"Science, Faculty of"@en . "Botany, Department of"@en . "DSpace"@en . "UBCV"@en . "Summerbell, Richard Charles"@en . "2010-03-26T22:31:30Z"@en . "1981"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "A search for members of the genus Leucospor idium (Ustilaginaceae) in and near southern British Columbia has yielded 147 isolates of L. scottii, and a single isolate of an undescribed species with apparent affinities in the genus. L. scottii was primarily found on decaying marine vegetation and driftwood, but isolates were also obtained from stream foam, snow, a decaying turnip root, bark mulch, and rain-derived stem flow over the trunk of a living tree. The species predominated in laboratory incubations of marine algal materials collected in the winter, spring, and late autumn. The majority of isolates obtained directly from natural substrates were also found during periods of cold seasonal conditions. It is suggested that low temperature is\r\ncharacteristic of L. scottii habitats.\r\nLocally-obtained L. scottii strains are all heterothallic, and are completely interfertile with mating test strains originating from the southern hemisphere. Morphological and known physiological and biochemical characters of the local strains are similar to those previously described for the species. However, mating studies of local isolates have revealed that the tetrapolar incompatibility system of L. scottii is distinguished by the possession of multiple alleles at the A locus. Similar modifications of the tetrapolar system are known in Tremella and other heterobasidiomycete genera, but have not previously been reported in species of the Leucosporidium-Rhodosporidium group. The strain thought to belong to an undescribed species of Leucosporidium was obtained from a sample of filamentous green algae growing in a vernal pond near a peat bog. The fungus is homothallic, and is morphologically and physiologically distinct from other Leucosporidium and Rhodosporidium species.\r\nOf the six known species of Leucosporidium, five (L. antarcticum, L. friqidum, L. gelidum, L. nivalis, and L. stokesii) were not found during the course of the present study. All five species are obligately psychrophilic and may not be able to remain established within the study area. However, a survey of local L. scottii isolates showed that 5 out of 147 isolates (3.4%) were obligately psychrophilic."@en . "https://circle.library.ubc.ca/rest/handle/2429/22693?expand=metadata"@en . "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.\u00E2\u0080\u00A2 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 \u00C2\u00B0C. 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\u00C2\u00B0C. 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\u00C2\u00B0C. 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 \u00C2\u00B0 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\u00C2\u00B0C. 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\u00C2\u00B0C. 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\u00C2\u00B0C. 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\u00C2\u00B0C. 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\u00C2\u00B0C. 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\u00E2\u0080\u0094 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\u00C2\u00B0C. 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\u00C2\u00B0C. , 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\u00C2\u00B0C. 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\u00C2\u00B0C. 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\u00C2\u00B0C. 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\u00C2\u00B0C. 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\u00C2\u00B0C. ( 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\u00C2\u00B0C. 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 \u00C2\u00BBim. 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\u00C2\u00B0C. 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 \u00C2\u00BB\u00C2\u00BBm. 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 \u00C2\u00BB\u00C2\u00BBm. ) , 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 \u00C2\u00B0 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 \u00E2\u0080\u00A2 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 \u00E2\u0080\u0094 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\u00C2\u00B0C. Colony appearance at 25\u00C2\u00B0C. 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\u00C2\u00B0C. True mycelium was formed at 25\u00C2\u00B0C. as w e l l as at 15\u00C2\u00B0C. 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\u00C2\u00B0C. 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\u00E2\u0080\u009E. 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\u00C2\u00B0N. 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\u00C2\u00B0C. (\"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\u00C2\u00B0C. . 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 \u00C2\u00B0 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) \u00E2\u0080\u009Em. as opposed to (7-20) X (7-13) m^. 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\u00C2\u00B0C. 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\u00C2\u00B0C. to 10\u00C2\u00B0C. 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\u00C2\u00B0C. 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\u00C2\u00B0C. 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\u00C2\u00B0C. (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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"Thesis/Dissertation"@en . "10.14288/1.0095481"@en . "eng"@en . "Botany"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "The genus Leucosporidium in Southern British Columbia, an area of temperate climate"@en . "Text"@en . "http://hdl.handle.net/2429/22693"@en .