni. I would l i k e t o extend my thanks t o him, as w e l l as to the o t h e r members of my graduate committee, Dr. G.C. Hughes, and Dr. G.H.N. Towers. Dr. J.W. F e l l k i n d l y p r o v i d e d c u l t u r e s of L. s c o t t i i f o r mating s t u d i e s . My thanks a l s o t o Dr. Bandoni, Andy Mackinnon, K e i t h S e i f e r t , and Tim Thompson f o r p r o v i d i n g s a m p l i n g m a t e r i a l s . Ross F r a s e r i s thanked f o r encouragement and f o r lon g hours spent w i t h " f l a k y " w o r d - p r o c e s s i n g s o f t w a r e . The a s s i s t a n c e of Dr. F. O b e r w i n k l e r i n sending r e f e r e n c e s i s much a p p r e c i a t e d . F i n a l l y , I w i s h t o acknowledge the f i n a n c i a l s u p port of the U n i v e r s i t y of B r i t i s h Columbia and the N a t u r a l S c i e n c e s and E n g i n e e r i n g Research C o u n c i l of Canada. 1 I . INTRODUCTION I t has l o n g been known t h a t a d i v e r s e group of f u n g i w i t h b a s i d i o m y c e t o u s a f f i n i t i e s go t h r o u g h a p o r t i o n of t h e i r l i f e h i s t o r i e s i n a y e a s t - l i k e budding phase. The f i r s t r e p o r t s of y e a s t - l i k e budding i n a b a s i d i o m y c e t e were those of Cornu and Roze (1875) and Cornu (1876). These a u t h o r s found t h a t the s p e r m a t i a of c e r t a i n r u s t s were c a p a b l e of " g e r m i n a t i n g " by budding i n the a l r e a d y well-known manner of Saccharomyces c e r e v i s i a e . P l o w r i g h t (1889) c o n f i r m e d the r e p o r t s and f i g u r e d the budding c e l l s . I n t e r e s t i n g l y , the budding c a p a b i l i t i e s of r u s t s p e r m a t i a a re l i t t l e - k n o w n t o d a y , d e s p i t e h a v i n g r e c e i v e d c o n s i d e r a b l e a t t e n t i o n i n e a r l y d i s c u s s i o n s about the proposed f u n c t i o n s of the c e l l s . These d i s c u s s i o n s were r e v i e w e d by C r a i g i e (1931) i n h i s c l a s s i c paper on r u s t s p e r m a t i z a t i o n . Soon a f t e r the d i s c o v e r y of budding i n r u s t s , B r e f e l d (1883) d i s c o v e r e d the same phenomenon i n members of s e v e r a l genera of U s t i l a g i n a c e a e , i n c l u d i n g Ust i l a g o . B r e f e l d c a r r i e d out thorough and comprehensive s t u d i e s of the l i f e h i s t o r i e s of these f u n g i , and r e p e a t e d l y demonstrated the a b i l i t y of the y e a s t - l i k e phase t o propagate i n d e f i n i t e l y on a r t i f i c i a l media. He a l s o demonstrated the remainder of the U s t i l a g i n a c e o u s l i f e - h i s t o r y : f u s i o n of y e a s t - l i k e c e l l s by means of c o n j u g a t i o n t u b e s , f o r m a t i o n i n host t i s s u e s of a p e n e t r a t i n g mycelium, p r o d u c t i o n of t e l i o s p o r e s by the c u l m i n a t i n g m y c e l i a l phase, g e r m i n a t i o n of the t e l i o s p o r e s t o produce e l o n g a t e , s e p t a t e " h e m i b a s i d i a " , or p r o m y c e l i a , and budding of the s p o r i d i a from the p r o m y c e l i a t o reproduce the budding phase. 2 B r e f e l d thought the above p r o c e s s a s e x u a l , and argued t h a t the y e a s t - l i k e s p o r i d i a c o u l d not be gametes because gametes were not known t o p e r p e t u a t e themselves a s e x u a l l y . DeBary (1887) argued o t h e r w i s e , and was e v e n t u a l l y v i n d i c a t e d by the c y t o l o g i c a l s t u d i e s of Dangeard (1892, 1894). These and r e l a t e d s t u d i e s showed t h a t budding c e l l s a re h a p l o i d , and fuse t o g i v e a mycelium t h a t i s b a s i c a l l y d i k a r y o t i c , a l t h o u g h t h e r e may be m u l t i n u c l e a t e c e l l s . Karyogamy t a k e s p l a c e i n the t e l i o s p o r e s as they form, and m e i o s i s o c c u r s d u r i n g the p r o c e s s of g e r m i n a t i o n . P r o d u c t s of m e i o s i s are s e q u e s t e r e d i n t o each of the f o u r c e l l s of the promycelium. These n u c l e i then d i v i d e i n the p r o d u c t i o n of s p o r i d i a . The genus T r e m e l l a , p r i m a r i l y known f o r i t s g e l a t i n o u s f r u i t i n g body w i t h c r u c i a t e l y s e p t a t e b a s i d i a , was the next fungus shown t o produce a y e a s t phase ( B r e f e l d , 1888). B r e f e l d showed t h a t the b a s i d i o s p o r e s of both T r e m e l l a and the now- synonymized (Ba n d o n i , 1961) genus N a e m a t e l i a gave r i s e t o a y e a s t phase which c o u l d be m a i n t a i n e d on a r t i f i c i a l media. Other members of the T r e m e l l a l e s have s i n c e been shown t o produce y e a s t - l i k e phases: M o l l e r (1895) demonstrated t h i s a b i l i t y f o r S i r o b a s i d i u m , w h i l e K o b a y a s i and Tubaki (1965) demonstrated i t f o r H o l t e r m a n n i a and Xenolachne. The r e c e n t l y - d e s c r i b e d genus F i b u l o b a s i d i u m Bandoni a l s o produces a y e a s t phase (Bandoni, 1979). S l o d k i et a l . (1966) s t u d i e d the e x t r a c e l l u l a r p o l y s a c c h a r i d e p r o d u c t i o n and carbon a s s i m i l a t i o n p a t t e r n s of T r e m e l l a y e a s t phases, and c o n c l u d e d t h a t the y e a s t s were s u r p r i s i n g l y s i m i l a r t o the common a s e x u a l y e a s t 3 s p e c i e s , C r y p t o c o c c u s l a u r e n t i i . Some members of the A u r i c u l a r i a l e s and the S e p t o b a s i d i a l e s a l s o produce a budding phase. Mycogloea c a r n o s a O l i v e produces c o p i o u s budding c e l l s when g e r m i n a t i n g b a s i d i a a re kept under water ( O l i v e , 1950). Donk (1972b) has observed t h a t the f i g u r e s i n which Couch (1938) d e p i c t e d b a s i d i o s p o r e g e r m i n a t i o n i n S e p t o b a s i d i u m g i v e e v i d e n c e of the i n i t i a t i o n of a budding phase. K o b a y a s i and Tubaki (1965) have c o n f i r m e d the e x i s t e n c e of a budding phase i n S e p t o b a s i d i u m as w e l l as i n Tj i b o d a s i a . M o l l e r ' s (1895) d e s c r i p t i o n of P l a t y g l o e a b l a s t o m y c e s M o l l , a l s o mentions the presence of budding c e l l s . K o b a y a s i (1952) and Tubaki and Kobayasi (1971) showed t h a t members of the anomalous f a m i l y G r a p h i o l a c e a e , a s m a l l group of s m u t - l i k e f u n g i , produce an extended budding phase i n c u l t u r e . As w i t h members of the U s t i l a g i n a c e a e , the budding stage of G r a p h i o l a s p e c i e s o r i g i n a t e s when h a p l o i d s p o r i d i a a re budded from a t e l i o s p o r e which has undergone m e i o s i s ( K i l l i a n , 1924). The G r a p h i o l a c e a e have been c l a s s e d i n the o r d e r U s t i l a g i n a l e s . F i n a l l y , the E x o b a s i d i a l e s , an o r d e r of h o l o b a s i d i a t e f u n g i , c o n t a i n s s p e c i e s which produce an i n d e f i n i t e l y - p r o p a g a t i n g , somewhat y e a s t - l i k e phase i n c u l t u r e . Branched c h a i n s of b l a s t i c " c o n i d i a " a re produced a f t e r b a s i d i o s p o r e s have been ge r m i n a t e d i n a r t i f i c i a l media ( G r a a f l a n d , 1953). These " c o n i d i a " c o n t i n u e t o reproduce u n t i l e n t i r e c o l o n i e s have been formed. Donk (1972a) compared t h i s p r o c e s s t o a budding phase. Nyland (1948, 1949) narrowed the gap between the 4 b a s i d i o m y c e t e s and the c o n v e n t i o n a l l y - d e f i n e d y e a s t s p e c i e s w i t h h i s p u b l i c a t i o n of the d e s c r i p t i o n of S p o r i d i o b o l u s j o h n s o n i i . The l i f e h i s t o r y of t h i s y e a s t was d e s c r i b e d i n d e t a i l by L a f f i n and C u t t e r (1959a, 1959b). S. j o h n s o n i i produces a d i p l o i d y e a s t phase which reproduces by budding or b a l l i s t o s p o r e p r o d u c t i o n . A d i k a r y o t i c m y c e l i a l phase i s s u b s e q u e n t l y formed. Chlamydospores are formed on the mycelium, and w i t h i n t h e s e s t r u c t u r e s karyogamy t a k e s p l a c e . The d i p l o i d phase i s r e g e n e r a t e d when the chlamydospore g e r m i n a t e s . M e i o s i s i n a d i p l o i d c e l l i n i t i a t e s the d i k a r y o p h a s e . I t was not u n t i l Banno's (1967) study of R h o d o t o r u l a q l u t i n i s t h a t a s p e c i e s o r i g i n a l l y d e s c r i b e d as an i m p e r f e c t y e a s t was shown t o have a b a s i d i o m y c e t o u s phase. Banno demonstrated t h a t the c a p s u l a t e d , c a r o t e n e - c o n t a i n i n g h a p l o i d y e a s t c e l l s c o n j u g a t e d t o produce a d i k a r y o t i c m y c e l i a l phase w i t h clamp c o n n e c t i o n s . T e r m i n a l and i n t e r c a l a r y t e l i o s p o r e s formed on the mycelium, and w i t h i n the t e l i o s p o r e s , karyogamy took p l a c e . G e r m i n a t i o n of the t e l i o s p o r e s by m e i o t i c d i v i s i o n gave r i s e t o a promycelium s i m i l a r t o those of members of the U s t i l a g i n a c e a e . T h i s promycelium became t r a n s v e r s e l y t h r e e - s e p t a t e , and h a p l o i d s p o r i d i a budded from each of the f o u r c e l l s . The d e s c r i p t i o n of the p e r f e c t genus Rhodosporidium was based on the s e r e s u l t s . F e l l et a l . (1970) suggested t h a t Rhodospor i d i u m i s a l l i e d t o the U s t i l a g i n a c e a e . F e l l et a l . (1969) found t h a t s i m i l a r l i f e - c y c l e s o c c u r r e d i n some members of the i m p e r f e c t y e a s t genus Candida , the members of which l a c k R h o d o t o r u l a - l i k e c a r o t e n e . The genus 5 L e u c o s p o r i d i u m was e r e c t e d t o c o n t a i n the p e r f e c t s t a t e s of s i x such s p e c i e s . A seventh s p e c i e s , the i m p e r f e c t s t a t e of which had been p l a c e d i n the T o r u l o p s i s grab-bag, was a l s o i n c l u d e d i n the genus. T h i s s p e c i e s , L. ca p s u l i g e n u m , was r e c o g n i z e d as an anomalous element i n the genus by R o d r i g u e s de Miranda (1972), and was t r a n s f e r r e d t o the genus F i l o b a s i d i u m (see below) . There are s e v e r a l o t h e r p u b l i s h e d r e p o r t s which a re s i g n i f i c a n t i n t h i s a r e a of knowledge. Among these a re t h r e e s t u d i e s (Bandoni et a l , 1971; Bandoni et a l . , 1975; F e l l and Ta l l m a n , 1980) which show the f o r m a t i o n of clamped mycelium and t e l i o s p o r e - l i k e s t r u c t u r e s i n matings of Sporobolomyces spp. R e c e n t l y , two s p e c i e s of Sporobolomyces, S. s a l m o n i c o l o r and S. s h i b a t a n u s have been shown t o have teleomorphs i n the genus S p o r i d i o b o l u s ( F e l l and T a l l m a n , 1981). Van der Walt (1970a) had e a r l i e r p u b l i s h e d the genus Aessosporon as a p u t a t i v e p e r f e c t s t a t e of S. s a l m o n i c o l o r , but t h i s genus was based on a m i s i n t e r p r e t a t i o n (see F e l l and T a l l m a n , 1980). S i m i l a r l y , S y r i n g o s p o r a , the proposed b a s i d i o m y c e t o u s stage of Candida a l b i c a n s (van der Walt, 1970b) has been d i s r e g a r d e d s i n c e i t was r e v e a l e d t h a t C. a l b i c a n s has ascomycetous a f f i n i t i e s (see Kreger v a n - R i j and V e e n h u i s , 1971; van der Walt and Hopsu-Havu, 1976). The f i n a l major group of d i m o r p h i c b a s i d i o m y c e t e s i s the F i l o b a s i d i a c e a e , a f a m i l y p l a c e by O l i v e (1968) i n the U s t i l a g i n a l e s , and by Cox (1976) i n the A p h y l l o p h o r a l e s . O l i v e (1968) f i r s t p u b l i c i z e d the e x i s t e n c e of the group by 6 d e s c r i b i n g the genus F i l o b a s i d i u r n , based on the f a c u l t a t i v e m y c o p a r a s i t e F. f l o r i f o r m e . T h i s s p e c i e s produces a h a p l o i d y e a s t phase-, a clamped d i k a r y o t i c m y c e l i a l phase, and a s e p t a t e a e r i a l b a s i d i a b e a r i n g s e s s i l e b a s i d i o s p o r e s a t t h e i r a p i c e s . L e u c o s p o r i d i u m capsuligenum was t r a n s f e r r e d t o F i l o b a s i d i u m a f t e r reassessment by R o d r i g u e s de Miranda (1972). The genus C h i o n o s p h a e r a , a fungus s i m i l a r t o F. f l o r i forme but l a c k i n g clamps and fo r m i n g synnematal b a s i d i o c a r p s , was d e s c r i b e d by Cox (1976). F i n a l l y , Kwon-Chung (1978) e r e c t e d the genus F i l o b a s i d i e l l a f o r the p e r f e c t s t a t e of the l o n g - n o t o r i o u s p a t h o g e n i c y e a s t C r y p t o c o c c u s neoformans. F i l o b a s i d i e l l a has a l i f e - c y c l e which resembles t h a t of F i l o b a s i d i u m . The v a s t m a j o r i t y of the d i m o r p h i c b a s i d i o m y c e t e s are poorly-known, and the l a r g e number of a s e x u a l y e a s t s which have been r e v e a l e d to have b a s i d i o m y c e t o u s a f f i n i t i e s (Nakase and Komagata, 1971a, 1971b; B a s t i d e e t a l . , 1975; van der Walt and Hopsu-Havu, 1976; von Arx and Weijman, 1978; H a g l e r and Ahearn, 1981; e t c . ) suggests t h a t many more e x i s t which are yet unknown. The e c o l o g y , g e n e t i c s , p h y s i o l o g y , and b i o c h e m i s t r y of a few p a r a s i t e s and pathogens have been e x t e n s i v e l y s t u d i e d , but l i t t l e i s known about a d i v e r s e c o l l e c t i o n of s a p r o b i c s p e c i e s . In the p r e s e n t s t u d y , I s e l e c t e d the genus L e u c o s p o r i d i u m f o r f u r t h e r e x a m i n a t i o n . The genus appears t o c o n s i s t e n t i r e l y of s a p r o b i c forms ( F e l l et a l . , 1969). The s i x s p e c i e s now r e c o g n i z e d i n L e u c o s p o r i d i u m a r e : L. a n t a r c t i c u m F e l l , S t a t z . , Hunt., & P h a f f , L. f r i g i d u m (DiMenna) F e l l , S t a t z . , Hunt., & P h a f f , L. gelidum (DiMenna) F e l l , 7 S t a t z . , Hunt., & P h a f f , L. n i v a l i s (DiMenna) F e l l , S t a t z . , Hunt., & P h a f f , L. s t o k e s i i F e l l , S t a t z . , Hunt., and P h a f f , and L. s c o t t i i ( D i d d . & Lodd.) F e l l , S t a t z . , Hunt., & P h a f f . These s p e c i e s d i f f e r i n many ways. F e l l e t a l . (1969) p r e s e n t e d a key which d i s t i n g u i s h e d between them on the b a s i s of n u t r i e n t a s s i m i l a t i o n c h a r a c t e r i s t i c s . However, the s p e c i e s a l s o d i f f e r i n the morphology of t h e i r c e l l s , mycelium, t e l i o s p o r e s , and p r o m y c e l i a , i n t h e i r s e x u a l c o m p a t i b i l i t y systems, and i n e x t r a c e l l u l a r s t a r c h p r o d u c t i o n and o t h e r b i o c h e m i c a l c h a r a c t e r s ( F e l l et a l . , 1969; F e l l , 1974b); i n t h e i r p h y s i o l o g i c a l a b i l i t y t o cope w i t h e n v i r o n m e n t a l heat ( F e l l et a l . , 1969); and i n t h e i r g e o g r a p h i c and e c o l o g i c a l d i s t r i b u t i o n (most r e c e n t r e v i e w : F e l l et a l . , 1969). In the p r e s e n t s t u d y , I e l e c t e d t o i n v e s t i g a t e two a s p e c t s of the b i o l o g y of L e u c o s p o r i d i u m spp. i n g r e a t e r d e t a i l : e c o l o g i c a l d i s t r i b u t i o n , and s e x u a l i n c o m p a t i b i l i t y system. A summation of p u b l i s h e d i n f o r m a t i o n on these s u b j e c t s i s g i v e n below. The e c o l o g i c a l d i s t r i b u t i o n of Leucospor i d i u m spp. must be un d e r s t o o d i n l i g h t of known p h y s i o l o g i c a l d i f f e r e n c e s among the s p e c i e s . A l l the s p e c i e s e x c e p t L. s c o t t i i a re o b l i g a t e p s y c h r o p h i l e s ; t h a t i s , they cannot grow above 19 C. ( F e l l e t a l . , 1969). L. s c o t t i i a l s o c o n t a i n s some o b l i g a t e l y p s y c h r o p h i 1 i c s t r a i n s . Not s u r p r i s i n g l y , the f i v e p s y c h r o p h i l i c s p e c i e s were a l l f i r s t i s o l a t e d from p o l a r l a t i t u d e s . Three of the f i v e p s y c h r o p h i l i c L e u c o s p o r i d i u m s p e c i e s had been d e s c r i b e d as i m p e r f e c t y e a s t s b e f o r e F e l l e t a l . (1969) 8 s e t up the genus. L. f r i q i d u m , L. g e l i d u m , and L. n i v a l i s were f i r s t d e s c r i b e d as Candida s p e c i e s by.DiMenna (1966a). DiMenna (1966b) i s o l a t e d s t r a i n s of a l l t h r e e s p e c i e s from A n t a r c t i c s o i l . She a l s o o b t a i n e d i s o l a t e s of C. g e l i d a from a s o i l sample from E a s t G r e e n l a n d , g i v i n g the y e a s t a known b i p o l a r d i s t r i b u t i o n . C. f r i g i d a has s u b s e q u e n t l y been i s o l a t e d o n l y once: Babyeva and A z i e v a (1980) r e p o r t o b t a i n i n g a s i n g l e s t r a i n from t u n d r a s o i l s i n West T a i m i r , U.S.S.R. The i s o l a t i o n of a s t r a i n of L. n i v a l i s was r e p o r t e d i n the same s t u d y . N e i t h e r s p e c i e s had p r e v i o u s l y been known from o u t s i d e the A n t a r c t i c . The presence of L. gelidum i n the n o r t h e r n hemisphere has been c o n f i r m e d by Babyeva et a l . (1976). However, the s t r a i n s o b t a i n e d i n t h a t study show some d i s s i m i l a r i t i e s w i t h the type s t r a i n . Golubev et a l . (1977) have r e p o r t e d a s i n g l e i s o l a t e from sap f l o w s of B e t u l a v e r r u c o s a (European b i r c h ) . F i n a l l y , Babyeva and A z i e v a (1980) have i s o l a t e d y e a s t s which they b e l i e v e t o be a s e x u a l v a r i a n t s of C. g e l i d a from a r c t i c t u n d r a s o i l s . L i t t l e i n f o r m a t i o n i s a v a i l a b l e on the d i s t r i b u t i o n s of the two L e u c o s p o r i d i u m s p e c i e s n e w l y - d e s c r i b e d i n F e l l et a l . ' s (1969) paper. L. a n t a r c t i c u m i s p r i m a r i l y known from pack i c e r e g i o n s near the n o r t h e a s t e r n end of the A n t a r c t i c p e n i n s u l a . ( F e l l e t a l . , 1969; F e l l , 1974a). However, Kockova- K r a t o c h v i l o v a et a l . (1972) have o b t a i n e d an i s o l a t e from the f r u i t of a w i l l o w , S a l i x a l b a , i n East Germany. The second s p e c i e s , L.• s t o k e s i i , was based on a s i n g l e s t r a i n i s o l a t e d by 9 S i n c l a i r and S tokes (1965) from A n t a r c t i c snow. No f u r t h e r i s o l a t e s have been r e p o r t e d . Compared t o the f i v e o b l i g a t e l y p s y c h r o p h i l i c s p e c i e s , L. s c o t t i i appears t o be much more w i d e l y d i s t r i b u t e d . Candida s c o t t i i , the i m p e r f e c t s t a t e d e s c r i b e d by Diddens and Lodder (1942), was based on i s o l a t e s o b t a i n e d by S c o t t (1936) i n A u s t r a l i a . S c o t t ' s i s o l a t e s were from r e f r i g e r a t e d beef and from s o i l near a meatworks. C l a r k et a l . (1954) i s o l a t e d a s i n g l e s t r a i n from the s u r f a c e of a Canadian a p p l e . Numerous i s o l a t e s were o b t a i n e d by DiMenna (1955, 1958, 1960a, 1960b, 1966b) from A n t a r c t i c , New Z e a l a n d , and E a s t G r e e n l a n d s o i l s , w h i l e K o c k o v a - K r a t o c h v i l o v a (1964) and K o c k o v a - K r a t o c h v i l o v a et a l . (1965) o b t a i n e d i s o l a t e s from p l a n t blossoms and mushroom f r u i t i n g b o d i e s , r e s p e c t i v e l y , i n C z e c h o s l o v a k i a . F e l l et a l . (1969) found the s p e c i e s t o be abundant i n the A n t a r c t i c seas (see a l s o F e l l , 1974a, 1976). The s p e c i e s was a g a i n found i n A n t a r c t i c s o i l s by Babyeva and Golubev (1969). A f t e r F e l l et a l . (1969) d e s c r i b e d the p e r f e c t s t a t e , L. s c o t t i i was found i n both temperate and a r c t i c r e g i o n s . Bruce and M o r r i s (1973) i s o l a t e d p s y c h r o p h i l i c s t r a i n s of C. s c o t t i i from l i v i n g marine f i s h i n waters near S c o t l a n d . Babyeva et a l . (1976) and Babyeva and A z i e v a (1980) r e p o r t e d f i n d i n g L. s c o t t i i i n A r c t i c t u n d r a s o i l s . A study of the y e a s t mycobiota of the F r e n c h r i v e r M o s e l l e by H i n z e l i n and L e c t a r d (1978) r e v e a l e d L. s c o t t i i as a minor component. S m a l l numbers of C. s c o t t i i i s o l a t e s have a l s o been found i n m o r e - o r - l e s s p o l l u t e d f l o w i n g f r e s h waters by W o o l l e t t and H e d r i c k (1970) and Simard 1 0 and Blackwood (1971). The former study was conducted on the Calumet R i v e r watershed i n I n d i a n a , w h i l e the l a t t e r i n v o l v e d s ampling the y e a s t mycobiota of the S t . Lawrence R i v e r i n Quebec, Canada. A L e u c o s p o r i d i u r n i s o l a t e o b t a i n e d from the s k i n of a h o s p i t a l p a t i e n t i n by Rose and Kurup (1978) may a l s o be L. s c o t t i i . There i s one r e p o r t of a L e u c o s p o r i d i u m s t r a i n not b e l o n g i n g t o any of the d e s c r i b e d t a x a . The s t r a i n was found i n t u n d r a s o i l by Babyeva and A z i e v a (1980). I t s h o u l d be noted t h a t numerous r e p o r t s i n the S o v i e t l i t e r a t u r e r e f e r r i n g t o C. s c o t t i i as a producer of i n d u s t r i a l s i n g l e - c e l l p r o t e i n are based on a m i s i d e n t i f i c a t i o n . The s t r a i n s i n q u e s t i o n have been i d e n t i f i e d as s t r a i n s of Candida m e s e n t e r i c a (Semushina et a l . , 1974). With r e g a r d t o mating g e n e t i c s , the genus L e u c o s p o r i d i u m i s d i v e r s e . A l l s p e c i e s have h o m o t h a l l i c ( s e l f - f e r t i l e ) s t r a i n s , and f o u r s p e c i e s are e x c l u s i v e l y h o m o t h a l l i c . These s p e c i e s a r e : L. f r i g i d u m , L. g e l i d u m , L. n i v a l i s , and L. s t o k e s i i ( F e l l et a l , 1969; F e l l 1974b). The observed h o m o t h a l l i s m i s i n a l l cases " p r i m a r y " ( t e r m i n o l o g y of Raper, 1966); t h a t i s , the organism remains monokaryotic throughout i t s l i f e h i s t o r y . L. a n t a r c t i c u m was b e l i e v e d by F e l l et a l . (1969) t o be e x c l u s i v e l y h o m o t h a l l i c , but was l a t e r shown t o have h e t e r o t h a l l i c s t r a i n s ( F e l l , 1974b). These s t r a i n s p o s s e s s a b i a l l e l i c , b i p o l a r mating system ( F e l l , 1974b) i n which the s e x u a l phase i s brought about by the c o n j u g a t i o n of two c o m p a t i b l e mating s t r a i n s , a and a l p h a . 11 H e t e r o t h a l l i c s t r a i n s of L. s c o t t i i p o s s e s s a t e t r a p o l a r mating system ( F e l l , 1974) i n which two genes, A an'd B, each e x i s t i n two a l l e l i c forms,A, and A 2, and B, and B 2. S t r a i n s are c o m p a t i b l e when they d i f f e r a t both l o c i ; t h a t i s , an A,B, s t r a i n w i l l o n l y mate w i t h an A 2 B 2 s t r a i n , w h i l e an A ^ s t r a i n w i l l o n l y mate w i t h an A 2B, s t r a i n . The l i f e c y c l e of L. s c o t t i i i s shown i n F i g u r e I . P u b l i s h e d work on mating systems of Leucospor i d i u m s p e c i e s has been the r e s u l t of study of s t r a i n s o b t a i n e d i n the s o u t h e r n hemisphere. The i n c o m p a t i b i l i t y g e n e t i c s of n o r t h e r n - hemisphere s t r a i n s have not been i n v e s t i g a t e d ; nor has t h e r e been an i n v e s t i g a t i o n of the r e l a t i o n s h i p of n o r t h e r n - and southern-hemisphere s t r a i n s . I t has been demonstrated t h a t some f u n g a l s p e c i e s p o s s e s s d i s t i n c t p o p u l a t i o n s of s t r a i n s d i s t i n g u i s h e d o n l y by mutual i n t e r s t e r i 1 i t y ( U l l r i c h , 1973; Anderson and U l l r i c h , 1979). Whether or not such groups of s t r a i n s occur i n Leucospor i d i u m spp. has not been examined. A l l t h a t i s known i s t h a t a l l southern-hemisphere s t r a i n s t e s t e d have proven t o be i n t e r f e r t i l e w i t h i n s p e c i e s , and not between s p e c i e s ( F e l l e t a l . , 1969). In o r d e r t o a c c o m p l i s h the aims of the p r e s e n t s t u d y , I pursued f o u r l i n e s of i n v e s t i g a t i o n . F i r s t l y , I undertook t o do a r e g i o n a l monographic study of Leucospor i d i u m spp. i n s o u t h w e s t e r n B r i t i s h Columbia and a d j a c e n t Washington. A wide v a r i e t y of s u b s t r a t e s and h a b i t a t s were sampled i n o r d e r t o p e r m i t c o l l e c t i o n of as d i v e r s e a range as p o s s i b l e of L e u c o s p o r i d i u m s t r a i n s . L a b o r a t o r y c u l t u r i n g t e c h n i q u e s were FIGURE 1: The l i f e cycle of Leucosporldium s c o t t i l ( a f t e r F e l l et a l . , 1969) HOMOTHALLISM HETEROTHALLISM te l i o s p o r e formation 1 3 d e s i g n e d t o permit d e t e c t i o n of new t a x a r e l a t e d t o Leucospor i d i u m spp. S e c o n d l y , i n c o n j u n c t i o n w i t h the r e g i o n a l monograph, an attempt was made t o determine the range of h a b i t a t s o c c u p i e d by Leucospor i d i u m spp. i n the study a r e a . I t was hoped t h a t by means of such a s t u d y , i n s i g h t would be gai n e d i n t o the r o l e s of Leucospor i d i u m spp. i n temperate h a b i t a t s i n g e n e r a l . T h i r d l y , i s o l a t e s o b t a i n e d from the study a r e a were screen e d f o r p s y c h r o p h i l y , i n or d e r t o determine the e c o l o g i c a l d i s t r i b u t i o n of p s y c h r o p h i l e s w i t h i n the a r e a . F i n a l l y , the mating systems of l o c a l l y - i s o l a t e d Leucospor idi u m s t r a i n s were examined, and compared w i t h those of s o u t h e r n - hemisphere s t r a i n s . A s e a r c h was made f o r i n t e r s t e r i l e groups of s t r a i n s . 1 4 II. MATERIALS AND METHODS C o l l e c t i o n of samples Samples of v a r i o u s m a t e r i a l s were c o l l e c t e d and t e s t e d f o r the p resence of L e u c o s p o r i d i u m spp. A l l m a t e r i a l s , w i t h the e x c e p t i o n s noted below, were c o l l e c t e d i n s t e r i l e p l a s t i c bags and r e t u r n e d t o the l a b o r a t o r y f o r f u r t h e r s t u d y . M a t e r i a l s t e s t e d a re l i s t e d below; they a r e grouped a c c o r d i n g t o the h a b i t a t s from which they o r i g i n a t e d . A) Marine environment 1. marine macroalgae i ) Fucus d i s t i c h u s L. i i ) U l v a l a c t u c a L. i i i ) M a c r o c y s t i s i n t e g r i f o l i a Bory. i v ) D e s m a r e s t i a i n t e r m e d i a P o s t . & Rupr. v) N e r e o c y s t i s l u e t k e a n a Mertens ( P o s t . & Rupr.) 2 . s e a g r a s s ( Z o s t e r a mar i n a L.) 3. c r a b p a r t s 4. d r i f t w o o d 5. m o i s t beach sand B) F r e s h w a t e r environment 1. stream water 2 . stream foam 3. submerged l o t i c mosses ( B r a c h y t h e c i u m sp.) 4. peat bog water 5. Sphagnum mosses from peat bog 6. d e c a y i n g peat bog v e g e t a t i o n 15 7. f r e s h w a t e r g r e e n a l g a e f r o m v e r n a l p o n d s ( U l o t h r i x , D r a p a r n a l d i a ) C) . T e r r e s t r i a l e n v i r o n m e n t 1. m i x e d b a r k m u l c h a n d s o i l 2. b a r k o f s t a n d i n g t r e e s ( P o p u l u s a n d A l n u s ) 3 . t e r r e s t r i a l m o s s e s 4. m a r s h v a s c u l a r p l a n t s ( J u n c u s a n d Sc i r p u s ) 5 . f r e s h m u s h r o o m s i ) C o l l y b i a s p . i i ) M e l a n o l e u c a m e l a l e u c a ( P e r s . ex F r . ) M u r r . i i i ) T u b a r i a f u r f u r a c e a ( P e r s . ex F r . ) G i l l e t . 6. d e c a y e d m u s h r o o m s 7. d e c a y e d t u r n i p ( B r a s s i c a r a p a L . ) 8 . w a x b e r r i e s ( S y m p h o r i c a r p o s a l b u s ( L . ) B l a k e ) 9 . s p o r e - d r o p ( T u l a s n e l l a s p . ) D) Snow 1. w i n t e r s n o w f a l l 2. a l p i n e s p r i n g a n d l a t e - s u m m e r s n o w p a c k s P r e c i s e d a t e s a n d l o c a t i o n s f o r c o l l e c t i o n s o f s a m p l e s a r e l i s t e d i n t h e ' R e s u l t s ' s e c t i o n o f t h i s r e p o r t . M o s t o f t h e m a r i n e m a t e r i a l s e x a m i n e d w e r e f r o m G e o r g i a S t r a i t , an a r m o f t h e n o r t h P a c i f i c n e a r V a n c o u v e r , B . C . T h e w a t e r s o f p a r t s o f t h e s t r a i t a r e b r a c k i s h i n n a t u r e , e s p e c i a l l y d u r i n g s p r i n g r u n o f f . Two o f t h e c o l l e c t i n g s i t e s f o r m a r i n e m a t e r i a l s , P t . G r e y a n d S q u a m i s h , B . C . , were n e a r e s t u a r i e s . A n o t h e r s i t e , O r c a s I s l a n d , W a s h i n g t o n , was a t a g r e a t e r d i s t a n c e f r o m s o u r c e s o f f r e s h w a t e r . T h e f i n a l two 1 6 m a r i n e s i t e s , P t . M c N e i l l a n d L o n g B e a c h , B . C . , w e r e on t h e o p e n P a c i f i c s e a c o a s t . M a r i n e m a c r o a l g a e were c o l l e c t e d b o t h a s a t t a c h e d a n d a s d e t a c h e d s p e c i m e n s , a l l o f w h i c h a p p e a r e d f r e s h when c o l l e c t e d . D e t a c h e d b l a d e s o f Z o s t e r a w e r e c o l l e c t e d f r o m d r i f t , b u t t h e s e b l a d e s h a d a l s o p r e s e r v e d a f r e s h a p p e a r a n c e . C r a b p a r t s l i k e w i s e l o o k e d a n d s m e l l e d f r e s h when c o l l e c t e d f r o m m o i s t b e a c h d r i f t . Two d r i f t w o o d s a m p l e s were c o l l e c t e d , one f r o m an e s t u a r i n e a r e a a n d a n o t h e r f r o m t h e o p e n c o a s t . T h e wood a p p e a r e d t o h a v e b e e n d e p o s i t e d on t h e b e a c h i m m e d i a t e l y b e f o r e i t was c o l l e c t e d , a n d i t s s u r f a c e was c o m p l e t e l y w a t e r - s o a k e d . One s a m p l e c o u l d n o t be c o l l e c t e d i n a s t e r i l e c o n t a i n e r , a n d a n o n - s t e r i l e g a r b a g e b a g was u s e d . S u b - s a m p l e s w e r e ' l a t e r t a k e n f o r f u r t h e r e x a m i n a t i o n f r o m a r e a s w h i c h h a d n o t come i n t o c o n t a c t w i t h t h e b a g . T h e f r e s h w a t e r m a t e r i a l s e x a m i n e d w e r e a l l f r o m t h e v i c i n i t y o f V a n c o u v e r , B . C . Foam was c o l l e c t e d f r o m s t r e a m s d u r i n g p e r i o d s o f r a i n f a l l a n d d u r i n g d r y p e r i o d s . On one o c c a s i o n , b o t h d r y a n d m o i s t foam w e r e c o l l e c t e d . O t h e r w i s e , t h e foam was m o i s t . S u b m e r g e d m o s s e s w e r e c o l l e c t e d f r o m a s t r e a m s w o l l e n w i t h r a i n . I t i s l i k e l y t h a t t h e a r e a i n w h i c h t h e m o s s e s w e r e g r o w i n g i s m a r s h y f o r m o s t o f t h e y e a r , b e c o m i n g p a r t o f t h e e x p a n d e d s t r e a m d u r i n g r a i n s a n d p o s s i b l y d r y i n g i n m i d s u m m e r . T h e v e r n a l p o n d f r o m w h i c h t h e s a m p l e o f U l o t h r i x s p . was c o l l e c t e d was on t h e m a r g i n o f a p e a t b o g . S a m p l e s o f t e r r e s t r i a l m a t e r i a l s a n d snow w e r e a l l c o l l e c t e d w i t h i n B . C . T h e b a r k c o m p o n e n t o f t h e m i x e d b a r k 1 7 mulch and s o i l sample was o u t e r bark of Pseudotsuga m e n z i e s i i ( M i r b e l ) F r a n c o . A water sample was o b t a i n e d from stem f l o w over the bark of two s t a n d i n g t r e e s as p a r t of an u n r e l a t e d study by Mackinnon ( u n p u b l i s h e d ) . R a i n f a l l was the source of the water. T e r r e s t r i a l mosses were sampled a f t e r they had been exposed t o a p r o l o n g e d p e r i o d of d a i l y r a i n s t o r m s . Dr. R . J . Bandoni s u p p l i e d a decayed sample of B o l e t e l l u s z e l l e r i (Murr.) S i n g . S n e l l & D i c k apud S n e l l , S i n g . & D i c k , and a y e a s t s t r e a k from the s u r f a c e of a t u r n i p which had been a l l o w e d t o decay i n a f i e l d near Vancouver, B.C. i n the w i n t e r . W a x b e r r i e s were p a r t l y decayed when c o l l e c t e d , but were s t i l l a t t a c h e d t o the s h r u b s . Yeast c o l o n i e s which grew among sp o r e s shed onto an agar p l a t e by a T u l a s n e l l a sp. f r u i t i n g body o b t a i n e d i n the w i l d were i n c o r p o r a t e d i n t o the s t u d y . A v a r i e t y of snow samples were c o l l e c t e d . W i n t e r snow was from a t r a n s i e n t s n o w f a l l which had been on the ground a p p r o x i m a t e l y ten days and was m e l t i n g when sampled. The a r e a from which the sample was taken was wooded w i t h deciduous t r e e s . S p r i n g a l p i n e snow was c o l l e c t e d from v a r i o u s e l e v a t i o n s on Mt. Seymour, near Vancouver. One sample was taken from d i s c o l o u r e d snow beneath a Tsuga m e r t e n s i a n a (Bong.) C a r r . (mountain hemlock) t r e e . S e v e r a l samples of " r e d snow", snow mixed w i t h r e d e n c y s t e d c e l l s of the f r e s h w a t e r C h l o r o p h y t e a l g a Chlamydomonas n i v a l i s (Bauer) W i l l e . , were examined. L a t e - summer snow was c o l l e c t e d from the Wavy Range, W e l l s - G r a y P r o v i n c i a l P a rk, B.C., a p p r o x i m a t e l y 600 km. from Vancouver. Both normal snow and " r e d snow" were c o l l e c t e d . 18 Winter and s p r i n g snow samples were processed, i n the l a b b e f o r e the snow had melted. The late-summer snow samples were m a i n t a i n e d a t a temperature below 15 °C. by means of i c e packs. T r a n s p o r t and p r o c e s s i n g of t h e s e samples were a c c o m p l i s h e d w i t h i n 72 h o u r s . Enr ichment i n c u b a t i o n s Bandoni ( u n p u b l i s h e d ) succeeded s e v e r a l y e a r s ago i n i s o l a t i n g Leucospor idium s c o t t i i from a Fucus d i s t i c h u s t h a l l u s b e i n g i n c u b a t e d i n the l a b o r a t o r y i n f r e s h water. In the p r e s e n t s t u d y , marine a l g a l samples were s u b j e c t e d t o s i m i l a r enrichment i n c u b a t i o n s . T h i s t e c h n i q u e was c a r r i e d out i n c o m b i n a t i o n w i t h d i r e c t s a m p l i n g of f r e s h l y - c o l l e c t e d a l g a l m a t e r i a l s f o r y e a s t s . In the e n r i c h m e n t s , a l g a l m a t e r i a l was submerged i n d i s t i l l e d water and a l l o w e d t o i n c u b a t e 3-40 days ( u s u a l l y 10-15) i n the l a b . In one c a s e , a sample of F. d i s t i c h u s was i n c u b a t e d i n f i l t e r - s t e r i l i z e d seawater. The r e s u l t s of t h i s i n c u b a t i o n were compared w i t h t h o s e of f r e s h - water i n c u b a t i o n s . F r e s h w a t e r a l g a e and bog v e g e t a t i o n were a l s o a l l o w e d t o i n c u b a t e i n f r e s h water b e f o r e samping f o r y e a s t s took p l a c e . As w i t h the marine a l g a e , however, some samples were p r o c e s s e d d i r e c t l y w i t h o u t i n c u b a t i o n . I n c u b a t i o n t e m p e r a t u r e s were 5°C. throughout most of the e x p e r i m e n t . D u r i n g the summer months of J u l y and August, however, samples were i n c u b a t e d a t 10°C. The l i g h t regime c o n s i s t e d of 12 h. l i g h t , 12 h. d a r k . 1 9 S t r e a k i n g of samples Two k i n d s of media were used f o r s t r e a k i n g from sample m a t e r i a l s : 1) S o r b o s e - y e a s t e x t r a c t medium, c o n t a i n i n g 4 g /1 L-sorbose 0.5 g /1 y e a s t e x t r a c t 8 g /1 ICN agar 0.1 g /1 t e t r a c y c l i n e 2) Cjm ( T r e m e l l a c o n j u g a t i o n medium), c o n t a i n i n g 2 g /1 d e x t r o s e 1 g /1 D i f c o soytone 0.5 g /1 y e a s t e x t r a c t 8 g /1 ICN agar 0.05 g /1 t e t r a c y c l i n e The former medium i s a m o d i f i c a t i o n of the s o r b o s e - c e l l u l o s e medium of Bandoni and B a r r (1976). I t s sorbose component i n d u c e s l i m i t e d , c o l o n i a l growth i n c e r t a i n Zygomycete and Hyphomycete s p e c i e s which t y p i c a l l y have a d i f f u s e , s p r e a d i n g growth form on a r t i f i c i a l media (Tatum et a l . , 1949). The l a t t e r medium i s a m o d i f i c a t i o n of t h r e e Cjm media used by Bandoni et a l . (1975). T e t r a c y c l i n e was added t o both media i n o r d e r t o reduce the growth of b a c t e r i a on i s o l a t i o n p l a t e s . S t r e a k s were made d i r e c t l y from sample m a t e r i a l s w i t h a w i r e l o o p , or by s t r e a k i n g m a t e r i a l s themselves on the agar s u r f a c e . In i n c u b a t i o n d i s h e s i n which a y e a s t scum had formed 20 on t h e w a t e r s u r f a c e , s t r e a k s w e r e made f r o m t h e scum a s w e l l a s f r o m t h e i n c u b a t e d m a t e r i a l . S a m p l e s c o n t a i n i n g s o i l were d i l u t e d i n s t e r i l e w a t e r , a n d s t r e a k s w e r e made f r o m t h e s u s p e n s i o n s . I n o r d e r t o i n c r e a s e t h e d i v e r s i t y o f y e a s t i s o l a t e s o b t a i n e d f r o m e a c h s a m p l e , t h e two i s o l a t i o n m e d i a were u s e d i n c o m b i n a t i o n . I s o l a t i o n p l a t e s w e r e i n c u b a t e d a t 5 ° C . , e x c e p t d u r i n g J u l y a n d A u g u s t , when t h e y w e r e k e p t a t 10°C. L i g h t r e g i m e was a s a b o v e . S e l e c t i o n o f y e a s t s f o r f u r t h e r t e s t i n g When y e a s t s h a d f o r m e d e a s i l y - d i s t i n g u i s h a b l e c o l o n i e s on i s o l a t i o n p l a t e s , a s t e r i l e l o o p was u s e d t o t r a n s f e r c e r t a i n o f t h e s t r a i n s t o new m e d i a f o r f u r t h e r s t u d y . S t r a i n s were c h o s e n p r i m a r i l y on t h e b a s i s o f a p p e a r i n g t o be p o t e n t i a l L e u c o s p o r i d i u r n s t r a i n s . A s t h e s t u d y p r o g r e s s e d , I became b e t t e r a b l e t o r e c o g n i z e L e u c o s p o r i d i u m s t r a i n s i n c u l t u r e . H o w e v e r , i n o r d e r t o c o m p e n s a t e f o r . p r e v i o u s l y - u n o b s e r v e d m o r p h o l o g i c a l v a r i a b i l i t y a n d t h e p o t e n t i a l o f new t a x a , w h i t e - c o l o u r e d y e a s t s t r a i n s w e r e w i d e l y s a m p l e d . I s o l a t i o n p l a t e s f r o m w h i c h t e s t s t r a i n s h a d b e e n s e l e c t e d w e r e r e t a i n e d f o r e x a m i n a t i o n a m o n t h o r m o r e l a t e r , when t h o s e y e a s t s r e m a i n i n g i n d i s t i n c t c o l o n i e s s h o w e d f u l l d e v e l o p m e n t o f c o l o n y m o r p h o l o g y a n d p i g m e n t a t i o n . A n y i n t e r e s t i n g c o l o n i e s w h i c h h a d n o t p r e v i o u s l y b e e n s e l e c t e d w e r e t r a n s f e r r e d f o r f u r t h e r s t u d y . T h e g e n e r a l - p u r p o s e m e d i u m u s e d f o r t h e t r a n s f e r s was MYPT 21 medium (Bandoni et a l . , 1975). MYPT medium 7 g /1 malt e x t r a c t 1 g /1 D i f c o soytone 0.5 g /1 y e a s t e x t r a c t 6 g /1 ICN agar 0.05 g /1 t e t r a c y c l i n e T r a n s f e r p l a t e s were i n c u b a t e d a t 10°C. Y e a s t s which d e v e l o p e d i n t e n s e c a r o t e n e p i g m e n t a t i o n or dark mycelium were not d e a l t w i t h f u r t h e r . C o l o n i e s v i s i b l y made up of more than one y e a s t type were suspended i n s t e r i l e d i s t i l l e d water and r e s t r e a k e d f o r r e - i s o l a t i o n . S t o r a g e of y e a s t s t o c k c u l t u r e s Y e a s t s on t r a n s f e r p l a t e s were s u b c u l t u r e d on MYP ( t e t r a c y c l i n e - f r e e ) tubes i n o r d e r t o pe r m i t r e t e n t i o n over a l o n g p e r i o d of tim e . Tubes were s t o r e d at 10°C. Diazonium b l u e B s t a i n i n g The d i a z o n i u m b l u e B s t a i n i n g t e c h n i q u e of Hopsu-Havu e t a l . (1967) was used t o d i f f e r e n t i a t e between ascomycetous and b a s i d i o m y c e t o u s y e a s t s . Y e a s t s w i t h b a s i d i o m y c e t o u s a f f i n i t i e s s t a i n r e d t o p u r p l e - r e d w i t h the r e a g e n t , w h i l e ascomycetous y e a s t s do not s t a i n or s t a i n weakly y e l l o w (van der Walt and Hopsu-Havu, 1976). For t e s t i n g , 1 mg/ml s t a b i l i z e d d i a z o n i u m 22 b l u e B s a l t was added t o a s t o c k s o l u t i o n c o n s i s t i n g of 15.8 g/1 Trizma HC1 b u f f e r (pH 7.0) which had been c h i l l e d t o 4°C. The s t a i n i n g s o l u t i o n was used i m m e d i a t e l y , b e f o r e s e l f - c o u p l i n g of the d i a z o n i u m s a l t c o u l d take p l a c e . An eyedropper was used t o p l a c e d r o p l e t s of s o l u t i o n i n t o y e a s t c o l o n i e s on two-week-old t r a n s f e r p l a t e s . Y e a s t s s t a i n i n g a r e d d i s h c o l o u r were presumed to be b a s i d i o m y c e t o u s . S t r a i n s showing ascomycetous c h a r a c t e r i s t i c s were r e c o r d e d , and these s t r a i n s were not s u b j e c t e d t o f u r t h e r t e s t i n g i n the p r e s e n t s t u d y . Yeast c o l o n i e s i n which s t a i n i n g was ambiguous were t r e a t e d as i f they were b a s i d i o m y c e t o u s . B a s i d i o m y c e t o u s y e a s t c o l o n y morphology Yeast s t r a i n s r e v e a l e d as b a s i d i o m y c e t o u s by d i a z o n i u m b l u e B s t a i n i n g were s t r e a k e d onto V-8 medium (Wickerham et a l . , 1946) f o r development of c o l o n y morphology and h o m o t h a l l i c s e x u a l s t a g e s . The V-8 medium was c o n s t i t u t e d as f o l l o w s : V-8 Medium 200 ml. V-8 v e g e t a b l e j u i c e (commercial p r o d u c t ) 3.0 g. CaC0 3 11 g. ICN agar 800 ml. d i s t i l l e d water P l a t e s were i n c u b a t e d a t 15°C. They were i n v e r t e d i n o r d e r t o p e r m i t d e t e c t i o n of b a l l i s t o s p o r e d i s c h a r g e . A f t e r 30 days of i n c u b a t i o n , c o l o n i e s were examined m i c r o s c o p i c a l l y f o r the 23 presence of mycelium, clamp c o n n e c t i o n s , and s e x u a l s t r u c t u r e s . A s e x u a l s t r a i n s which had c o l o n i a l or m i c r o s c o p i c c h a r a c t e r s markedly d i s s i m i l a r from those of Leucospor i d i u m spp. were not s u b j e c t e d t o f u r t h e r s t u d y . Those s u g g e s t i v e of Leucospor i d i u m spp. were r e t a i n e d f o r p h y s i o l o g i c a l and g e n e t i c t e s t i n g . C e l l s from s e x u a l l y - r e p r o d u c i n g c o l o n i e s were suspended i n s t e r i l e d i s t i l l e d water and r e s t r e a k e d t o s e p a r a t e p o s s i b l e h e t e r o t h a l l i c mating s t r a i n s . S i n g l e - c e l l i s o l a t e s of r e s t r e a k e d s t r a i n s were t r a n s f e r r e d t o V-8 medium t o determine whether or not they would reproduce the s e x u a l c y c l e . I n o s i t o l a s s i m i l a t i o n The a b i l i t y t o a s s i m i l a t e m y o - i n o s i t o l i s c h a r a c t e r i s t i c of members of the genus C r y p t o c o c c u s , as w e l l as of some Candida s p e c i e s of b a s i d i o m y c e t o u s a f f i n i t i e s , and of L e u c o s p o r i d i u m f r i g i d u m , L. g e l i d u m , L . n i v a l i s , and L. s t o k e s i i . (see Lodder, 1970). I used t h i s t e s t p r i m a r i l y t o q u i c k l y s o r t C r y p t o c o c c u s spp. s t r a i n s from L. s c o t t i i s t r a i n s . The medium used f o r the d e t e c t i o n of i n o s i t o l a s s i m i l a t i o n was t h a t of P a l i w a l et a l . (1979). 24 R a p i d t e s t f o r i n o s i t o l a s s i m i l a t i o n (pH 7.0) 10 g /1 i n o s i t o l 0.67 g /1 D i f c o y e a s t n i t r o g e n base 20 mg/1 b r o m o c r e s o l p u r p l e dye "~"\ F i f t e e n - m l . a l i q u o t s of t h i s medium were poured i n t o 12 5— ml. erlenmeyer f l a s k s . The opening of each f l a s k was s e a l e d w i t h a d o u b l e - t h i c k n e s s of paper t o w e l ; t h i s c o v e r i n g was made secure w i t h an e n c i r c l i n g e l a s t i c band. S u b s e q u e n t l y , the f l a s k s were a u t o c l a v e d at 15 p s i f o r 15 min. S t e r i l i z e d f l a s k s were i n o c u l a t e d w i t h y e a s t c o l o n i e s and i n c u b a t e d a t 25°C. on a New Brunswick Model G-2 L a b o r a t o r y R o t a t o r s e t a t 100 rpm. The h i g h i n c u b a t i o n temperature was chosen so t h a t s t r a i n s of the i n o s i t o l - a s s i m i l a t i n g L e u c o s p o r i d i u m s p e c i e s , a l l of which a r e o b l i g a t e l y p s y c h r o p h i 1 i c , would not be c o n f u s e d w i t h c r y p t o c o c c i and i n o s i t o l - a s s i m i l a t i n g Candida spp. A change i n c o l o u r of the i n d i c a t o r from p u r p l e t o y e l l o w was h e l d t o i n d i c a t e i n o s i t o l a s s i m i l a t i o n . In g e n e r a l , i n o s i t o l - a s s i m i l a t i n g s t r a i n s were not s u b j e c t e d t o f u r t h e r s t u d y . However, those s t r a i n s whose m o r p h o l o g i c a l f e a t u r e s were s t r o n g l y s u g g e s t i v e of Leucospor i d i u m spp. were t r e a t e d i n the manner of o t h e r p o t e n t i a l L e u c o s p o r i d i u m s t r a i n s . T h i s p o l i c y was n e c e s s a r y because of t h r e e p o s s i b i l i t i e s : 1) mutant i n o s i t o l - a s s i m i l a t i n g s t r a i n s of L. s c o t t i i ; 2) mutant m e s o p h i l i c s t r a i n s of L. f r i g i d u m , L. g e l i d u m , L. n i v a l i s , and L. s t o k e s i i ; and 3) p r e v i o u s l y - u n d e s c r i b e d m e s o p h i l i c i n o s i t o l - a s s i m i l a t i n g s p e c i e s 25 of L e u c o s p o r i d i u m . C r o s s i n g Y e a s t s r e v e a l e d by the above t e c h n i q u e s as p o t e n t i a l L e u c o s p o r i d i u m s t r a i n s were c r o s s e d w i t h L. s c o t t i i mating t y p e s o b t a i n e d from Dr. J . F e l l . C r o s s i n g was done on V-8 medium (see above). The p o t a t o - d e x t r o s e - c h a r c o a l medium of B u t l e r and B o l k a n (1973) was a l s o t e s t e d as a mating medium, but proved t o be u n s a t i s f a c t o r y . M a t i n g type s t r a i n s were s p o t t e d on p l a t e s , and each spot r e c e i v e d an admixture of c e l l s of the t e s t s t r a i n . An a d d i t i o n a l spot of the t e s t s t r a i n was p l a c e d on the same p l a t e t o s e r v e as a c o n t r o l . These p l a t e s were i n c u b a t e d f o r up t o 5 weeks a t 15°C. , and then were examined under the d i s s e c t i n g and compound m i c r o s c o p e s f o r the presence of clamped mycelium and t e l i o s p o r e s . C u l t u r e s which f a i l e d t o mate w i t h the L. s c o t t i i mating t y p e s were grouped a c c o r d i n g t o t h e i r m o r p h o l o g i c a l c h a r a c t e r i s t i c s and c r o s s e d amongst t h e m s e l v e s . The same media and t e c h n i q u e s were used. The purposes of these c r o s s e s were: 1) t o d e t e c t groups of L. s c o t t i i s t r a i n s i n t e r s t e r i l e w i t h Dr. F e l l ' s s t r a i n s , which have southern-hemisphere provenances; and 2 ) t o d e t e c t o t h e r h e t e r o t h a l l i c s p e c i e s i n L e u c o s p o r i d i u m and a l l i e d genera. C e r t a i n s t r a i n s which had c u l t u r a l and m o r p h o l o g i c a l c h a r a c t e r i s t i c s r e m i n i s c e n t of L. a n t a r c t i c u m were c r o s s e d w i t h mating t y p e s of t h a t s p e c i e s o b t a i n e d from Dr. J . F e l l . C r o s s e s were made on V-8 medium s t o r e d a t 10°C. S i x weeks were a l l o w e d 26 f o r m a t ing. E x t r a c e l l u l a r s t a r c h t e s t S t r a i n s of wh i t e y e a s t which produced t e l i o s p o r e s s p o n t a n e o u s l y were t e s t e d f o r e x t r a c e l l u l a r p r o d u c t i o n of s t a r c h - l i k e compounds. As F e l l (1974b) has p o i n t e d o u t , t h i s c h a r a c t e r i s t y p i c a l of C r y p t o c o c c u s s p e c i e s , F i l o b a s i d i u m s p e c i e s and the f o u r i n o s i t o l - a s s i m i l a t i n g s p e c i e s (see above) of L e u c o s p o r i d i u m . The f o l l o w i n g medium (Lodder and Kreger-van R i j , 1952) was used t o d e t e c t s t a r c h p r o d u c t i o n : Medium f o r d e t e c t i o n of e x t r a c e l l u l a r s t a r c h p r o d u c t i o n 1 g/1 ammonium s u l f a t e 1 g/1 p o t a s s i u m d i h y d r o g e n phosphate 0.5 g/1 magnesium s u l f a t e h e p t a h y d r a t e 0.5 g/1 y e a s t e x t r a c t 10 g/1 g l u c o s e 8 g/1 ICN agar Y e a s t s were i n o c u l a t e d and grown f o r one week a t 15°C. P r o d u c t i o n of s t a r c h - l i k e compounds was then d e t e c t e d w i t h i o d i n e - p o t a s s i u m i o d i d e s o l u t i o n . The t e c h n i q u e was t e s t e d w i t h a s t r a i n of Rhodosporidium ? c a p i t a t u m i n o r d e r t o ensure t h a t s t a r c h - p o s i t i v e s p e c i e s would produce the e x p e c t e d b l u e - v i o l e t c o l o u r w i t h I K I . 27 Fermentat ion of glucose F u r t h e r c h a r a c t e r i z a t i o n of t e l i o s p o r e - p r o d u c i n g s t r a i n s was c a r r i e d out by t e s t i n g f o r glucose fermentation. Neither L. s c o t t i i nor L. a n t a r c t icum can ferment glucose; the remainder of the Leucospor idium species are weak fermenters ( F e l l et a l . , 1969) . The t e s t was c a r r i e d out by i n o c u l a t i n g t e s t s t r a i n s i n t o standard Durham tubes c o n t a i n i n g the f o l l o w i n g medium: Glucose medium 18 g/1 dextrose 1 g/1 D i f c o soytone 0.5 g/1 yeast e x t r a c t Tubes were i n o c u l a t e d at 15°C. for 2 days with screw-caps s l i g h t l y open. The caps were then t i g h t e n e d shut and i n c u b a t i o n was continued f o r a f u r t h e r 15 days. The medium and technique were m o d i f i c a t i o n s of those of Santa Maria (1972). Formation of a gas bubble i n the i n s e r t was h e l d to i n d i c a t e fermentative act i v i t y . D e t e c t i o n of p s y c h r o p h i 1 i c s t r a i n s A l l confirmed Leucosporidiurn s t r a i n s and a number of suspected s t r a i n s were t e s t e d f o r a b i l i t y to grow at 25°C. The maximum temperature f o r growth of o b l i g a t e l y p s y c h r o p h i 1 i c Leucosporidium s p e c i e s i s g e n e r a l l y 17°C. ( F e l l and Phaff, 1970) . Test s t r a i n s were i n o c u l a t e d onto MYPT medium (see above) and kept at 25°C. f o r two weeks. Two known p s y c h r o p h i l i c 28 s t r a i n s , t h e two m a t i n g t y p e s t r a i n s of L. a n t a r c t i c u m , were t e s t e d i n t h e same manner. At t h e end of t h e week, c o l o n i e s showing n e g l i g i b l e or weak growth were n o t e d . 29 I I I . RESULTS D e s c r i p t i o n of L e u c o s p o r i d i u m s t r a i n s examined Of the 629 y e a s t s t r a i n s i s o l a t e d d u r i n g the c o u r s e of t h i s s t u d y , a t o t a l of 147 s t r a i n s were i d e n t i f i e d as L e u c o s p o r i d i u m s c o t t i i . A l l L. s c o t t i i s t r a i n s were i n t e r f e r t i l e w i t h the southern-hemisphere mating s t r a i n s used as t e s t e r s . In c r o s s e s between l o c a l i s o l a t e s and t e s t s t r a i n s , mating proceeded i n the u s u a l manner: clamped mycelium formed around and beneath the c o l o n i e s , and t e r m i n a l and i n t e r c a l a r y t e l i o s p o r e s were produced on t h a t mycelium 3-4 weeks a f t e r c r o s s i n g . The t e l i o s p o r e s were h y a l i n e and g r a n u l a r i n appearance under the m i c r o s c o p e , and were u s u a l l y 8-10 »im. i n s i z e . On a macroscopic s c a l e , they o f t e n formed h i g h l y - v i s i b l e golden-brown masses around c o l o n i e s where mating had taken p l a c e . O c c a s i o n a l l y , they were not produced i n the a r e a around the mixed y e a s t c o l o n y , but were i n s t e a d produced on i t s s u r f a c e , g i v i n g a m o t t l e d appearance. Where g e r m i n a t i o n was attempted w i t h t e l i o s p o r e s o b t a i n e d from matings of t e s t s t r a i n s and l o c a l i s o l a t e s , a p p a r e n t l y - n o r m a l p r o m y c e l i a were formed. L o c a l l y - i s o l a t e d L. s c o t t i i s t r a i n s d i d not a s s i m i l a t e i n o s i t o l , produce e x t r a c e l l u l a r s t a r c h - l i k e m a t e r i a l , or ferment g l u c o s e . In these r e s p e c t s , the s t r a i n s were s i m i l a r t o southern-hemisphere s t r a i n s (see F e l l et a l . , 1969). No comprehensive t e s t s of c a r b o n - or n i t r o g e n - a s s i m i l a t i o n p a t t e r n s were done. 30 Two d i s t i n c t groups of yeasts with s u p e r f i c i a l s i m i l a r i t y to L. s c o t t i i f a i l e d to c r o s s with t e s t s t r a i n s of that s p e c i e s . S t r a i n s of each of the two groups were i n t e r c r o s s e d , but with negative r e s u l t s . One group was l a t e r r e v e a l e d to c o n t a i n s t r a i n s of a Candida species of ascomycetous a f f i n i t i e s . T h i s group of s t r a i n s appeared to s t a i n red in diazonium blue B, but c l o s e r examination r e v e a l e d that the red c o l o u r was weak, and was d e r i v e d from c o u p l i n g of the dye with components in the medium beneath the c o l o n i e s . The c e l l s themselves were not s t a i n e d . One member of t h i s group of s t r a i n s was t e s t e d f o r fermentative a b i l i t y at 25°C. and gave a weak p o s i t i v e r e s u l t . The second group contained i n o s i t o l - a s s i m i l a t i n g s t r a i n s belonging to the genus Cryptococcus. A l s o , a t o t a l of 44 l o c a l l y obtained i s o l a t e s of v a r i o u s d e s c r i p t i o n s were c r o s s e d with mating t e s t s t r a i n s of L. a n t a r c t i c u m . A l l r e s u l t s were n e g a t i v e . F i v e l o c a l i s o l a t e s produced t e l i o s p o r e s spontaneously. Three were l a t e r r e v e a l e d to be mixed c u l t u r e s c o n t a i n i n g two compatible L. s c o t t i i mating types. A f o u r t h was contaminated with Rhodosporidium ?capitatum. The f i f t h s t r a i n , RCS-Y478, appeared to represent a h o m o t h a l l i c species of Leucosporidium, but the s t r a i n d i d not resemble those of any of the p r e v i o u s l y - d e s c r i b e d s p e c i e s . I s o l a t e RCS-Y478 was obtained from a d i r e c t streak of a sample of the freshwater green a l g a , U l o t h r i x sp. The a l g a l sample had been c o l l e c t e d Jan. 8, 1981 i n a v e r n a l pond near the margin of Camosun Bog, U n i v e r s i t y of B.C. Endowment Lands, 31 Vancouver. On V-8 medium, the s t r a i n produces o f f - w h i t e , creamy, domed c o l o n i e s surrounded by a prominent f r i n g e of a e r i a l mycelium, and a band of mycelium d e v e l o p i n g near the agar s u r f a c e . The mycelium l a c k s clamp c o n n e c t i o n s . Yeast c e l l s a r e budded from the mycelium i n w h o r l s around the s e p t a , or i n r o s e t t e s at the a p i c e s of s h o r t l a t e r a l branches. T e l i o s p o r e s are produced t e r m i n a l l y on s h o r t s i d e - b r a n c h e s of the mycelium. They are s p h e r i c a l to o b p y r i f o r m , s i n g l e or uncommonly d o u b l e , g r a n u l a r , and h y a l i n e . T h e i r s i z e i s u n u s u a l : 7-20 X 7-13 /iin. , as opposed t o 7-16 »»m. d i a m e t e r f o r L. s c o t t i i t e l i o s p o r e s (commonly 8-10 »»m. ) , and 5-7.5 X 5.5-11 tsm. f o r L. f r igidum t e l i o s p o r e s ( F e l l et a l . , 1969). The s t r a i n grows a t 2 5 ° C , ferments g l u c o s e , and f a i l s both t o a s s i m i l a t e i n o s i t o l and t o procduce e x t r a c e l l u l a r s t a r c h . T h i s c o m b i n a t i o n of c h a r a c t e r s i s unknown i n the genus. Attempts t o germinate the t e l i o s p o r e s have so f a r been u n s u c c e s s f u l . F u r t h e r c h a r a c t e r i z a t i o n of t h i s y e a s t needs t o be done b e f o r e i t s a f f i n i t i e s can be c o n c l u s i v e l y drawn. D i s t r i b u t i o n of L. s c o t t i i w i t h i n study area I s o l a t e s of L. s c o t t i i were o b t a i n e d from a v a r i e t y of h a b i t a t s (Tab. I ) . The l a r g e s t number of i s o l a t e s , 123 out of the t o t a l 147, were o b t a i n e d from marine s o u r c e s (Tab. I I ) . In f a c t , 52% of a l l marine w h i t e y e a s t s t r a i n s examined t u r n e d out t o be L. s c o t t i i s t r a i n s . Brown a l g a e (Phaeophyta) were good s o u r c e s of the y e a s t . Specimens of Fucus d i s t i c h u s , Desmarest i a i n t e r m e d i a , and 32 N e r e o c y s t i s l u e t k e a n a c o l l e c t e d d u r i n g t h e f a l l , w i n t e r , a n d s p r i n g m o n t h s y i e l d e d l a r g e n u m b e r s o f L . s c o t t i i s t r a i n s . H o w e v e r , L . s c o t t i i c o u l d n o t be i s o l a t e d f r o m F . d i s t i c h u s a n d M a c r o c y s t i s i n t e g r i f o l i a s a m p l e s c o l l e c t e d d u r i n g t h e summer m o n t h s . T h e a p p a r e n t s e a s o n a l o c c u r r e n c e o f L . s c o t t i i was r e f l e c t e d b o t h i n d i r e c t s t r e a k s f r o m f r e s h l y - c o l l e c t e d m a t e r i a l s , a n d i n s e l e c t i v e i n c u b a t i o n s . B o t h f r e s h w a t e r a n d s a l t w a t e r i n c u b a t i o n s o f F . d i s t i c h u s g e n e r a t e d l a r g e n u m b e r s o f L . s c o t t i i i s o l a t e s . D e v e l o p m e n t o f L . s c o t t i i i n s u c h i n c u b a t i o n s d o e s n o t a p p e a r t o be a f f e c t e d by s a l i n i t y . L. s c o t t i i was f o u n d i n s t r e a k s f r o m w a t e r - s u r f a c e scum i n i n c u b a t i o n d i s h e s a s w e l l a s i n s t r e a k s f r o m a l g a l m a t e r i a l s . T he g r e e n a l g a , U l v a l a c t u c a , was a l s o a g o o d s o u r c e o f L . s c o t t i i i s o l a t e s . No L . s c o t t i i was i s o l a t e d f r o m U . l a c t u c a t h a l l i c o l l e c t e d d u r i n g t h e summer m o n t h s , b u t a number o f i s o l a t e s w e r e o b t a i n e d f r o m f a l l c o l l e c t i o n s . S t r e a k s f r o m t h e m a r i n e v a s c u l a r p l a n t Z o s t e r a mar i n a p r o d u c e d an a b u n d a n c e o f L . s c o t t i i i s o l a t e s . L e a v e s o f t h i s p l a n t w e r e s t r e a k e d b o t h b e f o r e a n d a f t e r b e i n g i n c u b a t e d i n f r e s h w a t e r . D r i f t w o o d f r o m r e l a t i v e l y l o w - s a l i n i t y a r e a s o f t h e S t r a i t o f G e o r g i a , a n d wood f r o m t h e h i g h - s a l i n i t y w a t e r s o f t h e o p e n P a c i f i c c o a s t b o t h y i e l d e d a d i v e r s i t y o f w h i t e y e a s t s t r a i n s , m o r e t h a n h a l f o f w h i c h t u r n e d o u t t o be L . s c o t t i i . T h e s i n g l e t e s t e d s a m p l e s o f c r a b f r a g m e n t s a n d b e a c h s a n d y i e l d e d no y e a s t s o f a n y k i n d . 33 L. s c o t t i i was much l e s s f r e q u e n t l y found in freshwater h a b i t a t s than i n marine h a b i t a t s (Tab. I I I ) . Ten i s o l a t e s were obtained in t o t a l , a l l of • which came from stream foam. Bog water, freshwater green algae, decaying bog v e g e t a t i o n , and submerged mosses y i e l d e d no L. s c o t t i i s t r a i n s . However, note the Leucospor idium sp. s t r a i n , RCS-Y478, d e s c r i b e d above, which was i s o l a t e d from freshwater green algae in a v e r n a l pond. L. s c o t t i i i s o l a t e s were obtained in stream foam both i n warm and c o l d seasonal c o n d i t i o n s . There was no a p p r e c i a b l e i n c r e a s e i n numbers of i s o l a t e s from foam during r a i n y weather, perhaps i n d i c a t i n g that the i n f l u x of these yeasts i n t o the stream from t e r r e s t r i a l h a b i t a t s was i n s u b s t a n t i a l . A s i n g l e i s o l a t e was obtained from d e s i c c a t e d foam. Few i s o l a t e s of L. s c o t t i i were obtained from the t e r r e s t r i a l m a t e r i a l s sampled (Tab. IV). No i s o l a t e s were found on f r e s h and decaying mushrooms, on waxberries, on mosses, or on l i v i n g and dead rushes and sedges from a marsh. A s i n g l e i s o l a t e was obtained from the trunk of a l i v i n g deciduous t r e e . Two good sources of L. s c o t t i i i s o l a t e s were found: a streak made by Dr. R.J. Bandoni from the s u r f a c e of a r o t t i n g t u r n i p y i e l d e d f i v e i s o l a t e s , while f i v e more s t r a i n s were i s o l a t e d from mixed s o i l and Pseudotsuga menziesi i bark mulch i n a garden. The l a t t e r group of i s o l a t e s was obtained under moist winter c o n d i t i o n s . Of a l a r g e number of yeasts i s o l a t e d from snow (Tab. V), only three were i d e n t i f i e d as L. s c o t t i i . One i s o l a t e was o b t a i n e d from each o f : 1) a l p i n e summer snow, 2) subalpine 34 l a t e - s p r i n g snow, and 3) t r a n s i e n t w i n t e r snow. N e i t h e r of the two montane i s o l a t e s was from " r e d snow". 35 TABLE I : I s o l a t i o n o f L. s c o t t i i f r o m v a r i o u s e n v i r o n m e n t s i n and n e a r s o u t h e r n B r i t i s h C o l u m b i a P r o v e n a n c e o f i s o l a t e s t o t a l # o f w h i t e y e a s t s i s o l a t e d # L. s c o t t i i i s o l a t e s m a r i n e e n v i r o n m e n t f r e s h w a t e r e n v i r o n m e n t t e r r e s t r i a l h a b i t a t s snow 229 1 53 98 1 49 1 23 1 0 1 1 3 a l l h a b i t a t s 629 1 47 36 I n T a b l e I I ( o p p o s i t e ) r e s u l t s o f s e l e c t i v e i n c u b a t i o n s a n d o f d i r e c t s t r e a k s a r e c o m b i n e d . M o s t s a m p l e s o f m a r i n e a l g a e a n d g r a s s e s were i n c u b a t e d i n f r e s h w a t e r a f t e r d i r e c t s t r e a k i n g ; h o w e v e r , t h e y e a s t s i s o l a t e d f r o m F. d i s t i c h u s c o l l e c t e d 10 Nov. 1980 were o b t a i n e d f r o m a s a l t - w a t e r i n c u b a t i o n . 37 T A B L E I I : I s o l a t i o n o f L. s c o t t i i f r o m t h e m a r i n e e n v i r o n m e n t i n a n d n e a r s o u t h e r n B r i t i s h C o l u m b i a D a t e o f l o c a t i o n s u b s t r a t e t o t a l # Of L. c o l l e c t i o n # o f s c o t t i i whi t e i s o l a t e s y e a s t s 22 M a r . ' 80 S q u a m i s h R. F u c u s 7 6 B.C. d i s t i c h u s 21 M a r . ' 80 P t . G r e y , F . d i s t i c h u s 8 6 n e a r V a n - c o u v e r 22 J u l . ' 80 S q u a m i s h R. F . d i s t i c h u s 28 - 9 S e p t . '80 S q u a m i s h R. F. d i s t i c h u s 5 - 3 O c t . ' 80 P t . G r e y F. d i s t i c h u s 8 -2 Nov. ' 80 O r c a s I s . , F. d i s t i c h u s 8 7 Wash. 10 Nov. ' 80 P t . G r e y F. d i s t i c h u s 1 4 1 3 27 J u l . ' 80 P t . M c N e i l l , Mac r o c y s t i s 29 - V a n c o u v e r i n t e g r i f o l i a I s . , B . C . 2 Nov. ' 80 O r c a s I s . , N e r e o c y s t i s 26 25 Wash. l u e t k e a n a 2 Nov. ' 80 O r c a s I s . D e s m a r e s t i a 25 25 i n t e r m e d i a 8 A u g . ' 80 P t . G r e y U l v a l a c t u c a 4 - 3 O c t . *80 P t . G r e y U. l a c t u c a 9 2 2 Nov. ' 80 O r c a s I s . , U. l a c t u c a 5 5 Wash. 2 Nov. ' 80 O r c a s I s . Z o s t e r a 1 0 8 m a r i n a 15 D e c . ' 80 P t . G r e y d r i f t w o o d 22 1 5 22 F e b . '81 L o n g B e a c h , d r i f t w o o d 21 1 1 V a n c o u v e r I s . , B.C. 8 A u g . ' 80 P t . G r e y c r a b p a r t s - -3 O c t . ' 80 P t . G r e y b e a c h s a n d — T o t a l s 229 123 38 TABLE I I I : I s o l a t i o n of L. s c o t t i i from freshwater h a b i t a t s i n southern B r i t i s h Columbia Date of l o c a t i on s u b s t r a t e t o t a l # of L. c o l l e c t i o n #_of s c o t t i i whi te i s o l a t e s yeasts 2 Mar. ' 80 Tincan stream foam 1 - Creek near Van- couver 20 Jun. ' 80 Tincan Cr. stream foam 1 4 1 26 Jun. ' 80 Tincan Cr. stream foam 1 1 - 4 J u l . ' 80 Tincan Cr. stream foam 35 - 5 Sept . ' 80 Tincan Cr. stream foam 8 4 5 Sept . ' 80 Tincan Cr. dry stream 8 1 foam 27 Sept . ' 80 Tincan Cr. stream foam 2 - 17 Oct. ' 80 Tincan Cr. stream foam 9 - 17 Oct. ' 80 Tincan Cr. stream water 1 - 9 Nov. ' 80 Lynn Cr., stream foam 1 9 1 near Van- couver 4 Mar. '81 unnamed stream foam 9 3 stream, Vancouver 10 Feb. ' 80 Burns Bog, bog water 2 - Richmond, B.C. 8 Jan. '81 Camosun bog water 1 - Bog, near Vancouver 8 Jan. '81 Camosun submerged 1 5 - Bog r o t t i n g vegetat ion 7 Jan. '81 Vancouver D r a p a r n a l d i a 6 - sp. (Chlorophyta) 8 Jan. '81 Camosun U l o t h r i x sp. 4 - Bog (Chlorophyta) 9 Nov. ' 80 Lynn Cr., Brachythec ium 7 - Vancouver sp. (moss) 8 Jan. '81 Camosun Sphagnum spp. 1 - Bog T o t a l s 153 10 39 TABLE IV: I s o l a t i o n of L. s c o t t i i from t e r r e s t r i a l h a b i t a t s i n southern B r i t i s h Columbia Date of c o l l e c t ion l o c a t i o n s u b s t r a t e t o t a l # of whi te yeasts # of L. s c o t t i i i s o l a t e s 1 2 Oct. ' 80 Vancouver poplar trunk 1 1 1 3 Nov . ' 80 Vancouver a l d e r trunk 1 - 7 Jan. '81 Vancouver Symphori- carpos albus b e r r i e s 1 0 1 5 Jan. '81 Vancouver decaying t u r n i p 19 5 23 Jan. '81 Vancouver Juncus -- l i v e & dead p a r t s 9 23 Jan. '81 Vancouver Sc i rpus - - l i v e & dead 19 - 26 Jan. '81 Vancouver bark mulch and s o i l 1 3 5 26 Jan. '81 Vancouver mosses 10 - 1 Oct. ' 80 Vancouver decaying C o l l y b i a sp. 1 - 1 4 Dec . '80 Vancouver decaying B o l e t e l l u s z e l l e r i 23 Jan. '81 Vancouver spore drop T u l a s n e l l a sp. 2 27 Jan. '81 Vancouver Melano- leu c a melaleuca 3 4 Feb. '81' Vancouver decaying mushrooms 4 - T o t a l s 98 1 1 40 TABLE V: I s o l a t i o n of L. s c o t t i i from samples c o l l e c t e d i n southern B r i t i s h Columbia Date of c o l l e c t ion l o c a t ion s u b s t r a t e t o t a l # of whi te yeasts # Of L. s c o t t i i i s o l a t e s 21 June '80 Mt. Seymour snow 42 1 near Vancouver 14 Aug. '80 Wavy Range, snow 98 1 Wells-Gray Prov. Park, near C l e a r - water, B.C. 16 Dec. '80 Vancouver snow 9 1 T o t a l s 1 49 3 41 P s y c h r o p h i 1 i c s t r a i n s Only f i v e of the L.' s c o t t i i s t r a i n s i s o l a t e d in t h i s experiment proved to be o b l i g a t e l y p s y c h r o p h i 1 i c . Almost a l l i s o l a t e s grew w e l l at 25°C. Colony appearance at 25°C. was g e n e r a l l y f l a t t e n e d , opaque, and semi-glossy. Such c o l o n i e s were d i s t i n c t l y d i f f e r e n t from the rounded, creamy, semi- transparent c o l o n i e s t y p i c a l l y seen at 15°C. True mycelium was formed at 25°C. as w e l l as at 15°C. The f i v e p s y c h r o p h i 1 i c s t r a i n s a l l came from marine samples. One was from a sample of Ulva l a c t u c a , two were from Desmarestia intermedia, one was from Zostera marina, and the l a s t was from semi-estuarine d r i f t w o o d . A l l were obtained i n the f a l l and winter months. When cross e d with t e s t e r s , p s y c h r o p h i 1 i c s t r a i n s a l l mated with s t r a i n s of mating types A , B, and A 2B T . Mat ing genet i e s As mentioned p r e v i o u s l y , a l l l o c a l l y - i s o l a t e d L. s c o t t i i s t r a i n s were i n t e r f e r t i l e with mating t e s t s t r a i n s from the southern hemisphere. In mating new i s o l a t e s of L. s c o t t i i with t e s t s t r a i n s , i t was found that a l a r g e number (102 out of a t o t a l of 147) of new i s o l a t e s mated with two of the four known mating types ( f o r an e x p l a n a t i o n of the s p e c i e s ' i n c o m p a t i b i l i t y system, see ' I n t r o d u c t i o n ' s e c t i o n of t h i s r e p o r t ) . In order to determine whether or not t h i s phenomenon was due to mixed c u l t u r e s , c e l l s of ten s t r a i n s were suspended 42 in s t e r i l e water and r e s t r e a k e d . S i n g l e - c e l l i s o l a t e s were obtained from the streaked p l a t e s . When these i s o l a t e s were cross e d with mating t e s t s t r a i n s , a l l were again found to be compatible with two of the four t e s t e r s . In a l l cases, the t e s t s t r a i n s which mated with l o c a l i s o l a t e s had d i f f e r i n g A a l l e l e s , but i d e n t i c a l B a l l e l e s (see Table V I ) . It i s apparent that each of the ten l o c a l i s o l a t e s i n Table VI contained an A a l l e l e which was d i f f e r e n t from both the A, and A 2 a l l e l e s . A t e s t was conducted i n order to determine whether or not the new A a l l e l e s were a l l i d e n t i c a l . The ten l o c a l i s o l a t e s were intermated i n a l l p o s s i b l e combinations; the r e s u l t s of t h i s t e s t are shown i n Table V I I . Note that i f a l l the new A a l l e l e s were i d e n t i c a l , no mating of the s t r a i n s would be expected to occur. In Table V I I , i t i s c l e a r t h a t there i s more than one new A a l l e l e . If the A a l l e l e possessed by s t r a i n 37-3 i s a r b i t r a r i l y d esignated A 3, i t i s apparent that s t r a i n s 37-3 and 5-1 are A 3B, , while 23-4, 24-4, and 59-3 are A 3 B 2 . S t r a i n s 1-3, 2-3, 3-2, and 30-1 possess A a l l e l e s which are n e i t h e r A,, A 2, nor A 3. The r e s u l t f o r s t r a i n 31-3 i s anomalous: i t mates with only one of the proposed A 3B, s t r a i n s . Since two other A.B 2 s t r a i n s (A. designates an A a l l e l e or a l l e l e s not assigned a number) mate only weakly with one of the two A 3B! s t r a i n s , i t i s p o s s i b l e that genetic c h a r a c t e r i s t i c s other than mating type are a f f e c t i n g the success of mating. S t r a i n 31-3 i s m o r p h o l o g i c a l l y d i f f e r e n t from the other s t r a i n s , tending to form low, semi-glossy c o l o n i e s at 15°C. rather than the more 43 usual creamy, rounded c o l o n i e s . I would t e n t a t i v e l y suggest that s t r a i n 31-3 possessed mating type a l l e l e s compatible with those of both A 3B, s t r a i n s , but f a i l e d to mate with one s t r a i n because of an u n r e l a t e d g e n e t i c i n c o m p a t i b i l i t y . I t has not been determined whether or not the A. a l l e l e s possessed by s t r a i n s 1-3, 2-3, 3-2, 30-1, and probably 31-3 are i d e n t i c a l to each other. However, the A a l l e l e of s t r a i n 1-3 may be a r b i t r a r i l y designated A„. The number of confirmed a l l e l e s at the A locus in L. s c o t t i i i s thereby i n c r e a s e d from two to f o u r . Two other independent c r o s s e s of l o c a l l y - o b t a i n e d L. s c o t t i i i s o l a t e s were made, and s i m i l a r r e s u l t s were obtained on both o c c a s i o n s . I t should be noted that s t r a i n s mating with both A, and A 2 t e s t e r s were i s o l a t e d from marine, freshwater, and t e r r e s t r i a l h a b i t a t s , as w e l l as from snow. There were no s t r a i n s which mated with both B, and B 2 t e s t e r s . 44 TABLE VI: Mating of l o c a l i s o l a t e s of L. s c o t t i i s t r a i n # compatible mating t e s t s t r a i n s a ssigned mating type 1-3 A,B 1 ; A 2B, A.B 2 2-3 A,B, , A 2 B, A.B2 3-2 A , B , , A 2 B , A.B 2 5-1 Ai B 2 r A 2B 2 A.B, 23-4 A,B,, A 2B , A.B2 24-4 A,B, , A 2 B, A.B 2 30-1 A,B,, A 2B, A.B2 31-3 A 1 B , , A 2 B , A.B 2 37-3 A,B 2, A 2B 2 A.B, 59-3 AiB, , A 2 B , A.B 2 The symbol A. designates an A a l l e l e which i s n e i t h e r A, nor A 2 TABLE V I I : R e s u l t s of c r o s s i n g A.B, and A.B 2 s t r a i n s A.B, A.B 2 s t r a i n s s t r a i n s 1 -3 2-3 3-2 23-4 24-4 30-1 31-3 59-3 37-3 + + + + + 5-1 + +w +w + + and - symbolize r e s p e c t i v e l y . The react i o n . p o s i t i v e and negative mating r e a c t i o n s , symbol +w i n d i c a t e s a weak p o s i t i v e mating 45 IV. DISCUSSION Although the genus Leucosporidium i s best known from A n t a r c t i c ( F e l l et a l . , 1969; F e l l , 1974a, 1975) and A r c t i c h a b i t a t s (DiMenna, 1966b; Babyeva et a l . , 1976; Babyeva and A z i e v a , 1980), i t i s c l e a r that L. s c o t t i i , at l e a s t , i s r e a d i l y i s o l a t e d in the northern temperate zone. The p r o p o r t i o n of L. s c o t t i i i s o l a t e s obtained in t h i s study (147/629 t o t a l white yeast s t r a i n s examined, or 23.4%) i s not r e p r e s e n t a t i v e of the abundance of the s p e c i e s in nature, but the species i s e v i d e n t l y common and w i d e l y - d i s t r i b u t e d . L o c a l l y - o b t a i n e d i s o l a t e s are s i m i l a r c u l t u r a l l y and m o r p h o l o g i c a l l y to the southern-hemisphere i s o l a t e s d e s c r i b e d by F e l l et a l . (1969). Those bioch e m i c a l and p h y s i o l o g i c a l c h a r a c t e r s which have been examined -- diazonium blue B s t a i n i n g , i n o s i t o l a s s i m i l a t i o n , a b i l i t y to ferment glucose, p r o d u c t i o n of e x t r a c e l l u l a r s t a r c h - l i k e compounds, and, i n c i d e n t a l l y , e l a b o r a t i o n of a urease enzyme as d e t e c t e d by the t e s t s of S e e l i g e r (1956) -- a l l f i t p r e v i o u s l y - d e s c r i b e d a t t r i b u t e s of L. s c o t t i i (see F e l l et a l . , 1969; van der Walt and Hopsu-Havu, 1976). A l s o , l o c a l i s o l a t e s are completely i n t e r f e r t i l e with southern-hemisphere s t r a i n s , which suggests that p o p u l a t i o n s i n the two hemispheres are not i s o l a t e d from one another. F i v e of the s i x d e s c r i b e d s p e c i e s of Leucosporidium were not i s o l a t e d d u r i n g the course of t h i s study. Although four of the s p e c i e s -- L. antarcticum, L. f r i g i d u m , L. gelidum and L. n i v a l i s --have a l l been i s o l a t e d i n the northern hemisphere (see ' I n t r o d u c t i o n ' ) , the m a j o r i t y of the r e p o r t s are from 46 s t u d i e s of A r c t i c tundra s o i l s . Only a s i n g l e i s o l a t e of one of the s p e c i e s , an L. a n t a r c t i c u m i s o l a t e r e p o r t e d from the flowers of S a l i x alba in East Germany (Kockova-Kratochvilova et a l . , 1972), has been found in l a t i t u d e s comparable to those of the present study area ( i . e . , below 55°N. L a t . ) . The s p e c i e s i n q u e s t i o n are a l l o b l i g a t e l y p s y c h r o p h i l i c and may s u r v i v e p o o r l y o u t s i d e of a r c t i c and b o r e a l r e g i o n s . However, i t should be noted that o b l i g a t e l y p s y c h r o p h i l i c b a c t e r i a have been found i n marine waters o f f the coast of Oregon (Morita and Burton, 1970), approximately 500 km. south of the present study area. A small number of o b l i g a t e l y - p s y c h r o p h i l i c L. s c o t t i i s t r a i n s were i s o l a t e d form the marine environment in the present study. Moreover, b a c t e r i a with growth optima below 20°C. ("psychrophiles" as d e f i n e d by I n n i s s , 1975) have been i s o l a t e d from a wide v a r i e t y of temperate h a b i t a t s ( I n n i s s , 1975). I t remains p o s s i b l e that some or a l l of the p s y c h r o p h i l i c Leucosporidium s p e c i e s e x i s t w i t h i n the present study area. One l o c a l l y - i s o l a t e d Leucospor idium s t r a i n , RCS-Y478, cannot be p l a c e d i n any of the d e s c r i b e d s p e c i e s of the genus. T h i s s t r a i n was i s o l a t e d from a sample of the filamentous green a l g a U l o t h r i x sp. c o l l e c t e d i n a v e r n a l pond near a peat bog. The s t r a i n d i f f e r s from L. s c o t t i i and L. a n t a r c t icum in the s i z e , shape, and s i t e of formation of i t s t e l i o s p o r e s , as w e l l as i n i t s a b i l i t y to ferment g l u c o s e . From the remainder of the Leucospor idium s p e c i e s , i t d i f f e r s i n s i t e of formation and s i z e of t e l i o s p o r e s , i n i t s i n a b i l i t y to a s s i m i l a t e i n o s i t o l and to produce e x t r a c e l l u l a r s t a r c h , and i n i t s a b i l i t y to grow 47 at 25°C. . One p r e v i o u s l y - d e s c r i b e d heterobasidiomycetous yeast s p e c i e s bears some s i m i l a r i t y to s t r a i n RCS-Y478. That s p e c i e s i s Rhodospor idium malvinellum, d e s c r i b e d by F e l l (1970) from i s o l a t e s c o l l e c t e d in the southern P a c i f i c and Indian oceans. R. malvinellum produces a creamy-white streak a f t e r three days growth on malt agar at 1 2 ° C . T h i s s t r e a k , however, a c q u i r e s a mauve c o l o r a t i o n a f t e r approximately ten days growth, and "at one month the c o l o u r has i n t e n s i f i e d " ( F e l l , 1970). Subglobose t e l i o s p o r e s are borne t e r m i n a l l y on short l a t e r a l branches of the mycelium, as i n s t r a i n RCS-Y478, but they are s m a l l : (5.1- 10) X 7.1-12) „m. as opposed to (7-20) X (7-13) m̂. f o r Leucosporidium sp. N e i t h e r s p e c i e s a s s i m i l a t e s i n o s i t o l or produces e x t r a c e l l u l a r s t a r c h . U n l i k e R. malvinellum, s t r a i n RCS-Y478 i s able to ferment g l u c o s e . F i n a l l y , R. malvinellum has a b i a l l e l i c b i p o l a r mating system ( F e l l , 1974b) and clamp connections ( F e l l , 1970), while s t r a i n RCS-Y478 i s h o m o t h a l l i c and l a c k s clamps. The importance of c h a r a c t e r s r e l a t e d to i n c o m p a t i b i l i t y i s not s t r e s s e d , however: L. s c o t t i i , f o r example, has h e t e r o t h a l l i c s t r a i n s with clamped hyphae as w e l l as h o m t h a l l i c s t r a i n s with undamped hyphae. Some r e l a t i o n s h i p between Leucosporidium s t r a i n RCS-Y478 and R. malvinellum i s t e n t a t i v e l y suggested, but f u r t h e r s p e c u l a t i o n i s i d l e u n t i l the t e l i o s p o r e s of RCS-Y478 sp. have been germinated. The l a r g e s t number of L. s c o t t i i i s o l a t e s obtained i n t h i s study were from the marine environment. Although the most r e l i a b l e means of i s o l a t i n g l a r g e numbers of L. s c o t t i i s t r a i n s 48 was by c a r r y i n g out i n c u b a t i o n s of decaying marine v e g e t a t i o n , numerous s t r a i n s of the species were a l s o obtained from d i r e c t s t r e a k s of f r e s h l y - c o l l e c t e d m a t e r i a l s on i s o l a t i o n media. Incubations of phaeophyte algae o f t e n y i e l d e d e x t r a o r d i n a r y numbers of L. s c o t t i i i s o l a t e s . Previous s t u d i e s on decaying brown algae have not r e s u l t e d in p a r a l l e l f i n d i n g s . C a p r i o t t i (1962) examined Sargassum t h a l l i from the Miami, F l o r i d a area and r e p o r t e d Candida t r o p i c a l i s, P i c h i a fermentans, and Trichosporon cutaneum as predominant yeast s p e c i e s . M a c r o c y s t i s pyr i f e r a ( g i a n t kelp) from southern C a l i f o r n i a was found to be a r i c h source of Metschnikowia z o b e l i i and Candida (Metschnikowia) pulcherrima by van Uden and Castelo-Branco (1963). In the study of Siepmann and Hohnk (1962), Ascophyllum nodosum from the North A t l a n t i c appeared to serve as a h a b i t a t f o r Debaryomyces subglobosus, Tr ichosporon cutaneum, and Rhodotorula g l u t in i s. In Japan, Suehiro (i960) i s o l a t e d y e a sts from decaying t h a l l i of a number of a l g a l s p e c i e s . These algae were incubated in f r e s h seawater in the l a b o r a t o r y at 20°C. before being sampled fo r y e a s t s . Sargassum hemiphyllum was the a l g a l s p e c i e s which s u s t a i n e d the l a r g e s t p o p u l a t i o n s of c o l o n i z i n g y e a s t s . Predominant s p e c i e s were T o r u l o p s i s C a n d i d a , Candida a l b i c a n s , C. n a t a l e n s i s (C. sake), Tr ichosporon cutaneum, and Endomycopsis c h o d a t i . Suehiro and Tomiyasu (1962) c a r r i e d out s i m i l a r i n c u b a t i o n s i n seawater c o n t a i n i n g a n t i b a c t e r i a l agents. The predominant c o l o n i z e r of phaeophyte s p e c i e s i n t h i s experiment was Candida n a t a l e n s i s , although C. t r o p i c a l i s o f t e n 49 became predominant during the summer months. L i v i n g brown algae, i n c l u d i n g Sargassum and Padina, were sampled by Roth et a l . (1962) in F l o r i d a and the Bahamas. The a l g a l t h a l l i were found to be low in numbers of y e a s t s , although there were l a r g e numbers of yeasts in the surrounding waters. The most common s p e c i e s were members of the genus Rhodotorula. Candida p a r a p s i l o s i s and Rhodotorula rubra were i s o l a t e d from l i v i n g Fucus v e s i c u l o s u s t h a l l i by Seshadri and S i e b u r t h (1971). T h i s study was c a r r i e d out using algae from waters near Rhode I s l a n d . The same area was the s i t e of the most comprehensive study to date of yeasts growing on marine a l g a l t h a l l i . In t h i s study, Seshadri and S i e b u r t h (1975) used homogenization and chemical f i l t e r i n g techniques i n order to o b t a i n yeast i s o l a t e s from l i v i n g t h a l l i of Fucus v e s i c u l o s u s , Ascophyllum nodosum, Laminar i a d i q i t a t u m , L. l o n q i c r u r i s , and s e v e r a l non-phaeophyte s p e c i e s . Large numbers of Candida spp. i s o l a t e s were obtained, l e a d i n g the authors to conclude that seaweeds were r e s e r v o i r s of Candida y e a s t s . No confirmed i s o l a t e s of C. s c o t t i i were obtained; however, a l a r g e number of i s o l a t e s u n i d e n t i f i a b l e by c o n v e n t i o n a l means were grouped by a computer-generated c l u s t e r a n a l y s i s . Of these i s o l a t e s , 14% f e l l i n t o a group s i m i l a r to C. s c o t t i i , C. capsuligenum, C. m e l i n i i , C. mesenter i c a , and s e v e r a l other Candida s p e c i e s . U n f o r t u n a t e l y , some of the s p e c i e s l i s t e d , i n c l u d i n g C. m e l i n i i (see B a s t i d e et a l . , 1975), have ascomycetous a f f i n i t i e s . N e v e r t h e l e s s , i t i s p o s s i b l e that some of these i s o l a t e s were L. s c o t t i i s l i g h t l y d i s g u i s e d by heterodox a s s i m i l a t i v e 50 c h a r a c t e r i s t i c s . Seshadri and S i e b u r t h d i d not attempt mating s t u d i e s . In the present study, no i s o l a t e s of L. s c o t t i i were obtained from brown algae sampled during the summer months or during the e a r l y autumn. The yeast was absent both i n d i r e c t s t r e a k s and i n i n c u b a t i o n s . Since i n c u b a t i o n temperature was r a i s e d from 5°C. to 10°C. durin g the summer, i t i s d i f f i c u l t to compare the r e s u l t s of summer enrichment i n c u b a t i o n s with those from other seasons. However, the lack of L. s c o t t i i in d i r e c t s t r e a k s made durin g the summer, as w e l l as i n early-autumn i n c u b a t i o n s , must be regarded as s i g n i f i c a n t . The green a l g a Ulva l a c t u c a was a r e l i a b l e source of L. s c o t t i i i s o l a t e s in the present study, although again the yeast was not found dur i n g the summer months. L. s c o t t i i has not p r e v i o u s l y been r e p o r t e d from U. l a c t u c a or r e l a t e d algae. Chlorophytes i n c l u d e d i n the in c u b a t i o n s of Suehiro and Tomiyasu (1962) developed abundant p o p u l a t i o n s of Cryptococcus and Rhodotorula s p e c i e s . Seshadri and S i e b u r t h (1971) i s o l a t e d Rhodotorula l a c t o s a and Candida p a r a p s i l o s i s from U. l a c t u c a c o l l e c t e d near Rhode I s l a n d . Further study by Seshadri and S i e b u r t h (1975) r e v e a l e d that l i v i n g U. l a c t u c a was c o l o n i z e d by a v a r i e t y of Candida s t r a i n s , many u n i d e n t i f i a b l e , and by s e v e r a l s p e c i e s of Rhodotorula. F i n a l l y , P a t e l (1975) found that U. r i g i d a from the Indian coast was p r i m a r i l y c o l o n i z e d by Candida t r o p i c a l i s and two s p e c i e s of Saccharomyces, while two sp e c i e s of Enteromorpha were c o l o n i z e d by T o r u l o p s i s g l a b r a t a and T. Candida. 51 Zostera mar ina ( Z o s t e r a c e a e ) , was sampled once duri n g the present study and found to be h e a v i l y c o l o n i z e d by L. s c o t t i i . S i e b u r t h and Thomas (1973) have shown that 2. mar ina develops a t h i c k f o u l i n g c r u s t whose biomass can r i v a l that of the host. The yeast components of the c r u s t have not been c h a r a c t e r i z e d . Other seagrass s p e c i e s undergo s i m i l a r f o u l i n g processes (see S i e b u r t h , 1979, f o r review). Roth et a l . (1962) s t u d i e d beds of T h a l a s s i a testudinum, a s u b t r o p i c a l s p e c i e s , and found that waters in the v i c i n i t y of these p l a n t s were densely populated by Rhodotorula p i 1imanae and R. rubra. S p a r t i n a a l t e r n i f l o r a (cordgrass) s t u d i e d i n L o u i s i a n a marshlands by Ahearn et a l . (1970), was found to be c o l o n i z e d by P i c h i a s p a r t i n a e and Kluyveromyces drosophilarum. It i s d i f f i c u l t to formulate any general c o n c l u s i o n s about the ecology of L. s c o t t i i from the above r e s u l t s . One p o s s i b l e g e n e r a l i z a t i o n , however, i s that temperature i s an important f a c t o r i n the establishment and development of t h i s s p e c i e s on marine v e g e t a t i o n . The known d i s t r i b u t i o n of L. s c o t t i i i n marine waters can be c i t e d i n support of such an a s s e r t i o n . The s p e c i e s i s widely known from marine waters in the southern regions of the southern hemisphere ( F e l l , 1974a), and i t has been found in a study of o b l i g a t e l y p s y c h r o p h i 1 i c yeasts growing on f i s h near S c o t l a n d (Bruce and M o r r i s , 1973). In the present study, i t was i s o l a t e d from marine v e g e t a t i o n c o l l e c t e d d u r i n g c o l d seasonal c o n d i t i o n s . However, L. s c o t t i i has never been found in warm-temperate, s u b t r o p i c a l , or t r o p i c a l marine h a b i t a t s ; a l s o , i t has been absent from warm in c u b a t i o n s 52 ( s p e c i f i c a l l y , the 20°C. i n c u b a t i o n s of Suehiro, 1960, and Suehiro and Tomiyasu, 1962). Although many i n v e s t i g a t o r s have f a i l e d to f i n d L. s c o t t i i in northern marine waters (Shinano, 1962; K r i s s , 1963; Norkrans, 1966; Meyers et a l . , 1967; Combs et a l . , 1971; Yamasoto et a l . , 1974) and on p l a n t and animal m a t e r i a l s from those waters (Siepmann and Hohnk, 1962; Ross and M o r r i s , 1965; Seshadri and S i e b u r t h , 1975), the sp e c i e s may be a cosmopolitan i n h a b i t a n t of c o l d marine environments. The s e a s o n a l i t y manifested by l o c a l L. s c o t t i i p o p u l a t i o n s has p r e v i o u s l y been observed i n po p u l a t i o n s of marine y e a s t s . Seshadri and S i e b u r t h (1975) found a complex p a t t e r n of seasonal v a r i a t i o n in taxonomic composition of yeast f l o r a s on marine a l g a e . I n t e r e s t i n g l y , a group of u n i d e n t i f i e d yeasts n u t r i t i o n a l l y s i m i l a r to L. s c o t t i i reached a p o p u l a t i o n peak in midwinter. Suehiro and Tomiyasu (1962) a l s o found seasonal changes i n the yeast f l o r a s of t h e i r aforementioned i n c u b a t i o n s . Marine d r i f t w o o d y i e l d e d numerous i s o l a t e s of L. s c o t t i i in the present study. Almost nothing i s known about the yeast f l o r a of decaying marine wood (Johnson and Sparrow, 1961, have been the most recent authors to lament t h i s f a c t . ) . U n f o r t u n a t e l y , the techniques used i n the present study do not allow an assessment of whether or not the yeast was p a r t i c i p a t i n g i n the process of wood decay. The d e n s i t y of L. s c o t t i i c e l l s in the water to which the wood was exposed i s unknown. N e v e r t h e l e s s , i t i s p o s s i b l e that marine wood serves, d i r e c t l y or i n d i r e c t l y , as a s u b s t r a t e f o r t h i s y e a s t . S t r a i n s 53 of an u n i d e n t i f i e d Leucospor idium s p e c i e s have been shown to a s s i m i l a t e x y l ose ( B i e l y et a l . , 1978); thus, L. s c o t t i i may be able to p r o f i t by a s s o c i a t i o n with wood-decay microorganisms. The p o t e n t i a l a b i l i t y of L. s c o t t i i to a s s i m i l a t e products of l i g n i n degradation i s d i s c u s s e d below. The ease with which L. s c o t t i i was i s o l a t e d in the marine environment in t h i s study stands in c o n t r a s t to r e l a t i v e l y unrewarding examinations which were conducted with m a t e r i a l s from the freshwater environment. Only a small number of s t r a i n s were obtained from streams. Streams were mainly sampled by g a t h e r i n g foam: the branching s t r u c t u r e s t y p i c a l of the pseudomycelium of L. s c o t t i i were expected to r e s u l t in the s p e c i e s being accumulated i n t o foam in the manner of branched a q u a t i c c o n i d i a (Iqbal and Webster, 1973). From the r e s u l t s obtained, i t would appear that L. s c o t t i i i s a minor component of the yeast mycobiota of l o c a l streams, or enters those streams i n small q u a n t i t i e s from adjacent t e r r e s t r i a l h a b i t a t s . A l t e r n a t i v e l y , the foam-sampling method used may give m i s l e a d i n g r e s u l t s about stream p o p u l a t i o n s of yeasts which form branched s t r u c t u r e s . I t should be noted that p revious s t u d i e s i n which L. s c o t t i i or i t s anamorph have been found i n f r e s h l o t i c waters (Woollett and Hedrick, 1970; Simard and Blackwood, 1971; H i n z e l i n and L e c t a r d , 1978) have given s i m i l a r r e s u l t s . The anomalous Leucosporidium sp. s t r a i n RCS-Y478 ( d e s c r i b e d above), was i s o l a t e d from the freshwater green alga U l o t h r i x sp. i n a v e r n a l pond near a peat bog. I have been able 54 t o f i n d o n l y one p r e v i o u s r e p o r t i n t h e l i t e r a t u r e a b o u t a f r e s h w a t e r g r e e n a l g a h a v i n g b e e n s a m p l e d f o r y e a s t s . C o o k e a n d M a t s u u r a ( 1 9 6 3 ) , a l t h o u g h s t u d y i n g w a s t e s t a b i l i z a t i o n p o n d s , a l s o i s o l a t e d y e a s t s f r o m H y d r o d i c t y o n m a t s i n an u n p o l l u t e d f r e s h w a t e r s t r e a m . U n f o r t u n a t e l y , t h e i d e n t i t i e s o f many o f t h e y e a s t i s o l a t e s o b t a i n e d c o u l d n o t be d e t e r m i n e d . No C. s c o t t i i i s o l a t e s ( C . s c o t t i i was t h e o n l y L e u c o s p o r i d i u m a n a m o r p h t h e n d e s c r i b e d ) o r s i m i l a r y e a s t s were f o u n d . The y e a s t m y c o b i o t a o f p e a t b o g s i s a l s o p o o r l y known. L u n d ( 1 9 5 4 ) e x a m i n e d p e a t b o g s o i l s , a n d f o u n d t h a t t h e p r e d o m i n a n t y e a s t s w e r e S c h i z o b l a s t o s p o r i o n s t a r k e y i - h e n r i c i i a n d T o r u l o p s i s Candida. The d i s t r i b u t i o n o f L . s c o t t i i i n t h e t e r r e s t r i a l h a b i t a t s s t u d i e d i s o f g r e a t i n t e r e s t . Of a v a r i e t y o f h a b i t a t s e x a m i n e d , o n l y t h r e e y i e l d e d i s o l a t e s o f t h e y e a s t : r o t t i n g t u r n i p , b a r k m u l c h s o i l , a n d r a i n - d e r i v e d s t e m f l o w o v e r t h e t r u n k o f a l i v i n g t r e e . As c a n be s e e n , two o f t h e s e h a b i t a t s i n c l u d e d t h e b a r k o f t r e e s . A s L e u c o s p o r i d i u m s p p . a n d a n a m o r p h s a r e n o t known f r o m t h e p h y l l o p l a n e ( s e e , f o r e x a m p l e , L a s t a n d P r i c e , 1969; D i M e n n a , 1971; K v a s n i k o v e t a l . , 1 9 7 4 ) , t h e p r e s e n c e o f L . s c o t t i i i n s t e m f l o w i s o p e n t o i n t e r p r e t a t i o n . L . g e l i d u m h a s p r e v i o u s l y b e e n d e t e c t e d i n a b i r c h s l i m e f l u x ( G o l u b e v e t a l . , 1 9 7 7 ) . Th e p r e s e n c e o f L . s c o t t i i i n b a r k m u l c h s h o u l d p e r h a p s n o t h a v e b e e n u n e x p e c t e d . The s p e c i e s h a s b e e n f o u n d i n v a r i o u s s o i l s ( D i M e n n a , 1955, 1960a, 1960b, 1966b; S c o t t , 1936; B a b y e v a a n d G o l u b e v , 1969; B a b y e v a e t a l . , 1976; B a b y e v a a n d A z i e v a , 1 9 8 0 ) , a l t h o u g h o n l y s m a l l n u m b e r s o f i s o l a t e s h a v e b e e n 55 obtained o u t s i d e p o l a r r e g i o n s . The only a s s o c i a t i o n of any of the temperate i s o l a t e s with p l a n t m a t e r i a l i s found in the study of DiMenna (1960b). T h i s author found C. s c o t t i i i n f o r e s t l i t t e r c o n s i s t i n g of leaves intermixed with twigs. Once again, bark was present i n the h a b i t a t . These r e s u l t s suggest, a l b e i t tenuously, that L. s c o t t i i may be a s s o c i a t e d with decaying bark and woody m a t e r i a l . The mycobiota of bark i s very p o o r l y known. Lund (1954) attempted to i s o l a t e yeasts from the bark of s e v e r a l t r e e s p e c i e s i n Denmark. He found almost no yeasts present, although Saccharomyces fermentati was d i s c o v e r e d on the bark of an elm. Phaff and Knapp (1956) obtained Hansenula minuta from decaying Populus tremuloides bark i n C a l i f o r n i a . Decaying wood has a l s o r e c e i v e d l i t t l e a t t e n t i o n from yeast e c o l o g i s t s . T h i s s i t u a t i o n may change: the recent s t u d i e s of Blanchette and Shaw (1978) and Blanchette (1979) have r e v e a l e d that yeasts are an important part of the a s s o c i a t i o n of organisms which b r i n g s about wood decay. At present, only two s t u d i e s e x i s t i n which yeasts on decaying wood i n temperate regions have been i d e n t i f i e d to s p e c i e s . Shehata et a l . (1956) found Candida p u l c h e r r ima, C. c a t e n u l a t a , and C. c l a u s s e n i i on decaying wood in C a l i f o r n i a . A l s o i n C a l i f o r n i a , Phaff and Knapp (1956) found P i c h i a polymorpha on decaying Abies and Pinus l o g s . Previous to the present study, there had been no examination of the yeasts of bark or wood decaying under c o l d c o n d i t i o n s . The f i n d i n g of L. s c o t t i i i n the decaying s u r f a c e of a t u r n i p ( B r a s s i c a rapa) root i s unprecedented. There has been 56 scant work on yeasts in the decay o f . r o o t crops. The r o o t s of Daucus c a r o t a and Beta v u l g a r i s were both sampled f o r yeasts by Lund (1954). Among the yeasts commonly found were T o r u l o p s i s m o l i s c h i a n a , T. C a n d i d a , and Candida p a r a p s i l o s i s . Work on yeasts of the rhizosphere of crop p l a n t s has y i e l d e d i n t e r e s t i n g r e s u l t s . Babyeva and Savelyeva (1963) d i s c o v e r e d that the number of yeasts around the roots of l i v i n g cabbage p l a n t s v a s t l y exceeded the number a s s o c i a t e d with other crop p l a n t s , i n c l u d i n g c a r r o t s , sugarbeets, and c e r e a l s . The cabbage p l a n t , l i k e the t u r n i p , i s a member of the genus B r a s s i c a . Predominant s p e c i e s i n the cabbage rhizosphere were T o r u l o p s i s a e r i a (Cryptococcus a l b i d u s var. a e r i a ) , and Rhodotorula g l u t i n i s. Candida s p e c i e s were c o n s p i c u o u s l y absent from rh i z o s p h e r e s o i l s , although they f l o u r i s h e d in c o n t r o l s o i l s . A f u r t h e r study of crop r h i z o s p h e r e s by Kvasnikov et a l . (1974) r e v e a l e d that cabbage r h i z o s p h e r e s were mainly i n h a b i t e d by two Cryptococcus s p e c i e s , Oosporidium m a r g a r i t i f e r u m , T o r u l o p s i s C a n d i d a , and Metschnikowia p u l c h e r r ima. These r h i z o s p h e r e mycobiotas are q u i t e d i f f e r e n t from the cold-weather decay mycobiota i s o l a t e d in the present study. The decaying t u r n i p s were p r i m a r i l y c o l o n i z e d by L. s c o t t i i , Rhodospor idium ?capitatum, a basidiomycetous Candida s p e c i e s , and a basidiomycetous T r i c h o s p o r o n s p e c i e s . The f i n a l h a b i t a t i n which L. s c o t t i i was found i n t h i s study was snow. Only three of 149 i s o l a t e s from v a r i o u s bodies of snow turned out to be s t r a i n s of L. s c o t t i i . I t seems l i k e l y t h a t the snow sampled d i d not support indigenous p o p u l a t i o n s of 57 t h i s y e a s t . None of the s t r a i n s i s o l a t e d from snow was o b l i g a t e l y p s y c h r o p h i 1 i c . DiMenna (1955b) has recorded the i s o l a t i o n of a C. s c o t t i i s t r a i n from a i r i n s i d e a. b u i l d i n g ; such a i r b o r n e propagules probably account fo r the o c c a s i o n a l appearance of L. s c o t t i i in the snow from which I took samples. The i s o l a t i o n of an L. s c o t t i i s t r a i n from d r i e d stream foam in t h i s study p o i n t s to at l e a s t one mechanism by which the yeast may become a i r b o r n e . Breaking waves at sea are a l s o capable of r e l e a s i n g l a r g e numbers of yeast propagules i n t o the atmosphere ( S c h l i c h t i n g , 1974). L. s c o t t i i might t h e r e f o r e be expected to be a common component of the a i r b o r n e yeast mycobiota in c o a s t a l regions of B.C. I t i s d i f f i c u l t to suggest what f a c t o r s , i f any, t y p i f y an L. s c o t t i i h a b i t a t . One common f e a t u r e possessed by most of the h a b i t a t s i n which the s p e c i e s has been found i s low temperature. Since yeast e c o l o g i s t s i n g e n e r a l , myself r e g r e t f u l l y i n c l u d e d , have been remiss i n measuring the temperature of n a t u r a l yeast s u b s t r a t e s , p r e c i s e i n f o r m a t i o n cannot be given on t h i s p o i n t . The s e a s o n a l i t y of L. s c o t t i i on decaying marine v e g e t a t i o n i n t h i s study i s h i g h l y suggestive of an e c o l o g i c a l requirement f o r low temperature, but f u r t h e r work i s needed before a g e n e r a l i z a t i o n about t h i s s p e c i es can c o n f i d e n t l y be made. The s e l e c t i o n of L. s c o t t i i i n i n c u b a t i o n s of brown algae o f f e r s another p o t e n t i a l g e n e r a l i z a t i o n . I t i s apparent that f r e s h water does not provide the s e l e c t i v e f a c t o r i n the i n c u b a t i o n s , although t h i s i s what was supposed when the study 58 began. L. s c o t t i i predominated i n a s a l t water c o n t r o l i n c u b a t i o n , and hence may not be g r e a t l y a f f e c t e d by s a l i n i t y under enrichment c o n d i t i o n s . Temperature may be the s o l e s e l e c t i v e f a c t o r , but a second p o s s i b i l i t y i s that the yeast i s s e l e c t e d f o r by the presence of p h e n o l i c compounds. Phaeophytes r e l e a s e l a r g e q u a n t i t i e s of y e l l o w - c o l o u r e d m a t e r i a l s i n t o surrounding waters ( C r a i g i e and McLachlan, 1964); these m a t e r i a l s c o n s i s t of t a n n i n - l i k e polyphenols (Haug and Larsen, 1958). Many yeasts appear to be i n h i b i t e d by these compounds (Seshadri and S i e b u r t h , 1971). Water in brown a l g a l i n c u b a t i o n s in the present study r a p i d l y became yellow-brown. The abundance of L. s c o t t i i in these i n c u b a t i o n s suggests that i t i s e i t h e r i n s e n s i t i v e to the compounds, or i s capable of reaping some b e n e f i t from t h e i r presence. Numerous s t u d i e s have shown that yeasts are capable of a s s i m i l a t i n g phenols, benzoic a c i d compounds, f l a v o n o i d s , and l i g n i n breakdown products (Henderson, 1961; H a r r i s and R i c k e t t s , 1962; Westlake and Spencer, 1966; Cain et a l . , 1968; Spencer et a l . , 1971; M i l l s et a l . , 1971; Neujahr, 1978; P i n t o et a l . , 1979). The a b i l i t i e s of Leucospor idium spp. to a s s s i m i l a t e these aromatic compounds have never been t e s t e d ; however, c l o s e l y - r e l a t e d Rhodosporidium anamorphs are capable of m e t a b o l i z i n g a wide range of the compounds. For example, Rhodotorula g l u t i n i s , the anamorph of Rhodosporidium t o r u l o i d e s , uses v a r i o u s f l a v o n o i d s (Spencer and G o r i n , 1971). In the present study, many of the h a b i t a t s i n which L. s c o t t i i i s o l a t e s were obtained i n l a r g e numbers were h a b i t a t s 59 in which such compounds may have been present i n r e l a t i v e l y high c o n c e n t r a t i o n s . In p a r t i c u l a r , brown algae, decaying marine wood, and bark mulch can be expected to have a high content of aromatic compounds. Other L. s c o t t i i h a b i t a t s , l i k e decaying t u r n i p and decaying Ulva and Zostera, probably a l s o c o n t a i n such compounds: t a n n i n s , e s p e c i a l l y , are known to be a s s o c i a t e d with dying and dead p l a n t c e l l s (Goodwin and Mercer, 1972). Of the low-temperature h a b i t a t s in which L. s c o t t i i was i n f r e q u e n t l y or never found, there are few i n which l a r g e c o n c e n t r a t i o n s of p h e n o l i c s would be expected. It i s evident from s t u d i e s of L. s c o t t i i in p e l a g i c marine waters ( F e l l , 1974a) that high c o n c e n t r a t i o n s of aromatic compounds cannot be a p r e r e q u i s i t e f o r the s u r v i v a l of the y e a s t . These compounds may, however, provide an e s s e n t i a l r e d u c t i o n i n competition i n some h a b i t a t s . In the a r t i f i c i a l , e n c l o s e d environment of an i n c u b a t i o n v e s s e l , t h i s e f f e c t may be witnessed in a p a r t i c u l a r l y dramatic form. A l t e r n a t i v e l y , the compounds may provide a n u t r i e n t source which i s u n a v a i l a b l e to other s p e c i e s at L. s c o t t i i 's e c o l o g i c a l temperature optimum. Tests to determine whether or not L. s c o t t i i can a s s i m i l a t e t a n n i n s , phenols, and l i g n i n breakdown products would c l e a r l y be of i n t e r e s t . D espite the apparent a s s o c i a t i o n of L. s c o t t i i with low temperatures in t h i s study, only a small f r a c t i o n (5/147) of the t o t a l i s o l a t e s were found to be unable to grow at 25°C. A l l o b l i g a t e l y p s y c h r o p h i l i c s t r a i n s came from decaying marine v e g e t a t i o n and wood. Although 90% of the seas' water volume i s 60 below 5°C. (Morita, 1975), i t i s l i k e l y that t h i s s t a b i l i t y does not b e n e f i t the yeasts present in the much l e s s s t a b l e i n t e r t i d a l zone (Seshadri and S i e b u r t h , 1975). The deep waters of the open sea may serve as a r e s e r v o i r of o b l i g a t e l y p s y c h r o p h i 1 i c L. s c o t t i i s t r a i n s , but most c o l o n i z e r s of decaying i n t e r t i d a l or s u b t i d a l m a t e r i a l s are probably indigenous f a c u l t a t i v e l y p s y c h r o p h i 1 i c s t r a i n s . T h i s would seem p a r t i c u l a r l y l i k e l y i n areas as temperate as the present study area. Although l o c a l l y - i s o l a t e d L. s c o t t i i s t r a i n s were i n t e r f e r t i l e with southern-hemisphere mating t e s t s t r a i n s , i t would appear that many possessed mating a l l e l e s not found in those s t r a i n s . The d e s i g n a t i o n s A 3 and A a were assig n e d to two d i s t i n c t a l l e l e s at the A mating loc u s which were compatible with both A, and A 2 t e s t e r s in c r o s s e s . Other A a l l e l e s may a l s o have been present. Although the presence of the new mating a l l e l e s was only c o n c l u s i v e l y demonstrated i n ten l o c a l l y - obtained i s o l a t e s , many more undoubtedly possessed A a l l e l e s which were n e i t h e r A, nor A 2. A t o t a l of 102 out of 147 L. s c o t t i i s t r a i n s mated with both A, and A 2 t e s t e r s . The exact p r o p o r t i o n of these s t r a i n s which possessed the new a l l e l e s i s u n c e r t a i n : some of the s t r a i n s may have been mixed c u l t u r e s , r a t h e r than the progeny of s i n g l e - c e l l i s o l a t e s . However, the small number of s t r a i n s which underwent spontaneous mating (3/147) suggests that the number of mixed c u l t u r e s was low. Even the observed spontaneous matings may have been the r e s u l t of the s t r e a k i n g of t e l i o s p o r e s onto i s o l a t i o n media. I t 61 appears safe to conclude that L. s c o t t i i possesses a f e a t u r e t y p i c a l of fungi with t e t r a p o l a r mating systems (Whitehouse, 1949) -- that i s , m u l t i p l e mating f a c t o r s . The d i s c o v e r y of more than two A a l l e l e s in L. s c o t t i i renders the i n c o m p a t i b i l i t y g e n e t i c s of t h i s s p e c i e s unique i n the Leucosporidium-Rhodosporidium group of sp e c i e s (see F e l l , 1974b). The other s p e c i e s in the group possess b i a l l e l i c or m u l t i a l l e l i c b i p o l a r mating systems, or t e t r a p o l a r systems i n which m u l t i p l e a l l e l e s have not been demonstrated. M u l t i a l l e l i c t e t r a p o l a r mating systems are widely known in other f u n g i : for example, Raper, Krongelb, and Baxter (1958) examined 114 Schizophyllum commune s t r a i n s , and found 96 d i s t i n c t A f a c t o r s and 56 d i s t i n c t B f a c t o r s . Whitehouse (1949) has s t a t e d that t e t r a p o l a r mating systems with the minimum number of a l l e l e s would tend to be s e l e c t e d a g a i n s t . I t should be expected, t h e r e f o r e , that a l l t e t r a p o l a r mating systems w i l l possess at l e a s t one locu s with more than two a l l e l e s . The L. s c o t t i i i n c o m p a t i b i l i t y system i s s i m i l a r to those d e s c r i b e d f o r other heterobasidiomycetes. In Treme l l a mesenterica, f o r example, c o n j u g a t i o n i s c o n t r o l l e d by a s i n g l e p a i r of a l l e l e s , A and a, while the formation of a v i a b l e dikaryon i s c o n t r o l l e d by a number of B a l l e l e s (Bandoni, 1962). The i n c o m p a t i b i l i t y systems of Treme l l a globospora (Brough, 1974) and S i r o b a s i d i u m magnum (Flegel,1976) are s i m i l a r . A l s o , U s t i l a g o zeae has a comparable system (Rowell and de Vay, 1954; Rowell, 1955). 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